Contents

Membership of the lOth Pharmacopoeia Commission of the People' s Republic of China ·····•············ ID
Working Committee of Pharmacopeia of the People' s Republic of China (2015) ··························· VII
Editorial Board of Pharmacopeia of the People' s Republic of China (2015) Volume N ·················· VIIl
Preface •· · ··• ··· ·•· •· · · · · · · · · · · · •· ·· · · ·· · · · · · · ·· · ·· · · · · ··· · · · · ·· · · · ·· · · · · · · · · · · ·· · · · · · ·· · · · · · · · · · •· · · ·· ·· · ··· · ·· ·· · · · · · · · · · · · · · · · · IX
History of the Pharmacopoeia of the People' s Republic of China · · · ·•• ·· · · ·· · ·· · · · •·· · ·· · · · · · · · · · · · · · · · · · · · · · XIII
General No tices · · · · · · · •· · · · · · · · · · · · •· · · · · · · · •· · · · · · · · · · · · · · · •· · · · •· · · · · · · •· · · · · · · · · · · •· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · :xxl.V"
Contents of General Chapters ·· · ••• ··· · ·· •·· ·· · ··· · · · · · · ·· · · · · · · · · ·· · · · · · · · · · · ·· ·· · · ·· •·· •· · · ·· · ·· · · · · ·· · · · · · · · · · · · · · · · · ·· 1
Monographs Part l General Chapters ····································•······································ ······ 1
Monographs Part Il Pharmaceutical Excipients · ·• · ·· · · · ·· · · · · · ·· · · · · · · · ·• ·· · · · · · ·· ·· · · ·· · ·· · · · · · · · · · · · · · · · · · · · · · 477
Index ............................................................................................................................................. I -1
 Membership of the lOth Pharmacopoeia Commission
of the People' s Republic of China

Honorary Chairman:
SANG Guowei (~ffi¡J.!)

Chairman:
CHEN Zhu (~M")

Executive Vice Chairman:

SHAO Mingli rn~im.il.)

Vice Chairmen:
CHEN Xinnian C~~if::) YU Wenming CrXim) WU Zhen (~~)

(From below: arranged alphabetically according to the names)

Members of the Executive Committee:
CAO Hongxin Cl?tfüvO CAO Xuetao Clr~~) CHEN Kaixian C~~5'6)
CHEN Keji (~Jl]'J() CHEN Saijuan C~~OrD CHEN Xinnian C~~if::)
CHEN Zhinan C~;=.!;Wij) CHEN Zhu (~M") DING Jian CT{il)
DU Xiaoxi (f±~llft) GAO Runlin C~~fJ~) GU Jianren CJ®lfitÁ)
HOU Huimin C-f*;i!~) LI Dapeng C*:kR) LI Guoqing C$ffil.!J¿)
LI Lianda C*:i!~) LI Yunlong (*~:ft) LIU Changxiao C:XtJ lf1t 1J:)
PENG Dongping C~*Jfl-) QIAN Zhongzhi C~,[',1D QIU Guixing (~:f)t~)
SANG Guowei C~ffiJJ!) SHAO Mingli rn~im.fO SHEN Beifen Ctt#f~)
SUN Yan (~]\!~) WANGJu CI@-) WANG Lifeng CI.il.$)
W ANG Ningsheng CI r1:.) WANG Ping CIJf) WANG Yongyan CIJ)<.~)

WU Yiling C~~tlit) WU Zhen (~~) XIAO Peigen C~ !fHLü

YANG Baofeng (fm~~) YANG Zhe (fmtf) YAO Hong C:!&~1i)
YAO Shouzhuo (~j~~fttl) YAO Xinsheng (j&~~1:_) YU Dequan CriwHjü
YU Wenming CrXim) YU Yongxin Cwuk~) ZHANG Boli C~-lHá*U
ZHANG Wei (jf,E{t) ZHAO Kai C~f'B) ZHEN Yongsu CliJj(~)
ZHOU Fucheng C)WJffi,m(;) ZHUANG Hui CJ±~)

Members:
Ajiaikebaier • Aisa (~iiJ=$f)t5lff$ • )t~) Aowuliji C~.º1jJ~) BA Xinguo ( E!.f~f ffil)
BAI Zhengzhong ( ÉJI§t,[',) BAO Jiake C~~f-4) BI Kaishun ( ~ 7f J@D
CAI Baochang ( ~ ~ lf1t) CAI Meiming C~~im) CAI Shanying C~IJ-~)
CAI Shaoqing C~Y'W) CAO Hui C"fr~) CAO Xiaoyun Clr~~)
m
CHANG Junbiao C1jt~f¡~) CHEN Daijie CMH"t~) CHEN Daofeng C~:i]i~)
CHEN Gang (~tlXj) CHEN Guiliang (~~~) CHEN Huipeng C~mg)
CHEN Kaixian C~~)t) CHEN Nan (~~) CHEN Shengdi (~~~)
CHEN Shilin C~±l~O CHEN Wansheng (~JJ~) CHEN Wei (~Jlj)
CHENG Pengfei CWJ!I~) CHENG Yiyu CWJl*) CHENG Zuoyong Clif'Fffl)
DAI Hong (jl~I) DAI Zhong (ji,~,) DENG Kaiying (x~ff~)
DING Lixia CTBlf~) DING Tianming C~:Xim) DONG Guanmu (JI-**)
DONG Shunling (jiJ119!~) DU Guangli cm)fL) DU Guanhua (f±m~)
DU Zenghui Cf±~~) DUAN Jin' ao cm~Dif) DUO Jie (~~)
FAN Huihong C11UitD F AN Ying Cm:@!) FENG Fang 01?7%=)
FENG Li O'l?, 1919) FENG Yi C/1?,·tit) GAO Chun (~~)
GAO Hua (~~) GAO Liqin C~1L.llJ) GAO Qipin C~Jt.fit)
GAO Shen (~$) Gesangbazhu cm~E.~) Gesangsuolang cm~~M)
GU Zhengyi (}®!~~) GUO De'an C~~g() GUO Dianwu ($15~~)
GUO Hongzhu C$15#t:ffl) GUO Qing C$151f) HE Langchong ( ~~ flf:t)
HE Yanlin CfnJ~#) HE Zhonggui ( fnJ {lftjt) HONG Liya C#tflj:f9r)
HU Changqin (i¡J1~llJ) HU Xin (¡~JffrX) HUA Baojin C{t~~)

HUANG Luqi (Jl'$1*) HUANG Min CjU~) HUANG Yaozhou O!it~rfN)

HUANG Ying Cj{f:l) JI Shen (*$) JI Yang (ftl¡fm)
JIA Liqun C:~f\rlf) JIA Tianzhu CM:Xf-t) JIANG Hong (~~)
JIANG Liangduo (~~~) JIANG Lin C~]if*) JIANG Xiongping C~ktV-)
JIANG Yingqiao Cü~fj'f) JIANG Yun (Ü~) JIN Fang (~Jr)
JIN Shaohong (~j;'~) JIN Yulan C~r ~) JIN Zhengyu (~{¡E*)
KANG Shuanglong (*~~) KONG Lingyi GL~ JO KUANG Anren C~::t({=)
KUANG Haixue (~~~) LI Bo C*tlt) LI Dakui C*::kfl:)
LI Gao C$~) LI Huilin (*~#) LI Jingyun C*tiií~)
LI Jun (*~) LI Lingling (*~~) LI Ning c*r)
u Ping c*n) LI Qihan C*1*~) LI Suzhi C*lt~)
LI Wenli (*jcfij) LI Yongxue C*i!'§'') LI Yuhua (*.3i~)
LI Yunxia (*~!t) LI Yuzhen (*.3i$) LI Zhenguo C*11&1~D
LIAN Hongzhen (~!~11&) LIANG Maoxin C~~jf) LIANG Zhenglun (~$le)
LIN Mei C#f[i) LIN Na C#MID LIN Ruichao C#JffifíHD
LIN Wenhan C#)C~) LIUBaokui CXIJ~~) LIU Dawei CXIJ::k1'J)
LIU Haijing CXIJ~ft) LIU Haiqing CXIJ~W) LIU Hao CXIJ$)
LIU Hongning CXIJ~r) LIU Jianxun CXIJilWJ) LIU Juyan CXIJ~Mf)
LIU Ping CXIJ1JZ-) LIU Tonghua CX!Jlfii]~) LIU Youning CXIJ X r)
LIU Yuling

LUO Jianhui
CXIJ.3i~)
LU Qiuhong c•fkg:I)
(~}l~)
LU Jing c•rt)
LU Weixing
LUO Ping
e•
(~ n)
J!~)
LU Minyi CMffit{)O
LUO Guoan (~ 00::t()

LUO Yuehua (~Jllé~)

LUO Zhifu C~~:f¡) LUO Zhuoya (~~Ji) LÜ Aiping C§ ~1JZ-)
LÜ Peiyuan ( §~t)j) LÜ Yang ( §fm) MA Chen (!Ji~)

MA Rong C!bi!k) MA Shuangcheng C!Ji~,mt) MA Yunan C!b.3i~)

MAO Xiangjun (~roJ~) MEI Zhinan CftiZm) MI Yaxian ( *SW.il*J)
MING Quanzhong ( im ~,~,) NA Shengsang OJ~~~) NAN Nan Cm~)
N
NI Jian C@{it) NI Weifang (@gt~) NIE Xiaochun C1I1J\~)
NIMA Dunzhu (J~J~tf3*) PAN Weisan (ji]!-:::::) PAN Xiqiang Cili!Si)
PAN Yang Cil!SED PEI Xuetao (~*~) PENG Cheng (~,Qlt)

PU Jinhua C~+~t$) PU Xufeng Oil:Jtl!~) QI Zhongtian (~ 9=' EH)

QIAN Jiaming (~~P~) QIAN W eiqing ( t\ttm) QIU Moyan (~fjt~)

RAO Chunming (~~~) RUAN Li (~jJ) RUI Jing C~if)

SHEN Kunling ( $ Fe.~) SHEN Pingniang CttV-IU SHEN Qi Ctt~)
SHEN Xinliang (t,t,c,. ~) SHENG Jinfang (tlg~@) SHI Dazhuo (~jcJj!)

SHI Jiangong C1im:9J) SHI Yaqin Cñm;5W.~) Silafu • Aibai CWT1Jrm • )t É3)
Sulaiman • Halike ($*~ • PfiJJ1D SUN Jianning e#mr) SUN Lingling (pj\~~)
SUN Ningling (pJ\r~) SUN Piaoyang ( pj\lfiliJ.J) SUN Wenji (pj\)C~)

SUN Xiaobo CPN!ítrBO TAN Renxiang ( it f=ff) TANG Qisheng cmJa~)

TANG Suoqin cmiJJi!JJ) TANG Xudong cm1IIL*) TAO Qiaofeng (~JJJXt)
TIAN J iahe CEH g 7F:) TIAN Ruihua CEH Jfflf$) TONG Xiaolin (~Jj\;fi\)

TU Jiasheng (~~~) TU Pengfei CM!llllD WANG Bailu CIÉ3ji)

WANG Chengde CI*~) WANG Chunlong CI~jt) WANG Dayou CI:::kWO
WANG Guozhi CI 00 #t) WANG Hao CI~) WANG Jie CIIDf)
WANG Jie CI~) WANG J unzhi CI~~) WANG Qingguo CIJ)¿OO)
W ANG Qingquan ( IJJ¿~) WANG Qingzhou Ciiff.t) WANG Rongfu CI~ffi)
WANG Ruwei CI~U{t) WANG Tiejie Cif!k~) WANG Wei CI~lü
WANG Xiaoliang CIBfá f.$!.) WANG Xijun CI:&~) WANG Xuan CIJi)
WANG Yan CI~) WANG Yong CI~) WANG Youchun CI#i~)
WANG Yu (I.35.) WANG Yuesheng (.:E~~) WANG Yuming C.:E*OO)
WANG Zhengtao CIJI$~) WANG Zhibin CI~~) WANG Zhimin CifW~)
WANG Ziling CI~~) WEI J ialing CRA ~) WEI Lixin Cftil.!JT)
WEN J ingwei ( ¡1ij ~1t) WU Chuanbin (~f~~) WU Song (~f'~)
WU Yuzhang ( ~.35. ~) XIAO Wei ( ~{t) XIAO Xiaohe C~ 1NPJ)
XIAO Xinyue Cf:'f ¡f fJ ) XIE Guilin ( iti:ft#) XIE Ning Citir)
XIE Zhijie ( iti~il!f) XU Anlong (~g(jt) XU Binghe C~~rñJ)
XU Fei (~~) XU Lihua (~88$) XU Yucong (~.~JI;~)
XU Zhikai C~~®D YANG Dajian (fmjc~) YANG Huaxin (fm{t!JT)
YANG Huichuan (fmt[Jll) YANG Jianhong Cfmm~D YANG Liang (fm~)
YANG Ming (fm~) y ANG Shilin ( lm1tt#) YANG Xiaoming (fmlJfé~)
YANG Xiuwei (fm~{~) YANG Yongjian (fm7j(.) YAO Naili (~~]J;fL)
YE Jiuzhi CittAzJ YE Wencai (itf)C~) YE Zuguang ( itf t§.:J't)
YIN Chunhua (Ep~$) YIN Hongzhang (~!(~) YIN Jun (Jji~)

YOU Qidong (jtJa ~) YU Boyang C~{Él II8) YU Hui ( lfrr*-')

YU Li C-/kil.) YU Shishan (~1:(L1J) YUAN Jun Ca~)
ZENG Lingbing ( 1!l/ ~{;je) ZENG Ming ( 1!l/ im) ZENG Su C1!l/$)
ZHANG Aihua C5*~$) ZHANG Baoxian C5-K{~t¡tjX) ZHANG Boli C5-lHá;fL)
ZHANG Fengchun C5*~~) ZHANG Liqun ( 5*.1r.lf) ZHANG Lirong C5*88~)
ZHANG Manlai (*mí*) ZHANG Mei C5-KJ&) ZHANG Peipei C5*~~)
ZHANG Qiang C5*5i) ZHANG Qiming (*Ja~) ZHANG Qingbo C5*mtlf)
ZHANG Qiusheng C*fJc~) ZHANG Shumin C*Eff~) ZHANG Weidong (*JI*)
V
ZHANG Xiaoqian (jlE1j\1Hf) ZHANG Yajie (jlESlf~) ZHANG Yuying (jlE~~)
ZHANG Zhirong (jlE~5f¿) ZHANG Zunjian (jlE:@;m) ZHAO Jianbang C~m~n
ZHAO Kai (~fi3) ZHAO Ming (~f!ij) ZHAO Ming (~f!ij)

ZHAO Ruihua (~JW$) ZHAO Weiliang (~!fE~) ZHAO Zhongzhen (~q:t~)

ZHENG Tai (~fl) ZHENG Xiaoli (~~BliJ) ZHONG Bohua ({~{S$)

ZHONG Dafang C#:XJJX) ZHONG Gansheng (#-~) ZHONG Guoyue (#00~)

ZHONG Ping ( {~1f) ZHONG Ruijian ( #llAfm) ZHOU Jianping Cffllm-V-)
ZHOU Kai CfflJ®D ZHOU Lichun (JWJ.1l.w) ZHOU Xu (}WJJ1ª)
ZHU Jun (*~) ZHU Liguo (*.1l.00) ZHU Ming ( fJl f!ij)
ZHU Xiaoxin (*~lff) ZHU Yi (*~V ZOU Quanming (~~~f!ij)

VI
 Working Committee of Pharmacopeia of the People' s
Republic of China (2015)

Director in General:
ZHANG Wei C5{Ef~)

(From below: arranged alphabetically according to the names)

Deputy Director:
GUO De'an (~~1() JIN Shaohong (~Y'~) LAN Fen (~~)

LI Bo (*7Jf) LUO Guoan (~ 00$:.) QIAN Zhongzhi ( f&,i!U~D

W ANG Ping CE-V-) WANG Youchun (J:{ti~) W ANG Zhengtao C.I:tl$~)

Members:
CHEN Daofeng (~:J1!dl!) CHEN Gang C~m) CHEN Guiliang ( ~;lt ~)
CHENG Yiyu (~fi.f) GUO Shengqi (~~rJO GUO Zhongping (~ 9='-V-)
HONG Liya C#t~Jtsí) HONG Xiaoxu C#t1J\~) HU Changqin ( ijij §!t lb)
LI Huiyi C*llSO LU Jing e•*) LUO Zhuoya (~ Jiift)
LÜ Aiping (§~1f) MA Shuangcheng ( .Q, ~JVt) NAN Nan (~~)
SHI Jiuhua (~,!A~) SHI Shangmei (1j _t.ffi) TANG Sufang (}8~~)
TU Jiasheng (~*~) WANG Junzhi (J:~~) WANGYu C±~)

YE Min (ittíi{) YU Li (~j'[) ZHANG Mei C5fE:i&)

ZHANG Mian (*~) ZHANG Qiang ( 5-lt5i) ZHANG Weidong C5-LEJI*)
ZHANG Xiaoqun C5-Lt~iM) ZHU Ming C;fi~)

Vil
 Editorial Board of Pharmacopeia of the People' s
Republic of China (2015) Volume N

Editor-in-Chief:
LI Bo C$tJt) LUO Guoan C~m!g()

CFrom below: arranged alphabetically according to the names)

Associate Editor-in-Chief:
CHEN Gang c~m)
HONG Liya (ijtftjj{f)
LUO Zhuoya C~ ljift)
TU Jiasheng C~*~)
ZHANG Xiaoqun C5!E~ilff)
CHEN Guiliang C~{E J§!) CHENG Yiyu C~Jlf)
HONG Xiaoxu CiJt1N··~) HU Changqin C¡!jij l§ iJJ)
MA Shuangcheng C.:fb~n\t) T ANG Sufang cm~~)
WANG Yu C_I~) ZHANG Qiang C5-lE5i)

Members of Editorial Cmmnitlee:

CAO Xiaoyun ClfBfE~) CHEN Gang c~m) CHEN Guiliang C~{E~)
CHEN Ying C~~) CHENG Yiyu C~Jlf) FAN Xiaohui Cffl:~~)
GUO Hongzhu C~iJt*'L) HONG Liya CiJtfrjj{f) HONG Xiaoxu (ijt;j\f~)

HU Changqin C¡!jJ]t'§ltJ) JI Shen C*$) ]IN Guimin Cf)f f:E ~)

LI Bo C$tJt) LIANG Qionglin C~Jffi-) LIU Yanming CXIJRIP~)
LUO Guoan C~ ffi!~) LUO Zhuoya C~ ljift) MA Shuangcheng Clb ~n\t)
SHANG Yue CrAJ·m) SONG Zonghua C5K*$) SUN Chunmeng C~J,#"WJ)
SUN H uimin C~J,~fit) TANG Liming cm~~) T ANG Sufang cm~~)
TU Jiasheng C~*~) WANG Tiesong C.I/fkf't) WANG Yu C.I~)

WEI Feng Cft'tf) XU Huayu Ci'f'.$~) YANG Meiqin Cfm~~)

ZHANG Qiang C5*5i) ZHANG Xiaoqun C5-LE~ilff) ZHANG Xuan C5-lEfil)
ZHENG Jinqi C*'5~JJ0
Robert P. Barron

Co-workers:
CHEN Yue c~·m) CHONG Xiaomeng 0*1j\WJ) DAI Zhong C~.~,)
HONG Jianwen O;!t~)C) KANG Xiaobo C~~tf) XIE Yuanyuan CiJtki~)
YANG Chunyu C;fm#f:f:D YANG Meicheng C;fm~n\t) y ANG Rui cfmfJl.)

VIII
 Preface

The Pharmacopoeia of the Peo ple' s Republic of China 2015 Edition ( hereinafter referred to as the
"Chinese Pharmacopoeia" ) is the lOth edition of Chinese Pharmacopoeia. All members of the Chinese
Pharmacopoeia Commission (ChPC) and the staffs of its permanent institution have worked diligently to
complete the compilation of Chinese Pharmacopoeia 2015 edition in accordance wit.h the guiding concepts,
basic principies, objectives and requirements set by the Pharmacopoeia Compilation Outline adopted at the
Founding Ceremony and Plenary Session of the lOth Chinese Pharmacopoeia Commission under the
leadership of the China Food and Drug Administration ( CFDA) , the vigorous support and assistance of
drug control institutions, research i~stitutions and universities at various levels, as well as the proactive
participation and coordination of drug manufacturers. On February 4, 2015, the Plenary Session of the
Executive Committee of the lOth Chinese Pharmacopoeia Commission adopted this edition of
pharmacopoeia, which was approved by the CFDA on June 5, 2015 and carne into effect as of December
1, 2015.

The Chinese Pharmacopoeia (ChP) 2015 edition comprises volumes I , 11, fil and N and contains a
to.tal of 5, 608 monographs, including l, 082 new monographs. Volume I contains a total of 2, 598
monographs of medicinal materials and the prepared slices of Chinese crude drugs, vegetable, oil fat and
extracts, singie-item preparations, etc., ·inciuding 440 new monographs, 517 revisions and seven
rejections. Volume 11 contains a total of 2, 603 monographs of chemical drugs, antibiotics, biochemical
drugs and radioactive drugs, including 492 new monographs, 415 revisions and 28 rejections. Volume fil
contains a total of 137 biologics, including 13 new monographs, 105 revisions and six rejections. In arder
to address such problems as repetitive inclusion of testing methods and lack of coordination, consistency
and standardization among various methods, this edition of pharmacopoeia has integrated the common
appendices of various volumes of pharmacopoeia and renamed the original appendices into General
Chapters, including general requirements of preparations, testing methods, standard substances,
reagents and guidelines. A standard coding system has been established, the general chapters and
pharmaceutical excipients have been separately included into volume N of the Chinese Pharmacopoeia
Volume N contains a total of 317 general chapters, including 38 general requirements for preparations,
240 testing methods, 30 guidelines and nine standard substances and testing solutions and reagents; 270
monographs of pharmaceutical excipients, including 137 new monographs, 97 revisions and two
rejections.

This· edition of pharmacopoeia is characterized by the following features:

The number of included products has been s~gnificantly increased. The scope of products included has been
further expanded to initially achieve the full coverage of biologics in the Catalogue of National Essential
Medicines and more than 90 % of coverage for tradition Chinese medicine ( TCM), and chemical drugs.
Adjustment has been intensified for certain products with incomplete standards, suspension of manufacturing
for many years, excessive clinical adverse reactions and unreasonable dosage forms. This edition of
JX
pharmacopoeia no longer contains a total of 43 products originally included under the ChP 2010 edition.

The phannacopoeia standard system has been further improved. Various appendices of the previous version of
pharmacopoeia have been integrated into the volume N of this edition of pharmacopoeia. The revision has
improved the pharmacopoeia standard system with general notice, general chapters and monographs as
overall, basic and specific requirements respecti vely. F or the first time, it has incl uded "Guidelines for
Preparation of Pharmaceutical Standard Substances of China", "Guidelines for General Requirements for
Drug Packaging Materials and Containers" and "Guidelines for Pharmaceutical Glass Materials and
Containers", and created a more comprehensive pharmacopoeia standard system encompassing drug
substances and their preparations, pharmaceutical excipients, drug packaging materials and standard
substances.

The application of modem analytical technology has been expanded. On the basis of retaining conventional
testing methods, this edition of pharmacopoeia has further expanded the application of new technologies
and new testing methods to increase the sensitivity, specificity and stability of testing. The following
methods have been employed for the quality control of TCM, including liquid chromatography, tandem
mass spectrometry, high-performance liquid chromatography, inductively coupled plasma mass
spectroscopy, etc. The following methods of quality control have been employed for chemical drugs,
including supercritical fluid chromatography, liquid chromatography at critical condition, powder x-ray
diffraction, etc. In addition, the new edition of pharmacopoeia has also adopted capillary electrophoresis
for the testing oí molecular isomers of monoclonal antibody products, and adopted high-performance
liquid chromatography for the testing of molecular size distribution of antitoxin and antiserum product,
etc. For the reserves of testing technology, the new version of pharmacopoeia has established method for
DNA barcode molecular identification of Chinese herbal medicine, pigment testing method, method for
testing of fungal toxin in TCM, near-infrared spectrum method, drug evaluation technology based on
gene chip, etc.

Drug safety assurance has been further improved. The new edition of pharmacopoeia has improved "General
Principle for lnspection of Crude Drugs and Decoction Pieces", "The Processing of Crude Drugs" and
"General Requirements for Pharmaceutical Excipients"; and newly included "General requirements for
National Pharmaceutical Reíerence Standards", "Requirements for Quality Control of the Raw Materials
and Excipients Used for the Biologics Production", "General Monograph for Vaccines for Human Use",
"General Monograph for Recombinant Monoclonal Antibody Products", etc., and newly included relevant
guidelines for microparticle preparations, drug crystal form research and crystal form quality control,
establishment of limit for harmful residue of TCM. Volume I has set the limits for sulfur dioxide residues
in Chinese herbal medicines and prepared slices of Chinese crude drugs, established the limits for harmful
elements in marine drugs such as pearls and seaweeds, set inspection standards for 16 pesticide residues
including organic chlorine in ginseng and American ginseng products, and newly included the inspection
item and limit of "aflatoxin" for 14 Chinese herbal medicines and the prepared slices of Chinese crude drugs
including Platycladi Seed. Volume II has further strengthened the control of relevant substances,
enhanced the system applicability requirements oí testing methods, and included the structural
information of about 500 impurities; included the control of chiral impurity; included osmolarity testing
for intravenous infusion and eye drops and control requirements for antimicrobial agents in injections and
eye drops. Volume ID has enhanced quality control oí raw materials and excipients used for the biologics
production, standardized the use of antimicrobial agents, and strengthened the control of residual
X
solutions; included the osmolarity testing, revised the genome sequence testing of virus seed lot used for
the vaccine production and stricted the limits for bacterial endotoxin.

Drug efficacy control has been further improved. Testing methods have been revised in a comprehensive
manner. Volume I has included the specificity microscopic identification and inspection of Chinese herbal
medicines and the testing of characteristic amino acid content, etc. , established characteristic spectrums
for more than 30 standards including the root of red-rooted salvia. Volume 11 has adopted iron
chromatography for the testing of acid group content in sulfate or hydrochloride drug substances; adopted
methods with greater specificity and accuracy for the testing of preparation content; revised the methods
for the inspection of the solution and releasing rates, and strengthened control of oral solid dosage forms
and modified-release preparations.

Standards for pharmaceutical excipients have been significantly improved. This edi tion of pharmacopoeia has
included multiple specifications of pharmaceutical excipients in series to meet the needs of pharmaceuticals
manufacturing. twenty-one injection-grade excipients have been newly included. Safety control has been
enhanced for pharmaceutical excipients such as inclusion of control requirements for residual solutions.
Greater attention has been paid to the functional evaluation of excipients. For instance, such inspection
items as multi-porosity, powder fineness, powder flow ability, specific surface area and viscosity have
been included and the requirements for the research of applicability of standards for pharmaceutical
excipients have been enhanced.

Guiding effect of pharmacopoeia has been further strengthened. Guiding effect of this edition of
pharmacopoeia for drug quality control has been enhanced through the screening and adjustment of
products, adopting the advanced testing methods and formulation of technical guidelines; meanwhile, m
light of the tendencies of international drug quality control and standardization as well as the realities of
drug manufacturing in China, equal emphasis has been given to the safety and accessibility of medications
in the configuration of inspection items and limits in order to guide the sound and science-based
development of pharmaceutical industry in China.

This new edition of pharmacopoeia continues to follow the concepts of protecting the wildlife and
environment and adhering to the sustainable development and promotion of green standards for TCM. For
instance, newly included prescriptions no longer contain the Chinese patent medicines of endangered
species or fossils such as leopard bone, antelope' s horn, fossil fragments and dens draconis; replacements
of toxic solutions in testing reagents are advocated, e. g. , the use of reagents containing benzene and
mercury is abolished in order to reduce pollution to the environment and laboratory personnel.

The new edition of pharmacopoeia has been drafted in a more public, transparent, standardized and orderly
manner. Drafting process of this edition of pharmacopoeia has always adhered to the principies of openness,
fairness and justice. The permanent institution of the ChPC has introduced the IS09001 quality
management system ( QMS) into the whole-process management of pharmacopoeia standards
development, continuously improved administra ti ve system for ChPC and standardized working procedure
for pharmacopoeia drafting to ensure the quality of pharmacopoeia developing process. ChPC has
vigorously promoted drug standards and scientific research to ensure the progress and quality of
pharmacopoeia drafting. Efforts have been made to strictly follow the "working procedure for the
developments of ChP", improve the communication and coordination among professional committees, and
XI
enhance the review and publication of standards. All the additions and revisions of standards have been
published at the website of ChPC and the results of expert review and feedback comments have been
published as well.

On the basis of maintaining scientificity and standardization · of pharmacopoeia, · this edition of

pharmacopoeia emphasizes on enhancing the control requirements of drug safety and efficacy, referencing
internationally advanced quality control technologies and experiences, improving the level of this edition of
pharmacopoeia, and reflecting current situations on pharmaceutical development and testing technologies
in China. lt will also play ah important role on promoting drug quality improvement, accelerating
corporate technology progress and product upgrades, expediting the healthy development · of
pharmaceutical industry and increasing the authoritativeness and international influence of the Chinese
pharmacopoeia in China.

Chinese Pharmacopoeia Commission

June 2015.
 History of the Pharmacopoeia of the
People' s Republic of China

Chines e Pharmaco poeia 1953 Edition ( First Edition) The Chinese Communist Party and the Chinese
Government have attached great importance to medical and health-care for the Chinese people. The
People's Republic of China was founded on October 1, 1949 and right in November of that year, the
Ministry of Health convened a meeting of medical and pharmaceutical experts in Beijing on the compilation
of a pharmacopoeia. In January 1950, the Ministry of Health invited Professor Meng Mudi, a well-known
pharmacist from Shanghai, to take up the responsibility for the establishment of the Editorial Commission
of Pharmacopoeia of China and its secretariat to deal with daily work concerning the compilation of such a
compendium for new China.

In April 1950, a working semmar was held in Shanghai, at which the principies and guidelines on the
selection of monographs were discussed and the monographs to be included in the Pharmacopoeia were
decided. It was recommended under the direction of the Ministry of Health that the new Pharmacopoeia
should be compiled in such a way that it is in conformity with the Chinese situations and that it should be
nationalistic, scientific and popular in nature. Thereafter, The Ministry of Health invited 49 experts as
members and 35 as correspondent members of the Commission who were appointed to 8 panels
( nomenclature, chemicals, pharmaceutical preparations, medicaments of plant origins, biological
products, medicaments of animal origins, pharmacology, and dosage) respectively. Health Minister Li
Dequan served as the chairperson of the Commission. The first Editorial Commission of Pharmacopoeia of
the People' s Republic of China was thus formally established.

The first Editorial Commission meeting composed of all members was held in Beijing April 24-28, 1951,
where resolutions were made on the title of the Pharmacopoeia, list of selected monographs, the
nomenclatures, units of measurement and weights, format, the order of arrangement etc. Based on
recommendations from the Commission meeting, the draft of the pharmacopoeia was then revised by the
secretariat and submitted to the Ministry of Health for review and the Culture and Education Commission
of the State Council for approval at the end of 1952. The Ministry of Health published the first Chinese
Pharmacopoeia in 1953.

Chinese Pharmacopoeia 1953 edition contained 531 monographs of substances and articles, including 215
chemicals, 65 medicaments from plant origins, oils and fats, 13 medicaments from animal origins, 2
antibiotics, 25 biological products, and 211 pharmaceutical preparations. After the publication of the
Pharmacopoeia, the first addendum of the 1953 edition was published in 1957.

Chines e Pharmaco poeia 1963 Edition ( Second Edition) The Ministry of Health set up the second
Pharmacopoeia Commission in 1955, with 49 members and 68 correspondent members. Due to vanous
reasons, this Commission failed to fulfill its mission. The third Commission was established in 1957, with
80 members (no correspondent members appointed) and Professor Tang Tenghan, a well-known
pharmaceutical chemist, as its chairman. The first meeting of the third Commission was convened from
J uly 28 to August 5 of the same year. Health Minister Li Dequan pointed out at the meeting that it was a
big flaw that the first Pharmacopoeia did not cover Chinese traditional medicines that the Chinese people
were so used to. At the meeting principles in compiling a Pharmacopoeia were made and nature and
purpose of such a compendium were discussed and constitution of the Commission was revised. It was
agreed unanimously to admit well-defined Chinese traditional medicines to the Pharmacopoeia. On August
27, six expert committees anda panel under the Commission were approved and set up by the Ministry of
Health, namely, the committees of medicines, chemicals, pharmaceutical preparations, biochemicals,
pharmacognosy and biological products, and a panel of nomenclature. Under the Commission a Standing
Committee was organized, however, routine work in general was dealt with by its Secretariat.

In 1958, it was recommended by the Standing Committee and approved by the Ministry of Health to invite
8 doctors of Chinese traditional medicine and 3 experts of Chinese traditional medicaments as members of
an expert committee dealing with the quality specifications of crude drugs used as Chinese traditional
medicaments and Chinese patent preparations. Collaborative efforts were made by experts in this field in
many parts of this country to incorporate the theory and practica! experience of Chinese traditional
medicine into the monographs concerned.

The second meeting of this Commission was held in Beijing from June 25 to July 5, 1959. A list of
monographs being admitted to the new Pharmacopoeia was proposed and the draft texts reviewed in detail
by the expert committee concerned. The work was accomplished in 1962. The State Council approved the
publication of the Pharmacopoeia of the People's Republic of China 1963 edition. On January 26, 1965,
the Ministry of Health issued a document for the Chinese Pharmacopoeia 1963 edition and the relevant
provisions for its implementation.

The Chinese Pharmacopoeia 1963 edition contained 1310 monographs in its two volumes, each with
separated General Notices and relevant appendices. 446 monographs of commonly used Chinese traditional
medicaments and 197 monographs of Chinese traditional patent preparations were admitted to Volume I ,
and 667 monographs of chemical drugs were admitted to Volume II. Additionally, "therapeutic function
and chief indication" were stated in the monographs admitted to Volume I and "action and use" of those
admitted to Volume I1.

Oiinese Pharmacopoeia 1977 Edition (Third Edition) The Pharmacopoeia Commission stopped functioning in
1966 due to the turmoil caused by the "Cultural Revolution". On April 28, 1972, the State Council agreed to the
suggestion in the report of the Ministry of Health that "the Commission should be re-established with the
participation of Ministries of Health, Petroleum and Chemical industry, Commerce and the PLA's Ministry of
Health, headed by the Ministry of Health". A working meeting of the Pharmacopoeia Commission was convened
under the above direction from May 31 to June 10 of the same year in Beijing. 88 Representatives of various
competent authorities and organizations, including all Provincial (Autonomous regional or Municipality under the
Central Govemment) Departments of Drug Policy and Management, lnstitutes for Drug Control and others
attended the meeting. The focus of the meeting was on the guiding principle, working process, requirements and
objects of the editing of the national Pharmacopoeia. The revision plan was recommended after exchange of past
expenences in various aspects. Arrangements were made for the drafting of individual monographs by the
organizations concemed The second meeting of the Pharmacopoeia C,ommission was held in Beijing in April 1973.
Sorne guidelines, basic requirements of the Pharmacopoeia and sample monographs for Chinese traditional
medicaments and modem medicinal substance as well as the respective explanatory notes had been well discussed
and appropriate recommendations were made. The drafting of individual monographs was rearranged according to
the conditions of production of either crude drugs or pharmaceutical products. On October 4, 1979 the Ministry of
Health promulgated that the Chinese Pharmacopoeia 1977 edition would come into use on January 1, 1980.

There are 1925 of monographs contained in the 1977 edition. In Volume I , 1152 monographs were
admitted, including 882 monographs of Chinese herbal drugs in general use and used in the region of
national minorities, extracts of Chinese herbal medicines, oils and fats and sorne preparation made of
single medicinal ingredient. 270 monographs of Chinese traditional patent preparations also included in
Volume l. In Volume II , it contained 773 monographs of chemicals and biological products etc.

Chinese Pharmacopoeia 1985 Edition (Fourth Edition) In 1979 the Ministry of Health invited 112
experts as members to form the fourth Pharmacopoeia Commission, and Health Minister Qian Xinzhong
was its Chairman. The first plenary meeting of that Commission went from Nov. 22 to Nov. 28 in the same
year in Beijing. Discussion was made on the constitution of the commission, provisions for the management
of specifications for pharmaceutical preparations and its working plan. Ten specialized advisory groups
were appointed in the fields of Chinese traditional medicine, Chinese traditional medicaments, medicine
and pharmacology, chemicals, biochemicals, pharmaceutical preparations, antibiotics, biological
products, radiopharmaceutieals and nomenclature respectively. Monographs being admitted to the new
Pharmacopoeia were recommended by the advisory groups concerned. The advisory group on Chinese
traditional medicine had the responsibility to review and select the range of monographs to be included in
Volume I and the advisory group on medicine and pharmacology had the same responsibility for Volume
11. The institutes for drug control and competent authorities or organizations at the local level (provinces,
autonomous regions and municipalities directly under the central government), where the drug substance
concerned was produced provided draft text of individual, monograph with prominent experience and
excellent quality. Coordinated review and technical validation were organized by the secretariat. Sorne
monographs were drafted only after the completion of objective collaborative studies as required. Finally,
members of respective advisory groups and representatives of institutes for drug control and drug
manufacturers concerned reviewed the draft text, and then sent to the Ministry of Heath for approval. The
Chinese Pharmacopoeia 1985 edition was published in September 1985 as it was planned. The effective
date was set on April 1, 1986 as approved by the Ministry of Health.

In this edition of Pharmacopoeia, 1489 monographs of drugs were admitted. 506 monographs of Chinese
traditional medicaments, crude drugs, oils and fats and preparations of single ingredient, 207 monographs
of Chinese traditional patent preparations, totally amount to 713 monographs were admitted to Volume l.
776 monographs of chemicals, biological products etc. were admitted to Volume 11. The addendum to the
Chinese Pharmacopoeia 1985 edition was published in November 1987 with 23 new admissions and 172
specific monographs and 21 general monographs were revised or amended. Based on the Chinese
Pharmacopoeia 1985 edition, its first English version was formally published in October 1988. The
selected notes in Volume 11 were also published in that year.

The "Drug Administration Law of the People's Republic of China" carne into effect on July 1, 1985. It
stipulated that "the quality of drugs and medicines must comply with a national, provincial, autonomous
XV
regional or municipal standard", and that "the Pharmacopoeia of People's Republic of China and standards
for drugs and medicines published and promulgated by Ministry of Health of the State Council are national
standards for drugs and medicines". It further stated, "The Pharmacopoeia Commission subordinate to the
Ministry of Health of the State Council is responsible for the stipulation and revision of national standards
for drugs and medicines", it defines clearly the official nature of standards for drugs and medicines and
responsibilities of the Commission.

Chinese Pharmacopoeia 1990 Edition (Fifth Edition) In 1986 the Ministry of Health reorganized the
Pharmacopoeia Commission in accordance with its constitution and invited 150 experts as members of the
fifth Commission with Health Minister Cui Yueli as Chairman. The office dealing with routine work was
changed to a system with the Secretary General as its chief executive officer. The first meeting of this
Commission was convened on May 5-8 of the same year. The constitution of the Commission was revised.
Comments were made on the task of drug standardization during the seventh Five-year Plan for National
Reconstruction. The guidelines and principies of the 1990 edition of the pharmacopoeia were discussed and
agreed to at the meeting. Panel meetings on Chinese traditional medicaments, Chinese patent preparations,
chemicals, antibiotics, biochemicals and pharmacology were held respectively for the tasks of drafting
different parts of the next edition and necessary research projects to be carried out. By March 1989, the
draft text of the new pharmacopoeia was basically ready for comments through the efforts made by various
organizations and associations. The Executive Body of the Pharmacopoeia Commission was authorized to
organize for reviewing and editing. In December 1989, an extended meeting attended by the chairman,
vice-chairmen of the Commission and the chairmen of aii advisory groups was heid in Beijing. It was
recommended to send the draft text to the Ministry of Health for comments and approval. The Ministry of
Health then publishes it as the Chinese Pharmacopoeia 1990 edition, on December 3, 1990 with effective
date on July 1, 1991.

This edition of the pharmacopoeia was still published in two volumes containing 1751 monographs of
substances and articles, 784 monographs including 509 monographs of Chinese traditional medicaments
and 275 monographs of Chinese traditional patent preparations and single ingredient preparations were
admitted to Volume I , while 967 monographs on cheinicals, antibiotics, biological products and
pharmaceutical preparations were admitted to Volume II. In comparison with the preceding edition, 80
new monographs were admitted and 3 monographs deleted from Volume I; 213 new monographs were
admitted (including 5 monographs transferred from Volume I to Volume II) and 22 monographs deleted
from Volume I1. Appropriate changes had been made on titles of certain drug substances and articles as
required. The headings "Action and Use" and "Administration and Dose" were changed to "Category" and
"Dosage" respectively. "A Guide to Clinical Information" was published as a companion volume to the
Chinese Pharmacopoeia to serve as a guiding reference for medical and pharmaceutical practices. The
infrared reference spectra were deleted from the Appendix with the publication of a separate volume-the
Atlas of Infrared Spectra of Drugs.

Chinese Pharmacopoeia 1995 Edition ( Sixth Edition) The sixth Pharmacopoeia Commission was
organized in 1991, with 168 members, invited by the Ministry of Health, and Health Minister Chen
Minzhang as its chairman. The first meeting of that Commission took place on May 16 -18 of the same
year, attended by all members. In this meeting, the constitution of the Commission was further revised
and the working plan for compilation and editing of Chinese Pharmacopoeia 1995 edition were discussed
and substantial recommendations were made. A Standing Committee composed of the chairman, vice
XVI
chairman and 11 other experts was set up with 13 subcommittees in respective specialized fields, namely,
Chinese traditional medicine, Chinese traditional medicaments, Chinese traditional patent medicines,
Modem medicine, · Pharmacology, Chemicals l , 11 and 1II, Antibiotics, Biochemicals, Biological
products, Radiopharmaceuticals and Nomenclature. The subcommittees then convened extended meetings
in their specific fields, respectively, to work out the programs for revision of the Pharmacopoeia.

Drafts of the appendices of the 1995 edition were sent out in 1993 to relevant local organizations as a
reference for the compilation and revision of the new edition. By July 1994 almost all local drafting had
been finished and the subcommittees started organizing reviews. On Nov. 29, 1994 the drafts were
discussed and further reviewed atan extended meeting of the Standing Committee, and then submitted to
the Ministry of Health for approval. Chinese Pharmacopoeia 1995 edition carne into use as of April 1,
1996 as promulgated by the Ministry of Health.

There are 2375 monographs in that edition. 920 monographs, including 522 monographs of Chinese
traditional crude drugs and of oils and fats, 398 monographs of Chinese traditional patent medicines and of
preparations of single ingredient were admitted to Volume I. In Volume 11, it was composed of 1455
monographs of chemicals, antibiotics, biochemicals, radiopharmaceuficals, biological products and sorne
excipients. In comparison with the previous edition, 142 and 499 new admissions were admitted to Volume
l and 11 respectively. English titles of drugs and preparation were adopted in Volume 11 and the Latin
titles were deleted, while Chinese titles only use their official common names and no alternate names. The
first Volume (1995 edition) of "Atlas of Infrared Spectra of Drugs and Medicines" was also compiled.

"A Guide of Clinical Information" was revised, and published together with the 1995 edition of Chinese
Pharmacopoeia. Ministry of Health approved that the "lndication" and "Dosage" in the later should be
adopted as the basis for promotion of drug administration and management by competent authorities of
drug management and drug manufacturing.

The Sixth Pharmacopoeia Commission published the first and second addenda in 1997 and 1998,
respectively. It also published "Commentary to Volume 11 of Chinese Pharmacopoeia 1990 edition".
"Selected Commentary to Volume l of Chinese Pharmacopoeia 1990 edition", "Atlas of Traditional
Chinese Medicines", "Atlas of Thin Layer Chromatography of Chinese traditional Medicines" and
"Adopted names of Chinese pharmaceutical products" as references in series relevant to pharmacopoeia.
English version of Chinese Pharmacopoeia 1990 edition was published in July 1993.

To strengthen standatdization of drugs and medicines, the Ministry of Health decided, on May 21, 1993,
that the standing office of the Chinese Pharmacopoeia Commission separated from the National lnstitute
for the Control of Pharmaceutical and Biological Products and subordinated directly to the Ministry of
Health. That was an important reform measure in the restructuring of the Commission.

Chines e Pharmaco poeia 2000 Edition ( Seventh Edition) Approved by the Ministry of Health, the
seventh Pharmacopoeia Commission was set up in May 1996. The Ministry invited 204 members, including
18 honorary members, to form the Commission. Health Minister Chen Minzhang served as its Chairman.
In September 1998 as approved by Document No. 32 (1998) of Central Office of Organization, the name of
Pharmacopoeia Commission of the Ministry of Health was changed to the State Pharmacopoeia
Commission, and the administration of the Commission was transferred to the State Drug Administration
xvn
( SDA). Af ter change of the management system and passing away of Health Minister Ch en Minzhang, the
standing committee of the seventh Pharmacopoeia Commission decided to appoint chairman and vice
chairmen of the Commission, as so stipulated in its Constitution in December 1999. Mr. Zheng Xiaoyu
served as its Chairman Under that Commission there were 16 specialty subcommittees: Chinese traditional
medicine, Chinese traditional medicaments I , 1I , I1I, and N, modern medicine, nomenclature,
appendices, pharmaceutical preparations, pharmacology, chemicals I and 11, antibiotics, biochemicals,
radiopharmaceuticals and biological products.

The first meeting of the seventh Commission was held in 1996, at which the design plans for the 2000
edition of Chinese Pharmacopoeia were endorsed. The guiding principies were decided that the VolumeI
should have special features in content and its quality should be largely improved, and the Volume 11
should reflect the combination of improvement and suitability to specific situations in China as well as a
combination of advancement and characteristics. As scheduled in the plans, all subcommittees convened
their own meetings and carried out their tasks starting from October 1996. By the end of 1997, all revision
of the appendixes and general rules for drug and medicine preparations had been finished and sent to local
drafting organizations for comments. A first draft was finalized at the end of 1998, and after reviews by
related organizations in different parts of China; the 16 subcommittees further reviewed it by the end of
October 1999. Chinese Pharmacopoeia 2000 edition was finally reviewed and passed by the seventh
Pharmacopoeia Commission in December 1999, and submitted to the State Drug Administration for
approval of publication. This edition was published in January 2000 and carne into use as of J uly 1, 2000.

The 2000 edition contained a total of 2691 monographs, with 992 ones in Volume I and 1699 in Volume
11. There were 399 new monographs and 562 revised ones in this edition. Appendices were considerably
improved. There were 10 new and 31 revised appendices in Volume I , and 27 new and 32 revised ones in
Volume Il. The Volume 11 , for the first time, included 6 guidelines such as Validation of Analytical
Method adopted in Pharmaceutical Quality Specification etc., which will play a role in the standardization
and regulation of testing methods. Application of modern analytical techniques was further enhanced and
stressed in this edition.

The seventh Pharmacopoeia Commission has also compiled the 1997 and the 1998 addendum of the Chinese
Pharmacopoeia 1995 edition, the "Adopted Names of Chinese Pharmaceutical Products ( 1998
Addendum) ", "Atlas of Infrared Spectra of Drugs and Medicines" (Volume 1I) and the third edition of
"A Guide to Clinical Information". The English edition of Chinese Pharmacopoeia 1995 was published in
1997. To strengthen exchanges and cooperation, this Commission has decided to publish concurrently both
Chinese and English versions of Chinese Pharmacopoeia 2000 edition.

"Dosage" and "Precaution" in preceding editions were too simple to reflect accurately the actual clinical use
therefore deleted from this edition ( Volume 1I ) as so proposed in the design plans for the relevant
contents. The two headings were included in "A Guide to Clinical Information".

Chinese Pharmacopoeia 2005 Edition (Eighth Edition) Approved by the State Drug Administration
(restructured as the State Food and Drug Administration in September 2003), the eighth Pharmacopoeia
Commission was set up in October 2002. The State Drug Administration invited 312 experts as members of
the Commission and did not appoint any honorary member. The Commissioner of the State Drug
Administration, Mr. Zheng Xiaoyu served as its Chairman. The Standing Committee of the Commission
was assigned as Executive Committee. The plenary session of Pharmacopoeia Commission was authorized
to examine and approve Chinese Pharmacopoeia and the important items of the national specifications for
pharmaceutical preparations. 24 subcommittees of specific duty and/ or appointment were set up under the
Commission. On the basis of previous Commission, 3 new subcommittees were established: namely ethnic
medicines, microorganism, and packing materials and excipients; former subcommittee of biological
products was expanded to 6 ones, namely: blood products, viral products, bacteria! products, somatocyte
therapy and gene therapy, recombinant products and biological diagnostic reagents used in vitro.

The first meeting of the eighth Pharmacopoeia Commission and its Executive Committee took place in
October 2002, approved "the design plans for the 2005 edition of Chinese Pharmacopoeia ". The design
plans clearly indicated that the Pharmacopoeia should adhere to guiding principies of "succession and
development" and "theory combined with practice"; and editing principies of pharmacopoeia should be
adhered to scientific, practica! and specifical basis. The meeting decided that Requirements for Biologics of
the People's Republic of China, known as the Chinese Requirements for Biologics ( CBR), would be
integrated into the pharmacopoeia as its Volume ID; "A Guide to Clinical Information for Chinese
Traditional Patent Preparations" was to be compiled for the first time.

All designated subcommittee meetings had been convened since November 2002, to <leal with the assigned
duty recommended on the meeting in various aspects. By J uly 2003, the draft of appendices was
accomplished at first and sent to relevant authorities and institutions for comments. Early in 2004, the
first draft of appendices and text of pharmaceutical and biological products was basically ready and
consecutively published on the website of Pharmacopoeia Commission for 3 months, so as to get the
feedbacks from various organizations and associations. Subcommittees, one after the other, convened
meetings to review and revise the drafts from ]une to August, the Executive Committee of eighth
Pharmacopoeia Commission endorsed the Chinese Pharmacopoeia 2005 in September of the same year. In
December 2004, the draft text was sent to the Sta te Food and Drug Administration for approval and
promulgation. The Chinese Pharmacopoeia 2005 was published on January 2005 with effective date on July
1, 2005. The number of monographs in the Chinese Pharmacopoeia 2005 was considerably increased. It
contained up to 3217 monographs of drugs and other articles with 525 new admissions and 1032 revised.
Volume I contained 1146 monographs, with 154 new admissions and 453 revised; Volume 11 dealt with
1970 monographs, with 327 new admissions and 522 revised; Volume ID contained 101 monographs, with
44 new admissions and 5 7 revised.

The numbers of appendices in this edition were as follows: There were 98 monographs admitted in Volume
I , with 12 new admissions, 31 revised and 1 old monograph deleted. There were 137 monographs in
Appendix of Volume 11 with 13 new admissions, 65 revised and 1 deleted. There were 140 monographs in
Appendix of Volume ID with 62 new admissions, 78 revised and 1 deleted. Appropriate monographs
common to all three volumes were presented in each volume respectively in a harmonized and unified form.

In the Pharmacopoeia 2005 edition, the issue of pharmaceutical safety was emphasized particularly. In
Volume I , Atomic Absorption Spectrophotometry and Inductively Coupled Plasma Mass Spectrometry
were applied to determine the deleterious elements Oead, cadmium, mercury, arsenic and cupper) and the
limits of these elements had been stipulated; the guidelines of the safety test for the injections of Chinese
traditional medicines was also added to the Volume I. In Volume 11 , the Test for Particulate Matter was
applied to 126 injections intended for intravenous injections, and the number of monographs undergone the
Test for Bacteria! Endotoxin reached 112; The international harmonized and unified limit requirement of
residual solvent had been incorporated in the Determination of Residual Solvent, which was applied for 24
drug substances. In Volume 11, the guidelines for analyzing impurities of drugs, positron and "Guidelines
for quality control of technetium [ 99 mTc] rediopharmaceutical preparations" were also admitted. In Volume
III , Determination of reverse transcriptase activity, Test for aluminum residue of human blood albumin,
etc., and both Test for bovine serum albumin residue and Test for CHO cell protein residue also were
improved. In combination with status quo of medica! industry and practica! situation of drugs for clinical
usage, the "Detailed Regulations for Clarity Test and Criteria" formerly issued by the Ministry of Health
was replaced by the "Determination of Visible Particles" in this edition, so as to enhance the safety of
pharmaceutical injections.

According to the "diagnosis and treatment, based on an overall analysis of the illness and the patient's
condition" theory of Chinese traditional medicine, the Actions and the Indications under the Chinese
patent preparations had been scientifically standardized in this edition in arder to provide the assurance of
understanding. The Actions and the Indications precisely and making use of drugs rationally, and to
facilitate the healthy development of Chinese traditional medicine.

Volume III of this edition was originated from CBR. Six editions of the CBR had been promulgated for
implementation since 1951, i. e. in 1951and1952 revised editions were issued, and the edition 1959, 1979,
1990 and 1993 (for diagnostic products), 1995 and 2000 and its supplement were published respectively.
The English version of CBR had been translated and published far the first time in 2002.

The eighth Pharmacopoeia Commission also had completed the Addendum 2002 and Addendum 2004 of the
Chinese Pharmacopoeia 2000, the Adopted Names of Chinese Pharmaceutical Products (2005 edition),
Atlas of Infrared Spectra of Drugs and Medicines ( third volume), "Cuide to Clinical Information"
(first edition far Chinese traditional patent preparations and faurth edition far chemicals) and Chinese
Pharmacopoeia 2005 English Edition.

aúnese Pharmacopoeia 2010 Edition (Ninth Edition) The State Food and Drug Administration set up the ninth
Pharmacopoeia Commission in November 2007. In the selection new members of the Commission were elected first
through open choose, competitive election and secret ballot The Commission consisted of 323 members, 163
further appointment members and 160 new members. The Commissioner of the State Food and Drug
Administration, Mr. Shao Mingli served as its Chairman. An Executive Committee and 25 subcommittees of
specific duty and/ or appointment were set up under the Commission. On basis of previous Commission, 5 new
subcommittees, namely Ethnomedicine and Ethnopharmacy, Policy and Development, Standard Material,
Standard Information and Injection Improvement, were established. The subcommittee of Somatocyte and Gene
Therapy was canceled. Two subcommittees of In Vitro Diagnostic Biological Reagent and the Blood Products in
previous Commission were merged into the new subcommittee of Blood Products. Four subcommittees of
traditional Chinese medicine under previous Commission were reorganized as three, Traditional Chinese Medicine,
Natural Medicine and Chinese Medicinal Materials and the Slices.

The inaugural meeting and Plenary Session of the ninth Pharmacopoeia Commission took place in
December 2007, reviewed the draft revision of the Pharmacopoeia Commission Constitution and approved
"the design plans far the 2010 edition of Chinese Pharmacopoeia ". The design plans clearly indicated that
the Pharmacopoeia should adhere to guiding ideology, fundamental principies, development goals and main
XX
m1ss1on. Subsequently each subcommittee went on selecting monographs far the new edition, proposing of
scientific research projects and implementing tasks.

The Cornmission took a new approach towards organizational guarantee and scientific rnanagement in the
enacting process of the 2010 edition of Chinese Pharmacopoeia. Sorne research projects were assigned as
"the plan assignments of standard research subjects" in which the responsibility, the tasks of projects, the
research objectives, the indicators far performance appraisal and the scheduling requirements were
included. The first working meeting, all committee members attended to, took place in December 2008 in
the enacting process of the 2010 edition of Chinese Pharmacopoeia, and discussed problems in the editing
the Pharmacopoeia. Each subcomrnittee held the meeting, far reviewing the drafting monographs one after
another from March to August in 2009. The manuscripts were submitted far exarnination and were
approved in an enlarged meeting of the Executive Committee on August 27th, 2009. This edition was
published in January 2010 and come into use as of October 1, 2010.

Monographs adopted in this edition of Chinese Pharmacopoeia are significantly increased compared with
previous editions. It contains up to 4567 monographs of drugs and other articles with 1386 new admissions
and 2237 revised. Volume l contains 2165 monographs, with 1019 new admissions and 634 revised;
Volume 11 deals with 2271 monographs, with 330 new admissions and 1500 revised; Volume III contains
131 monographs, with 37 new admissions and 94 revised.

The numbers of appendices in this edition are as follows: There are 112 appendices admitted in Volume
I , with 14 new admissions, 47 revised. There are 152 articles in Appendix of Volume 11 with 15 new
adrnissions, 69 revised. There are 149 articles in Appendix of Volurne III with 18 new admissions, 39
revised. Appropriate articles common to all three volurnes are presented in each volume respectively in a
harrnonized and unified forrn.

Under the active leadership of the Chairman of Pharmacopoeia Comrnission, modern analysis technologies
have been further used in this edition. New technologies have been applied to sorne monographs. The issue
of pharrnaceutical safety is further strengthened. In sorne of the monographs, items of safety examination
have been added, even though general requirements of safety examination are specified in General Notices
and Appendices. Technical supports in controllability and effectiveness of drug quality are further
strengthened. In sorne of the rnonographs, iterns of effectiveness testing have been added, even though
exarnination methods and guidelines are newly admitted and revised in Appendices. Overall Requirernents
of pharrnaceutical excipients are written in general requirements far preparations far the need of drug
adrninistration. The edition also actively introduces the control requirements and limits of related
substances, and sterility test method suggested by International Conference of Harrnonization, <loes not
adrnit endangered natural Chinese medicinal herbs in arder to protect wild plant resources and develop
sustainably traditional Chinese medicine.

The ninth Pharrnacopoeia Cornmission also has completed the Addendum of the Chinese Pharmacopoeia
2005, "Atlas of Infrared S pectra of Drugs and Medicines" ( fourth volume) , "Guide to Clinical
Information" ( first edi tion far Chinese Medicinal Ma terials and Slices, second edi tion far Chinese
Traditional Patent Preparations and fifth edition far chernicals) "Atlas of microscopical identifications of
Chinese Medicinal Materials" and "Atlas of Thin Layer Chromatogram of Chinese Traditional
Medicines" (volume I and volume 11).
Chinese Pharmacopoeia 2015 Edition ( tenth Edition) In December 2010, the lOth sess10n Chinese
Pharmacopoeia Commission CChPC) was established by the State Food and Drug Administration (renamed
into the China Food and Drug Administration, CFDA on March 22, 2013). In accordance with the newly
revised Measures for the Screening of New Expert Members and Working Scheme Jor the Screening of
the lOth Session ChPC, the ChPC openly solicited membership candidates from the public and adopted
competitive elections and secret ballots for the election of new expert members. This Commission has a
total of 351 members, of whom 248 are re-appointed and 103 are newly appointed. Mr. Sang Guowei, the
former vice-chairman of the 11 th National People' s Congress Standing Committee, serves as the Honorary
Chairman, Mr. Chen Zhu, the former minister of Ministry of Health, serves as Chairman, and Mr. Shao
Mingli, the former director of the CFDA, serves as executive vice-chairman. This Commission has an
executive committee and 23 professional committees. The executive committee has a total of 67 members,
including 28 academician members, three senior experts, 20 directors of various professional committees,
four experts from relevant ministries and commissions, and seven leaders of relevant technical agencies of
the CFDA. In accordance with the needs of pharmacopoeia standardization development, this term of
commission has made appropriate adjustments to the creation of professional committees on the basis of
the configuration of the 9th ChPC; in arder to enhance the developing of chemical drug standards, the
Third Professional Committee for Chemical Drugs has been established and the number of expert members
on the chemical drug committees has been increased; meanwhile, in light of actual needs, the Policy and
Development Committee, Standard Information Working Committee and Injection Working Committee
have been abolished.

In December 2010, the Founding Ceremony and Plenary Conference of the lOth ChPC was held. The
conference deliberated and adopted the Compilation Outline for the Chinese Pharmacopoeia ( ChP) 2015
Edition, which specified guiding concepts, basic principles, development objectives and main tasks for
the developing of the ChP 2015 edition.

In accordance with the requirements of the 12th Five-Year Plan of National Drug Safety, the ChPC has
organized various professional committees and relevant institutions to carry out the drafting work of
pharmacopoeia on the basis of implementing the "action plan for national drug standard improvement."
The permanent institution of the ChPC has introduced the ISO 9001 quality management system ( QMS)
into the whole-process management of pharmacopoeia development, and conducted various activities
including product screening, project initiation, testing and research, standard development, review and
finalization, and steadily advanced various tasks for the development of this edition of pharmacopoeia. On
February 4, 2015, the ChP 2015 edition was approved by the Plenary Session of the Executive Committee
of the lOth ChPC and on J une 5, 2015, approved and enacted by the CFDA for official implementation as
of December 1, 2015.

This edition of pharmacopoeia has further expanded the admissions and revisions of drug products, with a
total of 5, 608 monographs. Volume I contains 2, 598 monographs, including 440 new monopraphs, 517
revisions and seven rejections. Volume II contains 2, 603 monographs, including 492 new monographs,
415 revisions and 28 rejections. Volume III contains 137 monographs, including 13 new monographs, 105
revisions, one newly added general chapter for biologics, three newly added general requirements for
biologics, and six rejections. For the first time, this edition of pharmacopoeia has integrated the
appendices of the previous edition of pharmacopoeia into the general chapters and included pharmaceutical
excipients as a separate Volume N of the ChP. Volume N contains 317 general chapters, including 38
general requirements for preparations, 240 for testing methods ( including 27 new methods) , 30
guidelines ( including 15 new guidelines), as well as nine general requirements for standard products,
standard substances and test solutions and substances. A total of 270 monographs of pharmaceutical
excipients have been contained, including 137 new monographs, 97 revisions and two rejections.

This edition of pharmacopoeia has improved the development of pharmacopoeia standard system,
enhanced quality control requirements in a comprehensive manner, further expanded the application of
advanced and sophisticated testing techniques, greatly increased the monographs of pharmaceutical
excipients, increased the rigor of quality requirements and safety control, and thus further enhanced the
guiding and technology orientation effects of the ChP.

In the process of drafting this edition of pharmacopoeia, we ha ve also completed the revision of the First,
Second and Third Supplements of the ChP 2010 Edition, the Atlas of lnfrared Spectra of Drugs (Volume
5), China Approved Drug Names, Working Handbook of National Drug Standards (4th edition) and the
Annotations of the ChP, and organized the drafting of the English version of ChP 2015 edition and the
lnstructions for Clinical Medications 2015 Edition.

XXIII
 General Notices

General Principies
l. The Pharmacopoeia of the People's Republic of China, known as Chinese Pharmacopoeia in
abbreviation, is enacted and promulgated in accordance with Drug Administration Law of The People' s
Republic of China. Where the Pharmacopoeia is issued for enforcement, the same drug standard of the
previous Pharmacopoeia or the original national drug standard shall not be used.
The Chinese Pharmacopoeia is composed of Volume l, Volume Il, Volume ID, Volume N and its
supplements. The Volume I contains traditional Chinese medicines, the Volume Il contains chemical
drugs, the Volume ID contains biologics, and the Volume N contains general charpters and
pharmaceutical excipients. When the Chinese Pharmacopoeia is quoted in this compendium, it denotes
the current edition of the Pharmacopoeia of the People' s Republic of China, except where a specific
edition is indicated.
This volume is the volume N of Chinese Pharmacopoeia.

2. National drug standards consist of General Notices, Monographs and referenced General Charpters from
which the monographs cite. The General Notices and the General Charpters in the current edition of
Chinese Pharmacopoeia are also official for other standards of drugs in addition to those con.tained in
Chinese Pharmacopoeia.

3. General Notices serve as the basic principles for the proper interpretation and application of the Chinese
Pharmacopoeia in quality control. It applies to any monographs, General Charpters and general
statements related to quality control of drugs so as to obviate replication in this compendium.

4. The wording "unless specified otherwise" adopted in General Notices and General Charpters indica tes
that appropriate requirement is admitted in the related monograph wherever the requirement is not
conform to that specified in General Notices or General Charpters.

5. The drugs cited in the mongraphs should be those admitted in this edition of Chinese Pharmacopoeia
and comply with the requirements of corresponding requirements.

6. The testing items established in monographs are specified for the medicaments manufactured in
accordance with the requirements of Good Manufacturing Practices (GMP). Any medicament produced
in the condition violating GMP does not comply with the monograph of the Pharmacopoeia in spite that
the testing results are in conformity with the Chinese Pharmacopoeia or no adulterated or related
substances are detected according to the Chinese Pharmacopoeia.

7. English name for the Chinese Pharmacopoeia is the Pharmacopoeia of the People' s Republic of China,
which is abbreviated as ChP.

Monographs
8. Full test under individual article in the "Chinese phocmacopocia" is monograph. The monographs are
 established based on physic-chemical characteristics and biological properties of the drugs, formulation
and processing, manufacturing techniques as well as conditions for storage and transportation that are
authorized officially, and are the technical requirements in order to determine if quality of drugs is for
medical use, stable and homogenous.

9. In general, the standard monographs of pharmaceutical excipients include the following items:
( 1) Title ( adopted Chinese names, Chinese phoneteic alphabet and English name) ; ( 2) Structural
formula of organics; ( 3) Molecular formula, molecular weight and CAS Number; ( 4) Sources;
(5) Production; (6) Description; (7) Identification; (8) Physic-chemical examination; (9) Content
determination; (10) Category; (11) Storage; (12) State.

General Charpters

10. General Requirements for Preparations, General Testing methods and guidelines are compiled in
General Charpters. General Requirements for Preparations classified on the basis of different
preparations are basic technical requirements depending on the characteristic of preparations. General
testing methods are established for those same tests specified in monographs concerned using same
apparatus, procedures, methods and limits. The Guidelines stated in General Charpters are not
considered as official requirements but used as statements in principles applied to the implementation
of Pharmacopoeia, monitoring of drug quality and revising or verifying of drug standards.

Titles and Arrangements

11. The Chinese titles of drugs admitted in the monographs are recommended according to the guideline
for the nomenclature of Chinese Approved Drug Names. The Chinese title of a drug is adopted as
official designation. Unless specified otherwise, the International Nonproprietary Name for
pharmaceutical substances CINN) is adopted as the English title.
Titles of organic chemical drugs are adopted on the basis of Guideline for the Nomenclature of
Organic Chemistry published by the Chinese Chemical Society. The selection of main part of its
chemical structure is in line with the rules of the International Union of Pure and Applied Chemistry
CIUPAC).

12. The chemical structure of the drugs are portrayed according to Guidelines for Graphic Representation
of Chemical Formulae recommended by the World Health Organization.

13. The monographs are arranged alphabetically on the English titles of drugs. General Charpters are
arranged into sections including general requirements for formulated preparations, general testing
methods and guidelines.

Specifications

14. The requirements of important pharmaceutical process and quality control are described in the item of
Productions of the monographs.
( 1) The pharmaceutical processes of all the drugs should be verified, approved by the drug regulatory
agency of the State Council and be in accordance with the requirements of GMP.
(2) The drugs extracted from animal tissues should be given with a clear indication of the animal
species. The visceral organs should be from healthy animals by quarantine inspection. The drugs
derived from cattle should be obtained using healthy cattle from the areas without Bovine Spongiform
Encephalopathy. The drugs extracted from human urine should be prepared with the urine of healthy
persons. The drugs mentioned above should have specific viral removal and viral inactivation processes
XXV
 and quality control.
(3) Microbial strain, seed cultures of viruses, cell from Human or Animal, engineering bacteria and
engineering cell of recombinant DNA technology used for pharmaceutical processes should be approved
by the drug regulatory authority of the State Council and comply with requirements for quality
control.

15. Description refers to the appearance, odour, taste, solubility and other physical constants of the drug,
which reflects the quality characters of the drug at a certain degree.
( 1) Appearance of a drug is the requirements for colour and externa! appearance.
( 2) Solubility refers to the physical property of a drug substance. Solvents described under the
monograph and the relevant solubility behaviors are stated for reference for purification or preparation
of solution of a drug substance. Requirement should be stated under the item of test of the drug, if
specific quality control is needed for the solubility behavior of the solvent accordingly.
Approximate solulilities of drugs indicated by the following descriptive phrases:
Very soluble refers to that 1 g (ml) of salute is soluble in less than 1 ml of solvent;
Freely soluble refers to that 1 g (ml) of solute is soluble in 1 ml to less than 10 ml of solvent;
Soluble refers to that 1 g (ml) of salute is soluble in 10 ml to less than 30 ml of solvent;
Sparingly soluble refers to that 1 g (ml) of solute is soluble in 30 ml to less than 100 ml of solvent;
Slightly soluble refers to that 1 g (ml) of solute is soluble in 100 ml to less than 1 000 ml of solvent;
Very slightly soluble refers to that 1 g (ml) of salute is soluble in 1 000 ml to less than 10 000 ml of
solvent;
Practica[ insoluble refers to that 1 g (ml) of salute is not soluble completely in 10 000 ml of solvent.
Testing method: Unless specified otherwise, weigh out finely powdered sample or measure an amount
of liquid sample, place in a certain volume of the solvent at a temperature of 25ºC ± 2ºC and shake
strongly for 30 seconds at an interval of 5 minutes. Observe the solubility behavior for 30 minutes. lt
is considered to be completely soluble if none of the particles or droplet of the salute is observed.
(3) Physical constants or parameters including relative density, distilling range, melting point or
melting range, congealing point, specific rotation, refractive index, viscosity, specific absorbance,
iodine value, saponification value and acid value etc. , are useful for identification and give sorne
indication of the drug purity. lt is one of the chief criteria for appraisal and assessment of drug quality.

16. Testing methods given in the identification section are intended to give sorne of the physical and
chemical characteristics of the drug, which are not designed to give a full confirmation of the chemical
structure of the drug.

17. Test section under the monograph includes the testing methods and acceptance criteria, uniformity and
purity requirements far manufacturing process, which are related to the safety and efficacy of the
drug. The specified testing items for impurities are framed from those possibly existed and produced
in the drug, which is manufactured according to the approved manufacturing process and stored under
normal condition, and those impurities are needed to be controlled, such as residual solvents and
related substances, etc. Revising to the relevant items may be necessary far where the manufacturing
process is changed.
The organic solvents introduced from manufacturing procedure should be effectively removed during
further processing. In addition to the "Residual solvents" listed in monographs should be tested the
other organic solvents unlisted in Residual Solvents, which are introduced during manufacturing or
remained in products should also be tested following "residual solvent" described in general chapters.
All the results should meet the limit of relevant requirement.
XXVI
 The drug substance intended for direct use in the preparation of sterile powders for injection should be
tested according to the corresponding requirements of the injection and should conform with the
req uiremen ts.
Preparation, unless specified otherwise, must comply with the requirements stated under the general
requirements for preparations of the Pharmacopoeia.

18. The testing methods specified under the item of Assay are designed to determine the content of active
ingredient of drug substance and preparations, chemical, instrumental or biological methods can
usually be adopted.

19. Category refers to main action and use of the drugs or its classified scientific designation. A drug is not
framed from using in other category on the basis of experiences in clinical practices.

20. Strength of preparations or dosage forms refers to the amount ( units of potency), labeled amount
(%) or content of active ingredient or ingredients in each ampoule, tablet or other unit container, or
preparation. For injections, the expression "1 ml : lOmg" indicates that the injection contains 10 mg
of active ingredient in 1 ml. Specification of weight or content may also be specified for the
monographs of the preparation in which the formula is listed or the concentration is indicated.

21. Storage refers to the basic conditions for storage and preservation of a drug in arder to avoid
contamination or degradation, which is stated by the following terms:
Protected from light refers to that a drug should be kept in light resistant container such as amber
coloured container, or a colourless transparent or semitransparent container wrapped with black
paper.
Protected from sunlight refers to avoiding the direct illumination by sunlight.
Well closed refers to that container is able to protect the container from extraneous matters or lose of
contents on normal handling condition.
Tightly closed refers to that container should be able to protect the contents from efflorescence,
deliquescence, volatilization or interference of extraneous matters.
Hermetically sealed or Tightly sealed refers to that container is sealed on fusion or sealed tightly with
suitable material to protect against contamination and from permeability of air and moisture.
Cool place refers to that the storage temperature is not exceeding 20ºC.
Cool and dark place refers to that container is kept in the dark place, protected from light and the
ambient temperature is not exceeding 20ºC.
Cold place refers to that container is kept at ambient temperature of 2-lOºC.
Normal temperature refers to that container is kept at ambient temperature of l0-30ºC.

22. The drug substances and excipients used in preparations or dosage forms should comply with the
requirements stated in the individual monographs of this edition of Pharmacopoeia; for those not
admitted in this edition of Pharmacopoeia, their specifications should be established, which should
comply with requirements for pharmaceutical use and be approved by the drug regulatory authority of
the State Council.
Where the same drug substance is used for different preparations, especially for preparations with
different administration routes, it is necessary to establish corresponding testing items for quality
control according to the clinical usage of the drug.
The excipients used for production of preparations should comply with the relevant requirements for
pharmaceutical excipients issued by the drug regulatory authority of the State Council as well as the
requirements in the Volume N <0251> of the Pharmacopoeia.
 The requirements for excipients included in this Pharmacopoeia are the basic requirements for the
excipientsof which the uses are stipulated under the item Category of individual monographs.
The excipients used by manufacturer should be corried out the suitability verification even if they meet
the requirements for excipients in this Pharmacopoeia.
The source and process of excipients, as well as the characters, administration route, target
population and dosage of preparations should be considered fully inverification of suitability
requirements for excipients.
The raw materials and process forproduction of excipients should be accepted by the national drug
regulatory authority. Any substances without permission by the national drug regulatory authority
should not be added during the production of excipients.
When the excipients in this Pharmacopoeia are used, the effect of other factors such as administration
route, usage, constitution of formula and dosage on the safety should be considered. The excipients of
the corresponding grades should be selected based on the risk degree of safety. Especially for the high
risk preparations such as injections and eye preparations, Excipients at injection grade should be
selected as far as possible under the premise that suitability, safety and stability meet the
requirements.
When the excipients m this Pharmacopoeia may possibly influence the suitability and safety of
preparations, the excipients meeting the relevant requirements should be used, and the corresponding
standards for excipients should be developed and approved by the drug regulatory authority.

Testing Methods and Limits

23. When the methods in this Pharmacopoeia are used, the applicability shall be confinmed.

24. The drug substances and preparations should be tested with official methods stated m the
Pharmacopoeia. It is not precluded from the application of alternative methods, if they have been
proved to be satisfactory in comparison with official methods accordingly. In case of doubt or dispute,
the methods of the Pharmacopoeia are authoritative for judgement.

25. Purity requirements and limits of purity of a drug substance as well as the weight ( or content)
variation of a preparation or dosage form stated in the monograph concerned include the values of
upper and lower limits and the medium value. Whether these values are expressed in percentage or in
absolute numerical value, the last decimal is a significant value.
In calculating of testing result, the last effective figure is measured in one decimal place more than the
significant decimal place indicated in the requirement and round up or clown to the specified decimal
place by the rule of commensuration, the value obtained is compared with the limits of the standard to
determine the conformity with the specified limits.

26. The percentage content of the drug substance is calculated by weight, unless specified otherwise. If an
upper limit of the content of a drug is stated as over 100 %, it refers to a value possibly obtained by the
assigned assay method in the monograph, representing the limit or permissible deviation stated in the
Ph~rmacopoeia. In case of no stated upper limit, the upper limit is considered to be not more than the
equivalent amount of 101. O%.
The limit range of content of a preparation or dosage form is assigned on the basis of contents of active
ingredient or ingredients, assay method being applied and possible change occurred in the process of
manufacturing and / or storage. A 100 % labelled amount of active ingredient or ingredients should be
used in manufacturing process. If a certain active ingredient is known to be lowering its content in
manufacturing process or in the storage period, sufficient amount of active ingredient concerned may
 be used to ensure that the content of the drug produced or being used in its shelf life complies with the
requirements of the Pharmacopoeia.

Reference Standards and Chemical Reference Substances

27. Reference standards and chemical reference substances refer to the standard materials used far
identification, test and assay. Reference standards are used far bioassay or determination of potency,
of which the characteristic value is expressed in units (or µg). Chemical reference substances are used
far identification, test and assay by physiochemical methods, of which the characteristic value is
expressed as purity ( %) in general.
The establishment or alteration in unit of potency or content of reference standards and chemical
reference substances should be conducted in comparison with the original reference standard, chemical
reference substances or International Reference Standard by collaborative standardization, and the
protocol and testing results should be reviewed and approved by audit process. Only the reference
standards and chemical reference substances, of which the qualities meet the requirements far
established uses may be used.
Reference standards and chemical reference substances are distributed with appropriate instruction
insert sheet to state the lot number, characteristic value, use, method for usage, condition for storage
and filling quantity in general.

Units of Measurement
28. The measuring apparatus used for tests and assays should comply with the relevant requirements
promulgated by the technical supervision authority of the State Council.

29. The units of measurement in this edition of the Pharmacopoeia

(1) The official names and symbols of units of measurement are listed as follows:
Units of length: meter ( m); decimeter ( dm); centimeter (cm); millimeter (mm); micrometer
( µm) ; nanometer ( nm)
Units of volume: liter (L); milliliter (mi): microliter (µD
Units of mass (weight): kilogram (kg); gram (g); milligram (mg); mtcrogram (µg); nanogram
(ng); pictogram (pg)
Units of amount of substance: mole (mol); millimole (mmoD
Units of pressure: megapascal (MPa); kilopascal (kPa); pascal (Pa)
Units of temperature: degree Celsius (°C)
Units of kinetic viscosity: pascal seconds (Pa • s); millipascal seconds (mPa • s)
Units of kinematic viscosity: square meter per second (m2 /s); square millimeter per second (mm2 /s)
Units of wave number: reciproca! of centimeter (cm-1 )
Units of density: kilogram per cubic meter (kg/m 3 ) ; gram per cubic centimeter (g/ cm 3 )
Units of radioactivity: gigabecquerel (GBq); megabecquerel CMBq); kilobecquerel (kBq); becquerel
(Bq)
(2) Where the strengths or concentrations of the volumetric solutions and test solutions are expressed
in terms of mol/L in this edition of Pharmacopoeia, the expression of "XXX volumetric solution
(YYY mol/L) " is adopted for the volumetric solution which should be accurately standardized. The
expression of "YYY mol/L XXX solution" is adopted for solutions of other purpose without specific
accuracy of their concentration.
(3) Temperature isdescribed by the following terms in general.
The temperature of a Water bath is 98-lOOºC, unless specified otherwise;
 Hot water refers to that at a temperature of 70-80ºC;
Slightly warm or Warm water refers to that at a temperature of 40-50ºC;
Room tem perature refers to that at a temperature of 10-30ºC;
Cold water refers to that at a temperature of 2-lOºC;
Ice bath refers to the bath temperature kept at about OºC;
Allow to cool refers to that the object is cooled to room temperature.
(4) The symbol used for the expression of percentage is %, usually by weight, but the percentage of
solutions, unless specified otherwise, refers to the number of grams of salute in 100 ml of the
solution. The percentage of ethanol refers to percentage by volume at 20ºC.
The following symbols may be used when needed:
% (g/g) expresses the number of grams of a salute in 100 g of solution;
% (ml/mD expresses the number of milliliters of a salute in 100 ml of solution;
% (ml/ g) expresses the number of milliliters of a salute in 100 g of solution;
% (g/ml) expresses the number of grams of a salute in 100 ml of solution.
(5) The abbreviation of "ppm" refers to parts per million by weight or volume.
(6) The abbreviation of "ppb" refers to parts per billion by weight or volume.
(7) The drop of a liquid refers to that l. O ml of water is equivalent to 20 drops at the temperature of
20ºC.
(8) The expression " 0-10) " stated under the solution refers to a solution of 10 ml produced by
addinga sufficient quantity of solvent to dissolve l. O g or l. O ml of a salute. lt is understood to be
aqueous solution, if the solvent is not specified. In case of two or more solvents are used as a mixture,
a hyphen is inserted between different solvents indicated by names and the parenthesis followed
expresses the proportion of each solvent by volume in the mixture.
(9) Sieves of Chinese National Standard R40/3 series are adopted in the Pharmacopoeia and the
numbers are assigned as follows:
Sieve No. Average internal diameter of aperture (µm) Mesh No.
1 2000+70 10
2 850±29 24
3 355+13 50
4 250±9. 9 65
5 180+7. 6 80
6 150±6. 6 100
7 125±5. 8 120
8 90+4. 6 150
9 75±4. 1 200
Powders are graded as follows:
Very coarse All particles pass through No. 1 sieve, not more than 20% pass through No. 3 sieve;
Coarse All particles pass through No. 2 sieve, not more than 40% pass through No. 4 sieve;
Medium All particles pass through No. 4 sieve, not more than 60% pass through No. 5 sieve;
Fine All particles pass through No. 5 sieve, not less than 95% pass through No. 6 sieve;
Very fine All particles pass through No. 6 sieve, not less than 95% pass through No. 7 sieve;
Ultra fine All particles pass through No. 8 sieve, not less than 95 % pass through No. 9 sieve.
(10) Ethanol refers to that of 95% (ml/mD in strength, unless specified otherwise.

30. The atomic weights adopted for calculating the molecular weights and the conversion factors are the
XXX
 values recently published by the lnternational Union of Pure and Applied Chemistry.

Precision and Accuracy

31. The accuracy of sampling quantity and precision of testing are defined m this edition of
Pharmacopoeia.
( 1) The quantity obtained by weighing or measuring the substance being examined and reagent being
used is expressed in Arabic figures. The required precision is expressed by the significant numerical
place. For example, the measurement of "O. 1 g" by weight refers to that O. 06-0. 14 g of the substance
may be weighed; for "2 g", l. 5-2. 5 g of the substance may be weighed; for "2. O g", it refers to that
l. 95-2. 05 g of the substance may be weighed; for "2. 00 g", it refers to that l. 995-2. 005 g of the
substance may be weighed.
Weigh accurately indica tes that the precision of measurement should he made to an accuracy of O. 1 %;
weigh indicates that an accuracy being made to 1 %. Measure accurately indica tes the accuracy of the
volume being measured complies with the national standard of pipet being used for the measurement of
required volume; Measure indicates that the measuring cylinder or other measuring apparatus being
used complies with requirements for the measurement of volume to the significant numerical place.
The word about states that the measuring quantity should not exceed ±10% of the specified quantity.
(2) Constant weight, unless specified otherwise, refers to that the drying or ignition of a substance or
material in two consecutive weighings do not differ by more than O. 3 mg. The second and subsequent
weighing are made after an additional hour of drying each time under the similar condition. The second
weighing of the substance or material made to constant weight by ignition is made after 30 minutes
under similar condition.
(3) The expression of calculated on the dried (anhydrous or solvent free) basis indicates that, unless
specified otherwise, the undried substance or solvent containing substance is used for the required
testing. The result of "Loss on dying (moisture or solvent) " should be subtracted from the amount
of substance.
( 4) Blank test refers to a test carried out in the similar manner without the substance being examined
or using same amount of solvent instead of the solution being tested. The statement of to make any
necessary correction of the result with a blank test refers to that the result is calculated by subtracting
number of milliliters of titrant used in blank test from that consumed in assay of the substance being
examined.
( 5) The temperature for a test is at room temperature whenever the temperature is not stated. In case
of that the temperature variation influences significantly to the testing result, the test should be
carried out at a temperature of 25ºC ±2ºC, unless specified otherwise.

Reagent, Test Solutions and lndicators

32. Reagents used in tests and assays, unless specified otherwise, should comply with the requirements
stated in the Appendix of the Pharmacopoeia. Different grades of reagents conform to national
standards or those issued by the competent authorities may be used for choice. Test solutions, buffer
solutions, indicator solutions, volumetric solutions and so on should comply with the statements or
be prepared as directed in the General Charpter of the Pharmacopoeia.

33. Water being used in tests and assays is purified water. Water being used for the test of acidity or
alkalinity is the water freshly boiled and cooled to room temperature.

34. Test for acidity or alkalinity of a solution without the statement of indicator being used refers to that
XXXI
 Litmus paper is used.

Animal Test

35. Laboratory animals used and managed in animal tests should comply with the requirements stipulated
and promulgated by competent authorities of the State Council.
The strain, age, sex etc. should comply with the requirements for quality control of drugs and
biological products.
Along with the improved level of purity of drugs, the animal tests should be adopted only in necessary
condition and become less popular where more precise chemical, physical or cellular method may be
applied to the quality control of drugs.

Insert Sheet, Package and Labelling

36. The insert sheet of drugs should be in conformity with the requirements of "The Drug Administration
Law of the People' s Republic of China", and provisions for insert sheet promulgated by the drug
regulatory authority of the State Council.

37. The immediate packaging materials and containers should be in conformity with the relevant provisions
promulgated by the drug regulatory authority of the State Council. The immediate packaging materials
and containers must be innocuous, clean, not interact with the drugs being packed, and do not affect
the drug quality in the containers.

38. Labelling of drugs must comply with the requirements of "The Drug Administration Law of the
People' s Republic of China", and the provisions for the package and labelling promulgated by the drug
regulatory authority of the State Council. The content of the labelling for different packaging should
be printed in accord with the above requirement, in which the drug information should be provided as
more as possible.

39. A stated mark should be printed on the insert sheet and the labelling of package for narcotics,
psychological drugs, toxic drugs for medicinal use, radioactive drugs, drugs for external use and
nonprescription drugs.

XXXII
 Contents of General Chapters

0300 ............................................................... 40
General Chapters 0301 General Identification Tests ........................ 40
0400 Spectrometry ............................................. 43
0100 General Requirements for Preparatioffi ·················· 3 0401 Ultraviolet-Visible Spectrophotometry ......... 45
0101 Tablets ··················································· 3 0402 lnfrared Spectrophotometry ........................ 46
0102 Injections ················································ 5 0405 Fluorescence Spectrophotometry .... · ........ ·.... 47
0103 Capsules ................................................ 7 0406 Atomic Absorption Spectrophotometry ......... 48
O104 Granules · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 8 0407 Flame Photometry •· · · · · · · · · · · · · · · · · · · · · · · ••· · · •· · •· · · 49
0105 Eye Preparations ··•···································· 9 0411 lnductively Coupled Plasma-Atomic Emission
0106 Nasal Preparations ································· 11 Spectrometry · · · .. · .. · .. · .. · · .. · · · · .... · .. · .. · .. •.. •.. · 49
0107 Suppositories ··· ··· ··· ··· ··· ··· ··· ········· ··· ··· ··· ··· 12 0412 lnductively Coupled Plasma-
0108 Pills ··················································· 13 Mass Spectrometry · · · · · · · · · · · · · · · · · · · · · · •· · · •· •· · · · 50
0109 Ointments, Creams ································· 14 0421 Raman Spectrometry ....................... ; ......... 55
0110 Pastes ................................................... 15 0431 Mass Spectrometry ·····················••···•··•··· 58
0111 Preparations for Inhalation ························ 15 0441 Nuclear Magnetic Resonance Spectrometry 61
0112 Sprays··················································· 19 0451 X-ray Diffraction .................................... 64
0113 Aerosols ··· ··· ··· ··· ··· ··· ··· ··· ········· ··· ··· ··· ··· ··· 20 0500 Chromatography .......................................... 66
0114 Gels ................................................... 22 0501 Paper Chromatography .. · · · .. · .. · .. · .............. " 66
0115 Powders ················································ 22 0502 Thin-Layer Chromatography ..................... 67
0116 Syrups ................................................ 23 0511 Column Chromatography ........................... 69
0117 Liniments ............................................. 24 0512 High Performance Liquid Chromatography ... 70
0118 Paints ................................................... 24 0513 Ion Chromatography ................................. 73
0119 Pigments ............................................. 24 0514 Size-Exclusion Chromatography · · · · · ••· ...... •.. • 74
0120 Tinctures ············································· 25 0521 Gas Chromatography ··· ........................... 75
0121 Patches ................................................ 25 0531 Supercritical Fluid Chromatography ............ 76
0122 Cataplasms ............................................. 26 0532 Liquid Chromatography at Critical Condition
0123 Oral Solutions, Oral Suspensions, Oral ............................................................ 77
Emulsions · .. · · · · · · · · · · .. · · · · · · · · · · .. •.. · · · · · · · · · .. · · · · 27 0541 Electrophoresis ....................................... 78
0124 lmplants ··· ··• ......... ··· ... ··· ... ··· .................. 28 0542 Capillary Electrophoresis · .. ·· •..... · · · · ·.. ...... ... 84
0125 Pellicles ................................................ 28 0600 Determination of Physical Constant .................. 87
0126 Ear Preparations .................................... 28 0601 Determination of Relative Density ............... 87
0127 Lotions ··· ··· ··· ··· ··· ··· ... ··· ··· ······ ...... ··· ··· ··· 29 0611 Determination of Distilling Range .... · · · .. · ·.... 88
0128 lrrigants ... ··• ··· ··· ··· ... ··· ······ ...... ··· ............ 30 0612 Determination of Melting Point .................. 88
0129 Enemas ................................................ 30 0613 Determination of Congealing Point ............... 89
0181 Mixtures ................................................ 30 0621 Determination of Optical Rotation ............... 90
0182 Troches ... ··· ··· ··· ··· ··· ...... ········· ...... ··· ··· ··· 31 0622 Determination of Refractive lndex ............... 91
0183 Concentrated Decoctions ........................... 31 0631 Determination of pH Value ........................ 91
0184 Glues ................................................... 31 0632 Determination of Osmolality ..................... 92
0185 Medicinal Wines .................................... 32 0633 Determination of Viscosity ... •· .. · · · ... · .... · ·· ... 93
0186 Plasters ··· ··· ... ··· ··· ··· ··· ... ······ ··· ......... ··· ... 32 0661 Thermal Analysis .................................... 96
0187 Medicinal Distillates ................................. 33 0681 Determination of Conductivity of Water for
0188 Medicinal Teas ....................................... 33 Pharmaceutical Use ····•··················•···•····· 98
0189 Liquid Extracts and Extracts ..................... 34 0682 Determination of Total Organic Carbon
0200 General Requirements for Other Phannaceutical in Water for Pharmaceutical Use ··············· 100
Materials · .. · · · · · · .. · · · · .. · · · · · .. · · · .. · · · .. · · · .... · .. · · · · · · · 34 0700 Other Determination Methods · · · · · · · · · · · · · · · · · · · · · · · · 100
0211 Sampling of Crude Drugs and Decoction 0701 Potentiometric Titration and Dead-stop
Pieces ................................................... 34 Titration ·········•··················•················ 100
0212 General Principie for lnspection of Crude 0702 Non-aqueous Titration · · · · · · · · · · · · · · · · · · · •· · · · · · · 102
Drugs and Decoction Pieces ........................ 35 0703 Oxygen-Flask Combustion · •· · •· · •· · · · · · · · · •· · · · · · 102
0213 The Processing of Crude Drugs .................. 36 0704 Determination of Nitrogen ....................... . 103
0251 General Requirements for Pharmaceutical 0711 Determination of Ethanol ····•••·····•····•••···· 104
Excipients · .... · · .. · · · · · · · .... · · · · · · · .. · .. · · .. · · · · · · ·.. 38 0712 Determination of Methoxy, Ethoxy and
0261 Water for Pharmaceutical Purposes ............ 39 Hydroxypropoxy Groups · · · · · · · · · · · · · · · •· •· · · · · · 106
0291 General Requirements for National 0713 Tests of Fats and Fatty Oils .................... . 108
Pharmaceutical Reference Standards ............ 39 0721 Vitamin A Assay ................................... . 110
1
 0722 Vitamin D Assay .................................... 111 1206Determination of Cytochrome C Activity ······ 192
0731 Determination of Protein Content ··············· 112 1207Assay of Hyaluronidase ........................... 192
0800 Limit Tests · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 115 1208Biological Assay of Heparin ..................... 193
0801 Limit Test far Chlorides ··························· 115 1209Biological Assay of Chorionic Gonadotrophin
0802 Limit Test far Sulfates ·····•········••·······•··· 116 ......................................................... 194
0803 Limit Test far Sulfides ········· ··•··· ······ ··· ··· 116 1210 Biological Assay of Oxytocin ..................... 194
0804 Limit Test far Selenium ··························· 116 1211 Biological Assay of Insulin ··· ··· ··· ...... ········· 195
0805 Limit Test far Fluorides ··························· 117 1212 Test far the Prolongation Effect of Protamine
0806 Determination of Cyanide · · · · · · · · · · · · · · · · · · · · · · · · 117 Zinc Insulin .......................................... 195
0807 Limit Test far Iron ································· 118 1213 Biological Assay of Protamine Sulfate ········· 196
0808 Limit Test far Ammonium ····················· 118 1214 Biological Assay of Digitalis ..................... 196
0821 Limit Test far Heavy Metals ······ ··· ········· ··· 118 1215 Toxicity Test of Sodium Stiboglu-conate ··· ··· 196
0822 Limit Test far Arsenic ·····•····················· 119 1216 Biological Assay of Follicle Stimulating
0831 Determination of Loss on Drying ··············· 120 Hormone ···········•································· 197
0832 Determination of Water ··························· 120 1217 Biological Assay of Luteinising Hormone ······ 197
0841 Determination of Residue on lgnition ········· 122 1218 Biological Assay of Calcitonin ··· ······••·••···• 198
0842 Limit Test far Readily Carbonizable Substances 1219 Biological Assay of Growth Hormone ········· 198
......................................................... 122 1401 The Test of Radiopharmaceutical Preparations
......................................................... 199
0861 Determination of Residual Solvents ············ 123
0871 Determination of Methanol ························ 128 1421 Methods of Sterilization ··························· 205
0872 Acetic Acid in Synthetic Peptides ··············· 129 1431 Statistical Methods far Biological Assays ·•···· 209
0873 Determination of 2-Ethylhexanoic Acid 129 2000 Special Methods for Traditional Chinease
0900 Tests for Special Properties or Other lndicators Medicines · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 227
............•............................................ 130 2001 Microscopical Identification ··················•·· 227
2101 Determination of Swelling Capacity ···•········ 229
0901 Colour of Solution ································· 130
2102 Determination of Plaster Softening Point ··· ··· 229
0902 Clarity of Solution ··· · · · ··· ··· ··· ··· ·•· ·· · · · · · ·· · · · 132
0903 Test far Particulate Matter in Injections ··· ··· 133 2201 Determination of Extractives ····················· 229
2202 Determination of Tannins ··· ··· ··· ··· ··· ······•·· 230
0904 Test far Visible Particles ························ 135
0921 Determination of Disintegration ·················· 138 2203 Determination of Cineol · · · · · · · · · · · · · · · · · · · · · · · · •· · 230
0922 Disintegration Test far Suppositories and Vaginal 2204 Determination of Volatile Oil ····················· 230
2301 Determination of Foreign Matter ······ ······•·· 231
T ablets · · · •· · •· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 140 2302 Determination of Ash .............................. 231
0923 Test far Tablet Friability ··· ··· ··· ··• ··· ··· ··· ··· 141 2303 Limit Tests of Rancidity ........................ 232
0931 Dissolution and Drug Release Test ······ ··· ··· 141
2321 Determination of Lead (Pb), Cadmium (Cd),
0941 Test far Content Unifarmity ····················· 145 Arsenic (As), Mercury (Hg) and Copper (Cu)
0942 Mínimum Fill ··· ··· ··· ··· ··· ··· ······ ··· ··· ··· ······ 146 ......................................................... 232
0951 Preparations Far Inhalation: Aerodynamic
2322 Determination of Mercury and Arsenic Speciation
Assessment of Fine Particles ····················· 146
and Their Valence States ························ 234
0952 Determination of Adhesion ························ 152 2331 Determination of Residue of Sulfur Dioxide
0981 Crystallinity ··· ··· ··· ··· ··· ··· ··· ··· ··· ··· ··· ··· ······ 154 ...............................•......................... 236
0982 Determination of Particle Size and Particle Size
2341 Determination of Pesticide Residues ·········•·· 237
Distribution · ·· · · · · · · ··· · · · ·· · ··· ··· ·· · ·· · ·· · · ·· ·· · ··· 154 2351 Determination of Aflatoxins ··················•·· 248
0983 Measurement of Consistency by Penetrometry
............•..........•................................. 156 2400 Determination of Materials in Injections ······ 249
3000 Special Mothods for Biological Products ············ 249
1100 Biological Tests · ·· ··· ··· ··· ··· ··· ··· ······ ··· ··· ··· ······ 158 3100 The Mothods for Content Detennination 249
1101 Sterility Tests ··· ··· ··· ··· ··· ··· ··· ··· ··· ··· ······ ··· 158 3101 Determination of Total Salid ····················· 249
1105 Microbiological E:xamination of Non-sterile 3102 Determination of Sialic Acid Content
Products: Microbial Enumeration Tests ······ 163 (Resorcinol Colourimetry) ············•········ 249
1106 Microbiological E:xamination of Non-sterile 3103 Determination of Phosphorus Content ·······•· 250
Products: Tests far Specified Microorganisms 3104 Determination of Ammonium Sulfate Content
...................................................•..... 168 ......................................................... 251
1107 Microbiological Acceptance Criteria of Non-sterile 3105 Determination of Sodium Bisulfite Content
Pharmaceutical Products ........................... 173 ...••••.....••••...••....••••..•..•....••••••.••••......• 251
1121 Antimicrobial Effectiveness Testing ······•····· 175 3106 Determination of Aluminium Hydroxide
1141 Test far Abnormal Toxicity ···············•····· 177 Cor Aluminium Phosphate)Content ···•········ 251
1142 Test far Pyrogens ································· 178 3107 Determination of Sodium Chloride Content
1143 Test far Bacteria! Endotoxin ····················· 179 ......................................................... 252
1144 Test far Vasopressor Substance ··············· 182 3108 Determination of Citrate Content ······•········ 253
1145 Test far Depressor Substances ··· ··· ··· ··· •·· ··· 183 3109 Determination of Potassium Content ············ 254
1146 Test far Histamine ··· ··· ··· ··· ······ ··· ······•·· ··· 183 3110 Determination of Sodium Content ··············· 254
1147 Test far Allergen ································· 184 3111 Determination of Sodium Caprylate ···•········ 254
1148 Test far Hemolysis and Agglomeration ······ 184 3112 Determination of Acetyltryptophan ············ 255
1200 Biological Activity ~ys · ·• ••••· •••• •• ••·• ••• •••••• 185 3113 Determination of Phenol Content ··············· 255
1201 Microbiological Assay of Antibiotics ····••······ 185 3114 Determination of Meta Creso! Content ··· ··· ··· 255
1202 Preparation of Penicillinase and Determination of 3115 Determination of Thimerosal Content •········ 256
Its Activity .......................................... 191 3116 Determination of Methyl Parahydroxybenzoate
1205 Biological Assay of Vasopressin ............... 191 and Propyl p-Hydroxybenzoate Contents ······ 257
2
 3117
Determination of 0-Acetyl Content ············ 257 (Pseudomonas) ··· ··· ••···· ·•• ······ ••· •·· ··· •·• ••· 284
3118
Determination of Adipic Dihydrazide Content 3414Determination of Residual Host Yeast Protein
......................................................... 258
··················•······································ 285
3119 Determination of Content in High Molecular 3415 Test for Blood Group A-Like Substance
Weight Conjugate ································· 258 (Hemagglutination lnhibition Assay) ········· 286
3120 Determination of Saccharide and Sugar Alcohol 3416 Determination of Residual Murine lgG ······•·· 286
Contents in Human Blood Products ············ 259 3417 ldentity Test for Acellular Pertussis Vaccines
3121 Determination of Polymer Content in Human (Enzyme Linked Immunosorbent Assay) 287
Albumin ··· · ·· · · · · ·· · · · · · · · · · · · · · ·· ··· · ·· ·· · · ·· · · · · ·· 259 3418 ldentity Test for Antitoxin and Antiserum
3122 Determination of lgG Monomer and Dimer in (Enzyme Linked Immunosorbent Assay) 287
Human lmmunoglobulins · · · · ·· ··· · ·· ··· · ·· · · · · ·· 260 3419 Determination of Molecular Size Distribution for
3123 Determination of Glycine Content in Human Group A Meningococcal Polysaccharide · · · · · · 288
lmmunoglobulin ···································· 260 3420 Determination of Molecular Size Distribution for
3124 Determination of Protein Content in Recombinant Typhoid Vi Polysaccharide ·· ................ · ..... 289
Human Granulocyte Colony-stimulating Factor 3421 Determination of Polysaccharide Content in
..................................•...................... 261 Haemophilus influenzae Type b Conjugate
3125 Determination of Free Histamine Phosphate in Vaccine ··········································•····· 289
Human Histamine lmmunoglobulin ············ 261 3422 Test for Human Thrombin Activity ··· ···•····· 290
3126 Determination of lgG Content (Ultraviolet- 3423 Test for Activated Coagulation Factor Activity
visible Spectrophotometry) · · · · · · · · · · · •·• · ··• ••· 262 ......................................................... 290
3127 Determination of Molecular Size Heterogeneities 3424 Determination of Heparin Content (Coagulation
in Monoclonal Antibodies ·· · · · · ·· · ·· · · · · ·· · ··· ·· · 262 Method) ················•·······•···················· 290
3200 Determination of Chemical Residues · · · · · · · · · · · · · · · 264 3425 Test for Anti-A and Anti-B Hemagglutinins
3201 Determination of Residual Ethanol Content Ondirect Anti-human Globulin Test) 291
( Conway Diff usion Method) · · · · · · · · · · · · · · · · · · · · · 264 3426 Determination of Human Erythrocyte Antibody
3202 Determination of Residual Polyethylene Glycol (Microtitre Plate Method) •·· ··· ··· ··· ········· 292
Content · ·· ··· · ·· · · · · ·· · · · · · · · · · · · · ··· · ·· · ·· ·· · · · · · · · ··· 264 3427 Determination of Human Platelet Antibody
3203 Determination of Residual Polysorbate 80 ....................................•.......•............ 292
Content · · · · ·· · · · · · · · · · · · · · · · · · · · · · · ·· ··· · · · ·•· · · · ·· · · ·· 264 3500 Test for Biological Activity/Potency ··············· 293
3204 Determination of Residual Glutaraldehyde 3501 In vitro Test for Relative Potency of
Content · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · •· · · · · · · · · · 265 Recombinant Hepatitis B Vaccine (Yeast)
3205 Determination of Residual Tributyl-phosphate ························································· 293
Content · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 265 3502 In vitro Test for Relative Potency of Hepatitis A
3206 Determination of Residual Carbodiimide Content V accine, lnacti va ted · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 293
......................................................... 266 3503 Potency Test on Rabies Vaccine for Human Use
3207 Determination of Free Formaldehyde Content (NIH Method) ··· ··· ··· ··· ... ··· ··· ··· ··· ··· ······ 293
......................................................... 266 3504 Potency Test on Adsorbed Tetanus Vaccine
3208 Determination of Residual Aluminium Content ......................................................... 294
in Human Albumin •································ 267 3505 Potency Test on Adsorbed Diphtheria Vaccine
3209 Determination of Residual Hydroxylamine · · · 268 ......................................................... 294
3300 Microbiological Test ···································· 268 3506 Determination of Flocculation Unit of Toxoid
3301 Test for Mycoplasma ······························ 268 ......................................................... 295
3302 Test for Adventitious Viruses ······ ······ ······ 270 3507 Potency Test on Diphtheria Antitoxin (Rabbit
3303 Test for Murine Viruses ··· ··· ··· ··· ··· ··· ······ ··· 271 Skin Test) ··· ··· ··· ··· ··· ······ · ·· ··· ··· ··· ··· ······ 296
3304 Test for Nucleotide Sequence of SV40 ········· 273 3508 Potency Test on Tetanus Antitoxin (Mouse
3305 Neurovirulence Test in Monkeys ··············· 273 Bioassay) ············································· 296
3306 Requirements for Virus Nucleic Acid Testing 3509 Potency Test for Gas-gangrene Antitoxins
on Source Plasma used for Blood Products (Mouse Bioassay) ································· 297
......................................................... 274 3510 Potency Test for Botulinum Antitoxin
3400 Bioassay Method · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 276 (Mouse Bioassay) ································· 298
3401 lmmunoblot ·········································· 276 3511 Potency Test for Antivenins (Mouse Bioassay)
3402 lmmunodot ··· ··· ········· ··· ··· ··· ··· ··· ··· ······ ··· 276 ......................................................... 299
3403 Double Immunodiffusion ·· · · · · ··· ·· · ·· · ··· · · · · · · 277 3512 Potency Test for Rabies lmmunoglobulin ······ 300
3404 lmmunoelectrophoresis · · · · · · · · · · · · · · · · · · · · · · · · · · · 277 3513 Potency Test for Diphtheria Antitoxin in Human
3405 Peptide Mapping ··• · · · · · · ·· · ··· · · · ··· ··· ·· · ··· · · · •· · 278 lmmunoglobulin .................................... 302
3406 Test for Losing Rate of Plasmid ··············· 278 3514 Test for Fe Function in Human lmmunoglobulin
3407 Determination of Residual Extraneous DNA ......................................................... 303
......................................................... 279 3515 Potency Test for Anti-human T Lymphocyte
3408 Determination of Residual Antibiotics (Culture Immunoglobulin (E Rosette Formation-
Method) · · · ··· ··· · · · · ·· · · · · ·· · ·· · ·· · · · ··· ··· ·· · ·· · · ·· 281 inhibition Test) ··· ................................. 304
3409 Determination of Prekallikrein Activator 3516 Potency Test for Anti-human T Lymphocyte
Content ................................................ 281 lmmunoglobulin (Lymphocytotoxicity Test)
3410 Test for Anticomplement Activity ············ 282 ......................................................... 304
3411 Determination of Residual Bovine Serum 3517 Potency Test for Human Coagulation Factor Il
Albumin Content .................................... 283 (One-step Method) .............................. 305
3412 Determination of Residual Host Bacteria! Protein 3518 Potency Test for Human Coagulation Factor VH
(E. coli) ··•···•···•·••••··••··•••••••·••··••··•••• 284 ( One-step Method) .. · · · · · .. · · · · .... · · · · · · · .. · · · · 305
3413 Determination of Residual Host Bacteria! Protein 3519 Potency Test for Human Coagulation Factor IX
3
 ( One-step Method) · · · · · · •· •·· · · · · •·· · · · •· · · · · · · · 306 9012 Guidelines far Validation of Quantitative
Potency Test far Human Coagulation Factor X
3520 Analytical Method of Biological Samples 387
( One-step Method) · · · · · · •· · · · · · · · · · · · · · · · · · · · · · · 306 9013 Guidelines far Sustained, Controlled and
3521 Potency Test far Human Coagulation Factor Vlll Delayed Release Preparations ············•········ 393
( One-step Method) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 307 9014 Guidelines far Microparticle Preparations ······ 396
3522 In vivo Test far Biological Activity of 9015 Guidelines far Studies and Quality Control
Recombinant Human Erythropoietin of Drug Polymorphism ··························· 397
(Reticulocyte Method) •··•••··•····•····•·•······ 307 9101 Guidelines far Validation of Analytical Method
3523 Biological Activity Test on lnterferon ··· ··· ··· 307 Adopted in Pharmaceutical Quality Specification
3524 Biological Activity Test far Recombinant ......................................................... 400
Human lnterleukin-2 (CTLL-2 Cell /MTT 9102 Guidelines far the Analysis of Impurities in
Colorimetric Method) · · · · · · · · · · · · · · · · · · · · · · · · · · · 309 Drugs ················•······························· 404
3525 Biological Activity Test far Recombinant Human 9103 Guidelines far Hygroscopicity ·· · ··· · ·· · ·· · · · ·· · 406
Granulocyte Colony-stimulating Factor (NFS-60 9104 Guideline far Near-infrared CNIR)
Cell Strain/MTT Colorimetric Method) · · · · · · 309 Spectrophotometry · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 406
3526 Biological Activity Test on Recombinant Human 9105 Guidelines far Bioactive Assays of Traditional
Granulocyte/Macrophage Colony-stimulating Chinese Medicine · · · · •· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 408
Factor (TF-1 Cell/MTT Colorimetric Method) 9106 Guidelines far Pharmaceutical Evaluation based
.....•................................................... 310 on Microarrays · · · · •· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 409
3527 Biological Activity Test far Recombinant Bovine 9107 Guidelines far Molecular DNA Barcoding of
Basic Fibroblast Growth Factor (Cell Chinese Materia Medica · · · · · · · · · · · · · · · · · · •· · · · · · · · 411
Proliferation/MTT Colorimetric Method) ······ 311 9201 Guidelines far Validation of Alternative
3528 Biological Activity Test far Recombinant Microbiological Methods far Pharmaceutical
Epidermal Growth Factor (Cell Proliferation/ Products ············································· 413
9202 The Guidelines far Microbiological Examination
MTT Colorimetric Method) · · · · · · · · · · · · · · · · · · · · · 311
3529 Biological Activity Test far Recombinant of Non-sterile Products ··················•········ 416
9203 Guidelines far Quality Management of
Streptokinase •· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 312 Microbiology Laboratory far Pharmaceutical
3530 Biological Activity Test far Mouse Nerve Growth
Products ············································· 417
Factor ·········•········•····························· 312 9204 Guidelines far Microbial Characterization,
3531 Biological Activity Test far Nimotuzumab
Identification, and Strain Typing ··············· 422
Inj ection •· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 313 9205 Guidelines far Microbiological Monitoring and
3532 Biological Activity Test far Recombinant Human
Control of Clean Rooms far Pharmaceutical
lnterleukin-11 (lnterleukin B9-11 Cell/MTT
Products · · · · · · · · · · · · · · · · · · · · · •· · · · · · · · · · · · · · · · · · · · · · · 425
Colorimetric Method) · · · · · · · · · · · · · · · · · · · · · · · · · · · 315 9206 Guidelines far Validation of Isolator Systems far
3533 Potency Test far Botulinum Toxin Type A
Use in Sterility Testing ··························· 428
(Method of Parallel Lines) ··•·················· 315 9301 Guidelines far Application of Safety Tests far
3600 Specific Biologica Raw Materials/ Animals · · · · · · · · · 316
Injections ·····················•······················· 430
3601 Test Requirements far Monitoring SPF Chick 9302 Guidelines far Establishment of Limit far
Embryos ······•······································ 316 Harmful Residue of Traditional Chinese
3602 Requirements far Microbiological Test on
Medicine ·····················•··············•········ 432
Laboratory Animals ······························ 316 9303 Guidelines far Determination of Pigments 434
3603 Requirements far Parasitological Test on 9304 Guidelines far Determination of Aluminium (Al),
Laboratory Animals ························•····· 318 Chromium (Cr), Iron (Fe) and Barium (Ba) in
3604 Test Requirements far Newborn Calf Serum Traditional Chinese Medicine····················· 437
......................................................... 318
9305 Guidelines far Determination of Mycotoxins in
3605 Culture Media far Biochemical Reactions of Traditional Chinese Medicine ····················· 437
Bacteria and Test Method ························ 320 9501 Guidelines far the Quality Control of Positron
3700 ............................................................ 323
Emission Tomographic Radiopharmaceutical
3701 List of National Standards far Biologics (far Preparation ·········································· 440
Potency, Titer, Activity, Concentration and 9502 Guidelines far the Quality Control of Technetium
[ m Te J Radiopharmaceutical Preparations
99
Content Tests) ·•·································· 323 · · · · · · 441
8000 Reagents and Reference Substance ·················· 325 9601 Guidelines far Research and Evaluation on
8001 Reagents ······ ··· ··· ··• ··· ··· ········· ······ ··· ··· ··· 325 Functionality-related Characteristics of
8002 Test Solutions ··· ··· ··• ··· ··· ······ ··· ··· ··· ······ ··· 346 Pharmaceutical Excipients ························ 443
8003 Test Papers ··· ········• ··· ··· ······ ······ ··· ··· ··· ··· 353 9621 Guidelines far General Requirements far Drug
8004 Buffer Solutions · ·· ··· ·· · · · · · · · · · · · · · · · · · ·· · · · •· · ·· · 354 Packaging Materials and Containers ············ 445
8005 Indicator Solutions ·· · ··· ·· · · ·· ·· · ··· · ·· · · · · · · · · · ··· 356 9622 Guidelines far Pharmaceutical Glass Materials
8006 Volumetric Solutions ······························ 358 and Containers ·····················•················· 447
8061 Reference Standards, Reference Drugs and 9901 Guideline far Preparation of Pharmaceutical
Reference Extractives ······························ 363 Standard Substances of China ·················· 449
8062 Reference Standards ·•· ············ ··· ··· ··· ······ 370 Annex table ······················································ 451
9000 ............................................................... 378 Atomic Weights of Elements · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 451
9001 Guidelines far the Stability Testing of Drug Ingredients of Patent Medicines not Included in the
Substances and Preparations ..................... 378 Pharmacopoeia ·· · · •· · · · ·· · · ·· ··· · · · · · · · · · · ·· ··· ··· · ·· · · · · · · 452
9011 Guidelines far in vivo Bioavailability and The list of the code number of the general Chapters in
Bioequivalence Studies far Drug Preparations Chp 2015 compared with that of the appendix in
.•........................•.•............................ 381 Chp 2010 ··· ·······•···· ··· ··· ··· ··· ············ ··· ··· · ·· ··· 459
4
MONOGRAPHS
PART I

0100 General Requirements for Preparations
l. Drug substances in the General Requirements for Prepa-
rations, are active ingredients for preparation, including
Chinese medicinal, chemical and biological active ingredients.
The Chinese medicinal substances are slices, vegetable oil &.
fat, extract, effective constituent or part. The chemical
substances are the active ingredients derived from chemical
synthesis, or are the substances extracted from natural
materials or prepared by biotechnology. The biological
substances are the biological bulk solutions or powders dried
from the biological bulk.
2. The preparation of the dosage forms or sub-dosage forms in
the General Requirements for Preparations should depend on the
characteristics of the drug substances, clinical demands, as well
as the safety, effectiveness, and stability of the drug products,
and might not be suitable for all drug substances.
3. The General Requirements are applicable to Chinese
medicine, chemical preparations and biologics for therapeutic
use ( including blood products, immune sera, cytokines,
monoclonal antibodies, immunoregulators, probiotics etc).
The biologics for prophylaxis should comply with the
requirements in relevant monographs of Volume ill.
4. Unless otherwise specified, biological products should be
stored and shipped at 2-BºC, protected from light.
0101 Tablets
Tablets are solid preparations of various shapes, round or
heteromorphic, and obtained by compressing uniform volumes of
particles consisting of drug substance with suitable excipients.
Traditional Chinese medicinal tablets also include extract
tablets, semi-extract tablets and powdered crude drug
tablets.
Tablets are mainly conventional oral tablets, but also include
lozenges, sublingual tablets, dental patches, chewable
tablets, dispersible tablets, soluble tablets, effervescent
tablets, vaginal tablets, effervescent vaginal tablets,
sustained-release tablets, controlled-release tablets, and
enteric-coated tablets, etc.
Lozenges Lozenges are tablets intended to be placed in the
mouth where they are slowly dissolved to exert local or
systemic action.
The drug substance in lozenges should be freely soluble in
water. Lozenges are intended mainly for local antiphlogistic,
antimicrobial, apocrustic, analgesic or local anesthetic
action.
Sublingual tablets Sublingual or buccal tablets are tablets,
intended to be inserted beneath the tongue, where they are
dissolved rapidly and the drug substances are absorbed
directly through mucosa to obtain a systemic effect.
The drug substances in sublingual tablets are directly
absorbed. Sublingual tablets are intended mainly for
treatment of emergencies.
Dental patches Dental patches are preparations that are
intended for adhesion to the mucous membrane of the mouth
for local or systemic effect.
They should comply with the requirements for Dissolution
and Drug Release Test <0931 ) .
0101 Tablets
Chewable Tablets Chewable tablets are tablets intended to
be chewed and then swallowed.
Mannitol, sorbitol, or sucrose, which are excipients freely
soluble in water, are usually utilized as fillers and binders.
The hardness of chewable tablets should be suitable.
Dispersible Tablets Dispersible tablets are tablets which are
intended to be dispersed rapidly in water giving a uniform
dispersion befare administration.
The drug substances in the dispersible tablets are usually
insoluble in water. Dispersible tablets may be administered
after being dispersed in water or sucked in the mouth or
swallowed whole.
Dispersible tablets should comply with the requirements for
the Dissolution and Drug Release Test < 0931 ) and
uniformity of dispersion.
Soluble tablets Soluble tablets are film-coated or uncoated
tablets which are dissolved in water befare administration.
Soluble tablets are freely soluble in water, and their solutions
may be slightly opalescent and are intended for oral admini-
stration, externa! application or gargling.
Effervescent tablets Effervescent tablets are tablets
containing sodium bicarbonate and organic acids which
release carbon dioxide giving an effervescent appearance when
dissolved in water.
The drug substances in effervescent tablets are freely soluble
in water. Organic acids such as citric acid, tartaric acid and
fumaric acid are usually used.
Vaginal tablets and effervescent vaginal tablets They are
tablets intended for administration to the vagina. Their
shape should be suitable for vaginal administration, and
may be inserted into the vagina with a suitable device.
Vaginal tablets are conventional tablets and may be readily
dissolved, dispersed, melted, or disintegrated in the
vagina, and the drug substances are released to obtain a
local antiphlogistic and antimicrobial effect. Sexual
hormones can also be administered in this way while the
drug substances with a local irritant effect should not be
prepared in such a way.
Vaginal tablets should comply with the Disintegration Test
for Suppositories and Vaginal Tablets <0922). Effervescent
vaginal tablets should comply with the requirements for the
test of effervescent volume.
Sustained-release tablets Sustained-release tablets are tablets
which release drug substances in a gradual, non-constant rate
way in a specified release medium. They should comply with
the related requirements for sustained-release preparations
(0913) and the Dissolution and Drug Release Test (0931 ).
Controlled-release tablets Controlled-release tablets are
tablets which release drug substances in a gradual, constant
rate way in a prescriptive release medium. They comply with
the related requirements for controlled-release preparations
(0913) and the Dissolution and Drug Release Test (0931 ).
Enteric-coated tablets Enteric-coated tablets are tablets
coated with enteric-coating material.
Tablets may be coated with gastro-resistant coating to
 0101 Tablets
prevent the drug substances from decomposition and failing
effectiveness or irritation to stomach, or to control the
targeted release of the drug substances in the intestinal fluid.
Tablets may be coated with colon-specific film for treatment
of colon diseases.
Enteric-coated tablets, unless otherwise stated, should
comply with the test for drug release.
Orally disintegrating tablets Orally disintegrating tablets are
tablets intended to be disintegrated or dissolved rapidly in the
mouth without given additional water.
Active ingredients at low dosages are suitable for preparing
orally disintegrating tablets which could be used for patients
with swallowing difficulty or uncooperative administration.
The orally disintegrating tablets are mainly prepared by
direct compression method and freeze-drying method.
Orally disintegrating tablets should be rapidly disintegrated
or dissolved in the mouth with acceptable taste and be easily
swallowed, without irritation to the mouth mucosa.
Except the orally disintegrating tablets prepared by
freezedrying method, the orally disintegrating tablets comply
with the Determination of Disintegration ( 0921 ). In
addition, orally disintegrating tablets with poor water soluble
active ingredients should also comply with the Dissolution
and Drug Release Test ( 0931 ). The orally disintegrating
tablets prepared by enteric-coated granules should also
comply with the Dissolution and Drug Release Test ( 0931 ) .
Orally disintegrating tablets prepared by freeze-drying
method may not comply with the test for friability.
The production and storage of tablets should comply with the
following requirements.
l. The drug substances should be mixed with the excipients
thoroughly. Tablets containing drug substances toxic or
potent in nature or those administered at small dosages are
dispersed uniformly in a way appropriate for the substances
concerned.
2. Tablets containing volatile, thermolabile or photosensitive
substances should be protected from light or heat during
production to avoid loss and failing effectiveness.
3. The moisture content of the drug substances or granules
used in the process should be controlled to meet the
requirements of processing and to prevent mold conta-
mination or deterioration during storage.
4. Excipients such as flavouring, aromatic and colouring
agents may be added to lozenges, dental patches, chewable
tablets, dispersible tablets, effervescent tablets and orally
disintegrating tablets, according to the compliance
requirement.
5. Tablets may be coated with sugar or film for a variety of
reasons, such as enhancement of stability, masking of
unpleasant tastes, and improvement of appearance. To
prevent the drug substances from decomposition and failing
effectiveness or irritation to stomach, or to control the
targeted release of the drug substance in the intestinal fluid,
tablets may be coated with gastro-resistant coating. If
necessary, film-coated tablets should be examined for
residual organic solvents.
6. Tablets have a clean, smooth appearance and uniformly
coloured surface. They possess suitable hardness and
wearability to protect them from damage or cracking during
packing and transportation. Unless otherwise specified, the
uncoated tablets should comply with the Test for Tablet
Friability ( 0923).
7. Tablets should comply with the requirements for
microbial limit.
8. Tablets should comply with the requirements for the tests
of dissolution, drug release, content uniformity according to
the characteristic of the active ingredient or the dosage
formulation, except those with complex active ingredients
from animal or plant origins, for which a suitable
determination method is difficult to develop.
9. Unless otherwise specified, tablets should be stored in
tightly closed containers. The biological product tablets shall
be stored and shipped in tightly closed containers at
2-8ºC. The origins and production procedures of source and
subsidiary materials used shall be controlled strictly and shall
comply with the requirements for tablets.
Unless otherwise specified, tablets should comply with the
following requirements.
Weight variation Tablets should comply with the following
requirements.
Average weight or labelled weight Weight variation limit
Less than O. 30 g ±7. 5%
Not less than O. 30 g ± 5. O%
Procedure Weigh accurately 20 tablets and calculate the
average weight; then weigh individually each of the 20
tablets and compare the weight of each tablets with the
average weight ( if assay is not required, the weight of each
tablet should be compared with the 'labelled weight). Not
more than 2 of the individual weights shall deviate from the
average weight by more than the weight variation limit
shown in the table, and none deviate by more than twice
the limit.
Befare being coated with sugar, the tablet cores should
comply with the test for weight variation. Sugar coated
tablets are not required to comply with the test for weight
variation while film coated tablets are required to comply
with.
Where the test for content uniformity is specified, the test
for weight variation may not be required.
Disintegration Unless otherwise specified, tablets should
comply with the Determination of Disintegration ( 0921 ) .
Lozenge tablets should comply with the Dissolubility Test
according to the Determination of Disintegration ( 0921 ) .
Sublingual tablets should comply with the Determination of
Disintegration ( 0921 ) .
Vaginal tablets should comply with the Disintegration Test
for Suppositories and Vaginal Tablets (0922).
Orally disintegrating tablets should comply with the
Determination of Disintegration ( 0921 ) .
Chewable tablets may not be required to comply with the test
for disintegration.
Where dissolution test or drug release test is specified,
disintegration test may not be required.
Effervescence volume Effervescent vaginal tablets should
comply with the following requirements.
Procedure Add 2 ml or 4 ml of water ( according to the
below table), accurately measured, to ten 25 ml graduated
test tubes with stoppers ( the intemal diameter is l. 5 cm; if
the tablet is large, the intemal diameter is 2. O cm)
separately, place in a water bath at 37ºC ± 1 ºC for 5
minutes. Add 1 tablet to each of the ten test tubes, stopper
the test tubes for 20 minutes, and examine the maximum
volume of effervescence. The average volume of
effervescence should be not less than 6 ml, and not more
than 2 of the tablets should be in average effervescence
volumes of of less than 4 ml.

Average weight Added water (ml)
Less than l. 5 g 2.0
Not less than 1. 5 g 4.0
Uniformity of dispersion Dispersible tablets should comply
with the following requirements.
Procedure Effervescent vaginal tables should comply with
the Determination of Disintegration ( 0921 ) . The intemal
diameter of stainless steel sieve is 710 µm while the water
temperature is 15-25ºC. Six tablets should be disintegrated
and should pass through the sieve within 3 minutes.
Microbial limit Tablets containing non-monomer active
ingredients from animal, plant herb or mineral origin;
biological product tablets; tablets far local administration
(such as buccal tablets, soluble tablets far extemal use,
vaginal tablets, effervescent vaginal tablets, etc. ) , should
comply with the test far Microbial Limit of Nonsterile
Products. The results should comply with the requirements
of the Microbiological Examination of Nonstable Product:
Microbial Enumeration Tests ( 1105 ) , Microbiological
Examination of Nonsterile Product: Tests far Specified
Microorganisms ( 1106 ) and Microbiological Acceptance
Criteria of Nonsterile Pharmaceutical Products ( 1107). Far
biological product tablets, where the test far contaminating
microorganisms is required, the microbial limit test may not
be carried out.
0102 lnjections
Injections are sterile products prepared using drug substances
with suitable excipients which are intended far administration
by injection into the body.
lnjections are classified as liquids far injection, sterilized
powders far injection and concentrated solutions far
injection.
Liquids for btjection Liquids far injection are solutions,
emulsions and suspenions, which can be applied to subcu-
taneous, intracutaneous, intramuscular and intravenous
injections, intravenous infusion, as well as intrathecal and
intraspinal injecting administrations. The large volume
injections ( unless otherwise specified, the volume is not less
than 100 ml, or not less than 50 ml far biological products)
far intravenous infusion are also called intravenous
transfusions. Normally, herb products are unfavorable far
preparing suspension injections.
Sterile powders for injection Sterile powders far injection are
sterilized powders or sterilized nubby materials containing
drug substances, which should be dispensed as liquids far
injection using suitable sterile solutions befare use. They are
prepared by filling under sterile conditions or by lyophili-
zation. They should be prepared by addition of a suitable
solvent far injection befare injection, or prepared by addition
of intravenous transfusion befare intravenous infusion. The
sterile powders of biological products prepared by
lyophilization are considered as freeze-dried preparations far
injection.
Concentrated solutions for injection Concentrated solutions
far injection are sterile concentrated solutions containing drug
substances which are intended far injection or intravenous
infusion after dilution.
The production and storage of injections should comply with
the following requirements.
0102 Injections
l. Aqueous solutíons far injectíon should be clear; unless
otherwise specífied, partícles contaíned in suspensíons far
injection should not exceed 15 µm in diameter, particles of
15-20 µm (rarely up to 20-50 µm) in díameter should not
exceed 10%. If a sedíment appears, the suspensions far
injection should be readíly dispersed on shaking. Suspensions
far injection can not be used far intravenous or vertebral
canula injection. Emulsions far injection should be stable and
show no evidence of phase seperation, and shall not be used
far vertebral canula injection. Over 90 % of the globules in
the emulsions far intravenous infusion should be less than
1 µm, and none is greater than 5 µm, in diameter. Unless
otherwise specified, intravenous infusions should be isotonic
with blood as far as possible.
2. Sources and manufacturing processes of drug substances
and excipients should be strictly controlled to comply with
the quality requirements far injection. Unless otherwise
specified, the drug substances far preparation of traditional
Chinese medicinal injection, such as prepared slices are
extracted, purified by the methods directed in individual
monographs to produce the semi-finished and finished
products which are subjected to overall control tests. The
production and quality control of bulk, final bulk and final
product of biologics should comply with the requirements in
individual monographs.
3. The solvents far injections should be safe and innocuous,
with good compatibility to other drug components, and shall
not affect the therapeutic efficacy and quality of active
ingredients. Aqueous solvents and non-aqueous solvents are
generally used as vehicles far injections.
(1) Water far injection is the most commonly used aqueous
solvents, O. 9 % sodium chloride solution or the aqueous
solutions of other suitable substances may also be used.
( 2) Vegeta ble oil is the most commonly used non-aqueous
solvent. Soya-bean oil for injection is commonly used as oil
solvent. Non-aqueous solvents also include solutions of
ethanol, propanediol and polyethylene glycol. Non-aqueous
solvents used far injection should be strictly controlled in
quantity and be examined as specified m individual
monographs.
4. Suitable additives may be added according to the need.
The common additives include the substances which are used
far regulating the osmotic pressure, adjusting the pH,
enhancing the solubility of the drug substances, and
antioxidants, antimicrobial preservatives, emulsifying agents,
and suspending agents, etc. The additives should not affect
the therapeutic efficacy, and not interfere with quality
testing. The concentration used should not cause toxicity or
obvious stimulation. Antioxidants commonly used are
sodium sulfite, sodium bisulfite and sodium pyrosulfite,
etc. , of which the concentrations used are generally O. 1 %-
0. 2 %; Multi-dose injections may contain an appropriate
concentration of suitable antimicrobial preservatives. The
concentration of antimicrobial preservatives should be enough
to prohibit the growth of micro-organisms in the injections.
Unless otherwise specified, the antimicrobial effect of these
injections in formula should comply with the Guide far
Antimicrobial Effectiveness Test ( 1121 ) . Sterilization
should be conducted far those injections with added
antimicrobial preservatives. It is not allowed to use
antimicrobial preservatives far injections to be administrated
by intravenous infusion, intracistemal injection, extradural
mJection, or vertebral canula injection. Antimicrobial
preservatives commonly used are O. 5% of phenol, O. 3% of
cresol and O. 5% of chlorobutanol, etc.
5. Containers commonly used far injections include glass
 0102 Injections
ampoules, glass bottles, plastic ampoules, plastic bottles
(bags) and prefilled syringes etc. The tightness of the
containers should be confirmed by suitable means. Unless
otherwise specified, containers should comply with the
related requirements of the National Standards for glass and
plastic containers for injections. The rubber closures,
especially the closures for multi-dose containers, should be
sufficiently firm and elastic, should comply with the related
requirements of the National Standards. Unless otherwise
specified, containers should be transparent enough for visual
inspection.
6. The time for manufacturing the injections should be
reduced as far as possible to avoid the contamination of
microorganisms, and pyrogens and to prevent the drug
substances from deterioration. The manufacture process of
intravenous transfusions should be controlled more strictly.
In the manufacture of emulsions for injection and suspensions
for injection, the necessary measures should be taken to
ensure that the particle size complies with the requirement of
quality standards. Sterile powders for injection should be
manufactured under aseptic conditions. If necessary, safety
examination should be conducted to comply with the
requirement, including abnormal toxicity, hypersensitivity,
hemolysis, agglutinability, depressor substances, etc.
7. Each container of injection is filled with a volume in slight
excess of the labelled quantity if labelled volume of injection
is 50 ml or less. The excess quantities are recommended in
the following table. A container may contain drug substances
of multiple doses. Unless otherwise specified, it should not
contain more than 10 doses, and the excess quantity in such
containers should be sufficient to ensure the administration of
the labelled quantity of each <lose.
Excess quantity/ml
Labelled
quantity/ml Mobile liquid Viscous liquid
0.5 o. 10 o. 12
1 o. 10 o. 15
2 o. 15 0.25
5 0.30 0.50
10 0.50 o. 70
20 0.60 0.90
50 l. o l. 5
The containers of injections should be sealed by fusion or
hermetically sealed as soon as possible after filling.
The containers for filling the drug substances, which are
readily deterioration on exposure to air, should be evacuated
and displaced by carbon dioxide or nitrogen, etc. , during
filling, and then they are sealed by fusion or hermetically
sealed.
For temperature-sensitive preparations, the temperature
during filling and sealing shall be controlled, and the
containers after sealing shall be stored at the specified
temperature immediately.
The freeze-dried preparations for injection after filling shall
be timely lyophilized. The residual moisture content in
preparations after lyophilization should comply with the
requirements in individual monographs.
The filling and lyophilization of biologics should comply with
the Requirements for Filling and Lyophilization of Biologics.
8. Af ter being sealed by fusion or hermetically sealed,
mJections are sterilized by appropriate method according to
the nature of the drug substances to ensure the sterility of the
finished products. Containers of injections should undergo a
leaker test by appropriate methods.
9. Unless otherwise specified, the storage of injections
should be protected from light. The production and quality
control of bulk, final bulk and final product of biologics
should comply with the requirements in individual mono-
graphs.
10. The excipients used for injections should be stated on the
label or package insert. Where the injections contain a
suitable antimicrobial preservative, the label or package
insert should include the name and concentration of the added
antimicrobial preservative. For sterile powders for injection,
the label or package insert should include the vehicle used for
injection.
Unless otherwise specified, injections should comply with the
following requirements.
Filling The volume of injections and concentrated solutions
for injection should comply with the following requirements.
Procedure Take 5 containers ( the labelled quantity is not
more than 2 mD or 3 containers ( the labelled quantity is
more than 2 ml and less than 50 mD. Open the containers
with caution to avoid any loss of the contents. Take up
individually the content of each container into a dry syringe,
then discharge the content of the syringe into a caliberated
cylinder (of such size that volume to be measured occupies at
least 40% of its rated volume, and the content in the needle
is not included) slowly and continuously, and measure the
volume at room temperature. For injections of oily liquids,
emulsions or suspensions, warm ( when necessary) and
thoroughly shake the containers before removing the
contents, then process as mentioned above. Cool to room
temperature before measuring the volume ( if the containers
are warmed previously). The content in each container is not
less than the labelled quantity of the injection.
Multidose test sample for biological products Take one
container of test sample and take up the content with a
syringe according to the labeled doses and the extractable
volume of each <lose, then measure the extractable volume of
each <lose by the procedure for single <lose test sample. The
extractable volume in each <lose shall be not less than the
labeled quantity of the injection.
lf the labelled volume is more than 50 ml, carry out the test
for Mínimum Fill ( 0942), and the result should comply
with the requirements.
The extractable volume may also be calculated through
dividing the weight by the relative density of test sample.
Measure the test sample accurately and weigh precisely to
calculate the weight of each milliter, i. e. the relative density
of sample. Precisely weigh the content drawn by dry syringe
or poured directly and slowly, and divide the result by the
relative density to calculate the extractable volume of test
sample.
Test samples in prefilled syringes and cartridge Take 5
containers of test sample if the labeled quantity is 2 ml or
less; 3 containers if the labeled quantity is more than 2 ml
but not more than 50 ml. Fit the syringes with needles, or
cartridge camber devices with pistons, then transfer the
contents into a calibrated cylinder slowly and continuously
( the liquid in syringe needle shall not be drained off) , and
measure by the procedure for single <lose test sample. The
extractable volume in each container shall be not less than the
labeled quantity of the injection.
Weight variation Unless otherwise specified, the weight

variation of powders for injection should comply with the
following requirements.
Procedure Take 5 containers, remove any adherent label
and the aluminium cover from the sealed container, wash the
outside with ethanol and dry thoroughly, weigh accurately
each container as soon as possible. Open the container with
caution to avoid foreign matter such as glass bits falling into
container. For the powders in glass containers, remove the
stopper with caution to equilibrate the gas pressures inside
and outside the container, then stopper tightly and weigh
accurately the whole container immediately. Remove the
content, wash the container with water or ethanol, dry under
a suitable condition and weigh accurately, calculate the
weight of each container and the average weight of 5
containers. The weight variation of each container shall not
deviate from the average weight ( or stated weight) by a
percentage greater than that shown in the table. If the weight
variation of content of one container <loes not comply with the
above requirements, repeat the operation with another 10
containers, all of them should comply with the requirements.
Average weight Weight variation limit
O. 05 g or less ±15%
More than O. 05 g to O. 15 g ±10%
More than O. 15 g to O. 50 g ±7%
More than O. 50 g ±5%
If the test for uniformity of content is specified for a sterile
powder for injection, the test for weight variation is generally
not required.
Osmolality Unless otherwise specified, the injections for
intravenous infusion or vertebral canula injections should
comply with the requirements of the Determination of
Osmolality ( 0632) specified in individual monographs.
Visible particles Unless otherwise specified, comply with
the requirements of the Test for Visible Particles ( 0904).
Particulate matter Unless otherwise specified, solutions for
intravenous injection, intravenous infusion and intrathecal or
intraspinal injection, sterilie powders for infusion and liquid
concentrates for InJection should comply with the
requirements of the Test for Particulate Matter ( 0903).
Related substance of Chinese medicine injection Carry out the
test stated in the individual monographs, comply with the
Determination of Materials in Injection ( 2400).
Residues of heavy metals and other harmful elements
Unless otherwise specified, carry out the Test for Residual
quantities of plum bum, cadmium, arsenic, mercury and
copper in Chinese medicinal injections ( 2321), according to
the maximum daily dosage of Chinese medicinal injection in
individual monographs. The residual plumbum content
should not exceed 12 µg; while cadmium content should not
exceed 3 µg; arsenic content should not exceed 6 µg;
mercury content should not exceed 2 µg and copper content
should not exceed 15 O µg.
Sterility Comply with the requirements of the Sterility Test
<1101>.
Bacterial endotoxin or pyrogens Unless otherwise specified,
the injections for intravenous infusion should comply with the
requirements of the Test for Bacteria! Endotoxin ( 1143) or
the Pyrogen Test ( 1142 ) as specified in individual
monographs.
0103 Capsules
0103 Capsules
Capsules are solid preparations consisting of drug substances
with or without excipients, filled in hollow hard capsules or
sealed in soft shells. They may be classified into hard, soft,
sustained-release, controlled-release, and enteric-coated
capsules. Capsules are mainly intended for oral
administration.
Hard capsules ( usually known as capsules) Hard capsules
are capsules containing a quantity of powders, granules,
minitablets, pellets, semisolids or liquids, ect. , with or
without suitable excipients, which are produced by suitable
preparative technology and enclosed in a hollow capsule
shell.
Soft capsules Soft capsules are capsules pre pared by
enclosing directly a quantity of liquid drug substances, or by
enclosing a solution, suspension, emulsion and semi-solid,
which are made by dissolving or dispersing solid drug
substances into spherical or elliptical soft capsule shells. The
soft capsule shells are generally made of gelatin, glycerin or/
and other suitable materials.
Sustained-release capsules Sustained-release capsules are
capsules which release drug substances in a gradual, non-
constant rate in a specified release medium. Sustained-release
capsules should comply with the Guidelines for Sustained,
Controlled and Delayed Release Preparations ( 9013) and the
Dissolution and Drug Release Test ( 0931 ) .
Controlled-release capsules Controlled-release capsules are
capsules which release drug substances in a gradual, constant
rate in a specified release medium. They should comply with
the Guidelines for Sustained, Controlled and Delayed Release
Preparations <9013) and the Dissolution and Drug Reiease
Test <0931).
Enteric-coated capsules Enteric-coated capsules are hard or
soft capsules of which the shell is prepared by suitable
enteric-coating material, or are hard or soft capsules filled
with granules or pills coated with enteric-coating material.
Enteric-coated capsules are insoluble in gastric fluid, but can
disintegrate in intestinal fluid to release active ingredients.
Unless otherwise specified, enteric-coated capsules should
comply with the Guidelines for Sustained, Controlled and
Delayed Release Preparations ( 9013) and the Dissolution
and Drug Release Test ( 0931 ) .
The production and storage of capsules should comply with
the following requirements.
l. The active ingredients or excipients enclosed in capsules
should not cause the deterioration of the shells.
2. Potent drug substances given in small doses are usually
mixed thoroughly with a suitable diluent before filling.
3. Hard capsules may be enclosed with the following
contents according to different preparative technologies.
( 1) uniform powders, granules or mini-tablets made of a
quantity of drug substances and suitable excipients such as
diluents, lubricants and disintegrating agents;
(2) pills, fast-release pills, sustained-release pills, controlled-
release pills or enteric-coated pills, in either simple form or
compound form, anda quantity of blank pills may be added
as the filler if necessary;
(3) medicinal powders without any excipients;
(4) inclusion complexes, solid dispersion, microcapsules or
microspheres of drug substances;
(5) solutions, suspensions, emulsions, etc. , filled by a
special machine, and sealed if necessary.
 O104 Granules
4. Capsules should have a clean, smooth surface and well
shaped without adhesion, deformation, leakage or breakage.
Capsules should not have foreign odour.
5. Capsules should comply with the requirements for tests of
microbial limit.
6. Capsules should comply with the requirements for tests of
dissolution, drug release, content uniformity and microbial
limit, except those which the active ingredients are derived
from animal or herb with complex ingredient without a
suitable determination method. If necessary, capsules with
coated films should be examined for residual organic
solvents.
7. Unless otherwise specified, capsules should be preserved
in tightly closed containers at temperatures not exceeding
30ºC and suitable humidity to protect from moisture, mold
contamination or deterioration. The biological product
capsules shall be stored and shipped in tightly closed
containers at 2-8ºC . The manufacture and specification
control of the bulk, final bulk and final product of biologics
should comply with the requirements m individual
monographs.
Unless otherwise specified, capsules should comply with the
following requirements.
Water Water determination for Chinese medicinal hard
capsules is required. The water content of the contents of the
hard capsule is determined by the methods described in
Determination of Water< 0832). Unless otherwise specified,
the water content is not more than 9. O per cent.
Water determination is not required where the contents of
hard capsules are liquids or semisolids.
Weight variation Capsules should comply with the following
weight variation limit.
Procedure Weigh accurately each of 20 capsules C10
capsules for Chinese medicine) , unless otherwise specified.
Open each capsule without loss of shell material, and remove
the content as completely as possible: for hard capsules,
clean the shell with a small brush; for soft capsules or the
hard capsules in which the contents are semisolids or liquids,
wash the shell with ether or other volatile solvents, and
allow to stand until the odour of the solvents is no longer
perceptible. Weigh the shell of each capsule. Calculate the
weight of content of each capsule and the average weight.
Not more than 2 of the individual weights should deviate
from the average eor labelled) weight by more than the
weight variation limit shown in the table, and none should
deviate by more than twice the limit.
Average or labelled weight Weight variation limit
Less than O. 30 g ±10%
±7. 5% (±10% for
O. 30 g or more
Chinese medicine)
Weight variation test may not be required for the capsules
where content uniformity test is specified.
Disintegration test Unless otherwise specified, capsules
should comply with the Determination of Disintegration
<0921 >.
Where a dissolution test or drug release test is specified, the
disintegration test may not be required.
Microbial limit Capsules containing non-monomer active
ingredients derived from animal, herb or minerals should
comply with the Microbiological Examination of Nonsterile
Products: Microbial Enumeration Tests < 1105 ) ,
Microbiological Examination of Nonsterile Products: Tests
for Specified Microorganisms < 1106 ) and Microbiological
Acceptance Criteria of Nonsterile Pharmaceutical Products
(1107). For biological product capsules, where the test for
contaminating microorganisms is specified, the microbial
limit test may not be required.
0104 Granules
Granules are dry granular preparations with appropriate
particle size made of drug substances and suitable excipients.
Granules may be classified as soluble granules Cusually as
known as granules ) , suspended granules, effervescent
granules, enteric-coated granules, sustained-release granules
and controlled-release granules.
Suspended granules Suspended granules are dry granules
with appropriate particle size made of insoluble solid drug
substances and suitable excipients. Water or other suitable
liquid is added with shaking before use to distribute the
contents and forma suspension.
Unless otherwise specified, suspended granules should
comply with Dissolution and Drug Release Test <0931 ) .
Effervescent granules Effervescent granules are granules
containing sodium bicarbonate and organic acids, which
release a quantity of effervescent gas when water is added.
The active ingredients in effervescent granules are freely
soluble in water and in the effervescent solution. Organic
acids such as citric acid, tartaric acid, etc. are usually used.
Enteric-coated granules Enteric-coated granules are granules
coated with enteric-coating material or made by other suitable
methods. Enteric-coated granules are resistant to gastric
acid, thus the active ingredients are released in intestinal
fluid or in specific site of the intestinal tract under control to
prevent from decomposition or irritation to the stomach.
Enteric granules should comply with Dissolution and Drug
Release Test <0931 ) .
Sustained-release granules Sustained-release granules are
granules which release drug substances in a gradual, non-
constant rate way in a specified release medium.
They should comply with the Guidelines for Sustained,
Controlled and Delayed Release Preparations < 9013) and
Dissolution and Drug Release Test <0931 ) .
Controlled-release granules Controlled-release granules are
granules which release drug substances in a gradual, constant
rate way in a specified release medium.
They should comply with the Guidelines for Sustained,
Controlled and Delayed Release Preparations < 9013) and
Dissolution and Drug Release Test (0931 ).
The production and storage of granules should comply with
the following requirements.
l. The drug substances and excipients should be mixed well.
Granules containing drug substances toxic or potent in nature
or those administered at small dosages are dispersed uniformly in
a way appropriate for the substances concerned.
Unless otherwise specified, the prepared slices should be
processed by extraction, purification and concentration to
form a thin extract with a required relative density as
described under individual monographs, dried by the appro-
priate methods, and pulverized to fine powder particles,
added with a quantity of excipients or finely powdered crude
drugs, mixed well and granulated. Also, a quantity of
excipients or finely powdered prepared slices may be added
into the thin extracts, mixed well and granulated. The
amount of excipients added should be controlled, which is

not more than 2 times of that of the dried extracts, or not
more than 5 times of that of the thin extracts in general.
2. A suitable temperature is controlled during the
manufacture of volatile or heat-labile drug substances, while
the drug substances unstable to light should be protected
from light.
3. The volatile oil should be sprayed evenly upon dried
granules, stored in well closed containers for the required
time, orbe added after being treated by the techniques such
as clathration, unless otherwise specified.
4. Suitable excipients such as diluents, adhesive, dispersing
agents, flavouring agents, colouring agents may be added to
granules needed.
5. Granules may also be film-coated to prevent moisture
absorption or cover up unpleasant odour. If necessary, solvent
residues should be examined for film-coated granules.
6. Granules should be dry, uniform in particle size and
colour, and show no evidence of moisture absorption,
agglomeration and deliquescence.
7. Granules should comply with the requirements for tests of
microbial limit.
8. Granules should comply with the requirements for the
tests of dissolution, drug release, content uniformity
according to the characteristics of the active ingredients or
the dosage formulation, except those which the active
ingredients are derived from animal or herb with complex
ingredients without a suitable determination method.
9. Unless otherwise specified, granules are preserved in
tightly closed containers, stored in a dry place and protected
from moisture. The production and quality control of bulk,
final bulk and final product of biologics should comply with
the requirements in individual monographs.
Unless otherwise specified, granules should comply with the
following requirements.
Particle size Unless otherwise specified, carry out the
Determination of Particle Size and Particle Size Distribution
( 0982, method 2 CTwo sieves ) ) . The total weight of
granules which can not pass through No. 1 sieve and can pass
through No. 5 sieve should not be more than 15% of the test
sample.
Detennination of water Carry out the method for the deter-
mination of water ( 0832) for Chinese medicinal granules.
The medicinal granules should contain not more than 8. O per
cent of water, unless otherwise specified.
Loss on drying Unless otherwise specified, carry out the
method for loss on drying ( 0831 ) on chemical products and
biological products. Dry the granules to a constant weight at
105ºC Cor at 80ºC under vacuum for granules containing sugar),
and the weight loss should not be more than 2. O percent.
Dispersion Unless otherwise specified, granules should
comply with the following requirements.
Procedure for soluble granules To 10 g granules C1 Unit
for single-dose packaged Chinese medicinal granules) , add
200 ml of hot water and stir for 5 minutes. The granules
should be completely dissolved or show slight turbidity.
Procedure for effervescent granules Take 3 packs of test
samples and transfer each to a 250 ml beaker containing
200 ml of 15-25ºC water. Bubbles of effervescent appearance
should be generated at once. Granules should be dispersed
completely or dissolved in the water in 5 minutes.
Examined by the methods above, granules should show no fareign
matter or burned charrings (far Chinese medicinal granules) etc.
Dispersion test may not be required for suspended granules or
granules that comply with Dissolution or Drug Release Test
( 0931) .
0105 Eye Preparations
Weight variation Weight variation limit of single-dose
granules should comply with the following requirements.
Procedure Take 10 packs Cbottles) of test samples, discard
the wrapper, and accurately weigh the content of each
separately. Calculate the weight of each pack Cbottle) and
the average weight. Compare the weight of each with the
average weight Cor the labelled weight for granules that assay
is not required). Not more than 2 individual values should
deviate more than the limit, and none should deviate more
than twice of the limit.
Average weight or labelled weight Weight variation limit
O. 1 g or less ±10%
More than O. 1 g to l. 5 g ±8%
More than l. 5 g to 6. O g ±7%
More than 6. O g ±5%
The test of weight variation may not be required for granules
where the Test for Content Uniformity is specified.
Filling Multi-dose granules should comply with the test of
Minimum Fill ( 0942).
Microbial limit Granules containing non-monomer active
ingredients derived from animal, herb or minerals and
biological product granules should comply with the Micro-
biological Examination of Nonsterile Products: Microbial
Enumeration Tests ( 1105), Microbiological Examination of
Nonsterile Product: Tests for Specified Microorganisms
( 1106) and Microbiological Acceptance Criteria of Nonsterile
Pharmaceutical Products ( 1107 ) . For biological product
granules where the test for contaminating microorganisms is
specified, the microbial limit test may not be required.
0105 Eye Preparations
Eye Preparations are sterile preparations intended for direct
application to the eye to display therapeutic effect.
Eye preparations may be classified into liquid eye
preparations Ceye drops, eye lotions, ophthalmic injections),
semi-solid eye preparations ( eye ointments, eye creams, eye
gels), solid eye preparations Ceye pellicles, eye pilules,
ophthalmic inserts) , etc. They can also be packed in a solid
form with a separate solvent, from which solutions or
suspensions are prepared before use.
Eye drops Eye drops are sterile liquid eye preparations in
the form of clear solutions, suspensions or emulsions made
of drug substances and suitable excipients, intended far
instillation onto the eye.
Eye lotiom Eye lotions are sterile aqueous solutions intended for
use in washing or bathing the eye to wash out the foreign
matter or secretions or to neutralize foreign chemical
materials.
Ophthalmic injections Ophthalmic injections are sterile
liquid eye preparations in the form of clear solution prepared
from drug substances and suitable excipients, intended for
injection into the tissues around the eye C including
subconjunctival injection, subtenon injection, and retrobulbar
injection, etc.) or into the eye ( including anterior chamber
injection, anterior chamber washing, intravitreous injection
and intravitreous perfusion, etc).
 0105 Eye Preparations
Eye ointments Eye ointments are semi-salid eye prepara-
tions in the pasty form of sterile solution or suspension made
of drug substances with suitable bases.
Eye creams Eye crearos are semi-salid eye preparations in
the form of sterile cream made of drug substances with
suitable bases.
Eye gels Eye gels are semi-solid eye preparations in the
form of sterile gel made of drug substances with suitable
bases.
Eye pellicles Eye pellicles are solid eye preparations in the
form of sterile film made of drug substances with suitable
polymer excipients, intended for use in the conjunctiva sac
for slow release of drug substances.
Eye pilules Eye pilules are salid eye preparations in the form
of a sterile sphere or almost sphere shape made of drug
substances with suitable excipients.
Ophthalmic inserts Ophthalmic inserts are sterile salid eye
preparations in suitable size and shape, made of drug
substances and suitable excipients, intended to be inserted in
the conjunctiva sac for slow release of drug substances.
The production and storage of eye preparations should
comply with the following requirements.
l. Eye preparations may contain auxiliary substances to
adjust the osmotic pressure, pH value and viscosity, or
excipients to increase the solubility of the drug substances or
to stabilize the preparation. Such additives should not
adversely affect the medicinal action or cause local irritation.
2. Unless otherwise specified, eye drops should be isotonic
with tears. Suspended eye drops may show a sediment which
is readily dispersed on shaking and should not be a cake
mass; their ratio of sedimenta! volume should be
determined. Unless otherwise specified, each container of
eye drops should not hold more than 1O ml.
3. Eye lotions are used in a large amount and should be
isotonic with tears as far as possible and have a similar pH
value to that of tears. Unless otherwise specified, each
container of eye lotions should not hold more than 200 ml.
4. Eye preparations packaged in multi-doses should contain
suitable bacteriostatics with ample safety. The label should
state the name and the amount of the added bacteriostatics.
Unless other specified, the antimicrobial effect of these
preparations should comply with the Guidelines for
Antimicrobial Effectiveness Test ( 1121 ) .
5. The selected bases of semi-salid eye preparations should
be filtered and sterilized, and the insoluble drug substances
should be finely powdered in advance. Eye ointments, eye
creams and eye gels should be homogenized, fine and
smooth. They should be applied easily to the eye for easy
dispersion and absorption of the drug substances without any
irritation. Unless otherwise specified, each container should
not hold more than 5 g.
6. Ophthalmic injections, ophthalmic inserts and eye
preparations for surgical procedures or emergency use should
not contain bacteriostatics, antioxidants or unsuitable
buffering agents. They should be packaged in sterile
disposable containers.
7. The container should be sterilie, and should not be broken
easily. The wall of the containers should be transparent enough
to carry out the test for Visible Particles in lnjections ( 0904).
8. Unless otherwise specified, eye preparations comply with
the general requirements for preparations of the dosage forms
concerned. For example, eye gels should comply with the
requirements for gels.
9. Unless otherwise specified, eye preparations should be
preserved in tightly closed containers protected from light.
10. Eye preparations must be discarded 4 weeks after
opening.
Unless otherwise specified, eye preparations should comply
with the following requirements.
Visible particles Unless otherwise specified, eye drops
should comply with the Test for Visible Particles ( 0904) of
eye drops; ophthalmic injections should comply with the
Test for Visible Particles ( 0904).
Particle size Unless otherwise specified, ophthalmic
preparations containing bulk powders of prepared slices and
suspended eye preparations should comply with the following
test.
Procedure for suspended eye drops Shake the liquid test
sample vigorously and transfer an appropriate quantity ( or a
quantity equivalent to about 10 /lg of the drug substances)
onto the slide in triplica te. Altematively, select 3 containers
of semisolid test samples, squeeze all the contents
thoroughly in an appropriate container and mix well. Take a
suitable quantity and put on the microscope slide, spread to
forma film, and cover with cover slide in triplicate. Carry
out the Determination of Particle Size and Particle Size
Distribution ( 0982 , method 1 ) . Not more 2 particles should
be greater than 50 11m ( except the test samples containing
the bulk powder of prepared slides) and no particle should be
greater than 90 11m in dimension.
Ratio of sedimental volume The ratio of sedimenta! volume
of suspended eye drops ( except the ophthalmic preparations
containing the fine powders of prepared slices) should comply
with the foilowing test and should be not less than O. 90.
Procedure Unless otherwise specified, shake vigorously
50 ml of the test sample in a stoppered cylinder for 1 minute,
and record the height H o of suspension at the beginning.
Allow to stand for 3 hours, and record the final height H.
Calculate the ratio of sedimenta! volume according to the
following equation:
Ratio of sedimenta! volume= H / H o
Metal particles Unless otherwise specified, semi-salid eye
preparations should comply with the following requirements.
Procedure Take 10 containers, extrude the content of each
container as completely as possible into culture dishes which
are 6 cm in diameter with flat bottom, free from gas bubbles
and visible particles. Cover the dishes, heat at 85ºC for 2
hours, unless otherwise specified, and allow the test sample
to distribute uniformly, cool at room temperature until the
ointment is congealed and invert each dish on the stage of a
suitable microscope. Each dish is illuminated from above
direction by a spotlight placed at an angle of 45º to the
bottom of the plate. Examine ata 30 magnification and count
the lustrous metal particles not less than 50 11m in
dimension. Not more than one container should be found to
contain more than 8 metal particles, and not more than 50
particles should be found in the 1O containers in total. If the
test does not comply with the requirements above, repeat the
test with another 20 containers. The requirements are met if
not more than 3 containers in the 30 containers are found to
contain more than 8 metal particles in each tube, and not
more than 150 particles are found in total.
Weight variation Unless otherwise specified, single-dose
packaged solid or semi-solid eye preparations should comply
with the following requirements.
Procedure Weigh accurately 20 test samples individually
and calculate the average weight. Not more than 2 of the
individual weights should deviate from the average weight Cor

the labelled weight) by more than ± 10 % and none more
than ±20%.
Where the test for content uniformity is specified, the test
for weight variation may not be required.
Filling Unless otherwise specified, single-dose packaged
liquid eye preparations should comply with the following
test.
Procedure Take 10 containers, then discharge the contents
into a caliberated cylinder ( or suitable container) , and
measure the volume. The content in each container should
not be less than the labelled quantity.
Multi-dose packaged liquid eye preparations should comply
with the test for Mínimum Fill ( 0942).
Osmolality Unless otherwise specified, aqueous eye drops,
eye lotions and ophthalmic injection should comply with the
requirements of the Determination of Osmolality ( 0632) as
specified in individual monographs.
Sterility Unless otherwise specified, eye preparations
should comply with Sterility Test ( 1101 >.
0106 Nasal Preparations
Nasal preparations are preparations intended for direct
administration to the nasal cavities to obtain a systemic or
local therapeutic effect.
Nasal preparations may be classified into liquid nasal
preparations (nasal drops, nasal washes, nasal sprays),
semi-so lid nasal preparations ( nasal ointments, nasal
creams, nasal gels) and salid nasal preparations (nasal
powders, nasal powders for spray, nasal sticks) , etc. Liquid
nasal preparations can also be packed in a salid form with a
solvent, from which solutions or suspensions are prepared
before use.
Nasal drops Nasal drops are liquid nasal preparations in the
form of clear solutions, suspensions or emulsions made of
drug substances and suitable excipients, intended for
administration to the nasal cavities.
Nasal washes Nasal washes are liquid nasal preparations
made of drug substances in isotonic aqueous solutions of
physiological pH value, intended for washing nasal cavities.
Nasal washes used for nasal injuries or surgical procedures
should be sterile.
Nasal aerosols Nasal aerosols are nasal preparations
prepared by sealing the active ingredients, additives and
suitable aerosol propellants in pressure resistant containers.
After spraying, the contents can be inhaled via the nostrils
and deposit in nasal cavities.
Nasal sprays Nasal sprays are liquid nasal preparations in
the form of clear solutions, suspensions or emulsions made
of drug substances and suitable excipients, intended for
administration to the nasal cavities through nebulization by
atomizing devices.
Nasal ointments Nasal ointments are semi-salid nasal
preparations in the pasty form of solution or suspension made
of drug substances with suitable bases.
Nasal e~ Nasal creams are semi-salid nasal preparations
in the form of cream made of uniform mixtures of drug
substances with suitable bases.
Nasal gels Nasal gels are semi-salid nasal preparations in the
form of gel made of drug substances with suitable bases.
Nasal powders Nasal powders are salid nasal preparations in
0106 Nasal Preparations l j"
the form of powder made of drug substances and suitable
excipients, intended for insufflation into nasal cavities with a
suitable tool.
Nasal powders for spray Nasal powders for spray are salid
nasal preparations in the form of powder made of drug
substances and suitable excipients, intended for spraying into
nasal cavities by a suitable valve system.
Nasal sticks Nasal sticks are salid nasal preparations in the
form of stick or almost stick shape made of drug substances
and suitable excipients, intended for inserting into nasal
cavities.
The production and storage of nasal preparations should
comply with the following requirements.
l. Nasal preparations may contain auxiliary substances to
adjust the viscosity and pH value, to increase the solubility
of the drug substances, to stabilize the preparation, or to
maintain the shape of the preparation. Unless otherwise
specified, aqueous nasal preparations of multi-doses should
contain bacteriostatics at a suitable concentration; the
antimicrobial effect of these nasal preparations should comply
with the test for antimicrobial effect effectiveness in the
processing of formulation ( 1121 ) ; bacteriostatics may not
be required when the preparation itself has sufficient
bacteriostatic effect.
2. Nasal preparations packaged in multi-doses should be
provided with an integral and appropriate device for
administration. The containers should be innocuous and
cleanly washed, and compatible with the drug substances and
excipients. The wall of the containers should be of a definite
and uniform thickness. Unless otherwise specified, the
containers should hold not more than 1O ml or 5 g of the
preparatioI1
3. Nasal solutions should be clear without sediments or
foreign matter. Nasal preparations in the form of suspen-
sions may show a sediment which is readily dispersed on
shaking. Nasal preparations in the form of emulsions may
show evidence of separation of oil and water phases but are
easily reformed on shaking. Nasal preparations in a semi-
solid form should be soft and smooth, and easy to be spread.
4. The particle size of the drug substances and excipients of
nasal powders for spray should be mostly in the range of 30-
150 µm. The size of droplets released by nasal aerosols and
nasal sprays should be mostly more than 10 µm.
5. Nasal preparations should be non-irritating and do not
adversely affect the function of the nasal mucosa and its cilia.
The pH value and osmolality of aqueous nasal preparations
should be adjusted.
6. Unless otherwise specified, nasal preparations should
comply with the general requirements for preparations of the
dosage forms concerned.
7. Unless otherwise specified, nasal preparations should be
preserved in well closed containers.
8. Nasal preparations packaged in multi-doses must be
discarded 4 weeks af ter opening.
Unless otherwise specified, nasal preparations should comply
with the following requirements.
Ratio of sedimental volume Unless otherwise specified, the
ratio of sedimenta! volume of suspended nasal drops should
comply with the following test, and should be not less than
o. 90.
Procedure Unless otherwise specified, shake vigorously
50 ml of the test sample in a stoppered cylinder for 1 minute,
and record the height H 0 of suspension in the beginning.
Allow to stand for 3 hours, record the final height H.
Calculate the ratio of sedimenta! volume according to the
 ,·· ... ,.,. . ·1
. !1.i} \ 0107 Suppositories
following equation:
Ratio of sedimenta! volume = H / H 0
Uniformity of delivered dose Metered-dose nasal aerosols,
metered-dose nasal sprays in the form of suspensions and
emulsions, and reservoir-type nasal powders for spray of
multi-dose packaging should comply with the following
requirement.
Procedure Take a container of tested sample, shake for 5
seconds and discard a puff; wait for 5 seconds at least, shake
for 5 seconds and discard a puff; repeat the process above
until 5 puffs discarded. Wait for 2 seconds, place the sample
in positive position, press the device and spray a puff
vertically ( or approximately vertically) into a collection
device. Collect the solution in the collection device with
specified solvent under individual monographs and determine
the amount of drug in the collecting liquid using to the
analytical method under individual monographs. Repeat the
procedure on 10 bottles in total.
Criteria for judgment The samples should comply with at
least one of the following requirements.
l. Not less than 9 of 10 testing results are between 75 % and
125 % of the average value, and all of the testing results are
between 65% and 135% of the average value.
2. Take another 20 containers of test samples if 2 or 3 of the
10 testing results lie outside the limit of 75 % to 125 % . Not
more than 3 of the 30 testing results should líe outside the
limit of 75 % to 125 % of the average value, and all of the
testing results should be between 65% and 135%.
Weight variation Unless otherwise specified, single-dose
packaged solid nasal preparations should comply with the
following requirements.
Procedure Weigh individually 20 test samples ( or their
contents) and calculate the average weight. Not more than 2
of the individual weights should deviate from the average
weight by more than ±10% and none more than ±20%.
Where the Test for Content Uniformity is specified, the test
for weight variation may not be required.
Filling Unless otherwise specified, liquid nasal preparations
in single-dose packages should comply with the following
requirements.
Procedure Transfer the contents of 10 tested samples
separately into 10 caliberaled cylinders, measure the volume
at room temperature and compare the packing quantity of
each sample with the labeled quantity. None should be found
to be less than the labeled quantity of the preparation.
Multi-dose packaging nasal preparations, should comply with
the test for Mínimum Fill ( 0942).
Sterility Nasal preparations for surgical procedures or
injuries should comply with be Sterility Test <1101 ).
Microbial limit Unless otherwise specified, nasal
preparations should comply with the requirements of
Microbiological Examination of Nonsterile Products:
Microbial Enumeration Tests ( 1105 ) , Microbiological
Examination of Nonsterile Products: Tests for Specified
Microorganisms ( 1106 ) and Microbiological Acceptance
Criteria of Nonsterile Pharmaceutical Products ( 1107).
0107 Suppositories
Suppositories are preparations made by incorporating drug
substances in suitable bases, intended for administration to
cavities.
Based on the different cavities they are intended to apply to,
suppositories may be classified as rectal suppositories,
vaginal suppositories and urethral suppositories. Rectal
suppositories are in the shape of a torpedo, corre or cylinder,
etc. Vaginal suppositories are in the shape of a drawing pen,
hall or oval. Urethral suppositories are usually rodlike.
The production and storage of suppositories should comply
with the following requirements.
l. Semi-synthetic fatty glycerides, cocoa butter, polyoxye-
thylene stearate, polyoxyethylene sorbitan fatty esters,
hydrogenated vegetable oíl, glycerogelatin, poloxamers,
polyethylene glycol and other suitable materials may be used
as suppository bases. Surfactants, diluents, lubricants and
preservatives, etc. may be added, if necessary. Unless other-
wise specified, the antimicrobial effect of these preparations
should comply with the Guidelines for Antimicrobial
Effectiveness Test <1121 ). Bases which are soluble in water
or able to mix with water are usually used for preparation of
vaginal suppositories.
2. Suppositories may be prepared by extrusion and molding
method. Salid drug substances, unless otherwise specified,
should be finely powdered or finest powder beforehand.
Suppositories may be made in to different shapes according to
their usage and the cavities they are intended to apply to.
3. The drug substances and bases in suppositories should be
thoroughly mixed. The suppositories should look intact and
smooth. Suppositories are non-irritating and able to be
melted, softened or dissolved when inserted into the cavity
and are miscible with body fluid to release the drug
substances gradually thus exert local or systemic effect.
Suppositories are sufficiently hard to withstand packaging or
storing without deformation.
4. Packing materials or containers used for suppositories
shall be non-toxic and do not interact physically or chemically
with the drug substances or bases.
5. Unless otherwise specified, suppositories should be
preserved and transported in well closed containers at a
temperature below 30ºC to protect from deformation, mould
contamination or deterioration due to excessive heat and
moisture. The bulk, final bulk and final product of biological
product suppositories should comply with the requirements in
individual monographs.
Unless otherwise specified, suppositories should comply with
the following requirements.
Weight variation Suppositories should comply with the
following requirements.
Average or labelled weight Weight variation limit
l. O g or less ±10%
More than l. O to 3. O g ±7.5%
More than 3. O g ±5.0%
Procedure Weigh accurately together 10 suppositories and
calculate the average weight; then weigh individually each of
the 10 suppositories. Not more than one of the individual
weights should deviate from the average weight ( or the
labeled weight) by more than the weight variation limit
shown in the table and none deviates from twice of the limit.
Where the test for content uniformity is specified, the test
for weight variation may not be required.
Disintegration test Suppositories should comply with the
requirements of Disintegration Test for Suppositories and
Vaginal Tablets (0922).

Microbial limit Unless other specified, suppositories should
comply with the Microbiological Examination of Nonsterile
Products: Microbial Enumeration Tests ( 1105), Micro-
biological Examination of Nonsterile Products: Tests for
Specified Microorganisms ( 1106 ) and Microbiological
Acceptance Criteria of Nonsterile Pharmaceutical Products
( 1107>.
0108 Pills
Pills are the solid preparations in spherical or almost
spherical shape made of the uniform mixture of the drug
substances and suitable excipients.
Chinese medicinal pills are classified into honeyed pills,
water-honeyed pills, watered pills, pasted pills, waxed pills,
concentrated pills and dripping pills, etc.
Chemical drug pills are classified into dripping pills and sugar
pills.
Honeyed pills Honeyed pills are made of fine powder of
prepared slices, using honey as binder. Among them, pills
weighing more than O. 5 g ( including O. 5 g) per pill are big
honeyed pills, while those weighing less than O. 5 g per pill
are small honeyed pills.
Water-honeyed pills Water-honeyed pills are made of fine
powder of prepared slices, using honey and water as binders.
Watered pills Watered pills are made of fine powder of
prepared slices, using water Cor yellow rice wine, vinegar,
dilute medicinal juice, dilute syrup, aqueous solution
containing less than 5 % honey etc. ) as binder.
Pasted pills Pasted pills are made of fine powder of prepared
slices, using rice powder, rice-paste or flour-paste as
binder.
Waxed pills Waxed pills are made of fine powder of prepared
slices, using beeswax as binder.
Concentrated pills Concentrated pills are made of condensed
extract of prepared slices or partial prepared slices, mixed
with appropriate excipient or fine powder of other prepared
slices, using water, honey or honey and water as binders.
They may be classified into concentrated watered pills,
concentrated honeyed pills and concentrated water-honeyed
pills based upon the different binders used in the production.
Dripping pills Dripping pills are the preparations made by
dripping a uniformly melted mixture of drug substances and
appropriate bases into an immiscible and non-interacting
cooling medium and congealing to pills in sphere or almost
sphere form.
Sugar pills Sugar pills are the preparations made of
appropriate sugar granules or base pills as the cores, sugar
powder or the mixture of other excipients as the powdering
materials, and suitable materials as binder or wetting agents.
The drug substances are encapsulated with an appropriate
method in divided quantities.
The production and storage of pills should comply with the
following requirements.
1. The powdered drugs for preparing pills are fine or very
fine powders, unless otherwise specified.
2. According to the degree of processing, honey is divided
in to primary processed honey, secondary processed and
tertiary processed honey, which may be selected and used to
prepare honeyed pills, depending on the climate and the
varieties of honeyed pills. They should appear oily-
moistened, with proper hardness.
·"1:·':a············••\.
't :':'·' ·. . ;·'. :· ~:. ·: :~
0108 Pills
3. The extract of prepared slices for preparing concentrated
pills should be made by extraction and concentration
according to the certain methods specified under the
monograph.
4. In preparing waxed pills, melt beewax by heating, then
allow to cool to a suitable temperature and add drug powder
proportionally, mix thoroughly.
5. Unless otherwise specified, water-honeyed pills, watered
pills or concentrated water-honeyed pills, concentrated watered
pills should be dried at a temperature below 80ºC; pills
containing a large amount of volatile constituents or starch,
including pasted pills, should be dried ata temperature below
60ºC. Thermolabile pills should be dried with other proper
methods.
6. The bases used for the preparation of dripping pills consist
of water-soluble and water-insoluble bases. Commonly used
bases include polyethylene glycols ( such as polyethylene
glycol 6000, polyethylene glycol 4000, etc. ) , poloxamers,
polyoxyl ( 40 ) sterate, gelatin, stearic acid, glycerin
monostearate, hydrogenated vegetable oil, etc.
7. The cooling medium for dripping pills must be innocuous
and does not interact with the drug substances. Liquid
paraffin, vegetable oil, methylsilicone oil and water are
commonly used for this purpose.
8. Unless otherwise specified, sugar pills should be dried
under suitable conditions before packing. They should be
sieved with sieves of suitable sieve number according to the
dimensions of the pills.
9. According to the characteristic of drug substances and the
requirements of usage and storage, the pills needed to be
coated and polished should be coated with the coating
materials and polished as specified under individual
monograph.
10. Oral dripping pills or pellets may be sugar-coated or
film-coated as required to suit for the propertie:s of the drug
substances and for the clinical usage and storage. If
necessary, film-coated pills should be examined for residual
organic solvents.
11. Unless otherwise specified, pills should be round,
integrate and uniform in appearance and colour, without
adhesion.
Waxed pills should be smooth in appearance without cracks,
and no drops or granules found inside the pills. The surface
cooling medium of dripping pills should be removed af ter
preparation.
12. The dripping pills and sugar pills of chemical drugs
comply with the requirements for the tests of content
uniformity and microbial limit.
13. Unless otherwise specified, pills should be kept in tightly
closed containers, protected from moisture, mold, insect
attack or deterioration.
Unless otherwise specified, the pills should comply with the
following requirements.
Detennination of water Carry out the method for the
Determination of Water ( 0832). Unless otherwise specified,
honeyed pills and concentrated honeyed pills contain not more
than 15. O%, while water-honeyed pills and concentrated
water-honeyed pills not more than 12. O%, and watered
pills, pasted pills and concentrated watered pills not more
than 9. O% of water.
No determination of water is required for waxed pills.
Weight variation (1) Unless otherwise specified, the limit
of weight variation for dripping pills should comply with the
following requirements.
Procedure Weigh accurately 20 pills and calculate the
 0109 Ointments, Creams

average weight, then weigh each of them accurately.
Compare the weight of each pill with the labeled or average
weight. Not more than 2 pills should deviate from the limit
of weight variation, and none should deviate from twice the
limit.
Procedure Take ten packs ( or vials) of pills, weigh
separately the content of each pack ( or vial) and compare
with the labelled weight. Not more than 2 packs ( vials)
should deviate from the limit of weight variation and none
should deviate from twice of the limit.

Labelled or average weight Weight variation limit Labelled weight Weight variation limit
O. 03 g or less ±15% O. 5 g or less ±12%
More than O. 03 g to O. 1 g ±12% more than O. 5 g to 1 g ±11%
More than O. 1 g to O. 3 g ±10% more than 1 g to 2 g ±10%
More than O. 3 g ±7.5% more than 2 g to 3 g ±8%

(2) Unless otherwise specified, the limit of weight more than 3 g to 6 g ±6%
variation for sugar pills should comply with the following more than 6 g to 9 g ±5%
requirements.
more than 9 g ±4%
Procedure Weigh accurately 20 pills and calculate the
average weight, then weigh each of them accurately. Filling The filling variation of multiple dose packed pills of
Compare the weight of each pill with the labeled or average
which filling is labelled in weight should comply with the test
weight. Not more than 2 pills should deviate from the limit
for Minimum Fill ( 0942).
of weight variation, and none should deviate from twice of
No filling test is required for multiple-dose packed pills of
the limit.
which filling is labelled in pill numbers.
Labelled or average weight Weight variation limit Disintegration test Unless otherwise specified, take 6 pills,
O. 03 g or less ±15% select a basket with proper porosity of sieve ( for pills at the
diameters of less than 2. 5 mm, 2. 5-3. 5 mm and more than
More than O. 03 g to O. 30 g ±10% 3. 5 mm, using sieves with pores of O. 42 mm, l. O mm and
More than O. 30 g ±7.5% 2. O mm in diameter respectively). Carry out the test as
described under the Determination of Disintegration ( 0921)
(3) Unless otherwise specified, the limit of weight using a disk. Small honeyed pills, water-honeyed pills and
variation for other pills should comply with the following watered pills should be completely disintegrated within 1
requirements. hour, while concentrated pills and pasted pills within 2
Procedure Take 10 pills as one part (1 pill as one part for hours. No disk is needed for dripping pills. Dripping pills
pills each weighing l. 5 g or more than l. 5 g). W eigh should be completely disintegrated within 30 minutes, while
separately 10 parts and compare with the labeled weight of the coated dripping pills within 1 hour. During the
each part Oabeled weight of each pill X the number of pills procedure, if pills adhere to the disk thus hinder the
weighed) ( if there is no labeled weight, compare the weight determination, take another 6 pills and carry out the
of each pill with the average weight calculated). Not more determination as described under the disintegration of tablets
than 2 parts should deviate from the limit of weight variation without disk.
and none should deviate from twice the limit. In the determination mentioned above, all the pills should
pass through the sieve within the specified time. The minute
Labeled or average weight Weight variation limit granulated masses which cannot pass the sieve but are
O. 05 g or less ±12% softened without hard core should be considered to comply
more than O. 05 g to O. 1 g with the requirements.
±11%
Waxed pills should comply with the Determination of
more than O. 1 g to O. 3 g ±10% Disintegration ( 0921) for tablets.
more than O. 3 g to l. 5 g ±9% Unless otherwise specified, no disintegration test is required
for big honeyed pills and the pills for grinding/ chewing
more than l. 5 g to 3 g ±8%
administration or the pills for administration after being
more than 3 g to 6 g ±7% dispersed with hot water/yellow rice wine.
more than 6 g to 9 g ±6% Microbial limit For pills containing non-monomer active
more than 9 g ±5% ingredients derived from animal, herb or minerals; biological
product pills, carry out the Microbiological Examination of
The core weight variation of sugar-coated pills should be Nonsterile Products: Microbial Enumeration Tests ( 1105),
examined befare coating, and should comply with the Microbiological Examination of Nonsterile Products: Tests
requirements in the table mentioned above. The examination for Specified Microorganisms ( 1106 ) and Microbiological
for weight variation is not required for sugar-coated pills after Acceptance Criteria of Nonsterile Pharmaceutical Products
coating, but is required for other-coated pills. <1107>.
Where the test for filling variation ( single-dose packed pills)
and the test for content uniformity (pills) are specified, the
test for weight variation may not be required. 0109 Ointments, Creams
Filling variation The filling variation of pills ( except sugar
pills) presented in single dose pack should comply with the Ointments Ointments are uniform semi-solid preparations
following requirements. made of drug substances and oleaginous or water-soluble

bases and are intended for external application to the skin.
Ointments may be divided into solution or suspended
ointments according to different dispersion states of the drug
substances in the bases. Solution ointments are the
ointments in which the drug substances are dissolved or
melted into the simple or compound bases; suspended
ointments are the ointments in which the fine powders of the
drug substances are dispersed into the bases evenly.
Creams Creams are uniform semi-solid preparations made
of emulsified bases in which drug substances are dissolved or
dispersed.
According to different bases, ointments may be divided
should into oil-in-water and water-in-oíl types.
The production and storage of the ointments and creams
comply with the following requirements.
l. Bases utilized for ointments and creams are selected based
on the characteristics of the preparation formulation,
properties of the drug substances, therapeutic effect of the
preparation and stability of the product. Mixed bases of
different types can also be used.
Bases for ointments are divided into oleaginous and water-
soluble bases. Oleaginous bases usually include vaseline,
paraffin, liquid paraffin, silicone oil, beeswax, stearic acid,
wool fat, etc. , while water-soluble bases mainly include
polyethylene glycols. Emulsifiers for creams are usually
divided into oil-in-water and water-in-oil types. Oil-in-water
emulsifiers include sodium soaps, triethanolamine soaps,
sodium fatty alcohol sulphate ( sodium lauryl sulphate) and
tweens, while water-in-oil emulsifiers include calcium soaps,
wool fat, mono glycerides, fatty alcohols, etc.
2. The bases of ointments and creams should be uniform,
fine and smooth without any irritation for use on skin or
mucous membranes. Insoluble solid drug substances in
suspended ointments should be finely powdered in advance
vúth suitable method to ensure the specified particle size.
3. If necessary, wetting agents, preservatives, thickening
agents, antioxidants and the transdermal accelerants may be
added to the ointments and creams. Unless otherwise
specified, the antimicrobial effect of these ointments or
creams should comply with the test for antimicrobial
effectiveness ( 1121) in the processing of the formulation.
4. Ointments and creams should be applied easily to the skin
or mucous membrane with proper viscosity but should not be
melted, and the viscosity should only change a little with
different temperatures.
5. Ointments and creams should show no evidence of
deterioration such as rancidity, foreign odour, dis-
colouration, hardening; and creams no evidence of bubbles
and separation of oíl and water.
6. Unless otherwise specified, ointments and creams should
be preserved in well closed containers, protected from light;
creams should be preserved below 25ºC but not frozen.
7. The immediate packaging materials for ointments and
creams shall not interact physically or chemically with the
drug substances or with the bases. The immediate packaging
materials for sterile preparations shall be sterile.
If ointments and creams used for burn treatment are non-
sterile products, their labels should be marked "non-sterile
preparation", and their product specifications should be
labeled "It is a non-sterile preparation". Meanwhile, "use for
minor bum" ( lst or minor 2nd degree) and "follow the
doctor' s advice" should be stated in indications and in
cautions, respectively.
Unless otherwise specified, ointments and creams should
comply with the following requirements.
Particle size For suspended ointments and ointments
0111 Preparations for lnhalation
containing powders of prepared slices, unless otherwise
specified, should comply with the following requirements.
Procedure Spread a quantity of the ointments onto three
microscope slides separately to form a thin layer of which the
area is equivalent to that of the cover-glass. Carry out the
Deterrnination of particle Size and particle Size distribution
(0982, method 1), no particle of greater than 180 µm m
dimension should be observed.
Filling Comply with the test of Mínimum Fill ( 0942).
Sterility Ointments and creams for burns ( except minor
burn of lst or minor 2nd degree) or grievous injuries should
comply with the Test for Sterility ( 1101 ).
Microbial limit Unless otherwise specified, ointments and
creams should comply with Microbiological Examination of
Nonsterile Products: Microbial Enumeration Tests ( 1105 ) ,
Microbiological Examination of Nonsterile Product: Tests for
Specified Microorganisms ( 1106 ) and Microbiological
Acceptance Criteria of Nonsterile Pharmaceutical Products
(1107>.
0110 Pastes
Pastes are semi-solid preparations which contain a large
amount ( usually more than 25 %) of drug substances
uniformly dispersed in suitable bases. Pastes may be divided
into homogeneous hydrogel and oleaginous pastes.
The production and storage of the pastes should comply with
the following requirements.
l. Bases utilized for pastes are selected based on the
characteristics of the preparation formulation, properties of
the drug substances, therapeutic effect of the preparation and
stability of the product. The bases of pastes should be
uniform, fine and smooth without any irritation for use on
skin or mucous membranes.
2. Pastes should show no evidence of deterioration such as
rancidity, foreign odour, discolouration, or hardening.
3. Unless otherwise specified, pastes should be preserved in
well closed containers, protected from light. Pastes should
be preserved below 25ºC but not frozen.
Unless otherwise specified, pastes should comply with the
following requirements.
Filling Comply with the test of Mínimum Fill ( 0942).
Microbial limit Unless otherwise specified, pastes should
comply with the requirements of Microbiological Examination
of Nonsterile Products: Microbial Enumeration Test
( 1105), Microbiological Examination of Nonsterile Product:
Tests for Specified Microorganisms ( 1106 ) and Micro-
biological Acceptance Criteria of Nonsterile Pharmaceutical
Products <1107>.
0111 Preparations for Inhalation
This general chapter is only used for general requirements of
manufacture and quality control of preparations for inhalation
(inhalation aerosols, inhalation powders, liquid preparations
for nebulization and preparations to be converted into
vapour), notas a single dosage form.
Preparations for inhalation are liquid or solid preparations
intended for administration as vapours or aerosols to the
lungs in order to obtain a local or systemic effect. They
contain one or more active substances that may be dissolved
 0111 Preparations for lnhalation

or dispersed in a suitable vehicle. Preparations for inhalation Apparatus The apparatus consists of a filter-support base
may, depending on the type of preparation, contain with an open-mesh filter--support, such as a stainless steel
propellants, cosolvents, diluents, antimicrobial preser-- screen, a collection tube that is clamped or screwed to the
vatives, solubilizing and stabilizing agents, etc. These filter--support base, and a mouthpiece adapter to ensure an
excipients do not adversely affect the functions of the mucosa airtight seal between the collection tube and the mouthpiece
of the respiratory tract or its cilia. Several categories of (Fig. D. The dose collection apparatus must be capable of
preparations for inhalation may be distinguished: inhalation quantitatively capturing the delivered dose.
aerosols, inhalation powders, liquid preparations for
nebulization, and preparations to be converted into vapour. screen
The manufacturing and storage of preparations for
~L Mmpleoollretiootube
inhalations should comply with the following requirements.
l. During the development of a preparation for inhalation
that contains an antimicrobial preservative, unless otherwise
specified, the effectiveness of the chosen preservative should
comply with requirements of Guidelines for Antimicrobial
Fat"'"- ' - -¡,---.l._
Filte<~
Effectiveness Test ( 1121 ) . Inhalation liquid preparations
should be sterile preparations.
2. In the processing of inhalation powders, suitable carriers
Mouth piece Metered-dose
or lubricants may be added to improve the flow-ability of the adapter
inhaler
powders. The added substances should be non-irritating and
non-toxic to mucosa of the respiratory tract or its cilia.
3. All the component units of the inhaler should be non-toxic,
non-irritating, stable and compatible with the contents.
4. Aerosol particles should be inhaled up to a certain
percentage to ensure adequate dosage can be deposited in sample
collection tube
lung. Fine particle <lose of preparations for inhalation should

-ffi---------~-
be characterized by appropriate methods.
5. The particle size of the active substances must be not
more than 10 µm, most of them should be less than 5 µm.
6. The delivered uniformity test of metered dese inhaler

~·~-~~~-@
preparation should be performed. In assessing the uniformity
of delivered dose of a multidose inhaler, it is not sufficient to
test a single inhaler. Manufacturers must substitute proce-
dures that take both ínter- and intra-inhaler dose uniformity
' 12.5
into account. Filler support base Cap
7. Monitor any leakage during manufacturering of inhalation
aerosol s. Fig. 1 Apparatus for determination of uniformity
8. For metered-dose preparations for inhalation, the of delivered dose for inhalation aerosols
following contents should be stated on the label:
The total number of deliveries per container; Use a mouthpiece adapter that ensures that the front face of
The content of active ingredient in a <lose; the inhaler mouthpiece is flush with the front face or the 2. 5
Where applicable, the number of deliveries from the inhaler
mm indented shoulder of the sample collection tube, as
to provide the mínimum recommended <lose;
appropriate. The filter--support base is designed to accom-
The label states, where applicable, the name of any added
modate 25 mm diameter filter disks. One end of the
antimicrobial preservative.
collection tube is designed to hold the filter disk tightly
9. For inhalation powders supplied in capsules or blisters the
against the filter-support base. The vacuum connector is
following contents should be stated on the label:
connected to a system comprising a vacuum source and a flow
The quantity of active ingredient contained in each capsule or
regulator. The source is adjusted to draw air through the
blister;
complete assembly, including the filter and the inhaler to be
The capsules are intended for use in an inhaler and are not to
tested; at 28. 3 L/min. Air should be drawn continuously
be swallowed;
through the apparatus to avoid loss of the active substance
The expiration date;
into the atmosphere. When assembled, the joints between
The storage conditions.
the components of the apparatus are airtight so that when a
lnhalation Aerosols vacuum is applied to the base of the filter, all of the air
lnhalation aerosols are solutions, suspensions or emulsions drawn through the collection tube passes through the inhaler.
supplied in containers equipped with a metering valve and
Procedure Select 1 inhaler, shake it for 5 s and discharge 1
which are held under pressure with (a) suitable propellant
delivery to waste. lnsert the inhaler into adapter, spray
(s). The preparations are released from the container as a
once, draw air for 5 s, and remove the inhaler. Repeat the
fine mist with the propellant pressure and inhaled into the
procedure until the number of deliveries that constitute the
lungs. Suitable auxiliary substances such as co-solvents, mínimum recommended dose have been sampled. Wash the
solubilizers and stabilizers may be added. filter and interior of the collection tube with suitable
Unless otherwise specified, inhalation aerosols should comply solution. Transfer the combined solution and washings to a
with the following requirements. suitable volumetric flask, and dilute to volume.
Uniformity of delivered dose Inhalation aerosols should Determine the beginning ( first 3 deliveries) , middle ( 4
com pl y with the following requirements. deliveries from n/2 delivered, n represents the total
 0111 Preparations far lnhalation

deliveries), last ( last 3 deliveries) of labeled deliveries. 10 Apparatus A dose collection apparatus similar to that
deliveried doses in total. described far the evaluation of pressurised metered-dose
Using methods as specified in the individual monograph, and inhalers may be used provided that the dimensions of the tube
calculate the content of active substance of each solution. and the filter can accommodate the measured flow rate.
Far preparations containing more than 1 active substance, Connect the tube to a flow system according to the scheme
carry out the test far unifarmity of delivered <lose far each specified in Fig. 2 and Table l.
active substance.
A
Criteria The preparation should comply with the test:
lf 9 out of 10 results lie between 75 per cent and 125 per cent
of the average value and all lie between 65 per cent and 135 F
per cent.
Vacuum
lf 2 or 3 of the 10 values lie outside the limits of 75 per cent pump
to 125 per cent, repeat the test far 2 more inhalers. Not
more than 3 of the 30 values lie outside the limits of 75 per
cent to 125 per cent and no value líes outside the limits of 65
per cent to 135 per cent.
Uniformity of delivered <lose of intra-inhaler should be
determined during the product release of inhalation aerosols.
Select 10 inhalers, far each inhaler collect 1 delivery that Fig. 2 Apparatus far determination of unifarmity
constitutes the mínimum recommended <lose have been of delivered dose far inhalation powders
sampled, using the procedure described above. Repeat the
procedure far a further 9 delivered doses. Table 1 Component units of apparatus for determination of
Criteria The preparation should comply with the test: uniformity of delivered dose for inhalation powders
If 9 out of 10 results lie between 75 per cent and 125 per cent Code Item Description
of the average value and all lie between 65 per cent and 135
per cent. A Sample Capable of quantitatively capturing
If 2 or 3 of the 10 values lie outside the limits of 75 per cent collection the delivered dos e, e. g. clase
to 125 per cent and no value lies outside the limits of 65 per tu be collection tube similar to that
cent to 135 per cent, repeat the test far 20 more inhalers. described in Figure 0671. -1 with
dimensions of 34. 85 mm ID X 12
Not more than 3 of the 30 values lie outside the limits of 75
cm length (e. g. product number
per cent to 125 per cent and no value líes outside the limits of XX40 04 7 00, Millipore
65 per cent to 135 per cent. Corporation. Bedfard, MA 01732,
Number of deliveries per inhaler Should comply with the USA with modified exit tube, ID;?
fallowing requirements. 8 mm, fitted with Gelman product
nurnber 61631), or equivalent.
Take 1 inhaler and discharge the contents to waste, actuating
the valve at intervals of not less than 5 s. The total number B Filter 4 7 mm filter, e. g. A/E glass fibre
of deliveries so discharged from the inhaler is not less than filter ( Gelman Sciences, Ann
the number stated on the la bel ( this test may be combined Arbor, MI 48106, USA), or
with the test far unifarmity of delivered dose). equivalent.
Fine particle dose Should comply with the requirements of e Connector ID;?8 mm, e. g. , short metal
the test far the Preparations far lnhalation: Aerodynamic coupling, with low-diameter branch
Assessment of Fine Particles ( 0951 ) . Using apparatus and to P3.
methods as specified in the individual monographs, calculate
the fine particle dose. Unless otherwise specified, the D Vacuum A length of suitable tubing having
percentage of fine partides should not be less than 15 % of tubing an ID;?8 mm and an interna!
volume of 25±5 ml.
the labeled amount per delivery.
For breath-triggered inhalation aerosols, the test conditions E 2-way A 2-way, 2-port solenold val ve
described above may need to be modified as directed in the solenold having a mínimum airflow
instructions to the patient. val ve resistance orífice with ID;?8 mm
Microbial limit Unless otherwise specified, comply with the andan opening time ~100 ms
(e. g. type 256-A08, Bürkert
Microbiological Examination of Nonsterile Products: GmbH, 74653 lngelfingen,
Microbial Enumeration Test ( 1105 ) , Microbiological Deutschland) , or equivalent.
Examination of Nonsterile Product: Tests far Specified
Microorganisms ( 1106 ) and Microbiological Acceptance F Vacuum Pump must be capable of drawing
Criteria of Nonsterile Pharmaceutical Products ( 1107>. pump the required flow rate through the
assembled apparatus with the
lnhalation Powders powder inhaler in the mouthplece
lnhalation powders are micronized powders of one or more adapter (e. g. product type 1023,
active ingredients together with or without carriers supplied 1423 or 2565, Gast Manufacturing
in capsules, blisters, or powder reservoirs. They are lnc. , Benton Harbar, MI 49022,
administered by dry-powder inhalers. After breathing by the USA) , or equivalent. Connect the
patient the powder is delivered to the lung. pump to the 2-way solenold valve
Unless otherwise specified, inhalation powders should using short and/or wide (;?10 mm
comply with the fallowing requirements. ID) vacuum tubing and connectors
to minimise pump capacity
Uniformity of delivered dose Inhalation powders should requirements.
comply with the fallowing requirements.
 0111 Preparations for Inhalation
1
continued
Code ltem Description
G Timer Timer capable of driving the 2-way
solenoid valve for the required time
period (e. g. type G814, RS
Components International, Cor by,
NNl 7 9RS, UK), or equivalent.
Pl Pressure 2. 2 mm ID, 3. 1 mm OD, flush
tap with internal surface of the sample
collection tube, centred and burr-
free, 59 mm from its inlet. The
pressure tap Pl must never be open
to the atmosphere. Differential
pressure to atmosphere is measured
at Pl.
P2 Pressure Absolute pressures.
P3 measurements
H Flow Adjustable regulating valve with
control maximum Cv~l (e. g. type
val ve 8FV12I. NSS, Parker Hannitin
ple. , Barnstaple, EX31 lNP,
UK) , or equivalent.
Prepare the inhaler for use and connect it to the inlet of the
apparatus using a mouthpiece adapter to ensure an airtight
seal. Use a mouthpiece adapter that ensures that the front
face of the inhaler mouthpiece is flush with the front face of
the sample collection tube. Put circular filter paper into the
base which should be fixed to one port of the sample
collection tube. Connect the base port and the vacuum pump,
and set up the apparatus. Insert the inhaler into adapter. Switch
on the pump, open the 2-way solenoid valve and adjust the flow
control val ve until the pressure drop ( P 1 ) across the inhaler is
4. O kPa as indicated by the differential pressure meter. Remove
the inhaler from the mouthpiece adapter and, without touching
the flow control valve, connect a flow meter to the inlet of the
sampling apparatus. Use a flow meter calibrated for the
volumetric flow leaving the meter (Qout ). For a meter calibrated
for the entering volumetric flow ( Q;n ) , use the following
expression:
Po= atmospheric pressure,
t::.P = pressure drop over the meter.
If the flow rate is above 100 L/min, adjust the flow control
valve to obtain a flow rate of 100 L/min C±S per cent). Note
the volumetric airflow rate exiting the meter and define this
as the test flow rate, Qout • Define the test flow duration,
T = ( 4 X 60) / ~ , in seconds. Record pressure reading
points P 2 and P 3 ; a ratio P 3 / P 2 of less than or equal to O. 5.
Prepare the inhaler as directed in the instructions to the
patient. Connect the inhaler to the apparatus using an
adapter that ensures a good seal. Switch on the pump, draw
air through the inhaler for T seconds. Switch off the pump
and remove the inhaler from the mouthpiece adapter. Repeat
the procedure until the number of deliveries that constitute
the mínimum recommended dose have been sampled. Wash
the filter and interior of the collection tube with suitable
solution. Transfer the combined solution and washings to a
suitable volumetric flask, and dilute to volume.
Inhalation powders supplied in capsules and blisters repeat
the procedure for a further 9 doses. For reservoir systems,
determine the beginning ( first 3 deliveries ) , middle ( 4
deliveries from n/2 delivered, n represents the total
deliveries), last Clast 3 deliveries) of labeled deliveries. 10
deliveried doses in total.
Using methods as specified in the individual monographs,
calculate the content of active substance of each solution.
For preparations containing more than 1 active substance,
carry out the test for uniformity of delivered dose for each
active substance.
Criteria Comply with the requirements as specified in
lnhalation Aerosols.
Uniformity of delivered dose of intra-inhaler for reservoir
systems should comply with the requirements as specified in
I nhalation Aerosols.
Fine particle dose Should comply with the requirements of
the test for the Preparations for lnhalation: Aerodynamic
Assessment of Fine Particles ( 0951 ) . Using apparatus and
methods as specified in the individual monographs, calculate
the fine particle dose. Unless otherwise specified, the
percentage of fine particle should not be less than 10 % of the
labeled amountper delivery.
Number of deliveries per inhaler for multidose inhalers
Discharge doses from the inhaler until empty, at the
predetermined flow rate. Record the deliveries discharged.
The total number of deliveries so discharged from the inhaler
is not less than the number stated on the label ( this test may
be combined with the test for uniformity of delivered dose).
Microbial limit Unless otherwise specified, comply with the
Microbiological Examination of Nonsterile Products:
Microbial Enumeration Tests ( 1105 ) , Microbiological
Examination of Nonsterile Products: Tests for Specified
Microorganisms ( 1106 ) and Microbioiogicai Acceptance
Criteria of Nonsterile Pharmaceutical Products ( 1107).
Liquid Preparations for Nebulization
Liquid preparations for nebulization are solutions,
suspensions or emulsions intended to be converted into
aerosols by consecutive or metered-dose nebulizers.
Consecutive and metered-dose nebulizers are devices that
convert liquids into aerosols by high-pressure gases,
ultrasonic vibration or other methods. The former are
liquid preparations for inhalation, which allow the dose to
be inhaled at an appropriate active--substance delivery rate
and with a particle size that ensures deposition of the
preparation in the lungs. The latter are metered-dose
sprays for inhalation, which could convert metered-dose
nebulization liquid preparations as aerosols inhaled with one
inspirationby patients.
Liquid preparations for consecutive nebulisers in concentrated
form are diluted to the prescribed volume with the prescribed
liquid before use. Liquid preparations for consecutive
nebulizers may also be prepared from powders. The pH of
liquid preparations for nebulization is not lower than 3 and
not higher than 8. 5. Suspensions and emulsions are readily
dispersible on shaking and they remain sufficiently stable to
enable the correct dose to be delivered. Liquid preparations
for nebulization supplied in multidose containers may contain
a suitable antimicrobial preservative at a suitable
concentration except where the preparation itself has
adequate antimicrobial properties. Unless otherwise
specified, during the development of a preparation, the
effectiveness should comply with requirements of Guidelines
for Antimicrobial Effectiveness Test ( 1121 ) .
Unless otherwise specified, liquid preparations for
nebulization should comply with the following requirements.
Active substance delivery rate and the total active substance
delivered Liquid preparations for inhalation should comply
with the following requirements.

Apparatus Composed of breathing simulator and filter
system. Breathing simulator should be able to generate the
breathing profiles specified in Table 2. Filter system should
be a suitably validated low-resistance filter, capable of
quantitatively collecting the aerosol and enabling recovery of
the active substance with an appropriate solvent. The dead
volume of the filter casing <loes not exceed 10 % of the tidal
volume used in the breath simulation.
e Test (1101 ).
A B
Fig. 3 Experimental set-up for determination
of delivery rate and the total active substance delivered
by liquid preparations for inhalation
A. lnhalation filter and filter holder
B. breathing simulator C. nebulizer
Table 2. Breathing simulator specifications
ltem Specification
Adult Neonate lnfant Child
Tidal 500 mL 25 mL 50 mL 155 mL
volume
Frequency 15 40 30 25
cycles/ min cycles/ min cycles/ min cycles/ min
W aveform sinusoidal sinusoidal sinusoidal sinusoidal
lnhalation/ 1: 1 1: 3 1:3 1 : 2
exhalation
ratio
Procedure Attach the filter ( contained in the filter holder)
(A) to the breathing simulator (B). Fill the nebulizer (C)
with the volume of the medicinal product as specified in the
patient instructions. Attach the mouthpiece of the nebulizer
to the inhalation filter using a mouthpiece adapter if required,
ensuring that connections are airtight. Make sure the
nebulizer is positioned in the same orientation as intended for
use; this may require tilting the breathing simulator and
filter holder. Set the breathing simulator to generate the
specified breathing pattern.
Start the breathing simulator then, at the beginning of an
inhalation cycle, start the nebulizer. Operate the nebulizer
for a defined initial time period. The time chosen, usually
60±1 s, must allow sufficient active substance deposition on
the inhalation filter to allow quantitative analysis. If the
quantity of active substance deposited on the inhalation filter
in 60 s is insufficient for this analysis, the length of the time
interval for aerosol collection can be increased. If the filter is
soaked with the preparation, this time can be decreased. At
the end of this initial period, stop the nebulizer.
Place a fresh filter and filter holder in position and continue
until nebulization ceases. lnterrupt nebulization and exchange
filter if necessary, to avoid filter saturation.
Criteria Use methods as specified in the individual mono-
graphs, determine the mass of active substance collected on
the filters and filter holders during each time interval.
Determine the active substance delivery rate by dividing the
mass of active substance collected on the first inhalation filter
by the time interval used for collection. Determine the total
0112 Sprays
mass of active substance delivered by summing the mass of
active substance collected on all inhalation filters and filter
holders.
Fine particle dose Should comply with the requirements of
the test for the Preparations for Inhalation: Aerodynamic
Assessment of Fine Particles ( 0951 ). Using apparatus and
methods as specified in the individual monograph, calculate
the fine particle dose.
Sterility Unless otherwise specified, liquid preparations for
inhalation should comply with requirements of the Sterility
Preparations to be Converted into Vapour
Preparations to be converted into vapour are solutions,
suspensions or solid preparations which could be converted
into vapour. They are usually added to hot water and the
vapour generated is inhaled.
Microbial limit Unless otherwise specified, comply with the
Microbiological Examination of Nonsterile Product:
Microbial Enumeration Tests ( 1105 ) , Microbiological
Examination of Nonsterile Product: Tests for Specified
Microorganisms ( 1106 ) and Microbiological Acceptance
Criteria of Nonsterile Pharmaceutical Products ( 1107).
0112 Sprays
Sprays are preparations of solutions, emulsions or suspen-
sions of one or more active substances in a suitable vehicle
(and/or excipients), supplied in special devices, which are
delivered from the container in the form of an aerosol with
pressure of hand pump, high pressure of gas, ultrasonic
vibration and others, to the deep respiratory tract, mucosa of
various body cavities or the skin. They may be classified as
sprays for inhaiation, nasai sprays, sprays for non-inhaiation
intended for topical or oromucosal use according to the route
of administration. They are presented as metered dose and
non-metered dose sprays.
Metered dose sprays for inhalation are preparations of
solutions, suspensions or emulsions, which release aerosols
intended for inhalation by metered-dose atomizing devices.
The production and storage of sprays should comply with the
following requirements.
l. Environmental conditions during the processing of sprays
should comply with the requirements specified in the
individual monographs, including a certain clarity,
sterilization conditions and low temperature environment,
etc.
2. In the processing of sprays, suitable auxiliary substances
may be added such as solvent, co-solvent, antioxidants,
antiseptics, surfactant or other additives. Unless otherwise
specified, the antimicrobial effect of these formulations
should comply with the Guidelines for Antimicrobial
Effectiveness Test ( 1121) in the processing of formulations.
All the auxiliary substances in the sprays should be non-
irritating to skin or mucosa membranes.
3. All the component units of the atomizing device should be
nontoxic, non-irritating, stable and compatible with the drug
substances.
4. The liquid in the solution sprays should be clear. Droplets
of the emulsion sprays should be dispersed uniformly in the
vehicle. The fine drug powder and the added substance in
suspension sprays should be well mixed, and prepared as a
stable spray. The particle size of the sprays for inhalation
should be not more than 10 µm, while most of them should
be less than 5 µm.
 ~" -~ ' ...,
'./~¡ ;¡i it,,~-\\{\t,id!{ 0113 Aerosols
5. Unless otherwise specified, the sprays should be stored in
well closed containers, and protected from light.
For non-sterile sprays intended for treating burns, the
product should be labeled "non-sterile preparation", the
product specification should be stated as "It is a non-sterile
product"; lndication should include "intended for the use of
minar burns Ost or minar 2nd degree) ". The precaution
should include "following the doctor' s advice".
Unless otherwise specified, the sprays should comply with
the following requirements.
Sprays for inhalation should comply with requirements of
both Sprays and lnhalations ( 0111 ). Nasal sprays should
comply with requirements of both Sprays and Nasal
Preparations ( 0106).
Total number of deliveries per container
For multi-dose sprays, the metered-dose sprays should
comply with the following requirements.
Procedure Select 4 containers, remove the caps and covers,
shake thoroughly, release the contents to the collection
container, and discharge the contents continuously until
exhausted ( the time interval is 5 seconds with gently
shaking) according to the insert-sheet. The total number of
deliveries available from each container should be not less
than the labelled number.
Delivery amount in a dose Unless otherwise specified, the
metered dose sprays should comply with the following
requirements.
Procedure Select 4 containers, discharge several doses
according to the package insert-sheet, clean the containers
and weigh accurately. Continuously discharge 3 doses, clean
the containers each time after discharging and weigh each
container accurately. Calculate the delivery amount in a dose
based on the 3 doses. Continuously discharge 10 doses, clean
the containers and weigh each container accurately. Repeat
the procedure and calculate the delivery amount in a dose
based on the other 3 doses. Continuously discharge 10 doses,
clean the containers and weigh each container accurately. Repeat
the procedure and calculate the delivery amount in one dose based
on another 4 doses. Calculate the average delivery amount in one
dose based on the 10 deliveries. Unless otherwise specified, the
result should be not less than 80 % and not more than 120 % of
the labelled amount in one delivery.
Where the test for content of active ingredient per spray is
specified, the test for delivered amount in a <lose may not be
required.
Content of active ingredient in a unit spray Unless otherwise
specified, the metered dose sprays should comply with the
following requirements.
Procedure Select 1 container, discharge 5 deliveries
according to the insert-sheet, wash the mouthpiece with a
suitable solvent and dry thoroughly. Discharge 10 or 20
deliveries ( the time interval is 5 seconds with gently
shaking), and the delivered substance is collected in a
quantity of absorbing solvent. Transfer the delivered
substance to a suitable volumetric flask, dilute to volume and
mix well. Determine the content, and divide the result by 10
or 20. The average content of active ingredient in a unit
spray should be not less than 80 % and not more than 120 %
of the labelled amount of ingredient in a unit spray.
Where the test for uniformity of delivered dose is specified,
the test for content of active ingredient per spray may not be
required.
Uniformity of delivered dose Unless otherwise specified, the
uniformities of delivered dose of the metered dose sprays for
inhalation, the emulsion nasal sprays and the suspension
nasal sprays should comply with the related requirements of
lnhalations ( 0111 ) and Nasal Preparations ( 0106).
Fine particle dose Unless otherwise specified, the fine
particle dose of the metered dose sprays for inhalation should
comply with the requirements of the test for the Preparations
for inhalation: Aerodynamic Assessment of Fine Particles
( 0951 ) . Use the methods as specified in the individual
monographs, and calculate the fine particle <lose.
Weight variation Unless otherwise specified, the weight
variation of single-dose sprays should comply with the
following requirements.
Procedure Weigh accurately 20 Units and calculate content
weight of each unit and the average weight according to the
individual monographs. Not more than 2 of the individual
weights should deviate from the average weight by more than
the weight variation limit shown in the table, and none
should deviate by more than twice the limit.
Average weight Weight variation limit
Less than O. 30 g ±10%
O. 30 g or more ±7.5%
Where the test for uniformity of delivered dose is specified,
the test for weight variation may not be required.
Filling Non-quantitative sprays should comply with
requirements of the test for Mínimum Fill ( 0942).
Sterility Unless otherwise specified, sprays intended to
treat burns [except minar burns Ost or minor 2nd degree) J,
severe wounds or those clinically required to be sterile should
comply with requirements of the Sterility Test ( 1101 ).
Microbial lirnit Unless otherwise specified, sprays should
comply with the requirements of Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests
( 1105), Microbiological Examination of Nonsterile Product:
Tests for Specified Microorganisms ( 1106) and Micro-
biological Acceptance Criteria of Nonsterile Pharmaceutical
Products ( 1107).
0113 Aerosols
Aerosols are preparations of active ingredients together with
or without additives supplied in containers equipped with a
specified val ve and which are held under pressure with (a)
suitable propellant ( s). U pon actuation of propellants, the
preparations are released from the container and the vapour
generated is inhaled into the lungs, on mucosa of the
respiratory tract or skin.
Preparations of which contents sprayed out in foamy or semi-
solid forms are called foams or gels/ creams. Aerosols may
be inhalation aerosols and non-inhalation aerosols according
to the route of administration. Aerosols may be two phase
aerosols (gas and liquid) or three phase aerosols (gas,
liquid, solid or liquid ) according to the formulation.
Aerosols may be metered-dose aerosols and non-metered dose
aerosols according to the delivery device.
Inhalation aerosols lnhalation aerosols are preparations
intended for oral inhalation to the lungs, which are more
commonly known as metered-dose inhalers or MDis.
lnhalation aerosols deliver a specified amount of active
ingredients upon activation of the valve system.

Nasal aerosols Nasal aerosols are preparations for local
application into the nasal passages and deliver a specified amount
of active ingredients upon activation of the valve system.
The manufacturing and storage of aerosols should comply
with the following requirements.
l. In the processing of aerosols, suitable additives may be
added such as solvents, co-solvents, antioxidants, antiseptics,
surfactants or other ingredients, as required. Unless other-
wise specified, antimicrobial effectiveness of the formulation
should comply with Guidelines for Antimicrobial Effecti-
veness Test ( 1121) when it is designed. All the additives in
inhalation aerosols should be non-irritating and non-toxic to
mucosa and cilia of the respiratory tract. All the additives in
non-inhalation aerosols should be non-irritating to skin or
mucous membrane.
2. Two phase aerosol is prepared as a clear solution
according to the formulation, which is filled in the containers
with the required amount. Three phase aerosol is a stable
suspension or emulsion prepared by mixing thoroughly the
micronized ( or emulsified) active ingredients and additives.
Fill in the containers after quality control of the preparation,
if necessary. In the process of preparation, if necessary,
water content should be controlled strictly to prevent from
water infiltration. The particle and droplet size of inhalation
aerosols should be not more than 10 µm, while most of them
should be not more than 5 µ m. Fine powder of prepared
slices is not generally recommended.
3. The propellants commonly used are liquids with low-boiling
range. Mixtures of appropriate proportions of two or more
propellants may be used to obtain the required pressure.
4. The containers are resistant to the intemal pressure.
Component units of the container should be compatible with
active ingredients and additives, and their precision of sizes
and swelling properties should comply with the relevant
requirements.
5. The content of drug substances released from metered-
dose aerosols should be accurate and homogeneous, the fine
mist particles should be uniform.
6. Aerosols should be tested for leakage to ensure the safety.
7. Aerosols should be stored in dark, cool place, not to be
exposed to heat or sunlight. The container should not be
punctured.
8. For metered-dose aerosols the following contents should
be stated on the label: (1) the total number of deliveries per
container; and (2) the content of active ingredients in an
actuation released from a metering valve and/or a mouth
splicer.
9. For aerosols which are non-sterile and used for bum
treatment, 'non-sterile preparations' should be stated on the
label, Product Specification should indicate "this product is a
non-sterile preparation". Indication for use should be clear '
for minar bum Clst or minar 2nd degree)'. Precaution should
specify 'use in accordance with the doctor' s advice'. Unless
otherwise specified, carry out the tests as follows.
In addition to the requirements of aerosols, inhalation
aerosols should comply with the relevant requirements of
Inhalations ( 0111 ) and nasal aerosols should comply with
the relevant requirements of Nasal Preparations ( 0106).
Nwnber of deliveries per inhaler The number of deliveries
per inhaler for metered-dose aerosols should comply with the
relevant requirements of Inhalations ( 0111 ) .
Unifonnity of delivered dose Uniformity of delivered dose
for metered-dose aerosols should comply with the relevant
requirements of Inhalations ( 0111 ).
Content of active ingredient delivered by actuation of the valve
0113 Aerosols
The content of active ingredient delivered from each actuation
of metered-dose aerosols should comply with the following
requirements.
Procedure Select 1 aerosol container, shake thoroughly and
remove the cap and cover. Discharge 5 deliveries wash the
mouthpiece with a suitable solvent and dry. Invert the
container in a beaker containing a quantity of absorbent
solution. Immerse the mouthpiece under the surface of the
absorbent solution ( at least 25 mm). Discharge 10 or 20
deliveries (Note -Discharge at intervals of 5 seconds and
shake gently). Remove the container, wash the exterior and
interior of the mouthpiece with the absorbent solution. Pool
above absorbent solution, transfer to a suitable volumetric
flask and dilute to volume. Determine the content of active
ingredient by the method described under the Assay in
individual monographs, and divide the result by the number
of deliveries. The average content of active ingredient
delivered from each actuation of the valve should not less
than 80% and not more than 120% of the labelled amount of
active ingredient delivered from each actuation of the valve.
Delivery rate The delivery rate of non-metered dose aerosols
should comply with the following requirements.
Procedure Select 4 aerosol containers, remove the caps and
covers, actuate each valve for a few seconds, and clean the
outside of the containers. Weigh each container accurately
and immerse in a constant temperature water bath ( 25ºC ±
1 ºC) for 30 minutes. Remove and wipe up the containers.
Unless otherwise specified, actuate each valve continually for
5 seconds. Wipe clean and weigh each container accurately.
Retum the containers to the constant temperature water bath
( 25ºC ± 1 ºC), and repeat above procedure for 3 times.
Calculate the average delivery rate (g/s) for each container,
the result should comply with the requirements described in
the monograph.
Total amount of spray The total amount of spray for non-
metered dose aerosols should comply with the following
requirements.
Procedure Select 4 aerosol containers, remove the caps and
covers. Weigh each container accurately, actuate each valve
continually in a container containing a quantity of absorbent
solution in a fume hood until the container is exhausted,
clean and weigh each container again. The total amount of
spray for each container should be not less than 85 % of the
labelled amount.
Delivered amount in a dose The delivered amount in a dose
of metered-dose aerosols should comply with the following
requirements.
Procedure Select 4 aerosol containers, remove the caps and
covers, actuate each valve to discharge several deliveries,
clean the containers, and weigh each container accurately.
Discharge 1 delivery, clean the containers, and weigh
accurately each container again. The weight diff erence
between the former and latter is 1 delivered amount.
Determine 3 delivered amounts continuously by the procedure
described above. Discharge deliveries continuously to waste
at intervals of 5 seconds, until n/2 deliveries remain.
Determine 4 delivered amounts continuously by the procedure
described above. Discharge deliveries continuously to waste,
until 3 deliveries remain. Determine these 3 delivered
amounts by the procedure described above. Calculate the
average delivery amount in adose based on the 10 deliveries.
Unless otherwise specified, the result should be not less than
80 % and not more than 120 % of the labelled amount of one
delivery.
 0114 Gels
Where the test for uniformity of delivered <lose is specified,
the test for delivered amount in a <lose may not be required.
Particle size Unless otherwise specified, the test for particle
size should be required for inhalation suspension of traditional
Chinese medicine if the test for fine particle <lose is not
specified.
Procedure Select 1 aerosol container, shake thoroughly and
remove the cap and cover. Discharge several deliveries and
wipe up the container. Discharge vertically 1 delivery onto a
clean and dry slide 5 cm away from the mouthpiece. The
slide is washed carefully with about 2 ml of carbon
tetrachloride. Blot up excessive carbon tetrachloride, dry the
slide and cover with a cover glass. Examine the slide under a
microscope equipped with a ocular micrometer, in 25 fields
of view, using 400 X magnification. The average particle size
of active ingredient should be less than 5 µm, and the
specimen should contain not more than 10 particles that are
grea ter than 10 µm.
Unless otherwise specified, the test for minimum fill should
be required for non-metered <lose aerosols.
Filling Non-metered <lose aerosols should comply with the
requirements of the test for Minimum Fill <0942).
Sterility Unless otherwise specified, aerosols intended to treat
bums except minor bum ( lst or minor 2nd degree), serious
wounds or those clinically required to be sterile should comply
with the requirements of the for Sterility Test <11O1 ) .
Microbial limit Unless otherwise specified, microbial limit
should comply with the requirements for microbiological
examination of nonsterile products, which inciude Micro-
biological Examination of Nonsterile Products: Microbial
Enumeration Test <1105), Microbiological Examination of
Nonsterile Product: Tests for Specified Microorganisms
<1106) and Microbiological Acceptance Criteria of Nonsterile
Pharmaceutical Products <1107).
0114 Gels
Gels are thick liquids or semi-solid preparations in the form
of solution, suspension or cream containing drug substances
with suitable gelling excipients. Unless otherwise specified,
gels are restricted to tropical use such as skin or body cavities
(nasal cavity, vagina and rectum, etc.).
Emulsified gels are also known as emulsion gels. Gels
consisting of polymer bases such as tracaganth are also
known as mucilages. A gel of small particles of inorganic
chemical drug substances ( e. g. aluminum hydroxide) ,
which consists of a network of discrete particles of drug
substances, is classified as a two-phase dispersion system,
and is also referred to as a magma. Magma may have
thixotropic property: a semi-solid is formed on standing
which becomes liquid on stirring or shaking.
Gel bases are single-phase dispersion systems and classified
into hydrophilic and hydrophobic ones. Hydrophilic gel bases
usually consist of water, glycerin or propylene glycol gelled
with cellulose derivative, carbomer and alginates,
tragacanth, gelatin, starch, etc. Hydrophobic gel bases
consist of liquid paraffin with polyethylene or fatty oil gelled
with colloidal silica or aluminum soap or zinc soap, etc.
The production and storage of gels should comply with the
following requirements.
l. Colloid particles in suspended gels should be dispersed
uniformly without sedimentation or caking.
2. Gels should be uniform, fine and smooth, and remain gel
form without drying up or liquefaction at room temperature.
3. Wetting agents, preservatives, antioxidants, emulsifiers,
thickening agents and percutaneous accelerators may be
added if necessary. Unless other specified, the antimicrobial
effect of these preparations should comply with the
Guidelines for Antimicrobial Effectiveness Test <1121 ).
4. Generally, the pH value of gels should be examined.
5. Unless otherwise specified, gels should be preserved in
tightly closed containers, protected from light, and not
allowed to freeze.
6. For non-sterile gels used for bum treatment, the label
should be marked with "non-sterile preparation", and the
product specification should indicate with "this product is a
non-sterile preparation". "Use for mild bum" Ost or minor
2nd degree) and "follow the doctor' s advice" should be stated
in indications and in cautions, respectively.
Unless otherwise specified, gels should comply with the
following requirements.
Particle size Unless otherwise specified, suspension gels
should comply with the requirement.
Procedure Take a suitable amount of suspended gels and
spread a quantity of the content to three microscope slides
separately to form a thin layer in an area approximately
equivalent to that of the cover-glass. Carry out the test for
Determination of Particle Size and Particle Size Distribution
<0982, metlwd 1), and no particle greater than 180 µm m
dimension is allowed.
Filling Comply with the test of Mínimum Fill <0942).
Sterility Unless otherwise specified, the gels intended for
treating bums except minor bum Ost or minor 2nd degree)
or serious wounds should comply with Sterility Test <1101 ).
Microbial limit Unless otherwise specified, the gels should
comply with the requirements of Microbiological Examination
of Nonsterile Products: Microbial Enumeration Test
<1105), Microbiological Examination of Nonsterile Product:
Tests for Specified Microorganisms < 1106) and Micro-
biological Acceptance Criteria of Nonsterile Pharmaceutical
Products 0107).
0115 Powders
Powders are preparations in the form of dry powder made of
pulverized and uniform mixtures of drug substances and
suitable excipients.
They may be classified into oral powders and powders for
topical use.
Oral powders are administered directly with water or after being
dissolved or dispersed in water, diluent or other liquids.
Powders for topical use may be applied to the skin, mouth,
throat, cavities, etc. The powders which are used parti-
cularly for therapeutic, prophylactic or lubricating purposes
may be called as dusting-powders or dusting preparations.
The production and storage of powders should comply with
the following requirements.
l. The ingredients for preparing powders should be finely
powdered. Unless otherwise specified, oral powders should
be fine powders, while powders for topical and pediatric use
should be very fine powder.
2. Powders should be dry, loose, mixed well and uniform in
appearance and colour. If they contain poisonous, potent
drug, or drug ata small dosage, they should be prepared or
compoundedly mixed well and sifted.
3. Powders are packaged as single-dose or multi-dose
preparations, and for latter a <lose dividing tool should be

attached. The powders containing poisonous drug should be
packaged as single-dose.
4. Powders are prepared with or without auxiliary
substances. Flavouring, colouring agents or aromatics may
be added in oral powders if necessary.
5. Unless otherwise specified, powders should be preserved
in well closed containers. Powders containing volatile or
hygroscopic drugs should be preserved in tightly closed
containers. Powders containing biological medicaments shall
be packaged with moisture-proof materials.
6. To prevent the destruction of active ingredients in powders by
gastric acid, the ingredients with a capacity of neutralizing
gastric acid may be added to the diluent of powders.
7. For non-sterile powders used for bum treatment, the
labels should be marked with "non-sterile preparation", and
the product specifications should be indicated with " this
product is a non-sterile preparation". Also, "use for minor
bum" Ost or minor 2nd degree) and "follow the doctor' s
advice" should be stated in indications and in cautions,
respectively.
Unless otherwise specified, powders should comply with the
following requirements.
Particle size Unless otherwise specified, powders for topical
use, the powders of chemical drugs used for bum or severe
trauma, as well as the powders of Chinese medicine for
paediatric use, should comply with the following
requirements.
Procedure Unless otherwise specified, weigh accurately
about 10 g of the powder. Carry out the Determination of
Particle Size and Particle Size Distribution ( 0982 , single
screening). Not less than 95 % of the powder should pass
though the sieve (No. 7 sieve for powders of chemical drugs,
and No. 6 for those of Chinese medicine).
Unifm mity of appearance
Procedure Spread evenly a sufficient quantity of powder in
an area of about 5 cm2 on a piece of smooth paper, press the
surface evenly, and observe the powder under a bright light.
It should be uniform in colouration with no discolourations
and colour stains.
Detennination of water for Chinese medicinal powder Carry
out the Determinatíon of Water <0832). The powders should
contain not more than 9. O per cent of water, unless
otherwise specified.
Los.s on drying For chemical drug powder and biological
product powder, unless otherwise specified, carry out the
test for Loss on Drying ( 0831 ) . Loss on drying to constant
weight at 105ºC should be not more than 2. 0%.
Weight variation The weight variation of powders packaged
in single-dose should comply with the requirements in the
following table.
Weight variation
limit (Chinese Weight variation
Average or labelled
medicine or limit (Biological
weight
chemical drug product powder)
powder)
O. 1 g or less
±15% ±15%
More than O. 1 g to O. 5 g ±10% ±10%
More than O. 5 g to l. 5 g ±8% ±7.5%
More than l. 5 g to 6. O g ±7% ±5%
More than O. 6 g ±5% ±3%
0116 Syrups
Procedure Unless otherwise specified, weigh accurately the
content of each 10 packs (bottles) of powders and calculate
the average content weight. Compare the content weight
with the average weight (or labelled weight). Not more than
2 packs should deviate from the average weight by more than
the weight variation limit, and none should deviate by more
than twice the limit.
Where the Test for Content Uniformity is specified, the test
for weight variation may not be required.
Filling Unless otherwise specified, multi-dose packaged
powders should comply with the test of Mínimum Fill
( 0942).
Sterility Unless otherwise specified, powders used for bum
except minor bum ( 1st or minor 2nd degree) , severe trauma
or powders for topical application required to be sterile in
clinic should comply with the Sterility Test ( 1101 ).
Microbial limit Unless otherwise specified, powders should
comply with the requirements of Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests
( 1105) , Microbiological Examination of Nonsterile Product:
Tests for Specified Microorganisms ( 1106 ) and Micro-
biological Acceptance Criteria of Nonsterile Pharmaceutical
Products ( 1107 ). Where the test for contaminating
microorganisms on powders is specified, the microbial limit
test may not be required.
0116 Syrups
Syrups are concentrated aqueous solutions of sucrose
containing drug substances.
The production and storage of syrups should comply with the
following requirements.
l. Syrups should contain not less than 45 % ( gÍ mi) oí
sucrose.
2. The drug substances should be dissolved with freshly
boiled water, and then added with simple syrup. The
prepared slices used as drug substance should be extracted,
purified and concentrated to a certain volume using the
appropriate methods specified under the individual mono-
graphs. If syrups are prepared with solid sucrose, water is
added, boiled and filtered, and a suitable amount of freshly
boiled water may be added to the filtrate to comply with the
labelled amount of formula described m individual
monographs.
3. Additives may be added to syrups if necessary. Unless
otherwise specified, if antimicrobial preservatives are
needed, the antimicrobial effect should comply with the
Guidelines for Antimicrobial Effect iveness Test ( 1121 ) . If
preservatives are needed, not more than O. 3% of sorbic acid
or benzoic acid (potassium and sodium salts are calculated to
their equivalent amount of the corresponding acid), or not
more than O. 05 % of hydroxyphenyl esters, may be used. If
other additives are added, the varieties and amount being
used should comply with the relevant requirements of the
National Standards, and do not affect the stability of the
product and quality control of the preparation concemed.
Suitable amounts of ethanol, glycerol or other polyhydric
alcohols may be added if necessary.
4. Syrups should be clear, unless otherwise specified.
Syrups should not be moldy or rancid, or generate gases and
other deteriorations during storage.
5. Generally, the relative density and pH value of syrups
should be examined.
6. Unless otherwise specified, syrups should be kept in
 0117 Liniments
tightly closed containers and protected from light.
Unless otherwise specified, syrups should comply with the
following requirements.
Filling Syrups in single-dose containers should comply with
the following requirements:
Procedure Take 5 containers, pour the contents into a
previously calibrated graduated cylinder as completely as
possible. Measure the volume of the contents in each cylinder
at room temperature. Not more than 1 of the volumes should
be less than labelled quantity, and none should be less than
95 % of labelled quantity.
Syrups in multi-dose containers should comply with the test
of Minimum Fill <0942).
Microbial limit Unless otherwise specified, they should
comply with the requirements of Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests
<1105), Microbiological Examination of Nonsterile Product:
Tests for Specified Microorganisms < 1106) and Micro-
biological Acceptance Criteria of Nonsterile Pharmaceutical
Products <1107).
0117 Liniments
Liniments are liquid preparations made of drug substances
and suitable solvents such as ethanol oil etc. , intend for
externa! use to rub on unbroken skin.
The production and storage of liniments should comply with
the following requirements.
l. Generally solvents used for liniments include water,
ethanol, liquid paraffin, glycerol or vegetable oil, etc.
2. During storage, liniments in the form of emulsions may
show evidence of separation of oil and water phases but are
easily reformed on shaking. Liniments in the form of
suspension may show a sediment which is readily dispersed
on shaking to give a suspension which remains sufficiently
stable to enable the correct dose to be delivered. Liniments
which deteriorate easily should be prepared befare use.
3. It is necessary to put the liniments on flannel or other soft
tissues first, and then apply it gently to the affected area.
The flannel or other soft tissues used must be clean.
4. Unless otherwise specified, test for relative density, and
pH value should be generally specified for the liniments using
water or diluted alcohol as a solvent. Test for the amount of
alcohol should be specified for those using alcohol as a
solvent. For those in which oils are used as solvents, there
should be no deterioration such as rancidity etc. , and test for
refractive index is required.
5. Liniments should be stable. If necessary, preservatives
and antioxidants may be added. Unless otherwise specified,
the antimicrobial effect of these formula should comply with
the Guidelines for Antimicrobial Effectiveness Test <1121 ).
6. Unless otherwise specified, liniments should be preserved
in well closed containers and protected from light.
Unless otherwise specified, liniments should comply with the
following requirements.
Filling Unless otherwise specified, liniments should comply
with the requirements of Minimum Fill <0942).
Microbial limit Unless otherwise specified, Liniments
should comply with the requirements of Microbiological
Examination of Nonsterile Products: Microbial Enumeration
Tests < 1105), Microbiological Examination of Nonsterile
Product: Tests for specified microorganisms < 1106 ) and
Microbiological Acceptance Criteria of Nonsterile Pharma-
ceutical Products <1107).
0118 Paints
Paints are liquid preparations in the form of aqueous or oily
solutions, suspensions or emulsions, intended to be smeared
on the skin or mucous membrane of mouth or throat with the
aid of sterile gauze or cotton impregnated with the liquid, or
as sterile freeze-dried powders which should be reconstituted
into solutions befare use for trauma treatment.
The production and storage of paints should comply with the
following requirements.
l. Paints are basically glycerol solutions of disinfectants or
anti-inflammatory agents, and ethanol or vegetable oil may
also be used as the solvent. For those in which oils are used
as solvents, there should be no deterioration such as rancidity
etc. , and test for refractive index is required.
If the drug substances are bulks of biologics, the manu-
facture and quality control of the bulk, find bulk and final
product should comply with the requirements in individual
monographs.
2. During storage, paints in the form of emulsions may show
evidence of separation of oil and water phases but are easily
reformed on shaking. Paints in the form of suspension may
show a sediment which is readily dispersed on shaking to give
a suspension which remains sufficiently stable to enable the
correct dose to be delivered. Paints which deteriorate easily
should be prepared befare use.
3. Paints should be stable. If necessary, preservatives and
antiox.idants may be added. Unless otherwise specified; the
antimicrobial effect of these formula should comply with the
Guidelines for Antimicrobial Effectiveness Test <1121 ) .
4. Unless otherwise specified, paints should be preserved in
well closed containers and protected from light. Temperature-
sensitive products should be stored and shipped at 2-8ºC.
5. Unless otherwise specified, paints must be discarded 4
weeks af ter opening.
6. For non-sterile paints used for bum treatment, the labels
should be marked with "non-sterile preparations", and the
instruction should be indicated with "this product is non-
sterile preparation". "Use for minor burn" Ost or minor 2nd
degree) and "follow the doctor' s advice" should be stated in
indications and in cautions, respectively.
Unless otherwise specified, paints should comply with the
following requirements.
Filling Unless otherwise specified, paints should comply
with the requirements of Mínimum Fill <0942).
Sterility Unless otherwise specified, paints intended for
treating burns except minor bum Ost or minor 2nd degree)
or grievous injuries should comply with the Sterility Test (1101 ).
Microbial limit Unless otherwise specified, paints should
comply with the requirements of Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests
<1105) , Microbiological Examination of Nonsterile Product:
Tests for specified microorganisms < 1106 ) and Micro-
biological Acceptance Criteria of Nonsterile Pharmaceutical
Products <1107>.
0119 Pigments
Pigments are liquid preparations made of drug substances
dissolved or dispersed in a solvent containing film-forming
material, intended for externa! use on the affected area to
forma film.

The production and storage of pigments should comply with
the following requirements.
l. Pigments are intended for smearing on the affected area.
After evaporation of the organic solvents, a film is generated
to protect the affected area, and the drug substances are
released slowly to exert therapeutic effect. Pigments are
usually used for injured dermatosis without exudate, etc.
2. Film-forming material generally used for pigments include
polyglycol, polyethylene pyrrolidone, ethylcellulose and
polyglycol aldehyde methylacetal, etc. , while plasticizers
used include glycerol, propylene glycol, dibutyl phthalate,
etc. , and solvents used include ethanol, etc. Suitable additives
nonirritant to skin or mucosa may be added if necessary.
3. Pigments should be stable. If necessary, preservati ves or
antioxidants may be added. Unless otherwise specified, the
antimicrobial effect of these formula should comply with the
Guidelines for Antimicrobial Effectiveness Test ( 1121 ) .
4. Unless otherwise specified, pigments should be preserved
in well closed containers and protected from light.
5. Unless otherwise specified, pigments must be discarded 4
weeks after opening.
6. For non-sterile pigments intended for bum treatment, the
labels should be marked with "non-sterile preparations", and
the instruction should be indicated with "this product is non-
sterile preparation". Meanwhile, "use for minor bum" Clst
or minor 2nd degree) and "follow the doctor' s advice" should
be stated in indications and in cautions.
Unless otherwise specified, pigments should comply with the
following requirements.
Filling Unless otherwise specified, pigments should comply
with the requirements of Minimum Fill ( 0942).
Sterility Unless otherwise specified, pigments intended for
treating burns except minar bum Clst or minar 2nd degree)
or serious wounds shouid compiy with the Steriiity Test
(1101).
Microbial limit Unless otherwise specified, pigments
should comply with the requirements of Microbiological
Examination of Nonsterile Products: Microbial Enumeration
Tests ( 1105), Microbiological Examination of Non sterile
Product: Tests for specified microorganisms ( 1106), and
Microbiological Acceptance Criteria of Non sterile Pharma-
ceutical Products (1107).
0120 Tinctures
Tinctures are clear liquid preparations of drug substances
macerated or dissolved in ethanol ata specified concentration
or made by diluting the fluid extracts. They are intended for
oral administration or externa! application.
The production and storage of tinctures should comply with
the following requirements.
l. Unless otherwise specified, 100 mi of the tinctures are
equivalent to 20 g of the prepared slices. Each 100 ml of
tinctures containing poisonous drugs are equivalent to 10 g of
the prepared slices. The active ingredients of a tincture
containing poisonous drugs may be determined by an assay of
its semi-finished product and then the potency is adjusted to
produce a finished product that complies with the require-
ments described under the individual monographs.
2. Tinctures may be prepared by dissolution, dilution,
maceration or percolation.
(1) Dissolution or dilution Dissolve a quantity of drug
powder or dilute the liquid extracts with a quantity of ethanol
0121 Patches
at specified concentration, allow to stand, filter if necessary.
( 2 ) Maceration Macerate the powdered drugs with a
quantity of solvent in a closed vessel for 3-5 days or a
specified time, stirring or shaking occasionally. Separate the
supernatant liquid, to the residue add a quantity of the
solvent and continue maceration in the same way until the
active ingredients are completely extracted. Pool the extracts
and add solvent to the required volume, allow to stand and
fil ter.
(3) Percolation Percolate with a quantity of solvent as
described under Liquid Extracts and Extracts ( 0189), until
sufficient quantity of the percolate is obtained as required,
allow to stand and filter.
3. Unless otherwise specified, tinctures should be clear.
Precipitates produced after prolonged standing of the tincture
should be dispersed easily on shaking.
4. Tinctures should be preserved in light-resistant, tightly
closed containers and preserved in a cool place.
Tinctures should comply with the following requirements,
unless otherwise specified.
Ethanol content Tinctures should comply with the
requirements of the Determination of Ethanol ( 0711 ) .
Methanol content Tinctures should comply with the
requirements of the Determination of Methanol ( 0871 ) .
Filling Comply with the test for Minimum Fill ( 0942).
Microbial limit Unless otherwise specified, tinctures should
comply with the requirements of Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests
( 1105) , Microbiological Examination of Nonsterile Product:
Tests for specified Microorganisms ( 1106), and Micro-
biological Acceptance Criteria of Non sterile Pharmaceutical
Products ( 1107).
0121 Patches
Patches are a class of laminar pharmaceutical preparations
applied to skin to produce systemic or topical effect.
Patches normally consist of an outer covering, a drug
reservoir, a contact adhesive and a protective liner for
removal before applying the patch to the skin. Patches are
intended to be applied to the surface of the unbroken skin,
and also the surface of the injured skin or the broken skin.
Transdermal patches are intended to be applied to the surface
of the unbroken skin in arder to deliver the active substance
(s) to the systemic circulation after passing through the skin
barrier.
There are severa! diffusion patterns for transdermal patches.
The active substance( s) maybe delivered to the skin and the
systemic circulation directly through a reservoir. Perhaps the
active substance ( s) may be delivered to the skin and the
systemic circulation through the rate controlling membrane
and the adhesive liner. The activity duration of the active
ingredient( s) is determined by its ( their) quantity in the
patch and the penetrating rate.
The reservoir of transdermal patches can be in the matrix
style or in the controlling membrane style.
The protective liner works on preventing the adhesion and
protecting the preparation, which generally is preventing
adhesion paper, a sheet of plastic or metal material. When
removed, the protective liner <loes not detach the preparation
reservoir or the adhesive liner from the patch. The outer
covering of transdermal patches is impermeable to the active
substance( s) and normally impermeable to water.
When applied to the dried, clean and unbroken skin, the
 0122 Cataplasms

patches adheres firmly to the skin by gentle pressure of the
hand or the fingers and can be peeled off without causing
appreciable injury to the skin or detachment of the
preparation from the outer covering.
The patch must not be irritant or sensitizing to the skin, even
after repeated applications.
The production and storage of patches should comply with
the following requirements.
l. The materials and excipients of patches should comply
with the relevant requirements of national standards, and are
innocuous, non-irritating, stable and compatible with active
ingredient( s ). The commonly used materials include
aluminum foil-ethylene composite membrane, adhesion-
preventive paper, ethylene-vinyl acetate copolymer, acrylic
acid or polyisobutylene pressure-sensitive adhesive, silicone
rubber and polyethylene glycol, etc.
2. Additives such as surfactant, emulsifier, humectant,
preservative, antioxidant or transdermal absorption promoter
may be added to patches if necessary.
3. The appearance of the patches should be intact and clean,
the applying area should be uniform, and the cut border
should be smooth without sharp edges.
4. Active ingredient ( s) may be dissolved in solvent and
filled into the reservoir. No air bubble is allowed to be
contained in the reservoir which is sealed without leakage.
The active ingredient ( s), suspended in the preparation,
should be homogenous, stable and spread evenly.
5. Pressure-sensitive adhesive should be spreaded evenly.
Organic solvent residues should be examined when organic
solvent is used in the processing.
6. It should be stated on the label that the patches should not
be applied to individuals with a history of allergy to solvent
such as ethanol etc.
7. The adhesive force of the patches should comply with the
requirements.
8. Unless otherwise specified, patches should be preserved in
well closed containers.
9. The quantity of active substance (s) per patch, the total
activity duration and the effective area of the releasing surface
should be stated on the label of patches.
Unless otherwise specified, patches should comply with the
following requirements.
Content uniformity Patches should comply with the Test for
Content Uniformity <0941 ) .
Drug release test Patches should comply with the
Dissolution and Drug Release Test ( 0931 , method 4 or 5).
Microbial limit Unless otherwise specified, patches should
comply with the requirements of Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests
( 1105 ) , Microbiological Examination of Nonsterile Product:
Tests for specified microorganisms ( 1106 ) and micro-
biological Acceptance Criteria of Non-sterile Pharmaceutical
Products ( 11O7).
0122 Cataplasms
Cataplasms are sheet preparations made of drug substances
and appropriate bases, intended for application to skin, may
produce local or systemic effect, including hydrogel plasters
(Babu plasters) and adhesive plasters.
Hydrogel plasters Hydrogel plasters are cataplasms prepared
by well mixing the drug substances with appropriate
hydrophilic bases, then spreading to the backing materials.
The commonly used bases include sodium polyacrylate,
sodium carboxymethylcellulose, gelatin, glycerine and
micronizing silica gel etc..
Adhesive plasters Adhesive plasters are cataplasms prepared
by mixing the drug substances with bases to the backing
materials. The preparation methods of this agent include
solvent method and hot-pressing method. The commonly
used solvents are gasoline and n-hexane, while the commonly
used bases include rubber, hot-plastic rubber, rosin, rosin
derivatives, vaseline, lanoline and zinc oxide etc.. Other
appropriate solvents and bases are applicable.
The commonly used backing materials for cataplasms include
calico, non-weaving cloth and paper etc. ; commonly used
covering materials include anti-sticking paper, plastic film,
aluminum foil, polyethylene compound film and hard gauze
etc..
The production and storage of cataplasms should comply
with the following requirements.
l. Surfactants, emulsifier, hygroscopic agents, preservatives
or antioxidants may be added in cataplasms, if necessary.
2. The plasters of cataplasms should be spread uniformly,
while the surface should be smooth and clean, uniform in
colour, not unglued and not viscous. Backing liner should be
smooth and clean, no leaking plaster. When organic solvents
are used in spreading, residual solvents should be examined if
necessary.
3. It should be stated on the label that the cataplasms should
not be applied to individuals with a history of allergy to
solvent such as ethanol etc..
4. Cataplasms should comply with the requirements for the
tests of content uniformity, drug release and adhesive force
according to the characteristic of active ingredients or the
preparation, except those in which the active ingredients are
derived from animal or herbs with complex ingredients
without a suitable determination method.
5. Unless otherwise specified, cataplasms should be
preserved in tightly closed containers.
Unless otherwise specified, cataplasms should comply with
the following requirements.
Extractives in plaster ~ Adhesive plasters are tested by
method 1, while hydrogel plasters by method 2.
Method 1 Take 2 pieces of cataplasm ( take 35 cm2 for the
cataplasms larger than 35 cm2 in area each piece), remove
the covering liner, weigh accurately and put into a glass
container with cover, macerate in suitable quantity of organic
solvent ( chloroform, ether etc. ) and shake over and again
until cataplasm separates from the cloth. Take out the cloth
and wash with above solvents until no cataplasm remains on
it. Remove the solvent and dry the cloth ata temperature of
105ºC for 30 minutes, transfer the cloth to the desiccator.
After cooling for 30 minutes, weigh accurately the cloth.
The difference between the weighings is the weight of
cataplasm. Calculate the weight per 100 cm2 of the cataplasm
by the labelled area. The result should comply with the
requirement specified in individual monographs.
Method 2 Take one piece of cataplasm, remove the covering
liner, weigh accurately and put into a beaker, add a suitable
quantity of water and heat to boíl until cataplasm separates
from the liner cloth. Take out the cloth, wash with water
until no cataplasm remains on it. Allow to stand to dry, then
put the cloth into an oven at a temperature of 105ºC for 30
minutes, and transfer to a desiccator. After cooling for 30
minutes, weigh accurately the cloth. The difference between
the weighings is the weight of cataplasm. Calculate the
weight per 100 cm2 of the cataplasm by the labelled area.
 0123 Oral Solutions, Oral Suspensions, Oral Emulsions
The result should comply with the requirement specified in
individual monographs.
Heat-resistance
Unless othrwise specified, take 2 pieces of adhesive plasters,
remove the covering liner, and heat ata temperature of 60ºC
for 2 hours. Af ter cooling, the back of the cataplasm should
not be oily; the surface of the cataplasm should have patina
and still be viscous when touch with the finger.
Excipient property
Place one piece of hydrogel plaster into the constant T & H
chamber ata temperature of 37°C and a relative humidity of
64 % for 30 minutes, then take out the piece and fix to a
smooth steel plane using a clip. The steel plane is placed at
an angle of 60º with the horizontal plane, allow to stand for
24 hours. No moving of the adhesive side should occur.
Adhesive property Unless otherwise specified, hydrogel
plasters should comply with the requirements stated under
Determination of Adhesion < 0952 , method 1 ) , while
adhesive plasters should comply with the requirements stated
under Determination of Adhesion <0952, method 2).
Content uniformity Hydrogel plasters ( except those in
which the active ingredients are derived from animal or herb
with complex ingredient without a suitable determination
method) should comply with the requirements for the Test
for Content Uniformity <0941 ).
Microbial limit Unless otherwise specified, hydrogel
plasters and adhesive plasters should comply with the
requirements of Microbiological Examination of Nonsterile
Products: Microbial Enumeration Tests < 1105), Micro-
biological Examination of Nonsterile Product: Tests for
Specified Microorganisms ( 1106 ) and Microbiological
Acceptance Criteria of Nonsterile Pharmaceutical Products
< 1107 ). No Staphylococcus aureus or Pseudorrwnas
aeruginosa should be found per 10 cm2 for adhesive plasters.
0123 Oral Solutions,
Oral Suspensions, Oral Emulsions
Oral solutions Oral solutions are clear liquid preparations
made of soluble drug substances dissolved in a suitable
solvent, intended for oral administration.
Oral suspensions Oral suspensions are suspended liquid
preparatíons made of insoluble salid drug substances
dispersed in a liquid medium, intended for oral adminis-
tration. Oral suspensions include also dried suspensions or
concentrated suspensions.
Oral emulsions Oral emulsions are emulsified preparations
of stable oíl-in-water emulsions, made of two immiscible
liquids.
Oral solutions, oral suspensions or oral emulsions measured
in small quantities with a suitable measuring device or in
drops are called oral droppings.
The production and storage of oral solutions, oral suspen-
sions and oral emulsions should comply with the following
requirements.
l. Unless otherwise specified, purified water is commonly
used as the solvent for oral solutions or dispersion medíum
for oral suspensions.
2. Suitable auxiliary substances such as preservatives,
dispersion agents, suspending agents, thickening agents,
solubilizing agents, wetting agents, buffering agents,
emulsifiers, stabilizing agents, flavouring agents and
pigments, etc. may be added. The varieties and amounts of
the selected auxiliary substances should comply with relevant
requirements of the National Standards. Unless otherwise
specified, the antimicrobial effects of these formula should
comply with the Guidelines for Antimicrobial Effectiveness
Test <1121 ).
3. Oral solutions, oral suspensíons and oral emulsions should
be stable and nonirritating, should not be contaminated by
mould, should not be rancid or discoloured, and no foreign
material, gases or other deterioration phenomena should
appear.
4. The package of oral droppings should usually include a
dropper with aspirating bulb or other measuring device.
5. Unless otherwise specified, oral solutions, oral suspen-
sions and oral emulsions should be preserved in tightly closed
containers and protected from light.
6. Oral emulsions should be uniformly opal coloured. When
they are centrifuged for 15 minutes at 4 000 revolutions per
minute (about 1 800 X g) using a centrifuge with a radius of
10 cm, no phase should be separated. Emulsions may show
evidence of separation but are easily reformed on shaking.
7. The particles of oral suspensions should be dispersed
uniformly and redispersed easily on shaking when a sediment
is produced on standing.
"Shaking befare use" should be designated on the label of
oral suspensions. The number of drops per milliliter or per
gram should be designated on the label of oral droppings.
Unless otherwise specified, oral solutions, oral suspensions and
oral emulsions should comply with the following requirements.
Filling Unless otherwise specified, single-dose oral
solutions, oral suspensíons and oral emulsions should comply
with the following requirements.
Procedure Take 10 test samples, transfer the content as
completely as possible into a calibrated cylinder individually,
and measure the volume. The content in each container
should not be less than the labeled quantity of the solutions,
suspensions and emulsions.
Where the test for content uniformity is specified, the test
for filling may not be required.
Multi-dose oral solutions, oral suspensions, oral emulsions
and oral droppings should comply with the test of Mínimum
Fill <0942 >.
Weight variation Unless otherwise specified, single-dose dry
suspensions should comply with the following requirements.
Procedure Weigh individually the contents of 20 test
samples and calculate the average content weight. Not more
than 2 of the individual weights should deviate from the
average weight by more than ± 10 % and none more than ±
20%.
Where the Test for Content Uniformity is specified, the test
for weight variation may not be required.
~ on drying Unless otherwise specified, dry suspensions
should comply with the test for Loss on Drying <0831 ) . The
weight loss should be not more than 2. O%.
Ratio of sedimental volume Oral suspensions should comply
with the following requirements. The ratio of sedimental
volume of oral suspension should be not less than O. 90.
Procedure Unless otherwise specified, shake vigorously
50 ml of the test sample in a stoppered cylinder for 1 minute,
and record height H o at the beginning. Allow to stand for 3
hours, record the final height H. Calculate the ratio of
sedimental volume according to the following equation:
Ratio of sedimental volume= H / H o
Oral dried suspensions should be dispersed uniformly on
 0124 lmplants
shaking after a certain proportion of water is added according
to the requirements in individual monographs. The ratio of
sedimental volume is determined and should comply with the
above requirements.
Microbial limit Unless otherwise specified, oral solutions,
oral suspensions and oral emulsions should comply with the
requirements of Microbiological Examination of Nonsterile
Products: Microbial Enumeration Tests ( 1105 ) , Micro-
biological Examination of Nonsterile Product: Tests for
Specified Microorganisms ( 1106 ) and Microbiological
Acceptance Criteria of Non sterile Pharmaceutical Products
<1107>.
0124 Implants
lmplants are the sterile solid preparations contammg drug
substances and excipients, which will be implanted into the
body. lmplants are commonly implanted with the special
syringe, or embedded by surgical opening. They can
continuously release the active substances over an extended
period of time.
The production and storage of implants should comply with
the following requirements.
l. The excipients of the implants should be biocompatible.
Both unbiodegradable materials such as silica gel and
biodegradable materials may be used. Unbiodegradable,
materials should be removed after a designed time period.
2. lmplants should comply with the requirements of Drug
Release Test.
3. lmplants should be packaged in a single-dose sterilized
container.
4. lmplants should be preserved in tightly closed container,
protected from light.
Unless otherwise specified, implants should comply with the
following requirements.
Weight variation Unless otherwise specified, the weight
variation of implants should comply with the following
requirements.
Procedure Take 5 containers, remove any adherent label
and the aluminium cover from the sealed container, wash the
outside with ethanol and dry thoroughly. Open the container
with caution to avoid foreign matter such as glass bits falling
into container, and weigh accurately each of the whole
containers immediately. Remove the contents, wash the
container with water or ethanol, dry under a suitable
condition and weight accurately. Calculate the weight of each
container and the average weight of 5 containers. The weight
variation of each container should not deviate from the
average weight by a percentage greater than the limit shown
in the table. If the weight variation of contents of one
container <loes not comply with the above requirements,
repeat the procedure with another 10 containers, all of them
should comply with the requirements.
Average weight Weight variation limit
O. 05 g or less ±15%
More than O. 05 g to O. 15 g ±10%
More than O. 15 g to O. 50 g ±7%
More than O. 50 g ±5%
Sterility Comply with the requirements of the Sterility Test
(1101>.
0125 Pellicles
Pellicles are preparations in pellicular form processed by the
drug substances with appropriate pellicular materials. They
are intended for oral use or topical use on external mucous
membranes.
The production and storage of pellicles should comply with
the following requirements.
l. Pellicular materials and other excipients should be
innocuous, non-irritating, stable and compatible with the
drug substances. Pellicular materials generally used are
polyvinyl alcohol or polyacrylic acid resins and cellulose with
high molecular weights.
2. Water-soluble drug substances may be prepared into a
solution of suitable viscosity by adding pellicular material.
Water-insoluble drug substances should be finely powdered and
prepared into a uniform mixture with the pellicular material.
3. Pellicles should be intact and smooth, uniform in colour
and thickness, and contain no obvious remaining bubble.
Pellicles of multiple <lose are prepared with clear and uniform
subdivision by pressing and can be separated along the
pressed mark.
4. The packaging materials used for pellicles should be
innocuous, convenient to use, efficient in prevention from
contamination and compatible with the drug substances and
pellicular material.
5. Unless otherwise specified, pellicles are preserved in
tightiy closed containers, protected from moisture, rnold and
prevented from deterioration.
Unless otherwise specified, pellicles should comply with the
following requirements.
Weight variation The weight variation should comply with
the following requirements.
Average weight Limit of weight variation
O. 02 g or less ±15%
More than O. 02 g to O. 20 g ±10%
More than O. 20 g ±7.5%
Procedure Unless otherwise specified, take at random 20
test samples, weigh accurately the total weight and the
weight of each. Calculate the average weight of the samples
and compare with the weight of individual sample. Not more
than 2 samples should deviate from the average weight by the
limit of weight variation, and none should deviate by more
than twice of the limit.
Where the test for content uniformity is specified, the test
for weight variation may not be required.
Microbial limit Unless otherwise specified, pellicles should
comply with the requirements of Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests
( 1105), Microbiological Examination of Nonsterile Product:
Tests for Specified Microorganisms ( 1106 ) and Micro-
biological Acceptance Criteria of Nonsterile Pharmaceutical
Products 0107).
0126 Ear Preparations
Ear preparations are preparations intended for direct appli-
cation to the auditory meatus to produce a topical therapeutic
effect.
Ear preparations may be classified into liquid ear preparations

( ear drops, ear washes, ear sprays ) , semi-solid ear
preparations (ear ointments, ear creams, ear gels, etc.) and
solid ear preparations (ear powders, ear pilules, etc. ) , etc.
They can also be packed in a solid form with a solvent, from
which solutions or suspensions are prepared befare use.
Ear drops Ear drops are clear solutions, suspensions or
emulsions made of drug substances, suitable exicipients and
suitable solvents such as water or glycerol, etc. , intended
for instillation in external auditory meatus.
Ear washes Ear washes are liquid ear preparations in the
form of clear aqueous solutions made of drug substances and
suitable excipients, intended for cleaning the external
auditory meatus. They are usually aqueous solutions of
physiological pH value; when used for injuries and surgical
procedures, they should be sterile.
Ear sprays Ear sprays are liquid ear preparations in the
form of clear solutions, suspensions or emulsions made of
drug substances and suitable excipients and are intended for
administration to the auditory meatus through nebulization
by atomizing devices.
Ear ointments Ear ointments are semi-solid ear preparations
in the pasty form of solution or suspension made of uniform
mixtures of drug substances and suitable excipients.
Ear creams Ear creams are semi-solid ear preparations in
the form of cream made of uniform mixtures of drug
substances and suitable excipients.
Ear gels Ear gels are semi-solid ear preparations in the form
of gel made of drug substances and suitable excipients.
Ear tampons Ear tampons are semi-solid ear preparations
made of drug substances and suitable excipients, intended to
be inserted into the external auditory meatus.
Ear powders Ear powders are so lid ear prepara tions in the
form of powder made of drug substances and suitable
excipients, intended to be put or insufflated into the external
auditory meatus.
Ear pilules Ear pilules are solid ear preparations in the form
of sphere or almost spherical shape made of drug substances
and suitable excipients, intended to be applied in the external
or medium auditory meatus.
The production and storage of ear preparations should
comply with the following requirements.
l. Ear preparations may contain a variety of excipients used
for adjusting tension, viscosity or pH value of the prepa-
rations, increasing the solubility of active ingredients,
stablilizing the preparation and providing adequate anti-
microbial properties. These excipients should not adversely
affect the intended medicinal action of the preparation, and
should not be nocuous or topically irritant. Solvents ( such as
water, glycerol and fatty oil, etc.) should not exert harmful
pressure on the ear-drum. Unless otherwise specified, multi-
dose packaged aqueous ear preparations should contain
antimicrobial preservatives at suitable concentrations; anti-
microbial preservatives may not be added if the preparation
itself has sufficient antimicrobial effect. Unless otherwise
specified, if antimicrobial preservatives are needed, the
antimicrobial effect of these formula should comply with the
test for antimicrobial effectiveness in the processing of the
formulation (]121 ).
2. Unless otherwise specified, for ear preparations packaged
in multi-dose, an integral dropper or suitable device should
be provided, usually consisting of a screw cap incorporating a
dropper and rubber or plastic teat. The container should be
innocuous and properly cleaned, and compatible with the
0127 Lotions
1
drug substances and exc1p1ents. The wall of containers
should be uniform and of definite thickness. The containers
hold not more than 10 ml or 5 g of the preparation.
3. Ear solutions should be clear and without sediments or
foreign matters. Ear suspensions may show a sediment
which is readily dispersed on shaking. Ear emulsions may
show evidence of separation of oil and water phases but are
easily reformed on shaking. Semi-solid ear preparations
should be smooth and fine, and should be applied easily to
the skin.
4. Unless otherwise specified, ear preparations should comply
with the general requirements for preparations of the dosage
forms concerned. For example, eye ointments should comply
with the requirements for ointments, while eye sprays should
comply with the requirements for sprays.
5. Unless otherwise specified, ear preparations should be
preserved in well closed containers.
6. Ear preparations must be discarded 4 weeks after opening.
Unless otherwise specified, ear preparations should comply
with the following requirements.
Ratio of sedimenta) volume The ratio of sedimenta! volume
of suspended ear drops should be not less than O. 90.
Procedure Unless otherwise specified, shake vigourously
50 ml of the test sample in a stoppered cylinder for 1 minute,
and record height H o at the beginning. Allow to stand for 3
hours, record the final height H. Calculate the ratio of
sedimenta! volume according to the following equation:
Ratio of sedimenta! volume= H / H 0
Weight variation Unless otherwise specified, single-dose
packaged solid ear preparations should comply with the
following requirements.
Procedure Weigh individually 20 test samples ( or their
contents) and calculate the average weight. Not more than 2
of the individual weights should deviate from the average
weight by more than ±10% and none more than ±20%.
Where the Test for Content Uniformity is specified, the test
for weight variation may not be required.
Filling Multi-dose packaged ear preparations should comply
with the test of Minimum Fill <0942).
Sterility Ear drops and ear washes for surgical procedures,
injured ear and perforated ear-drum should comply with for
Sterility Test (]101 ).
Microbial limit Unless otherwise specified, ear preparations
should comply with the requirements of Microbiological
Examination of Nonsterile Products: Microbial Enumeration
Tests < 1105), Microbiological Examination of Nonsterile
Product: Tests for Specified Microorganisms < 1106) and
Microbiological Acceptance Criteria of Nonsterile Pharma-
ceutical Products <11O7).
0127 Lotions
Lotions are liquid preparations m the form of solutions,
suspensions or emulsions containing drug substances,
intended for bathing the unbroken skin or cavities.
The production and storage of lotions should comply with the
following requirements.
l. They should be innocuous and without local irritation.
2. During storage, lotions in the form of an emulsion may
show evidence of separation of oil and water phases but are
easily reformed on shaking. Lotions in the form of a
suspension may show a sediment which is readily dispersed
on shaking to give a suspension which remains sufficiently
 0128 lrrigants
stable to enable the correct <lose to be delivered. Lotions
which deteriorate easily should be prepared befare use.
3. Unless otherwise specified, test for pH value should be
generally specified for the lotions using water or diluted
alcohol as a solvent. Test for the amount of alcohol should be
specified for those using alcohol a solvent <0711 ) .
4. Unless otherwise specified, lotions are preserved in well
closed containers.
Unless otherwise specified, lotions should comply with the
following requirements.
Filling Unless otherwise specified, lotions should comply
with the test of Mínimum Fill <0942).
Microbial limit Unless otherwise specified, lotions should
comply with the requirements of Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests
<1105), Microbiological Examination of Nonsterile Product:
Tests for Specified Microorganisms ( 1106) and Micro-
biological Acceptance Criteria of Nonsterile Pharmaceutical
Products (1107).
0128 Irrigants
Irrigants are sterile solution intended for irrigating open
injuries or cavities.
The production and storage of irrigants should comply with
the following requirements.
l. Irrigants should be sterile, innocuous and without local
irritation.
2. Irrigants may be made of drug substances, electrolytes or
isotonic modifiers dissolved in water for injection. lrrigants
may also be water for injection, of which "for irrigation"
should be designated on the label. lrrigants are usually
isotonic. Irrigants should be clear when examined visually
under suitable conditions. The containers of irrigants should
comply with the requirements for those of injections.
3. Irrigants should be used as soon as possible after opening
the seal. The remainder should be discarded.
4. Unless otherwise specified, irrigants are preserved in
tightly closed containers.
Unless otherwise specified, irrigants should comply with the
following requirements.
Filling Unless otherwise specified, irrigants should comply
with the test of Minimum Fill ( 0942).
Sterility Irrigants should comply with the Test for Sterility
(1101).
Bacteria) endotoxin or Pyrogens Unless otherwise specified,
irrigants should comply with the requirements of the Test for
Bacteria! Endotoxin ( 1143) or Pyrogens Test ( 1142 ). The
bacteria! endotoxin per milliliter should be less than O. 50
EU.
lrrigants which can not be examined by the test for bacterial
endotoxin should comply with the requirements for the test
of pyrogens. Unless otherwise specified, the dose is 10 ml
per kilogram body weight of rabbit.
0129 Enemas
Enemas are liquid preparations of aqueous or oily solutions,
emulsions or suspensions, intended to be perfused in to the
rectum for therapy, diagnosis or nutrition.
The production and storage of enemas should comply with
the following requirements.
l. They should be innocuous and without local irritation.
2. Unless otherwise specified, enemas are preserved in
tightly closed containers.
Unless otherwise specified, enemas should comply with the
following requirements.
Filling Unless otherwise specified, enamas should comply
with the test of Minimum Fill <0942).
Microbial limit Unless otherwise specified, enemas should
comply with the requirements of Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests
( 1105), Microbiological Examination of Nonsterile Product:
Tests for Specified Microorganisms ( 1106) and Micro-
biological Acceptance Criteria of Nonsterile Pharmaceutical
Products <1107>.
0181 Mixtures
Mixtures are liquid preparations intended for oral adminis-
tration, prepared by extracting the prepared slices with water
or other solvents in suitable ways ( the single dose packaged
mixtures are also known as "oral solutions" ).
The production and storage of mixtures should comply with
the following requirements.
l. The pre pared slices should be extracted, purified and
concentrated to a certain volume by the methods as described
under individual monographs.
2. Suitable additives may be added to mixtures as required.
Unless otherwise specified, the antimicrobial effect of these
formula should comply with the Guidelines for Antimicrobial
Effectiveness Test <1121 ) . If preservatives are needed, the
amount of sorbic acid and benzoic acid should not be more
than O. 3% (its potassium or sodium salt should be taken by
the acid amount), or the amount of p-hydroxybenzoic acid
esters, not more than O. 05%. If other additives are added,
the variety and quantity to be used should comply with the
requirements of the national standard, and not affect the
stability or interfere with the tests for mixtures. If
necessary, mixtures could also contain a proper quantity of
alcohol.
3. If sucrose is used as an additive in mixtures, unless
otherwise specified, its content is not more than 20 %
(g/ml).
4. Unless otherwise specified, mixtures should be clear,
show no evidence of mold contamination, rancidity, foreign
matters, colour changing, gas or other deterioration during
storage, but a small amount of precipitates easily dispersed
on shaking allowed.
5. In general, relative density and pH value etc. should be
determined.
6. Unless otherwise specified, mixtures should be preserved
in tightly closed containers and stored in a cool place.
Unless otherwise specified, mixtures should comply with the
following requirements.
Filling Single-dose packaged mixtures should comply with
the following test.
Procedure Take 5 containers of a mixture, pour the
contents into the calibrated graduated cylinders individually,
and examine the volume at room temperature. Compare the
filling volume of each with the labelled amount. Not more
than 1 of the volumes should be less than the labelled
amount, and none should less than 95 per cent of the labelled
amo un t.
Mixtures filled in multiple dose should comply with the
requirements stated in Mínimum Fill <0942).
Microbial limit Unless otherwise specified, mixtures should

comply with the requirements of Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests
( 1105), Microbiological Examination of Nonsterile Product:
Tests for Specified Microorganisms ( 1106 ) and Micro-
biological Acceptance Criteria of Nonsterile Pharmaceutical
Products ( 1107>.
0182 Troches
Troches are solid preparations of various shapes made from
fine powders of prepared slices and suitable binders ( or using
the binding property of fine powder of prepared slices
themselves).
The production and storage of troches should comply with
the following requirements.
l. The honey, powder of glutinous rice etc. used for troches
should be treated by the specified methods.
2. Troches should be prepared with binders or by the binding
property of fine powders of prepared slices themselves as
described under individual monographs, and formed by a
moulding ora kneading method, then renovated and dried in
the shade.
3. Troches may be coated or polished, if necessary, using
the coating materials stated under the individual monograph
of the preparation.
4. Troches should be neat, smooth, uniform in colour with
no wrinkle, breach, gap, cavity and change in form.
5. Troches should be preserved in well closed containers and
stored in a cool and dry place unless otherwise specified.
Unless otherwise specified, troches should comply with the
following requirements.
Weight variation Unless otherwise specified, troches should
comply with the test for Weight variation stated under Pills.
Microbial limit Comply with the requirements of Micro-
biological Examination of Nonsterile Products: Microbial
Enumeration Tests ( 1105), Microbiological Examination of
Nonsterile Product: Tests for Specified Microorganisms
( 1106) and Microbiological Acceptance Criteria of Nonsterile
Pharmaceutical Products ( 1107>.
0183 Concentrated Decoctions
Concentrated decoctions are semi-fluid preparations prepared
by decocting the prepared slices in water, concentrating the
decoction and adding honey or sugar (or invert sugar).
The production and storage of concentrated decoctions should
comply with the following requirements.
1. The prepared slices should be decocted by the methods as
described under individual monographs and filtered. The thin
extract is obtained by concentrating the filtrate to a specified
relative density.
2. Unless otherwise specified, the fine powder of prepared
slices should be added when the powder is needed as
required.
3. The thin extract is concentrated by adding specified
quantity of processed honey or sugar (or invert sugar). The
fine powder of prepared slices should be added into a cold
concentrated decoction, stirred and mixed well. The quantity
of honey or sugar ( or invert sugar) added is generally not
more than 3 times of that of the thin extract, unless specified
otherwise.
4. Concentrated decoctions should have no burnt or other
abnormal odour, and produce no sugar crystals.
5. Concentrated decoctions should be preserved in tightly
0184 Glues
closed containers, stored in a cool place, unless otherwise
specified.
Unless otherwise specified, concentrated decoctions should
comply with the following requirements.
Relative demity Unless otherwise specified, weigh accurately an
appropriate quantity of concentrated decoctions being
examined, dilute with double quantities of water, weigh
accurately and mix well. Carry out the Determination of
Relative Density ( 0601 ) , and calculate by the following
formula. The result should comply with the requirements
under individual monographs.
. d . f dd . W1 -W1 Xf
R e1at1ve ens1ty o concentrate ecoctlons Wz-Wz Xf
Where W1 is the weight (g) of concentrated decoctions in
pycnometer;
W2 is the weight (g) of water in pycnometer.
weight of water added in concentrated decoctions
f weight of concentrated decoction+weight of water
added in concentrated decoctions
It is not necessary to examine the relative density when the
concentrated decoctions contain fine powder of prepared
slices.
Insoluble matter To 5 g of the concentrated decoctions add
200 ml of hot water, stir to dissolve, allow to stand for 3
minutes. No foreign matters such as scorched masses, etc.
should be observed.
The concentrated decoctions containing fine powder of
prepared slices should be examined and should comply with
the requirements before the powder is added. It is not
necessary to examine the insoluble matters after the powder
is added.
Filling Comply with the requirements for Mínimum Fill
( 0942).
Microbial limit Comply with the requirements of Micro-
biological Examination of Nonsterile Products: Microbial
Enumeration Tests ( 1105), Microbiological Examination of
Nonsterile Product: Tests for Specified Microorganisms
( 1106) and Microbiological Acceptance Criteria of Non
sterile Pharmaceutical Products ( 1107>.
0184 Glues
Glues are solid lump preparations intended for interna!
administration, prepared by decocting animal skin, bone,
shell or horn with water, concentrating into thick gelatinous
mass and then drying.
The production and storage of glues should comply with the
following requirements.
l. Soak and rinse the raw materials for the preparation of
glues in water, remove foreign matters or non-medicinal
parts, cut into small lumps or saw into small pieces, then
wash clean.
2. Decoct with water for several times until the decoction
becomes light, pool the decoctions, allow to stand, filter and
concentrate the filtrate. The concentrated gelatinous liquid
should be able to coagulate under room temperature.
3. Before the glues begin to coagulate, add a suitable amount
of excipients ( yellow rice wine, crystal sugar, edible
vegetable oil, etc. ) , according to the requirements for
processing specified in individual monographs.
4. After the glues coagulate, cut into lumps by weight as
required and dry in the shade.
5. Glues should be translucent, uniform in appearance and
colour, without abnormal odour.
 0185 Medicinal Wines
6. In general, total ash, heavy metals, arsenic salts etc.
should be determined.
7. Glues should be preserved in well closed containers and
protected from moisture.
Unless otherwise specified, glues should comply with the
following requirements.
Water Put 1 g of glue into the flat weighing bottle, weigh
accurately, add 2 ml of water, heat in the water bath to
dissolve, allow to dry. Make sure the thickness is not more
than 2 mm. Carry out the method for Determination of
Water ( 0832 , method 2). The glues should contain not more
than 15. O% of water.
Microbial limit Comply with the requirements of Micro-
biological Examination of Nonsterile Products: Microbial
Enumeration Tests ( 1105 ) , Microbiological Examination of
Nonsterile Product: Tests for Specified Microorganisms
( 1106) and Microbiological Acceptance Criteria of Nonsterile
Pharmaceutical Products ( 1107).
0185 Medicinal Wines
Medicinal wines are clear liquid preparations prepared by
maceration and extraction of prepared slices with distilled
wme.
The production and storage of medicinal wines should comply
with the following requirements.
l. Generally, the prepared slices for producing medicinal
wines should be properly ground.
2. Medicinal wines used for oral administration should be
prepared with wines made from grains.
3. Medicinal wines may be prepared by maceration,
percolation or other suitable methods. The concentration and
quantity of distilled wine, temperature and duration for
maceration as well as speed of percolation should comply
with the requirements for preparation described under
individual monographs.
4. Medicinal wines may be flavoured by adding a sufficient
quantity of sugar or honey as required.
5. Medicinal wines being prepared should be allowed to stand
for clarifying then filtered and packed in clean containers. A
small amount of dispersibe precipitates during storage are
allowed.
6. Ethanol and methanol contents should be generally
determined for medicinal wines.
7. Unless otherwise specified, medicinal wines should be
preserved in tightly closed containers and stored in a cool
place.
Unless otherwise specified, medicinal wines should comply
with the following requirements.
Total solids Medicinal wines containing sugar or honey
should be examined by method 1, sugar or honey free
medicinal wines should be examined by method 2.
Method 1 Transfer accurately 50 ml of the supematant of
medicinal wine to an evaporating dish and evaporate to a
thick extract on a water bath. Unless otherwise specified,
extract the residue with 4 quantities, each of 10 ml, of
dehydrated ethanol with stirring, and filter. Pool the filtrates
to another evaporating dish which have been dried to a
constant weight and evaporate nearly to dryness on a water
bath, add accurately 1 g of diatomite ( dry at 105°C for 3
hours and cool in a desiccator for 30 minutes) , stir
thoroughly, dry at 105ºC for 3 hours, cool the dish in a
desiccator for 30 minutes, and weigh immediately. Deduct
the weight of added diatomite, the weight of residue should
comply with the requirements stated under the individual
monographs.
Method 2 Transfer accurately 50 ml of the supematant of
medicinal wine to an evaporating dish which have been dried
to a constant weight and evaporate to dryness on a water
bath, then dry at 105ºC for 3 hours. Cool the dish containing
the residue in a desiccator for 30 minutes and weigh
immediately. The weight of residue should comply with the
requirements stated under the individual monographs.
Ethanol content Carry out the method for Determination of
Ethanol ( 0711 ). The result should comply with the
requirements described under the individual monograph.
Metbanol content Carry out the method for Determination
of Methanol ( 0871 ) . The result should comply with the
requirements described under the individual monographs.
Filling Comply with the test for Mínimum Fill ( 0942).
Microbial limit Comply with the requirements of Micro-
biological Examination of Nonsterile Products: Microbial
Enumeration Tests ( 1105), Microbiological Examination of
Nonsterile Product: Tests for Specified Microorganisms
( 1106) and Microbiological Acceptance Criteria of Nonsterile
Pharmaceutical Products ( 1107 ) . The number of bacteria
should not exceed 500 cfu per ml, while the number of
mildew and microzyme should not exceed 100 cfu per ml and
other indexes should comply with the requirements.
0186 Plasters
Plasters are preparations intended for extemal application to
the skin, prepared by refining prepared slices, edible
vegetable oíl and red lead oxide or ceruse as plaster and
spread on the backing material. The forrner is called black
plasters, the latter white plasters.
The productíon and storage of plasters should comply with
the following requirernents.
l. The prepared slices should be broken or cut appropriately,
and fried to char as described under the individual
monographs; the prepared slices which are light in texture
and intolerable to frying, should be added after other
prepared slices are fried to charred yellow. Prepared slices
containing volatile substances, mineral drugs and precious
drugs should be ground into fine powders and added just
before spreading at a temperature not higher than 70ºC.
2. Red lead oxide or ceruse used should be dry, without
agglomeration because of moisture absorption.
3. The oil af ter frying the pre pared slices should be refined to
"pearl form on dropping". Add red lead oxide or ceruse and
stir to mix well and spray with clean water. The plaster
lumps are formed and soaked in clean water.
4. Plasters should be greasy, delicate and bright. They
should be done appropriately and spread evenly, with no
breach or gap, and they should be a ble to stick on the skin
without moving after being heated. Black plasters should be
black, with no red spots; white plasters should be free of
white spots.
5. Plasters should be preserved in tightly closed containers
and stored in a cool place, unless otherwise specified.
Unless otherwise specified, plasters should comply with the
following requirements.
Softening point Carry out the method for Determination of
Plaster Softening Point ( 2102 ) and comply with the
requirements stated under the individual monographs.
Weight variation Weigh separately 5 pieces of plasters, clip

a unit area (cm2 ) of backing material and weigh. Calculate
the weight of backing material of each piece of the plaster.
The weight of drugs on the p1aster is calculated by
subtracting the weight of backing material from the total
weight of the plaster. The limit of weight variation far
plasters should comply with the requirements in the
following table.
Labelled weight Weight variation limit
3 g or less ±10%
more than 3 g to 12 g ±7%
more than 12 g to 30 g ±6%
more than 30 g ±5%
0187 Medicinal Distillates
Medicinal distillates are the aromatic waters prepared by
steam distillation of the prepares slices containing volatile
constituents.
The production and storage of medicinal distillates should
comply with the following requirements.
l. Prepares slices should be macerated in water far a certain
time befare steam distillation, distillates collected should be
stored in sterilized, clean and dry containers in time.
2. Collection of distillates and procedures of filling should be
carried out under circumstances with required cleanliness.
3. Suitable preservatives and taste-masking agents may be
added to the medicinal distillates if necessary, the variety and
quantity used should comply with the requirements stated in
notional standards. Unless otherwise specified, the
antimicrobial effect of these formula should comply with the
Guidelines far Antimicrobial Effectiveness Test ( 1121 ) .
4. Medicinal distillates should be clear, and show no
evidence of deterioration such as foreign matter, rancidity.
5. Determination of pH value is required in general.
6. Medicinal distillates should be preserved in tightly closed
containers, stored in a cool place, unless otherwise specified.
Unless otherwise specified, medicinal distillates should
comply with the following requirements.
Filling Comply with the test far Minimum Fill ( 0942).
Microbial lirnit Comply with the requirements of Micro-
biological Examination of Nonsterile Products: Microbial
Enumeration Tests ( 1105), Microbiological Examination of
Nonsterile Product: Tests far Specified Microorganisms
( 1106) and Microbiological Acceptance Criteria of Nonsterile
Pharmaceutical Products ( 1107).
0188 Medicinal Teas
Medicinal teas are preparations intended far oral adminis-
tration, which are made by mixing prepared slices or extracts
( liquid extractives) of prepared slices with tea or other
excipients. They consist of medicinal Tea lumps, medicinal
bag-packed teas and medicinal teas far decoction.
Medicinal tea lumps are classified into sugar free tea lumps
and sugar containing tea lumps. Sugar free tea lumps are
those prepared by compacting the coarse powder, broken
pieces of prepared slices, and tea or suitable binder. Sugar
containing tea lumps are those prepared by compacting
extracts or fine powders of prepared slices and suitable
0188 Medicinal Teas
excipients such as sugar etc..
Medicinal bag-packed teas are those prepared by packing bags
with tea, coarse powder of prepared slices or partial prepared
slices absorbing liquid extractives and then dried. The
medicinal bag-packed teas packed into drinking bags are
called tea bags.
Medicinal teas far decoction are those prepared by packing
bags with slices, pieces, sections, slivers or coarse powder
of the pre pared slices, and decocted far drinking.
The production and storage of medicinal teas should comply
with the following requirements.
l. The prepared slices to be used should be processed into
slices, pieces, sections, slivers or coarse powders according
to the requirements, and mixed well. The liquid extractives
should be sprayed evenly. Where prepared slices and extracts
of prepared slices are added with suitable binder or excipients
such as sugar etc. , a uniform mixture is required.
2. Drying should be carried out ata temperature below 80ºC
generally; medicinal teas containing a large amount of
volatile constituents should be dried at a temperature below
60ºC and the thermolabile drugs should be dried with proper
methods.
3. Tea and drinking tea bags should comply with the
specified requirements of drinking tea.
4. Medicinal teas should be preserved in well closed
containers, medicinal teas containing volatile and moisture
absorbing drugs should be preserved in tightly closed
containers.
Unless otherwise specified, medicinal teas should comply
with the following requirements.
Water Sugar-Jree tea lum ps Carry out the method far
Determination of Water ( 0832). Unless otherwise specified,
water content should be not more than 12. 0%.
Sugar containing tea lumps Grind the tea lump into
particles about 3 mm in diameter. Carry out the method far
Determination of Water ( 0832). Unless otherwise specified,
water content should not be more than 3. O%.
Bag-packed teas and teas for decoction Carry out the
method far Determination of Water ( 0832 ). Unless
otherwise specified, water content should be not more than
12.0%.
Dispersibility Sugar containing tea lumps should comply
with the following test.
Procedure Take one lump, add hot water at 20 times of its
weight and stir far 5 minutes. The tea lump should be
completely dissolved without bumed charings, etc. ( slight
turbidity is acceptable) .
Weight variation Medicinal tea lumps should comply with
the following test.
Procedure Weigh 10 lumps of medicinal tea respectively,
and compare the weight of each with the labelled weight.
Sugar-free tea lumps should comply with the requirements
stated in table 1 and sugar-containing lump tea lumps with
those in table 2. Not more than 2 lumps should deviate from
the limit, and none should deviate by more than twice of the
limit.
Table 1
Labelled weight or filling Weight or Filling variation limit
2 g or less ±15%
more than 2 g to 5 g ±12%
 0189 Liquid Extracts and Extracts
continued
Labelled weight or filling Weight or Filling variation limit
more than 5 g to 10 g ±10%
more than 10 g to 20 g ±6%
more than 20 g to 40 g ±5%
more than 40 g ±4%
Table 2
Labelled weight Weight variation limit
6 g or less ±7%
more than 6 g ±5%
Filling variation Unless otherwise specified, medicinal bag-
packed teas and medicinal teas far decoction should comply
with the fallowing test.
Procedure Weigh the contents of 10 packs respectively,
and compare the filling of each pack with the labelled filling.
Not more than 2 packs should deviate from the limit given in
Table 1 and none should deviate by more than twice of the
limit.
Microbial limit Except teas far decoction, comply with the
requirements of Microbiological Examination of Nonsterilo
Products: Microbial Enumeration Tests ( 1105), Micro-
biological füc..a.mination of Nonsterile Product: Tests for
Specified Microorganisms ( 1106 ) and Microbiological
Acceptance Criteria of Nonsterile Pharmaceutical Products
(1107>.
0189 Liquid Extracts and Extracts
Liquid extracts or extracts are preparations made by soaking
prepared slices in suitable solvents to extract the active
ingredients and evaporating the solvents partially or
completely to a specified concentration.
Unless otherwise specified, 1 ml of a liquid extract is
equivalent to 1 g of the prepared slices concerned; 1 g of an
extract is equivalent to 2-5 g of the prepared slices
concerned.
The production and storage of liquid extracts or extracts
should comply with the fallowing requirements.
l. Unless otherwise specified, liquid extracts are prepared by
the percolation method or by the dilution of an extract with a
specified solvent. Extracts are prepared by decoction or
percolation method. All the extracts should be concentrated to a
thick extract at a low temperature and then added with diluting
0200 General Requirements f or Other Pharmaceutical Materials
0211 Sampling of Crude Drugs
and Decoction Pieces
Sampling of crude drugs and decoction pieces refers to the
agents or further concentrated to the specified amount.
The fallowing requirements should be observed m a
percolating process.
(1) Either a cylindrical or a conical percolator may be used,
depending on the nature of the prepared slices.
(2) Befare it is packed into the percolator, the prepared
slices should be pulverized properly, moistened unifarmly
with the specified solvent and allowed to stand in a well
closed vessel far a required time.
(3) The prepared slices should be packed with unifarm
compactness and the solvent added to the percolator to expel
most of the air in the gap of crude drug. The top of the
column of prepared slices should be well covered by the
solvent, allowed to stand far sorne time befare the
percolation takes place.
( 4) The rate of percolation should comply with the
requirements specified in the individual monographs.
(5) When an amount of the initial percolate equivalent to
85 % of the prepared slices in the percolator has been
collected, it is set aside. The rest of the percolate should be
concentrated ata low temperature and the mixed products of
percolation and concentration are made up to the required
volume with the solvent. Allow to stand, and use the
supernatant liquid as the final product far packing.
2. Precipitates produced on prolonged standing of the liquid
extracts may be removed by filtration, but the content of
ethanol and active ingredients should comply with the
requirements specified in the individual monographs.
3. Unless otherwise specified, liquid extracts should be
preserved in tightly closed containers, protected from light,
and extracts should be stored in a cool place.
Unless otherwise specified, liquid extracts and extracts
should comply with the fallowing requirements.
Detennination of ethanol Unless otherwise specified, liquid
extracts contammg ethanol should comply with the
Determination of Ethanol ( 0711 ) , and the result should
comply with the requirement described in the individual
monographs.
Detennination of methanol Unless otherwise specified,
liquid extracts containing ethanol should comply with the
Determination of Methanol ( 0871 ) , and the result should
comply with the requirement described in the individual
monographs.
Filling Comply with the test far Minimum Fill ( 0942).
Microbial limit Comply with the requirements of Micro-
biological Examination of Nonsterile Products: Microbial
Enumeration Tests ( 1105), Microbiological Examination of
Nonsterile Products: Tests far Specified Microorganisms
( 1106) and Microbiological Acceptance Criteria of Nonsterile
Pharmaceutical Products ( 1107>.
method used to sample crude drugs and decoction pieces far
examination.
During sampling, the fallowing requirements should be
complied with.
l. Befare sampling, check the name, source of material,
specification, and packaging of the cargo. Examine the
 0212 General Principie for Inspection of Crude Drugs and Decoction Pieces
intactness, cleanliness of the package, and if there is water
stain, the contamination of moulds and other contami-
nations. Make notes in detail. The abnormal packages should
be examined separately and photographed.
2. The following rules should be followed when sampling
crude drugs and decoction pieces of the same batch:
For less than 5 packages, sample each package;
For 5-99 packages, sample 5 packages at random;
For 100-1000 packages, sample 5% of the packages;
For more than 1000 packages, sample 1% of the part m
excess of 1000 packages;
For precious crude drugs and decoction pieces, sample each
package, regardless of the number of package,
3. Samples from at least 2-3 parts of the package should be
collected for examination; For large packages, samples
should be collected from different parts of the package at
least 10 cm deep from the surface; For crude drugs and
decoction pieces in crushed or powdered form less than 1 cm
in size, use a special sampling tool ( "probe" ) to collect the
samples; For very large packages or crude drugs,
appropriately collect representative samples according to the
individual situation.
The quantity of samples collected from each package:
Ordinary drugs and decoction pieces, 100-500 g;
Powdered drugs and decoction pieces, 25-50 g;
Precious drugs and decoction pieces, 5-1O g.
4. Mix the collected samples thoroughly to make the total
quantity of samples for examination. If the total quantity of
samples exceed several times of that required for the tests,
take an average sample by quartering, i. e. , spread the
sample as a square, draw diagonal lines to divide the sample
in to four parts, and use samples from two parts in opposite
positions. Keep this quartering repeatedly until the finally
obtained sample is just sufficient for the tests.
5. The finally obtained sample for exarnination should be at
least three times the quantity required for the tests, using
one third for laboratory analysis, one third for verification,
and the remaining one third as reserve specimen.
0212 General Principie for Inspection of
Crude Drugs and Decoction Pieces
General principie for inspection of crude drugs and decoction
pieces includes the "Description", "Identification", "Test",
"Determination of extractives", and "Assay" of crude drugs
and decoction pieces. When carrying out the examination,
the following requirements should be taken into
consideration.
l. Collect samples according to the requirements under
Sampling of Crude Drugs and Decoction Pieces ( 0211 ) .
2. Whenever necessary, use a reference drug specimen which
complies with the requirements from individual monographs
for correct analysis.
3. If the samples being examined are pulverized or ground,
they should comply with the whole requirements in the
monograph, except those described under "Description" and
"Microscopical identification".
4. "Description" refers to the form, size, colour, surface
characters, texture ( including cut surface or fracture
characters) , odour, and tas te of the crude drugs and
decoction pieces.
(1) "Form" refers to the shape of crude drugs and decoction
pieces. Generally the samples are not treated, but when the
samples are wrinkled herbs, lea ves, or flowers, they need to
be moistened, softened, and spread. Sorne special fruits and
seeds can be softened, if necessary, and then remo ve the
pericarp or seed coat to examine the inner characters.
( 2) "Size" refers to the length, diameter, and thickness of
crude drugs and decoction pieces. In general, a number of
samples should be measured, and a few values falling out of
the specified range are permitted. Use a millimetre ruler for
the measurement. For fine seeds or fruits, arrange 10 grains
seed in a row, measure with a millimeter, and calculate the
average value.
(3) "Surface" refers to the colour and glossiness of crude
drugs and decoction pieces observed in daylight. If the colour
is described in a combination of two colours, the main colour
is the latter one. For example, the main colour is brown in
yellowish-brown. Surface characters also include wrinkles,
smooth, rough, hole appearance, and appendages of crude
drugs or decoction pieces without pretreatment.
( 4 ) "Texture" refers to the sensation of crude drugs or
decoction pieces when broken by hand.
Cut surface refers to the colour and glossiness, and fracture
surface characters of crude drugs and decoction pieces
observed in da.ylight. If the striations of fracture surface are
difficult to be observed, it can be cut evenly.
(5) "Odour" refers to the sense of smell and taste of crude
drugs and decoction pieces. When examining the odour of
crude drugs or decoction pieces, smell it directly, or in the
time of fracture, crush, or rub the samples to do it. When
necessary, moisten the sample with hot water befare the
examination. When examining the taste of crude drugs or
decoction pieces, taste a small quantity of the samples
directly, or macerate the samples in hot water and taste the
extractive. Be cautious of poisoning when tasting poisonous
crude drugs or decoction pieces.
( 6 ) Moth, mould, and other contamination by foreign
matters should not be observed on the crude drugs and
decoction pieces.
5. "ldentification" refers to the method used to examine the
authenticity of crude drugs and decoction pieces, including
traditional empirical identification, microscopical identifica-
tion, and physico-chemical identification.
( 1) Empirical identification uses easy and simple traditional
means to observe characteristics, for example, colour
change, sinking or floating in water, and bursting sound or
flame colour on burning the samples.
( 2 ) Microscopical identification refers to observing the
tissue, cell, or cell content characters of sections, powder,
or surface of the samples under a microscope. Carry out the
method for microscopical identification ( 2001) and prepare
the slides appropriately.
(3) Physico-chemical identification refers to testing certain
chemical constituents in the samples by physical and chemical
methods.
CD For fluorescent identification, examine the fluorescence
produced by the samples (including fractures or extractives)
directly or after being treated with an acid or base, under
ultraviolt light (placed about 10 cm away from the light
source). Unless otherwise specified, the ultraviolet light
wavelength is 365 nm.
@Far microsublimate identification, place a metallic or glass
slide on an asbestos plate, and place a metallic ring, 8 mm
high, on the slide. Put an appropriate quantity of the powder
being examined in the ring, and cover the ring with a glass
slide. Heat the asbestos plate gently with an alcohol burner
until the powder is charred. Remove the fire and allow to
cool. Sublimates can be observed on the glass slide. Put the
slide under a microscope, and examine the shape and colour
of crystals. Alternatively, add test solutions to the slide and
 0213 The Processing of Crude Drugs
examine the colour change.
® Ultraviolet-visible spectrophotometry, infrared spectro-
photometry, thin layer chromatography, high performance
liquid chromatography, and gas chromatography are usually
used for spectroscopic and chromatographic identifications.
( 4) Polymerase chain reaction identification method refers to
identify crude drugs or decoction pieces by comparing their
DNA differences.
6. "Test" refers to purity and soluble substance tests, as
well as limit test of harmful or poisonous substances,
including the content of water, ash, foreign matters,
poisonous ingredients, heavy metals, harmful elements,
pesticides residues, aflatoxins, and so on.
Unless otherwise specified, decoction pieces contain not more
than 13% of water, not more than 3% of crude drug crumbs
and foreign matters, and not more than 150 mg/kg of sulfur
dioxide residues.
7. "Determination of extractives" refers to determining the
content of soluble substances in crude drugs and decoction
pieces extracted with water or other appropriate solvents.
8. "Assay" refers to determining certain constituents in the
sample by chemical, physical or biological methods.
Note: ( 1) When the crude drugs and decoction pieces need
to be powdered before determination, pulverize the sample as
described under individual monographs, sift and mix well.
(2) Carry out the determination and assay as described under
individual monographs or specified appendices.
( 3) For prepared drugs which are only required to remove
foreign matters, examine the samples as crude drugs, unless
otherwise specified.
0213 The Processing of Crude Drugs
The proc<;!ssing of Chinese herbal medicines is a unique
pharmaceutical technology that processes crude drugs
according to the Traditional Chinese Medicine theory and
individual crude drugs' nature, and the requirements of drug
dispensing, pharmaceutical preparation, and clinical use.
Once crude drugs are processed like cleaning, cutting, and
stir-baking, they are called "decoction pieces". Crude drugs
should be cleaned before cutting or stir-baking.
The format of decoction pieces specified in this pharma-
copoeia refers to the format for clinical preparation. The
format of decoction pieces used for pharmaceutical prepa-
ration should comply with the requirements for individual
practical manufacturing procedures.
Use drinking water for drug processing.
Unless otherwise specified, the processing should comply
with the following requirements.
l. Cleaning It refers to cleansing the crude drugs as
required. The methods include sorting, sifting, winnowing,
washing, cutting, scraping, paring, rejecting, enzymatic
hydrolysis, peeling, squeezing, blanching, brushing, rubbing,
grinding, cauterizing, and rinsing.
2. Cutting Unless cut in fresh or dry form, crude drugs
should be moistened to soften befo re cutting, by
consperging, showering, washing quickly, soaking,
moistening, rinsing, boiling, or stewing. Alternatively, use
softening devices such as rotary decompressed infiltration
cans or gas phase replacement infiltration chambers. Crude
drugs should be treated separately and appropriately
according to their size, thickness, and hardness, at specified
temperature, with specified quantity of water, and for
specified duration. Moistening is preferable to soaking
because it avoids the loss of active principies. The prepared
drugs should be dried immediately to guarantee their quality.
Crude drugs may be cut into slices, sections, pieces, and
slivers, etc. Their size, extent, and thickness are generally
as follows:
Slices: Less than O. 5 mm in thickness for very thin slices,
1-2 mm for thin slices, 2-4 mm for thick slices;
Sections: 5-1O mm in length for short sections, 10-15 mm
for long sections;
Pieces: Cubes of 8-12 mm;
Slivers: 2-3 mm in width for narrow slivers, 5-10 mm for
broad slivers.
Crude drugs that are not appropriate for cutting are generally
pounded or crushed.
3. Roasting and Broiling Unless otherwise specified, the
general methods and requirements are as follows.
( 1) Stir-baking It is divided into simple stir-baking and
stir-baking with excipients. While baking, stir frequently on
a homogeneous fire and control the temperature, duration,
and extent.
Simple stir-baking Place clean crude drugs in a pot, stir-
bake on a gentle fire to the required condition, remove the
drugs, and allow to cool. For crude drugs which need to be
baked to charring, stir-bake on a medium fire until the
surface turns brown and the fractures become dark in colour,
remove the drugs, and allow to cool. For inflammable crude
drugs, spray a little water while stir-baking to char, and then
bake to dryness or dry in the sun.
Stir-baking with bran Put bran in a hot pot and heat until
the bran smokes. Put the clean crude drugs into the pot, stir
quickly until the surface of crude drugs turns yellow or dark,
take them out, remove the bran by sifting, and allow to
cool.
Unless otherwise specified, use 10-15 kg of bran for each
100 kg of clean crude drugs.
Stir-baking with sand Put clean sand in a stir-baking pot,
heat on a strong fire until the sand can move smoothly in the
pot, add the crude drugs, stir frequently, and keep baking
until the drugs expanded crisply or to a specified extent.
Take the drugs out, remove the sand by sifting, and allow to
cool.
Unless otherwise specified, use an appropriate quantity of
sand just enough to cover the crude drugs.
If required to be tempered with vinegar, sift out the sand,
dip the crude drugs into vinegar while hot until they become
crisp.
Stir-baking with powdered clarn-shell Put a quantity of clam-
shell, freely powdered and sifted, in a pot, and heat on a
medium fine until the powders can move smoothly in the
pot. Add the crude drugs, keep stir-baking until the drugs
expanded crisply, the surface becomes yellow, or to a
specified extent. Take the crude drugs out immediately, remove
the powdered clam-shell by sifting, and allow to cool.
Unless otherwise specified, use 30-50 kg of powdered clam-
shell for each 100 kg of crude drugs.
Stir-baking with tale Puta quantity of tale in a pot, and heat
on a medium fire until the powders can move smoothly in the
pot. Add the crude drugs, keep stir-baking until the drugs
expanded crisply, the surface becomes yellow, or to a
specified extent. Take the crude drugs out immediately,
remove the tale by sifting, and allow to cool.
Unless otherwise specified, use 40-50 kg of tale for each
100 kg of crude drugs.

(2) Stir-baking with liquid excipent Mix crude drugs with
liquid excipient until the drugs are moistened, then stir-bake
to a specified extent.
Stir-baking with wine Mix clean crude drugs with wine, and
allow to stand for a while until the drugs are completely
moistened. Put the drugs into a stir-baking pot, heat on a
gentle fire to a specified extent. Take out the drugs, and
allow to cool.
Unless otherwise specified, use yellow rice wine. For each
100 kg of crude drugs, use 10-20 kg of wine.
Stir-baking with vinegar Mix clean crude drugs with
vinegar, and allow to stand for a while until the drugs are
completely moistened. Put the drugs into a stir-baking pot,
heat on a gentle fire to a specified extent. Take out the
drugs, and allow to cool.
Unless otherwise specified, use rice vinegar. For each 100 kg
of crude drugs, use 20 kg of vinegar.
Stir-baking with salt-water Mix clean crude drugs with salt-
water, and allow to stand for a while until the drugs are
completely moistened. Put the drugs into a stir-baking pot,
heat on a gentle fire to a specified extent. Take out the
drugs, and allow to cool.
To prepare salt-water, dissolve salt in an appropriate quantity of
water, and filter. Unless otherwise specified, use 2 kg of
salt for each 100 kg of crude drugs.
Stir-baking with ginger juice Crush fresh clean ginger into a
paste, add a quantity of water, squeeze to obtain the juice.
Add a quantity of water to the ginger residue and squeeze
again. Combine the juice for use. The yield of ginger juice to
fresh ginger is 1 : l.
Put the crude drugs, mixed well with ginger juice, in a pot,
heat on a gentle fire until the ginger juice is completely
absorbed, or to a specified extent, take the drugs out, and
allow to cool.
Unless otherwise specified, use 10 kg of fresh ginger for each
100 kg of crude drugs.
Stir-baking with honey Dilute refined honey with an
appropriate quantity of boiling water, add the crude drugs,
mix well, and allow to stand until the drugs are completely
moistened. Put the drugs in a pot, heat on a gentle fire to a
specified extent. Take the drugs out, and allow to cool.
Use refined honey for the stir-baking. Unless otherwise
specified, use 25 kg of refined honey for each 100 kg of crude
drugs.
Stir-baking with fat Heat sebum fat in a pot until it melts,
remove the residue, add crude drugs, and mix well. Heat on
a gentle fire until the fat is completely absorbed and the
surface is bright, spread the drugs, and allow to cool.
( 3 } Carbonizing When crude drugs are carbonized, the
nature should be preserved. Avoid ashing and combustion.
Carbonizing by stir-baking Place crude drugs in a hot pot,
keep stir-baking on a strong fire until the surfaces become
brownish-black, and the inner parts become dark brown orto
a specified extent. Spray a little water to extinguish any
spark. Take the drugs out and allow to cool.
Carbonizing by calcining Place crude drugs in a calcining
pot, close tightly, heat to a specified extent, allow to cool,
and take the drugs out.
( 4) Calcining Calcine crude drugs until they become crisp
and easily crushed.
Calcining openly Crack crude drugs in to small pieces, put in
0213 The Processing of Crude Drugs
a suitable container, and calcine until the drugs become
crisp, brittle, or red. Take the drugs out, cool, and grind to
a powder.
Mineral salts containing crystalling water do not need to be
calcined to red. Make sure to evaporate all the crystalling
water, or calcinate until the drugs become honeycomh-like
solid masses.
Calcining and quenching Calcine crude drugs to red and dip
them quickly into a specified liquid excipient so that the
minerals become crisp. If the drugs are not crisp, repeat this
process until they are crisp enough. Take the drugs out,
dry, and grind in to a powder.
( 5} Steaming Classify crude drugs according to their size,
add water or liquid excipient as described under individual
monographs, mix well, allow to stand until the drugs are
moistened, and put the drugs in a suitable steaming
container, and steam to a specified extent. Take the drugs
out, cool, mix with the steaming liquid, put in air until
60%-70% dry, cut into slices or sections, and allow to dry.
( 6) Boiling Classify crude drugs according to their size,
add water or liquid excipient as described under individual
monographs, and keep boiling until no white colour is
observed in the center of drugs. Take the drugs out, put in
air until 60 % dry, cut in to slices, and allow to dry.
( 7 } Stewing To crude drugs add liquid excipient as
described under individual monographs, put in a suitable
tightly closed container, stew thoroughly on a water bath or
by steaming, or keep stewing until the excipients are
completely absorbed, allow to cool, take the drugs out, put
in air to 60 % dry, cut in to slices, and allow to dry.
For steaming, boiling, and stewing, use 20-30 kg of water of
specified excipients for each 100 kg of crude drugs, unless
otherwise specified.
( 8} Roasting Wrap crude drugs with wet flour or wet
paper, or separate the crude drugs into uniform layers with
absorbent paper, and heat the drugs. Alternatively, put the
crude drugs together with bran in a stir-baking container,
heat on a gentle fire to a specified extent, and allow to cool.
Unless otherwise specified, use 50 kg of bran for each 100 kg
of crude drugs.
4. Other pr~ing methods
(1) Blanching Put crude drugs in boiling water, stir for a
short time, and take them out. For certain seeds, blanch
until the wrinkled tessta become smooth and easily
removable, take them out, put in cool water, remove the
tessta, and dry in the sun.
( 2) Frosty Process by removing the oil from crude drugs.
Unless otherwise specified, crush the crude drugs into a
paste, heat gently, and squeeze out majority of the oil until
the residual oil content complies with the specified require-
ments. Pulverize the residue into a fairly dispersible powder
as required in individual monographs.
( 3} Levigating Put crude drugs in a container, add an
appropriate quantity of water, and keep grinding until the
sample becomes a paste. Add an appropriate quantity of
water, agitate, and decant the supernatant suspension.
Repeat the operation with the precipitate for several times.
Combine the supernatant suspensions, allow to stand,
separate the precipitate, dry, and pulverize into a free
powder.
( 4} Sprouting Put crude drugs in a container, soak in an
appropriate quantity of water, and then take the drugs out.
Let the samples sprout to a specified extent at a suitable
 0251 General Requirements for Pharmaceutical Excipients
humidity and temperature. Dry it in the sun or dry it ata low
temperature. Avoid the contamination of grease and getting
rotten. The sprouts are generally not longer than 1 cm.
( 5) Fennenting Mix well the crude drugs with specified
excipients, make a specific shape, put ata suitable humidity
and temperature, and let microorganisms grow until the
enzyme content reaches a specified range. Dry it in the sun or
dry at a low temperature. The fermented products cannot be
used when flavus is observed during the fermentation.
0251 General Requirements for
Pharmaceutical Excipients
Pharmaceutical excipients refer to the vehicles and additives
used for drug production and prescription dispensing,
including those used for controlled drug release, delivery
systems, substances that may be added in preparation
process but indicated to be removed, and materials additional
to active components or precursors, with a reasonable
evaluation in terms of safety and contained in pharmaceutical
preparation. In certain cases, sorne pharmaceutical excipients
can be active ingredients and then should be in accordance
with the requirements of drugs. As non-active substances,
pharmaceutical excipients also ha ve solubilizing, complexing,
sustained and controlled release and other important func-
tions in addition to acting as vehicles, improving stability,
which are important components that may affect the quality,
safety and efficacy of formulation. Therefore, attention
should be given to the safety of pharmaceutical excipients
themselves, as well as drug-excipient interactions and their
safety.
Pharmaceutical excipients can be classified according to their
source, chemical structure, usage, dosage form and route of
administration.
According to their source, Pharmaceutical excipients can be
classified into natural, semi-synthetic and fully-synthetic
materials.
According to different dosage forms, Pharmaceutical excipients
can be used to prepare solution, mixture, emulsion, eye
drops, nasal drops, tablets, capsules, suppositories,
granules, pills, pellicles, injection, aerosol, etc.
According to their usage, Pharmaceutical excipients can be
classified into solvent, propellant, solubilizer, cosolvent,
emulsifier, colourant, adhesive, binders, anticaking agent
disintegrating agent, filler, emollient, wetting agent, osmotic
pressure regulator, stabilizer, glidant, pressure aid agent, taste
rnasking agents, preservative sweetener, suspending agent,
flavoring additive coating, arornatic agent, antiadhesive,
antioxidant, antioxidant synergist, chelating agent, skin
permeation enhancer, air replaced agent, pH regulator,
adsorbent, plasticizer, surfactant, foaming agent, defoaming
agent, thickening agent, protective, humectant, softener,
absorbent, diluent, flocculant and anti flocculant, filter aid,
printing ink, pressure-sensitive adhesive, water-proof agent
lyophilized filler, lyoprotectant, vacant capsule, colloid
stabilizer, emollient vaccine adjuvant, etc.
According to route of administration, Pharmaceutical
excipients can be classified into those uses for oral, mucosal,
percutaneous or topical administration, injection, nasal or
inhalation administration, ocular administration, etc.
The same pharmaceutical excipient can be used for
preparation of different routes of administration, dosage
form, and used for different purposes.
During production, storage and application, pharmaceutical
excipients shall comply with the following requirements.
-i. Pharmaceutical excipients for the production of drugs shall
meet the standards required for pharmaceutical use, i. e. raw
materials for production are confirmed to meet the require-
ments through demonstration, compliance with production
quality specifications of pharmaceutical excipients and supply
chain security.
2. After a reasonable assessment of the application appro-
aches and usage amount, pharmaceutical excipients shall be:
no toxic effects on human body, stable in chemical
properties, not easily affected by temperature, pH value and
preservation time; no incompatibility with the main
ingredients, no effect on <lose, efficacy and test preparation
of the main ingredients, especially no effect on the safety;
and excipients with functions meeting the requirements
should be selected as much as possible to play a larger role
with a smaller amount after screening.
3. The national standards of pharmaceutical excipients shall
be established on the basis of production conditions,
production technology and sources of raw materials
confirmed by the drug administration regulatory authorities
of the State Council of China, and produced in accordance
with the GMP of pharmaceutical excipients. It should be
reconfirmed when any change in these factors occurs, to
confirm the applicability of the pharmaceutical excipients
standard.
4. Pharmaceutical excipients can be used for preparation of a
variety of routes of administration, when the same pharma-
ceutical excipient is used for the formulations with different
routes of administration, the corresponding quality control
program shall be developed m accordance with the
requirements of clinical medicine. The quality standard
program shall set safety indicators that need to be focused
on. The quality standard for pharmaceutical excipients can be
set "label" item, to mark its specifications, i. e. pharma-
ceutical excipients for injection, etc.
5. Pharmaceutical excipients used for preparation of different
routes of administration or purposes have different quality
requirements. In developing standards of excipients, both
the safety of excipients themselves and the properties
affecting preparation production, quality, safety and efficacy
should be considered. The content of pharmaceutical
excipients test mainly includes two parts: CD production
processes and safety-related routine testing, such as
identification, appearances, test, assay and other items;
(2) functional indicators affecting the performance of
preparations, such as viscosity, particle size and so on.
6. The residual solvent, microbial limits, pyrogens,
bacteria! endotoxins, sterility of pharmaceutical excipients
shall comply with the relevant requirements. The pharma-
ceutical excipients of injection, eye drops and other sterile
preparations shall meet the requirements of injection
excipients or ophthalmic preparations, and the bacteria!
endotoxins of injection excipients shall comply with the
relevant requirements ( 1143), the injection excipients used
to withstand terminal sterilization process shall comply with
the requirements of microbial limit and control bacteria
<1143 ) , the injection excipients for sterile production
process, preparation of sterile process shall comply with the
sterility requirements ( 11O1 ) .
7. For the same pharmaceutical excipient with multiple uses,
it is described as "pharmaceutical excipients" in the
[ Category] of text uniformly, no specific categories are listed
again.
8. "Pharmaceutical Excipients" should be indicated on the
packaging of pharmaceutical excipients, and the scope of
application ( route of administration ) , packaging
 0291 General Requirements for National Pharmaceutical Reference Standards
specifications and storage requirements shall be clarified on
the packaging; the pharmaceutical excipients used in the
drug shall be written into the drug instructions.
0261 Water for Pharmaceutical Purposes
Water is the excipient used largely and widely in the
production process and the preparation of medicines in
pharmaceutical industry.
In this edition of the Pharmacopoeia of the People' s Republic
of China, water for pharmaceutical purposes includes
drinking water, purified water, water for injection and sterile
water for injection according to the purposes for which it is to
be used. Water for pharmaceutical purposes should be
suitably selected according to manufacturing processes or
usage purposes and requirements. The quality of water for
pharmaceutical purposes should comply with the
requirements of expected usage.
The source water used for water for pharmaceutical purposes
is usually drinking water.
The system for water preparation should be verified, systems
of daily monitoring, analysis and report should be
established, and complete original data should be kept for
reference. The system for water preparation should be
cleaned and sterilized periodically. Sterilization could be
conducted using thermal or chemical processes. The.
processes and the removal of disinfectant should be verified.
Drinking water Drinking water is prepared by purification of
natural water and its quality complies with the current
National Standard of the People' s Republic of China
( "Hygienic Standard of Living Drinking Water" ).
Drinking Water may be used for washing crude drugs before
purification and for preliminarily washing pharmaceutical
appiiances. Uniess otherwise specified, drinking water may
also be used as the solvent for extraction of medicine material
crude slices.
Purified water Purified water is prepared from drinking
water by distillation, ion exchange, reverse osmosis or by
means of any other appropriate methods. lt contains no
additives and its quality complies with the requirements of
the monograph of Purified water.
Purified water may be used as the solvent for preparation of
ordinary pharmaceutical preparations, or as a reagent in the
test, or as the solvent for extraction of medicine material
crude slices for preparation of sterile preparations such as
injections or eye drops of traditional Chinese drugs, oras the
solvent or diluent for preparation of oral preparations or
preparations for externa! use, or as the water for precisely
washing appliances for nonsterile preparations. lt may also
be used as the solvent for extraction of medicine material
crude slices for nonsterile preparations. Purified water should
not be used for preparation of injections or as their diluent.
Purified water can be produced by various methods. Every
step of the production should be monitored to prevent from
microbial contamination and to control the quality of the
water when used.
Water for injection Water for injection is water prepared by
distillation of purified water. lt complies with the requirements
for the test of bacteria! endotoxins. Bacteria! endotoxins must be
prevented during the production process, storage and
distribution. The quality of water for injection complies with the
requirements of the monograph of Water for injection.
Water for injection may be used as the solvent or diluent for
preparation of injections and eye drops, etc. , or for precisely
washing their containers.
In arder to guarantee the quality of water for injection,
bacteria! endotoxins in the source water should be mini-
mized, every step of the production process of preparation of
water for injection by distillation should be monitored, and
the microbial contamination should be prevented. The
system for water preparation should be cleaned and sterilized
periodically. Storage conditions and periods for water for
injection should be verified to ensure the quality water for
injection comply with the requirements. Water for injection
should generally be kept at a temperature exceeding 80ºC,
kept circulating at 70ºC or preserved on sterile condition
below 4ºC.
Sterile water for injection Sterile Water for Injection is
prepared from water for injection according to the techno-
logical conditions under which injections are prepared. lt
contains no additives and is mainly used as the solvent for
sterile powders for injection or as diluent for injections. lts
quality complies with the requirements of the monograph of
sterile water for injection.
The specification of filling for Sterile water for injection
should meet the clinical requirements. Contamination due to
large volume or multi-use must be avoided.
0291 General Requirements for National
Pharmaceutical Reference Standards
National Pharmaceutical Reference Standards ( NPRS) refer
to the substances with defined characteristics or values,
which are used for physical, chemical and biological tests on
drugs in national drug standards for calibration of instru-
ments, evaluation of test methods, as well as value
assignment and identification of test drugs.
NPRS shouid be stabie, homogenous and accurate.
The following statutory requirements should be complied
with NPRS regarding their grade and classification,
establishment, use, stability monitoring, labeling, storage,
and distribution.
l. Grading and Classification of NPRS
There are two grades of NPRS.
NPRS Grade 1 : NPRS of grade I show high quality
properties, of which the characteristic values may be
measured by definition or other precise and reliable methods.
NPRS Grade ll: NPRS of grade II show high quality
properties, of which the characteristic values may be
measured by accurate and reliable methods or comparison
with NPRS of grade l.
There are five classes of NPRS.
Biological Reference Standards: Standard substances refer to
the NPRS that consist of single or mixed components, which
are used for biological identification as well as determination
of potency, toxicity or content of antibiotics or biochemical
drugs. The biological activities of the substances are
represented by international unit ( IU), unit (U), or mass
unit (g, mg, µg).
Chemical Reference Standards: Reference substances refer to
the NPRS that consist of single, combined or mixed
components, which are used for the determination of
potency, toxicity or content of chemical drugs, antibiotics,
parts of biochemical drugs, pharmaceutical excipients,
Chinese crude drug ( including prepared slices), extraction,
Chinese patent medicine and biologics (physiochemical test),
etc. , as well as calibration of instruments.
 0301 General ldentification Tests
Reference Extract: Reference extract refer to the national
pharmaceutical reference standard that consist of multiple
bioactive ingredients or representative ingredients, which are
extracted by specified extraction process, and are used for
identification or quantitation of Chinese crude drug (including
prepared slices), extract, Chinese patent medicine, etc.
Reference Crude Drug: Reference crude drug refer to the
NPRS as properly treated Chinese crude drug with clear
origin, accurate medicinal part and high quality, which are
used for identification of Chinese crude drug ( including
prepared slices), extract, and Chinese patent medicine,
etc.
Biological reference materials: Reference materials refer to
the national of drug reference standards used for qualitative
identification of microorganisms or their products, or for
quantitative determination of biological potency and bio-
activity of sorne products, of which the potencies are
represented by the specific activity unit. The materials can
also refer to the substances used for disease diagnosis, which
are prepared from biological reagents, biomaterials, or
specific antiserum.
2. Establishment of NPRS
The establishment of NPRS includes determination of the
drugs needing new reference standards, obtaining of
reference standard candidates, determination of calibration
protocol, analysis of calibration, data review and approval,
filling and packaging.
2. 1 Detennination of the kind of new reference standards
Unless specified otherwise, the kind of new reference
standards is determined according to requirements from
development or amendment of national drug standards.
2. 2 Obtaining of reference standard candidates
Candidates for biological reference standards, chemical
reference standards and biological reference materials should
be a batch of product by routine procedure with high quality,
or extracted from Chinese crude drug ( including prepared
slices).
Ref erence extract candidates are extracted from Chinese
crude drugs (including prepared slices) with clear origin, or
from animal or plant origins.
Reference crude drug candidates are selected from Chinese
crude drugs with clear origin and medicinal parts.
2. 3 Calibration of NPRS
The calibration of NPRS must be accomplished collabo-
ratively by at least three laboratories accredited by the China
Food and Drug Administration. Laboratories participating in
the calibration should employ the identical protocol, methods
and document formats. The calibration results should be
0300
0301 General ldentification Tests
Acetates
(1) Heat a quantity of the substance being examined with
sulfuric acid and ethanol, the characteristic odour of ethyl
acetate is liberated.
processed by statistical analysis ( at least five independent
results are needed). The mean value of calibration results
from all laboratories is usually served as the calibration value of
NPRS.
Calibration of NPRS includes qualitative identification,
structural identification, purity analysis, value assignment,
and stability evaluation.
2. 4 Filling and packaging
The requirements of GMP should apply to the conditions for
filling and packaging of NPRS. Primary influencing factors,
including temperature, humidity, light and safety, should be
controlled.
NPRS should be packaged in a single-dose form to ensure
reliability in application. The material used for packaging
containers should guarantee the quality of NPRS.
3. The Use of NPRS
NPRS are used for the implementation of national drug
standards, including calibration of instrument, evaluation of
test methods, or identification/assignment of test drugs.
The assigned values of NPRS are only effective for specified
uses. The applicability for other purpose should be
determined by the users themselves.
Generally, one packaging unit is only for one-time use and
the solution of reference standard should be prepared freshly
before use. Otherwise, the applicability should be proved by
users.
4. Stability Monitoring of NPRS
Distributing department of NPRS should establish routine
quality assurance system to regularly monitor the distributed
NPRS and ensure their quality under normal storage
condition. If quality problems are found in reference
standards, the distributing department should publicize
stopping the use of problematic batches.
5. The Storage of NPRS
The storage conditions are determined by physicochemical
properties of reference standards. Unless otherwise specified,
NPRS are generally stored at room temperature.
6. Labels and Package Insert of NPRS
The label of national pharmaceutical standard substances
should include the information about name, serial number,
batch number, filling quantity, use, storage conditions,
supplier and etc. . Moreover, the information on content
should be stated on the label of standard substances used for
content determination.
In addition to the information indicated on the label, the
instruction of NPRS should also provide the composition,
structure, origin, etc. , and information on reference
spectrum if necessary.
(2) To a neutral solution of the substance being examined
add 1 drop of ferric chloride TS, a deep red colour is
produced which disappears on addition of dilute mineral
acid.
Aluminium Salts
(1) Add sodium hydroxide TS to a solution of the substance
being examined, a gelatinous white precipitate appears which
is soluble in an excess of sodium hydroxide TS.

(2) Add ammonia TS to a solution of the substance being
examined until a gelatinous white precipitate is formed. Add
a few drops of sodium alizarinsulfonate IS, the precipitate
becomes cherry red in colour.
Ammonium Salts
( 1) Heat a quantity of the substance being examined with an
excess of sodium hydroxide TS, the characteristic odour of
ammonia is perceived, the vapour turns moistened red litmus
paper to blue and blackens a strip of filter paper moistened
with mercurous nitrate TS.
(2) To a solution of the substance being examined add 1
drop of alkaline mercuric potassium iodide TS, a reddish
brown precipitate is produced.
Antimony Salts
( 1) Acidify a solution of the substance being examined with
acetic acid, heat on a water bath, and to the hot solution add
a few drops of sodium thiosulfate TS, an orange red
precipitate is produced gradually.
(2) Acidify a solution of the substance being examined with
hydrochloric acid, pass hydrogen sulfide gas into the
solution, an orange precipitate, soluble in ammonium sulfide
TS or sodium sulfide TS is produced.
Barium Salts
(1) Moisten the substance being examined with hydro-
chloric acid on a platinum wire, it imparts a yellowish green
colour in a nonluminous flame, ora blue colour when viewed
through a green glass plate.
(2) Add dilute sulfuric acid to a solution of the substance
being examined, a white precipitate is produced which is
insoluble in hydrochloric acid or nitric acid.
Benzoates
( 1 ) Add ferric chloride TS to a neutral solution of the
substance being examined, a dull yellow precipitate is formed
which turns to white on addition of dilute hydrochloric acid.
(2) Introduce a quantity of the substance being examined
into a dry test tube, add sulfuric acid and heat gently, no
charring occurs, a white sublimate of benzoic acid is
deposited on the inner wall of the test tube.
Bisrnuth Salts
( 1) Add potassium iodide TS to a solution of the substance
being examined, a reddish brown colour or dark brown
precipitate is produced, the precipitate is soluble in an excess
of the reagent, forming a yellowish brown solution. Dilute
the solution with water; an orange precipitate is produced.
(2) Acidify a solution of the substance being examined with
dilute sulfuric acid, add a quantity of 10 % thiourea solution,
an intense yellow colour is produced.
Borates
( 1) A solution of the substance being examined, acidified
with hydrochloric acid, turns turmeric paper to brownish
red, the colour deepens on drying and becomes greenish
black when moistened with ammonia TS.
(2) Mix a quantity of the substance being examined with
sulfuric acid and add methanol, when the mixture is ignited,
it burns with a green-bordered flame.
Bromides
(1) Add silver nitrate TS to a solution of the substance being
examined, a curdy pale yellow precipitate is formed which is
slightly soluble in ammonia TS and practically insoluble in
nitric acid.
(2) Add chlorine TS dropwise to a solution of the substance
being examined, bromine is liberated, add chloroform and
shake, a yellow or reddish brown colour is developed in the
chloroform layer.
0301 General Identification Tests
Calcium Salts
( 1) Moisten the substance being examined with hydrochloric
acid on a platinum wire, it imparts a brick red colour to a
nonluminous flame.
( 2 ) Add 2 drops of methyl red IS to a solution of the
substance being examined Cl-20), neutralize with ammonia
TS and then acidify with hydrochloric acid. Add ammonium
oxalate TS, a white precipitate is produced which is soluble
in hydrochloric acid but insoluble in acetic acid.
Carbonates and Bicarbonates
( 1 ) Add dilute acid to a solution of the substance being
examined, it effervesces with the evolution of carbon dioxide,
producing a white precipitate when passed into calcium
hydroxide TS.
(2) Add magnesium sulfate TS to a solution of the substance
being examined, a white precipitate is produced immediately
(carbonates) or on boiling (bicarbonates).
(3) A solution of the substance being examined is colourless
or only slightly coloured on the addition of phenolphthalein IS
( bicarbonates), or an intense red colour is produced
(carbonates).
Chlorides
( 1) Acidify a solution of the substance being examined with
nitric acid and add silver nitrate TS, a curdy white precipitate
is formed which is soluble in ammonia TS and reprecipitated
on addition of nitric acid. Organic bases should be removed
by the addition of ammonia TS and filtration prior to the
test.
(2) Mix a small quantity of the substance being examined
with an equal part of manganese dioxide, moisten with
sulfuric acid and heat gently, chlorine is evolved which tums
a strip of moistened starch-potassium iodide paper to blue.
Citrates
(1) To 2 mi oí a solution oí the substance being examined
equivalent to about 10 mg of citric acid, add a few drops of
dilute sulfuric acid and heat to boiling, then add a few drops
of potassium permanganate TS and shake, the violet colour
disappears. Divide the solution into two portions, to one
portian add a drop of mercuric sulfate TS, to the other
portian add a few drops of bromine TS, a white precipitate is
produced in both solutions.
(2) To about 5 mg of the substance being examined add
about 5 ml of pyridine-acetic anhydride ( 3 : 1), shake, a
yellow to red or violet-red colour is produced.
Copper Salts
(1) Add a few drops of ammonia TS to a solution of the
substance being examined, a light blue precipitate is
produced which is soluble in an excess of the reagent,
forming a dark blue solution.
(2) Add potassium ferrocyanide TS to a solution of the
substance being examined, a reddish brown colour or
precipitate is produced.
Ferric Salts
( 1) Add potassium ferrocyanide TS to a solution of the
substance being examined, a dark blue precipitate is formed
which is insoluble in dilute hydrochloric acid, it decampases
to form a brown precipitate on addition of sodium hydroxide
TS.
(2) Add ammonium thiocyanate TS to a solution of the
substance being examined, a red colour is produced.
Ferrous Salts
( 1 ) Add potassium ferricyanide TS to a solution of the
substance being examined, a dark blue precipitate is formed
which is insoluble in dilute hydrochloric acid, it decomposes
 0301 General ldentification Tests
to form a brown precipitate on addition of sodium hydroxide
TS.
(2) To a solution of the substance being examined add a few
drops of a 1 % solution of o-phenanthroline in ethanol, a deep
red colour is produced.
lodides
(1) Add silver nitrate TS to a solution of the substance being
examined, a curdy yellow precipitate is produced which is
insoluble in nitric acid or ammonia TS.
(2) Add chlorine TS dropwise to a solution of the substance
being examined, iodine is liberated, add chloroform and
shake, a violet colour is produced in the chloroform layer, if
starch IS is added instead of chloroform, a blue colour is
produced.
Lactates
To 5 ml of a solution of the substance being examined
equivalent to about 5 mg of lactic acid in a test tube, add 1
ml of bromine TS and O. 5 ml of dilute sulfuric acid. Heat on
a water bath with stirring until the colour is discharged, add
4 g of ammonium sulfate and mix well, add dropwíse along
the inner wall of the tube O. 2 ml of a 10 % solution of sodium
nitroprusside in dilute sulfuric acid and 1 ml of concentrated
ammonia TS, allow to stand far 30 minutes without mixing,
a dark green ring is developed at the interface of the two
layers.
Lithium Salts
( 1) Alkalize the solution of the substance being examined
with sodium hydroxide TS, add sodium carbonate TS, a
white precipitate is produced on boiling which is soluble in
ammonium chloride TS.
(2) Moisten the substance being examined with hydrochloric
acid on a platinum wire, it imparts a crimson colour in a
nonluminous flame.
(3) To a quantity of the substance being examined add dilute
sulfuric acid or soluble sulfates solution, no precipitate is
produced (distinction from strontium).
Magnesium Salts
( 1) Add ammonia TS to a solution of the substance being
examined, a white precipitate is produced which redissolves
on addition of ammonium chloride TS. Add 1 drop of
disodium hydrogen phosphate TS and shake, a white
precipitate insoluble in ammonia TS is produced.
(2) Add sodium hydroxide TS to a solution of the substance
being examined, a white precipitate is produced, filter, the
precipitate is insoluble in an excess of sodium hydroxide TS
but is coloured reddish brown on addition of iodine TS.
Malonylureas
(1) Dissolve about O. 1 g of the substance being examined in
1 ml of sodium carbonate TS and 10 ml of water, shake far 2
minutes and filter. To the filtrate add a few drops of silver
nitrate TS, a white precipitate is produced which redissolves
on shaking, but it is insoluble in an excess of the reagent.
(2) Dissolve about 50 mg of the substance being examined in
5 ml of pyrídine solution ( 1 - 10), add 1 ml of copper
pyridine TS, a violet colour or precipitate is produced.
Mercuric Salts
(1) Add sodium hydroxide TS to a solution of the substance
being examined, a yellow precipitate is produced.
(2) Add potassium iodide TS to a neutral solution of the
substance being examined, a scarlet precipitate is produced
which is soluble in an excess of the reagent. To the solution
add sodium hydroxide TS andan ammonium salt, a reddish
brown precipitate is produced.
(3) When applied to bright copper foil, solutions of mercury
salts, free from an excess of aitric acid, yield a deposit that
upon rubbing, becomes bright and silvery in appearance.
Mercurous Salts
( 1) To a quantity of the substance being examined add
ammonia TS or sodium hydroxide TS, a black colour is
developed.
( 2) To a quantity of the substance being examined add
potassium iodide TS and shake, a yellowish green precipitate
is produced, changing to greyish green rapidly and then to
greyish black gradually.
Nitrates
( 1 ) Mix cautiously a solution of the substance being
examined with an equal volume of sulfuric acid and allow to
cool, add ferrous sulfate TS along the inner wall of the test
tube, a brown ring is developed at the interface of the two
layers.
( 2) To a solution of the substance being examined add
cautiously sulfuric acid and metallic copper; a reddish brown
fume is evolved on heating.
(3) Add dropwise potassium permanganate TS to a solution
of the substance being examined, the violet colour <loes not
disappear (distinction from nitrites).
Organic Fluorinated Compounds
Weigh about 7 mg of the substance being examined, carry
out the method far oxygen flask combustion ( 0703), using
20 ml of water and 6. 5 ml of O. Olml/L sodium hydroxide
solution as the absorbing liquid. To 2 ml of the resultíng
solution add O. 5 ml of alizarin fluorine blue TS and O. 2 ml of
a solution containing 12% of sodium acetate in dilute acetic
acid, dilute with water to 4 ml and add O. 5 ml of cerous
nitrate TS, a bluish violet colour is produced. Perform a
blank determination in the same manner.
Phosphates
( 1 ) Add sil ver ni trate TS to a neutral solution of the
substance being examined, a light yellow precipitate is
formed which is freely soluble in ammonia TS or dilute nitric
acid.
(2) Add magnesium ammonium chloride TS to a solution·of
the substance being examined, a white crystalline precipitate
is produced.
( 3) To a solution of the substance being examined add
ammonium molybdate TS and nitric acid, a yellow precipitate
soluble in ammonia TS is produced on heating.
Po~ium Salts
( 1) Moisten the substance being examined with hydrochloric
acid on a platinum wire, it imparts a violet colour in a
nonluminous flame. If sodium is also present, the yellow
colour can be screened out by viewing through a cobalt glass
plate.
(2) lgnite the substance being examined to remove any
ammonium salt contaminated, cool, dissolve it in water, add
acetic acid anda O. 1% solution of sodium tetraphenylborate,
a white precipitate is produced.
Primary Aromatic Amines
To about 50 mg of the substance being examined add 1 ml of
dilute hydrochloric acid and boíl gently to effect dissolution if
necessary, cool, add a few drops of O. 1 mol/L sodium nitrite
solution and alkaline p-naphthol TS, a precipitate coloured
from orange yellow to scarlet is formed depending on the
identity of the substance being examined.
Salicylates
(1) To a dilute solution of the substance being examined add
1 drop of ferric chloride TS, a violet colour is produced.

( 2 ) Add dilute hydrochloric acid to a solution of the
substance being examined, a white precipitate of salicylic
acid is produced which is soluble in ammonium acetate TS.
Silver Salts
( 1 ) Add dilute hydrochloric acid to a solution of the
substance being examined, a curdy white precipitate is
produced which is soluble in ammonia TS and reprecipitated
on addition of nitric acid.
(2) Add potassium chromate TS to a solution of the
substance being examined, a brick red precipitate is produced
which is soluble in nitric acid.
Sodium Salts
( 1) Moisten the substance being examined with hydrochloric
acid on a platinum wire, it imparts an intense yellow colour
in a nonluminous flame.
(2) To about 100 mg of the substance being examined in a
10 ml test tube, add 2 ml of water to dissolve, add 2 ml of
15% potassium carbonate, and heat to boiling, no precipitate
is formed, add 4 ml of potassium pyroantimonate TS, and
heat to boiling, allow to cool in ice water, if necessary, rub
the inside of the test tube with a glass rod, a dense
precipitate is formed.
Stannous salts
One drop of an aqueous solution of the substance being examined
turns ammonium molybdophosphate test paper to blue.
Sulfates
( 1) Add barium chloride TS to a solution of the substance
being examined, a white precipitate is formed which is
insoluble in hydrochloric acid or nitric acid.
(2) Add lead acetate TS to a solution of the substance being
examined, a white precipitate is formed which is soluble in
ammonium acetate TS or sodium hydroxide TS.
( 3) Add hydrochloric acid to a solution of the substance
0400 Spectrometry
Spectroscopic methods may mainly refer to the measure-
ments of the frequencies and intensities of the radiation
(light) emitted, absorbed and scatted by matter undergoing
transitions between quantized energy levels in the interaction
of matter and electromagnetic radiation. With different
classification strategies, these methods can be divided into
emission, absorption and scattering spectrometry, or into
atomic and molecular spectrometry, or into nuclear level,
electron, vibration, rotation, electron spin, nuclear spin
spectrometry, etc.
Mass spectrometry ( MS) is the analytical technique by
ionizing the molecules in a sample to generate gaseous
charged molecules or molecule fragments, measuring the
mass-to-charge ratios (MS) and their abundance, elucidating
the chemical structures of molecules and quantifying the
amount of compositions in the sample. Strictly speaking,
Mass spectrometry <loes not belong to the category of optic
spectroscopy. However, Mass spectrometry is hereby
grouped into spectroscopic methods based on their similar
manner in characterizing the spectra.
Optic spectrometry, or spectrophotometry, the most
important part of spectrometry, is the measurement of an
interaction between electromagnetic radiation and the
molecules, or atoms, of a chemical substance. Techniques
0400 Spectrometry
lp , .., ...... ,····
:1,j>;.;
.··<·
being examined, no white precipitate is produced (distinction
from thiosulphates).
Sulfites and Bisulfites
(1) Add hydrochloric acid to the substance being examined,
the pungent odour of sulfur dioxide is perceived, the vapour
blackens a strip of filter paper moistened with mercurous
nitrate TS.
(2) Add iodine TS dropwise to a solution of the substance
being examined, the colour of iodine is discharged.
Tartrates
(1) To a neutral solution of the substance being examined in
a clean test tube, add a few drops of ammoniacal silver
nitrate TS and heat in a water bath, silver is deposited on the
inner wall of the test tube as a mirror.
(2) Acidify a solution of the substance being examined with
acetic acid, add 1 drop of ferrous sulfate TS and 1 drop of
hydrogen peroxide TS, make the solution alkaline with
sodium hydroxide TS when the colour is discharged, a violet
colour is produced.
Tropane Alkaloids
To about 10 mg of the substance being examined add 5 drops
of fuming nitric acid, evaporate to dryness on a water bath, a
yellow residue is obtained. Cool, moisten the residue with
2-3 drops of ethanol, add a small pellet of potassium
hydroxide, an intense violet colour is produced.
Zinc Salts
(1) Add potassium ferrocyanide TS to a solution of the
substance being examined, a white precipitate is formed
which is insoluble in dilute hydrochloric acid.
(2) To a neutral or alkalic solution of the substance being
examined, add soldium sulfide TS, a white precipitate is
produced.
frequently employed include ultraviolet-visible, infrared,
fluorescence, and atomic absorption spectrometry. Spectro-
photometric measurement in the visible region was formerly
referred to as colourimetry.
Light-Scattering involves measurement of the light scattered
because of submicroscopic optical density inhomogeneities of
solutions and is useful in the determination of weight-average
molecular weights of polydisperse systems in the molecular
weight range from 1000 to several hundred million. Raman
spectroscopy is an inelastic light-scattering process in which
the specimen under examination is irradiated with intense
monochromatic light ( usually laser light) and the light
scattered from the specimen is analyzed for frequency shifts.
The wavelength range available for these measurements
extends from the short wavelengths of the UV through the
IR. For convenience of reference, this spectral range is
roughly divided into the ultraviolet (UV) region (190 to 400
nm), the visible ( 400 to 760 nm), the near- infrared (NIR)
region (760 to 2500 nm), and the infrared (IR) region (2. 5
to 40 µmor 4000 to 250 cm- 1 ). These instruments are called
Ultraviolet-Visible Spectrophotometer, Near Infrared
Speciation-Photometry, Atomic Absorption Spectrophoto-
meter, Light scattering meter and Raman Spectrometer
respectively. In order to ensure the accuracy and precision of
 0400 Spectrometry
measure ment, the instruments should be calibrated
periodically according to the national metrology verification
regulations or the corresponding principles of spectrometry in
the pharmacopoeia.
Theory and Tenns
When monochromatic radiation passes through an absorbing
medium, the absorbance of the radiation is proportional to
the concentration of the absorbing substance and the
thickness of the absorbing medium within a certain
absorption range, this relation is expressed by Lambert-Beer'
s law with the following equation:
1 band intensity is linearly proportional to the concentration of
A= lg- =ECL
T
Where A is the absorbance, T is the transmittance, E is the
absorption coefficient, C is the concentration of the
substance expressed in g per 100 ml, calculated on the dried
or dehydrated basis and L is the absorption path length
expressed in cm. The term E}~ is used in this pharmacopoeia
to denote the absorbance of a 1% solution in a 1 cm cell.
The molar absorption coefficiente (or molar absorptivity) is
also used to denote the absorbance of a 1 mol/L solution in a
1 cm cell. Its value at the wavelength of maximum
absorption is expressed as emax .
The wavelength of the selective absorption and the
corresponding absorption coefficient are physical constants of
the substance being examined. For most systems used in
absorption spectrophotometry, the absorption coefficient of a
substance is a constant independent of the intensity of the
incident radiation, the interna! cell length, and the concen-
tration. When the absorption coefficient of a substance is
known, its content can be calculated from the above
equation. In the visible region, the content of a colourless
substance can be determined colourimetrically af ter the
addition of a colour developing agent or any other treatment.
Deviations from Lambert-Beer' s law may be caused by either
chemical or instrumental variables. Apparent failure of Beer'
s law may result from a concentration change in solute
molecules because of association between solute molecules or
between solute and solvent molecules, or dissociation or
ionization. Other deviations might be caused by instrumental
effects such as polychromatic radiation, slit-width effects, or
stray light.
Atomic absorption processes do follow the Beer relationship
in principie. The absorbance is directly proportional to the
number of absorbing atoms. On this basis, calibration curves
may be constructed to permit evaluation of the absorption
values in terms of concentration of the element in solution.
The fluorescence emission spectrum is a graphical presen-
tation of the spectral distribution of radiation emitted by an
activated substance, showing intensity of emitted radiation as
ordinate, and wavelength as abscissa. The fluorescence
excitation spectrum is a graphical presentation of the
activation spectrum, showing intensity of radiation emitted
by an activated substance as ordinate, and wavelength of the
incident ( activating) radiation as abscissa. As in absorption
spectrophotometry, the important regions of the electroma-
gnetic spectrum encompassed by the fluorescence of organic
compounds are the UV, visible, and near-IR, i. e. , the
region from 250 to 800 nm. Af ter a molecule has absorbed
radiation, the energy can be lost as heat or released in the
form of radiation of the same or longer wavelength as the
absorbed radiation. Both absorption and emission of radiation
are due to the transitions of electrons between different
energy levels, or orbitals, of the molecule or atom. There is
a time delay between the absorption and emission of light;
this interval, the duration of the excited state, has been
measured to be about 10- 9 second to 10-s second for most
organic fluorescent solutions. The short lifetime of
fluorescence distinguishes this type of luminescence from
phosphorescence, which is a long-lived afterglow having a
lifetime of 10- 3 second up to several minutes.
Raman Scattering Activity - The molecular property (in
units of cm4 per g) governing the intensity of an observed
Raman band for a randomly oriented specimen. The
scattering activity is determined from the derivative of the
molecular polarizability with respect to the molecular motion
giving rise to the Raman shifted band. In general, the Raman
the analyte.
Where values for the peaks of IR spectra or Raman spectra
are given in an individual monograph, the letters s , m, and
w signify strong, medium, and weak peak, respectively; sh
signifies a shoulder, bd signifies a band, and v means very.
Comparative utility of spectral methods
For many pharmaceutical substances, spectroscopic
measurements can be made in the UV and visible regions
with greater accuracy and sensitivity than in the near-IR and
IR. The UV and visible spectra of substances generally do
not have a high degree of specificity as other spectra.
Nevertheless, they are highly suitable for quantitative
assays, and for many substances they are useful as additional
means of identification. There has been increasing interest in
the use of near-IR spectroscopy in pharmaceutical analysis,
especially for rapid identification of large numbers of
samples, and also for water determination. The near-IR
region is especially suitable for the determination of -OH
and -NH groups, such as water in alcohol, -OH in the
presence of amines, alcohols in hydrocarbons, and primary
and secondary amines in the presence of tertiary amines.
The IR spectrum is unique for any given chemical compound
with the exception of optical isomers, which have identical
spectra. However, polymorphism may occasionally be
responsible for a difference in the IR spectrum of a given
compound in the solid state. Frequently, small differences in
structure result in significant differences in the spectra.
Because of the large number of maxima in an IR absorption
spectrum, it is sometimes possible to quantitatively measure
the individual components of a mixture of known qualitative
composition without prior separation.
Raman and IR spectroscopy exhibit different relative
sensitivities for different functional groups, e. g. , Raman
spectroscopy is particularly sensitive to c-s and e-e
multiple bonds. And sorne aromatic compounds are more
easily identified by means of their Raman spectra. Water has
a highly intense IR absorption spectrum, but a particularly
weak Raman spectrum. Therefore, water is almost
completely transparent to Raman scattering and useful as
solvent for salute identification. The two major limitations of
Raman spectroscopy are that the mínimum detectable
concentration of specimen is typically 10- 1 M to 10- 2 M and
that the impurities in many substances fluoresce and interfere
with the detection of the Raman scattered signal.
Optical reflectance measurements provide spectral informa-
tion similar to that obtained by transmission measurements.
Since reflectance measurements probe only the surface
composition of the specimen, difficulties associated with the
optical thickness and the light-scattering properties of the
substance are eliminated. Thus, reflectance measurements
are frequently more simple to perform on intensely absorbing
materials. A particularly common technique used for IR
reflectance measurements is termed attenuated total reflec-
tance ( ATR), also known as multiple internal reflectance
 0401
(MIR). The ATR technique provides excellent sensitivity,
but yields poor reproducibility. It is not a reliable quanti-
tative technique unless an interna! standard is intimately
mixed with each test specimen.
Fluorescence spectrophotometry is often more sensitive than
absorption spectrophotometry. In fluorescence spectrophoto--
metry, the solvent blank has low rather than high output, so
that the background radiation that may interfere with
determinations is much less. Whereas few compounds can be
determined conveniently at concentrations below 10-s M by
light absorption, it is not unusual to employ concentrations
of 10- 7 M to 10-s M in fluorescence spectrophotometry.
Use of reference standards
In identifications, tests and determination, a reference
standard may be used for comparison. lt is to ensure that the
measured conditions are identical for the test specimen and
the reference substance. These conditions include wavelength
setting, slit-width adjustment, cell placement and correc-
tion, and transmittance levels. Cells may differ considerably
in transmittance at different wavelengths. Appropriate cell
corrections should be established and used where required.
The expressions, " similar preparation" and " similar
solution", indicate that the reference specimen, generally a
reference standard, is to be prepared and observed in a
manner identical for all practical purposes to that used for the
test specimen. Usually in making up the solution of the
specified reference standard, a solution of about the desired
concentration ( i. e. , within 10%) is prepared and the
absorbance is calculated on the basis of the exact amount
weighed out; if a previously dried specimen of the reference
standard has not been used, the absorbance is calculated on
the anhydrous basis.
The expressions, "concomitantly determine" and "concomi-
tantly measured", indicate that the absorbances of both the
solution containing the test specimen and the solution
containing the reference specimen, relative to the specified
test blank, are to be measured in immediate succession.
0401 Ultraviolet-Visible
Spectrophotometry
Ultraviolet-visible spectrophotometry is a method to measure
the degree of absorption of light between the wavelengths ·of
190 nm and 800 nm by substances for assay and for the tests
of their identity and purity. When a light beam passes
through a solution, the absorbance by the sample varíes with
the wavelength. So, an absorption spectrum is obtained by
determining the absorbance of a substance at various
wavelengths and by graphically plotting the absorbance
versus the wavelength. From the absorption spectrum, it is
possible to determine the wavelength of maximum absorption
Amax and that of mínimum absorption Amin • The absorption
spectrum of a substance in the solution is characteristic,
depending on its chemical structure. Therefore, it is possible
to identify a substance by comparing the spectrum of a
sample within the specified wavelength range with the
Reference Spectrum or the spectrum of Reference Standard,
by determining the wavelengths of maximum absorption, or
by measuring the ratio of absorbances at two specified
wavelengths. For the purpose of assay, measure the
absorbance of a sample solution with a certain concentration
at the wavelength of the maximum absorption Amax , and
compare it with the absorbance of a standard solution with a
certain concen-tration, or with the absorption coefficient to
calculate the content of substance in sample solution.
Ultraviolet-Visible Spectrophotometry
Calibration and Perfonnance Test of the lnstrument
l. Wavelengh The change of environment may affect the
mechanical parts of the spectrophotometer and cause a drift
of the wavelength scale. Therefore, the spectrophotometer
should be calibrated regularly, and the wavelength scale must
be verified immediately before the measurement. Mercury
lamp with the following spectral lines: 237. 83, 253. 65,
275.28, 296.73, 313. 16, 334. 15, 365.02, 404.66,
435. 83, 546. 07 and 576. 96 nm, is often used as the light
source for this purpose. The wavelength scale may also be
calibrated by means of the 486. 02 nm and 656. 10 nm lines of
deuterium discharge lamp in spectrophotometer. Holmium
glass filter exhibiting sharp absorption peaks at 279. 4,
287.5, 333.7, 360.9, 418.5, 460.0, 484.5, 536.2 and
637. 5nm may be suitable for the calibration wavelength.
However, the observed wavelengths of these peaks may
change slightly depending on the commercial source of the
filter or along with the time going by. Recently, the
holmium perchlorate solution is widely used to verify the dual
beam apparatus. The absorption maxima of solution
containing 4 % of holmium oxide ( Ho 2 Ü3) , prepared by
using 10% of perchloric acid solution, peaks at 241. 13,
278. 10, 287. 18, 333.44, 345.47, 361.31, 416.28,
451. 30, 485. 29, 536. 64 and 640. 52 nm, respectively.
The permitted tolerance of the apparatus wavelength is
± 1 nm for the ultraviolet region, and ± 2 nm nearby 500
nm.
2. Control of absorbance Check the absorbance with a
solution of potassium dichromate in sulfuric acid. Dissolve
about 60 mg of potassium dichromate primary standard,
previously dried to constant weight at 120ºC and accurately
weighed, in O. 005 mol/L sulfuric acid solution to make 1000
ml. Check the absorbance at wavelengths indicated in the
following table and calculate the absorption coefficient which
shouid be in accord with the specified range in the table when
compared with the specified absorption coefficient.
235 257 313 350
Wavelength/ nm
(min) (max) (min) (max)
Specific absorbance 124.5 144.0 48.6 106.6
(El~)
Maximum tolerance 123. 0- 142. 8- 47. o- 105. 5-
126.0 146.2 50.3 108.5
3. Lirnit of stray light Stray light may be detected at the
given wavelength with suitable solutions as indicated in the
following table. The transmittance of these solutions
measured in a 1 cm quartz cell against water should be in
accord with the limit specified in the table.
Concentration/ % Wavelength/ Transmittance/
Reagent
(g/mD nm %
Sodium l. 00 220 <0.8
Iodide
Sodium 5.00 340 <0.8
Nitrite
Requirements for the Solvents
The organic solvents containing hetero atoms usually have
strong absorption at the lower wavelength. Thus, they
should be used in the ranges greater than the cut off
wavelength. For example, the cut off wavelength of
methanol and ethanol is 205 nm. Otherwise, impurity of
solvents would enhance the interference of absorption. The
 .._iFI<. ·•.· •· .·····
· .· . :·. ·.•··¡.
·;;:;''.. ,.. ,
0402 Infrared Spectrophotometry
solvent used in spectrophotometric determinations should be
checked for any interfering absorption peak around the
selected wavelength for the measurement of the absorbance
being examined. The absorbance of a solvent should not
exceed O. 40 in the range of 220 to 240 nm, O. 20 in the range
of 241 to 250 nm, O. 10 in the range of 251 to 300 nm and
O. 05 at wavelengths above 300 nm, when measured in a 1
cm quartz cell against air.
Procedure
Unless otherwise specified, the same batch of solvent used to
prepare the solution of the substance being examined should
be used as the blank in matched 1 cm quartz cells. The
absorbance of the substance being examined should be
measured at the specified wavelength within a range of ± 2
nm, or the spectrum be plotted in the vicinity of the specified
wavelength, to check whether the wavelength of the
absorption maximum is correct or not. Unless otherwise
specified, the absorption maximum must be within ± 2 nm
as specified in the monograph. The assay should be carried
out at the wavelength of maximum absorption. The
concentration of the solution should be adjusted to give an
absorbance reading of O. 3 to O. 7. The width of the spectral
slit should be smaller than the tenth of half-height width of
the absorption band so that low absorbance will not be
resulted. The slit width is appropriate if further reduction
does not result in an increase in absorbance reading. The
absorbance of an unmatched cell with the solvent concerned
as a blank must be subtracted from the absorbance of the
substance being examined or be automatically deducted by
the spectrophotometer.
When the pH value affects the results determined, the pH of
the test preparation should be adjusted to be equal to that of
the reference preparation.
l. ldentification and Quality Tests Carry out the method
described in the items specified under the individual
monograph.
2. Assay Usually as the following methods.
(1) Reference suhstance comparison method Prepare
separately the solutions of the substance being examined and
CRS according to the method described under ítem of the
individual monograph. The content of the CRS in the
solution should be within 100 % ± 10 % of the labelled
amount of the solution prepared with substance being
examined and with solvent of the same batch. Determine the
absorbances of solutions of test preparation and of reference
preparation at specified wavelength, calculate the
concentration of the test preparation in the solution according
to the following equation:
Ax
Cx = -CR
AR
where Cx is the concentration of the test preparation; Ax is
the absorbance of the test preparation; CR is the
concentration of the reference preparation and AR is the
absorbance of the reference preparation.
(2) Absorption coefficient method Prepare the solutions of
the substance being examined according to the method
described under the individual monograph, determine the
absorbance at specified wavelength and calculate the
concentration of the test preparation with the absorption
coefficient specified in the monograph concerned. Usually,
absorption coefficient should be more than 100. Pay attention
to the calibration and check of the instrument being used.
( 3 ) Chemometric methods Carry out the procedure as
described under the specified monograph. When absorbances
are measured at wavelengths of the ascending or descending
position of the absorption curve, the minar variation of the
wavelength may affect the result significantly. lt is essential
to determine the reference and test preparations under the
same condition.
Commonly, chemometric method is not suitable for assay.
( 4) Colourimetry Colourimetry is used with the addition of
suitable developer befare determination, to move the
absorption maxima of reaction products to visible region
when the substance being examined has no strong absorption
in the ultraviolet-visible region, or to avoid the interference
or increase the sensitivity.
Colourimetric determination should be carried out with a
reference substance concomitantly. Unless otherwise
specified, an equal volume of solvent, added with the same
reagent and treated in the same manner, is used as blank.
Calculate the concentration of test preparation as " ( 1)
Reference substance comparison method" described under the
above method ( 1).
If the linear relation between absorbance and concentration is
not good enough, the absorbance of a series of preparations
containing gradient amounts of the reference substance
should be measured, and a calibration curve should be
produced by plotting the absorbance against concentration.
The concentration of the test preparation can be determined
by interpolating its absorbance on the calibration curve.
0402 Infrared Spectrophotometry
Infrared spectrophotometry is the measurement of absorption
of mid-infrared light in the region of 4000 to 400 cm- 1 by a
chemical substance. As few compounds ha ve the same
infrared spectra except sorne optical isomers and sorne long
chain alkane homologues, infrared spectrophotometry
therefore is used for qualitative and structural analysis of
components, including identification, polymorphism
inspection and limit test of the isomers. The relationship
between the infrared spectral absorption and the
concentration of a compound complies with the law of
Lambert-Beer, which is the basis of quantitative
measurement by infrared spectrophotometry.
Equipment and Calibration
Fourier-transform infrared spectrophotometers or dispersive
infrared spectrophotometers are often used for pharmaceutical
analysis . The wave-number scale of an infrared spectropho-
tometer may be calibrated by use of a polystyrene film of O. 04
mm thickness. The absorption peaks at 3027, 2851, 1601, 1028
and 907 cm- l are used for the calibration. Tolerance should be
not more than ± 5 cm- 1 in the vicinity of 3000 cm- 1 and not
more than ± 1 cm- l in the vicinity of 1000 cm- l for the Fourier-
transform infrared spectrophotometer.
When a polystyrene film is used for calibration, the
resolution of the instrument in the region 3110-2850 cm- 1 is
required, so that seven peaks should be clearly isolated, that
the depth of the resolution from the maximum absorption at
about 2851 cm- 1 to the mínimum at about 2870 cm- 1 should
be not less than 18% transmittance, and that from the
maximum at about 1583 cm- 1 to the mínimum at about
1589 cm- 1 should be not less than 12% transmittance.
Unless otherwise specified, the nominal resolution factor of
the instrument should not be less than 2 cm- 1 •
Preparation and Detennination
The methods, such as clise, mull, film, solution and gas
absorption cell for the preparation of test specimens and
Reference substances, are generally used in infrared
spectrophotometry. For those samples which have strong
absorption or have covering materials on their opaque

surface, sorne infrared spectral methods are preferred such as
attenuated total reflection, diffuse reflectance and emission
and so on. For those samples which have only trace amounts
or need micro pro be analysis, infrared micro-spectroscopy
could be used.
1. ldentification f or drug substances U nless otherwise
specified, for the identification test the substance being
examined must be prepared according to the method
described in each volume of "Atlas of Infrared Spectra of
Drugs ", edited by Chinese Pharmacopoeia Commission.
Detailed procedures see also the lntroduction of the Atlas.
If the spectrum of the substance being examined and the
reference spectrum show differences dueto polymorphism of
solid specimen, in this case, after excluding the possibility of
externa! or human factors, the substance being examined
should be pre-treated according to the method for specified
substance described in "Atlas of the Infrared Spectra of
Drugs" or individual monograph, and then the spectrum be
collected again. If the crystalline form and suitable pre-
treatment method are not specified, the reference substance
may be used, the substance being examined and the reference
substance are re-crystallized using suitable solvent under the
same condition, and then the collected spectra for both
sample and reference may be compared. If the crystalline
form is specified, the reference substance with corresponding
crystalline form should be used.
When the solid sample preparation technique is not
satisfactory for identification, a solution preparation in a
suitable solvent may be an altemative method used for
comparison of the sample spectrum with reference substance
spectrum obtained under the same condition.
2. Identification for preparations The specimen should be
prepared as described in the individual monograph, and
solvent extraction method is often used. In order to minimize
the interference of excipients and avoid the potentiai change
of crystalline form, suitable solvent should be chosen for the
extraction procedure, and then carry out the test for
identification after the extract specimen has been dried by a
suitable method.
3. Identification for multi-component drug substances If it is
not applicable to compare the spectra in the region of 4000-
400 cm- 1 , the method described in the notes "2 (3)" may
be used, i. e. , carry out the identification test by comparison
of main bands of major constituent provided that the
substance being examined has a fixed composition.
4. Polymorphism inspection, limit test of the isomers and
assay The method of sample preparation and determination
is performed according to the procedure described in the
individual monograph.
Notes
l. The reference infrared spectrum, which the infrared
absorption spectrum of sample being examined should be
concordant with, prescribed in the monograph, refers to the
spectrum specified in the "Atlas of Infrared Spectra of
Drugs", consisted of Vol. I (1995 edition), Vol. Il (2000
edition) and Vol. ill (2005 edition). If the spectra for the
same compound are described in different volume, the latter
one should be taken as the reference.
2. The spectrum of the substance obtained from the
formulated preparations by extraction method is compared by
one of the following approaches:
( 1) If the excipients in a formulated preparation do not
interfere with the spectrum comparison and there is no
change in the crystalline form of the substance being
examined after the extraction, compare the recorded
0405 Fluorescence Spectrophotometry
spectrum with the reference spectrum of its drug substance.
(2) If the excipients in a formulated preparation do not
interfere with the spectrum comparison, however, there is
difference in the crystalline form of the substance being
examined after the extraction, use the reference substance.
The extracted sample and the reference substance should be
treated under the same condition and then the spectra be
collected and compared.
(3) If there is no change in the crystalline form for substance
and the excipients have somewhat interference with the
spectrum comparison, select 3-5 specific bands of absorption
in the fingerprint region based on the reference spectrum of
its drug substances, where excipients have no interference for
the comparison, and perform the spectrum comparison refer
to the above criteria for identification. In the identification,
the deviation of the specified wavenumber from that of
measured should be not more than ± 5 cm (O. 5 %) .
( 4) If there is difference in the crystalline form of the
substance being examined after the extraction, and excipients
also interfere with the comparison after treatment, infrared
spectrophotometry is not recommended for the identification
test dueto the complicated conditions.
3. Severa! factors, such as difference in instrument
performance of various models, inadequate or successive
grinding in preparation of the clise, moisture affect or halide
carrier, may give rise to unsatisfactory spectrum in shape.
Those factors need to be considered when using the reference
spectrum for comparison purposes.
0405 Fluorescence Spectrophotometry
When substances are exposed to ultraviolet or visible radiation,
sorne of them may emit fluorescence at a wavelength longer
than that of the exciting radiation. The excitation and
emission spectra of a substance can be used for qualitative
analysis. When the intensity and wavelength of the exciting
radiation, the solvent and the temperature are constant, the
intensity of the fluorescence emitted by a substance is directly
proportional to the concentration of the substance within a
definite range, therefore, this relation can be used for
quantitative determinations. The sensitivity of fluorescence
spectrophotometry is usually higher than that of UV and
visible spectrophotometry. However, if a solution is too
concentrated, a self-quenching effect andan absorption of the
exciting radiation near the surface may result in a decrease of
the intensity of emitted radiation, and the intensity of
fluorescence emitted is not directly proportional to the
concentration. Therefore, fluorescence spectrophotometry
should be carried out only in dilute solutions.
Procedure
The instrument used is fluorimeter or fluorimetry spectro-
photometer. Select the excitation and emission bands and
prepare the reference and the test solutions as specified in the
monograph.
Measurements are usually carried out with a properly
selected reference substance to determine the linear relation
of fluorescence intensity and concentration. When the linear
relation is good, adjust the sensitivity of the instrument with
appropriate dilutions of the reference solution before each
test, and then record the fluorescence intensities of the test
preparation and reference preparation and their corresponding
blanks. Calculate the concentration Cx of the substance in the
test preparation, using the formula:
ex Rx -Rcb C
Rr -Rrb X r
 0406 Atomic Absorption Spectrophotometry
where Cx is the concentration of the test preparation, Cr is
the concentration of the reference preparation, Rx is the
fluorescence intensity of the test preparation, Rr is the
fluorescence intensity of the reference preparation, R:rh and
Rrb are the fluorescence intensity of the corresponding
blanks.
The range within which the fluorescence intensity is directly
proportional to the concentration of the substance is usually
very narrow, therefore, the ratio (Rx - R:rh)/(Rr - Rrb)
should not be less than O. 5 or more than 2. Otherwise the
concentration of the solutions should be adjusted and the
measurements made again. If the fluorescence intensity is not
strictly proportional to the concentration, the previously
drawn calibration curve under the same conditions should be
used.
For a substance which decomposes on exposure to light or its
relaxation time is too long, in order to avoid the fluorescence
intensity being affected by multi-irradiation of the exciting
radiation, the sensitivity of the instrument may be checked
with a stable reference solution of another fluorescent
substance with excitation and emission bands similar to those
of the substance being examined in place of ref erence
substance of the substance being examined. For instance,
quinine in dilute sulfuric acid is usually used for blue
fluorescence, sodium fluorescence for green fluorescence and
rhodamine B for red fluorescence.
Notes
The interferences of fluorimetry are great due to high
sensitivity.
( 1) The purity of solvent rnay rnarkedly affect the intensity
of fluorescence, blank test should be carried out and the
solvent distilled in a glass distillator before use if necessary.
(2) The presence of suspended particles may cause the light
to be scattered, therefore, it is necessary to eliminate such
particles by centrifugation or filtration with a sintered glass
filter.
( 3) All glasswares and cells must be cleaned thoroughly.
( 4) It is also important to regula te the temperature because it
would notably affect the fluorescence intensity.
(5) Oxygen dissolved in the solution, having a strong
quenching effect, can be removed by passing inert gas
through the solution if necessary.
(6) The effect on the fluorescence intensity by the pH value
of the solution and the purity of the reagents should be
noticed during the determination.
0406 Atomic Absorption
Spectrophotometry
Atomic absorption spectrophotometry is used in the deter-
mination of metal elements and sorne non-metal elements in
the atomic state.
The light of characteristic wavelength emitted from a
cathodic discharge lamp is absorbed when it passes through
the atomic vapor generated from sample containing the
element being examined atomized to the ground state. The
assay of the element being examined is tested by determing
the decreased degree of light intensity of radiation. Atomic
absorption spectrophotometry obeys the rule for absorption
spectrophotometry. The assay is carried out by comparing
the absorbance of the test preparation with that of the
reference preparation.
Apparatus
An atomic absorption spectrophotometer consists of a light
source, an atomic generator, a monochromator, a
background compensation system, automatic sampling
system, anda detector system etc.
l. Light source A hollow-cathode discharge lamp is
usually used. The cathode is made of the element being
examined.
2. Atomic generator There are four main types: flame
atomizer, graphite furnace atomizer, hydride-generated
atomizer and cold vapor atomizer.
(1) Flame atomizer It mainly consists of a nebulizer anda
burner. Its function is to nebulize the test solution into an
aerosol which is then mixed with combustion gas. And the
mixture is introduced into the flame generated by the burner.
So that the substance being examined is to be dried,
evaporated to form the ground state atoms of the element
being examined. The burning flame is generated by diff erent
mixtures of gases, acetylene-air is mostly used. By
modifying the proportion of combustion gas, the temperature
of the flame can be controlled, and a better stability and a
better sensitivity can be obtained.
(2) Furnace atomizer It consists of electric furnace and a
power supply. Its function is to dry and incinerate the subs-
tance being examined. During the stage of high temperature
atomization, the ground state atoms of the element being
examined are to be formed. Graphite is commonly used as
the heater. Protection gas is introduced into the furnace to
avoid oxidation and used to transfer the sample vapor.
(3) Hydride-generated atomizer It consists of hydride
generator and atomic absorption cell. It is used for the
determination of the elements such as arsenic, germanium,
lead, cadmium, selenium, stannum and antimony etc. Its
function is to reduce the element to be examined in acidic
medium to the low-boiling and easily pyrolyzed hydride. And
then the hydride is swept by a stream of carrier gas into the
atomic absorption cell which consists of quartz tube and
heater, etc. , in which the hydride is pyrolyzed by heating to
form the ground-state atom.
( 4) Cold vapor atomizer It consists of a mercury vapor
atomizer and an absorption cell. It is suitable for the
determination of mercury. Its function is to reduce the
mercuric ion in to free mercury, then the mercury vapor is
swept into the quartz absorption cell by carrier gas.
3. Monochromator Its function is to separate the specified
wavelength radiation from the electromagnetic radiations
emitted from the light source. The optical path of the
apparatus should assure the good spectra resolution and has
the ability to work well at the condition of narrow spectral
band (0. 2 nm). The commonly used wavelength region is
190. 0-900. O nm.
4. Background compensation system Background interfe-
rences often happen in the determination of absorption.
Background absorption may arise from the thermal emission,
optical absorption and light scattering of the co-existed
component of the sample, and the secondary-molecular or
atoms generated from atomizing. The interferences should be
avoided in the stage of the design of apparatus. Continuous
spectrum sources (a deuterium lamp is often used in the UV
region), the Zeeman effect, the self-inversion phenomena
and non absorption line can be utilized for background
compensation.
5. Detector system lt consists of a detector, a signal
processor and a recording system. It should have relatively
higher sensitivity and better stability, and can follow the
rapid change of the signal absorption.
In the analysis using atomic absorption spectrophotometry,
the interference to determination caused by background and
other reasons should be noticed. Changes of sorne
 0411 lnductively Coupled Plasma-Atomic Emission Spectrometry
experimental conditions, such as the wavelength, the slit
width, the atomizing condition, etc. , may affect the
sensitivity, the stability and the interference. In the analysis
with flame atomizer, the interference may be eliminated by
the selection of the suitable wavelength, slit width and flame
temperature, the addition of complex agents and releasing
agents, and the use of Standard addition method. In the
analysis with furnace atomizer, the interference may be
removed by the selection of suitable background compen-
sation system and the addition of suitable matrix modifying
agents, etc. Background compensation method should be
selected as specified in the individual monograph.
Procedure
Method 1 ( Direct calibration method) U nless otherwise
specified, prepare not fewer than 5 reference solutions of the
element being examined of different concentrations, covering
the range recommended by the instrument manufacturer and
add separately the corresponding reagents as that far the test
solution and prepare the blank reference solution with the
corresponding reagents. Measure the absorbances of the
blank reference solution and each reference solution of
different concentrations separately, record the readings and
prepare a calibration curve with the average value of 3
readings of each concentration on the ordinate and the
corresponding concentration on the abscissa.
Prepare a test solution of the substance being examined as
specified in the monograph, and adjust the concentration to
fall within the concentration range of the reference solution.
Measure the absorbance 3 times, record the readings and
calculate the average value. lnterpolate the mean value of the
readings on the calibration curve to determine the concentration
of the element. The standard calibration curve can be generally
drawn by linear regression, or by nonlinear regression.
Method 2 (Standard addition method) Place equal volumes
of the test preparation prepared as specified in the individual
monograph in each of 4 similar volumetric flasks, add to all but
the first one of the flasks an accurately measured amount of the
reference solution containing increasing amounts of the element
being determined. Dilute the contents of each flask to volume
with deionized water and proceed as described in the direct
calibration ( method 1). Measure the absorbances and record the
readings. Plot the mean val ues of each group of 3 readings
against the corresponding concentration of the element
contributed by the reference solution, and extrapolate the straight
line to intersect with the axis of zero absorbance. The
interception represents the concentration of the element
contributed by the test preparation ( as Fig. ) . Calculate the
concentration of the element in the test preparation from the
result so obtained.
When the test used far impurities, prepare two test prepa-
rations of the same concentration as specified in the
monograph. To one of the test preparation add an amount of
the reference substance equivalent to the limit of the element
specified in the monograph. Proceed as directed above and
measure this solution to give an appropriate reading a ; then
measure the test preparation without the addition of the
reference substance under the same condition and record the
reading b; b is not greater than (a-b).
0407 Flame Photometry
When a test solution containing the element of alkali metals
and alkaline-earth metals being examined is introduced into a
flame in the farm of an aerosol by the equipment of
nebulization, the element being examined is atomised by the
heat energy of the flame to excite its characteristic spectrum.
The content of the element being examined is determined by
measuring the light intensity of the element using
photoelectric detector and comparing the light intensity of the
reference solution and the test solution.
Apparatus Flame photometer consists of a combustion
system including nebulizer-burner, combustion lamp,
combustion gas and supply of assisted combustion gas, a
monochromator anda detector.
The combustion gas is often a mixture of air-coal gas or air-
liquefied petroleum gas ( LPG) using air as the assisted-
combustion gas and coal gas or LPG as combustion gas.
Any variation of the experimental condition, such as type and
state of flame, the pressure supplied by air-compressor may
affect and interfere with the sensitivity and steadiness of the
instrument, so they should be selected as specified in the
monograph.
Procedure When used far assay and limit testing of
impurities, flame photometry is carried out respectively as
method 1 and 2 described under Atomic Absorption
Spectrophotometry ( 0406 ) .
0411 lnductively Coupled Plasma-
Atomic Emission Spectrometry
This method is an atomic emission spectrometric analysis
technique employing plasma as the excitation source. It can
be used far simultaneous determination of multiple elements.
After introduced into the nebulization system by the carrier
gas (argon gas), the sample enters the center of plasma zone
in aerosol farm. There it is desolvated, atomized, and
excited in the high-temperature and inert atmosphere. All
elements involved emit their respective characteristic spectral
line. The existence of the characteristic spectral lines can be
used to identify the corresponding element in samples
( qualitative analysis). The intensity of the characteristic
spectral lines can be used to determine the quantity of the
corresponding element in samples (quantitative analysis).
This method is applicable far analysis of elements in various
pharmaceuticals from trace to macro quantities. It is
especially suitable far qualitative and quantitative determi-
nation of elements in mineral chinese medicine and nutrition
supplements.
l. General requirements for instruments
An inductively-coupled plasma atomic emission spectrometer
consists of sample-introduction systern, inductively-coupled
plasma (ICP) light source, dispersion system, and detection
system, etc. It is also equipped with computer control and
data processing system, cooling system, and gas control
system, etc.
Sample-introduction system Same as ( 0412) lnductively
coupled plasma-mass spectrometry.
Inductively coupled plasma exdtation source To ignite and
sustain the inductively coupled plasma source, constant and
stable ultra-pure argon gas flow, plasma torch, induction coil,
high frequency generator, and cooling system are required.
Sample aerosols are introduced to the high temperature plasina
source, undergoing desolvation, evaporation, dissociation,
atomization, excitation and subsequent light emission at
temperatures of 6000 to 10 000 K Emitted light can be collected
from either the axial or radial view of the ICP source, where
dual-view ICP source achieves both axial and radial view. The
selection of appropriate viewing model should depend on sample
matrix, elemental analyte, wavelength, sensitivity, etc.
 0412 Inductively Coupled Plasma-Mass Spectrometry
Dispersion System The Monochromator of an inductively
coupled plasma-atomic em1ss1on spectrometry usually
employs a grating or a combination of a prism and a grating.
Via the dispersion system, the light emitted from the plasma
is disposed into spectral lines according to wavelength,
thereby forming the spectrum.
Detection system The detection system used in inductively
coupled plasma-atomic em1ss1on spectrometers are
opticalelectrical converters. Based on the photoelectric
effect, the radiation energy of light of different wavelengths
is transferred to photoelectric signals. The photoelectric
converters commonly used include photomultipliers and solid-
state detectors. The latter is a kind of focal plane array
detector made from photosensitive elements. Its basic
material is semiconductor silicon wafer and it is composed of
multi array integrated circuit. Solid-state detectors are
further categorized into charge coupled devices ( CCD) and
charge injection devices (CID), both capable of simultaneous
multi-line determination, and providing rapid analysis, wide
response range, high sensitivity. The detection system shall
possess the following characteristics: stable performance,
rapid determination speed, wide dynamic range, good
sensitivity and resolution.
Cooling and gas control system Cooling system includes
ventilation system and circulating water system, whose
function is to discharge the heat inside the instrument. The
temperature of the circulating water and vent should be
controlled to fall in the range of instrument requirement.
Operation of the gas control system should be stable, and the
purity of argon gas should be no less than 99. 99 %.
2. Interference and calibration
lnterferences that exist in inductively coupled plasma-atomic
emission spectrometry can be divided into two main
categories: one is spectral interferences, including continuous
background and line overlap, etc; the other is non-spectral
interferences, including chemical interference, ionization
interference, physical interference, etc.
Methods of interference elimination and calibration include
blank calibration, dilution calibration, interna! standard
calibration, background subtraction calibration, interference
coefficient calibration, standard addition, etc.
3. Preparation of sample solution
Same as < 0412 ) lnductively coupled plasma-mass
spectrometry.
4. Procedures
The general principie far selecting analysis spectral lines is to
choose the line with lowest interference and highest
sensitivity; however the objective of analysis should also be
considered. Far analysis of trace elements, a sensitive line
should be used, while determination of high concentration
elements may require the use of a less sensitive line.
Qualitative analysis In atomic emission spectrometry, the
existense of characteristic spectral lines of an element
indicates the presence of that element in samples. In the
characteristic spectrum of the element, the spectral line with
highest intensity is called the sensitive line. In sample
spectrum, the detection limit of the sensitive line of a certain
element is the detection limit of that element.
Quantitative analysis Same as <0412) lnductively coupled
plasma-mass spectrometry.
Selection principies of an internal standard element and
reference line:
Selection of internal standard elements Elements selected
far use as an internal standard should not exist in analysis
samples, or be present in concentrations low enough to
neglect. If the matrix element in samples is stable in
concentration, it could also be used as an interna} standard.
lnternal standard elements should have similar characteristics
to the test elements, which means elements of same group
and similar ionization energy are a better choice.
Selection of a reference line A reference line should be
selected so that its excitation energy, wavelength and
intensity are clase to those of the analysis line; however,
analyzed elements should not interfere with the reference
line. The reference line should also exhibit no self-absorption
and have minimal background.
5. lirnit of detection and quantitation
Same as < 0412 ) lnductively coupled plasma-mass
spectrometry.
0412 lnductively Coupled Plasma-
Mass Spectrometry
This method is a mass spectrometric elemental analysis
technique employing an argon plasma as the ionization
source. It is mainly used far simultaneous determination of
multiple elements, and it can also be coupled with
chromatographic separation techniques to analyze elemental
species.
The sample is introduced into the plasma vía a nebulization
system Cin the case of liquid samples) and transported to the
plasma by a carrier gas Cargan gas). The sample is converted
to fine droplets (aerosol) which are carried to the central
zone of the plasma. In the high temperature of the plasma,
the aerosol is desolvated, evaporated, and ionized, to farm
positively charged ions, then passed to the mass spectro-
meter vía a vacuum interface and ion focusing system. The
mass spectrometer separates the ions according to mass-to-
charge ratio, and the quantity of corresponding elements in
the sample can be determined by referring to the intensity of
the elemental mass peaks.
This method possesses high sensitivity, and is applicable far
the analysis of elements in pharmaceuticals from trace to
minute quantity. It is especially suitable far determination of
trace heavy metal elements.
l. General requirements for instrument
An inductively coupled plasma-mass spectrometer consists of
sample introduction system, inductively coupled plasma
CICP) ion source, interface, ion lens, quadrupole mass
analyzer, detector, etc. Other supporting systems include
vacuum system, cooling system, gas control system,
computer control, data processing system, etc.
Sample-introduction system According to the state of the
sample, a sample-introduction system suitable far liquid, gas
or salid samples may be used, among which liquid sampling
is the most commonly employed. The liquid sample
introduction system consists of two main components:
sample introduction and sample nebulization into a spray
chamber. The component far sample introduction is usually a
peristaltic pump to deliver the sample to the nebulizer, or the
sample may be delivered using a self-aspiration nebulizer. It
is required that the peristaltic pump has a stable rotation
speed, and the pump pipe is flexible and in good condition,
so that sample solution can be pumped at a steady rate, with
the waste also being pumped smoothly from the spray
chamber drain. Components far sample nebulization include
the nebulizer and a spray chamber. The sample solution
 0412 lnductively Coupled Plasma-Mass Spectrometry
enters the nebulizer by pumping or self-aspiration, where the
sample is nebulized by the carrier gas, to form aerosol
droplets, which then enter the spray chamber. The spray
chamber removes the bigger droplets from the aerosol cloud,
as larger droplets are excluded after encountering the wall of
the spray chamber. Only droplets small enough to be fully
decomposed in the plasma can pass through the spray
chamber and enter the plasma ion source. The nebulizer
must have high nebulization efficiency, good stability, low
memory effects, and be resistant to typical chemicals used in
sample stabilization. The spray chamber must be maintained
under constant temperature, and should also be efficiently
rinsed between samples. Several types of solution nebulizer
are commonly used, including concentric nebulizers, cross
flow nebulizer, etc. The commonly employed spray chamber
types include double-pass type and cyclonic type. In practica!
applications, the most suitable type of nebulizer and spray
chamber should be selected based on factors such as sample
matrix, elements of interest, and required sensitivityl
detection limits.
Inductively coupled plasma ion source The inductively
coupled plasma ion source is a high temperature ( 6000 to
10 000 K) electrical discharge sustained by inductive coupling
of a radio frequency ( RF) current in a flow of ultra-pure
argon (purity should be no less than 99. 99%). Components
include plasma torch, induction coil, radio-frequency
generator, and cooling system. Sample aerosol is introduced
into the central channel of the plasma ion source, where the
aerosol rapidly undergoes desolvation, evaporation, dissocia-
tion, atomization, and ionization due to the high plasma
temperature. Most elements are finally transformed with
almost 100 % efficiency in to positively charged ions. The
specific settings of operating parameters such as RF power,
carrier gas flow rate, torch axis position, and peristaltic
pump flow rate are typically optimized for high sensitivity
and low interferences, based on the specific requirements of
the sample analysis.
Interface system The function of the interface system is to
efficiently transmit sample ions from the plasma into the
mass spectrometer. The crucial parts of the interface include
the sampling cone and skimmer cone; these components
should be cleaned regularly as part of the routine mainte-
nance. Attention should also be paid to avoid damage to the
eones' orifices, in order to maintain high performance.
Ion lens system The ion lens is an electrostatic lens
positioned in the intermediate vacuum zone behind the
skimmer cone. Its function is to efficiently focus the ions into
the mass filter, and prevent neutral atoms and photons from
entering. Ion lens voltages should be set appropriately so that
high sensitivity can be achieved for low, medium and high
mass 10ns.
Quadrupole mass analyzer The mass analyzer used in ICP-
MS is generally a scanning quadrupole analyzer, which
separates elemental ions based on their characteristic mass to
charge (miz). Mass selection is achieved based on the
specific electric fields generated in the space between the four
electrodes, due to the voltages applied to the opposing pairs
of rods. Only ions with certain miz can maintain a stable
path along the axis of the electrodes, pass through the
quadrupole, and exit the mass filter to be passed to the
detector. lons with any other miz would be deflected away
from a stable central path, collide with the electrodes, get
neutralized then lost. Mass selection is thereby accomp-
lished. During instrument Startup prior to analysis, the
operator should set appropriate parameters for quadrupole
mass analyzer, optimize mass resolution, response and
calibrate the mass axis.
Detector A discrete dynode, dual mode electron multiplier
is the most commonly used detector. The ions that emerge
from the quadrupole mass analyzer are focused into the
detector. The detector registers each ion impact as an electric
pulse, which is counted by the integration circuit. A dual-
mode detector uses a combination of pulse counting and
analog mode to simultaneously determine elements of either
low or high concentration in one measurement of the sample.
When determining low intensity signals from trace elements,
the detector employs pulse mode, directly recording the
individual ions that strike the detector. When the ion count is
high, the detector automatically switches to analog mode.
This mode allows high signals to be measured without
damaging the detector, extending the measurement range of
the instrument, and prolonging detector lifetime. Approp-
riate detector parameters should be set to optimize
sensitivity, and a cross-calibration should be carried out to
ensure linear response across dual-mode detection signals
(pulse and analog).
Other supporting systems The vacuum system consists of a
mechanical rotary vane pump anda turbo molecular pump, in
order to keep the multistage vacuum required by the
instrument to maintain operation of mass analyzer. The
vacuum pressure in each chamber should meet the required
value for the instrument. The cooling system includes an
exhaust system, and a circulating water system, whose
function is to remove the heat from components inside the
instrument. The temperature of circulating water and inletl
exhaust vent should be controlled to fall in the range of
instrument requirement. Operation of gas control system should
be stable, and the purity of argon gas should be no less than
99. 99%.
2. Interference and calibration
lnterference during inductively coupled plasma-mass spectro-
metry falls to two main categories: spectral interferences and
non-spectral interferences. Mass spectral interferences
including isobaric overlaps, polyatomic ions, double-charged
ions, etc. ; non-spectral interferences include physical
interference, signal suppression, matrix effect, memory
effect, etc.
Methods of interference elimination and calibration include
optimizing instrument parameters, interna! standard
calibration, interference equation calibration, collisionl
reaction cell technique, isotope dilution calibration, standard
addition, etc.
3. Preparation of sample solution
Reagents usually used for sample digestion are mineral acids,
including nitric acid, hydrochloric acid, perchloric acid,
sulfuric acid, hydrofluoric acid, and mixed acid [such as the
mixture of nitric acid and hydrochloric acid ( 4 : 1) J.
Hydrogen peroxide can also be used effectively for sorne
sample types. Nitric acid is the preference acid for sample
preparation due to its minimum interference. All reagents
should be guaranteed reagent or higher level, and deionized
water should be employed ( resistivity should be no less than
18M!l).
During preparation of sample solution, blank solution should
also be prepared. The matrix and acidity of standard solution
should be matched to the sample solution.
Solid sample Unless otherwise specified, accurately weigh
an appropriate amount (O. 1 to 3 g) of sample, and select
appropriate digestion method according to laboratory
conditions and sample matrix. Digestion methods include
 0412 Inductively Coupled Plasma-Mass Spectrometry
open-vessel digestion, closed-vessel digestion and microwave
digestion. Microwave digestion uses fewer reagents and
provides faster digestion with higher efficiency. It is
recommended as it decreases the reagent blank value,
reduces contamination, prevents the evaporation loss of test
elements and benefits for environmental protection during
sample preparation. After sample digestion, dilute the
solution to appropriate volume according to the levels of test
elements, and the matrix level, and the sample is then ready
for mass spectrometry determination.
Liquid sample On the basis of sample matrix, organics
content and test elements content, different determination
methods could be selected such as direct analysis, analysis
after dilution or pre-concentration, analysis after solubiliza-
tion in an appropriate solvent, digestion, etc.
4. Procedures
For each elements to be measured, selection of the preferred
isotope is usually simply based on the most abundant isotope
that is free from direct isobaric overlap. It is also necessary
to consider the potential for interferences that might occur in
the sample matrix. Isotopes with least interference and
highest abundance should be selected for determination.
lnterference equations may be employed during calibration
for sorne isotopes. Under circumstances where the interfe-
rence is not clear, multiple isotopes of an element could be
selected and compared for determination. Commonly used
methods for test element calibration and quantitation
include:
( 1) Standard curve method Under the analysis conditions
selected for the samples, measure a series of solutions
containing different known concentrations of the elements
(matrix and acidity of the standard solution should match the
sample). Draw a standard curve with response value as Y-
axis and concentration as X-axis to calculate regression
equations. The calibration should be linear with a correlation
coefficient of no less than O. 99.
Measure a blank solution (e. g. digest blank) under the same
analysis condition, and subtract the blank interference
according to instrument manual, to correct for any
contamination form the digest vessels or reagents.
lnternal standard corrected calibration standard curve
method
Prepare a solution of internal standard ( ISTD) elements and
add a spike at the same concentration in every sample
( including standard solution, sample solution and blank
solution). Draw a standard curve for each test element with
the ratio of response value of test elements in standard
solutions to that of internal standard elements as Y-axis and
concentration as X -axis. Calcula te regression equations.
Obtain the ratio of response value of analysis peak in sample
solution to that of internal standard elements reference peak,
subtract blank, determine the corresponding concentrations
from standard curve or regression equation, and calculate the
content of test elements in the sample. The use of ISTD
correction is effective to correct the fluctuation of signals,
and the ISTD calibrated standard curve method is the most
commonly used determination method.
The following factors should be considered when selecting
the ISTD. the test sample should not contain this element;
its mass number and ionization energy is close to test
elements; chemical characteristics of the element. ISTD
could be added respectively i11to every sample and standard
solutions, or through peristaltic pump online.
( 2) Standard addition method Transfer a same volume of
the test solution into one of four volumetric flasks with same
volume respectively. Except for the first volumetric flask,
add precise elemental analyte standard solution at different
concentrations to the other three volumetric flasks. Dilute
them to scale respectively, shake well, and use the series
solutions for analysis. Perform sample analysis and
determination of the test elements under the selected analysis
condition. Draw a standard curve for each test element with
the measured elemental signal as the Y-axis, and the added
concentration of the elements to be measured as the X -axis.
Calculate regression equations and extrapolate the calibration
line until it intercepts the X -axis to the left of the origin.
The distance between the intercept and origin is the
concentration of the element determined in the unspiked
prepared samples. Then, the concentration of each test
element can be calculated.
S. Lirnit of detection and quantitation
Determine 7 or more blank sample solutions under the
optimum experimental conditions. Calculate the concentra-
tion of 3 times the standard deviations (3SD) of the response
values of the blank sample solutions to give the limit of
detection. Calculate the corresponding concentration of 10
times the standard deviation (lOSD) of the response values of
the blank sample solutions to give the limit of quantitation.
6. High perfonnance liquid chromatography /Inductively
coupled plasma-mass spectrometry ( HPLC/ICP-MS)
The method is used for the online detection of elemental
species or oxidation states, by separation of the different
chemical forms with high performance liquid chromatography
(HPLC), followed by detection with inductively coupled
plasma-mass spectrometry (ICP-MS). HPLC-ICP-MS can be
used for elemental speciation analysis of arsenic, mercury,
selenium, antimony, lead, tin, chromium, bromine, iodine
and other elements.
To carry out elemental sample analysis, samples are
separated by HPLC, and introduced with the mobile phase
into the ICP-MS for detection. The qualitative determination
of each separated elemental speciation is based on the elution
order, chromatographic retention time, and signal for each
separated peak measured by ICP-MS, The quantitative
determination of the separated elemental speciation is based
on the chromatographic peak area or peak height.
( 1 ) General requirements for instrument
Besides the inductively coupled plasma-mass spectrometer
system described previously, the HPLC-ICP-MS system also
includes the high performance liquid chromatography
( HPLC) system, the interface system and the data
processing system. The HPLC system should be connected
to the inductively coupled plasma-mass spectrometry via an
appropriate interface. The operating software for the
combined HPLC-ICP-MS system should have the function to
setup configuration, method settings, run controls, injection
parameters, and data analysis.
High performance liquid chromatographic system The
system includes high pressure chromatography pump
system, degasser, autosampler, m1ection system, and
appropriate separation columns. Temperature control for
samples and column, and UV detector are optional as
required by the method. All parts should be certificated
periodically to meet related regulations.
High performance liquid chromatography used for elemental
speciation analysis could be categorized according to the
separation principle, including ion-exchange chromato-
graphy, reversed phase ion pair chromatography, partition
chromatography, exclusion chromatography and chiral liquid
chromatography, among others. Choose an appropriate
 0412 lnductively Coupled Plasma-Mass Spectrometry
column and mobile phase to separate the target elemental
species according to their properties.
Ion exchange chromatographic columns and reverse phase
chromatography columns are commonly used, with
methanol, acetonitrile, water and inorganic salt buffer
solution as mobile phase. Binary and quatemary gradient
pump are commonly used to mix organic modifier with water
as mobile phase. In terms of high ionization energy elements
esuch as arsenic, selenium, bromine, iodine, mercury,
etc. ) , the degree of elemental ionization in the plasma
environment would be improved significantly, and the
sensitivity would be enhanced, if a certain amount of carbon
exists in the center channel of plasma. Therefore, it is
suggested to add certain proportion of organic modifier to the
mobile phase. The proportion depends on the elements to be
measured and the carbon chain length of the organic
modifier. When measuring mobile phases with a high propor-
tion of organic modifier (such as over 20% methanol or 10%
acetonitrile), the ICP-MS should be equipped with a special
organic solvent sampling system, including solvent resistant
tubing, additional mass flow controller to add an oxygen/
argon gas mix to the plasma, platinum-tipped interface
eones, organic-type plasma torch ewith an injector tube with
the inside diameter of l. 5 mm or l. O mm) and organic
resistant drain system for the spray chamber waste.
The working conditions of the inductively coupled plasma-
mass spectrometer must be appropriate for the composition
of the mobile phase used for the chosen high performance
liquid chromatography method. The working conditions
should be optimized according to the practica! situation. To
achieve the required chromatographic separation, the flow
rate of the mobile phase will usually be set in the range from
O. 1 ml to 1 ml per minute. It is recommended to apply post-
chromatographic split in the sample transfer line if the flow
rate is too high (over l. 5 ml per minute). It may be required
to add a make-up liquid to the sample solution channel, or
use a special low-flow nebulizer to ensure that sample can be
nebulized efficiently' if the flow rate is very low eless than
O. 1 ml per minute).
Interface system PTFE sample uptake tu be Cwith the
inside diameter of O. 12 mm to O. 18 mm) is usually used to
transfer the sample solution from the HPLC column exit into
the nebulizer of the ICP-MS. The connecting tubes should be
as short as possible to avoid broadening of the chromato-
graphic peaks. The connection between the sample uptake
tube and the nebulizer should have a suitable low dead
volume fitting.
The self-aspiration nebulizer (for example the Micromist and
the PFA concentric nebulizer) was applied to interface
system due to its high efficiency and low dead volume. The
nebulizer tube directly connects to the HPLC column exit or
chromatographic detector exit in the case chromatographic
detector is used.
Many HPLC mobile phase consists of high salt compounds
and organic solvents, and use columns of smaller particle size
is the trend of interface system. The main hyphenated
technique interface systems including ultrasonic nebulizer,
hydride generation, directly injection, micro-concentric
nebulizer, thermo spray nebulizer, electrothermal vapori-
zation and hydraulic pressure nebulizer.
Inductively coupled plasma-mass spectrometry system All
conditions of the inductively coupled plasma-mass spectro-
metry system should be optimized before analysis, to ensure
sensitivity and prec1s10n when combined with high
performance liquid chromatography.
When the mobile phase contains a high proportion of
inorganic salt or organic solvent, the inorganic salt matrix or
carbon could be deposited on the sampling cone and skimmer
cone, which might clog the cone orifice, leading to baseline
drift and reduced sensitivity of the instrument; In addition,
inorganic salt or organic solvent in the mobile phase can
overload to ICP; if the ICP uses too much energy to break
clown the mobile phase matrix, there will be insufficient
energy to fully ionize the target element atoms, leading to
lower test element sensitivity, known as suppression. Ideally
the optimization should be carried out while aspirating a
solution that matches the composition of the HPLC mobile
phase. If acceptable separation and sensitivity can be
achieved using a different mobile phase chemistry, then it
may be better to adjust the mobile phase to suit the ICP-MS
conditions, rather than the other way round.
When the organic mobile phase cannot be avoided, and the
quantity exceeds a certain proportion, it is necessary to use
an organic solvent setup for the ICP-MS. This includes
setting appropriate analysis parameters, adding an additional
flow of oxygen to the carrier gas, and fitting platinum-tipped
eones. Due to the safety concem, it is recommended that a
combination gas of oxygen in argon e with a certain
proportion such as 1 : 4 or 1 : 1) is used for the oxygen
make-up gas, instead of pure oxygen.
When gradient elution is required, the mobile phase changes
composition through the HPLC run, which leads to variation
in the matrix effect on the inductively coupled plasma. Often
it is possible to optimize the tuning conditions separately for
each change in the gradient mobile phase composition, to
ensure the optimal sensitivity and matrix tolerance throug-
hout the run. An alternative approach is to compensate for
the changing mobile phase composition by adding a post-
chromatographic make up flow, which follows the reverse
composition change of the HPLC mobile phase gradient.
For the transient signai anaiysis used in chromatography
measurements, the ICP-MS acquisition parameters should be
set so that each analyte mass is acquired using one point per
mass. The integration time should give consideration to both
signal intensity and the number of points that will be
measured across the chromatographic peaks ( the number of
measured points across a chromatographic peak is in
proportion to the width of chromatographic peak, and in
inverse proportion to the integration time). The number of
points measured across the chromatographic peaks should be
more than 15 points per peak.
Data processing system It should be easy to operate, able
to tune the instrument to the optimal conditions and maintain
stable operation and have the function of observing chroma-
tographic peaks and mass spectrum peaks simultaneously.
In conventional spectrum measurement of a continuous
steady-state signal, inductively coupled plasma-mass spectro-
metry just integrates (sums) the total intensity count of the
elements collected during the measurement. However, high
performance liquid chromatography requires to record the
retention time and integrated peak area. In order to put the
two together, when HPLC is combined with ICP-MS, the
data processing system should have the function of
synchronization control (instrument and run control for both
the HP LC and ICP-MS), real time peak display and
chromatographic separation during acquisition. In the
meantime, the data processing system should also be
compatible with the analysis of other chromatographic
detector signals Csuch as ultraviolet) and ICP-MS signals
synchronously, such as spectrogram overlap, integration and
working curve etc. in order to effectively process qualitative
and quantitative analysis.
 0412 Inductively Coupled Plasma-Mass Spectrometry
( 2) System suitability
System suitability tests are mainly used to confirm that the
analytical system and parameters are generally applicable.
The testing items and methods used for HPLC-ICP-MS are
the same as those used for HPLC. The parameters could be
set based on the parameters used for HPLC, such as
repeatability, capacity factor, tailing factor, linear range, limit
of detection and quantitation, etc. Detailed list of test parameters
and performance requirements should in accordance with the
monograph. Due to the characteristics of ICP-MS, the
repeatability RSD could be broadened to 10. 0%.
( 3) Interference and calibration
Full consideration should be given to the possibility of
interference from mobile phase and the process of sample
pretreatment in the test. Necessary methods should be
adopted to eliminate such interferences. If possible, it is
recommended to avoid the use of correction equations to
mathematically correct for the contribution from interfe-
rences, as such equations require the measurement of
additional masses besides the isotopes of the test elements to
be measured; this extends the total data acquisition time for
each measurement, so reduces the number of measurements
across each chromatographic peak. Where interferences do
occur, they can often be avoided by optimizing the chro-
matographic conditions ( such as pH, type or concentration
of mobile phase, etc. ) to separate the retention time of the
interfering ion and the test element ion.
An alternative approach when the interfering ion cannot be
separated by optimizing the chromatographic conditions is to
use collision/ reaction technique. When the interference from
mobile phase increases the base line of the instrument and
affects the detection limit, it is suggested to investigate an
alternative mobile phase chemistry.
If the mobile phase contains a high level of salts, the ICP-MS
signal can drif t during a long sequence of measurements. It is
suggested that the sensitivity is monitored with quality
control samples or a periodic recalibration solution (reference
substance) , or interna! standard correction can be used to
compensate for gradual signal changes over time.
( 4) Sample pretreatment
When target elemental compounds are present at very low
levels, or the sample matrix is complex, sample pretreat-
ment such as separation and concentration is necessary. In
order to maintain a representative concentration and
composition of the different compounds in the original
sample, the pretreatment method should separate or extract
the elemental species from the sample matrix, but not lead to
degradation, loss or transformation of the individual species.
Reagent to be used should be guaranteed reagent or higher
level. Vessels to be used should be soaked in 10%-20%
nitric acid solutions over night, then washed with deionized
water and dried out. Reagent blank should be prepared at the
same time, using the same procedure. The solution
(solvent) of the standards or reference substance solutions
should be the same as the sample solutions, and have no
obvious solvent effects.
Besides routine pretreatment methods ( extraction, ion
exchange, ultrafiltration, centrifugation and co-precipitation,
etc. ) , elemental speciation analysis usually adopts separation
methods such as enzyme hydrolysis, ultrasonic-assisted
extraction, microwave-assisted extraction, solid phase
extraction, and accelerated solvent extraction.
(5 ) Procedures
The isotopes of the target elements should be selected so as
to avoid possible interference from mobile phase and sample
matrix, as far as possible. If spectral interferences cannot be
avoided, choose HPLC conditions that ensure the retention
time of the interference ion is separated chromatographically
(elutes at a different time) from the test element species to
be measured. An alterna ti ve approach when the interfering
ion cannot be separated by opt1m1zmg the HPLC
chromatographic conditions is to select an alternative isotope
for the test element, where the contribution of the
interference is less. Finally, as for normal ICP-MS analysis,
interferences can be corrected using inter-element equations,
although these add extra time to the measurement, so it is
important to confirm that at least 15 measurements are
acquired across each chromatographic peak. Quantitative
analysis in elemental speciation analysis usually adopts a
standard curve method, and the two most common
approaches are externa! calibration method and interna!
calibration method. Less frequently, standard addition
calibration may be adopted.
External calibration method Measure standard solutions
that contain the different elemental species to be measured.
Preferably at least four different concentration levels should
be prepared for each compound. The standard solutions
should be prepared in the same solvent as the sample
solutions. Measure the standards using the selected analysis
conditions, then draw a standard curve with chromatographic
peak areas (or heights) as the ordinate and the corresponding
concentrations as the abscissa to calculate regression
equations. The correlation coefficient should be no less than
O. 99. Measure the sample solutions and find out the
corresponding concentrations for each test compound from
the standard curve or regression equation, then calculate the
content of elemental species measured in the sample.
Perform blank experiment under same analysis condition,
and subtract the blank contribution.
Internal standard correction Whichever calibration approach is
selected, interna! standards can be used to correct for
fluctuations of signal response, and reduce or eliminate any
difference in matrix effect between samples and standard
solutions. Interna! standard correction in elemental speciation
analysis can be divided into the following 3 methods
according to the method of adding the interna! standards and
correcting the measured signals.
A. Sample addition method Add the interna! standard
compounds into the samples or sample solutions. The
interna! standard compounds should contain the elements to
be measured, but in a different speciation from the target
compounds. The method requires that the interna! standard
compounds are stable, absent from the samples, and the
interna! standard species must be able to be chromato-
graphically separated from the target species found in the
samples. Moreover, if added to the sample prior to any
extraction steps, the extraction efficiency should be identical.
B. Online real time interna) standard calibration T wo
methods of interna! standard addition can be adopted, one is
to add interna! standard elements in to the mobile phase, and
the other is to add the interna! standard solution online into
the eluent stream, using a separate peristaltic pump. Interna!
standard correction using online real time correction collects
interna! standard signals corresponding to every data
collection point. Point-to-point calibration is used, where the
chromatographic peaks are based on the measured counts for
the target elements measured at each point on the
chromatogram, corrected by the interna! standard counts
measured at that data collection point. The data processing
software of the HPLC-ICP-MS system should possess

corresponding functions to allow this point-to-point
correction to be applied.
Online real time internal standard calibration can prevent the
problem of inaccuracy due to signal drif t. It should be noticed
that selected internal standard substances should possess
similar mass number and ionization energy to the elements to
be measured. The selected elements should also not be
present in the sample.
C. Valve switch method The valve-switch method injects a
separate internal standard solution after every sample, using
a switching valve to add the solution containing the internal
standard compound to the eluent prior to the nebulizer. This
approach may be useful when it is hard to find a suitable
internal standard compound to allow interna} standard
correction of the compounds of interest in every sample.
Measure the standards using the selected analysis conditions,
then draw a standard curve with the ratio of chromatographic
peak areas (or heights) of test elements in standard solutions
to that of internal standard elements or the ratio of
chromatographic peak areas ( or heights) after point-to-point
correction as the ordinate and the corresponding concen-
trations as the abscissa to calculate regression equations. The
correlation coefficient should be no less than O. 99. Measure
the sample solutions and obtain the ratio of chromatographic
peak areas Cor heights) of test elements in sample solutions
to that of internal standard elements or the ratio of
chromatographic peak areas (or heights) after point-to-point
correction and find out the corresponding concentration for
each test element from the standard curve or regression
equation, then calculate the content of elemental species
measured in the sample. Perform blank experiment under
same analysis condition, and subtract the blank contribution.
Standard addition method Standard addition method can
effectively eliminate matrix effects, as the calibration is
constructed from calibration solution additions spiked into the
sample matrix itself. The results are more accurate and
reliable dueto the fact that the matrix of the spiked samples
are almost identical . The concentration of the elemental
species added to the sample should be similar to or slightly
more than the expected quantity in the samples, in arder to
give a signal increase that can be easily measured. Therefore,
it is very beneficial to know the approximate concentrations
in the samples in arder to select appropriate concentrations
for the additions. Select three or more concentrations to draw
standard curve. The response of the elements to be measured
should be linear within the concentration range of the
additions.
0421 Raman Spectrometry
Raman spectrometry is one of the important analytical
methods for qualitation and quantitation, which deals with
the radiations scattered by molecule exposed to mono-
chromatic light, and the relationship to the energy difference
between the incident light and the scattered radiation, and to
the molecule vibrational and rotational frequency.
Similar to Infrared spectrophotometry, Raman is a vibra-
tional spectroscopic technique. The difference is that Infrared
absorption relates to the dipole moment change in the
molecules vibration, while the Raman effect itself arises as a
result of a change in the polarizability of a molecular bond.
A Raman spectrum is generated by exciting the sample of
interest to a virtual state with a monochromatic source,
typically a laser. If the sample relaxes to a vibrational energy
level differing from the initial state, the scattered radiation is
0421 Raman Spectrometry
shifted in energy. This "inelastically scattered" light is
referred to as Raman scatter. Light elastically scattered (no
change in wavelength) is known as Rayleigh scatter. If the
Raman scattered photon is of lower energy, it is referred to
as Stokes scattering. If it is of higher energy, it is referred to
as anti-Stokes scattering. In practice, nearly all analytically
useful Raman measurements make use of Stokes-shifted
Raman scatter.
The intensities, or the number of Raman photons counted,
are plotted against the shifted energies. The spectrum is
interpreted in the same manner as the commensurate
absorption IR spectrum. The positions of the shift frequen-
cies for a given bond in an analyte are similar to their
respective absorption frequencies in an IR spectrum.
However, the peaks emphasized in a Raman spectrum are
often de-emphasized in an IR spectrum and vice versa.
Therefore, the two spectroscopic techniques are often used to
be complementary.
Raman spectroscopy is advantageous because quick and
accurate measurements can often be made without destroying
the sample (salid, semi-salid, liquid, or gas) and with
minimal orno sample preparation. The signal is typically in
the visible or NIR range, allowing efficient coupling to fiber
optics. This also means that a signal can be obtained from
any medium transparent to the laser, such as glass, plastics,
or samples in aqueous media. Modern instrumental systems
are easier to use, rapider ( seconds to several minutes) and
more reliable. Therefore, the analysis modeling may be
simpler than that associated with other spectroscopic
techniques (both univariate and multivariate methods can be
used).
In addition to normal Raman spectroscopy, there are a
number of more specialized Raman techniques. These include
resonance Raman ( RR ) , surface-enhanced Raman
spectroscopy ( SERS), Raman ontir;:il artivitv (ROA).
coherent anti-Stokes Raman spect;~~c~~y -(-CARS) ~ - Ram~~
gain or loss spectroscopy, and hyper-Raman spectroscopy.
Resonance Raman and surface-enhanced Raman may have
found rather more wide applications in pharmaceutical
analysis than other techniques.
Qualitative and Quanitative
l. Qualitative Raman measurements
Raman spectrum can give accurate spectral information about
the vibrational bands present in the sample. The contrast of
the spectrum of the test sample with the standard sample
collected in the same measurement conditions should be
carried out, if the two spectra matche in configurations and
intensities, the two samples may be identical except they are
stereoisomers. In this case, the sample should analyzed by
the method specified in lnfrared spectrophotometry.
2. Quantitative Raman measurements
Quantitative Raman measurements follow a relationship
comparable to Beer' s law:
Iv=KLCI 0
in which Iv is the peak intensity ata given wave number, K
represents instrument and sample constants, L is the path
length, C is the molar concentration of a particular
component in the sample, and Io is the laser intensity.
In practice, path length is more accurately described as sampling
volume, which is an instrumental variable described by the focus
of the laser and the collection optics. The relationship is the basis
far the majority of quantitative Raman applications.
3. Factors affecting quantification
The most important sample-based factors are fluorescence,
sample heating, and matrix absorption. Fluorescence is
 0421 Raman Spectrometry
typically observed as a broad sloping background underlying
the Raman spectrum. The effect on quantification is
therefore that of an unstable baseline and decreased signal-to-
noise ratio. The exact wavelength and intensity are
dependent on the identity and concentration of the fluorescing
material. Because fluorescence is generally a much more
efficient process, even very minor amounts of fluorescent
impurities can lead to significant Raman signal degradation.
Fluorescence can be minimized by using longer wavelength
excitation sources such as 785 nm or 1064 nm. However,
the intensity of the Raman signal is proportional to A. - 4 ,
where A. is the excitation wavelength. The optimum signal-to-
noise ratio will be obtained by balancing fluorescence
rejection, signal strength, and detector response.
Fluorescence in solids can also be mitigated by exposing the
sample to the laser source for a period of time before
measurement. This process is called photobleaching, and
operated by degrading the highly absorbing species.
Photobleaching is less effective in liquids, where the sample
is mobile, or if the amount of fluorescent material is more
than a trace.
Sample heating can cause a variety of issues, such as physical
form change ( melting), polymorph conversion, or sample
burning. This is usually an issue for coloured, highly
absorbing species, or very small particles that have low heat
transfer. The effects of sample heating are usually observable
either as changes in the Raman spectrum over time or by
visual inspection of the sample. Besides decreasing the laser
flux, a variety of methods can be employed to diminish
heating, such as moving the sample or laser during the
measurement or improving the heat transfer of the sample
with thermal contact or liquid immersion.
Absorption of the Raman signal by the matrix or the sample
itself can also occur. In long wavelength FT-Raman
systems, Raman signal can overlap with a NIR overtone
absorption. This effect will be dependent on the optics of the
system as well as on the sample presentation. Associated
with this effect is variability from scattering in solids as a
result of packing and particle size differences. The magnitude
of all of these effects, however, is typically less severe than
in NIR because of the limited depth of penetration and the
relatively narrower wavelength region sampled in Raman
spectroscopy.
Quantitative Raman spectroscopy differs from many other
spectroscopic techniques in that it is a single beam
Table 1 Typical lasers used in pharmaceutical applications
Laser A., nm
Typical Power
( nearest whole Type
at Laser
number)<D
NIR Lasers
1064 Solid state CNd: YAG) Up to 3 W
Up to
785 Diode
500 MW
UV-Visible Lasers
Ion gas and solid state
488-632. 8 Up to 1 W
frequency doubled lasers
UV-Visible dye laser Tuna ble
Note CDThe wavelength of lasers equipped in different instruments may differ slightly from that listed in the table.
lasers can reduce fluorescence risks somewhat likely.
measurement with no background. Careful instrument design
and sampling can minimize this variation but not entirely
remove it. Thus the absolute Raman signal intensity is very
difficult to use for direct quantification of an analyte. Among
the potential sources of variation are changes in sample
opacity, sample heterogeneity, changes in laser power at the
sample, and changes in optical collection geometry or sample
position. These effects can be minimized by sampling in a
reproducible, representative manner.
An internal reference is most commonly used for eliminating
variations due to absolute intensity fluctuations. There are
several choices for this approach. An internal standard can be
deliberately added, and isolated peaks can be employed. In a
solution, an isolated solvent band can be employed because
the solvent will remain relatively unchanged from sample to
sample. Also, in a formulation, an excipient peak can be
used if it is in substantial excess compared to the analyte.
The entire spectrum can also be used as a reference, with the
assumption that laser and sample orientation changes will
affect the entire spectrum equally.
Another important sampling-based factor to consider is
spectral contamination. Raman scatter is a weak effect that
can be masked by a number of external sources. Common
contamination sources include sample holder artifacts
(container or substrate) and ambient light. Typically, these
issues can be identified and resolved by careful
experimenta tion.
Apparatus
According to the manner of getting spectrum, Raman
instruments can be divided into FT-Raman spectrometer and
dispersive Raman spectrometer. However, all modern
Raman measurements involve irradiating a sample with a
laser, collecting the scattered radiation, rejecting the
Rayleigh scattered light, differentiating the Raman photons
by wavelength, and detecting the resulting Raman spectrum.
All commercial Raman instruments therefore share the
following common features to perform these functions:
1 Excitation source ( laser)
Table 1 identifies several common lasers used for
pharmaceutical applications. UV lasers have also been used
for specialized applications but have various drawbacks that
severely limit their utility for general analytical measu-
rements.
Wavelength Range, nm
(Stokes Region, 100 cm- 1 to Comments
3000 cm- 1 shift)
Commonly used in Fourier
1075-1563
transform instruments
Most ubiquitous dispersive
791-1027
Raman laser
488-781 Fluorescence risks
Tunable in the region Fluorescence risks@
® UV

2 Sampling device
A wide variety of sampling arrangements are possible,
including direct optical interfaces, microscopes, fiber optic-
based pro bes ( either noncontact or immersion optics), and
sample chambers ( including special sample holders and
automated sample changers). The sampling optics may also
be designed to obtain the polarization-dependent Raman
spectrum, which often contains additional infarmation.
Selection of the sampling device will often be dictated by the
analyte. However, considerations such as sampling volume,
speed of the measurement, laser safety, and reproducibility
of sample presentation should be evaluated to optimize the
sampling device far any given application.
3 Filtering device
Scattered light at the laser wavelength ( Rayleigh) is many
orders of magnitude greater than the Raman signal and must
be rejected prior to the detector. Notch filters are almost
universally used far this purpose and provide excellent
rejection and stability combined with small size. In addition,
various filters or physical barriers to shield the sample from
external radiation sources (e. g. , room lights, laser plasma
lines) may be required depending on the collection geometry
of the instrument.
4 Wavelength processing unit
The wavelength may be processed by either dispersion or
interferometry (Fourier transfarm). Any properly qualified
instruments should be suitable far qualitative measurements.
However, care must be taken when selecting an instrument
far quantitative measurements, as dispersion and response
linearity may not be unifarm across the full spectral range
(far example, when using an echelle spectrograph).
5 Detector
The silicon-based charge-coupled device ( CCD) is the most
common detector far dispersive instruments. The cooled
array detector allows fast, full-spectrum measurements with
low noise. It also has peak wavelength responsivity when
matched to the commonly used 785-nm diode laser. Fourier
transfarm instruments typically use single-channel
germanium or indium-gallium-arsenide ( lnGaAs) detectors
responsive in the NIR to match neodymium: yttrium-
aluminum-garnet (Nd: YAG) 1064-nm excitation.
Instrument Calibration
Raman instrument calibration consists of three components:
primary wavelength ( x-axis), laser wavelength, and
intensity (y-axis).
In all Raman systems suitable far analytical Raman
measurements, the vendar will offer a procedure of
calibration that can be perfarmed by the user. Unless
otherwise specified, user may compase a detailed SOP
(standard operation procedure) according to the proce-
dure of calibration offered by vendar, and should strictly
fallow the SOP to perform the validation of the relative
parameters of the instruments.
It must be noticed that laser wavelength variation can impact
both the wavelength prec1s1on and the photometric
(intensity) precision of a given instrument. Even the most
stable current lasers can vary slightly in their measured
wavelength output. The laser wavelength must therefare be
confirmed to ensure that the Raman shift positions are
accurate far Raman instruments. Spectrophotometric grade
material can be purchased from appropriate suppliers far this
use. Certain instruments may use an internal Raman
standard separated from the primary optical path. External
calibration devices exactly reproduce the optical path taken by
the scattered radiation.
0421 Raman Spectrometry
Detailed functional validation employing external reference
standards is recommended to demonstrate instrumental
suitability far laboratory instruments.
Method Validation
It is necessary to carry out the validation of Raman methods,
at least far main criteria such as accuracy, precision, etc.
However, several of these criteria are aff ected by variables
specific to Raman. Fluorescence is the primary variable that
can affect the suitability of a method. The presence of
fluorescent impurities in samples can be quite variable on the
acceptability of a material. The method must be flexible
enough to accommodate different sampling regimes that may
be necessary to minimize the effects of these impurities.
Detector linearity must be confirmed over the range of
possible signal levels. Fluorescence may drive the signal
baseline higher than that used in the validation, in which case
the fluorescence must be decreased, or the method validated
to accommodate the higher fluorescence levels. This is also
true far the precision, LOD, and LOQ of the method, as
increased baseline noise will negatively impact all of these
values.
Because fluorescence may also affect quantification due to
baseline shifts, confirmation of acceptable quantification at
different levels of photobleaching, when used, should also be
obtained.
The impact of the laser on the sample must be determined.
Visual inspection of the sample and qualitative inspection of
the Raman spectrum far measurements with differing laser
powers and exposure times will confirm that the sample is
not being altered ( other than by photobleaching). Specific
variables to confirm in the spectrum are shifts in peak
position, changes in peak intensity and band width, and
unexpected changes in background intensity.
Method precision must also encompass sample position. The
sample presentation is a critical factor for both soiids and
liquids, and must be either tightly controlled or accounted far
in the calibration model. Sample position sensitivity can often
be minimized by appropriate sample preparation or sample
holder geometry, but will vary from instrument to
instrument based on excitation and collection optical
configura tion.
Procedures
Collecting Raman spectra can be carried out in any of the
fallowing substance states: crystals, amorphous, liquids,
gases and plasms.
Liquids can be directly measured in a glass tube or a quartz
tube. Amorphous and micro-crystal solids can be filled in the
glass tube or the quartz tube far the direct measurement. To
achieve the stronger Raman scattering, the angle crossing the
incident laser onto the sample with the Raman scattering
light to be detected is often made into Oº, 90º and 180º. The
manners of placing the sample cell may be varied.
Unless otherwise specified, the samples needn' t be prepared
far identification. When the sample is used far the crystalline
identification, the limit test of isomers and the quantitative
assay, the procedures of the sample preparation and the
assay methods are carried out to meet the specifications
indicated in monograph.
A few of special sampling techniques are used in the measu-
rement of SERS or Confacal Micro-Raman Spectro-scopy.
To prevent the samples from decomposing, one of the
means is rotating the sample. With a practical device the
relative movement of the surface of sample against the
focus of laser beam can be obtained so as to avoid the
localized over on the sample. This procedure also increases
the assay sensitivity.
 0431 Mass Spectrometry
Use of an internal reference is the most common and robust
method. The added internal standards, under the irradiation
of laser, will give Raman spectroscopy peaks, one of which
could be selected for the reference standard. Quantitative
analysis is calibrated by ratio of the intensity of Raman peak
of the sample to that of the internal standard, commonly
with the peak area or peak height. The interference factors
from the internal standard and the sample will compensate
each other because of the same experiment condition taken.
The internal standard may be required to meet the following
specifications: CDit is chemically relative stable, not reacting
with the components of sample or other material. @ The
peak as the reference standard do not interfere the analysis
peak of the sample. ®Its purity is high enough to contain no
obvious impurity. In non-aqueous solution, Carbon tetra-
chloride (459 cm- 1 ) is one of the best internal standards. In
water solution, the ion peak of either of sodium nitrate (1050
cm- 1 ) or of perchlorate is often selected as the internal
standard. As to solid samples, the selected Raman peak of
the sample itself can be used as the internal standard line.
Raman spectrum of the sample being examined may differ
from that of reference standard or from that of reference
spectrum, due to the polymorphous solid sample. In this
case, the sample being examined, or both of the sample
being examined and the reference standard, should be treated
by the method specified in the monograph before the spectra
are recollected and compared.
The spectrum configuration also depends on the instrument
mode or its capability, the laser wavelength, the sample
status and the amount of water containing in the sample, and
etc. Therefore, the various effects should be considered
when the spectra are compared for structural elucidation and
interpretation.
0431 Mass Spectrometry
Mass spectrometry is based on the direct measurement of the
ratio of the mass to the number of elementary charges of ions
(m/z) in the gas phase obtained from the substance to be
analyzed. The limit of detection is 10- 15 -10- 12 mol. Mass
spectrometry is used to obtain molecular mass and relevant
structural information. Internal standard or external standard
methods are preferred for quantitative analysis.
Major components of a mass spectrometer are illustrated as
following Fig. The ions, produced in the ion source of the
apparatus, are accelerated and then separated by the analyzer
before reaching the detector. All of these operations take
place in a chamber where a pumping system maintains a
vacuum of 10- 3 to 10- 6 Pa. The computer system is used to
control the instrument, acquire and manipulate data, and
compare spectra to reference libraries.
Computer System
Sample
Ion source Analyzer Detector
lntroduction
Fig. Components of mass spectrometer
1. Sarnple Introduction System
When samples are introduced in to a mass spectrometry, the
vacuum degree should not be aff ected. The means of sample
introduction depend on the property, purity and ionization
modes of samples.
( 1 ) Direct Introduction
A gaseous or liquid sample can be admitted to the ion source
as the form of a neutral vapor vía a controllable leak system
at room temperature and normal pressure. Volatile
substances adsorbed in solids or dissolved in liquids can be
concentrated with a headspace analyzer, captured by an
adsorption column, desorbed vía temperature programmed
process, and delivered into the mass spectrometer through a
capillary connection.
Direct introduction by an insertion probe is usually adopted
for solid samples. The sample is introduced in a small
crucible at the tip of the probe, which is heated and gasified
under high vacuum in close proximity to the ion source.
Coupled with desorption ionization methods, the introduction
mode could be used for ionization of poorly volatile or heat
sensitive samples when they are gasified.
Most separation methods can be used in tandem with mass
spectrometry. After being separated and purified, the
analyte is admitted into the mass spectrometer by a
compatible interface.
( 2) Gas Chromatography/Mass Spectrometry ( GC/MS)
With the application of capillary GC and high capacity
vacuum pumps for mass spectrometers, the gas chroma-
tographic effluents can be fed directly into the ion source.
( 3) Liquid Chromatography/Mass Spectrometry ( LC/MS)
Suitable LC/MS interfaces encompass a number of
approaches to separating the compound of interest from the
liquid chromatographic mobile phase and transforming it into
an ionized species suitable for mass spectrometry. Buffer salt
and addition reagents should be volatile and as little as
possible to reduce pollution, and avoid chemical noise and
ionization depression.
Particle beam interface ( PBI)
The solvent is removed from an aerosol of the liquid
chromatographic effluent, and the resulting neutral analyte
molecules are introduced into the ion source of the mass
spectrometer where they are ionized by electron impact ( ED
ionization or chemical ionization ( CI). And the resulting
spectra are called El or CI spectra. PBI is best suited for
small organic molecules with low polarity, thermal stability
and molecular mass less than 1000 Da. and El spectra
usually contain a wealth of structural information.
Moving belt interface ( MBI)
The liquid chromatographic effluent is deposited in a uniform
manner over the moving belt with the flow rate of O. 5-
1. 5 ml/min, and then the solvent is evaporated. The analyte
is ionized and introduced into the mass spectrometer. MBI is
not suitable for high polar or heat labile compounds, and
spectra are obtained by electron impact ionization, chemical
ionization or fast atom bombardment (FAB) ionization.
Atmospheric pressure ionization ( API)
Atmospheric pressure ionization is a kind of interfaces widely
used in liquid chromatography/mass spectrometry. These
interfaces will be described in the section of ion source due to
the function of ionization.
Supercritical fluid chromatography/mass spectrometry ( SFC/
MS)
Electron impact ionization and chemical ionization are widely
used for supercritical fluid chromatography/mass
spectrometry. The mobile phase enters the gas state after
passing a heated restrictor between the column and the ion
source. The analyte is then ionized and introduced into mass
spectrometer.
Capillary electrophoresis/mass spectrometry ( CE/MS)
Almost all of the capillary electrophoresis apparatus can be in
tandem with mass spectrometers by a suitable interface.

However, it should be paid attention that CE has low flow
rate and volatile buffers must be used when CE is coupled
with a mass spectrometer. Electron impact ionization is
widely used for capillary electrophoresisl mass spectrometry.
2. Ion Source
According to the property of the sample under analysis and
the information wanted, different ionization methods could
be used.
( 1 ) Electron Irnpact (El) Ionization
After entry in to the ionization chamber, gaseous sample
molecules are ionized by a beam of electrons whose energy
( usually 70 e V) is greater than the ionization energy of the
molecules. In addition to the molecular ion M+ , fragments
characteristic of the molecular structure are observed. El
spectra provide the most abundant structural information of
the analytes and the study on the El fragmentation pattern is
also the most comprehensive. Electron impact ionization is
suitable for samples with good volatility and thermal
stability, and widely used for ionization of GC-MS. It can
also be used in LC-MS when PBI or MBI is adopted.
( 2) Chemical Ionization ( CI)
Reagent gas (such as methane, iso-butane and ammonia) in
the ion source is ionized by a high energy electron beam or
discharge. The ions resulted react further with reagent gas
molecules, or have ion-molecule reaction with sample
molecules. The spectrum is characterized by ions of the
[M+l]+ or [ M-1]- types, or adduct ions formed from the
analyte and the reagent molecules. Fewer fragments are
produced than with El. CI is suitable for analysis of
thermally stable and volatile compounds which fail to yield
molecular ion signal under the El mode.
( 3) Fast Atom Bombardrnent ( FAB) and Liquid Secondary Ion
Mas.s Spectrometer ( LSIMS) lonization
The sample, dissolved in a viscous matrix such as glycerol,
is applied to a metal surface and ionized by a beam of neutral
atoms ( such as argon) or high-kinetic-energy cesium ions.
Ions of the [M+l]+ or [M-1]-types, or adduct ions formed
from the matrix or the sample are produced. F AB and
LSIMS technique have the advantage of good ionization
efficiency and is well suited to analyze polar and thermally
labile compounds for mass determination and structural
characterization. It is widely used in the analysis of
peptides, antibiotics, nucleotides, lipids, organometallic
compounds and surfactants with molecular masses of up to
10 000 Da.
F AB or LSIMS can be combined with LC-MS by adding 1 %-
10 % glycerol to the mobile phase, however, the flow rates
must be very low ( 1 µl/ min-1 O µl/ min).
( 4) Matrix-As.sisted Laser Desorption lonization ( MALDI)
Sample dissolved in a suitable matrix is deposited on a metal
support, and then ionized by a pulsed laser beam of high
intensity whose wavelength may range from UV to IR. This
mode of ionization plays an essential role in the analysis of
very high molecular mass compounds (more than 100 000 Da)
and is very compatible with a time-of-flight mass spectrometer.
( S) Electro Spray Ionization ( ESI)
The ionization is carried out at atmospheric pressure. The
samples ( such as the liquid chromatographic effluent) are
introduced into the source through a capillary tube, the end
of which has a potential of the order of several kilovolts. A
gas can be used to facilitate nebulization. Desolvation of the
resulting microdroplets produces singly or multiply charged
ions in the gas phase, and the ions are transported from the
atmospheric-pressure region to the high vacuum of the mass
'' u'·:h
0431 Mass Spectrometry :··.::59:····
analyzer via a series of pressure-reduction stages. The flow
rates can vary from 1 to 1000 µl/min. ESI is suited to polar
compounds and biomolecules with molecular masses of up to
100 000 Da. It is the most successful interface for LC-MS
and CE-MS analysis.
( 6) Atmospheric Pressure Chemical Ionization ( APCI)
The principle of APCI mode is similar to that of CI, but
APCI is carried out at atmospheric pressure. The solvent
molecules in mobile phase, which is nebulized both by
thermal effects and by the use of a stream of nitrogen, are
ionized by the action of an electrode maintained ata potential
of several kilovolts and placed in the path of the mobile
phase. The resulting ions carring a single charge are of the
[M+ H]+ type in positive mode and the [M-H]- type in
negative mode. APCI can afford the flow rate of 2 ml/min
and is an ideal interface for coupling to liquid chroma-
tography. APCI is widely used for the analysis of small
molecules or weak polar compounds with mass less than
1500 Da.
( 7) Atmospheric Pressure Photo Ionization ( APPI)
Different from APCI, APPI ionize the gaseous sample
molecules with a beam of photons of known energy. APPI is
mainly used for the analysis of non-polar compounds as a
supplement of ESI and APCI. APPI is sensitive to the
experimental conditions. Properties of solvent, types of
dopant or buffer components could strongly influence the
selectivity or sensitivity of the detection of analytes.
3. Mas.s analyzer
The two main technical parameters of a mass analyzer are the
mass range and the resolution. The mass range indicates the
limit of miz over which the mass analyzer can measure ions,
and the resolution shows the ability of a mass spectrometer
to distinguish between two neighboring ions that differ only
slightly in their mass. Although different types of mass
analyzers have different definition of resolution, high
resolution is usually used to describe the analyzers with a
resolving power greater than 104 •
( 1 ) Magnetic Sector Analyzer
Ions produced in the ion source are accelerated by a voltage V
and focused towards a sector-type magnetic field ( magnetic
field strength, B). Given the difference in miz ratios, these
ions will have specific radius of curvature (r) in their motion
paths in the sector field.
mlz=B 2 r 2 l2V
By varying the strength of magnetic field, ions passing
through the small slit sequentially are detected. Spatial
separation of the ions is then realized and the spectra
obtained. Magnetic sector analyzer can detect single charged
ion with molecular masses of up to 15 000 Da. The resolution
could be up to 105 , when a proper combination of an
electrostatic field analyzer with a magnetic analyzer to forma
double-focusing mass spectrometer.
( 2) Quadrupole Analyzer
A quadrupole analyzer gets its name from the four parallel
metal rods it comprises of. The ion beam is focused on the
axis parallel to the rods. A direct current voltage (DC) anda
radio frequency (RF) voltage are applied to the rods, making
the two pairs of rods in opposite electrical voltage. For given
combination of DC and RF voltage, ions of a specific miz
ratio travel steadily along the axis, while ions of other miz
ratios are deflected to the sides and lost. By varying the
voltage applied to the rods so that the ratio of continuous
voltage to alternating one remains constant, mass scanning of
a compound could be achieved. Quadrupole analyzers exhibit
a relatively higher sensitivity in selected ion determination.
 0431 Mass Spectrometry
Quadrupole analyzers usually have a mass range of 1 to 4000
Da, with a resolution of about 10 3 •
(3) Ion Trap Analyzer
Quadrupole ion trap CQIT) consists of a quadrupole-like ring
electrode between two end cap electrodes. The end cap
electrodes are held at ground potential while an RF voltage is
applied to the ring electrode and then three-dimensional
quadrupole field is formed. The ion trap can capture ions
within certain mass range, depending on the RF voltage
level. Mass analysis is accomplished through use of the
"mass selective instability'' mode of operation. By raising the
RF voltage on the ring electrode, ions of successively higher
mass are ejected from the ion trap into an electron multiplier
detector, ionization and mass analysis of volatile compounds
under test can be achieved in the same quadrupole field. With
a pre-set time sequence, a single ion trap can achieve the
function of multiple-stage mass spectrometry CMSn ).
Linear ion trap, a two-dimensional quadrupole ion trap, has
the same structure as the quadrupole mass analyzer, while
has the similar operation as the three-dimensional ion trap.
Quadrupole linear ion trap has better ionic storage efficiency
and capacity. It possesses improvable efficiency of ion spray,
quicker scanning speed and higher detection sensitivity.
Ion trap analyzer has the similar upper limit of mass and
resolution as that of quadrupole analyzer.
( 4) Time of Flight ( TOF) Analyzer
Ions with the same kinetic energy but different masses are
separated according to their flight velocities. For a fixed
distance of ion flight, the flight time of ion is proportional to
the square root of miz ratio, and ions of smaller masses will
reach the detector first. Ions are introduced into the mass
spectrometer in discrete packets so that their flights start at
the same point and time of flight can be measured sequen-
tially. Ion packets are either through a pulsed ionization
process i. e. MALDI or through a gating system in which
ions are produced continuously, but are introduced only at
given times into the flight tube.
Time of flight analyzer has wider analyzable mass range
Cupper limit is 15 000 Da), higher ion transmission efficiency
Cespecially for obtaining spectrum) and mass resolution
greater than 104 •
( 5) Ion Cyclotron Resonance ( ICR) Analyzer
Under very low pressures Cof the order of 10- 1 Pa), ions
move in circular orbits in superconducting magnetic field,
and their paths of movement are restricted by a resonant
crossover electric field. When the crossover electric field
frequency equals the cyclotron frequency, ions will be
accelerated steadily, with the corresponding increases to their
path radius and kinetic energy level. As soon as the electric
field disappears, the orbiting ions will create an alternate
current on the electrodes and their masses can be obtained by
analyzing the signal frequencies. Thus, the Fourier
transform of the time domain transient signal yields the
corresponding frequency spectrum from which the mass
spectrum is computed.
Ionization and mass analysis are simultaneous processed in
one analyzer. It is suitable for the compounds of mass up to
10 000 Da and has high resolution Cup to 10 6 ) , yielding miz
accurate to about one thousandth of a Da. Ion cyclotron
resonance analyzer can be used for tandem mass
spectrometry.
( 6) Tandem Mass Spectrometry ( MS/MS)
Tandem mass spectrometry CMSIMS) refers to the coupling
of two stages of mass analysis by time or space. The
experiments can determine the mass relationship between
precursor ions in the first-stage mass analysis CMS1 ) and
product ion in the second-stage mass analysis CM~ ) .
Tandem mass spectrometry is not restricted to two stages of
mass analysis, it is also possible to perform multiple-stage
MS experiments, abbreviated as MSn.
Product-ion sean
It is used to detect the miz and relative intensity of all the
fragment ions in M~ within a certain mass range, generated
from the precursor ion with a selected miz ratio in MS1 .
And the mass spectrum of the precursor ion is obtained.
Precursor-ion sean
It is a method for scanning in MS1 the precursor ions, from
which the product ions in M~ with a specific miz ratio are
generated by dissociation, within a certain mass range.
Neutral-loss sean
All the precursor ions that undergo the loss of a specified
common neutral are monitored. To obtain this information,
both MSl and MS2 are scanned simultaneously with a mass
offset that correlates with the mass of the specified neutral,
to find compounds or homologues which lose the specific
neutral fragments Clike carbon dioxide) dueto dissociation.
Selected-reaction monitoring ( SRM)
The method detects the precursor ion with a specific mass-to-
charge ratio Cmiz) 1 in MS1 and its product ion with a
specific ma ss-to-charge ratio Cmlz) 2 in M~, and it is
useful in quantitative measurement of trace analyte present in
a complex mixtures.
Multi~le-reaction monitoring ( MRM)
Multiple-reaction monitoring detects two or more precursor
ion-product ion pairs of the anaytes simultaneously. it is also
useful in quantitative measurement of analytes present in a
complex mixtures.
4. Procedures
Before sample analysis, mass calibration should be carried
out for either single-stage mass spectrometer or tandem mass
spectrometer. In general, this can be done using a reference
substance. The calibration may be external Cacquisition file
separate from the analysis) or internal C the reference
substance Cs) are mixed with the substance to be examined
and appear on the same acquisition file).
( 1 ) Qualitative Analysis
The mass spectrum of a compound is a plot of miz ratios
Con the x-axis) of all ions Ci. e. , the molecular ion and its
fragment ions) that reach the detector versus their
abundances Con the y-axis). Accurate molecular mass of the
compound could be determined using a high-resolution mass
spectrometer.
Sample molecules or reference ones can be introduced into the
same mass spectrometer which is operated by direct mode or
flow injection mode, under the same conditions, and each of
the mass spectra will be determined. Comparing the ions
with specific miz ratios in both of the spectra, effective
substances, impurities, illegal additives can be identified
respectively. Product-ion sean could be used for identification
of polar macromolecules. For the analysis of a compound in a
complex matrix, chromatographylmass spectrometry or
tandem mass spectrometry is best to be adopted.
In a mass spectrum, ions with certain miz ratios and relative
abundance reflect the structure features of the analyte. And
the molecular structure of the compound might be deduced or
confirmed when the analytical results from tandem mass
spectrometry are taken into account. When using electron
impact ionization, a compound could be identified conve-
niently by matching the determined spectrum with the
 0441
standard spectrum in the eXIstmg collection of spectra
library. Far structure elucidation of an unknown compound,
comprehen-sive application of different mass spectrometry is
usually needed and knowledge of sample is also essential.
The other test infarmation from elemental analysis,
spectroanalysis (i. e. , NMR, IR, UV and X-ray diffraction)
should be utilized far the final judgment, if necessary.
{ 2) Quantitative Analysis
External or internal standard methods may be used to
determine the analyte using selected ion monitoring ( SIM)
mode or SRM or MRM mode. Far mass spectrometry,
internal standards may be either structural or stable isotope
( i. e. , 2 H, 13 C, 15 N) labeled analogs of the analyte.
Prepare certain concentration of test solution and impurity
reference solution, then analyze by chromatography-mass
spectrometry. If the response ( or sum of response) of the
characteristic ion at certain miz ratio obtained from test
solution is lower than that obtained from impurity reference
solution, the impurities contained in the test sample meets
the requirements.
Chromatography-mass spectrometry with standard curve
method is employed to determine the toxic and hazardous
substances, illegal additives, traces drugs and its metabolites
in complex sample. Standard curve and the regression
equation can be obtained by the determination of the response
of ions at certain miz ratio in series standard solutions with
the same volume. Prepare the test solution according to
method described in monograph, determine the response of
ion at certain miz, obtain the concentration of analyte by
calculation with the standard curve or the regression
equation. Add an equal amount of interna! standard in series
standard solution, determine the response of ions of analyte
and internal standards at each certain miz ratio, calculate
the regression equation by taking the ratio of both responses
as ordinate, concentration of anaiyte as abscissa and standard
curve calibrated by internal standard are accomplished.
Better precision and accuracy of analysis can be obtained
when stable isotope labeling compounds are selected as
internal standards.
0441 Nuclear Magnetic
Resonance Spectrometry
Nuclear Magnetic Resonance ( NMR) Spectrometry, an
analytical method based on the phenomenon that specific
atomic nuclei absorbs radiation frequency field energy under
the applied magnetic field, corresponds to their split energy
level difference and then generate resonance. Qualitative
structural infarmation such as connection ways and relative
spatial orientation of atoms in the molecule is derived from
the NMR spectrum that can be described by chemical shifts,
multiplicity, coupling constants, relative intensity and
correlated peaks in various two-dimension and multi-
dimension spectra. Far NMR quantitative analysis based on
structural analysis, identification of the molecular structure
of a compound is first carried out befare quantitative
analysis, then quantification is perfarmed according to the
relationship between the number of protons in certain
functional groups and corresponding peak areas.
When a positively charged atomic nucleus is spinning, it
produces a magnetic field and an angular momentum. The
magnetic property is described in nuclear magnetic moment
µ. Angular momentum P is related to spin quantum number
I (if the mass number is odd, I is 112 or an integer plus
ll2; otherwise, I is O or a whole number). Further, its
Nuclear Magnetic Resonance Spectrometry
spatial orientation is quantized. µ is also a vector, with the
same direction with P, its spatial orientation is also
quantized. lt is decided according to the value of magnetic
quantum number m (m = I, I -1, · · ·, - I, 2I + 1 possible
values in total). Far nuclei such as 1 H, 13 C with I = 112,
there are only two orientations, corresponding to two
different energy states. The particle undergoes transition
between the two energy states through the absorption or
emission of corresponding energy.
When placed in a unifarm external static magnetic filed of
strength, H o , nuclei with spin quantum number I is spin*º
around the external magnetic field on the strength of magnetic
field. Meanwhile, the nuclei keep their self-spinning. This
kind of motion mode is called Larmor precession. The
precession frequency of the atomic nuclei is determined by:
wo=rHo
in which y is the gyromagnetic ratio and one of the basic
properties of atomic nuclei.
Different nuclei cause y in different, where the bigger its
value, the stronger the magnetic property of the nuclei, and
then it would be easier to be detected in NMR. If a radio
frequently field is introduced, its frequency v meets:
D.E=hv=µHoll
where h is Planck' s constant, and
v=wol27r=rHal27r
Thus, when the frequency of the radio frequently field is
equal to the nuclei precession frequency in magnetic field
H o, a nuclear magnetic resonance, which transits between
two energy states by absorption of the corresponding amount
of energy, is achieved.
NMR is a kind of technique with high specificity but relatively
low sensitivity. The basic reason far the low sensitivity is the
energy between excited and ground states have little
difference. Actually, the general difference between two
energy states is only a fev: particles per hundred thousand
( when the externa! magnetic field is approximate to 2T).
NMR spectrometer
There are two types of NMR spectrometers: the classical
continuous wave ( CW) instruments and the modern pulse
Fourier-transfarm ( PFT) instruments, where the latter is
more commonly used recently. lt consists of superconductive
magnet, radio frequency pulse emission system, NMR signal
reception system and the computer system far data
collection, storage, processing and spectrometer control (see
Fig. ).
Network
----. 1 NMR workstation 1
Supereonductive
magnet
Data
acquisition system
Shimming and
Transmitter
control unit locking control 1 .--~__,
Acquisition ---+-----------"'----- Low energy
control RF system
Fig. Components of NMR spectrometer
In pulsed NMR spectrometers, a pulse covering RF energy of
all resonance nuclei is used to simultaneously activate all
nuclei. When the excited nuclei return to the lower energy
level, they generate free induction decay ( FID) signal
containing time domain infarmation. After analog-to-digital
 0441 Nuclear Magnetic Resonance Spectrometry

conversion through Fourier transformation by computer, the field strength. Coupling could also occur between hydrogen
frequency spectrum is obtained. and other nuclei (1 #O) , such as 19 F, 13 C, and 31 P etc.
In the experiment, spectrometer parameters such as pulse Another characteristic of the NMR signal is its intensity.
dumping angle, corresponding pulse intensity, pulse Under suitable experimental conditions ( see the section
interval, data collection point ( resolution), sampling time Determination Method) , the area or intensity of a signal is
etc. , are set according to instrument operation manual. directly proportional to the number of protons giving rise to
After acquisition of enough FID data transformated by the signal. Therefore, it is possible to determine the relative
computers, phase adjustment to obtain absorption peaks with ratio of the different kinds of protons or other nuclei in a
high purity and chemical shift calibration by reference specimen or to perform NMR quantitative analysis with the
materials, a spectrum is recorded by the output equipment. aid of an interna! standard.
NMR spectrum Determination Method
The signals ( peaks) in an NMR spectrum can provide four In spite of being familiar with NMR theory, analyst should
important spectral parameters: chemical shift, multiplicity, also be familiar with sample property. Operator should
coupling constant and relative intensity. The same nucleus, strictly follow the operation manual and correctly operate the
when located in different molecular environments, exhibit instrument. Inadequate specimen preparation or incorrect
different resonance frequencies, it is due to the fact that the instrumental adjustment on parameters may lead to poor
effective field experienced by a particular nucleus is a resolution, decreased sensitivity, spectral artifacts and even
composite of the external field provided by the instrument erroneous data. The sample in NMR is commonly solution,
and the field generated by the circulation of the surrounding the solid sample only can be analysed in NMR with specific
electrons. ( The latter is generally opposed to the externa! unit.
field and this phenomenon is called "shielding" ). For atomic The most commonly used is 1H ( proton) NMR spectro-
nuclei in diff erent chemical environments, beca use of the metry, and others include 19 F, 31 P, 13 C NMR spectrometry
small difference in resonance frequencies caused by shielding, and various two-dimension spectrometry. Before determi-
it is hard to measure accurately the absolute values of nation, it is preferable to prepare sample into proper
transition frequencies. In practice, with certain reference solutions.
standards as benchmarks, the difference between the Selection of Solvent In addition to having good solubility
resonance frequency of the nucleus in test samples and that of properties for the sample, suitable solvents do not exhibit
a reference standard is accurately measured. In an NMR resonance peaks that obscure resonance peaks of the
spectrum, the position of a signal is described by its deviation specimenanalyzed. Deuterated solvents also provide signals
from another resonance signaL This deviation is caiied
for heteronuclear system lock. High purity and high isotopic
chemical shift.
purity solvent should be used and attention should be paid to
The resonance frequency is proportional to the externa!
the coupling deuterium caused on other atomic signals.
magnetic field strength H 0 • When magnetic field strength is
Deuterated solvents commonly used in the NMR spectro-
different, the nuclei resonance frequency in the same
metry, and their chemical shift of residual proton signal are
chemical environment is different. In order to solve this
shown in the following table.
problem, chemical shift constant is employed to describe
chemical shift: Table Deuterated Solvents Commonly Used for Proton NMR
and Their Chemical Shifts of the Residual Proton Signals
8 = (J.Js -J.Jr) +Br
J.lo Residual Possible
in which J.Js is the resonance frequency of nuclei in sample; J.Jr Molecular Pro ton Residual
is the resonance frequency of nuclei in reference; J.Jo is the Solvents
formula Signal Water Signal
output frequency of instrument, in MHz; and Br is the 8 (ppm) 8 (ppm)
chemical shift value of reference.
Besides, residual proton signal in deuterated solvent could Deuterium
COCb 7.26 l. 56
also be used as chemical shift reference value. Chloroform
The most widely used chemical shift reference is tetrame- Deuterium
CD30D 3. 31 4.87
thylsilane (TMS). It is chemically inert, exhibits a singlet at Methanol
a higher field, not interfering with most signals from the test Deuterium
substance because of overlap, and has low boiling point (CD3)2CO 2.05 2.84
Acetone
(27°C), thus allowing for easy to eliminate and benefit of
Deuterium
specimen recovery. For aqueous solutions, 3- ( trimethyl-
Dimethyl DMSO-d6 2. 50 3.33
silyl) propionate sodium ( TSP) or 2, 2-dimethyl-2-
Sulfoxide
silapentane-5-sulfonate sodium ( DSS) are also commonly
Deuterium
used since their chemical shift values are clase to zero. DSS CD3CN l. 94 2. 13
Acetonitrile
has the disadvantage of containing protons in three methylene
Deuterium
groups that may interfere with signals from the test C6D6 7. 16 /
Benzene
substance, therefore it is suitable for external reference.
Chemical shift only describes the electronic environment of Deuterium
Heavy Water
D20 / 4.79
nuclei, i. e. the shielding of extra-nuclear electron cloud on
nuclei, <loes not involve the interaction between nuclei in the Deuterium
Dioxane
Dioxane-ds 3.55 /
same molecule. This interaction is very small, having no
effects on chemical shift, but have significant effects on peak Deuterium
Acetic acid
CD3C02D 2.05, 8.5* /
shape. This mutual interference between nuclei is called spin-
spin coupling, and the induced peak multiplicity is called Deuterium
spin-spin split. The split separation ( in hertz) is called T rifl uoroacetic CF3C02D 12.5* /
coupling constant J which is not related to external magnetic acid
 0441
continued
Residual Possible
Molecular Pro ton Residual
Solvents
formula Signal Water Signal
8 (ppm) 8 (ppm)
Deuterium 7. 18, 7. 55,
CsDsN 4.8
Pyridine 8. 70
Deuterium N,
2. 77' 2. 93,
N-Dimethylfor- DMF-d1 / often necessary to use 1H, 13 C NMR as well as two-
8.05
mamide
* Chemical shift of active protons is variable, depending
upon variation in temperature and solvents.
For 19 F NMR, most solvents used in proton NMR may also
be employed, the most common ones being CDCb, CD30D,
D2 O, DMSO-d6, DMF-d1, acids and bases. In general, any
non-fluorinated solvent may be used. It should be noted that
fluorine atom in fluorinated sample may induce J-coupling on
other nuclei.
Specirnen Preparation Directions are usually given in
individual monographs. Solution concentration depends on
objective of the experiment and the type of instrument.
Detection of minor contaminants may require higher
concentrations. The sample volume required depends on size
of the tu be and geometry of the instrument. Generally, the
level of the solution in tube must be two times higher than
the height of coils. NMR tubes that meet the requirement of
quantitative determination should be used, and commonly
used are those with outer diameters of 5 mm or 10 mm and
length of 15 cm or 20 cm. Micro NMR tube might be
employed when the quantity of sample specimen is small.
Procedure Place the sample tube in the spectrometer, first
perform tuning of sample and instrument, then shim the
spectrometer carefully and adjust the instrument to its
optimum operating state. Set appropriate experiment
parameters, collect sample signal, perform spectrum
processing af ter the experiments and then break clown to
sections and integrate.
One experiment may simultaneously generate qualitative and
quantitative data. In quantitative NMR analysis, special
precautions must be taken to set experimental parameters, in
order to ensure the signal area is proportional to the number
of protons. The delays between pulses must be long enough
to allow complete relaxation of all excited nuclei, which
results in a considerable increase in the analysis time for
quantitative analysis.
Qualitative and Quantitative Analysis NMR spectroscopy has
been used for a wide range of applications such as structure
elucidation, thermodynamic, kinetic and mechanistic studies, as
well as quantitative analysis.
l. Qualitative Analysis
NMR spectroscopy is a very useful tool for structure elucidation.
Chernical shift provides information on atornic nuclei
environment. Peak multiplicity provides information on adjacent
groups and stereochernistry. Value of coupling constant can be
used to determine the substitute of certain groups. Peak intensity
( or integration area) can be used to determine number of protons
in functional groups. Several special techniques, such as double
resonance, chernical exchange, use of shift reagents, two-
dimensional analysis, etc. are available to simplify the relative
complex spectra, identify certain functional groups and determine
the coupling correlations.
For the specimens with simple structure, their structure
could be determined directly by the numeric value of chemical
Nuclear Magnetic Resonance Spectrometry
shift, coupling situation ( number of coupling peaks and
coupling constant) and the number of protons under each
signa!. Sample structure, as well as the presence of
impurities, could also be determined through comparison
with literature data (spectrum). When compared to literature
data ( spectrum), effect of experimental details should be
noted, such as the variety of solvent used, specimen
concentration, chemical shift reference and tested tempe-
rature. For sample with complex or unknown structure, it is
dimension, 19 F, 31 P spectromery, and to couple with other
analytical techniques such as mass spectrometry to identify
its structure.
2. Quantitative Analysis
Compared with other nuclei, 1H NMR is more suitable for
quantitative analysis. Under suitable experimental condi-
tions, the ratio of areas ( or intensi ti es) of two signals are
proportional to that of the total number of protons generating
the signals.
(1)
where A1, A2 is the integrating area ( or intensity) of
corresponding signal; N1, N2 is the number of protons of
corresponding signal.
If the two signals originate from two functional groups on the
same molecule, the equation can be simplified to:
A1 n1
(2)
n2
m which n1 and n2 are the number of protons in the
respective functional groups.
If the two signals originate from different molecular species,
then:
(3)
n2m2
where m1 and m2 are the number of moiecuies; W1 and W 2
are the masses; and M1 and M2 are the molecular weights of
compounds 1 and 2, respectively.
Equations 2 and 3 show that NMR quantitative analysis can
be performed either in an absolute or a relative manner.
Absolute quantification use reference standards to directly
determine the contents of components in test specimens;
relative quantification is bases on integrals of specific signals
arising from each component in test specimens to determine
the relative contents of specific compounds being tested.
In the absolute method, an accurately weighted interna!
standard is added to the specimen and a resonance peak area
arising from the test substance is compared with a resonance
peak area from the internal standard. The absolute quantity
( purity) of the substance may therefore be calculated. A
good internal standard possesses the following properties: a
reference resonance peak, preferably a singlet with proper
width, the reference peak at a field position separated from
all specimen peaks, soluble in the analytical solvent, its
proton having equivalent weight, low ratio of molecular mass
to the number of protons of reference peak, and not
interacting with the compound being tested. Typical
examples of useful standards are 1, 2, 4, 5-tetrachlo-
robenzene, 1, 4-dinitrobenzene, hydroquinone, tetraphthalate,
benzyl benzoate and maleic acid. The selection of a reference
standard depends on properties of the specimen.
The relative method may be used to determine the molar
fraction of impurities in test substance (or the components in
mixture) as calculated by Equation 3.
(1) Absolute Method of Quantitation
Solvent, Internal Standard, and NMR Reference Use as
directed in the individual monograph.
 0451 X-ray Diffraction
Test Sample Preparation Transfer appropriate amount of
accurately weighed test sample and interna! standard
( directed in the individual monograph) to a glass-stopper
centrifuge tube. Accurately add appropriate amount of
solvent, shake to dissolve completely. Add appropriate
amount of NMR reference, mixto dissolve.
Determination method Transfer the test preparation to a
NMR tube, set the instrumental parameters, adjust the spin
rate so that no spinning side bands interfere with the peaks of
interest, and then record the spectrum. Measure the area
under each characteristic and interna! standard peaks
specified in the individual monograph by integrating no fewer
than five times. Record the average area, and calculate the
quantity of the analyte W., in the test preparation by
formula:
A. E,
W, = Wr X Ar X Er
in which Wr is the weight of interna! standard, A, and Ar are
the average peak area of specimen peak and interna! standard
peak, respectively. E, and Er are the proton equivalent
weights (i. e. , the molecular weights divided by the number
of protons giving rise to the reference peak) of the analyte
and the interna! standard, respectively.
(2) Relative Method of Quantitation
Solvent, NMR Reference, Test Preparation and
Determination Method Use as directed in the individual
monograph and refer to the " Absolute Method of
Quantitation".
Calculate the mole percent of the analyte m the test
preparation by formula:
Ailn1 X lOO
Ai/n1 +Adn2
In which A1 and Az are the average area resulting from the
resonances of the groups designated in the individual
monograph, n1 and nz are, respectively, the numbers of
protons in the designated groups.
In NMR quantitative analysis, special precautions must be
taken for the setting and optimization of experimental
parameters, such as the weight of test substances and
accuracy, the concentration of test specimens and interna!
standards, the solubility of test substances, the NMR
excitation pulse, acquisition time, temperature, phase and
baseline correction, integration parameters, etc.
0451 X-ray Diffraction
X-ray diffraction ( XRD) is a kind of analysis method to
characterize the three dimensional structure of crystalline
materials, including chirality, crystal form, hydrate or
solvate, or to analyze the components of materials, such as
major component and impurities, crystal form and contents,
by applying a collimated beam of monochromatic X-ray upon
the specimen.
One crystal can be analyzed by X-ray single crystal diffraction
(XRSD), while the specimen analyzed by X-ray powder
diffraction ( XRPD ) is randomly oriented solid micro-
particles, which may be crystal or amorphous solids.
One of the methods could be chosen depending on the
detection requirements, objects and results.
A so lid substance can be classified as crystalline ( also called
crystaD or non-crystalline ( also called amorphous solids,
glasses or etc. ) .
The molecules, atoms or ions in crystalline materials
( crystal) are ordered periodically in a three dimensional
array, the smallest repeating unit is called lattice. A lattice is
a parallelepiped with three axles (a, b, c, unit: Á) and three
angles (a , f3, y, unit: º) , the axes and angles are called
lattice constants. The lattices form the crystal in order along
three dimensions (x, y, z) infinitely.
The molecules, atoms or ions in non-crystalline material,
amorphous solids, glasses, etc. , aren' t ordered periodically
in a three dimensional arra y, they orient out of order within
the solid particles.
The principle of XRD is as follows:
A collimated beam of monochromatic X-ray is diffracted in
various directions when it is impinged upon a rotating crystal
or randomly oriented powdered crystal. The crystal acts as a
three-dimensional diffraction grating to this radiation. The
condition by which the diffraction can occur should conform
to the Bragg equation:
dhkt --~
2•sin8
Where dhkl denotes the inter-planar spacings (hkl are indices
of crystallographic plane).
n is diff raction series.
il is the wavelength of X-ray.
(} is the grazing angle.
Metallic copper (Cu) and molybdenum (Mo) are frequently
used as X-ray anode when organic specimen is analyzed. The
wavelength of Cu anode is l. 541 78 Á; the wavelength of Mo
anode is O. 710 73 Á. X-ray is composed of Ka and K~.
Usually Kª is used as the characteristic X-ray both in XRSD
and XRPD to analyze the structure, component or crystal
form.
When crystalline materials are impinged upon by X-ray,
diffraction occurred. While non-crystalline materials don' t
have the same result. This is the fundamental principle of
crystal' s qualitative and quantitative analysis.
X-ray diffraction equipment usually comprise four main
parts: X-ray source ( high direct current power, vacuum
tube, metal anode), collimation system (collimator, sample
holder) , instrument control system ( instruction control,
data control) , and cooling system.
Method 1 X-ray single crystal diffraction (XRSD)
XRSD is used for a single crystal to obtain the information of
molecular configuration and conformation. The information
includes space group, lattice parameters, molecular formula,
structural formula, atomic coordinate, bond length and
angels of the bonding atoms, intermolecular and intramole-
cular hydrogen bond, salt bond, and coordinate bond, etc.
XRSD is an absolute method to quantitatively analyze the
components and three-dimensional structure of specimen. It
can be used independently in the analysis of chirality or
stereo-isomer, eutectic substance (hydrate or solvate), pure
crystal substance ( changes of molecular arrangement
regular), etc. Since only one crystal being used in the
experiment, the result can give the information of pure
crystal form.
Twice Fourier transforms are needed to complete the crystal
structure analysis in XRSD. This method is suitable for the
structure characterization and crystal form analysis of
crystalline materials. Cu anode is applied for the absolute
configuration measurement, and Mo anode is used for the
relative configuration of compounds except those specimens
which contain halogen or metal atom.
Specimen preparation and related experimental techniques
Specimen preparation a suitable single crystal usually is
prepared by recrystallization. The size of the crystal is

between O. 1 mm and l. O mm. The single crystal should be
transparent, bubbles-free, flawless and without any impu-
rities. The appearance of single crystal could be massive,
plate-like or columnar. Approximately spherical or massive
crystals are the best appearance of crystal far tests because
they have similar absorption of X-ray in every direction.
The diff racting power of crystal is under the influence from
internal or external substance. The internal influencing
factors are attributed to chemical element species existed in a
crystal, structure type, rule of molecular symmetrical
arrangement, force distribution, and mass of single crystal.
The external influencing factors include the power of X-ray
emitter and the kind of anode.
When Cu anode is used, the 2() region to record the
diffraction pattern should be beyond 114º; when Mo anode is
used, the 2() region should be extended to more than 54 º.
The errors of lattice constants, three axles (a, b, c, unit:
Á) and three angles (a, ~' y, unit: º), are the third and
second digits after the decimal point, respectively. The
errors of relative atomic coordinate, bond length and bond
angel are the fourth, third and first digits after the decimal
point, separately.
This method is applicable to the analysis of crystalline
materials' three dimensional structure, chirality, crystal
form, water and solvent of crystallisation.
Instrument calibration lt is advisable to calibrate the
instrument periodically with reference material provided by
the instrument manufacturer.
Method 2 X-ray powder diffraction (XRPD)
XRPD is used far qualitative and quantitative chemical phase
analysis. Every kind of chemical material, with confirmed
chemical composition and salid state ( crystal form) , has
characteristic X-ray diffraction pattern and data. The
information included in the X-ray diffraction pattem are
diffraction peak number, the anguiar position of diffraction
peak (2() value or d value), the intensities of diffraction peak
( relative intensity or absolute intensity), diffraction peak
profiles (ratio of different diffraction peaks).
XRPD is applicable to the qualitative quantitative analysis of
crystalline or non- crystalline materials, and usually far
examination of salid' s crystallization degree, polymorphic form
and purity. In the experiments, Cu ancx:le is in general use.
There are tens of or hundreds of sharp diffraction peaks
(narrow peaks) in crystalline material' s X-ray diffraction
pattern). While there are less diffraction peaks in non-
crystalline material' s X-ray diffraction pattern, the peaks
appear diffusion ( broadened peak or steamed buns peak).
The intensity of crystalline and non-crystalline material' s
peaks at same position varíes greatly in quantitative analysis.
When the chemical substance has two or more than two kinds
of salid status, the phenomena is called polymorphism or
allomorphism. Polymorphism is usually caused by changes of
molecular configuration, molecular conformation, molecular
arrangement arder, molecular acting force, and sometimes
also caused by the join of crystal water or crystal solvent
(amount and species). Each crystal form has its characte-
ristic X-ray diffraction pattern.
When the specimens being measured have same chemical
structure, but in their X-ray diffraction patterns the
diffraction peaks' number and 2() position, absolute intensity
or geometric topology are different. This means that
polymorphism exists in this kind of specimens.
Specirnen preparation and related experimental techniques
Specirnen preparation In general, the shapes of many
crystalline particles tend to give a specimen that exhibits
0451 X-ray Diffraction ¡ ,·t '~f.':/;j'
sorne degree of preferred orientation in the sample holder.
This is especially evident far needle-like or plate-like
crystals. Preferred orientation in the specimen influences the
relative intensities of various diffractions. To improve
randomness in the orientation of crystallites, the specimen
may be ground carefully in a mortar to a fine powder and be
sifted ( usually use 100 mesh sifter).
Amount of specirnen used In quantitative analysis, the
specimen needs to be accurately weighed and put onto the
sample holder, the upper surface of specimen parallels with
the sample holder.
The data collected range When Cu anode is used, the 2()
region to record the diffraction pattern should be set from 3º
to 60º, sometimes from 1º to 80º.
Quantitative analysis method Commonly used is a standard
curve method, including externa} standard method, interna!
standard method and standard addition method.
In quantitative analysis, one characteristic diffraction peak
should be chosen. In the internal standard method, the linear
relationship should be established between the mass of
interna! standard and the intensity of diff raction peak. The
diffraction pattern of internal standard selected should not
overlap with that of the specimen but have approximately the
same density as the specimen. The standard curve should be
made by mixing different amounts of internal standard with
specimen. The content of specimen being examined should be
in the linear range of standard curve. In the externa! standard
method, linear relationship should be also established
between the mass of reference material and the intensity of
diffraction peak, different amounts of reference materials are
used in the preparation of standard curve. Similarly, the
content of the specimen being examined by the external
method should be in the linear range of standard curve. In
the standard addition method, the peak intensity of the
standard should be clase to that of the specimen.
In quantitative analysis, every specimen should be deter-
mined three times, and the arithmetic average value is taken.
When the phase transformation may be induced by grinding
pressure and resulting in change of diffraction pattern, the
quantitative analysis method should be used cautiously.
When the diff raction number peak and 2() position in the
diffraction patterns of different polymorphs are almost the
same, the number of characteristic diff raction peak should be
increased from 1 to 3-5 to certify the liner relationship
between the content of polymorph and the intensity of
characteristic diffraction peaks.
Under the same experiment condition, the error range of 2 {)
between specimen and reference material is ±O. 2º when the
samples are prepared in the same way and have the same
mass. The error range of peak' s relative intensity between
specimen and reference material is 5 % . Otherwise, the
experiment should be repeated; there is a possibility that the
polymorphic form might exist.
This method is applicable to crystalline analysis, to test the
similarities and differences of specimen and reference
material, to monitor the stability of production process, to
analyze the chemical purity qualitatively and quantitatively,
to identify the crystal form identification, to analyze the
crystal purity quantitatively ( It can be detected in the
diffraction spectrum if the content of an impurity more than
1 % of the substance) , etc.
Instrument calibration It is advisable to calibrate the
instrument periodically with reference material Alz 0 3 or
monocrystalline silicon powder.
 0501 Paper Chromatography
0500 Chromatography
Depending on the mechanism of the separation process,
chromatography can be classified into adsorption chromato-
graphy, partition chromatography, ion-exchange chromato-
graphy, size-exclusion chromatography etc.
Adsorption chromatography is based on the different affinity
of individual components to the adsorbent, so that they can
be eluted successively with a solvent or gas. The adsorbents
commonly used are aluminum oxide, silica gel, polyamide
powder etc.
Partition chromatography is based on the distribution of
individual components between two phases. The stationary
phase is coated on or chemically bonded to a salid support of
large surface area. The mobile phase is a liquid or a gas. The
supports commonly used are silica gel, diatomaceous earth,
florisil ( silicicacid magnesium salt) and cellulose powder
etc.
lon-exchange chromatography is based on the different
exchange capacity of individual components to the resin. In
ion-exchange chromatography, the stationary phase is either
a cation exchange resin or an anion exchange resin. The
mobile phase is usually an aqueous buffer solution,
sometimes a definite quantity of organic solvent is added to
modify the exchange property.
Size exclusion chromatography is also known as gel per-
meation chromatography or gel filtration chroma tography.
lt is based on the diff erent permeability of components of
different molecular size into a support of definite pore size.
Molecular sieves, cross-linked polystyrene gels, glucosan
gels, silica gel and porous glass beads are commonly used as
the stationary phase. Water or organic solvent is used as the
mobile phase, depending on the chemical characteristics of
the support and the substance being examined.
Chromatography may be classified into paper chromatog
raphy, thin layer chromatography, column chromatography,
gas chromatography, high performance liquid chromato-
graphy and so on based on the method of separation.
Solvents used in chromatography must be of high purity and
must not react with the substance being examined. Except
gas chromatography, the operation is carried out at room
temperature, unless otherwise specified.
The separated substance should be detected by the method
specified in the monograph. In column chromatography,
paper chromatography and thin layer chromatography, the
coloured zones can be detected visually, colourless substances
can be detected under ultra-violet radiation of 254 nm or
365 nm. In paper chromatography or thin layer chromato-
graphy, colourless substances can also be detected by
spraying with a visualization reagent. Silica gel plates
containing a fluorescent substance are sometimes used in thin
layer chromatography, so that colourless substance can be
detected by the fluorescence quenching method.
In column chromatography, gas chromatography and high
performance liquid chromatography, the components can be
detected by a suitable detector connected to the outlet of the
column. In column chromatography, the components
sometimes can be determined quantitatively by a suitable
method after fractionation.
0501 Paper Chromatography
Paper chromatography is a partition chromatographic technique,
in which paper is used as a support and the water or other
substance contained in the paper is used as a stationary
phase. The substance being examined is developed by the
mobile phase, after which the retardation factor (Rf) could
be used to denoted the location of each component (Rf value
is defined as the ratio of the distance from the point of
application to the centre of the spot and the distance from the
point of application to the mobile phase front). In practice,
Rf value may vary considerably due to experimental
conditions. Therefore, the identification of a compound is
usually carried out by comparing the behavior of the
compound to be identified with that of a reference substance
under the same conditions. For drug identification, the
position and colour ( or fluorescence) of the main spots of
the substance being examined on the chromatogram should
be identical with those of the reference substances. For
purity inspection, the number or colour intensity ( or
fluorescence intensity) of the impurities contained should be
examined according the requirements specified under the
monograph, after a certain amount of substance being
examined has been developed. For assay, the main
chromatographic spot could be cut out from the paper, eluted
with a solvent and subjected to a suitable measurement.
l. Apparatus and materials
( 1 ) Developing chamber lt is usually a glass chamber,
cylindrical or rectangular, which could be tightly closed with
a gound glass lid. When it is used for descending method, a
separator could be inserted through the lid with a hole on it
for the addition of the mobile phase. A solvent trough
supported by a rack is placed inside near the top of the
chamber, a glass rod is used for holding the chromatographic
paper, on both sides of the trough, there are two glass rods
used to guide the paper. When it is used for ascending
method, the hole on the lid is blocked by a stopper with a
glass hook where the paper is suspended. The hook is
capable of being lowered without opening the chamber.
Remove the trough and rack.
(2) Sample applicator Microsyringes or quantitative capillary
tubes (no burr fin) with a rack are often used, which could
ensure a correct and compact position of the applicated spot.
(3) Chromatographic filler paper Fil ter paper should be
clean and uniform in texture and thickness, and has a tensile
strength. lt should contain no impurities which may affect
developing process or react with visualizing reagent
employed, and affect the separation or identification. lt may
be pretreated befare use if necessary. For descending
method, cut a piece of filter paper along the grain direction
into strips of sufficient length and a convenient width, and
draw a fine pencil line horizontally across the paper at such a
distance from one end to make the line a few centimeters
below the guide rod. When this end is secured in the solvent
trough and the remainder of the paper is hanging freely
outside the trough. The lower end of the paper may be cut

into saw-toothed form to facilitate the mobile phase to move
clown homogeneously, if necessary. For ascending method,
the length of the paper is about 25 cm, the width of the
paper is varied as required. Sometimes the paper can be
rolled into cylindrical form to save space, the spotting line is
about 2. 5 cm from the lower edge of the paper.
2. Procedure
( 1} Descending method Dissolve the substance being examined
in a suitable solvent or solvent mixture to prepare a solution
of specified concentration. Measure the solution by using
microsyringe or quantitative capillary tube, and apply it on
the baseline of application. The amount of the application
should be not more than 10 µl at one time. If the amount of
the application is too big, then apply the solution in portions
on the baseline, allow it to dry in air orina stream of warm
air befare the next application. The spots are commonly
2-4 mm in diameter, and spaced about l. 5-2. O cm apart
from each other in circular shape.
Place the spotted end of paper in the solvent trough and hang
the paper freely over the guide rod. Put a Petri dish
containing the specified solvent or a strip of chromatographic
filter paper moistened with the specified solvent into the
chromatographic chamber to pre-equilibrate the chamber
with the vapour of the specified solvent. Introduce a
sufficient quantity of the mobile phase into the solvent trough
and allow it to move along the chromatographic paper for the
prescribed distance. The filter paper should be protected
from bright light during the developing process. Remove the
chromatographic paper from the chromatographic chamber,
mark the position of the solvent frontal and visualize the
chromatogram prescribed in the monograph when the solvent
is volatilized.
(2) Ascending method The solution of substance being
examined is aoolied on the baseline as directed in the
descending method. The chamber is saturated with the
vapour of a specified solvent or the mobile phase placed in it.
Lower the hook so that the paper is immersed in the mobile
phase to a depth of 1 cm, allow the mobile phase to ascend
for about 15 cm, unless specified otherwise. Remove the
paper from the chamber, mark the position of the solvent
frontal and visualize the chromatogram as prescribed in the
monograph apeer the solvent is volatilized.
One dimensional chromatography is that the development
proceeds along one direction. Sometimes two-dimensional
chromatography may be performed by turning the filter paper
at right angle and then continue the development with the
original mobile phase or a different solvent system. Other
chromatographic techniques, such as multiple development,
continuous development etc. , may also be used.
0502 Thin-Layer Chromatography
The thin-layer chromatography ( TLC) is a separation
technique in which the test solutions are deposited on the
thin-layer plate and developed by the mobile phase in a
chromatographic chamber to separate the components of the
substance being examined. The chromatogram obtained is
compared with that of the appropriate reference substances
under the same conditions. The TLC scanning instrument
can be further used. The result can be used for identifi-
cation, inspection and assay.
l. Apparatus and materials
(1) Plates
According to the types of support materials, the thin-layer
0502 Thin-Layer Chromatography
plates are classified into glass plates, plastic plates and metal
(aluminum) plates. According to the types of stationary
phase, the thin-layer plates are classified into silica gel
plates, chemically bonded silica gel plates, microcrystalline
cellulose plates, polyamide plates and aluminum oxide plates
etc. Binder or fluorescer may be added to the stationary
phase. The most widely used stationary phases are silica gel
G, silica gel GF2s4 , silica gel H, silica gel HF2s 4etc. G or H
indicates the stationary phases with or without gypsum as
binder. F2s4 is a kind of fluorescer which shows green
background under UV light of wavelength 254nm. According
to the particle size of stationary phases, the thin-layer plates
are classified into normal thin-layer plates (10-40 µm) and
high performance thin-layer plates ( 5-10 µm).
Under the premise of ensuring the quality of chromato-
graphy, the plates could be special treated and chemically
modified in arder to meet the separation requirements and the
home-made plates could be used. The particle size of
stationary phases is generally 10-40 µm in diameter. The
surface of the glass plate should be smooth and flat, no bead
on it after being washed.
( 2) Sample applicator Milrosyringe, Calibrated disposable
capillaries or other manual operated, semi-automatic, full
automatic applicators are used generally.
(3) Chromatographic chamber For ascending development,
a chamber of suitable size for the plates, with a flat bottom
or twin trough and a tightly fitting lid, should be used
generally, while for horizontal development, a chamber
specially designed for horizontal development should be used.
( 4 ) Visualization device Glass mister sprayer or special
mister sprayer should be used during spray visualization.
Compressed gas should be used to eject the visualization
reagent in the form of homogeneous mist. Special glass
apparatus or appropriate glass chamber can be used instead
during the immersion visualization. Twin trough glass
chamber or desiccator with appropriately size could be used
during the fumigation visualization.
(5) Detection device It is a camera obscura equipped with
visible light and ultraviolet light of wavelength 254 nm and
365 nm, and corresponding filter. Additional camera
equipment could be used to take picture. The light resource
should have enough intensity of illumination.
( 6) TLC scanning instrument It is used to sean the spot
(s), which can absorb ultraviolet, visible light or emit
fluorescence after being excited. Thus obtained scanning
profiles of the chromatogram and the intergration data can be
used for qualitative and quantitative analysis.
2. Procedure
( 1 ) Preparation of TLC plates
Cmnmercial Pre-coated plates The plates should be usually
activated in an oven at l lOºC for 30 minutes befare use. The
polyamide thin-layer need not be activated. The aluminium
film thin-layer plates and plastic thin-layer plates can be cut
to meet the need. Pay attention to the silica gel layer at the
lower edge of TLC plate, which should not be broken after
cutting. It may be necessary to pre-wash the plates in the
way of ascending development by chloroform, methanol or
the mixed solution of the above two reagents in the
chromatographic chamber if the plates have been polluted by
the impurities in air during storage. The plates are then
activated at llOºC and stored in a desiccator for later use.
Home-made TLC plates Unless otherwise specified,
triturate the mixture of one part of stationary phase and three
parts of water ( or aqueous solution with binder, such as
 . - ;5¡f;; ,i 1 0502 Thin-Layer Chromatography
O. 2%-0. 5% carboxymethyl cellulose sodium solution, or
the modifying agent solution with prescribed concentrations)
in one direction to a homogeneous paste, remove gas bubbles
on the surface, and put the paste in the spreader, move the
spreader smoothly and steadily over the glass plates ( the
thickness of the layer is generally O. 2-0. 3 mm). Allow the
plate to be dried on a horizontal plane at the room
temperature. Heat it at llOºC for 30 minutes and then store
it in a desiccator for later use. Inspect the degree of the
homogeneity under both the reflected light and the
transmitted light, the surface of the plates should be
uniform, flat, smooth without rough portian, gas bubble,
breakage or contamination.
( 2) Sample application Unless otherwise specified, the
samples are applied on the TLC plates, usually in the form of
circular spots or narrow bands, with special capillary or
semi-automatic and automatic sample applicators under a
clean and dry environment. The distance of the sample zone
from the lower edge of TLC plate is 10-15 mm, while that of
HPTLC plates is 8-10 mm. The diameter of the circular
spots is generally not more than 4 mm for TLC, while not
more than 2 mm for HPTLC. Pay attention do not damage
the surface of the thin-layer where the sample applied. The
width of the bands is generally 5-10 mm for TLC, while 4-8
mm for HPTLC. Sample could also be applied by spraying
method with special semi-automatic or automatic sample
applicators. Spots intervals should be adjusted according to
the diffusion of the spots and with no interference between
neighboring spots. Usually, the interval is not less than 8
mm for TLC, not less than 5 mm for HPTLC.
(3) Development Place the plate loaded with sample into
the chromatographic chamber, keeping a distance of about
5 mm from the line of applying sample to the edge of solvent
in the chamber. Close the chamber tightly. Unless otherwise
specified, for ascending development, usually 8-15 cm will
be developed for TLC, while 5-8 cm for HPTLC. When the
mobile phase has moved over the prescribed development
distance, remove the plate from the chamber, mark the
mobile phase front and dry it for later detection.
If the chamber should be pre-equilibrated before develop--
ment, add appropriate amount of mobile phase into the
chromatographic chamber. Close the chamber tightly and
usually maintain for 15-30 minutes. After the vapor of
solvent has reach equilibrium, place the plate loaded with
samples quickly into the chamber and close it immediately
tightly for development. If the vapor of the solvent should be
saturated in the chromatographic chamber, filter paper of the
same dimension with the inner diameter of the chamber
should be laid on the inner wall of the chamber and one end
of the filter gaper is immersed into the mobile phese. Keep
the chamber tightly for a while and allow it to be fully
saturated before developing.
If necessary, secondary development and two-dimension
development could be applied. The residual mobile phase on
the thin-layer plates should be evaporated before the second
development.
( 4) Visualization and detection The coloured substances
could be detected directly under visible light. The colourless
substancs may be visualized with appropriate visualization
reagent by the way of spraying or immersion. They could
also be visualized by being heated and detected directly under
the visible light. The fluorescence spots of the fluorescent
substances or the substances which may excite fluorescence
may be visualized under the UV lamp ( 365 nm or 254 nm).
The silica gel plates containing fluorescer ( such as silica gel
GF2s4 plates) could be used if the components have
ultraviolet absorption. The spots formed by the quenching of
the fluorescent substance on the plate can be observed under
the UV lamp (254 nm).
( S) Documentation The image of the TLC could be taken
by camera and saved in the form of optical picture or
electronic image. The TLC scanning imstrument and other
suitable method could also record the corresponding
chromatogram.
3. System suitability test The system suitability test should
be carried out for the experiment conditions according to the
requirements specified under the monograph. The test
solution and the reference solution are used to test and adjust
the experiment condition in order to meet the prescribed
requirements.
( 1 ) The retardation factor (R r) lt is defined as the ratio
of the distance from the point of application to the center of
the spot and the distance from the point of application to the
mobile phase front.
the distance from the point of application
R _ to the center of the spot
e - the distance from the point of application
to the mobile phase front
Unless otherwise specified, when impurities are tested, for
spots of impurities, the retardation factor (Re) should be
controlled within O. 2-0. 8.
( 2) Detection Limit It is the minimum concentration or
amount of the substance being examined which can be
detected in the test solution in limit test or impurity test.
Usually the test solution or reference standard solution with
known concentrations, and series dilute self-control reference
standard solution are applied, developed and visualized on the
same TLC plate under the prescribed chromatographic
conditions. The concentration or amount of the reference
standard solution when clear and distinguishable spot is
exhibited is the detection limit.
(3) Resolution ( or Separating power) For identification
testing, the main spot (s) in the reference solution and in
the test solution should be clearly separated. For limit test or
assay, the peak used for quantification should be well
separated with the adjacent peak. The formula for calculating
the resolution (R) is:
R = 2(d2 -di)
(W1 +W2)
Where d 2 is the distance between the latter of the two
adjacent peaks and the original point;
d 1 is the distance between the former of the two
adjacent peaks and the original point;
W1 and W2 is the peak width of the two adjacent
peaks respectively.
Unless otherwise specified, the resolution should be more
than l. O.
For the selection of the impurities test methods of chemical
drug, the reference substance of the impurity may be dis-
solved with the diluted self-control solution to prepare the mixed
reference solution The reference substance of the impurity may
also be dissolved with the reference solution of the substance
being examined to prepare the mixed reference solution The
mixed reference solution may be obtained by an appropriate
degradation method of the substance being examined . In the
chromatogram developed after solutions described above are
applied, clearly separated spots should be exhibited.
( 4) Relative standard deviation
For the assay by TLC Scanning method, when the same test

solution is applied in parallel on the same thin-layer plate,
the relative standard deviation of the peak areas of the
ingredients being examined should be not more than 5. O%.
The relative standard deviation for those which should be
determined after visualization or on different plates should be
not more than 10. 0%.
4. Measurement
( 1) ldentification The reference solution and the test
solution of appropriate concentration are prepared, applied,
developed and visualized on the same TLC plate with the
method prescribed under the monograph. The colour ( or
fluorescence) and the position of the main spot ( s) of the
test solution should be identical with those of the spot ( s) of
the reference solution. If necessary, the test solution and the
reference solution may be mixed, applied and developed for
chemical drugs. Compared with the corresponding spot of
the reference solution, the spot should be single and
compact.
(2) Limit test and lmpurity test The reference solution and
the test solution of appropriate concentration are prepared,
applied, developed and visualized on the same TLC plate
with the method prescribed under the monograph. The
colour Cor fluorescence) of the spot being examined in the
chromatogram of the test solution should be not more intense
compared to the corresponding spot of the reference solution.
Or, the peak area may be determined according to the
method of TLC scanning and the peak area of the
corresponding spot in the chromatogram of the test solution
should be not more than the peak area of the reference
solution. The content limit test should be determined
according to the requirements.
The impurity reference solution, the diluted test solution or
both can be adopted for impurity test for chemical drugs.
The spots other than the principal spot of the test solution
should not be more intense than the corresponding spot of the
impurity reference solution or its series solution, or the
corresponding spot of the diluted test solution or its series
solution, when comparison is made. The number of the
impurity spots and the amount of the single impurity should
be specified. The total content of impurities may be
estimated using series diluted reference solutions.
( 3 ) ~y According to the TLC scanning method, the
reference solution and the test solution of appropriate
concentration are prepared, applied, developed and scanned
on the same TLC plate with the method prescribed under the
monograph. Or, determine with suitable method after the
chromatogram spot is scarped and eluted.
S. TLC scanning method It is to sean the spots, which can
absorb ultraviolet light or visible light or which can emit
fluorescence after being excited, exposing the TLC plate to
light of a certain wavelength. Thus obtained chromatograms
and integral data are used to identify, test or assay.
According to the structural characteristics of different scanning
instrument, the spots may be scanned and determined based
on specified method. Generally, reflective scanning mode in
the absorbance method or fluorescence method is freqrently
used. Unless otherwise specified, the commercial pre-coated
TLC plates should be used in the assay.
Single-wavelength scanning or dual-wavelength scanning
method could be used. For dual-wavelength scanning, the
wavelength at which the spots being examined have no
absorption or the mínimum absorption should be used as a
reference wavelength. In the chromatogram of the test
solution, the retardation factor ( Rf) of the spots being
examined the absorption spectrum obtained from the
;.:··: .:·
.:,sa··
, .... .' ..,,.,
0511 Column Chromatography
11
spectrum scanning or the obtained maximum absorption and
minimum absorption of the spectrum should be identical with
those of the reference solution to ensure the accuracy of the
determination results. For the quantitative analysis by using
the TLC scanning method, it should be ensured that the
amount of the spot being examined is within the linear range.
If necessary, the amount of the test solution being applied on
the plate could be adjusted appropriately. The test solution
should be applied, developed, scanned, measured and
calculated on the same plate with the reference solution.
When the TLC scanning method is used for assay, linear
regression two-point method is usually adopted for
calculation. If the linear range is very narrow, multinomial
regression of multiple level of standard calibration could be
used. The test solution and the reference solution should be
applied alternatively on the same TLC plate. The number of
the spots of the test solution should not be less than 2 and
that of the reference solution should not be less than 2 for
each concentration. Sean the plate along the direction of
development, but not horizontally.
0511 Column Chromatography
l. Adsorption colwnn chromatography
The chromatographic tube is made of borosilicate glass with
uniform internal diameter. A sintered glass disk or a wad of
glass wool is placed at the contracted lower end of the tube to
keep the adsorbent in the column. Adsorbents of uniform
size are required in arder to get good seperation. Unless
otherwise specified, adsorbents of a uniform size O. 07-0. 15
mm in diameter are commonly used. The size of the tube,
the type and quantity of the adsorbent to be used, and the
composition and flow rate of the mobile phase are specified
under individual monograph.
( 1) Packing the adsorbent into the colwnn The column can
be packed either by the dry method or the wet method.
CDDry method Fill the chromatographic tube with the dry
adsorbent, tap the tube gently to form a compact column,
then add the mobile phase along the inner wall of the tube.
Alternatively, the chromatographic tube can be filled with
the mobile phase first, and then the adsorbent added in small
portions until a homogeneous bed of the adsorbent is formed.
During the whole process, the upper surface of the adsorbent
must be covered by a layer of the mobile phase.
@Wet method Mix the adsorbent with the mobile phase,
agitate to expel air bubbles and slowly add the mixture as a
slurry into the chromatographic tube. Wash clown the
adsorbent adhering to the inner wall of the tube with the
mobile phase, open the outlet of the tube and allow the
mobile phase to flow slowly until the surface of the liquid is
almost on the same level as the top of the column. The
column is now ready for loading of the substance being
examined.
( 2) Loading the substance being examined Unless otherwise
specified, the substance being examined is dissolved in the
mobile phase and added slowly along the inner wall of the
tube, care should be taken that the adsorbent is not
disturbed. The substance being examined can also be
dissolved in a suitable solvent, mixed thoroughly with a
small quantity of the adsorbent and added on top of the
column when the solvent is volatilized. If the substance being
examined is insoluble in the common solvents, mix it with
the adsorbent in a mortar and add the mixture on top of the
column.
 0512 High Performance Liquid Chromatography
( 3) Elution It is usually done by changing the type or the
composition of the mobile phase in the sequence of increasing
order of elution strength, unless otherwise specified. Collect
the eluate fractionally until the component disappears from
the eluate or the amount eluted decreases markedly, then
change the mobile phase and continue the elution as described
above. It is important to keep sufficient mobile phase on top
of the adsorbent during the whole process.
2. Partition column chromatography
The procedure is similar to that of adsorption column
chromatography. The support, mixed with the liquid
stationary phase, is added into the chromatographic tube in
small portions, pressed with the flat end of a glass rod after
each addition. The substance being examined may be
dissolved in the liquid stationary phase, mixed with a small
quantity of the support, and then loaded on top of the
column. The mobile phase is saturated with the liquid
stationary phase previously so as to prevent the change of
distribution coefficient or degree of partition between two phases
during the elution process.
0512 High Performance
Liquid Chromatography
High performance liquid chromatography ( HPLC) is a
method of chromatographic separation of the substance being
examined, in which the mobile phase is pumped into a
column packed with the stationary phase by a high-pressure
pump system. The test solution injected is carried into the
column by the mobile phase. All the components are
separated in the column and flow through the detector. The
integrator or data acquisition system thus records the
chromatographic signals.
l. General requirements for apparatus and chromatographic
conditions
The apparatus consists of a high-pressure pumping system,
an injector, a chromatographic column, a detector anda data
acqmslt1on system. Generally, the interna! diameter of
columns is 3. 9-4. 6 mm and the particle size of the stationary
phases is 3-10 µm. Ultra-high performance liquid chromato-
graphy, with ultra-high pressure resistance, small injection
volume, low hold-up volume and high sensitivity detection,
is suitable for the column packed with small particle size
(about 2 µm) of stationary phase.
( 1) Chromatographic column Reversed-phase chromato-
graphic column: the column packed with stationary phases
using bonded non-polar groups as supports. The most widely
used of supports are silica gel, polymer composite silica gel and
polymers etc, and commonly used packing materials include
octadecyl silane bonded silica gel, octylsilance bonded silica gel and
phenyl bonded silica gel etc.
Normal-phase chromatographic column: the column packed
with stationary phases of silica gel or bonded polar group
silica gel. The most widely used of stationary phases include
silica gel, amino group bonded silica gel and cyano group
bonded silica gel. Amino group and cyano group bonded
silica gel can also be used in reversed-phase chromatographic
column.
Ion-exchange chromatographic column: the column packed
with ion-exchanged stationary phases, including cation
exchange chromatographic column and anion exchange
chromato graphic column.
Chiral chromatographic column: the column packed with
chiral stationary phases.
The interna! diameter and the length of columns, the shape,
the particle size and distribution, the pare diameter, the
surface area, the surface coverage of bonded functional
groups and the residual surface groups of stationary phases,
the density and uniform of packing, etc. affect the
performance of columns. The appropriate chromatographic
column should be selected based on the characteristics of the
substance being separated.
The temperature can affect separation. Unless otherwise
specified in the monograph, the temperature of columns
refers to room temperature and the effect of room
temperature change should be paid attention to. Columns
may be heated to give higher separation efficiency. It is
recommended that columns should not be heated above 60ºC.
Far silica gel columns with residual unblocked silicon
hydroxyl group, the pH value of the mobile phase is
generally controlled within 2-8. Far columns with residual
blocked silicon hydroxyl group, or polymer composite silica
gel or polymers, the mobile phase with more extensive pH
value is employed. The mobile phase with pH value less than
2 or more than 8 is preferred.
(2) Detectors Ultraviolet-visible CUV) spectrophotometry
detector, including diode arra y detector, is the most
commonly used detectors. Fluorescence spectrophotometry
detector, evaporative light scattering detector, differential
refractometer, electrochemical detector, mass spectrometry
detector may also be used.
UV spectrophotometry detector, fluorescence spectrophoto-
metry detector and electrochemical detector are all selective
detectors. The response value is related to the quantity of the
substance being examined as well as its structure. Evapo-
rative light scattering detector and differential refractometer
are general-purpose detectors, which respond to all compounds.
The response value of evaporative light scattering detector for
compounds with similar structure is almost only related to
the quantity of the substance being examined.
The response values of the UV spectrophotometers, fluores-
cence spectrophotometers, electrochemical detectors and
differential refractometers are linear with the amount of
substance being examined within a certain range, while the
relationship between the response value of the evaporative
light scattering detector and the amount of the substance
being examined is usually exponential. Therefore it should be
logarithmically transferred befare calculation.
Different detectors have different requirements far the mobile
phase. The mobile phase used in UV-vis spectrophotometry
detector complies with the requirements far solvents under
Ultraviolet-visible Spectrophotometry ( 0401 ) . When the
light of short wavelength is applied for detection, the cut-off
wavelength of the organic solvents should be considered and
the organic solvent with chromatographic grade should be
used. The mobile phase containing non-volatile salts cannot
be used far evaporative light scattering detector and mass
spectrometry detector.
( 3 ) Mobile phase In reversed-phase chromatographic
system, the preferred mobile phase is methanol-water
system and acetonitrile-water system. In case of using the
ultraviolet end of the wavelength detection, acetonitrile-
water system is used as priority. The mobile phase containing
buffer salts should be used as little as possible, if necessary,
the low concentration of buffer salts may be used. When a
column packed with octadecylsilane bonded silica gel is used,
the concentration of the organic solvents in the mobile phase
is not less than 5 %. Otherwise it easily results in the decline
of column efficiency and instability of the chromatographic
system.
 os12
Two or more than two organic solvents such as dichlorome-
thane and n-hexane etc. are commonly used as the mobile
phase for normal-phase chromatographic system.
The type of the stationary phase, the composition of the
mobile phase and the mode of detector specified in the
monograph should not be changed, other items, including
internal diameter and length of column, particle size of the
stationary phase, flow rate and ratio of components in the
mobile phase, temperature of column, injection volume and
the sensitivity of detector, can be changed appropriately to
meet the requirements of the system suitability test. When
the ratio of components in mobile phase is adjusted, if the
percentage of X component with small proportion is not more
than 33 % , the range of O. 7X-1. 3X is val id; if the percentage
of component X is more than 33%, the range of X±lü% is
valid.
The small particle size ( 2 µm) of the stationary phase used
should be compatible with the properties of the pumping system,
injection volume, volume of the detection pool and hold-up
volume, of the system the chromatographic conditions should be
adjusted correspondingly if necessary. The determination result
obtained under the chromatographic conditions described in the
monograph is valid if dispute is generated.
The column with specified brand being used should be stated
clearly in the monograph in order to satis{y the requirements
of separation.
2. The system suitability test
The suitability test of the chromatographic system generally
includes five parameters, which are the number of theoretical
plates, resolution, sensitivity, tailing factor and repeatability.
Carry out the system suitability test in accordance with the
requirements specified in the monograph, using specified
reference solution or system suitability test solution under
the specified chromatographic conditions, which could be
adjusted to compiy with the requirements if necessary.
( 1 ) Number of Theoretical plates of the column ( n ) 1t is
used to evaluate the separation performance of columns. Due
to different chromatographic behaviors of diff erent subs-
tances on the same column, using the number of theoretical
plates as the parameter to evaluate the column performance,
the substance being examined should be stated. Number of
theoretical plates usually refers to that of the substance being
examined or the internal standard.
lnject the test solution or the internal standard solution specified
in the monograph into the column under the prescribed
chromatographic conditions. Record the chroma-togram and
measure the retention time lR and width or width at half-height
(Wh;2) of the principle peak obtained with the test solution or
the internal standard solution. Calculate number of theoretical
plates of the column, using the following equation:
n = 16(tR/W) 2 or n =5. 54(tR/Wh;2 ) 2
The values of lR, W and Wh; 2 can be expressed in minutes or
in length ( same as below) but must be expressed in the
same units.
(2) Resolution (R) It is used to evaluate the resolution
between the substance being examined and the substance
being separated and it is a key parameter for evaluating the
separation performance of chromatographic system. The
separation performance can be evaluated and adjusted by the
resolution between the substance being examined and known
impurities, or by the resolution between the substance being
examined and a certain index component ( such as the
internal standard or other substance being separated with
difficultly), or by the resolution between the substance being
examined and a certain degraded product obtained with the
High Performance Liquid Chromatography ls. . '.' f j ·•.: ..
degradation of the substance being examined or the reference
substance using an appropriate method.
No matter in qualitative or quantitative determination, good
resolution between peaks of the substance being examined
and the internal standard or specified impurities or other
substances should be achieved. Unless otherwise specified,
the resolution between the peak of the substance being
examined and the adjacent peaks is more than l. 5. Calculate
the resolution using the following equation:
R = 2 X (tRZ - lR1) or R = 2 X (tR2 - lR1)
+
W1 W2 +
l. 70 X CWi. h/2 W2, h/2)
Where lRZ is the retention time of the latter of the two
adjacent peaks;
lR1 is the retention time of the former of the two
adjacent peaks;
W1 , W2 , W1, h/2, W2, h/2 are the peak width and peak width
at half height of the two adjacent peaks separately (as in
Fig. below).
Fig.
Number of theoretical plates of the column ( n) and
resolution (R ) are calculated as peak width ( W) if the
determination result is disputed.
(3) Sensitivity It is used to evaluate the capability of the
chromatographic system for determining minute substances,
usually expressed in signal-to-noise ratio ( S / N) , which is
obtained by determining the test solution or the reference
solution with a series of different concentrations. For quanti-
tative determination, signal-to-noise ratio is not less than 10;
for qualitative determination, signal-to-noise ratio is not less
than 3. The sensitivity test solution is used for evaluating the
detection capability of the chromatographic system.
(4) Tailing factor (T) It is used to evaluate the symmetry
of chromatographic peaks. Calculate the tailing factor using
the following equation:
T= Wo.osh
2d1
Where Wo.osh is the peak width at 5 % of the peak height;
d1 is the distance between the perpendicular
dropped from the peak maximum and the
leading edge of the peak at 5 % of the peak
height (as Figure).
 0512 High Performance Liquid Chromatography
Unless otherwise specified, the T value should be within
O. 95-1. 05 by using peak height as the quantitative parameter.
Using peak area as the quantitative parameter, general peak
tailing or fronting <loes not affect the integration of peak
area. But serious peak tailing will affect the judgment of
baseline and the front and the end of peak as well as the
accuracy of the integration of peak area. Thus tailing factor
should be stated in the monograph.
( S) Repeatability It is used to evaluate repeatability of the
response value of chromatographic system for consecutive
injections. Using the extemal standard method, inject the
reference solution described in the monograph for 5 times
consecutively, unless otherwise specified, the relative
standard deviation of peak area measurement is not more
than 2. 0%. Using intemal standard method, prepare a
series of reference solutions equivalent to 80%, 100% and
120 % of the concentration of the substance being examined,
add specified volume of the interna} standard solution
separately to produce solutions with three different concen-
trations, inject each solution at least twice, calculate the
average correction factor and corresponding relative standard
deviation is not more than 2. 0%.
3. Procedure
( 1 ) Interna) standard method Prepare solutions containing
an accurately weighed quantity of the reference substance and
the internal standard respective as specified in the
monograph. Measure an accurately volume of each solution,
mix well to produce the reference solution used for
determination of the correction factor. Inject a certain volume
of solution into the equipment and record the chromatogram.
Measure the peak area or the peak height of the reference
substance and that of the internal standard, calculate the
correction factor, using the following equation:
. As/Cs
Correctlon factor (f) = AR/CR
Where As is the peak area or peak height of the interna!
standard;
AR is the peak area or peak height of the reference
substance;
Cs is the concentration of the internal standard
solution;
CR is the concentration of the reference solution.
Inject the test solution containing the internal standard as
described in the monograph and record the chromatogram.
Measure the peak area or the peak height of the substance
being examined and that of the intemal standard. Calculate
the content, using the following equation:
Ax
Content (Cx) = f X As' /Cs'
Where Ax is the peak area or peak height of the substance
being examined;
Cx is the concentration of the solution of the
substance being examined;
A~ is the peak area or the peak height of the
internal standard;
c ~ is the concentration of the internal standard
solution;
f is the correction factor.
Using the internal standard method, the effects on
determination result due to sample pre-treatment and
injection volume errors can be avoided.
( 2) External standard method Prepare solutions containing
an accurately weighed quantity of the reference substance and
the substance being examined respectively. lnject a certain
volume of each solution and record the chromatograms.
Measure the peak area or peak height of the substance being
examined in the reference solution and the test solution
separately. Calculate the content, using the following
equation:
Ax
Content (Cx) = CR X -
AR
Where each symbol has the same meaning as mentioned
abo ve.
As the microsyringe is not easy to control the injection
volume precisely, if using external standard method, the
manual fixed-loop or automatic sampler may be used.
( 3 ) Corrected peak areas of impurities compared with that
produced by the main peak of the substance being examined
This method is used for determination of contents of
impurities. In the establishment of a method, prepare
solutions containing an accurately weighed quantity of the
reference substances of impurities and the reference
component as specified in the monograph, mix well to
produce the solution for determination of the correction
factor. Inject a volume into the column and record the
chromatogram. Calculate the correction factor of impurities
being examined, using the following equation:
. CA/AA
Correct1on factor= CB/AB
Where: CA is the concentration of the solution of
impurities being examined;
AA is the peak area or peak height of impurities
being examined;
C8 is the concentration of the reference com-
ponent solution;
AB is the peak area or peak height of the
ref erence componen t.
Also prepare solutions containing an accurately weighed
quantity of the main component CRS and impurity CRS with
different concentrations. Inject a volume and record the
chromatogram, plot regression curves of the concentration of
the main component and impurities to corresponding peak
areas. Calculate the correction factor, using the ratio of
regression line slope of the main component to that of
impurities.
The correction factor can be directly stated in the monograph
and it is used for correction of the measured peak areas of
impurities. The value of impurity to be corrected should be
stated in the monograph, using the main component as the
reference component and the relative retention time as the
location.
To determine the content of impurity, dilute the test solution
to equivalent concentration of impurity limit solution, in
accordance with the impurity limit indicated in the
monograph, use as the reference solution. lnject a volume
and record the chromatogram. If necessary, adjust the
ordinate range ( limited by the noise level that can be
accepted) until the peak height of the main component
obtained in the reference solution is about 10 %-25 % of the
full scale. Unless otherwise specified, for the impurity with
content less than O. 5%, relative standard deviation (RSD)
of the peak area should be less than 10 %; for the impurity
with content within O. 5%-2%, RSD of the peak area should
be less than 5 %; for the impurity with content more than
2 %, RSD of the peak area should be less than 2 %. lnject a
volume of the test solution and the reference solution into the
column separately, unless otherwise specified, the record
time of the test solution is twice the retention time of the
principle peak. Measure the peak areas of all impurities on
the chromatogram obtained with the test solution, multiply
by the corresponding correction factor separately, with

respect to the peak area of the main component obtained with
the reference solution, calculate the content of impurities.
( 4) Peak areas of impurities compared with that produced by
the main peak of the substance being examined
If correction factor of the impurities being examined is not
achieved or is disregarded, this method is used to determine
the content of impurities. Prepare the reference solution as
indicated in above Method 3. Adjust the ordinate range and
calculate RSD of peak area. Inject separately an appropriate
volume of the test solution and the reference solution into the
column, unless otherwise specified, the record time of the
test solution is twice the retention time of the peak of the
main component. Measure the peak areas of all impurities
obtained in the test solution with respect to the peak area of
the main component obtained in the reference solution,
calculate the content of impurities.
( 5) Peak area nonnalization method
Prepare the test solution as specified in the monograph.
Inject a certain volume into the column and record the
chromatogram. Measure the peak area of each peak and all
peaks other than solvent peak. Calculate the percentage of
each peak area in the total area of all peaks. Due to linear
limit of apparatus response, the peak area normalization
method is not suitable for determination of minute impurity.
0513 Ion Chromatography
Ion chromatographic ( IC) is a method of chromatographic
separation of the dissociated substances using a high-pressure
pumping system in which the specified eluent is pumped into
a column packed with the stationary phase. The components
to be examined are separated on the stationary phase and each
component passes through a detector ( Pass through a
suppressor or a post-column derivatisation system, if
necessary) , then a chromatogram is thus recorded by the
recorder or a data processing system. IC can be applied to
qualitative and quantitative determination of inorganic anions
and cations, organic acids, sugar alcohols, aminoglycosides,
amino acids, proteins, glycoproteins and other analytes etc.
The principie separation mechanism is ion-exchange, based
on a reversible exchange between ions dissociated from ion
exchange resin and those with the same charges from salutes
in the eluent. Other separation mechanisms of IC include ion-
pair and ion-exclusion etc.
l. General requirements for apparatus
Tubing and other parts that are directly in contact with
eluents and samples should be made of inert materials such as
polyetheretherketone ( PEEK ) . High performance liquid
chromatography instruments can be used, as long as its parts
of the instruments adapt to the eluent and sample solution.
Instruments should be periodically validated according to the
requirements.
( 1) Column There are two types of packing materials used
as stationary phase, organic polymers-based and silica gel-
based. Organic polymers are predominately used as support
materials for IC, such as styrene-divinylbenzene copolymers,
ethylstyrene-divinylbenzene copolymers, polymethacrylate
and polyvinyl resins etc. By chemical reaction, the surface of
the support materials is bonded with latex-particles con-
taining a high amount of anion-exchange functional groups
(such as alkyl quaternary ammonium group, alkanol quater-
nary ammonium group etc. ) or cation-exchange functional
groups ( such as sulfonic group, carboxylic group, carboxylic-
phosphoric group, carboxylic-phosphoric-crown ether group
0513 Ion Chromatography
etc. ) which can be used for exchange of anions or cations,
respectively. Organic polymer based packing materials have a
high stability in a wide pH range (0-14) and are tolerated of
corrosive by organic solvents.
lnorganic packing material usually refers to silica gel-based
support material. The surface of silica gel is chemically
bonded with anion-exchange groups such as quaternary
ammonium groups or cation-exchange groups such as sulfonic
groups, carboxylic groups etc, which is used for the
separation of anions or cations, respectively. Silica gel based
support materials have good mechanical stability and do not
swell or shrink in organic solvents. The silica gel based
packing materials are stable in eluents at pH 2-8, and
typically used for the separation of cations.
( 2) Eluent The separation of complicated analytes by IC
relies mainly on the packing materials, the eluent is simple.
Diluted alkaline solvents and carbonate buffers are the typical
eluent for separation of anions, while diluted methanesulfonic
acid solution for the separation of cations. The separation
strength of eluents can be adjusted by changing the pH value
or ion strength. By adding a portian of organic modifiers
such as methanol and acetonitrile, the peak shape can be
improved . The deionized water used for preparation of
eluents should be purified and the electrical resistivity should
be more than 18 Mn •cm. The eluent should be degassed,
typically degassed on-line with helium, or by off-line
ultrasonication, vacuum filtration and refrigeration.
(3) Detector Conductivity detector is the most commonly
used detector in IC. Other detectors in IC include UV
detector, amperometry detector, evaporative light scattering
detector etc.
The conductivity detector is commonly used in the testing of
inorganic anions, inorganic cations, and sorne polar organic
compounds such as carboxylic acid. In suppressed IC, the
ionic eluent with high conductivity is converted to solution
with lower conductivity by the suppressor, therefore the
sensitivity is significantly increased.
Amperometry detector is usually used in the testing of
compounds with weak ability of dissocaiton and property of
oxidation or reduction. Direct current amperometry detector
is applied in the testing of 1- , SCN- and other phenolic
compounds. lntegration and pulsed amperometry detector is
applied in the testing of carbohydrates and amino acids.
UV detector is suitable for testing trace amount of Br-,
N02, N03 in presence of high concentration of c1-, and
other compounds with strong UV absorption. Post-column
derivatization followed by UV/VIS detection mode is applied
to determine transition-metal ions and Lanthanide-metal
ions.
AAS, AES ( include ICP-AES), MS ( include ICP-MS)
detection can also be employed as IC detectors. When ELSD
or (and) MSD are coupled with IC, a suppressor is usually
equipped in the system.
2. Sample preparation
The clarified aqueous samples in simple matrix should be
diluted, filtered with O. 45 µm membrane and subsequenthy
directly injected into system. Far samples with complicated
matrix, remove the interference substances using microwave
digestion, UV degradation, solid-phase extraction techniques
and then inject into the system.
3. System suitability test
Complies with the requirements for High Performance Liquid
Chromatography ( 0512).
4. Procedure
( 1) lnternal standard method
 0514 Size-Exclusion Chromatography
(2) External standard method
(3) Peak area normalization method
The above method ( 1 ) - ( 3 ) are the same as described
under High Performance Liquid Chromatography ( 0512).
( 4) Calibration curve method
Weigh accurately a quantity of the reference substance and
prepare stock solution according to the method specified in
the monograph. Measure a volume of stock solution and
prepare a series of reference solutions with gradient
concentrations. Inject an appropriate volume of the reference
solutions into the chromatograph, record chromatograms,
and measure the peak area or peak height of the components
being examined. T aking the peak area or peak height as the
vertical coordinate, the concentration of the reference
solution as the horizontal coordinate, the calibration curve is
calculated by regression the formula as follows:
AR=aX cR+ b
Where AR is the peak area or peak height of the
components being examined in the reference
solution;
CR is the concentration of the reference
solution;
a is the slop of the standard curve;
b is the intercept of the standard curve.
Inject the sample solution described in the monographs,
record the chromatograms, and measure the peak area or
peak height of the components being examined, calculate the
concentration with the linear equation, using the formula:
As-b
Cs=---
a
Where As is the peak area or peak height of the com-
ponents being examined m the sample
solution;
Cs is the concentration of the sample solution;
The meanings of a and b are the same as that described
above.
The external standard method and calibration curve method
are the most commonly used methods.
0514 Size-Exclusion Chromatography
Size-exclusion chromatography is a liquid chromatographic
technique that separates molecules in solution according to
their size. The separation of size-exclusion chromatography
is based on molecule sieve mechanism of the gel column.
Hydrophilic silica gel, gel or modified gel, such as sephadex
and sepherose, etc. , are usually used as the packing material
of the column. The different size pares are distributed in the
surface of the packing material. When the sample is injected
into the column, the molecules apparently larger than the
maximum pare size of the packing material migrate along the
column only through the spaces between the particles of the
packing material without being retained and elute earliest by
the mobile phase, their retention time is the shortest. The
molecules which are smaller than all pares size penetrate all
the pare spaces and elute latest, their retention time is the
longest. Other molecules elute through the column in
sequence according to their molecule size.
l. General requirements for apparatus
The injector and detector are as same as those far High
Performance Liquid Chromatography ( 0512 ). Pumping
systems typically include normal pressure pump, middle
pressure pump and high pressure pump. High performance
size-exclusion chromatography ( HPSEC) is the most
frequently adopted in pharmaceutical analysis, especially in
determination of molecular weight and molecular weight
distribution. The packing material adopted should be
compatible far the molecular weight of samples. The mobile
phase is usually aqueous solution or buffer solution, the pH
value of which should not exceed tolerance of the packing
material and is better between 2 and 8. A suitable quantity of
organic modifier can be added in the mobile phase, but the
concentration should not be high and usually not exceed
30 %. Flow rate should not be fast and is usually O. 5-1. O ml
per minute.
2. System suitability test
Generally the determination method of the number of
theoretical plates ( n), resolution, repeatability and tailing
factor of the column far system suitability test is the same as
the method stated in High Performance Liquid Chroma-
tography ( 0512 ). But if the monomer and dimer of sorne
drug molecule cannot be separated by baseline during
determi-nation of macromolecule impurities, resolution is
calculated by the following formula:
R = peak height of dimer
valley height between monomer and dimer
Resolution is more than 2. O, unless otherwise specified m
the monograph.
3. Procedure
( 1 ) Determination of molecular weight The method is
generally suitable far determination of molecular weight of
protein and peptide. Carry out the method stated in the
monograph, using a column and reference substance suitable
for the molecular weight of sample. Reference substance and
substance being examined should both be processed by
dithiothreitol (DTT) and sodium lauryl sulfate (SDS) in
arder to break disulfide bond and make the molecule confi-
guration and conformation concord. Usually the processed
protein and peptides which have been turned to a straight line
are separated. Plot a graph of the retention time of the
reference substance as a function of the logarithm molecular
weight and calculate the equation of linear regression,
lgMw =a + btR. Calculate molecular weight or subunit
molecular weight using the regression equation.
( 2) Detennination of molecular weight ami molecular weight
distribution of biopolymers Molecular weight of biopoly-
mers, such as amylose, nucleic acid and collagen, is usually
unhomogeneous. Molecular weight and molecular weight
distribution of the biopolymer is a key index. To determine
the molecular weight and molecular weight distribution of
biopolymers, it is important to adopt reference substances
with similar structures and properties to the samples.
Carry out the method stated in the monograph, unless otherwise
specified, use the molecular weight reference substance and
suitable GPC software, plot a graph of the retention time of the
reference substance as a function of the logarithm of weight-
average molecular weight and calculate the equation of linear
regression lgMw=a +btR. Process the result with suitable GPC
software and calculate the molecular weight and molecular weight
distribution of substance being examined.
Mn = LiRL/Li (RJ¡/M¡)
Mw= °Li(Rl¡/M¡)/°LiRf¡
D=Mw/Mn
Where Mn is the number-average molecular weight;
Mw is the weight-average molecular weight;
D is the distribution coefficient;
Rl; is the peak height of substance being examined
at retention time i ;
M; is the molecular weight of substance being
examined at retention time i.

( 3 ) Determination of macromolecule impurities Macro-
molecule impurities are the impurities with higher molecular
weight than drug molecule, which are produced during
procedure of manufacture or storage and cannot be removed
completely ( sensitizing polymers).
Separation is carried out according to the chromatographic
condition stated in the monograph.
Quantitative method
©Peak areas of impurities compared with those produced by
the main peak of the sample See the section of High
Performance Liquid Chromatogram ( 0512 ). The method is
typically used far determination of macromolecule impurities
with low content in the samples.
@Peak area nonnalization method See the section of High
Performance Liquid Chromatogram ( 0512).
@Retention time limit method Unless otherwise specified,
it is specified that components with shorter retention time
than that of the main peak are not allowed to be detected.
This method is typically used to control macromolecule
impurities in the mixture.
@ External standard method by the substance to be examined
This method is typically used far determination of macro-
molecule impurities in ,B-lactam antibiotics by Sephadex G-10
gel chromatography system. Except far sorne oligomers,
macromolecule impurities in ,B-lactam antibiotics are not
retained in this system and only appear to be single peak.
Calculate content of the macromolecule impurities by external
standard method, using the substance to be examined as
reference substance.
[Appendix] Processing method for Sephadex G-10
Packing cllromatographic colmnn Soak about 15 g of Sephadex
G-10 with water far 48 hours befare packing and allow it
swelling thoroughly, stir to expel air bubbles. Slowly add
the mixture as a slurry into the chromatographic tube or
other column with suitable material. Wash clown the
Sephadex G-10 adhering to the inner wall of the tube with
water, smooth the surface of the column. Newly packed
column should be eluted with water far 4-6 hours to expel air
bubbles.
Loading the substance being examined Both an automatic
inject valve and manual loading can be adopted to load the
substance being examined into the column. Add the test
solution slowly along the inner wall of the tube, care should
be taken that the packing material is not disturbed. Wash
clown the samples adhering to the inner wall of the tube with
3-5 ml of the mobile phase af ter substance being examined
solution permeate through the column surface.
0521 Gas Chromatography
Gas chromatography ( GC) is a separation technique in
which the mobile phase, an inert gas known as carrier gas, is
passing through the chromatographic column packed with
packing materials far separation and determination. The
substance or its derivatives are injected into the vaporizer
with a micro-syringe and vaporized, separated on the stationary
phase, each component passes through the detector in
succession and a chromatogram is thus recorded by data
acquisition system.
l. General requirements for apparatus
The apparatus consists of a carrier gas source, an injector, a
chromatographic column contained in an oven, a detector and
a data acquisition system, etc. The temperature of the
0521 Gas Chromatography
injection port, column, and detector are suitably set according to
the analysis requirements.
( 1) Carrier gas source The mobile phase of gas chromato-
graphy is gas, known as carrier gas. Carrier gases used are
usually helium, nitrogen, and hydrogen. The gas is supplied
by a high-pressure steel cylinder or high-purity gas generator
and passes through suitable pressure-reducing valves and a
flow meter to the injector port and column. The gas selection
depends on the properties of the substances being examined
and the species of the detector. The commonly used carrier
gas is nitrogen, unless otherwise specified.
( 2) Injection port Direct injections or automatic injections
of solutions and headspace injection are the usual modes of
injection.
Direct injection may be carried out by using a syringe or an
injection valve, or into a vaporization chamber which may be
equipped with a stream splitter. The temperature of the
vaporizer is usually 30-50ºC higher than that of the column
when using direct injection or automatic injections. The
volume of solution injected is not more than several µl. The
smaller the column diameter the less volume of injected
solutionis. Capillary column is used with injectors which is
able to split samples into two fractions to avoid overloading.
Headspace injectors are suitable far separation and deter-
mination of volatile component in salid or liquid substance
being examined. The test solutions produced with salid or
liquid substance being examined and stored in tightly closed
containers are heated in the chamber far a period of time,
allowing the volatile components in the test solution to reach
a equilibrium between the liquid phase and the gaseous
phase. A predetermined amount of the head-space of the vial
is flushed into the column by automatic syringe.
( 3 ) The chromatographic column The chromatographic
columns are classified into the packed columns and capillary
columns. The packed columns, which are made of stainless
steel or glass, are 2-,4 mm in internal diameter and 2-4 m in
length. The columns are packed with the sorbent, porous
polymer bead or the supports coated with liquid phase,
particle size of which is O. 18-0. 25 mm, O. 15-0. 18 mm or
O. 125-0. 15 mm. The support usually used is acid washed
and silanized diatomaceous earth or porous polymer beads;
the liquid phase commonly used is methylpolysiloxane,
polyethyleneglycol etc. The capillary columns are made of
glass or quartz. The inner wall of the column is coated or
cross-linked with stationary liquid, the internal diameter is
usually O. 25 mm, O. 32 mm or O. 53 mm, the length is 5-60
m, and the film thickness of the stationary liquid is O. 1-5. O
µm. The stationary liquid commonly used are methyl poly
siloxane, phenylmethylpoly-siloxane with different ratio of
composition and carbowax, etc.
New packed and capillary column must be conditioned befare
use to remove oxygen and residual solvents. The chromato-
graphic column must be conditioned befare use until the
baseline is stable if the column is not in use long time.
( 4) The column oven The temperature-controlled precision
of the oven should be± 1 ºC and fluctuation of temperature
should be less than O. 1 ºC per hour, as the temperature
fluctuation of oven will influence the reproducibility of
chromatographic analysis result. Temperature-controlled
system may be classified into constant temperature and
temperature programming.
( 5) Detector The detectors suitable far the gas chromato-
graphy include flame-ionization detector ( FID), thermal
conductivity detector (TCD), nitrogen-phosphorus detector
( NPD ) , flame-photometer detector ( FPD ) , electron-
 0531 Supercritical Fluid Chromatography
capture detector ( ECD ) , mass spectrometric detector
(MSD) etc. FID responses well to hydrocarbon and is
suitable far the determination of most drug compounds; NPD
is sensitive to organic nitrogen and phosphorus compounds;
FPD is sens1t1ve to organic sulfur and phosphorus
compounds; ECD is suitable far determination of halogen
compounds; MSD can offer chemical construction infar-
mation of the compounds which is useful far structure
verification. Unless otherwise specified, the detector should
be FID which employs hydrogen as combustion gas and air as
combustion-supporting gas. When FID is used, its
temperature is higher than that of the column and not less
than 150ºC to prevent condensation of the vapour, which
usually is 250-350ºC.
( 6) Data process system It is classified in to recorder, integ-
rator and computer work station etc.
If necessary, except the kind of the detector, the type of the
stationary phase and the material of the column, other
parameters specified under the monograph, such as the
intenal diameter and length of the column, brand and size of
the support, concentration of the stationary phase, the flow
rate of carrier gas, the temperature of the column, the
injection volume, the sensitivity of the detector etc, may be
varied to meet the requirment of the system suitability test.
Usually the chromatogram is completed within 30 minutes.
2. System suitability test
The requirements are the same as described under High
Performance Liquid Chromatography ( 0512 ) , unless
othePNise specified.
3. Procedure
(1) lnternal standard method.
(2) Externa! standard method.
( 3) Peak area normalized method.
The specific content of ( 1) - ( 3) is the same as described
under High Performance Liquid Chromatography ( 0512).
( 4) Standard addition method. Dissolved an accurately
weighed quantity of impurities or reference substance of the
substance being examined to produce a reference solution
with a suitable concentration. Measure accurately the
solution and add to the test solution, then calculate the
content of individual impurities or the main component by
interna! standard method or externa! standard method.
Deduct the content of added standard solution and gain the
content of individual impurities or the main component in the
test solution.
The content may also be calculated by the following equation.
The correction factor is the same as that of adding the
reference solution.
A¡. Cx +.1Cx
Ax Cx
The concentration Cx of component being examined may be
calculated by the fallowing equation.
Cx = !:!l.Cx
(A¡s/Ax) -1
Where Cx is the concentration of component X being
examined;
Ax is the peak area of component X being
examined;
.1Cx is the concentration of added reference subs-
tance of component being examined.
A;. is the peak area of component X after adding
reference substance of component being
examined.
Because the amount of sample injected is usually several
microliter in gas chromatography, a majar source of error is
the irreproducibility in the amount of sample injected, which
is aff ected by retaining time of syringe and room
temperature, notably when manual injections are carried out
with a syringe. The effects of variability can be minimized by
the interna! standard. Automatic injectors greatly improve
the reproducibility of sample injections and reduce the need
far interna! standards. When headspace injectors are
equipped, the effect of matrix may be eliminated by standard
addition method because the test solution and reference
solution are in different matrix. When the quantitative result
of standard addition method is different from others, the
result of standard addition method should be adopted.
0531 Supercritical Fluid
Chromatography
Supercritical Fluid Chromatography ( SFC) is a method of
chromatographic separation in which the mobile phase is
supercritical fluid.
Supercritical fluid is a state of substance. Sorne substances
have triple points and supercritical points. Gaseous, liquid
and salid are at an equilibrium stage at the triple point.
Gaseous phase and liquid phase are of same density under the
supercritical temperature. When temperature is above super-
critical temperature gas will not turn into liquid with
increased pressure. Substance is in the state of supercritical
fluid when substance condition is above critical temperature
and critical pressure, its density however will change with
the increase of tempcrature and pressure in the supercritical
stage. Supercritical fluids refer to sorne substances which are
neither gas nor liquid to the extent that their physical
properties fall in between gas and liquid. The critical
temperature is generally higher than substance boiling point
or triple point.
Supercritical fluids have physical properties extremely
favorable far separation. These physical properties fall
exactly in between gas and liquid, which lead to supercritical
fluid chromatography endowed with the characteristics of
both gas chromatography and liquid chromatography. The
diffusion coefficient and viscosity of supercritical fluids are
clase to a gas, thus mass transfer resistance of salute is low,
quick and efficient separation can be achieved. On the other
hand, supercritical fluids with similar density to liquid and
high solvency can be used to separate uneasily volatile and
thermally unstable substances with relatively large molecular
mass under the low temperature.
In addition, physical and chemical properties of supercritical
fluids such as diffusion, viscosity and solvency etc. are the
functions of density. Therefare, the properties of the fluids
varíes with the change of density, no gas-liquid equilibrium
curve is required far changing from gas state to liquid state.
The separation can be optimized by adjustment of
temperature and pressure and change of the density of fluids.
Control accurately the temperature and pressure in arder to
keep the fluids in a stable state, and convert to gas, liquid or
supercritical fluids befare entering the detector during the
process of separation.
l. General requirements for apparatus
Many parts of the instrument used far supercritical fluid
chromatography, similar to those of high performance liquid
chromatography ( HPLC), mainly consists of three parts,
high-pressure pump (fluid delivery unit), analytical unit and
control system. In generally the high-pressure pump system
with high precision and stability are required to deliver the
pulse-free supercritical fluids at an accurate and stable flow
 0532 Liquid Chromatography at Critical Condition

rate. Analytical unit is mainly composed of injection valve, continued

chromatographic column, resistor and detector. The control
system is designed to control the pump in order to maintain Critical
the stability of the temperature of the column oven, and pressure
Molecular Critical Critical
process and display data. MPa
Substance mass temperature density
(standard
( 1) Chromatographic colmnn Chromatographic column used in g/mol K g/cm3
atmospheric
supercritical fluid chromatography can either be packed pressure)
column or capillary column, which respectively corres-
ponding to packed column supercritical fluid chromatography Methane 16.04 190.4 4.60 o. 162
( pSFC) or capillary column supercritical fluid chroma- (CH4) (45.4)
tography ( cSFC). Depending on the properties of substance
being examined, different chromatographic columns are Ethane 30.07 305.3 4.87 0.203
chosen to be used for supercritical fluid chromatography. (C2 H6) (48. 1)
Almost all the columns for high performance liquid chro- Propane 44.09 369.8 4.25 0.217
matography can be used for supercritical fluids chroma- (C3 Hs) (41.9)
tography. Common chromatographic columns include silica
gel column (SIL), amino column ( NH 2 ), cyano column Ethylene 28.05 282.4 5.04 0.215
(CN), 2-ethyl pyridine column (2-EP) and kinds of chiral (C2H4) (49. 7)
chromatographic columns etc. ; reversed phase columns such
as C1s and Cs etc. and various capillary chromatographic Propylene 42.08 364.9 4.60 0.232
columns may also be used for sorne applications. (C3 H6) (45.4)

( 2) Mobile phase The most widely used mobile phase in Methanol 32.04 512.6 8.09 0.272
supercritical fluid chromatography is liquid carbon dioxide (CH3QH) (79. 8)
( C02 ) . C02 is colourless, odorless, non-toxic, readily
available and inexpensive, and it is a good solvent with high Ethanol 46.07 513.9 6. 14 0.276
solubility for various organic molecules; it is transparent (C2HsOH) (60.6)
without absorption in ultraviolet region; its supercritical
Aceto ne 58.08 508. 1 4.70 0.278
temperature and supercritical pressure is 31 ºC and 7. 38 X
(C3H60) (46.4)
10 6 Pa, respectively. In chromatographic separation, the
temperature and pressure of C02 fluid have wide selection
( 3) Detector Detectors commonly used in high performance
range. As most drugs have polarity, sometimes polar
liquid chromatograph, such as ultraviolet detector,
modifiers may be introduced into the fluid according to the
evaporative light-scattering detector etc. can be applied in
polarity of the substance being examined. The selection of
supercritical fluid chromatography. Flame ionization detector
polar modifiers is based on the practical experiments. The
( FID), nitrogen phosphorus detector ( NPD) in gas
most commonly used modifier is methanol and the ratio of
chromatography ( GC) can also be used in supercritical fluid
modifiers is usually not more than 40 %, for example, 1 %-
chromatography, and supercritical fluid chromatography can
30 % of methanol is added to improve selectivity factor a
be used together with mass spectrometry (MS) and nuclear
value for separation. Besides methanol, 2-propanol or
magnetic resonance (NMR) etc. Compared with HPLC and
acetonitrile can also be used. In addition, trace amount of
NMR combination technique, C0 2 as the mobile phase which
additives may be added, such as trifluoroacetic acid, acetic
is free from hydrogen signal, thus water peak suppression
acid, triethylamine and isopropanolamine to improve the
does not need to be considered.
chromatographic peak shape and separation performance,
and increase the elution and solvency of the mobile phase. 2. System suitability test
Besides C0 2 fluid, ethane, pentane, ammonia, nitrous Complies with the requirements for high performance liquid
oxide, dichlorodifluoromethane, diethyl ether and chromatography <0512).
tetrahydrofuran etc. may also be used as the mobile phase. 3. Procedure
Physical properties of sorne substances generally used as the ( 1) Internal standard method.
mobile phase in supercritical fluid chromatography are listed (2) External standard method.
in the following Ta ble. (3) Peak area normalization method.
Table Critical pressure, temperature and The details of above procedures comply with the requi-
density of sorne substances rements for high performance liquid chromatography <0512 ).
The internal standard method and the external standard
Critical
method are most commonly used.
pressure
Molecular Critical Critical
MPa
Substance mass temperature (standard density
0532 Liquid Chromatography
g/mol K g/cm3
atmospheric at Critica) Condition
pressure)

Car bon 44.01 304. 1 7.38 0.469 Liquid chromatography at critical condition (LCCC) lS a
dioxide (72. 8) liquid chromatographic technique tha t separa tes polymers
(C02) according to different functional groups and segmented
structures. The principie of LCCC is based on scaling theory
Water 18.015 647.096 22.064 0.322 of above, under and near the critical points. With porous
(Hz O) (217. 755) packing materials as the stationary phase, the separation
mechanisms of size exclusion chromatography ( SEC) and
 0541 Electrophoresis
interaction chromatography (IC) react simultaneously when
separation occurs. Under the specified chromatographic
conditions ( the components of the stationary phase and the
mobile phase, and temperature), the critica} point of two
separation mechanisms exists, which is called enthalpy and
entropy complementary point, or chromatographic critica}
conditions, or critica} adsorption point (CAP). At this
point, polymers separation depends on differences of
terminal functional groups or segmented structure, rega-
rdless of molar mass (molecular weight). Elution volumes of
polymers are equal to the space volumes of the column. The
long chains of polymers turn to chromatographically
invisible.
SEC separation can only determine the molecular weight
distribution of polymers; therefore LCCC is a necessary
supplement to SEC separation.
1. General requirements for apparatus and chromatographic
conditions
(1) Apparatus The apparatus ( the injector, pumping systems
and detector) are the same as those described under high
performance liquid chromatography ( 0512).
(2) Column Non-polar packing materials commonly used far
fat soluble polymers, the most widely used is chemically
bonded silica gel, such as octadecylsilane bonded silica gel,
polystyrene divinyl benzen. Chromatographic system applied
to water soluble polymers uses polar packing materials, the
frequently used columns are HILIC column, Dial column,
etc.
The pare size of the support affects directly the separation
performance. Generally speaking, packing materials can be
selected with reference to the principie of HPLC but due to
different topological structure of polymers, packing materials
can be chosen by the characteristics of monograph and
experiments in actual application.
( 3) Mobile phase Generally, non-aqueous solvents and the
suitable proportion of mixture as the mobile phase are
employed to separate fat soluble polymers, which shall be
absolutely free of water. The preferred mobile phase adopted
far water soluble polymers is a mixture of water and
methanol or acetonitrile, if necessary several additives can be
added, such as buffer salt.
(4) Column temperature Column temperature is of a great
significance in determination of CAP. The highest
temperature used of the bonded stationary phase with silica
gel as support is not more than 60ºC, therefore the polymers
of polystyrene divinyl benzene as the stationary phase can be
used with the highest temperature of lOOºC.
2. Determination of chromatographic conditions for critica)
adsorption point (CAP)
To determine the chromatographic conditions of CAP, the
chromatographic conditions should be optimized sequentially
from three factors, the stationary phase, the mobile phase
(in different proportion) and column temperature, affecting
enthalpy and entropy changes of polymers.
The first step of optimization is to determine the range of the
stationary phase and the mobile phase. Firstly the pare size
of the column should be compatible with the molecular
weight of the components to be determined so that the
components are within the grading range of the column
preventing from being complete size exclusion molecule.
Secondly the mobile phase should have elution strength so
that the components have a certain capacity factor, and a
suitable retention time.
In the case of optimization near the critical point, the
chromatographic retention behavior of the polymers with
different degree of polymerization is observed, indicating the
tautomerizing of the SEC and IC, the dispersed chromatographic
peaks of polymers with different degree of polymerization merge
together and then combine to a single sharp chromatographic
peak.
3. System suitability test and procedure
Complies with the requirements far high performance liquid
chromatography ( 0512).
0541 Electrophoresis
Electrophoresis refers to the migration of electrically charged
proteins, colloids, large molecules, or other particles
dissolved or suspended in an electrolyte towards the electrode
of opposite sign. Electrophoresis is a method permitting
separation of components in test sample that are capable of
acquiring an electrical charge in a conducting electrolyte. The
components move at different speeds towards the corres-
ponding electrode under applied electric field by using cations and
anions with different magnitude of charge in the electrolyte
solution. The separation is recorded or calculated by an
appropriate detection method and the objective of the analysis is
achieved. There are two categories of electrophoresis; one is free
solution electrophoresis or moving boundary electrophoresis and
the other is zone electrophoresis.
Moving boundary electrophoresis is electrophoresis in a free
solution without supporting medium, so it is also called free
solution electrophoresis, which is particularly suitable far
substances of high molecular weight. Zone electrophoresis is
electrophoresis with supporting medium. The eiectricaHy
charged particles in test samples ( large molecules such as
protein and nucleic acid or other particles, etc. ) migrate at
different speeds towards the electrode of opposite sign in the
inert supporting medium (such as paper, cellulose acetate,
agarose gel and polyacrylamide, etc. ) under the application
of an electric field and thus shall be separated into narrow
zones. Different supporting medium can be selected far zone
electrophoresis, and corresponding electrophoretogram is
recorded by using appropriate detection method to calculate
the content of components ( %) in the test samples. U nless
otherwise specified, carry out zone electrophoresis with
different supporting medium as below methods. Please refer
to the instructions of the instrument when using a full
automatic electrophoresis system and an automatic scanner or
gel imager to obtain testing results.
Method 1 Paper Electrophoresis
Paper electrophoresis uses filter paper far chromatography as
supporting medium with large pare size and thus no
molecular sieving effect is produced. This method separates
different components in test samples with different electrical
charge and is suitable far the determination of substances
with similar properties like nucleotides etc.
l. Apparatus
The apparatus consists of two parts, electrophoretic chamber
and a direct current power source.
The horizontal electrophoresis chamber shown in figure (as
Fig. ) , consists of two troughs A and a hermeticable lid B
made of glass ( or appropriate material). Both troughs are
separated respectively into two compartments by plexiglass
plate C ( or appropriate material). In the outer compart-
ments, there are platinum electrodes D with a diameter of
O. 5-0. 8 cm connected to the outside power supply by
insulated cables through the trough walls; and in the inner
compartments, there are movable plexiglass stands sup-
porting the filter papers.

Fig. The horizontal electrophoresis chamber
The power source supplying a direct current is composed
with a voltage stabilizer. Ordinary voltage electrophoresis
refers to that proceeded at 100-500 V, and high voltage
electrophoresis refers to that proceeded at 500-10 000 V.
2. Procedure
( 1 ) Electrophoretic buffer Citrate buffer solution ( pH
3. O): Dissolve 39. 04 g of citric acid (C6 H 8 01• H 2 0) and
4. 12 g of sodium citrate (C6 Hs Na3 01• 2H2 0) in 4000 ml of
water.
( 2) Filter paper Immerse a chromatographic filter paper in
formic acid solution (l mol/L) not less than 12 hours, and
then wash with water until the pH value of elute is not lower
than 4. Dry the filter paper at 60ºC and preserve far use.
The filter paper can be cut into strips of 27 cm in length and
18 cm in width, or appropriate far the size of electrophoresis
chamber. Draw a líne about 5-8 cm from the bottom of the
paper and, alone the line, mark points every 2. 5-3 cm apart
far application of the solution being examined.
( 3 ) Loading the samples It may be divided in to two
categories, wet applying and dry applying. In wet applying,
immerse the trimmed paper entirely into the citrate buffer
( pH 3. O) , take out and remove the surplus buffer solution
with filter paper. Place the paper in the stand and place the
application line near the cathode and the both ends of the
paper immersed into the buffer solution. Apply 10 µl of the
test solution, accurately measured, to 3 marked points using
micro injector. Leave two marked points for blank control.
Dry applying is suitable far dilute samples. In this method,
apply the test solution on the paper, after drying, apply
again and repeat these steps until all the sample solution has
been completed. Wet the filter paper with sprayer and allow
the application spots wet at the end.
( 4) Electrophoresis Fill the troughs of the cham her with
the electrolyte solution to submerge the platinum electrode.
Connect the cables to the power source with a constant
voltage. Adjust the voltage gradient to about 18-20 volts
per cm of the paper and allow electrophoresis to proceed far
about one hour and forty-five minutes. Remove the paper,
dry immediately in a current of air, examine under
ultraviolet light ( 254 nm) and mark the violet spot with
pencil.
( 5) Assay Cut the spots containing the test sample in the
filter paper, as well as a piece of blank filter paper with a
similar area, into slim strips and place into test tubes
respectively. Add accurately measured 5 ml of hydrochloric
acid solution (O. 01 mol/L) to each tube, shake thoroughly
and allow to stand for 1 hour. Filter through a No. 3 sintered
glass filter and collect the filtrate, or collect the supernatant
by natural sedimentation or centrifugation. Measure the
absorbance of the collected solutions at the wavelength as
specified in the monograph and calculate the content.
Method 2 Cellulose Acetate Film Electrophoresis
Cellulose acetate film electrophoresis uses cellulose acetate
film as supporting medium with large pore size and thus no
0541 Electrophoresis
molecular sieving effect is produced. This method separates
different components in test samples with different electrical
charge and is suitable far the determination of serum protein,
immunoglobulin, lipoprotein, glycoprotein, steroid hormone
and isoenzyme.
l. Apparatus
The electrophoretic chamber and power source supplying
direct current are the same as that of paper electrophoresis.
2. Reagents
( 1) Barbital buffer solution ( pH 8. 6) Dissolve 2. 76 g of
barbitone and 15. 45 g of barbital sodium in water to produce
1000 ml.
( 2) Staining solution The following staining solutions are
commonly used, one of them can be selected as requirements
specified in the monograph.
©Amino black staining solution Dissolve O. 5 g of amino
black 1O B in a mixture of 50 ml of methanol, 1O ml of
glacial acetic acid and 40 ml of water.
(Í)Ponceau staining solution Dissolve 9. 04 g of ponceau and
6 g of trichloroacetic acid in water and dilute to 100 ml.
@Ponceau staining solution containing acetic acid Dissolve
O. 1 g of ponceau and 5 ml of acetic acid and dilute with water
to 100 ml. Store at 4ºC.
© Ponceau staining solution containing trichloroacetic acid and
5-sulfosalicylic acid Dissolve 2 g of ponceau, 30 g of
trichloroacetic acid and 30 g of 5-sulfosalicylic acid in water
and dilute to 100 ml.
{3) Destaining solution Mix 45 ml of ethanol, 5 ml of
glacial acetic acid and 50 ml of water thoroughly.
( 4) Hyalinization solution Mix together 25 ml of glacial
acetic acid and 75 ml of dehydrated ethanol.
3. Procedure
( 1) Cellulose acetate film Cut the cellulose aceta te foil into
strips with a dimention of 2 cm X 8 cm, immerse in the
barbital buffer solution ( pH 8. 6) , with the glossy side
upwards . After steeping, take out the strip and press
between filter paper to remove excess buffer solution. Place
the strip on the electrophoretic support with the glossy side
downwards, immerse into the barbital buffer solution ( pH
8. 6) through filter paper bridge.
( 2) Application of sample and electrophoresis Apply to the
foil as a band at 2 cm from the cathode edge 2-3 µl of test
solution with a protein content of about 5 % . Allow the
electrophoresis to proceed at a constant voltage of 10-
12 V/ cm or current O. 4-0. 6 mA/ cm ( total current =
current (mA/cm) X width of the film X number of the
film) until electrophoretic bands are 4-5 cm apart.
During the assay of sample like human serum albumin and
immunoglobulin, using fresh human serum as the reference.
Allow electrophoresis to proceed until albumin and immuno-
globulin have moved apart in 2 cm distance.
( 3) Staining After electrophoresis, remove the strip and
immerse in amino black staining solution or ponceau far 2-3
minutes, wash with destaining solution until the background
is free from colour.
( 4) Hyalinization Immerse the washed and dry foil in the
hyalinization solution far 10-15 minutes, soak totally, then
remove and place evenly on a clean glass plate. After drying,
it will become a transparent film and ready to be measured
far relative content, purity testing or in a spectrophotometer
and be kept as specimen for long-term preservation.
 0541 Electrophoresis
(5) ~y The electrophoretogram of the cellulose acetate
foil that is untreated for hyalinization can be examined by the
method specified in the monograph. Generally, the elution
method or scanning method is used to determine the relative
percentage content of every protein component in the sample.
Elution method Dry the washed foil with filter paper, cut
out every electrophoretic band derived from the test solution
and immerse separately in l. 6 % solution of sodium
hydroxide, shake for severa} times until the elution is
complete. Measure the absorbance of the eluent by
ultraviolet-visible spectrophotometry ( 0401 ) at the
wavelength specified in the monogragh. At the same time,
cut out a corresponding band of foil without proteins, treated
in the same manner as blank. Calculate the total absorbance
and the percentage content of each protein component in the
sample.
Scanning method Sean the dry cellulose acetate foil with
chromatographic scanner by means of reflection ( for non-
transparent film) or transmission ( for transparent film).
The scanning chromatogram for every protein component is
recorded automatically with the length of foil as abscissa and
the absorbance as ordinate. Calculate the percentage content
for each protein component in the sample, which also could
be performed by integrates.
During the analysis of samples, human serum albumin and
immunoglobulin, human serum should be set as the refe-
rence. Calculate the content of the each protein component
by peak area (%).
Method 3 Agarose-gel Electrophoresis
Agarose gel electrophoresis uses agarose as supporting
medium. An agarose is a catenarian polysaccharide generally
extracted from agar, which is a disaccharide made up of
D-galactose and 3, 6-anhydro-L-galactopyranose. Many
agarose chains are intertwined into rope agarose bundles and
constitute gel with large pare size. Such network structure
produces molecular sieving effect and separate particles by
not only property and number of electrostatic charges but
also molecular size, by which the resolution is further
increased and thus its capability of separation is improved.
This method is suitable for the separation, identification and
purification of immune complexes, nucleic acid and nuclear
protein.
Effects of electric charge and molecular sieving exist during
the migration of DNA molecular in agarose gel. DNA
molecular moves toward anode with negative charge in the
solution with higher pH than its isoelectric point. Because of
the repeatable structure of the sugar-phosphate backbone,
double-strand DNAs move toward the anode at the same
speed with similar electrostatic charges. Electrophoretic
mobility of DNA molecular is inversely proportional to
common logarithm of molecular weight in a certain
concentration of agarose gel medium. Molecular confi-
guration impacts the electrophoretic mobility, e. g. covalent
closed circular DNA (ccc DNA) >linear DNA>open circle
double-strand DNA. Such method is suitable for the
determination of DNA and the DNA electrophoresis in PCR
reaction. The method is given under each monograph.
Agarose-gel Electrophoresis-Method 1
l. Apparatus
The electrophoretic chamber and power source supplying
direct current are the same as that of paper electrophoresis.
2. Reagents
( 1) Acetic acid-Iithium salt buffer solution ( pH 3. O) Mix
50 mi of glacial acetic acid and 800 ml of water, adjust pH to
3. O with lithium hydroxide and dilute to 1000 mi with water.
( 2) Toluidine blue solution Dissolve O. 1 g of toluidine blue
in 100 ml of water.
3. Procedure
( 1) Gel preparation Add about O. 2 g of agarose to 10 mi
of water, heat in a water-bath to swell completely, add 10 ml
of hot acetic acid-lithium salt buffer solution ( pH 3. O) and
shake thoroughly, spread on a horizontal glass plate with an
appropriate dimention (2. 5 cmX7. 5 cm or 4 cmX9 cm) to
yield a layer of about 3 mm thick while it is still warm.
Allow it to congeal and to form a uniform thickness gel
without bubbles.
( 2 ) Preparation of the referenre solution and the test solution
Prepare the solutions as described in the monograph.
(3) Loading the sample and electrophoresis Fill the electro-
phoresis troughs with acetic acid-lithium salt buffer solution
(pH 3. O), place the gel plate on the electrophoretic support
and immerse into the electrophoretic buffer solution through
filter paper. Apply separately 1 µl of sample solutions to the
gel at the cathode edge. Connect the electrodes to the power
supply immediately and allow the electrophoresis to proceed
at voltage gradient of about 30 V/ cm, and a current of 1-2
mA/ cm for about 20 minutes. Switch off the current.
( 4) Staining and destaining Remove the gel plate and stain
the gel with toluidine blue solution. Destain the excess by
washing with water until the background being colourless.
Agarose-gel Electrophoresis-Method 2
l. Apparatus
The electrophoretic chamber and power source supplying
direct current are the same as that of paper electrophoresis.
2. Reagents
( 1) Barbital buffer solution ( pH 8. 6) Dissolve 4. 14 g of
barbital and 23. 18 g of sodium barbital in a volume of water
by heating. Cool to a room temperature and add O. 15 g of
sodium azide. After sodium azide is dissolved, dilute the
solution to 1500 mi with water.
( 2) l. 5 % Agarose solution Add 50 ml of water and 50 ml
of barbital buffer solution ( pH 8. 6) to l. 5 g of agarose.
Heat the mixture until the agarose is completely swollen.
(3) O. 5 %Amino black solution Dissolve O. 5 g of amino
black lOB in a mixture of 50 ml of methanol, 10 mi of glacial
acetic acid and 40 mi of water.
( 4) Destaining solution Mix 45 mi of ethanol and 5 ml of
glacial acetic acid with 50 ml of water thoroughly.
( 5) Bromophenol blue indicator solution Dissolve 50 mg of
bromophenol blue in water and dilute to 100 ml.
3. Procedure
( 1) Gel preparation Pour the hot l. 5 % agarose solution
onto a horizontal glass plate of suitable size to a thickness of
3 mm. Allow to stand to form a thin and even agarose gel
plate which is free of bubble.
( 2) Reference solution and test solution
Reference substance Normal human serum or other suitable
reference substances can be used.
Preparation of test solution Dilute the substance being
examined to a protein concentration of 1%-2% with physio-
logical saline.
(3) Loading the sample and electrophoresis Add the barbital
buffer solution ( pH 8. 6) into the electrophoresis trough;
punch wells at the one-third length of the agarosegel plate
apart from negative pole. The diameter of each well is 2-3
mm, put the plate onto the electrophoresis trough. Connect

the two ends of the gel plate to barbital buffer solution (pH 8. 6)
through three layers of filter paper. Add a quantity of the
test solution and one drop of bromopenol blue indicator
solution into the test well. Add a quantity of the reference
substance and one drop of bromophenol blue indicator
solution into the reference well. Perform electrophoresis for
2 hours at a constant voltage of 100 V until the indicator
migrates to the forward position, turn off the power source.
( 4) Staining and destaining Remove the gel plate and stain
the gel with O. 5 % amino black solution after electrophoresis
and then decolourize with destaining solution until the
background being colourless.
Method 4 Polyacrylamide-gel Electrophoresis
Polyacrylamide gel electrophoresis uses polyacrylamide gel as
supporting medium. Polyacrylamide is cross-linked by
methylenebisacrylamide under the effect of the catalyst to
form three-dimensional network after polymerization. The
pore size of the gel varíes from different concentration of
monomer or different ratio of the monomer to the crosslink
agents. Biomacromolecule remains native state and its
electrophoretic mobility depends on its charge density as well
as its size and shape when performing an electrophoresis
using polyacrylamide gel as supporting medium. It is
commonly used for characterization of biomacromolecules
such as the properties of electric charge, molecule weight,
isoelectric point etc. There are three electrophoresis methods
in place including horizontal plate electrophoresis, vertical
plate electrophoresis and clise plate electrophoresis according
to the difference of apparatus and two electrophoresis
methods including continuous and discontinuous electro-
phoresis on the basis of different gel preparation methods.
l. Apparatus
It consists of power source supplying constant current and
clise or plate electrophoretic troughs. The electrophoretic
chamber is assembled with upper trough and lower trough,
in which platinum electrodes are fixed and connected by the
insulated cables to the power supply at constant current
mode. Refer to the SDS--polyacrylamide electrophoresis
method when analyzed by using vertical plate electrophoretic
trough. See the following analytical procedure by using clise
electrophoretic troughs.
2. Reagents
{ 1) Solution A Dissolve 36. 6 g of trihydroxymethyl amino-
methane, O. 23 ml of tetramethylethylene diamine and 48 ml
of hydrocholric acid solution ( 1 mol/L) in water to produce
100 ml. Place in an amber coloured bottle and store in
refrigerator.
{2) Solution B Dissolve 30. O g of acrylamide and O. 74 g of
methylene bisacrylamide in water to produce 100 ml, filter
and place in an amber coloured bottle, store in refrigerator.
(3) Electrode buffer solution {pH 8. 3) Dissolve 6 g of
trihydroxymethyl aminomethane, 28. 8 g of glycine in water
to produce 1000 ml, store in refrigerator. Dilute to 10
volumes befare use.
{ 4) Bromophenol blue indicator solution Dissolve O. 1 g of
bromophenol blue in 3. O ml of sodium hydroxide solution
(O. 05 mol/L) and 5 ml of 90% ethanol solution by gentle
heating, add 20 % ethanol solution to produce 250 ml.
{ 5 ) Staining solution To 2. 5 ml of O. 25 % ( g/ ml )
coomassie brilliant blue G250 solution add 12. 5% (g/mD
trichloroacetic acid solution to produce 10 ml.
{ 6) Dilute staining solution Dilute 2 ml of staining solution with
12. 5 % (g/ mD trichloroacetic acid solution to produce 10 ml.
0541 Electrophoresis
{7) Destaining solution 7 % acetic acid solution.
3. Procedure
{ 1) Gel preparation Dissolve 2. 9 g of urea in 2 ml of
solution A and 5. 4 ml of solution B, add 4 ml of water, mix
well and evacuate to remove air bubbles. Add 2 ml of
O. 56 % ammonium persulphate to form gel solution.
Introduce the gel solution along the wall of the tubes using a
syringe fitted with long needle or a thin pipette into glass
tubes ( 10 cm X O. 5 cm) with rubber stopper at the
bottom, until the gel column raises up to 6-7 cm. Air
bubbles must not be trapped at the bottom of the tube. Cover
the gel mixture in the tubes with a layer of water and allow to
set for about 30 minutes. Gel polymerization is complete
when a sharp interface form between the gel and water layer.
Remove the water layer.
{ 2) Preparation of the reference substance/molecular weight
standard solution and the test solution.
Prepare the solutions as prescribed in the monograph.
{ 3) Electrophoresis Fit the tubes with prepared gel in to the
clise electrophoresis trough. Apply separately 50-100 µl of
the test solution or reference/ standard solution to the tubes.
To each tube add 1-2 drops of glycerol or 40% solution of
sucrose and 1 drop of O. 04 % bromophenol blue indicator
solution to prevent from diffusion. Alternatively, several
drops of O. 04 % bromophenol blue indicator solution could be
added directly to the buffer solution in upper trough. Fill the
top of glass tubes with electrolyte buffer solution. Connect
the electrodes to the power supply and allow electrophoresis
to proceed ata constant current of 1 mA per tube for several
minutes, and then increase the current to 2-3 mA per tube.
Switch off the current when the bands of bromophenol blue
migrate to 1 cm from the bottom of glass tube.
{ 4) Staining and destaining After electrophoresis, extrude
the gel by injecting water between the gel and the wall of the
tube from the bottom by means of a syringe fitted with a fine
needle. Immerse the gel strips for 10-30 minutes in staining
solution or 10-12 hours in dilute staining solution. Wash with
water and decolourize in destaining solution until the
background of the gel in the zone without protein is
transparent.
4. Results judgment Examine the gel strips under lamp.
Determination is made by comparing the position and colour
of the bands between the reference/ standard substance and
the substance being examined.
{ 1) Relative mobility The electrophoretic bands of the test
sample and the reference/ standard substance can be
compared by means of relative mobility ( R 'm ) , which can
be calcula ted as below:
distance from origin to
substance being examined
zone or reference /
standard su bstance zone
Relative mobility (R'm) =
distance from origin to
bromophenol blue zone
{ 2) Scanning Sean the clear gel strips in a thin layer scanner
with double wavelength or gel electrophoretic scanner, calculate
the percentage content of individual component based on its
peak area.
Method 5 SDS-polyacrylamide Gel Electrophoresis
SDS--polyacrylamide electrophoresis is electrophoresis by
using denatured polyacrylamide as supporting medium.
Determining the molecular weight of proteins by the way of
SDS--polyacrylamide gel electrophoresis ( SDS--PAGE) is
 0541 Electrophoresis
1

based on the experience that the great majority of proteins ( 14) Silver staining solution ( silver staining method A)
can bind with sodium dodecyl sulfate ( SDS), a kind of Dissolve 2. 04 g of silver nitrate in water and dilute to
cation surfactant, to form complex. The negative charges 1000 ml.
carried by the protein complexes are much higher than that of Silver nitrate solution ( silver staining method B) Dilute O. 8 g
the natural proteins, resulting in eliminating the discrepancy of silver nitrate to 4. O ml of water. Add dropwise to a
in charge effect derived from diff erent proteins. The proteins mixture of 20 mi of O. 1 mol/L sodium hydroxide and l. 5 ml
can be separated according to their molecular size. of 25 % ammonium solution, mixed well and dilute to 100 ml
l. Apparatus with water.
It consists of power source supplying constant voltage or ( 15 ) Developing solution ( silver staining method A )
constant current, horizontal plate or clise electrophoretic Dissolve 30 g of sodium carbonate in a quantity of water.
troughs and gel preparation mould. Add O. 5 mi of formaldhyde solution and dilute to 1000 ml
2. Reagents with water.
( 1) Water (Water conductivity shall be not less than 18. 2 Developing solution ( silver staining method B) Add 2. 5 ml
MO•cm). of 1 % citric acid solution and 270 µl of 37 % formaldehyde
solution and dilute to 500 mi with water.
(2) Solution A l. 5 mol/L trihydroxymethyl aminomethane-
hydrochloride ( tris-HCD buffer solution. Dissolve 18. 15 g ( 16) Stopping solution ( silver staining method A) Dilute
of T trihydroxymethyl aminomethane in a volumn of water. 10 ml of glacial acetic acid to 1000 ml with water.
Adjust pH to 8. 8 with hydrochloric acid and dilute to 100 mi Stopping solution ( silver staining method B) Dilute 100 ml
with water. of glacial acetic acid to 1000 mi with water.
(3) Solution B 30% acrylamide-0. 8% N, N'- methylene ( 17) Coomassie brilliant blue staining solution Dissolve 1 g
bisacrylamide solution (protected from light). of coomassie brilliant blue R250 in a mixture of 200 ml of
(4) Solution C 1% sodium dodecyl sulfate CSDS) solution. methanol, 50 mi of glacial acetic acid and 250 mi of water
thoroughly.
( 5) Solution D 1O% N, N, N', N' -tetramethyl-
ethylenediamine solution. ( 18) Coomassie brilliant blue destaining solution Mix 400 mi
of methanol, 100 ml of glacial acetic acid and 500 mi of water
( 6) Solution E 10 % ammonium persulfate solution, prepared thoroughly.
befare use.
( 19) Preserving solution Add 75 ml of glacial acetic acid
( I j Solution F O. 5 moi/L trihydroxymethyi aminomethane. and dilute to 1000 ml with water and mixed well.
hydrochloride ( tris-HCD buffer solution. Dissolve 6. 05 g of
Preparation of the test solution Mix three volumes of the
trihydroxymethyl aminomethane in a volumn of water.
sample with one volume of sample buffer solution thorou-
Adjust pH to 6. 8 with hydrochloric acid and dilute to 100 ml
ghly, or prepared as methods described in the monograph.
with water.
Unless otherwise specified, it will be heated in water bath far
(8) Electrode buffer solution Dissolve 3 g of trihydroxy- 3-5 minutes. Reference/ standard substance shall be prepared
methyl aminomethane, 14. 4 g of glycine and 1 g of sodium by the same method.
dodecyl sulfate in a quantity of water. Adjust pH to 8. 3 with 3. Procedure
hydrochloric acid and dilute to 1000 mi with water. ( 1 ) Preparation of separation gel Base on the molecular
( 9) Sample buffer solution Dissolve O. 303 g of trihydroxy- weight, to prepare the separation gel according to the
methyl aminomethane, 2 mg of bromophenol blue, O. 8 g of compositions listed in the following table and inject the gel
sodium dodecyl sulfate, O. 189 ml of hydrochloric acid and into the electrophoresis trough to a certain height. Cover the
4 ml of glycerol in water and dilute to 10 ml. This solution is top with water and polymerize at room temperature. The
sued far non-reducing SDS-PAGE. Far reducing SDS- polymerization time varies at different room temperatures.
PAGE, add 2 mi of ¡1-mercapto ethanol into the solution.
Type of Gel Separation Gel
( 10) Molecular weight standard The molecular weight of
the sample shall be in the range of molecular weight of Gel Concentration 5% 7. 5% 10% 12. 5% 15% 17. 5% 4. 5%
s tandards used.
Solution A 4 4 4 4 4 4
( 11 ) Fixing solution ( Coomassie blue staining method )
Solution B 2. 7 4 5.4 6. 7 8 9. 4 l. 35
Dissolve 5 g of trichloroacetic acid in 200 ml of water, then
add 200 ml of methanol and diluted with water to 500 ml. solution/ Solution e l. 6 l. 6 l. 6 l. 6 l. 6 l. 6 0.9
Fixing solution ( silver staining method A) Dilute 250 mi of mi Solution D o. 1 0.1 0.1 o. 1 o. 1 0.1 0.07
methanol and 60 mi of glacial acetic acid to 500 ml with
water. Solution E 0.1 0.1 0.1 o. 1 0.1 0.1 0.07
Fixing solution ( silver staining method B) Dilute 50 ml of Solution F 2.25
methanol and 54 µl of 37 % formaldehyde solution to 100 ml
with water. Hz O 7. 3 4.88 3.3 2.28 0.88 4.33

( 12) Rinsing solution ( silver staining method A) Dilute
100 mi of ethanol and 50 ml of glacial acetic acid to 1000 ml
with water.
( 13 ) Auxiliary staining solution ( silver staining method A)
Dissolve 10 g of potassium dichromate in a quantity of water.
Add 2 ml of nitric acid and dilute to 200 mi with water.
Make 40-fold dilution befare use.
( 2) Preparation of stacking gel After the separation gel is
polymerized, blot out the water on its top with filter paper
and inject stacking gel ( see formula in the above table).
Insert the sample comb carefully to avoid bubbles.
( 3 ) Sample loading Remove the sample comb after the
stacking gel is polymerized. Fill both front and rear electro-
phoretic troughs with electrode buffer solution. Add 5 µg

(intended for silver staining) of the sample and reference/
standard substance or more than 10 µg (intended for Coomassie
brilliant blue staining) of the sample into the samples loading
well.
( 4} Electrophoresis Vertical plate electrophoresis: Constant
voltage mode, starts at 80 V, then turns to 150-200 V when
the test solution moves into the separating gel. The gel shall
not be terminated until bromophenol blue has migrated to the
bottom of the gel. Or constant current mode, starts at 10
mA, then turns to 20 mA when the test socution moves into
the separating gel. And keep the current at 20 mA until
electro-phoresis is completed.
Disc electrophoresis: Adjust the current to 8 mA per tube.
( 5) Fixing and staining
Ci)Coomassie brilliant bine staining method After electro-
phoresis, remove the gel and immerse it in fixing solution for
30 minutes, then immerse it in staining solution for 1-2
hours. Decolourize the gel with destaining solution until the
background is transparent and store in preserving solution.
®Silver staining method Silver staining method is not used
for quantitative determination unless otherwise specified.
The amount of sample loaded may be properly increased for
qualitative tests. It is recommended to use Coomassie
brilliant blue staining method when the amount-effect
relationship is not proportional for a purity test.
Silver staining method A Immerse the gel after electro-
phoresis in fixing solution for 10-12 hours. Take out the gel
and rinse 3 times, 10 minutes for each time, with destaining
solution at temperature not lower than 25ºC. After rinsing,
immerse the gel in auxiliary staining solution for 7-10
minutes. Take out the gel and wash 3 times by immersing in
water, 2 minutes for each time. After washing, immerse the
gel in silver staining solution. Expose to relatively strong
sunlight or similar light source for 30 minutes, allow to
stand under indoor light for 20 minutes. Take out the gel
from silver staining solution and wash twice by immersing in
water, 1 minute for each time. After washing, immerse the
gel in developing solution. Change the solution every 2
minutes until all the protein bands develop completely.
Immerse the gel in stopping solution for 10 minutes and then
preserve in water.
Silver staining method B Immerse the gel slice for 2 hours at
least in fixing solution, discard the fixing solution; wash the
gel with water for 1 hour at least; place the gel slice in 1%
glutaraldehyde solution for 15 minutes; wash twice the gel
slice with water, 15 minutes for each time, place in silver
nitrate solution for 15 minutes, wash three times the gel slice
with water, 15 minutes for each time. Immerse the gel slice
in developing solution until all bands is examined, put the gel
slice in stopping solution.
4. Results judgment
Measure the migration distance of the bromophenol blue indicator
and the protein using calipers or scanning method for location
(For clise electrophoresis, the length of gel strip befare and after
staining should be measured. The thickness of gel from vertical
plate electrophoresis is less than 1 mm, so the length of gel strip
almost remains unchanged befare and after staining). Calculate
the relative mobility by the following formula:
Relative mobility =
migration distance the length of gel
of the protein zone X strip befare staining
the length of gel migration distance
strip after destaining of the dye zone
( 1) Mobility of main component in the test solution is
0541 Electrophoresis
1
identical to that of reference solution.
(2) Molecular-weight Plot graphically in semi-log coordinate
paper by taking the R' m as abscissa and the log of the
molecular weight of the standard proteins as ordinate.
Calculate the molecular weight of the substance being
examined from the standard curve.
(3) Purity Sean the clear gel in a thin layer scanner and
calculate the percentage content of individual component
using its peak area normalization method.
If commercially available SDS-polyacrylamide precast gels
electrophoresis systems are used, manufacturers may provide
gels of diff erent surface area and thickness. Perform the
electrophoresis under the conditions recommended by the
manufacturer. Electrophoresis running time and current/
voltage need to be adjusted as described in manufacturer
instruction in arder to achieve optimized separation.
[Annotation] Silver staining method A should be adopted,
when the biological products execute Chinese Pharmacopoeia
Volume fil.
Method 6 Isoelectric Focusing Electrophoresis
Ampholytes formed a pH gradient in an electrophoretic field.
Protein, as an ampholyte, carries the electric charges related
to the pH value of the medium. The charged protein will
migrate towards the electrode bearing the opposite polarity
during electrophoresis and stop at the position with a
mínimum migration force where the pH value is equal to its
isoelectric point ( at this point, the specified protein no
longer carries electric charge). The method is used for the
determination of the isoelectric point of protein and peptides
samples.
Isoelectric Focusing Electrophoresis-Method 1
l. Apparatus
It consists of power source supplying constant voltage or
constant current, vertical plate electrophoretic troughs with
cooling equipment and gel preparation mould.
2. Reagents
( 1} Water (Water conductivity shall be not less than 18. 2
M!l•cm).
( 2) Solution A Dissolve 29. 1 g of acrylamide and O. 9 g of
methylene-bisacrylamide in a quantity of water and dilute to
100 ml. Filter with double layers of filter paper and store
protected from light.
( 3 ) Solution B 10 % ammomum persulfate solution,
prepared befare use.
( 4 ) Sample buffer solution ( 4 times of concentration )
Dilute 8 ml of glycerin and 4 ml of 40 % ampholytes solution
(pH 3-10) with water to 20 ml. Add 20 µl of 0.1% methyl
red solution.
( 5 ) Isoelectric point standard The pl range of selected
standards should generally cover pl of all test samples.
( 6) Fixing solution Dissolve 34. 5 g of trichloroacetic acid and
1O. 4 g of sulfosalicylic acid in water and dilute to 300 ml.
(7) Destaining solution (Balancing solution) Mix 500 ml of
95 % ethanol and 160 ml of glacial acetic acid with water and
dilute to 2000 ml.
( 8) Staining solution To O. 35 g of Coomassie brilliant blue
G250 (or R250), add 300 ml of destaining solution. Heat in
60-70ºC water bath until it is dissolved.
( 9) Preserving solution Mix 30 ml of glycerin with 300 ml
of destaining solution thoroughly.
( 10) Cathode solution (O. 01 mol/L phosphoric acid solution)
Dilute lml of phosphoric acid to 1800 ml with water.
 0542 Capillary Electrophoresis
( 11) Anode solution (O. 01 mol/L sodimn hydroxide solution)
Dissolve O. 4 g of sodium hydroxide in water and dilute to
1000 ml.
3. Procedure
( 1 ) Gel preparation Set up the vertical electrophoresis
trough and squeeze out water. Add lml of 60 % glycerin
between cellophane paper and glass plate. Mix 12 ml of
water, 2 ml of glycerin, 4. O ml of solution A and l. O ml of
ampholyte solution with a range of pH 3-10 ( or other
ampholytes) thoroughly. Mix well and <legas. Add 72 µl of
solution B and 3µ1 of N, N, N', N' -tetramethyl
ethylenediamine, and mix well. lnject the mixture into the
trough. lnsert the sample comb carefully to avoid bubbles.
( 2) Test solution preparation Desalt the substance being
examined by dialyzing against water, or by other methods,
and mix well with the sample buffer solution at a volume
ratio of 3 : l. The final concentration of the test solution
shall be more than O. 5 mg per ml or prepare the test solution
as described in each monograph.
( 3 ) Electrophoresis Remove the sample comb carefully
after polymerisation achieved. Add the electrode buffer
solution to front and back electrophoresis troughs. Add 20 µl
of test solution buffer to each sample well. Connect cooling
water circulating system and perform electrophoresis for 30
minutes under 250 V (about 10 mA) at lOºC. Add 20 µl of
test solution and pi standard solution separately to each well and
perform electrophoresis for 3. 5 hours under 500 V (about
10 mA). The voltage should not be more than 2000 V.
( 4) Fixing and staining After electrophoresis, fix the gel
more than 20 minutes in fixing solution and transfer to
balancing solution for 20-30 minutes. Then, immerse the gel
in staining solution for 40-60 minutes. Decolourize the gel
with destaining solution until the background is colorless and
transfer to preserving solution for 30 minutes. The gel may
also be preserved as a dry film.
4. Results judgment
( 1) Identification The principie bands of eletropherogram
obtained with the test solution correspond in position to the
principie bands of eletropherogram obtained with the
reference solution.
( 2) lsoelectric point A linear regression equation is obtained by
regressing the pi values of the isoelectric focusing markers
with the corresponding migration distances. The isoelectric
point of substance being examined is obtained by inserting
the migration distance of substance being examined into the
equation.
Isoelectric Focusing Electrophoresis -Method 2
l. Apparatus
It consists of power source supplying constant voltage or
constant current, horizontal plate electrophoretic troughs
with cooling equipment and gel preparation mould.
2. Reagents
( 1) Water (Water conductivity shall be not less than 18. 2
M!l•cm)
( 2) Solution A Dissolve 29. 1 g of acrylamide and O. 9 g of
methylene-bisacrylamide in a quantity of water and dilute to
100 ml. Filter with double layers of filter paper and store
protected from light.
( 3) Solution B 10 % ammonium persulfate solution, prepared
befare use.
( 4 ) Isoelectric point standard The pi range of selected
standards should generally cover pi of all test samples.
( 5) Fixing solution Dissolve 34. 5 g of trichloroacetic acid
and 10. 4 g of sulfosalicylic acid in water and dilute to
300 ml.
( 6) Destaining solution ( Balancing solution) Mix 500 ml of
95% ethanol and 160 ml of glacial acetic acid with water and
dilute to 2000 ml.
(7) Staining solution To O. 35 g of Coomassie brilliant blue
G250 (or R250), add 300 ml of destaining solution. Heat in
60-70ºC water bath until it is dissolved.
( 8) Preserving solution Mix 30 ml of glycerin with 300 ml
of destaining solution thoroughly.
( 9) Cathode solution (O. 5 mol/L phosphoric acid solution)
Dilute 50 ml of phosphoric acid to 1800 ml with water.
( 10) Anode solution ( O. 2 mol/L sodimn hydroxide solution)
Dissolve 8 g of sodium hydroxide in water and dilute to 1000
ml.
3. Procedure
( 1 ) Gel preparation Mix well 6. 25 ml of solution A,
l. 5 ml of ampholyte solution with a range of pH 3-10 ( or
other ampholytes) and 17. 1 ml of water. Degas the above
solution for 5-10 minutes. Then, add 175 µl of solution B
and 20 µl of N, N, N', N' -tetramethyl ethylenediamine
(adjust the volumn of solvents according to the speed of
gel), and mix well. Inject the mixture slowly into the space
of horizontal plate mould and polymerize at room
temperature. Put the polymerized polyacrylamide gel on the
cooling plate, coat liquid paraffin or kerosene, avoid
bubbles.
( 2 ) Test solution preparation Desalt the substance being
examined by dialyzing against water or by other methods, to
adjust the concentration of sample to O. 5-5 mg of protein or
peptide per ml. Or prepare the solutions as described in the
monograph.
( 3) Electrophoresis Impregna te the cathode strip with
cathode solution and anode strip with anode solution, and
then put them onto the cathode and anode respectively.
Place filter paper for sample loading on the gel. Load 5-
30 µl of test solution and pi standard solution respectively.
Lay the electrodes at the center of electrode strips and lid a
cover. Perform the electrophoresis for 2. 5 hours at 4 ºC
under a voltage with an upper limit of 2000 V and a
current with an upper limit of 5 O mA. The power is 1 W
per 1 cm gel. Remove the filter paper for sample loading
after 3 O minutes of electrophoresis. Perform the
electrophoresis for 3 O minutes under the starting voltage
200 V, if necessary.
( 4) Fixing and staining The same as Vertical Gel Isoelectric
Focusing.
4. Results judgment The same as Vertical Gel Isoelectric
Focusing.
0542 Capillary Electrophoresis
Capillary electrophoresis is a separation technique using
elastic quartz capillary as the separation channel and high
voltage direct current electric field as the driving force. The
components in the sample are separated according to the
difference of electrophoretic mobility ( migration velocity
under the unit electric field strength) and ( or) phase
partition.
When a fused-quartz capillary is filled with an operating
buffer, silanol groups on the inner wall of the capillary

release hydrogen ions to the buffer, which cause the wall' s
surface to carry the negative charges, and then form an
electrical double layer with the solution ( s electric
potential) , even when the buffer is in a fairly low pH value.
The solution with positive charges will move to the cathode
by applying direct current voltage on the capillary. This
movement of the solution under the force of the electrical
field is called the electroosmotic flow (EOF). The ionization
degree of silanol groups on the capillary inner wall is related
to the pH value of the operating buffer and the additive
modifiers. When the pH value decreases, the silanol groups
generally will have a low ionization degree, meanwhile the
EOF decreases. When the pH value increases, the silanol
groups will generally have a high ionization degree and the
EOF increases. The addition of organic modifiers sometimes
could inhibit the ionization degree of the interna! surface,
then it will result in the decreasing of the EOF. In the
buffer, the charged particles migrate towards the opposite
direction to their respective charge with different velocity
under the action of electric field, resulting in electrophoresis.
The migration velocity of the charged particles is equal to the
vector sum of its electrophoretic mobility and eletroosmotic
mobility. The EOF is usually greater than the electrophoretic
mobility; thus, even anions are swept to the cathode and the
detector. Besides lower pH value or the addition of organic
modifiers, coated capillaries can also decrease the EOF and
reduce the absorption of macromolecules on silica surfaces.
l. Separation mode
There are several modes of capillary electrophoresis as follow
in terms of hollow capillary tube as separation carrier.
( 1 ) Capillary zone electrophoresis ( CZE) In CZE, after the
sample being introduced into the capillary, apply direct
current voltage on the capillary. Every components will be
separated depending on the differences in the vector sum of
its electrophoretic and eletroosmotic mobility. The
components will pass the detector in the order of the cation,
the neutral particle, the anion according to their charge and
size. Neutro-moleculars can' t be isolated from each other.
The elution time of certain component in the electro-
phoretogram is called migration time Ctm), equivalent to the
retention time in HPLC and GC.
( 2 ) Capillary isotachophoresis ( CITP ) It employs two
buffers, which endose the analyte zones between them.
Either anions or cations can be analyzed in sharply separated
zones and pass through the detector one by one.
( 3) Capillary isoelectric focusing electrophoresis ( CIEF )
Electroosmotic flow is minimized by coating the capillary
inner wall and the mixture of sample and amphoteric
electrolyte is injected into the capillary. Acid and alkali
solutions are separately added in two electrode reservoirs.
The pH gradient is formed in the capillary af ter high voltage
is applied. The solutes migrate to their isoelectric point
respectively in the capillary, forming discrete zones. After
focusing, the salutes pass through the detector by applying
pressure or changing the pH value of the solutions in the
electrode reservoirs at the end of the detector or using full-
column imaging detection mode.
( 4) Micell electrokinetic capillary electrophoresis ( MEKC)
Ionic surfactants are added to the operating buffer at a
concentration above their critical micelle concentration. The
micelles provide a pseudostationary phase. The substances
are distributed between the aqueous phase and the micelle
phase, solutes are separated according to the difference of
partition coefficient. With common anionic surfactant,
sodium lauryl sulfate, strong hydrophilic component can not
0542 Capillary Electrophoresis
enter the micelle after injection. It flows towards the detector
along with the buffer solution (capacity factor k'=O). But
strong hydrophobic components enter the micelle core and
reach the detector lastly ( k' = oo). Commonly used mi celle
reagents still include cationic surfactant, such as cetrimide
and cholic acid. Amphipathic polymer, especially the block
copolymer may form micelle structure in solution with
different polarity, which plays a role similar to the
surfactant.
( 5 ) Affinity capillary electrophoresis ( ACE ) It is carried
out by loading affinity reagent into buffer or capillary tube to
achieve substances separation. For example, the component
in test sample specifically bound with the pre-fixed protein
(antigen or antibody) in the capillary can be separated based
on high specificity of antibody-antigen reaction, high
efficiency and fast separation of the capillary electrophoresis,
and high sensitivity of laser induced fluorescence detector.
There are severa! modes of capillary electrophoresis as follow
in terms of filled capillary tube as separation carrier.
( 6) Capillary gel electrophoresis ( CGE) For analysis of
protein, DNA and other large molecules, capillary chemical
gel electrophoresis is carried out by loading monomers into
the capillary and initiating monomers to form the gel for
analysis, such as polyacrylamide-gel and agarose-gel. The other
kind of operation, called capillary physical gel electrophoresis, is
carried out by loading the solution of polymer with sieving
action, such as dextran, into the capillary for analysis.
( 7) Capillary electrochromatography ( CEC ) It is carried
out by loading the micro particle size stationary phase in the
capillary or coating onto the inner wall. Or prepare
monolithic columns in the capillary by in situ cross-linked
polymerization of polymers. Use electroosmotic flow as the
driving force for the buffer solution. ( Sometimes supple-
mented with pressure). The analytical method can be
classified into normal phase, reversed phase and ion exchange
chromatography according to different packing materials.
Besides above modes commonly used single capillary, there
is capillary array electrophoresis which is carried out with one
more capillaries. Also chipbased capillary electrophoresis is
used for separation.
( 8) Capillary array electrophoresis (CAE) Generally, only
one sample is analyzed by a single capillary analysis. High-
throughput analysis of samples requires more capillaries
which is called capillary array electrophoresis. Capillary
array electrophoresis system mainly employs laser-induced
fluorescence detection which includes scanning detection and
imaging detection. It is mainly used for the sequence analysis
of DNA.
( 9) Chip based capillary electrophoresis ( Chip CE) Chip
based capillary electrophoresis technique is transferring
routine capillary electrophoresis to the chip. Micron levels of
channels are established by processing glass, quartz and
various polymeric materials. lnjection, separation and
detection of the sample can be performed by using high
voltage direct current electrostatic field as the driving force.
Chip based capillary electrophoresis has the same mechanism as
routine capillary electrophoresis, and also has several advantages
such as short separation time, high efficiency separation, small
size and ease to integrate different operational units. It has
certain advantages in the separation of biomacromolecule
samples.
Among the above modes, (1) and (4) are commonly used.
The mechanisms of modes (5) and (7) are mainly
chromatographic mechanism combined with electrophoresis.
Severa! additive agents added into the operating buffer
 0542 Capillary Electrophoresis
•r·······••I
solution can achieve kinds of separation efficiency. For the
separation of chiral compounds, such as cyclodextrins,
modified cyclodextrins, crown ethers, polysaccharides,
proteins, chleolates, ionic liquid or sorne kinds of antibiotics,
is added into the operating buffer. Organic solvents are added
to improve separation of certain compounds, which can be
analyzed in non aqueous solution.
2. General requirement for the apparatus
The main assembly unit of capillary electrophoresis apparatus
and their function are described as follows:
( 1) Capillary Using an elastic quartz capillary with two
commonly used internal diameters of 50 µm and 75 µm
(capillary with larger internal diameter is sometimes used in
CEC). Smaller capillary diameter has advantages of better
resolution and lower Joule heat, so it permits a higher
voltage. However, smaller capillary diameter has disad-
vantage of greater LOD due to the shorter light length
detected on column. Length of the capillary is called total
length. Capillaries of 20-100 cm can be chosen according to
the separating requirement; length between injector and
detector is called effective length. Capillary is usually housed
in a cartridge to control the Joule heat, viscosity of the
operating buffer and electrical conductivity, which are very
important to the repeatability of the measurements.
( 2 ) High-voltage direct-current power supply A power
supplying controllable direct-current of 0-30 kV (or similar)
can supply direct-current of about 300 µA and provide
constant voltage and constant currency.
( 3) Electrode and electrode reservoirs Add operating buffer
into two electrode reservoirs, insert two ends of the capillary
and platinum electrodes into the operating buffer; connect
platinum electrodes with high-voltage direct-current power
supply, the polarity of electrodes can change with each
other. For many types of instruments, sample bottles also
serve as electrode reservoirs.
( 4 ) Auto-flash and injection system Capillary should be
flushed with different solutions each time before injection. It
is convenient to flush the capillary by auto--flush and inject
system. Modes of sample injection into the capillary include
electromigration ( electrokinetic mode ) and negative-and
positive-pressure injection (hydrostatic mode). The injected
sample volume is controlled by pressure or voltage and the
time.
( 5 ) Detection system CE system can use UV-visible
detector, laser-induced fluorescence (LIF) detector, electro--
chemical detector, mass spectrometric detector, nuclear
magnetic resonance detector, chemoluminescence detector,
LED detector and resonance rayleigh scattering spectra etc.
UV-visible detectors, including single wavelength, program-
ming wavelength and diode arra y detector, are most widely
applied. Strip polymer from the outer layer of the capillary
about 2 mm in length and make the quartz tubal wall to be
exposed. Put a quartz hall on both sides of the capillary so
that the light can be focused on and pass through the
capillary and reach the photovoltaic cell. To detect solute
without absorption ( or fluorescence ) , ultraviolet or
fluorescence derivatization reagent can be selected approp--
riately to react with test samples pre-column, on-column and
post-column to achieve separation and detection of the given
solute. lndirect test method may be adopted to detect non-
absorption ( or fluorescent) solute. Additives which can
absorb light (or fluorescence) may be added to the operating
buffer so that reversal peak will appear as solute reach
detection windows.
( 6 ) Data processing system It is generally the same as
common chromatographic data processing system.
3. System suitability test
System suitability test, such as repeatability ( relative
standard devia tion, RSD) , capaci ty factor ( k ') , theoretical
plate number of capillary (n) , resolution (R) , tailing factor
(T), linearity range, limit of detection (LOD) and limit of
quantitation (LOQ), together with calculating equation and
requirements are the same as those described under HPLC or
GC in order to investigate the configuration of the capillary
electrophoresis system and the setting of parameters. The
index should meet the requirements specified in the
monograph, especially the effect on the precision of the
analysis produced by the precision of the sample introduction
and the difference in the migration rate of different charged
analytes.
4. Procedure
(1) Switch on the instrument, pre-heat, input parameters,
such as capillary temperature, operating voltage, detection
wavelength and flush procedure, etc. Filtrate and <legas the
operating buffer. Fill the flush solution and buffer in the
sample bottles, put onto the injector in order.
(2) The treatment of the capillary has large effect on the
detection result. Using of a new uncoated fused-silica
capillary usually requires a regeneration procedure to actívate
the surface silanol groups. This procedure may include an
extended rinse with 1 mol/L sodium hydroxide solution at
60ºC and then with O. 1 mol/L sodium hydroxide solution, water
and operating buffer, each for a few minutes. In the intermission
of runs, the capillary may be rinsed with the operating buffer.
But if the efficiency of separation changed, the capillary should
be rinsed with O. 1 mol/L sodium hydroxide solution, even with
concentrated hydroxide solution at high temperature. Gel
capillary, coated capillary and packed capillary may be rinsed
following their guidelines. Put the rinsing solution onto the
injector in order, set the order and time program.
(3) Type of operating buffer, pH value, concentration and
additive (used for increasing solubility and (or) controlling the
degree of dissociation and separating the chiral components,
etc.) have large effect on the detection result. They may be
prepared according to the procedure specified in the monograph
and adjusted and optimized according to the result.
( 4) Put the bottles filled with samples onto the injector, set
operating parameters, such as pressure ( voltage in electro--
kinetic mode), injecting time, injecting terminal ( positive
electrode or negative electrode) , operating voltage or current
and detection parameter, etc. , begin testing. Adjust the
operating parameters and operating buffer according to the
electrophoretogram.
(5) After analysis, the capillary is rinsed with water and the
ends of the capillary are preserved in water. For long time
preservation, it should be flushed with nitrogen gas to dry
before shutting the instrument.
( 6 ) The internal standard method is suitable for the
quantitative determination. Viscosity of test solution will
affect injection volume with negative and positive-pressure
injection. It is necessary to keep a uniform viscosity of the
test solution and the reference solution. When the
electrokinetic mode is used, electric discrimination
phenomenon and ionic strength of solution will affect the
migration amount of component in the solution.

0600 Determination of Physical Constant
0601 Determination of Relative Density
Relative density is defined as the ratio of the mass density of
a substance to that of water under the same conditions of
temperature and pressure. Unless otherwise specified, the
measuring temperature is 20ºC.
Relative density of the pure substance is a constant under a
particular condition. However, when the substance is not
purified, the determined value of relative density changes
with the purity. So relative density is indicative of the
identity and purity of the substance being examined.
The relative density of a liquid is determined with a
pycnometer ( as Fig. 1 ) . W estphal balance ( as Fig. 2 ) is
used for the determination of the relative density of a volatile
liquid.
The temperature of determination environment of the
pycnometer and balance should be a little bit below 20ºC or
that specified under individual monograph.
l. Pycnometric method
(1) Fill a clean, dry and accurately weighed pycnometer (as
Fig. la) with the substance being examined ( keep the
temperature below 20ºC or that specified under individual
monograph) , and fix the thermometer (no air bubble is left
in the pycnometer). Place the pycnometer in a water bath
maintained at 20ºC or the specified temperature, allow to
stand in the water bath for severai minutes, so as to raise the
temperature of the content to 20ºC or to the temperature
specified in the monograph. Wipe away the liquid overflown
from the side tube with a piece of filter paper and put the cap
on immediately. Remove the pycnometer from the water
bath and clean off any material on the outside with a piece of
filter paper. Weigh the pycnometer accurately and subtract
the weight of empty pycnometer from the result to calculate
the weight of its content.
4
3
i
o
2 Adjust the temperature to 20ºC or that specified under
a b
Fig. 1 Pycnometer
1-body of the pycnometer; 2-side tube; 3-side hole;
4-cap; 5-thermometer; 6-glass ground joint
Discard the content of the pycnometer and wash the
pycnometer with water, fill it with freshly boiled and cooled
0601 Determination of Relative Density
water. Determine the weight of the water in the pycnometer
in a similar manner. The relative density of the substance
being examined is calculated by the following formula:
weight of the substance
b eing examined
Relative density = - - - - - - - - - -
weight of water
(2) Fill a clean, dry and accurately weighed pycnometer (as
Fig. 1b ) with the substance being examined ( keep the
temperature below 20ºC or that specified under individual
monograph). lnsert the capillary stopper and wipe away the
overflown liquid with a piece of filter paper. Place the
pycnometer in a water bath maintained at 20ºC or the specified
temperature, allow to stand in the water bath for several
minutes.
Sorne of the liquid will spill out through the hole of capillary
stopper constantly with the rise of temperature of the content.
Wipe away the overflown liquid with a piece of filter paper at any
time until the fluid does not overflow anymore. Remove the
pycnometer from the water bath rapidly. Proceed as described in
method (1), starting from the words "clean off any material on
the outside with a piece of filter paper... ".
2. Westphal balance method
Fill the glass cylinder of W estphal balance (as Fig. 2) , of
which the relative density is 1 at 20ºC, with freshly boiled
and cooled water to about 80 % of the volume. Place the
cylinder and stir the content of it in a water bath maintained
at 20ºC or the specified temperature in the monograph.
9
10
Fig. 2 W estphal balance
1-support; 2-adjuster; 3-pointer; 4-beam; 5-cut;
6-rider; 7-hooklet; 8-platinum wire; 9-plunger;
10-glass cylinder; 11-adjusting screw
individual monograph. Dip the plunger hung on the end of
the beam into the water in the cylinder, place a rider at the
notch marked l. 0000 and adjust the equilibrating screw until
the beam is horizontal. Discard the water and wipe away any
water left in the cylinder, fill the cylinder with the substance
being examined to about 80 % of the volume and adjust the
temperature as mentioned above. Dip the plunger into the
substance being examined and equilibrate again by placing
suitable riders at appropriate notches along the beam. The
reading gives directly the relative density of the substance
being examined.
 0611 Determination of Distilling Range
If the Westphal balance is standardized to give a relative
density of l. 0000 at 4 ºC, the riders should be placed at
appropriate notches to give a reading of O. 9982 when
calibrating with water, and the relative density of the
substance being examined, measured at 20ºC, should be
divided by O. 9982.
0611 Determination of Distilling Range
The distilling range of a liquid is the temperature range,
corrected for a pressure of 101. 3 kPa (760 mmHg), within
which the liquid distils under the following conditions. The
lower limit of the range is the temperature at which the fifth
drop of condensate leaves the tip of the condenser and the
upper limit is the temperature at which all the liquid except
3-4 ml has been distilled, or when a specified amount of
distillate has been collected.
Sorne liquid drugs have specified distilling ranges, which is
indicative of the identity and purity of the substance being
examined.
Apparatus Use a set of distillation apparatus No. 19, with
standard ground joint rnade in China (as Fig.), A, a distillation
flask; B, a condenser cooled by water for the liquids distilling
below 130ºC, and by air for the liquids distilling above 130ºC;
C, a 25 ml glass cylinder, graduated in O. 5 ml increments; D, a
partial-immersion thermometer, graduated in O. 2ºC subdivisions
and calibrated. The upper end of the mercury bulb is level with
the lower wall of the side-tube of distillation flask. Select a
heater according to the distilling range oí the substance being
examined. For liquids of distilling range below 80ºC, a water
bath is used as the heater; for liquids of distilling range above
80ºC , use an open flame or an electric heater.
~D
Fig. Distillation Apparatus
Procedure Pour 25 mi of the substance being examined into
the dried distillation flask through a dry funnel with a long
stem. Add a few enamel-free porcelain fragments and insert
the thermometer with ground joint. Attach a 25 mi cylinder
as collector to the lower end of the condenser. If heating with
an open flame, place the distillation flask on the hole in the
center of an asbestos plate (in a width of 12-15 cm, a
thickness of O. 3-0. 5 cm andan aperture of 2. 5-3. O cm), and
the bottom of the flask should be fitted tightly into the
perforation to avoid the excessive heating of the vapour of the
liquid. Heat with an open flame to boíl the substance being
examined and adjust the heating level to distil the liquid at a
rate of 2-3 ml per minute. Record the temperature at which
the fifth drop of condensate leaves the tip of the condenser
and the temperature at which all the liquid except 3-4 mi has
been distilled, or when a specified volume of distillate has
been collected. The temperature range exhibited on the
thermometer is defined as the distilling range of the
substance being examined.
When not less than 90 % of the liquid is to be distilled within
the distilling range, use a 100 ml distillation flask with 50 ml
of the substance being examined, and a 50 mi glass cylinder
as the collector.
Correct the observed temperature readings for any variation
in the barometric pressure from the normal ( 101. 3 kPa or
760 mmHg) by subtracting or adding O. 1 ºC for every O. 36
kPa ( 2. 7 mmHg) increase or decrease of the pressure.
0612 Detennination of Melting Point
Three methods for the determination of melting point are
given herein, varying in accordance with the nature of the
substance being examined. When no specified method is
designated in the monograph, use method l.
Method 1 For Pulverizable solid substances
A. Heating liquid method
Pulverize the substance being examined to a fine powder, dry
ata temperature as specified in the monograph under the test
for Loss on drying, unless otherwise specified. Dry the
substance at 105ºC if it melts above 135ºC without decom-
position, or no test for Loss on drying is required; if the
substance melts below 135ºC or decomposes on heating, dry
it overnight over phosphorus pentoxide or with any suitable
method, e. g. in vacuum ata constant temperature.
Transfer an appropriate amount of the dried powder to a
capillary tube ( made of neutral hard glass with one end
sealed, O. 9-1. 1 mm in internal diameter with a wall
thickness of O. 10-0. 15 mm and at least 9 cm in length.
When more than 6 cm of the capillary tube is immersed into
the heating liquid, longer tube should be used to ensure that
not less than 3 cm of the tube is above the surface of the
liquid) and pack the powder by tapping the tube wall slightly
or dropping from the top of a clear glass tube placed
vertically on the watch glass or other appropriate hard object
so as to form a tightly packed column of 3 mm in height. Put
the thermometer (a partial-immersion thermometer graduated in
divisions of O. 5ºC and calibrated with a reference substance
for melting point determination) into the glass vessel with
heating liquid ( the heating liquid is water for the substances
with melting points below 80ºC, and silicone oil or paraffin
for those with melting points above 80ºC) , the bottom of its
bulb is at least 2. 5 cm away from the bottom of the vessel
( when the vessel equipped with internal heating is used, the
bulb of the thermometer is at least 2. 5 cm away from the
surface of the heating source). Add the appropriate liquid to
the vessel until the surface of the liquid reaches the
immersion line of the thermometer when it is heated. Heat
the bath until the temperature is about lOºC below the lower
limit of the expected melting range, insert the capillary tube
into the bath and fix to the thermometer by using a rubber
ring or capillary clamp to make sure the content of the tube is
kept close to the middle of the bulb. Regulate the rate of
temperature rising to l. 0-1. 5ºC per minute, and keep the
temperature uniform in the bath by constant stirring. Record
the temperatures from the beginning to the end of melting.
Repeat the test for 3 times and calculate the mean value of
results.
"Beginning of melting" is the temperature at which partial
liquefaction of the substance in the capillary tube occurs and
definite liquid drop just appears.
"End of melting" is the temperature at which liquefaction of
 0613 Determination of Congealing Point

the substance takes place just completely. Method 3 For Vaseline or other similar substances
For a substance which melts with decomposition, the melting Take an appropriate amount of the substance being
point is determined in the same way as described above examined, stir slowly and heat until the temperature reaches
except that the rate of temperature rising is regulated to 2. 5- 90-92°C , then add to a heat-resistant vessel with flat bottom
3. OºC per minute. The temperature at which liquefaction to a thickness of 12 mm± 1 mm, and cool clown to a
occurs ( or frothing begins) is served as the beginning of temperature of 8-lOºC above the upper limit of specified
melting, while that at which salid phase disappears as the melting point. Cool a thermometer graduated in divisions of
end of melting. If the salid phase does not disappear O. 2°C with mercury bulb 18-28 mm in length and 5-6 mm in
distinctly, the temperature at which expanding and rising of diameter, of which the upper end is previously stopperred by
decom-position product occur is served as the "end of a cork with a groove cut in the side, to 5°C, then wipe to dry
melting". If there is no distinction between the beginning and
and insert vertically into the molten substance to the bottom of
the ending of melting, the temperature at which a sudden
the vessel (a length of 12 mm is immersed). Take out the
change of the substance occurs should be served as the
thermometer immediately and hold vertically until the surface of
melting point.
the substance adherent to the bulb becomes turbid, then immerse
B. Electric heater method into a water bath ata temperature below 16ºC for 5 minutes, take
Automatic melting point analyzer is applied. Either trans- out, insert into a test tube which is about 25 mm in externa!
mission light or reflected light is used for photometry in an diameter and 150 mm in length, and stopper tightly. Fix the
automatic melting point analyzer. For sorne analyzers, both thennometer securely in the test tube so that the bulb of
the lights are used. Most of the analyzers may be equipped
thennometer is about 15 mm above the bottom of the test tube.
with several capillary tubes for simultaneous test.
Suspend the test tube in a water bath of about 16ºC, adjust the
Transfer an appropriate amount of dried substance being
height of the test tube to make the partial-immersion line of the
examined ( the same as that in Method A) to a capillary
thennometer at the surface of water; raise the temperature of the
tube. Heat the heater of the automatic melting point analyzer
bath at the rate of 2°C per minutes to 38ºC , then change to a rate
to a temperature about lOºC lower than the lower limit of
expected melting point and insert the capillary tube into the of 1ºC per minute and record the temperature at which the first
heater. Regulate the rate of temperature rising to l. 0-1. 5ºC drop of melted substance leaves the thermometer, as the
per minute. Repeat the test for 3 times and calculate the approxirnate melting point of the substances being examined.
mean value of results. Repeat the test for several times. H the variation of three test
For a substance which melts with decomposition, the melting results is not more than 1ºC , take the mean of the three results as
point is determined in the same way as described above the melting point If the variation of three test results is more than
except that the rate of temperature rising is regulated to 2. 5- 1ºC , repeat the test for another 2 times and take the mean of the
3. üºC per minute. five test results.
Far a coloured powder, a substance which melts with
decomposition, a substance in which salid phase disappears
unobviously and at an enlarged volume caused by the 0613 Determination of Congealing Point
formation of decomposed products, ora substance containing
crystal water, instrumental parameters may be adjusted to The congealing point of a substance, determined as described
improve the accuracy of judgment of the change of the below, is the highest temperature, which remains constant for a
melting point. When the photometry by transmission light short time, occurring during the solidification of the liquid
and reflected light is disturbed significantly, visual Sorne drugs have specified congealing point which changes
observation of the change of melting point is permitted. The with the changing purity. Congealing point is indicative of
melting course is recorded and evaluated retrospectively by a the identity and purity of the substance being examined.
camera system. The accuracy of test result should be verified
by Method A if necessary. e
The stated value of temperature of automatic melting point
analyzer should be calibrated regularly with the standard for D
melting point, followed by correction of substance being
examined with the standard.
If the test result by Method Bis uncertainty, Method A is prefer.
Method 2 For Substances not easy to be pulverized ( such as
fat and f atty acids, hard paraffin, lanolin etc)
Melt the substance being examined ata temperature as low as
possible, and then suck the liquid up to a height of about
10 mm in a capillary tube which is the same as that used in
Method 1 but left open at both ends. Cool the tube at lOºC or
lower far 24 hours or on ice for not less than 2 hours. Attach
the tube to the thermometer by means of a rubber band so
that the column of substance in the tube is kept clase to the
middle of the bulb of the thermometer (as method D. Place
the thermometer and capillary tube into the heating bath, mm
and the distance between the upper end of the column of Fig. Apparatus for determination of congealing point
substance and the level of heating liquid is about 10 mm.
Heat the bath with caution until the temperature is about 5°C Apparatus As shown in figure, a dry test tube CA) about
below the lower limit of the expected melting point, and 25 mm in internal diameter and 170 mm in length is fixed by
regulate the rate of temperature rise to not more than O. 5ºC
a bored cork inside a larger tu be ( B) about 40 mm in
per minute. Record the temperature at which the substance
internal diameter and 160 mm in length. The distance
in the capillary tube begins to move upwards.
 0621 Determination of Optical Rotation
between the bottoms of the two tubes is about 10 mm. The
inner tube is stoppered by a bored cork which carries a
thermometer (C) graduated in O. 1 ºC anda stirrer CD). The
bottom of thermometer is about 10 mm above the bottom of
the inner tube. Stirrer (D) is a glass rod slightly bent at the
upper end, while the other end is in the form of a loop which
is about 18 mm in diameter at a right angle to the rod. The
whole assembly is placed in a 1 000 ml beaker containing
water or other suitable cooling liquid and the surface of the
cooling liquid is about 20 mm from the mouth of the beaker.
Procedure Take the substance being examined (15 ml for
liquid, 15-20 g for salid. The salid substance is melted by
heating slightly) into the inner tube and determine the
approximate congealing point by cooling rapidly. Place the
inner tube in a water bath of about 5-lOºC above the
approximate congealing point until all the solidified substance
but the last trace is melted. Fill the beaker with water or
other suitable cooling liquid ata temperature about 5ºC below
the approximate congealing point and fix the apparatus as
described above. Stir the liquefied substance continuously
and read the temperature every 30 seconds until it begins to
solidify. Stop stirring and read the temperature every 5-10
seconds until the temperature remains constant or becomes
constant after a slight elevation for about 1 minute. The
highest temperature observed is regarded as the congealing
point of the substance being examined.
Annotations If the substance being examined does not start
to solidify under normal condition, congelation may be
induced by adding a small amount oí the substance
crystallized by cooling to a lower temperature.
0621 Determination of Optical Rotation
The plane of polarized light can be rotated clockwise or
counterclockwise when plane polarized light passes through
sorne liquids or solutions of compounds which are optically
active. Optical rotation is expressed in degrees by which the
plane of polarization is rotated. Specific optical rotation is
defined as the optical rotation measured under given
wavelength and temperature, when polarized light passes
through a layer of a solution, at a thickness of 1 dm
containing 1 g of optically active substance per ml. Specific
optical rotation ( or optical rotation) is indicative of the
identity and purity of optically active drugs, and may be used
for determination of content of the drugs.
Enantiomer is defined as the stereo-isomer with mirror-image
relationship while is non-overlapping in space structure.
Except the same degree and opposite direction of deflection of
plane polarized light, the physicochemical properties of
enantiomers of chiral materials are identical in non-chiral
environment. In general, biomacromolecules such as
enzymes and biological receptors have the stereoselectivity to
an enantiomer, so the enantimers may be diff erent in
pharmacology and toxicology. Most of materials from nature
origin, such as amino acid, protein, alkaloid, antibody,
glucoside and saccharide, exist in a form of enantiomer.
Racemeate usually consists of equal qrantity of enantiomers,
the net optical rotation is zero, and the physical property may
be different from those of its enantiomer.
The most common light source is the D line ( 589. 3 nm) in
visible regían of sodium lamp. Use of lower wavelengths,
such as those available with the mercury lamp lines isolated
by means of filters of maximum transmittance at
approximately 578, 546, 436, 405, and 365 nm in a
photoelectric polarimeter, has been found to pro vide
advantages in sensitivity with a consequent reduction in the
concentration of the test compound. Other light sources,
such as xenon lamp with an appropriate optical filter, may
also be used.
Unless otherwise specified, the values of optical rotation
cited in this method are measured at 20ºC with sodium D line
(589. 3 nm), in a tube of 1 dm. Conversion factor should be
applied if the tube used is not of the appropriate length.
Optical rotation is measured with a calibrated polarimeter
accurately read to O. 01 º.
In general, optical rotation should be determined within 30
minutes after preparation of solution. Rinse the polarimeter
tube several times with the liquid or solution of substance
being examined prepared as described in the individual
monograph. Fill the polarimeter tube slowly with the liquid
or solution, taking care to avoid creating or leaving air
bubbles. Put the tube into the polarimeter and read the
degrees of optical rotation. Substances are described as
dextrorotatory or levorotatory according to whether the plane
of polarization is rotated clockwise or counter lockwise,
respectively, as viewed toward the light source. Dextro-
rotation is designated as "+" while levorotation as " - ".
Carry out three measurements, take the mean and calculate
the specific optical rotation by one of the following equations:
For liquids [ a JDI -_ lda
, -, 1 _ lOOa
For solid substances La Jn -----¡;;-
Where [a] is specific optical rotation;
D is sodium D line;
is temperature for measurement, ºC;
is the length of polarimeter tube, dm;
a is the observed optical rotation in angular
degrees;
d is the relative density of the liquid;
e is the weight of substance being examined (g)
in 100 ml of the solution, calculated on the
dried basis or anhydrous basis.
Standard quartz polarimetric tube may be used to calibrate
the polarimeter, and the deviation of reading should comply
with the requirements.
Announcements
( 1) Blank tests should be performed to check the zero point
befare and after each measurement. If the deviation of
reading exceeds ± O. 01 after measurement, repeat the
measurement.
(2) The temperature of the solution being tested should be
kept constant at 20ºC ± O. 5ºC ( or at the temperature
specified in the individual monograph).
( 3) Liquids or solutions of a salid substance should be
completely dissolved, and the test solution should be clear.
( 4) The optical rotation of a liquid or a substance in solution
is affected by several factors including light source,
wavelength, solvent, concentration and temperature. When
the optical rotation is described, the condition for
measurement should be provided.
(5) For the substances with known racemization or optical
inversion, measures should be taken to specify the time for
preparation of test solution and time interval of filling the
solution to the polarimeter tube.
 0631 Determination of pH Value
. . . . . .• • . 11·.··
., ..... ,, ..... , .. ···>'

the constitute of the solution to be examined is nearly equal

to that of the standard buffer.
0622 Determination of Refractive lndex The pH value of a solution is determined by an acidimeter.
The pH value of an aqueous solution is determined by a pH
Refraction takes place when a beam of light is transmitted meter using a glass electrode as the indicator electrode, anda
from a transparent medium into another transparent saturated calomel electrode or a silver-silver chloride
medium, since the velocities of light are different in two electrode as the reference electrode. Metrological verification
media. The refractive index of a substance is the ratio of the of the pH meter should be carried out at regular intervals to
velocity of light in air to its velocity in the substance. The meet the related national requirements. Befare each
refractive index may also be defined as the ratio of the sine of measurement, the pH meter should be calibrated with the
the angle of incidence to the sine of the angle of refraction: standard buffer solutions prepared as fallows, or those of a
sin i declared pH value accurate to O. 01 pH unit distributed by
n= sin r national administrative department of certified reference
Where: n is the refractive index; material (CRM).
sin i is the sine of angle of incidence; l. Standard buffer solutions used far the calibration of pH
sin r is the sine of angle of refraction. meters
The refractive index varies with the temperature of the (1) Standard tetraoxalate ~ Weigh accurately 12. 71 g of
substance being examined and the wavelength of incident potassium tetraoxalate, previously dried at 54 ºC ± 3ºC far 4-5
light. It decreases with the increase of temperature. The hours, dissolve in water and dilute to a volume of 1000 ml.
shorter the wavelength of incident light is, the larger the
(2) Standard biphthalate ~ Weigh accurately 10. 21 g of
refractive index is. Refractive indices are usually stated in
potassium biphthalate, previously dried at 115ºC ± 5ºC far
terms of sodium line Data temperature of t and symbolized
2-3 hours, dissolve in water and dilute to a volume of 1000
by nt v . The measurement of refractive index is employed to
ml.
establish the identity of oils and test far purity of the
substance being examined. (3) Standard phosphate ~ Weigh accurately 3. 55 g of
Unless otherwise specified, the values of refractive index anhydrous disodium hydrogen phosphate and 3. 40 g of
cited in this method are measured at 20ºC with sodium line D potassium dihydrogen phosphate, previously dried at 115ºC
( 589. 3 nm) against air ( white light may be used if an Abble ±5ºC far 2-3 hours, dissolve in water and dilute to a volume
refractometer is available). of 1000 ml.
The refractometer should be able to give readings accurate to ( 4) Standard sodium tetraborate ~ Weigh accurately
O. 0001 in the range of l. 3-1. 7. If an Abble refractometer or 3. 81 g of sodium tetraborate (avoid efflorescence), dissolve
other equivalent instrument is used, the measurement should in water to a volume of 1000 ml. Preserve the solution in
be conducted at 20ºC ±O. 5ºC ( or in accordance with the well closed polyethylene containers, protected from carbon
temperature stated under individual monograph). Three dioxide in the air.
readings should be taken and the mean value is used as the
( 5 ) Standard calcium hydroxide ~ Shake an excess of
refractive index of the substance being examined.
calcium hydroxide with carbon dioxide-free water at 25ºC to
The readings of the refractometer should be calibrated befare
prepare a saturated solution, and collect the supernatant far
use with a prism or against water. The refractive index of
use. The buffer solution is a saturated solution of calcium
water is l. 3330 at 20ºC, l. 3325 at 25ºC and l. 3305 at
hydroxide at 25ºC . Make sure if the temperature of the
40ºC.
solution is 25ºC befare use. If not, adjust the temperature to
25ºC and let calcium hydroxide dissolve again till appearance
0631 Determination of pH Value of dissolution equilibrium and then collect the supernatant far
use. Store protected from carbon dioxide in the air. Discard
and repeat the preparation if the solution becomes turbid.
The pH value represents the hydrogen ion concentration of
Standard buffer solutions mentioned above must be
an aqueous solution, which is defined as the negative
prepared from the certified reagents far pH determination.
logarithm of hydroaqueous of the solution, i. e. pH = - lg
The exact pH values of the standard buffer solutions at
a Ht • However, the hydrogen ion concentration is difficult to
different temperatures mentioned above are given in the
be accurately determined by experiments. For practical
fallowing table.
purposes, the pH of a solution is determined by the fallowing
equation: Standard
pH=pHs-(E-Es)/k Standard calcium
Where: E is the potential of the cell containing the solution Tempera- Standard Standard Standard
sodium hydroxide BS
to be examined ( pH) , V; tu re tetraoxa- biphtha- phosphate
tetraborate (saturated
Es is the potential of the cell containing the C°C) late BS late BS BS
BS solution
standard buffer solution of known pH ( pHs), at 25ºC)
V;
k is the constant concerned with temperatureºC. o l. 67 4.01 6.98 9. 46 13.43
k =O. 059 16+0. 000 198(t-25).
l. 67 4.00 6. 95 9.40 13. 21
Since numbers of factors, such as ionization constant of the
solution to be examined, dielectric constant of the medium 10 l. 67 4. 00 6. 92 9.33 13.00
and liquid junction potential etc. , may influence the accurate
15 l. 67 4. 00 6. 90 9.27 12. 81
determination of pH value, the practical value cannot strictly
present the hydrogen ion concentration of the aqueous 20 l. 68 4.00 6.88 9.22 12. 63
solution. In spite of this, the pH value calculated from the
equation above is nearly equal to the real one of the solution if 25 l. 68 4.01 6. 86 9. 18 12. 45
 0632 Determination of Osmolality

continued
Standard 0632 Determination of Osmolality
Standard calcium
Tempera- Standard Standard Standard
sodium hydroxide BS
ture tetraoxa- biphtha- phosphate Biomembrane, such as cell membrane or capillary wall of
tetraborate (saturated
CC) late BS late BS BS human body, has the properties of a semi-permeable
BS solution
at 25°C) membrane. The phenomenon that a solvent diffuses through
a semi-permeable membrane from a solution at a low
30 l. 68 4. 01 6.85 9.14 12.30 concentration to that at a high concentration is called
osmosis. The pressure that is needed to prevent osmosis is
35 l. 69 4.02 6.84 9.10 12. 14 called osmotic pressure. Osmotic pressure plays a critical
40 l. 69 4.04 6.84 9.06 ll. 98 role in all biological processes that involves diffusion of
salutes or transfer of fluids through biomembranes. Thus,
45 l. 70 4.05 6.83 9. 04 ll. 84 much attention should be paid to the osmotic pressure in
producing pharmaceutical preparations such as injections and
50 l. 71 4.06 6.83 9. 01 ll. 71
ophthalmic liquid preparations. Osmolalties of the prepa-
55 l. 72 4.08 6.83 8.99 11. 57 rations with osmotic pressure regulators in their formula
should be controlled.
60 l. 72 4.09 6.84 8.96 ll. 45 The declaration of osmolality on the label of intravenous
infusion, nutrient ( s), electrolyte ( s) or osmotic diuretic
2. Notes Operate the pH meter according to the manufac- agents, such as Mannitol lnjection, are required to guide the
turer' s instructions and pay attention to the following clinical doctors to handle appropriately according to the
precautions when pH value is determined. practical needs, for example, dilute the preparations. The
(1) Select two standard buffer solutions with difference in normal osmolality of human blood ranges from 285 to 310
pH value of 3 units befare determining test solution, and the mOsmol/kg. The osmolality of O. 9 % sodium chloride or
pH value of the test solution is between that of two standard 5 % glucose solution is equal to that of human blood. As one
buffer solutions. of the colligative properties of a solution, osmotic pressure
( 2) Calibra te the apparatus using the primary standard depends on the number of salute particles in the solution,
buffer solution whose pH value is closer to that of test and is usually expressed in Osmolality. lt reflects the total
soiution, adjusting the meter ( fixed position) to read the contribution of various salutes in a solution to its osmotic
appropriate pH value given in table mentioned above. pressure.
(3) Calibrate the apparatus using the second standard buffer The units of osmolar concentration are usually expressed as
solution. The deviation should not be more than ±O. 02 pH milliosmoles (mOsmoD of salute per kilogram of solution.
units, otherwise the slope should be adjusted carefully to The milliosmole concentration may be calculated by the
make the observed pH value meet the value in the table following equation.
mentioned above. Repeat the adjusting procedure for fixed Milliosmole concentration (müsmol/kg)
position and slope until the difference between the observed = Weight of salute in each kilogram of solvent(g) X
value on the apparatus and the value of standard buffer molecular weight(g)
solution is not more than O. 02 pH units. Otherwise the n X 1000
apparatus should be examined or the electrode should be Where: n is the number of particles produced when a
exchanged till complying with the requirement. molecule of salute is dissolved or dissociated.
(4) The electrode should be rinsed with water and dried (or In ideal solutions, for example, n = 1 for
rinsed with the solution being examined) befare each glucose, n = 2 for sodium chloride or
measurement. magnesium sulfate, n =3 for calcium chloride,
(5) Care should be taken to the error caused by alkalinity and n =4 for sodium citrate.
when the solutions being examined solutions and standard Deviations from ideal condition is usually slight in solutions
buffer are determined. Appropriate glass electrode should be within the physiological range and for more dilute solutions,
used if necessary. but for highly concentrated solutions, the actual osmolalities
(6) Unless otherwise specified, the determination of the pH may be appreciably lower than ideal values. For example,
value of a liquid with weak buffering capacity or a liquid the ideal osmolality of O. 9 % sodium chloride injection
without a buffer effect should be conducted after the pH calculated by the above equation is 2 X 1000 X 9/58. 4 = 308
meter is calibrated with standard biphthalate BS, and mOsmol/kg, but in fact, its n value is slightly less than 2,
repeated until the shift of readings is not more than ±O. 05 and the actually measured osmolality is 286 mOsmol/kg.
unit within one minute. Calibrate the pH meter with The reason for this is that, at this concentration, two ions
standard sodium tetraborate BS and repeat the dissociated from one sodium chloride molecule may associate
determination. The pH value of the liquid being examined is at a certain degree, decreasing the number of effective ions.
the mean of the two readings provided that they do not differ The theoretical osmolality of a complex mixture, such as
by more than O. 1 unit. Protein Hydrolysate Injection, is not easy to be calculated,
(7) Freshly boiled and cooled distilled water with a pH value so the actually measured value is usually used.
of 5. 5-7. O should be used for preparing standard buffer
l. Detennination of Osmolality
solutions and dissolving the substance to be examined. Osmolality is usually determined by measuring the
(8) Usually, the standard buffer solutions can be kept for depression of freezing point of solution. In ideally dilute
2-3 months, but should not be used if any turbidity, mold or solution, the depression of freezing point should comply with
precipitate is discovered. the following equation: D.T¡ = K 1 • m, where D.T¡ is the
depression value of freezing point, K 1 is the depression

constant of freezing point ( K = l. 86 when the solvent is
water), m is the weight mole concentration. Osmolality is
calculated by the following equation: Po= Ko•m, where Po
is the osmolality, Kº is the constant of osmolality, and m is
the weight mole concentration. Due to the same meaning of
concentrations in the two equations mentioned above,
osmolality can be determined by measuring the depression of
freezing point.
Apparatus The apparatus designed based on the principie of
depression of freezing point usually consists of a cooling
system, a thermistor used for determination of current or
potential difference and an oscillator ( or metal pro be).
lmmerse the sensor into the center of test solution and lower
into the cooling container. Switch on the cooling system,
when the temperature of test solution decreases under the
freezing point, the instrument induces the solution freezing
by an oscillator (ora metal probe), and the temperature of
the depression of freezing point is recorded automatically.
The determination result may be displayed as either the
temperature of depression of freezing point or the osmolality.
Preparation of standard solution for calibration of osmometer
Dry sodium chloride ( primary reference substance) at
500-650ºC for 40-50 minutes and cool to room temperature in
a desiccator containing silica gel. Weigh accurately an
appropriate amount of sodium chloride as described in the
Table, dissolve in 1 kg of water and mix well to obtain the
standard solution.
Table Standard solution for calibration of osmometer
Weights of sodium The depression
Milliosmolali ty
chloride in 1 kg of of freezing point
(müsmol. kg- 1 )
water (g) 6.T ce)
3.087 100 o. 186
6.260 200 0.372
9.463 300 0.558
12.684 400 o. 744
15.916 500 0.930
19.147 600 l. 116
22.380 700 l. 302
Test solution Unless otherwise specified, the substance to
be tested should be determined directly or prepared into a
solution by the methods in individual monograph based on its
clinical application, of which the osmolality should be within
the range specified in the Table. For example, the sterile
powders for injection may be determined after dissolution and
dilution with the solvent specified on label ( or in instruc-
tion). Special attention should be paid when the interaction
between particles in diluted solution is different from that in
the original solution, so the osmolality of original solution
could not be calculated by multiplying the value of deter-
mined osmolality of diluted solution by the dilution factor.
Procedure Operate according to the instructions of
osmometer. Adjust the zero point of osmometer by using an
appropriate volume of freshly boiled and cooled water, and
calibrate the osmometer by using two standard solutions in the
Table, then determine the osmolality or the depression of
freezing point of the test solution. The osmolality of the test
solution should be between those of the two standard solutions.
0633 Determination of Viscosity
2. Determination of osmolality ratio
The ratio of osmolality of the test solution to that of O. 9 %
( g/ ml ) sodium chloride standard solution is defined as
osmolality ratio. Determine the osmolalities of the test
solution COr) and O. 9% sodium chloride standard solution
(Os) using osmometer respectively by the method the same
as that for determination of osmolality. Calculate the
osmolality ratio by the following equation:
Osmolality ratio = Or /Os
Preparation of standard solution for measurement of osmolality
ratio Dry sodium chloride ( primary standard reagent) at
500-650ºC for 40-50 minutes and cool to room temperature in
a desiccator containing silica gel, then weigh accurately
O. 900 g, dissolve with water and dilute to 100 ml and mix
well.
0633 Determination of Viscosity
Viscosity is the property of a liquid related to resistance to
flow. It is defined in terms of kinetic viscosity, kinematic
viscosity or intrinsic viscosity.
The kinetic viscosity is also known as viscosity coefficient
(r¡). The shearing stress (r) is the tangential force per unit
area and expressed in Pa, which cause the movement of the
hypothetical parallel layers of liquid with gradient velocity.
The shearing velocity ( D) is the velocity gradient with
respect to the direction rectangular to that of the movement
per unit length and expressed in s- 1 • The kinetic viscosity is
the ratio of shearing stress to shearing velocity and its
expression is r¡= dr/ dD. The unit of kinetic viscosity is Pa •s.
The most commonly used unit is mPa • s because it is more
convenient to use.
The relevance of the shearing velocity and shearing stress of
liquids can reflect their rheological properties. There are two
majar kinds of fluids: Newtonian and non-Newtonian,
divided by the above relevance. There is a linear variation of
shearing stress with shearing velocity of Newtonian fluids
without the yield strength. Pure liquids and solutions of low
molecular weight substances belong to this category. For
non-Newtonian fluids, there is a nonlinear variation of
shearing stress with shearing velocity. This category of liquid
consists of high polymer solutions, suspensions, emulsifying
dispersive liquids and surfactant solutions. The kinetic
viscosity of a Newtonian fluid is a constant value at a certain
temperature which <loes not change as the shearing velocity is
changing. Since the kinetic viscosity of a non-Newtonian fluid
varíes with its shearing velocity, the kinetic viscosity
measured at a certain shearing velocity is called an apparent
viscosity.
The kinematic viscosity of a Newtonian fluid is the ratio of
the kinetic viscosity to the density at the same temperature
and expressed in m 2 /s. The most commonly used unit is
mm2 / s because it is more convenient to use.
The viscosity of solvent, r¡o, usually increases with the
dissolution of polymers of high molecular weight. The ratio
of the viscosity of a solution ( r¡) , to that of the solvent
( r¡o ) , is termed as "relative viscosity ( r¡r ) ", which is
usually expressed by the ratio of flow time ( T /To )
measured with the Ubbelohde-type viscosimeter. When the
concentration of high polymer is low, the ratio of logarithmic
value of relative viscosity to the concentration of polymer
solution is the intrinsic viscosity ( r¡) of that polymer. The
mean molecular weight of the polymer concerned may be
estimated from its intrinsic viscosity.
Viscosity can be determined with viscosimeters. Many types
 0633 Determination of Viscosity

of viscosimeters can be used for this purpose. In this the mark mz . Remove the viscosimeter and immediately
method, Ostwald-type capillary viscosimeter, Ubbelohde- revert it, wipe away the liquid adhering to the exterior of
type capillary viscosimeter and rotating viscosimeter are tube 1 and disconnect the rubber tubing from side tube F and
adopted . The capillary viscosimeter is suitable for the connect it to tube l. Place the viscosimeter upright in the
kinematic viscosity determination of Newtonian fluids. The water bath so that the water surface is above the middle of
rotating viscosimeter is suitable for the kinetic viscosity bulb C. Allow to stand in the water bath for 15 minutes,
determination of Newtonian or non-Newtonian fluids. suck from the other end of the rubber tubing until the liquid
or solution fills the bulb A and rises above the mark
Method 1 Ostwald-type capillary viscosimeter method
The time required for a certain volume of a liquid to drop m 1 • Release the suction and allow the liquid to drop
through the capillary by gravity is measured to determine the spontaneously. Record the time required for the liquid
kinetic viscosity or kinematic viscosity of the liquid. surface to drop from m1 to mz. Repeat this measurement 3
times using the same sample, the dropping time recorded in
Apparatus each case should not deviate from the mean value by more
Constant tem perature -water bath A glass or plexiglass than ± O. 25 % . Perform another series of measurements
vessel, 30 cm or more in internal diameter, 40 cm or more in with a second sample. Calculate the kinetic viscosity or
height, equipped with a mechanical stirrer and an electric kinematic viscosity of the sample being examined using the
heating device. The fluctuation of the constant temperature average of the results of the two samplings as follows:
is within ± O. 1 ºC . Unless otherwise specified, kinetic v=Kt
viscosity or kinematic viscosity is measured at 20ºC ±0. 1 ºC. r¡= 10- 6 Kt • p
Thermometer Graduated in divisions of O. 1ºC . Checked Where: K is constant of the viscosimeter determined using
regularly and comply with the relevant requirements. standard liquid of known viscosity, mm2 / s2 ;
Stopivatch Graduated in divisions of O. 2 second. Checked is the mean dropping time, s;
regularly and comply with the relevant requirements. p is the density of the sample being examined,
Ostivald-type Viscosimeter (as Fig. 1) The suitable inside measured at the same temperature, g/ cm3 •
diameter of the capillary viscosimeter can be chosen according Unless otherwise specified, the viscosity is
to the viscosity range of the sample (Table 1). Checked or determined at 20ºC ± O. 1 ºC , thus p = d~8 X
calibrated regularly and comply with the relevant O. 9982, d~8 is the relative density of the sample
requirements. K , constant of the capillary viscosimeter, is at 20ºC.
obtained.
.! 2 Table 1 Specifications of the
Ostwald-type capillary viscosimeter
lnternal Volume of
2 Nominal
Viscosity diameter bulb
Size Constant of
range of tube E (cm3 )
F number viscosimeter
(mm2 /s 3 ) (mm) (±5%)
(mm2 /s 3 )
(±2%) e A
o o. 001 7 o. 6-1. 7 0.40 3. 7 3. 7
1 o. 008 5 l. 7-8. 5 0.60 3.7 3. 7
2 0.027 5. 4-27 0.80 3. 7 3. 7
mz
3 0.065 13-65 l. 00 3. 7 3. 7

E 4 o. 14 28-400 l. 20 3. 7 3. 7
5 0.35 70-350 l. 50 3. 7 3. 7
6 l. o 200-1000 2.00 3.7 3. 7
7 2.6 520-2600 2.50 3.7 3. 7
8 5.3 1060-5300 3.00 3. 7 3. 7
Fig. 1 Ostwald-type Fig. 2 Ubbelohde-type
capillary viscosimeter capillary viscosimeter 9 9. 9 1980-9900 3.50 3. 7 3. 7
1-main tube; 2-wide tube; 1-main tube; 2-wide tube; 10 17 3400-17 000 4.00 3. 7 3. 7
3-bending tube; A-meas- 3-side tube; 4-bending tube;
uring bulb; B-reservoir; A-measuring bulb; B-reser- Note: the minimum dropping time is 200 s, except that of
C-buffering bulb; E-capil- voir; C-buffering bulb; D- Size O Ostwald-type capillary viscosimeter is 350 s.
lary tube; F-side tube; m1, suspended-level reservoir; E- Method 2 Ubbelohde-type capillary viscosimeter method
mz-circular mark capillary tube; m1, mz-circu-
Ubbelohde-type capillary viscosimeter is usually used to
lar mark
determine the intrinsic viscosity of a high polymer solution of
an extremely low concentration.
Procedure Select a suitable Ostwald-type capillary viscosi-
meter with required internal diameter of the capillary tube as Apparatus
described under individual monograph. Connect a rubber Constant tem perature -water bath A glass or plexiglass
tubing to the side tube F of the Ostwald-type viscosimeter, vessel, 30 cm or more in internal diameter, 40 cm or more in
block the mouth of tube 2 with a finger and turn the height, equipped with a mechanical stirrer and an electric
viscosimeter upside clown so that tube 1 is plunged into the heating device. The fluctuation of the constant temperature
liquid or the solution being examined. Suck from the other is within ± O. 1 ºC . Unless otherwise specified, kinetic
end of the rubber tubing until the liquid or solution is filled to viscosity or kinematic viscosity is measured at 20ºC ±0. 1 ºC.
 0633 Determination of Viscosity JM•. · ·
Thermometer Graduated in divisions of O. 1 ºC. Checked (n), angular velocity (w) or shearing velocity is called an
regularly and comply with the relevant requirements. apparent viscosity.
StopUXltch Graduated in divisions of O. 2 second. Checked Rotating viscosimeters can be classified into three types:
regularly and comply with the relevant requirements. coaxial cylinder rotating viscosimeter, cone-plate viscosi-
Ubbelohde-type capillary Viscosimeter (as Fig. 2) The meter and rotating spindle viscosimeter. It also can be
suitable inside diameter of the capillary viscosimeter can be divided into 2 groups, namely absolute and relative viscosi-
chosen according to the viscosity range of the sample (Table meters. In absolute viscosimeters the measuring geometry is
2). Checked or calibrated regularly and comply with the well defined, thus the measurements result in absolute
relevant requirements. K, constant of the capillary viscosity values, which can be compared with any other
viscosimeter, is obtained. absolute values. Coaxial cylinder rotating viscosimeter and
cone-plate viscosimeter belong to this category. In relative
Table 2 Specifications of the Ubbelohde -type
viscosimeters the measuring geometry is not defined. The
capillary viscosimeter
measurements result in relative viscosity values which are
Interna! V l f Interna! obtained through comparing with that of a certified viscosity
Nominal . o ume o d"
Size Viscosity diameter bulb A iameter liquid. It cannot be compared with absolute values or other
Constant of
num- viscosimeter range of tu be E ( cm3 ) of tu be 4 relative values unless using the same viscosimeter and rotor
her (mm2 /s) (mm) % (mm) under the same measurement conditions. Rotating spindle
(mm2 /s 2 ) (±2%) (±5 o) (±5%) viscosimeter belongs to this category.
(1) Coaxial cylinder rotating viscosimeter (absolute viscosi-
oc 0.003 o. 6-3 0.36 2. o 6.0
meter)
1 0.01 2-10 0.58 4.0 6.0 There are two majar kinds of coaxial cylinder rotating
1B 0.05 10-50 0.88 4.0 6.0 viscosimeter: the inner cylinder rotating viscosimeter (Searle
2 o. 1 20-100 l. 03 4.0 6.0 type viscosimeter, Fig. 3) and the outer cylinder rotating
viscosimeter ( Couette type viscosimeter, Fig. 4 ) . Both
2B 0.5 100-500 l. 55 4.0 6.0
viscosimeters mentioned share the same measuring method
3 l. o 200-1000 l. 83 4.0 6. o and formula, but the inner cylinder rotating viscosimeter is
Note: the minimum dropping time is 200 s. more commonly used. lnject the sample or solution of
appropriate concentration as described under individual
Procedure Dissolve the substance being examined in a
solvent to produce a solution of appropriate concentration as monograph into the outer cylinder. Immerse the inner
cylinder in the sample inside the outer cylinder to the
described under individual monograph, filter through a No. 3
described hight. Make the inner or outer cylinder to rotate
sintered glass filter and discard the initial filtra te ( about 1
driven by motor and measure the angular velocity (w) and
mD. Fill not less than 7 ml of the successive filtra te along the
torque ( M) acting on the cylinder surface. The kinetic
inner wall of tube 2 into bulb Bofa clean and dry Ubbelohde-
viscosity is given by the following formula:
type capillary viscosimeter. Place the viscosimeter upright in
the constant temperature \"later bath so that the middle of ~ - _!__ M . ( J_ - J_ \
'I - w - 4rr·h- \Rr R~ J
bulb eis entirely immersed under the surface of water.
Allow it to stand in the water bath for 15 minutes. Connect a Where: r¡ is the kinetic viscosity, Pa • s;
rubber tubing to tube 1 and 3 and clamp the rubber tubing M is the torque acting on the cylinder surface,
connected to tube 3. Suck from the end of the rubber tubing N•m;
connected to tube 1 until the solution rises to the middle of h is the height of immersion of the inner cylinder
in the liquid medium, m;
bulb C. Release the suction and remove the clamps of tube 3
w is the angular velocity of the inner cylinder,
and tube 1 in succession and allow the solution to drop
rad•s~ 1 ;
spontaneously. Record the time required for the surface of
the solution to drop from IIli to m2 . Repeat this
Ri and Ro are radius of the inner and outer cylinder
respectively, m.
measurement 2 times using the same sample, the dropping
The a hove formula can simplified as:
time recorded in each case should not deviate from the mean
M
value by more than ±O. 5 % and take the mean value as the r¡ = K·-
dropping time ( T). Perform the same determination with w
the solvent previously filtered through a No. 3 sintered glass Where
filter. Repeat the operation once more. The values of the two
measurements should be the same and it is taken as the w
dropping time of the solvent (To). Calculate the intrinsic
viscosity as follows:
Intrinsic viscosity [r¡] = lnr¡r /e M
Where 1/r is T /To;
e is the concentration of the solution being
examined, g/ ml.
Method 3 Rotating viscosimeter method
Rotating viscosimeters for the determination of kinetic
viscosity of a liquid are based on the measurement of the
torque (M) acting on a rotor when it rotates at a constant
angular velocity (w) in the liquid. Rotating viscosimeters
are used for measuring the kinetic viscosity of Newtonian
liquids ( shear-independent viscosity) or non-Newtonian
liquids (shear dependent viscosity). The kinetic viscosity of a
non-Newtonian fluid measured at a certain rotational speed Fig. 3 Searle type viscosimeter
 0661 Thermal Analysis
Fig. 4 Couette type viscosimeter
If only the size of the measuring system and the angular
velocity (or rotational speed) are described under individual
monograph when using cylinder rheometer, shearing stress
or shearing velocity can be calculated by the following formula:
M Rr +R~
r = 4TCh X RrR~
D Rr +R~ Rr +R~
TC
= Rr - R~ X w = Rr - R~ X 30n
Where: r is the shearing stress, Pa;
D is the shearing velocity, s- 1 ;
w is the angular velocity of the mner cylinder,
rad•s- 1 ;
n is the rotational speed of the mner cylinder,
r/min;
the meaning and unit of other parameters are the same as
ahove.
(2) Cone-plate viscosimeter (absolute viscosimeter)
Cone-plate viscosimeter is consisted of a flat clise (Fig. 5) and
a cone (Fig. 6) forming a define angle (a). A viscous liquid
or semisolid sample is introduced into the gap between the
flat clise and cone. When either the cone or the flat clise is
rotated at a certain angular velocity (w) driven by motor,
resulting in the shearing force perpendicular to the normal
direction acting on the viscous fluid, the torque ( M )
generated by motor are measured. The kinetic viscosity is
given by the following formula:
3aM
r¡ = 27íR 3 w
Where: r¡ is the kinetic viscosity, Pa•s;
a is the angle between the flat clise and the cone,
rad;
M is the torque, N •m;
R is the radius of the cone, m;
w is the angular velocity of the cone or the flat
clise, rad • s- 1 •
a
Fig. 5 Cone-plate viscosimeter (cone-rotated)
The above formula can simplified as:
M
r¡ = K·-
w
3a
Where: K = ZTCR. 3
Fig. 6 Cone-plate viscosimeter (flat disc-rotated)
If only the size of the measuring system and the angular velocity
( or rotational speed) are described under individual monograph
when using cone-plate rheometer, shearing stress or shearing
velocity can be calculated by the following formula:
3M
T = 2TCR_3
D = ~.n
= !:!!._
a 30a
Where: r is the shearing stress, Pa;
D is the shearing velocity, s- 1 ;
w is the angular velocity of the inner cylinder, rad
·s-1;
n is the rotationai speed of the inner cyiinder,
r/min;
the meaning and unit of other parameters are the same as above.
(3) Rotating spindle viscosimeter (relative viscosimeter)
Select a proper spindle ( there are so many types of spindle,
only sorne of them are shown in Fig. 7 ) , immerse the
spindle in to the sample being examined, make the spindle
rotate at a certain angular velocity ( w) , and measure the
torque (M) generated by the motor. The viscosity is given
by the following formula:
M
r¡=KX-
w
In a general way, the constant K of the apparatus is
determined using a certified viscosimeter calibration liquid,
thus the viscosity determined is relative viscosity.
Fig. 7 the spindles for rotating spindle viscosimeter
0661 Thermal Analysis
Thermal analysis is a group of techniques in which the

physical-chemical properties of a substance being examined
are precisely recorded as a function of temperature according
to a controlled temperature program. And the physical
vanat1on, i. e. crystalline structure inversions, fusion,
evaporation, dehydration or the chemical changes, i. e.
decomposition, oxidation etc. , and the accompanied varation
of temperature, energy and weight are studied.
Absorption or release of energy accompanies phase change or
chemical reaction when a substance is heated or cooled.
According to the phase rule, temperature during phase
inversion C such as melting and boiling points ) keeps
constant. A pure substance has a specific phase inversion
temperature and enthalpy change Ct::.H ). These constants
can be used for qualitative analysis, and the variation or
variation extent of the measured values from them can
indicate the purity of a substance being examined.
Thermal analysis is widely used in the studies of
polymorphism, phase inversion, crystal water, crystalline
solvent, and decomposition of a substance, and purity,
consistency and stability of a drug.
l. Thennogravimetry
Thermogravimetry CTGA) is a technique in which the
weight of a substance being examined is determined as a
function of temperature according to a controlled temperature
program. The thermogravimetry CTGA) curve is recorded
as a graph of relationship between the variation of weight and
temperature CT) or time Ct). The TGA curve is usually in
the shape of a stair, for the temperature during phase
inversion Csuch as loss of crystal water or crystalline
solvent, decomposition etc. ) keeps constant, and the range
in which the weight of the substance being examined basically
keeps unchanged is called a plateau. It is easy to discriminate
the absorbed water Cor absorbed solvent) from the crystal
water Cor crystal solvent) contained in a substance being
examined, and the molecular ratio of crystal water ( or
crystal solvent) can be calculated from the weight loss
percentage between plateaus.
Usually, when heated, loss of absorbed water Cor absorbed
solvent) is a gradual process, while loss of crystal water Cor
crystal solvent) occurs only at a specified temperature point
or range Crelated to heating rate) where the TGA curve is in
the stair shape due to the jump of weight loss.
Thermogravimetry can be used to measure the loss on drying
of certain drugs. When the method is selected to measure the
moisture content, no other volatile compositions should be
contained in the substance being examined.
The temperature and scale of instrument should be calibrated
according to the operating instructions with the standard
substance with certificate Csuch as highly pure indium or zinc
for temperature and calcium oxalate monohydrate for scale)
to ensure the accuracy of determination results.
2. Differential thermal analysis and differential scanning
calorirnetry
When heated simultaneously, temperature difference Ct::.T)
is produced between the substance being examined and the
heat-inert reference material due to the energy effect resulting
from certain physical or chemical changes of the former. As
a function of temperature Cor time) according to a controlled
temperature program, differential thermal analysis CDTA)
and differential scanning calorimetry CDSC) measure the
temperature difference and the conveyed energy difference
C dQ/dT), separately, between the substance being
examined and the reference material.
Differential scanning calorimeter includes powercom-pensated
differential scanning calorimeter and heat flux differential
scanning calorimeter. Powercompensated differential scanning
0661 Thermal Analysis
calorimeter automatically calibrate the heating power
conveyed to the substance being examined to compensate the
energy effect, and keep the temperature constant between the
substance being examined and the reference material Ct::.T =
O). Since t::.T = O, there is no additional heat conduction
between the substance being examined and the ref erence
material. Heat flux differential scanning calorimeter
measures the temperature difference Ct::. T ) between the
substance being examined and the reference material to which
the same heating powers are conveyed, and the t::.T can be
converted to energy difference CdQ/ dT ) by heat flux
equation. Heat flux differential scanning calori-meter is used
widely. The accuracy of quantitative determi-nation by
differential scanning calorimetry is higher than that by
diff erential thermal analysis in general.
The shape of DTA curve is similar to that of DSC, with
temperature or time on the abscissa for both of them, but on
the ordinate t::.T for the former and dQ/dT for the latter. On
both curves, there are specific endothermic or exothermic
peaks corresponding to a certain substance being examined.
a-Aluminium oxide may be used as an inert reference for
DTA and DSC. Empty a-aluminum oxide crucible or the
crucibles made of other inert materials are used in general.
The temperature of instrument should be calibrated according
to the operating instructions with the standard substance
with certifica te Csuch as highly pure indium or zinc) to
ensure the accuracy of determination results.
DTA and DSC can be used to measure the following
parameters.
2. 1 Inversion Temperature
DTA and DSC objectively record the temperature where the
substance state inverse. For example, fusion curve indicates
the onset and peak temperatures within the fusion process.
However, both temperatures may not be in accordance with
the meiting point because of the influence by the factors such
as heating rate.
2. 2 Inversion Enthalpy
The area of endothermal or exothermic peak is directly
proportional to the enthalpy change expressed as the
following equation.
M•t::.H=K•A
Where: M is the mass;
t::.H is the inversion enthalpy of a unit mass;
A is the measured peak area;
K is the instrument constant.
The instrument constant K can be determined using a
standard substance with a known t::.H, then the experimental
inversion enthalpy of a substance being examined can be
calculated from the above equation.
When the chernical compositions of different samples were
identical, while the measured values of the inversion temperatures
or inversion enthalpies by differential thermal analysis or
differential scanning calorimetry change, there are differences
among the crystal solid substance states of the substances.
2. 3 Purity
Theoretically, a pure solid substance melts at a specified
temperature point or infinity narrow temperaute range,
absorbing a certain amount of energy Cfusion enthalpy
t::.H f). A broadening of the melting range or lowering of
melting point means decrease purity. Lowering of melting
point induced by impurity can be expressed as the following
Van' t Hoff equation.
dT/dX = RT2· Ck - 1) C1)
z t::.H¡
Where: T is the thermodynamic temperature, K;
 0681 Determination of Conductivity of Water far Pharmaceutical Use
X2 is the concentration ( molar fraction ) of
impurity;
~H f is the molar fusion enthalpy of pure subs-
tance;
R is the gas constant;
k is the distribution coefficient of impurity
between the salid and liquid phases at fusion.
Perhaps no salid solution farms at fusion, k is equal to O and
the equation (1) can be integrated to give:
X - (To- Tm)~H¡ (2)
z- RT5
Where To is the melting point of pure substance, K;
T m is the measured melting point of the substance
being examined, K.
~Hf, To and T m can be experimentally measured, then the
content of impurity can be calculated by the equation (2).
lndefinite salid ( or amorphous salid) substances may have
no definite melting point ( To ) or have a wide melting
range, and the width of melting range was unrelated to the
chemical or crystalline purities of the substances. The state
of an indefinite substance <loes not meet the principie in Van' t
Hoff equation.
3. Hot stage microscope
Hot stage microscope may be used far observation of phase
change course of substance being examined. The change of
substance under programmed temperature control is observed
and recorded by optical or polarizing microscope.
The observation result by hot stage microscope may provide
more intuitive infarmation on phase change far thermog-
ravimetry, differential thermal analysis and differential
scanning calorimetry. The parts of hot stage microscope far
temperature control needs to be calibrated.
4. Procedure
The thermal analysis procedures of thermogravimetry,
differential thermal analysis, differential scanning caroli-
metry and hot stage microsope are given by each instru-
mental instruction in detail. To obtain an objective, accurate
and reproducible thermogram or phase change principie, it is
appropriate to process a preliminary examination overa wide
temperature range (from room temperature to a temperature
10-20ºC higher the decomposition temperature or melting
point) at a higher heating rate ( 10-20ºC per minute), then
replicate the examination deliberately over a narrow
temperature range at a lower heating rate (e. g. 1 ºC per
minute if necessary) to obtain a satisfactory result of thermal
analysis.
A thermal analysis report should be attached with a complete
description of the determination conditions, including
instrument model, calibrated temperature value, sampling
amount and preparation method of the substance being
examined, atmosphere pressure, direction and rate of
temperature change and the sensitivity of instrument.
It is needed to point out that purity determination using Van' t
Hoff equation is based on the assumption that impurity <loes
not lead to farmation of salid solution. The application of the
method in accurately measuring the chemical or crystalline
purity is limited, especially when the substance being
examined is polymorphism ( the melting points of mixtures
of different crystal forms show no difference) or decampases
at fusion point.
0681 Determination of Conductivity of
Water for Pharmaceutical Use
This method is used for the monitor of total amount of
electrolyte present in water far pharmaceutical use by a
measure of water conductivity.
Conductivity is a physical quantity which represents a
material' s ability to conduct an electric current. The
conductivity of a solution is, by definition, the reciproca! of
resistivity. The unit of conductivity is S/cm (Siemens) or
µS/cm.
Far purified water, to sorne extent, water molecules
dissociate into hydrogen ions and hydroxide ions, and the
conductivity could be detected irrespective of pure water' s
poor ability to conduct electricity. Water conductivity is
closely correlated to the purity of water, the purer the water,
the lower the conductivity, and vice versa. Sorne
atmospheric gases, such as carbon dioxide, readily dissolve
in water and interact to form ions, which raises the
conductivity. Water conductivity is also raised in the
presence of other extraneous ions. Additionally, the water
conductivity varíes as a function of pH and temperature.
Instrument specifications and operating parameters
Water conductivity must be measured accurately using
calibrated instrument. The conductivity cell contains two
parallel electrodes, which are generally protected by a glass
tube. Other types of cells may also be used. The instrument
should be recalibrated regularly according to the its designed
function and degree of use. The cell constant can be
calibrated directly by using a standard solution, or indirectly
by comparing the instrument reading taken with the cell in
question to the readings from a cell of known or certified cell
constant. The conductivity cell constant must be known
n/ r .1 • r· r · . . ~
•. 1 •
wnmn 1
:r c.n 7o 1 1 1 1
OI rne specmea vame OI msrrumenr. r.acn scaie
on the meter may require separate calibration prior to use.
The instrument must have a minimum resolution of
O. 1 µS/ cm on the lowest range and the instrument accuracy
must be ±0. 1 µS/cm.
Because temperature has a substantial impact on conductivity
readings, many instruments automatically correct the actual
reading to display the value theoretically. This temperature
compensation algorithm may not be accurate and is not
applied for the water conductivity. Conductivity values used
in this method are nontemperature-compensated measure-
ments. The accuracy of the temperature measurement must
be within ±2ºC.
Procedure
l. Purified Water
Determine the conductivity off-line or on-line and record the
temperature. The corresponding conductivity value at the
recorded temperature in Table 1 is the limit. Far tempe-
ratures not listed in Table 1, calculate the maximal permitted
conductivity by linear interpolation. If the measured
conductivity is not greater than the conductivity require-
ments, the water to be examined meets the requirements of
the test for conductivity. If the measured conductivity is
greater than this value, the water to be examined <loes not
meet the requirements of the test for conductivity.
Table 1 Temperature and conductivity
requirements ( purified water)
Temperature/ºC Conductivity/ µS•cm- 1
o 2. 4
10 3. 6
20 4.3
25 5. 1
30 5.4
40 6. 5
 0681 Determination of Conductivity of Water for Pharmaceutical Use

continued (2) Transfer a sufficient amount of the water to be examined

Temperature/ºC Conductivity/ µS• cm- 1 000 ml or more) to a suitable container, and stir the test
sample. Adjust the temperature to 25ºC, stir vigorously and
50 7. 1 measure the conductivity every 5 minutes. When the change
60 8. 1 in conductivity is less than O. 1 µS/ cm, record the
conductivity. If the conductivity is not greater than 2. 1 µS/
70 9. 1
cm, the water to be examined meets the requirements of the
75 9. 7 test for conductivity. If the conductivity is greater than
80 9. 7 2. 1 µS/cm, proceed with stage 3.
(3) Perform this test within 5 minutes of the conductivity
90 9. 7 determination under stage 2. Adjust the sample temperature
100 10.2 to 25ºC, add a saturated solution of potassium chloride (0. 3
ml per 100 ml of the test sample), and determine the pH
lnterpolation is given by the expression: value ( 0631 ) to the nearest O. 1 pH unit. Look up the
T-To) corresponding conductivity limit at the measured pH value in
1C = ( T1 -To X (1e1 -1eo) +1eo Table 3. If the measured conductivity under stage 2 is not
Where 1C is the corresponding conductivity at the greater than the conductivity requirements for the pH value
measured temperature; determined, the water to be examined meets the require-
1e 1 is the corresponding conductivity at the next ments of the test for conductivity. If either the measured
higher temperature on the table 1; conductivity is greater than this value or the pH value is
"º is the corresponding conductivity at the next outside the range of 5. 0-7. O, the water to be examined <loes
lower temperature on the table 1 ; not meet the requirements of the test for conductivity.
T is the measured temperature; Table 3 Limits of pH value and conductivity
T1 is the next higher temperature on the table
pH value Conductivity/ µS• cm- 1
1;
T2 is the next lower temperature on the table l. 5.0 4. 7
2. Water for lnjection 5. 1 4. 1
( 1) Determine the conductivity off-line or on-line. The
5. 2 3. 6
conductivity value corresponding to the closest temperature
value that is not greater than the measured temperature in 5.3 3. 3
Table 2 is the limit at that temperature. If the measured 5.4 3.0
conductivity is not greater than the value in Table 2, the
water to be examined meets the requirements of the test for 5. 5 2.8
conductivity. If the conductivity is greater than the value in 5. 6 2. 6
Tabie 2, proceed with stage 2.
5. 7 2. 5
Table 2 Temperature and conductivity requirements
5.8 2.4
(water for injection)
Temperature/ºC Conductivity/ µS • cm- 1 5. 9 2.4
o 0.6 6.0 2.4
5 0.8 6. 1 2.4
10 0.9 6. 2 2.5
15 l. o 6. 3 2.4
20 l. 1
6.4 2.3
25 l. 3
6.5 2.2
30 l. 4
6.6 2. 1
35 l. 5
40 l. 7 6. 7 2.6

45 l. 8 6.8 3. 1
50 l. 9 6.9 3.8
55 2. 1 7.0 4.6
60 2. 2
3. Sterile Water for lnjection
65 2.4 Use off-line equipment and maintain the sample temperature
70 2. 5 at 25ºC. The limit is maximum 25 µS/cm for containers with
75 2.7 a nominal volume of 10 ml or less, while a maximum
5 µS/cm for those with a nominal volume greater than 10
80 2.7
ml. If the measured conductivity is not greater than the
85 2. 7 conductivity requirements, the water to be examined meets
90 2.7 the requirements of the test for conductivity. If the measured
95 2. 9 conductivity is greater than the limit, the water to be
examined does not meet the requirements of the test for
100 3. 1
conductivity.
 0682 Determination of Total Organic Carbon in Water for Pharmaceutical Use
0682 Determination of Total Organic
Carbon in Water for Pharmaceutical Use
This method is used fol the indirect moni toring of organic
substances present in water for pharmaceutical use by a measure
of total organic carbon ( TOC). TOC can also be used as a
process control attribute to monitor the performance of unit
operations comprising the purification and distribution system.
The organic substances present in water for pharmaceutical use
are generally from a water source, water supply system including
purification, storage and distribution, and the velum growth in
water system.
Usually use sucrose as the easy to oxidige substance, and
1,4-benzoquinone as the difficult to oxidige substance.
Prepare the standard solutions according to the requirements and
determine their response values on the instrument to test the
oxidizable ability of the instrument and system suitability.
General requirements for apparatus A variety of methods is
available for measure of TOC. Any method may be used to
measure TOC in water as long as it could meet the following
requirements:
(1) Discriminate between the inorganic carbon, which may
be present in the water due to dissolved carbon dioxide and
decomposition of bicarbonate, and the carbon dioxide
generated from the oxidation of organic molecules in the
sample, and suppress the interference of inorganic carbon to
the determination of organic carbon.
(2) Comply with the requirements for system suitability test.
(3) To be of adequate sensitivity ( with a limit of detection
of O. 05 mg or less of carbon per litre).
This test method is performed either as an on-line test oras
an off-line laboratory test using a calibrated instrument. It is
convenient for the on-line test system to achieve the real-time
monitor and in-process control on water system; quite a few
problems, such as contamination caused by sampling
process, sampling container or less-controlled environmental
factors (including vapor of organic substances), may arise
from the off-line test system. Adoption of an on-line or an
off-line test system depends on the condition of water
production or is justified in a particular case, since batch-size
quantities of the water can be prepared using a continuous
manufacturing process.
Water for TOC determination Use highly purified water
with a TOC level of not more than O. 10 mg per L and a
conductivity of not more than l. O µS/cm at 25ºC . The
prepared water used for standard and system suitability test
solution prepared should share the same container.
Preparation of standard solution Sucrose standard solution
Unless otherwise specified, dissolve an accurately weighed
0700 Other Determination Methods
0701 Potentiometric Titration and
Dead-stop Titration
The potentiometric method and dead-stop method may be
used for the confirmation of end-point or the colour change of
quantity of sucrose CRS, dried to constant weight at 105ºC,
in water for TOC determination to produce a solution
containing l. 20 mg per L (O. 50 mg of carbon per L).
1, 4-benzoquinone standard solution Unless otherwise
specified, dissolve an accurately weighed quantity of
1, 4-benzoquinone CRS in water for TOC determination to
produce a solution containing O. 75 mg per L (O. 50 mg of
car bon per L).
Test solution Off-line test system Extreme caution shall
be taken when collecting samples for TOC analysis, since
water samples can be easily contaminated during the process
of sampling and transportation. In order to ensure the
accuracy of the measured result, collect the test solution in a
tightly closed container with minimal head space, and carry
out the test as rapidly as possible to minimize the impact of
organic contamination and absorbed carbon dioxide. Glass-
ware must be scrupulously cleaned to remove the organic
residue and rinsed with water for TOC determination.
On-line test system Properly connect the water-production
system to the on-line detection devices for TOC determi-
nation. futh sampling and measurement systems must be
adequately cleaned.
System suitability test Test the water for TOC determi-
nation, sucrose standard solution and 1, 4-benzoquinone
standard solution separately in the apparatus and record the
response values. Calculate the percentage response efficiency
using the following expression. The response efficiency is not
less than 85 % and not more than 115 %
rss - rw X 100
rs - rw
Where rw is the response vaule of water for TOC
determina tion;
is the response value of sucrose standard
solution;
is the response value of 1, 4-benzoquinone
standard solution.
Procedure To a volume of the water for pharmaceutical use,
carry out the test as described in instrument standard
operation procedure, run the test and record the response
value ( r u). Unless otherwise specified, the response vaule of
specimen of water for pharmaceutical use ( r u ) is not more
than r s -r w( o. 50 mg/L).
The method can be applied using an on-line or off-line
instrument that has been previously calibrated and verified by
system suitability test. The measured quality attributes of
water by such on-line or off-line instrument is closely
correlated to the sampling location (s) in the water system.
The sampling location ( s) must actually reflect the quality
of the water for pharmaceutical use.
indicators at the end-point. Using a suitable electrode
system, these methods can be applied to oxidation-reduction,
neutralization ( aqueous solution or non-aqueous solution) ,
precipitation titrations, nitrite titrations or the method 1 of
determination of water.
Two different electrodes are used in potentiometric titration,
one is an indicator electrode, whose potential is sensitive to
 0701 Potentiometric Titration and Dead-stop Titration

the ionic concentration of the substance being examined, the continued

other is a reference electrode, whose potential is constant.
Electrode
At the end-point of the titration, the ionic concentration of Procedure Note
system
the substance being examined changes sharply, causing an
abrupt increase or decrease of the potential ( a potential Use a saturated solution of
jump) of the indicator electrode. Neutraliza tion KCl in dehydrated methanol
In the dead-stop titration, a low voltage of about 50 m V is titration in Glass- as the salt bridge. W ash the
applied across two same platinum electrodes. Since the nonaqueous calomel glass electrode after each
electrodes are polarized, there is no current or only a very solution titration and immerse m
weak current flows. When the end-point is reached, a slight water while not in use
excess of the titrant depolarizes the electrodes and a current
flows, causing a permanent deflection of the ammeter Silver-glass
needle. On the other hand, if non-polarized electrodes are Argento- Silver-KN03 Silver electrode cleaned with
polarized, the deflected ammeter needle turns back to zero metrictitration salt bridge- dilute nitric acid quickly
permanently. calomel
Apparatus
In potentiometric titration, a pH meter or potentiometer, or Hydrogen Glass-KN03
any model of a potentiometric titration apparatus may be displacement salt bridge-
used. In dead-stop titration, any model of a dead-stop in-C==CH calomel
titration apparatus or an assembly illustrated in the following
Platinum electrode rinsed
figure may be used (as Fig. ).
with water after being
Mercury Platinum-
immersed in 10% (g/ml)
1.5 V dry cell
potentio- mercury-
sodium thiosulfate. Mercury-
--4 metric mercurous
mercurous sulfate electrode
titration sulfate
6(}-70 n rinsed with water after being
immersed in dilute nitric acid

Cleaned with nitric acid

R galvanometer containing a small quantity of
Dead-stop Platinum-
ferric chloride, or with
titration platinum
chromic acid cleaning
solution

Potentiometric end-point detection The end-point of the

titration can be determined by the plotting method or
calculation method. The plotting method is to plot a graph of
the potential (E) as a function of the volume of the titrant
Fig. Dead-stop titration apparatus added (V). The mid-point of this linear vertical and the
The value of R is approximately equivalent to the critical inflection points of the E-V curve the end-point. The
damping resistance of the galvanometer. calculation method is determined as following steps. The
first derivative till/ t:N (The ratio of till to AV, when till is
Unless otherwise specified, the sensitivity of galvanometer is the potential difference between two successive measure-
10-s A per graduation for the determination of water, and ments and AV is the volume of titrant added corres-
10- 9 A per graduation for nitrite titration. The electrode pondingly) and the second derivative A2 E/ AV2 (The ratio
system applied are shown in the following table. of the difference of successive till /AV to AV) can be
Titration calculated successively. Make a list of results for the
( 1) Potentiometric Titration Place a beaker containing the measured values (E, V) and the calculated values, plot a
solution of the substance being examined on a magnetic curve with the till/ AV or the A2 E/ AV2 as ordinate and the
stirrer, clip the electrodes into the solution and start the corresponding volume of titrant (V) as abscissa. The end-
stirrer. Add the titrant from a burette in portions, record the point occurs at the volume V where till/ AV has the
potential after each addition. When the end-point is nearly maximum value or A2 E/ AV2 is zero (Curve of zero-
reached, add the titrant dropwise and record the potential. crossing). The former is known as the first derivative
Add a few drops more af ter the potential j ump and record the method. The volume of titrant at end-point can also be
potential value. obtained by calculation. It is the average of the adjacent
volumes where the till/ AV has the maximum value. The
Electrode latter is known as the second derivative method. The volume
Procedure Note
system of titrant at end-point can also be obtained by linear
interpolation method performed over the points adjacent to
Platinum electrode cleaned
the zero crossing point:
with nitric acid containing a
Redox titration Platinum- a
small quantity of ferric Vo =V+ a+ b X AV
in aq. solution calomel
chloride, or with chromic
Where: Vo is the volume of titrant at end-point;
acid cleaning solution
a is the absolute value of the second derivative
Neutraliza tion before zero crossing point;
titration in Glass-calomel b is the absolute value of the second derivative
aq. solution behind zero crossing point;
 0702 Non-aqueous Titration
V is the volume of titrant corresponding to point a;
~V is the volume of titrant added between point a
to point b.
The second derivative is the most usual method because of
the accuracy.
By automatic electrometric titrimeter, titration data or
titration curve can be obtained conveniently.
If the colour changes of an indicator at the end-point is to be
checked, add the indicator before the titration and observe
the colour changes in the vicinity of the end-point.
( 2) Dead-stop Titration For the indication of the end-point
of diazotization reaction by a nitrite titration, apply a voltage
of about 50 m V across the electrodes by adjusting R1. Place
an accurately weighed quantity of the substance being
examined in a beaker on a magnetic stirrer, add 40 ml of
water and 15 ml of hydrochloric acid solution Cl-2), unless
otherwise specified, stir to dissolve. Add 2 g of potassium
bromide, clip the electrodes into the solution, plunge the
burette into the solution so that about 2/3 of the tip is under
the liquid surface, ti trate with sodium nitrite (O. 1 or O. 05
mol/L) VS quickly with stirring. Withdraw the burette tip
when the end-point is nearly reached, rinse it with water and
add the washings to the solution, continue the titration slowly
until the galvanometer needle is permanently deflected.
For the indication of the end-point of the method 1 of
determination of water, adjust the resistance R 1 until the
galvanometer shows an initial current of 5-10 µA, titrate
until the current rises to 50-150 µA abruptly and the
deflection of galvanometer needle persists for a few minutes.
0702 Non-aqueous Titration
Non-aqueous titration is carried out in a non-aqueous solvent
and used mainly for the determination of organic bases;
organic salts of hydrohalic acid, phosphoric acid and sulfuric
acid or organic acids and the alkali metal salts of organic
acids, as well as sorne weak organic acids.
Non-aqueous Solvents
(1) Acidic solvents The relative alkalinity of weak organic
bases can be remarkably enhanced in acidic solvents. The
most commonly used acidic solvent is glacial acetic acid.
(2) Basic solvents The relative acidity of weak organic
acids can be remarkably enhanced in basic solvents. The
most commonly used basic solvent is dimethylformamide.
( 3) Amphiprotic solvents They have the properties of
acids and bases. The most commonly used amphiprotic
solvent is methanol.
( 4) Aprotic solvents They are neither acidic nor basic,
such as benzene, chloroform etc.
Method 1
Unless otherwise specified, dissolve an accurately weighed
quantity, equivalent to about 8 ml of perchloric acid
(O. 1 mol/L) VS, of the substance being examined in 10-30 ml
of glacial acetic acid, add 1-2 drops of the indicator as specified in
the monograph and ti trate with perchloric acid (O. 1 mol/L)
VS. The colour change of the indicator should be checked by
potentiometric titration. Carry out a blank titration when
necessary.
If the temperature at which the titration is performed differs
by more than lOºC from the temperature at which the
perchloric acid VS was standardized, the titrant must be
standardized again. If the difference <loes not exceed lOºC,
the concentration of the titrant can be corrected as follows:
No
1 +O. OOllCt1 - to)
Where: O. 0011 is the volume expansion coefficient of
glacial acetic acid;
to is the temperature at which perchloric
acid VS was standardized;
is the temperature at which the titration ·
is performed;
is the concentration of perchloric acid VS
atto;
is the concentration of perchloric acid VS
at t1.
When the substance being examined is a salt of hydrohalic
acids, unless otherwise specified, the titration can be carried
out after the addition of 3-5 ml of mercuric acetate TS. The
mercuric acetate TS should be minimally used because of its
toxity when the titration method is established. Phosphates
and sulfates can be titrated directly, but in the case of
sulfates, the reaction stops when bisulfate is formed. In the
titration of nitrates, the end point should be determined by
potentiometric titration, because the indicator can be
discoloured by nitric acid.
In non-aqueous potentiometric titration, the indicator
electrode is a glass electrode and the reference electrode is a
saturated calomel electrode ( using a saturated solution of
potassium chloride in dehydrated methanol as the salt bridge)
or silver-silver chloride, or use the combined electrode.
Method 2
Unless otherwise specified, dissolve an accurately weighed
quantity, equivalent to 8 ml of the titrant (O. 1 mol/L), of the
substance being examined in a solvent specified in the
monograph, add 1-2 drops oí the speciíied indicator and titrate
with the specified basic titrant (O. 1 mol/L). The colour change
of the indicator should be checked by potentiometric titration.
Carry out a blank titration when necessary.
In the course of the titration, protect the solvent and titrant
from atmospheric carbon dioxide and moisture, and avoid the
evaporation of the titrant.
The electrodes used in potentiometric titration are the same
as that described in method l.
0703 Oxygen-Flask Combustion
In the method, organic substances contammg halogen or
sulfurare burned in a combustion flask filled with oxygen,
the combustion products are absorbed with an absorbing
liquid which is then analysed to determine the content of
halides, sulfur, etc.
Apparatus A 500 ml, 1000 ml or 2000 ml hard glass conical
flask with a tightly fitted ground glass stopper is used as the
combustion flask. One end of a platinum wire ( 1 mm in
diameter) is fused in to the bottom of the stopper, the other
end of the wire is twisted into a helix or reticulation. The
length of the wire is about two-thirds of the length of the
bottle (as Fig. 1).
Fig. 1 Apparatus

Procedure Weigh accurately a specified quantity of the
substance being examined (solid material should be powered
finely ). Unless otherwise specified, the substance being
examined is wraped in a piece of ashless filter paper as shown
in Fig. 2a and 2b, then insert the platinum helix or
reticulation, leaving the narrow strip out. If the substance
being examined is a liquid, stick a piece of ashless filter paper
(16 mm X 6 mm) onto the center of a piece of cellophane
adhesive paper and another piece ( 6 mm X 35 mm) to the
protruding part (as Fig. 2c and as Fig. 2d). Fold the paper
as shown in Fig. 2e, tightly adhere to the bottom and the
other side, leaving the upper side open. Weigh accurately,
then add the liquid being examined dropwise to the filter
paper from the top, close the upper side immediately and
weigh again accurately. The difference of two weighings
0704 Detennination of Nitrogen 1 ¡~t~--~
25-30 mg of nitrogen in a 500 ml dry Kjeldahl flask. Solid or
semi-solid substances may be wrapped in a sheet of filter
paper and then transferred to the Kjeldahl flask. Add
successively 10 g of potassium sulfate or anhydrous sodium
sulfate, O. 5 g of powdered cupric sulfate, then add slowly 20
ml of sulfuric acid along the inner wall of the Kjeldahl flask
puta funnel at the top of Kjeldahl flask inclined atan angle of
45º. Heat the mixture gently, and keep the temperature
below boiling point until forthing has ceased. lncrease the
temperature until there is vigorous boiling. Af ter a clear
green solution is obtained, continue the heating for 30
minutes unless otherwise specified. Cool, add cautiously 250
ml of water along the inner side of the flask, mix well and
allow to cool. Add 75 ml of 40% sodium hydroxide solution
along the inner side of the flask to form a layer beneath the

0
indicate the weight of the substance being examined. acid solution. Add a few pieces of zinc granules, connect one
so 1
end of a nitrogen bulb to the flask and the other end to a
2 condenser. Add 50 ml of 2% boric acid solution and 10 drops
1
1
1
1
lti- º of a mixed methyl red-bromocresol green IS to a 500 ml
o<"".
---1----1---
1

1
: :
1
___ 1L ___ 1L __
1
o 1
narrow strip -
M
'°
conical flask. Dip the end of the condenser into the boric acid
solution, mix the contents of the Kjeldahl flask by gentle
30 25 rotation and distil. When 250 ml of distillate is collected,
draw the end of the condenser above the surface of the
a b e distillate. Continue the distillation for about 1 minute and
rinse the tip of the condenser with water. Titrate the
distillate with sulfuric acid (O. 05 mol/L) VS until the
16X6
fil ter colour changes from bluish green to greyish-violet. Perform
: óX3S a blank determination and make any necessary correction.
1
1
Each ml of sulf uric acid (O. 05 mol/L) VS is equivalent to
d e l. 401 mg of N.
(mm)
Method 2 (Semi-micro Method) The distillation apparatus
Fig. 2 The diagrams to fold the filter paper (Unit: mm)
(As Fig. ). The apparatus consists of a 1000 ml round
bottom flask A, a safety tube B, a distillator C with a
Secure the package containing the substance being examined,
nitrogen bulb, a funnel D, a condenser E and a 100 ml
in the platinum helix or reticulation, leaving the narrow strip
conical flask F. G and H are two damps.
out. Introduce the specified absorbing liquid into the flask,
Add a few drops of methyl red IS and a quantity of water in
moisten the neck of the flask with water, displace the air
flask A and acidify with dilute sulfuric acid, add a few pieces
with oxygen by means of a tube having its end just above the
of glass beads or zeolite and connect flask A to tube B. To
liquid for 1 minute and cover the flask with a watch glass.
distillator C add about 50 ml of water through funnel D,
Ignite the free end of the narrow strip of filter paper and
close clamp G, turn on the water and allow the water to flow
immediately insert the flask. Keep the flask firmly closed
through the condenser and heat the water in flask A to
with the stopper by adding a little water around the rim of
boiling. Stop heating when steam condenses and drips out of
the mouth of the flask during the combustion. As soon as the
the condenser. The water in distillator C backflushes to tube
combustion is completed, shake the flask vigorously until the
B on the closure of clamp H. Open clamp G and drain the
combustion products is completely absorbed. Allow to stand
water intube B. Close the stopcock of tube B and clamp G,
for 15 minutes, wash the stopper and the platinum wire with
dip the end of the condenser into about 50 ml of water which
a little water and combine the washings and the absorbing
backflushes to C and B in turn, again drain the water intube
liquid. Carry out a blank test in the same conditions. Then
B. Repeat this process 2-3 times so that the apparatus inside
proceed with the test or assay as specified in the monograph.
is washed clean.
Annotations Protective measures should be taken against
explosion. D

0704 Determination of Nitrogen

The method is based on the principle that nitrogen-containing

organic substances form ammonium sulfate af ter digestion
with sulfuric acid. ammonium sulfate is decomposed by
sodium hydroxide and ammonia is released. The released
ammonia is evaporated by steam distillation and absorbed by
boric acid solution to form ammonium borate. The formed
ammonium borate solution is titratied with strong acid. the
nitrogen content of the test sample can be calculated from the
volume of strong acid consumed.
Method 1 (Macro Method) Place an accurately weighed Fig. Distillation Apparatus
quantity of the substance being examined equivalent to about
 0711 Determínation of Ethanol
Placean accurately weíghed quantity of the substance being
examined equivalent to l. 0-2. O mg of nitrogen in a 30-50 ml
dry Kjeldahl flask, add O. 3 g of potassium sulfate or
anhydrous sodium sulfate and 5 drops of 30 % cupríc sulfate
solutíon, then add dropwise 2. O ml of sulfuric acid along the
ínner side of the flask. Place a funnel on top of the Kjeldahl
flask ínclined at an angle of about 45º, heat the mixture
gently, keep the temperature below boíling point until
farthing has ceased. Raise the temperature and boíl the
mixture briskly until the solution has been clear green in
colour. Continue the heating far a further 10 minutes, unless
otherwise specified, allow it to cool and add 2 ml of water.
To the conical flask F add 10 ml of 2% boric acid solution
and 5 drops of a mixed methyl red-bromocresol green IS.
Keep the tip of the condenser below the level of the acid.
Transfer the content of the Kjeldahl flask into the distillator
C through funnel D, rinse the Kjeldahl flask and funnel
several times with small amounts of water. Add 10 ml of
40 % sodium hydroxide solution and again wash the funnel
several times with small amounts of water. Clase clamp G
and heat the water in flask A and distill with steam until the
colour of the boric acid solution changes from red to bluish
green. Continue the distillation far a further 10 minutes, lift
the end of the condenser above the surface of the distillate,
flash the condenser with steam far about 1 minute and stop
the distillation and wash the tip of the condenser with water.
Titrate the distillate with sulfuric acid (O. 005 mol/L) VS
until the colour changes from bluish green to greyish violet.
Carry out a blank titration when necessary ( the volume of
distillate collected in the blank determination should be
approximately the same as that collected in the determination
made with the substance being examíned, i. e. about 70-75
mD. Each ml of sulfuric acid O. 005 mol/L VS is equivalent
to O. 1401 mg of N. The amount of sulfuric acid should be
increased when the substance being examined is more than
O. 1 g so as to make the digestion complete, and the amount
of sodium hydroxide solution should also be increased
proportionally.
[AnnotationsJ
(1) The distillator should be rinsed by steam distillation far
more than 15 minutes befare distillation.
(2) Sulfuric acid (0. 005 mol/L) VS preparation Transfer
accurately 100 ml of sulfuric acid (O. 05 mol/L) VS in a
1000 ml volumetric flask, dilute with water to volume and
mix well.
Method 3 ( Azotometer Method)
This method can be used far the determination of nitrogen in
organic substances which contain nitrogen range from semi-
micro to macro.
Semi-automatic azotometer is composed of digestive apparatus
and automatic distillator; while automatic azotometer is
composed of digestive apparatus, automatic distillator and
titrator.
Place a quantity of the substance being examined in the
digestion flask of the apparatus as described in method 1 or
method 2, add potassium sulfate, cupric sulfate and sulfuric
acid successively. Digest according to instructions, usually
keeping the temperature at 150ºC far 5 minutes to remove
water, then increase the temperature to 350ºC Celase to the
boiling point of sulfuric acid) far 5 minutes, and continue
the heating at 400ºC far 60-80 minutes. When the solution
has been clear green in color or almost colourless, continue
the heating far another 10 minutes, remove the digestion
flask and cool.
Transfer the alkali solution, absorption solution and
volumetric solution prepared previously to the corresponding
vessels of the automatic distillator, respectively. Then
successively place the digestion flask in the correct position,
clase the protective <loor, turn on the water and allow the
water to flow through the condenser, edit the parameters of
apparatus such as the volume of reagents, the time of
distillation and the cleaning program, etc. Press the start
button to begin distillation and titration. Distillate collected
by semi-automatic azotometer should be titrated according to
method 1 or method 2.
0711 Determination of Ethanol
l. Gas Chromatography
The gas chromatographic method ( 0521 ) is intended far the
determination of the ethanol ( C2 Hs OH ) content ( %)
(ml/mD in different preparations at 20ºC unless otherwise
specified.
Method 1 (Capillary Colwnn Method)
Chromatographic system and system suitability test U se a
capillary column with 6 % Cyanopropylphenyl -94 %
dimethylpolysiloxane as stationary phase. The initial
temperature is 40ºC and maintained far 2 min. Raise the
temperature at a rate of 3ºC per minute to 65ºC, then raise
the temperature at a rate of 25ºC per minute to 200ºC and
maintained far 10 min. The temperature of the injection port
is 200ºC and the temperature of the detector ( FID) is
220ºC. Split injection with a split ratio of 1 : l. Headspace
injection is equilibrated at 85ºC far 20 min. The number of
theoretical plates of the column is not less than 10 000,
calculated far the peak due to ethanol, and the resolution
factor between the peaks corresponding to ethanol and
n-propanol is more than 2. O.
Determination of correction factor Measure accurately 5 ml
of dehydrated ethanol maintained at 20ºC to a 100 ml
volumetric flask, add 5 ml of n-propanol maintained at 20ºC,
as interna} standard and dilute with water to 100 ml, mix
well. Measure accurately 1 ml and dilute with water to 100
ml, mix well ( dil ute further, if necessary) , as reference
solutions. 2 solutions prepared in parallel, Measure
accurately 3 ml of the solutions into a 10 ml headspace vial
and tightly sealed. lnject a volume of each of the reference
solutions far 3 times successively. Calculate the correction
factors according to the peak area of each injection, the
relative standard deviation of these correction factors is not
more than 2. 0%.
Procedure Measure accurately a volume of the solution to be
examined ( equivalent to about 5 ml of ethanoD , maintained
at 20ºC, to a 100 ml volumetric flask, add 5 ml of accurately
measured n-propanol, maintained at 20ºC, dilute with water
to 100 ml and mix well. Measure accurately 1 ml, dilute
with water to 100 ml and mix well ( dilute further, if
necessary) as the test solution. Transfer 3 ml, accurately
measured, into a 10 ml headspace vial, seal, perfarm
headspace injection, measure the peak area, and calculate the
content by the internal standard method.
[Annotations]
It is recommended to choose a capillary column with large
diameter and thick film (30 m X O. 53 mm, 3. 00 µm).
Method ll (Packed Colwnn Method)
Chromatographic system and system suitability test U se
porous polymer beads of ethylvinylbenzene cross-linked with
divinylbenzene O. 18-0. 25 mm in diameter as the support.
The temperature of the column is maintained at a range of
120-150ºC. The number of theoretical plates of the column is

not less than 700 calculated for the peak due to n-propanol
and the resolution factor between the peaks corresponding to
ethanol and n-propanol is more than 2. O.
Detennination of correction factor Measure accurately 4 ml,
5 ml, and 6 ml of dehydrated ethanol, maintained at 20ºC,
to a 100 ml volumetric flask, add respectively 5 ml of
n-propanol, which is maintained at 20ºC, as internal standard,
dilute with water to 100 ml and mix well ( dilute further, if
necessary) as solutions. Inject a volume of each of the solu-
tions for 3 times. Calcula te the correction factors according
to the peak area of each injection, the relative standard
deviation of these correction factors is not more than 2. O%.
Procedure Measure accurately a volume of the solution to be
examined ( equivalent to about 5 ml of ethanoD , maintained
at 20ºC, to a 100 ml volumetric flask, add 5 ml of
n-propanol, maintained at 20ºC, dilute with water to 100 ml
and mix well (dilute further, if necessary). Inject a volume
into the column, and record the chromatograms. Calculate
the content of ethanol by the internal standard method.
[Annotations]
( 1) In the chromatogram obtained from the test solution in
absence of internal standard, no other peak due to impurity
appears at the same position corresponding to the internal
standard peak.
(2) Unless otherwise specified, if the results obtained by the
distillation method are inconsistent with those obtained by
the gas chromatographic method, the latter prevails.
2. Distillation Method
Distillation method is intended to determine the content ( %)
(ml/mD of ethanol (C2 Hs OH) in various preparations at
20ºC according to the relative density after distillation, and
can be subdivided into the following three methods based on
the different characters of preparations.
Method 1
This method is designated to determine the content of ethanol
in the majority of fluid extracts, tinctures, and preparations
containing glycerin. Based on the content of ethanol, there
are two situations.
{1 ) For preparations presumed to contain 30 % of ethanol or
·~
Adjust the temperature of a quantity of the substance being
examined to 20ºC, measure accurately 25 ml to a 150-200 ml
distillation flask, add approximately 25 ml of water, add a
few glass beads or zeolite or equivalent material, connect to a
condenser, directly heat, and slowly distill at a speed of distillate
collected drop by drop. Drain the distillate into a 25 ml
volumetric flask. Stop distillation upon approximately 23 ml of
distillate collected and adjust the distillate to 20ºC. Dilute to
volume with water at 20ºC, mix well, and determine the relative
density at 20ºC by the method under Determination of Relative
Density (0601 ). Find out the content of ethanol C%) (ml/ml)
in the Table of the Relative Density of Ethanol.
{2) For preparations presumed to contain more than 30 % of
ethanol
Adjust the temperature of a quantity of the substance being
examined to 20ºC, measure accurately 25 ml to a 150-200 ml
distillation flask, add approximately 50 ml of water, and
distill as above. Drain the distillate into a 50 ml volumetric
flask. Stop distillation upon approximately 48 ml of distillate
collected. Dilute to volume with water at 20ºC, mix well, and
determine the relative density by method mentioned above.
Multiply the content of ethanol ( %) (ml/ml) found out in the
Table of Relative Density of Ethanol by 2 to obtain the content of
ethanol ( %) ( ml/ ml) in the substance being examined.
0711 Determination of Ethanol
Method ll
This method is designated to determine the content of ethanol
in tinctures, spirits and other preparations containing volatile
substances, such as volatile oils, chloroform, ethyl ether,
camphor, etc. Also, there are two situations depending on
the content of ethanol.
{1 ) For preparations pmrumed to contain 30% of ethanol or ~
Adjust the temperature of a quantity of the substance being
examined to 20ºC, measure accurately 25 ml to a 150 ml
separating funnel, add an equivalent volume of water, add a
sufficient quantity of sodium chloride to produce a saturated
solution, then add petroleum ether, shake and extract for 1-3
times, 25 ml each time. Allow the volatile substances
dissolve in petroleum ether to eliminate interference in the
determination. Once the liquids separate, transfer the lower
aqueous solution layer into a 150-200 ml distillation flask,
wash the petroleum ether layer with saturated solution of
sodium chloride for 3 times, 10 ml each time. Combine the
washings into the distillation flask, carry out the distillation
as in Method l ( Part 1) and test.
{2) For preparations presunul to oontain nue than 30% of ethanol
Adjust the temperature of a quantity of the substance being
examined to 20ºC, measure accurately 25 ml to a 250 ml
separating funnel, add 50 ml of water, add sodium chloride
as the above method to produce a saturated solution, exact
with petroleum ether for 1-3 times, separate the lower
aqueous solution layer, then carry out the distillation as in
Method I (Part 2) and test.
After adding petroleum ether into the substance being
examined, if emulsification occurs after shaking or the
distillate is still turbid af ter processing with petroleum ether,
use another aliquot of the substance being examined, dilute
with water, carry out the distillation as in Method l , the
distillate is processed as this method. then distill and test.
If the substance being examined is water glue cotton, use
water instead of saturated sodium chloride solution.
Method ][
This method is intended to determine the ethanol content in
preparations containing free ammonia or volatile acid. For
the substance containing free ammonia, adjust with dilute
sulfuric acid to weak acidity. For the substance containing
volatile acid, adjust with sodium hydroxide TS to weak
alkaline. Then carry out the method for distillation in
Method I and test. For the substance also containing
volatile oil at the same time, process as the above method
followed by the Method II . For the substance containing
soap, add an excess volume of sulf uric acid to decompose the
soap, and then carry out the method.
[Annotations]
( 1) If the distillate is turbid in all of the above methods, add
suitable quantity of talcum powder or calcium carbonate, mix
well, filter, to make the solution clear, and then determine
the relative density.
(2) If the test solution produces foams in distillation, add
suitable volume of sulfuric acid or phosphoric acid to strong
acidity, or add an excess volume of calcium chloride TS, or
add a small quantity of paraffin wax, and then distill.
Table the Relative Density of Ethanol
Relative Relative
Concentration Concentra tion
density density
C%) (ml/mD C%) (ml/mD
C20ºC) C20ºC)
o. 9992 0.5 0.9693 25.5
0.9985 l. o 0.9687 26.0
 0712 Determination of Methoxy, Ethoxy and Hydroxypropoxy Groups

continued continued
Relative Relative Relative Relative
Concentration Concentration Concentra tion Concentra tion
density density density density
C%) (ml/mD (%) (ml/mD (%) (ml/mD (%) (ml/mD
(20ºC) (20ºC) C20ºC) (20ºC)

0.9978 l. 5 0.9681 26.5 0.9732 22.0 o. 9376 47.0

0.9970 2.0 0.9675 27.0 0.9726 22.5 0.9366 47.5
0.9968 2. 5 0.9670 27.5 0.9721 23.0 0.9357 48.0
0.9956 3.0 0.9664 28.0
o. 9715 23.5 0.9347 48.5
0.9949 3. 5 0.9658 28.5
o. 9710 24.0 0.9338 49.0
0.9942 4.0 0.9652 29.0
0.9704 24.5 0.9328 49.5
0.9935 4. 5 0.9646 29.5
0.9698 25.0 0.9318 50.0
0.9928 5. o 0.9640 30.0
0.9922 5. 5 o. 9633 30.5
0.9915 6.0 0.9627 31. o 0712 Determination of Methoxy,
0.9908 6. 5 0.9621 31. 5 Ethoxy and Hydroxypropoxy Groups
0.9902 7.0 0.9614 32.0 Gas chromatography ( 0521 ) or volumetry is used for
0.9896 7. 5 0.9608 32.5 determination of the content of methoxy ( -0CH3 ) , ethoxy
( -OC2 Hs) and hydroxypropoxy ( -OCH2 CHOHCH3 )
0.9889 8.0 0.9601 33.0 groups in the excipients such as methylcellulose, ethyl-
0.9883 8. 5 0.9594 33.5 cellulose, hyprolose or hypromellose. Both gas chroma-
tography ( Method 1 ) and volumetry ( Method 2) can be
0.9877 9.0 0.9587 34.0 used. When the result of method 2 doesn' t comply with the
0.9871 9. 5 0.9580 34.5 limit test, the last result should be based on method l.
Method 1 (Gas Cbromatography)
0.9865 10.0 0.9573 35.0
Cbromatographic conditions and system suitability test The
0.9859 10.5 0.9566 35.5 gas chromatograph is equipped with a hydrogen
flameionization detector ( FID) or thermal conductivity
0.9853 11. o 0.9558 36.0
detector CTCD). Use a packed column impregnated with
0.9847 11. 5 0.9551 36.5 20% of 25% phenyl-75% methylpolysiloxane as stationary
phase or a capillary column coated with 6 %
0.9841 12.0 0.9544 37.0 cyanopropylphenyl-94 % dimethylsiloxane ( or similar
0.9835 12. 5 0.9536 37.5 polarity stationary phase) as stationary phase. The initial
temperature of lOOºC lasts for 8 min, then raise the
0.9830 13. o 0.9529 38.0 temperature at a rate of 50ºC per min to 230ºC and maintain
0.9824 13. 5 0.9521 38.5 for 2 min. The temperature of the injection port is 200ºC and
the temperature of the detector is 250ºC. The number of
0.9818 14.0 0.9513 39.0 theoretical plates of the column is not less than 1500 (packed
0.9813 14.5 0.9505 39.5 column) or 10 000 ( capillary column), calculated for the
peak dueto n-octane, and the resolution factor between the
0.9807 15.0 0.9497 40.0 peaks corresponding to reference substance and internal
0.9802 15. 5 0.9489 40.5 standard substance complies with the related requirement.
Inject 1 µl of the solution of reference substance into column
0.9796 16.0 0.9481 41. o with 5 replicate, calculate the correction factors, the relative
0.9790 16. 5 0.9473 41. 5 standard deviation CRSD) is not more than 3. 0%.
Procedure Transfer about 65 mg of dried sample, accurately
0.9785 17.0 0.9465 42.0
weighed, to a previously weighed airtight reaction vial
0.9780 17. 5 0.9456 42.5 equipped with a pressure-tight septum-type closure ( 10 ml
0.9447 43.0 headspace vial can be used), add 80 mg of adipic acid' then
0.9774 18.0
pipet 2. O ml of interna! standard solution ( dissolve about
0.9769 18. 5 0.9439 43.5 O. 5 g of n-octane in a 100 ml volumetric flask with o-xylene
0.9764 19.0 0.9430 44.0 and dilute to volume, mix well) into the vial. Cautiously
pipet 2. O ml of 57 % hydriodic acid into the mixture.
0.9758 19. 5 0.9421 44.5 lmmediately cap the vial tightly, and weigh accurately.
0.9753 20.0 0.9412 45.0 Shake for 60 min at 130-150ºC. or heat at 130-150ºC for 30
min, then shake vigorously for 5 min by hand, continue to
0.9748 20.5 0.9403 45.5 heat at 130-150ºC for 30 min. Allow it to cool and weigh
0.9743 21. o 0.9394 46.0 accurately. If the weight loss is not more than O. 50 % of the
contents in the vial and without evidence of leakage, use the
0.9737 21. 5 0.9385 46.5 upper layer of the mixture as test solution. If the weight loss
 0712 Determination of Methoxy, Ethoxy and Hydroxypropoxy Groups
is more than O. 50 % of the contents in the vial, discard the
mixture and prepare another tset solution. Separately
transfer 80 mg of adipic acid to a reaction vial previously
weighed, then pipet 2. O ml of internal standard solution and
2. O ml of 57 % hydriodic acid in to the vial. Immediately close
the vial securely with a suitable septum stopper and weigh.
Add an amount of methyl iodide ( ethyl iodide or isopropyl
iodide ) reference substance based on the content of
methoxy, ethoxy or hydroxypropoxy groups calculated from
the test sample through the septum with a syringe, weigh
again, and calculate the weight of reference substance added,
by subtraction operation. Shake the reaction vial for about
30 s and allow the layers to separate, use the upper layer as
standard solution.
Separately inject about 1 µl of standard solution and test
solution into the column, and record the chromatograms.
Calculate the content with respect to the area obtained in the
chromatogram by the internal standard method, multiply the
result by the coefficient [ O. 2186 is the coefficient of methyl
iodide (molecular weight 141. 94) into methoxyl group
(molecular weight 31. 03), O. 2889 is the coefficient of ethyl
iodide ( molecular weight 155. 97 ) into ethoxyl group
(molecular weight 45. 06), O. 4417 is the coefficient of
isopropyl iodide ( molecular weight 169. 99 ) into
hydroxypropoxy group (molecular weight 75. 09) J.
Method 2 ( Volumetry}
( 1} Determination of Hydroxypropoxy Groups
Apparatus The apparatus is shown in Fig. l. D is the 25 ml
boiling flask provided with a side arm, the outlet is fitted
with an aluminium foil-jacketed fractionating column E of
95 mm long; e is an adapter bleeder tube, with a capillary
tip of O. 25-1. 25 mm in inner diameter inserting into the
distilling flask; Bis the steam generating tube (25mmX150
mm) with capillary tip of O. 25-1. 25 mm in inner diameter,
attached to the bleeder tube C; F is a condenser with a jacket
of 100 mm long attached to E. G is a graduated, stoppered
conical flask of 125 ml in capacity to collect the distillate. D
and B are immersed in an electric heated oil bath A,
adjusting to keep the temperature at 155ºC.
Fig. l
Procedure Place a quantity of the substance being
examined, specified under individual monograph and
accurately weighed, in the distilling flask D, add 10 ml of
30% (g/g) solution of chromium trioxide. Fill the steam
generator B with water almost to the bottom of the joint,
assemble the apparatus. lmmerse B and D in the oil bath
(glycerin may be used) until the liquid surface in the bath
reach the level of the chromium trioxide solution in D. Start
the cooling water in condenser, pass nitrogen gas through the
flask at a rate of 1 bubble per second if necessary. Raise the
temperature of the oil bath to 155ºC within 30 min and
maintain at the temperature throughout the determination to
collect about 50 ml of the distillate. Detach the condenser
from the fractionating column and rinse with water. Combine
the washings with the distillate, add 2 drops of
phenolphthalein IS, titrate with sodium hydroxide (O. 02
mol/L) VS to a pH of 6. 9-7. 1 ( determined by the pH
meter). Record the volume consumed, V 1 (ml), add O. 5 g
of sodium bicarbonate and 10 ml of dilute sulfuric acid, allow
the solution to stand until the evolution of carbon dioxide
ceases. Add l. O g of potassium iodide, stopper the flask,
shake thoroughly and allow it to stand in the dark for 5 min.
Titrate with sodium thiosulfate (0. 02 mol/L) VS to the end
point, using 1 ml of starch IS, and record the consumed
volume of sodium thiosulfate (O. 02 mol/L) VS, V2 CmD.
Carry out a blank test and record the consumed volumes of
sodium hydroxide (O. 02 mol/L) VS and sodium thiosulfate
(O. 02 mol/L) VS as V. and Vb respectively. Calculate the
percentage content of hydroxypropoxy groups using the
following expression:
CV1M1 -KV2M2) X (0. 0751/W) XlOO%
K is the blank correction coefficient M1 V./M2 Vb;
W is the weight of the substance being examined, g;
M1 is the concentration of sodium hydroxide VS, mol/L;
M2 is the concentration of sodium thiosulfate VS, mol/L;
O. 0751 is the millimolar mass of hydroxypropoxy group.
( 2} Determination of Methoxy Groups
Apparatus As Fig. 2. The apparatus consists of a 50 ml
round bottom flask (A) with a capillary side arm of 1 mm
in inner diameter to provide an inlet for a stream of carbon
dioxide or nitrogen. The flask is fitted with an upright air
condenser (E) about 25 cm long and about 9 mm in interna!
diameter, bent at the top into an outlet which is contracted
into a glass capillary of 2 mm in diameter, dipping in to a
small scrubber ( B) containing about 2 ml of water. The
outlet of the scrubber is a tube of about 7 mm in diameter
with a removable tube of 4 mm in diameter as the
termination, dipping below the surface of the liquid in the
first (C) of two receivers CC and D) connected in series.
receiver
interior diameter 1
exterior diameter 6
(mm)
A
Fig. 2
Procedure Place a quantity of the dried substance being
examined equivalent to 10 mg of methoxyl groups, accurately
weighed, in the boiling flask. Add 2. 5 ml of melted phenol
and 5 ml of hydroiodic acid and connect the apparatus in their
right position. Add 6 ml and 4 ml respectively of a solution
of 10 % potassium aceta te in glacial acetic acid and O. 2 ml of
bromine to the two receivers. Pass a slow, uniform stream
of carbon dioxide or nitrogen through the side arm into the
flask (1-2 bubbles per second are suitable), heat the liquid
gently at such a rate that the boiling liquid vapour rises
halfway up the condenser ( the temperature of oil bath rises
to 135-140ºC in about 30 min). Usually, the reaction is
 0713 Tests of Fats and Fatty Oils
completed in 45 min at the temperature ( the time of heating
is determined by the characteristic and/or structure of
substance being examined. If it contains more than two
methoxyl groups, the time of heating should be extended to
1-3 hours ). Disconnect the apparatus and transfer the
contents of two receivers into a 250 ml stoppered conical
flask containing 5 ml of 25% sodium acetate solution. Wash
the inside of two receivers with water up to about 125 ml in
total volume. Add O. 3 ml of formic acid, rotate the flask
until the colour of bromine is discharged. Add further O. 6 ml
of formic acid, stopper the flask, shake thoroughly to
remove the excess bromine and allow to stand for 1-2 min.
Add l. O g of potassium iodide and 5 ml of dilute sulfuric
acid, titrate with sodium thiosulfate (O. lmol/L) VS. Carry
out a blank test to make any necessary correction. 1 ml of
sodium thiosulfate (O. 1 mol/L) VS is equivalent to O. 5172
mg of methoxyl groups.
Notes:
l. Methyl iodide, ethyl iodide and isopropyl iodide are all
volatile materials. The upper layer of the standard solution
and test solution should be transferred in to the sampling
bottle immediately after opening the seal of the reaction vial.
The bottle and vial should be airtight as well.
2. Methyl iodide, ethyl iodide and isopropyl iodide should be
kept protected from light. The colour of methyl iodide, ethyl
iodide and isopropyl iodide will deepen due to the releasing of
iodine on standing. Determine the content befare use ( see
annotation) and make any necessary correction.
3. A commercially available reagent of 57 % hydroiodic acid
can be used. 57 % hydroiodic acid can also be obtained by
purifying hydroiodic acid from a commercial source. The
procedure for purification is as follows: Transfer hydroiodic
acid from the commercial source into a whole glass apparatus
and add a quantity of hypophosphorous acid until the colour
of hydroiodic acid changes from brown to colourless, heat
and pass nitrogen slowly, collect the fraction of the distillate
of 126-127°C . The purified hydroiodic acid is stored in an
airtight amber-coloured glass bottle under nitrogen.
Annotation Detennination of methyl iodide, ethyl iodide or
isopropyl iodide
l. Purity test (gas chromatography)
Carry out the test protected from light. Gas chromatography
( 0521) is used. The gas chromatograph is equipped with a
hydrogen flame-ionization detector ( FID) or thermal
conductivity detector (TCD). Use a capillary column coated
with 6 % cyanopropylphenyl-94 % dimethylpolysiloxane ( or
similar polarity stationary phase) as stationary phase. The
initial temperature of 60ºC lasts for 8 min, then raise the
temperature at a rate of lOºC per minute to 150ºC and
maintain for 10 min. The temperature of the injection port is
200ºC and the temperature of the detector is 250ºC. Inject
about 1 µl into the column and record the chromatogram. In
the chromatogram, calculate the percentage content of the
main peak by normalization. It should be not less than 99. 5%.
2. ~y {volumetry)
Carry out the test protected from light. Transfer 10 ml of
ethanol into a 100 ml volumetric flask, weigh accurately, add
1 ml of methyl iodide ( ethyl iodide or isopropyl iodide) , and
weigh accurately again. Add ethanol to volume and mix well,
pipet accurately 20 ml of this solution into a second 100 ml
volumetric flask, add accurately 50 ml of silver nitrate
(0. 1 mol/L) VS and 2 ml of nitric acid, stopper, shake
frequently for 2 hours, and allow to stand overnight
protected from light, shake frequently for additional 2 hours,
add water to volume, mix well, filter and discard 20 ml of
the initial filtrate, pipet accurately 50 ml of the successive
filtrate, add 2 ml of ferric ammonium sulfate IS and titrate
with ammonium thiocyanate (O. 1 mol/L) VS. Carry out a
blank test to make any necessary correction. 1 ml of silver
nitrate (0. 1 mol/L) VS is equivalent to 14. 19 mg of methyl
iodide (CH3 D [15. 60 mg of ethyl iodide CC2 Hs D, or
17. 00 mg of isopropyl iodide (C3 H1 D ]. It contains not less
than 98. O% of methyl iodide ( ethyl iodide or isopropyl iodide).
0713 Tests of Fats and Fatty Oils
If a liquid sample being examined shows turbidity owing to
the separation of stearin, warm the container on a water bath
at 50ºC until the sample melts to form a clear liquid. If it
does not become clear on warming, centrifuge or filter
through a dry filter with hot-water jacket. Mix while hot and
not solidified, weigh as many portions as needed for the
various determinations, using preferably a weighting bottle
having a pipette dropper, ora weighing beaker having a glass
spoon. Salid sample is previously melted at a temperature
not more than lOºC above its melting range, centrifuge or
fil ter and then weigh.
Determination of relative density Carry out the method for
Determination of relative density <0601).
Detennination of refractive index Carry out the method for
Determination of refractive index <0622).
Determination of melting range Carry out the method for
Determination of melting range ( 0612, rP...ethod 2).
Detennination of congealing point of fatty acids
{1) Preparation of the fatty acids Heat 75 g of 20 % per
cent g/ g solution of potassium hydroxide in glycerin in
glycerin and 50 g of the substance being examined, in a 800
ml beaker, at 150ºC with constant stirring for about 15
minutes until saponification is complete. Cool to about
lOOºC, add 500 ml of carbon dioxide-free water, stir and mix
well, add slowly 50 ml of sulfuric acid solution 0-4), and
heat the solution until the fatty acids separate clearly as a
transparent layer. Transfer the fatty acids to another beaker
whilehot, wash the acids with freshly boiled water repeatedly
until the washing shows yellow colour to methyl orange IS.
Collect the clear fatty acids in a dry small beaker while hot,
add 5 ml of dehydrated ethanol, mix well and heat gently
until no small bubbles evolved.
{2) Determination of congealing point Carry out the method
for Determination of congealing point <0613), using the dry
fatty acid obtained above.
Detennination of acid value Acid value is the number that
expresses in milligrams the quantity of potassium hydroxide
required to neutralise the free acid in 1 g of fat, fatty oil or
other analogs. In its determination, sodium hydroxide (O. 1
mol/L) VS may also be used.
Quantity of substance
Presumed acid value
being examined/ g
0.5 10
1 5
10 4
50 2
100 1
200 0.5
300 0.4

Unless otherwise specified, weigh accurately a quantity of
the substance being examined as indicated in the above table,
in a 250 ml conical flask, add 50 ml of a mixture of ethanol-
ether ( 1 : 1) which has been neutralised with sodium
hydroxide (O. 1 mol/L) VS befo re use, using l. O ml of
phenolphthalein IS. Shake to dissolve the substance being
examined ( heat under reflux gently, if necessary, until the
substance is completely dissolved). Ti trate the resulting
solution with sodium hydroxide (O. 1 mol/L) VS until a
pink colour which persists for 30 seconds is obtained. Take
the number of ml of sodium hydroxide (O. 1 mol/L) VS
required as A and weight of substance being examined (g)
as W, calculate the acid value from the expression:
A c1·¿ va lue = AXW5. 61
Use semi-micro burette of 10 ml in capacity for the titration
when the acid value is less than 10.
Detennination of saponification value The saponification
value is the number that expresses in milligrams the quantity
of potassium hydroxide required to neutralise the free acids
and to saponify the esters present in 1 g of fat, fatty oil or
other related substances.
Weigh accurately a quantity of the substance, equivalent to
about 250 or maximum saponification value of the substance,
in a 250 ml conical flask, add 25 ml, accurately measured, of
O. 5 mol/L ethanolic potassium hydroxide solution, heat
under reflux for 30 minutes, cool. Rinse the inner wall of
condenser and the lower part of stopper with 10 ml of
ethanol, add 1 ml of phenolphthalein IS, titrate with
hydrochloric acid (0. 5 mol/L) VS until the pink colour just
disappears. Heat the solution to boiling, continue the
titration if pink colour reappears. Take the volume (ml) of
hydrochloric acid (O. 5 mol/L) VS required in titration for
the substance being examined as ( A). Perform a blank
determination and take the volume (ml) of hydrochloric acid
(O. 5 mol/L) VS required in the blank determination as (B).
Calculate the saponification value from the expression:
.f. . l (B- A) X 28. 05
Sapom 1cat1on va ue = W
Where W is the weight of the substance being examined, g.
Detennination of hydroxyl value The hydroxyl value is the
number that expresses in milligrams the quantity of
potassium hydroxide equivalent to the hydroxyl group
contained in 1 g of the substance being examined after
acetylation.
Quantity of substance
Presumed hydroxyl value
being examined/ g
10-100 2.0
100-150 l. 5
150-200 l. o sodium thiosulfate (O. 01 mol/L) VS as A. Carry out a
200-250 o. 75
250-300 0.60
Unless otherwise specified, weigh accurately a quantity of
the substance being examined as indicated in the table in a
250 ml dry conical flask fitted with a ground-glass stopper,
add 5 ml, accurately measured, of acetylating reagent
(prepared by dissolving 14. 4 g of p-toluene-sulfonic acid in
360 ml of ethyl acetate in a 500 ml conical flask, then add
slowly 120 ml of acetic anhydride, mix well and allow to
stand for 3 days). Moisten the glass stopper with a small
0713 Tests of Fats and Fatty Oils 1 •.
quantity of pyridine, stopper the flask tightly and shake
gently to effect solution. Place the flask in a water bath at
50°C±l°C for 25 minute (shake gently at every 10 minutes
intervaD. Cool, add 20 ml of a mixture of pyridine-water
(3 : 5). After 5 minutes, add 8-10 drops of cresol red-
thymol blue mixed indicator solution, titrate with potassium
hydroxide (or sodium hydroxide) (1 mol/L) VS until the
colour of solution changes to greyish-blue or blue. Take the
volume ( ml ) of potassium hydroxide ( or sodium
hydroxide) ( 1 mol/L) VS required as A, perform a blank
determination and take the volume ( ml ) of potassium
hydroxide (or sodium hydroxide) (1 mol/L) VS required as
B. Calculate the hydroxyl value from the expression:
(B-A) X 56.1
Hydroxyl value = W D +
Where: W is the weight of the substance being examined, g;
D is the acid value of the substance being examined.
Detennination of iodine value lodine value is the weight of
iodine in g, required to halogenate 100 g of fat, fatty oil or
other related substances.
Weigh accurately a quantity of the substance (g), equivalent
to 25 or maximum iodine value of the substance being
examined, in a 250 ml dry flask fitted with a ground-glass
stopper. Dissolve it in 10 ml of chloroform, add 25 ml of
iodine bromide solution, accurately measured, stopper the
flask, mix well and keep it in the dark for 30 minutes. Then
add 10 ml of freshly prepared potassium iodide TS and
100 ml of water, mix well. Titrate with sodium thiosulfate
(0. 1 mol/L) VS with constant shaking until the colour
changes to pale yellow, add 1 ml of starch IS, and continue
to titrate until the blue colour just disappears. Take the
volume ( mD of sodium thiosulfate (O. 1 mol/L) VS as A.
Perform a blank determination, and take the volume ( ml)
of sodium thiosulfate (O. 1 mol/L) VS as B. Calcula te the
iodine value from the expression:
. l (B-A) X l. 269
1odme va ue = W
Where: W is the weight of the substance being examined, g.
Detennination of peroxide value The peroxide value is the
number that expresses in milliequivalents of active oxygen
the quantity of peroxide contained in 1000 g of the substance.
Unless otherwise specified, place accurately weighed 5 g of
the substance being examined in a 250 ml iodine flask fitted
with a ground-glass stopper. Add 30 ml of a mixture of 2
volumes of chloroform and 3 volumes of glacial acetic acid.
Shake to dissolve the substance and add O. 5 ml of potassium
iodine TS. Shake for exactly 1 min, then add 30 ml of
water. Ti trate with sodium thiosulfate (O. 01 mol/L) VS,
adding the titrant slowly with continuous vigorous shaking,
until the yellow colour is almost discharged. Add 5 ml of
starch IS and continue the titration, shaking vigorously, until
the blue colour is discharged. Take the volume ( ml) of
blank test under the same conditions, and take the volume
(ml) of sodium thiosulfate (O. 01 mol/L) VS as B and
weight of substance being examined (g) as W. The volume
of sodium thiosulfate (0. 01 mol/L) VS used in the blank
titration must not exceed O. 1 ml. Calculate the peroxide
value from the expression:
. lO(A-B)
Peroxide value = W
Heating test Heat about 50 ml of the substance being
examined, in a beaker, on a sand bath to about 280ºC at a
rate of lOºC per min. Note the change in colour or other
character of the oil.
 0721 Vitamin A Assay
Foreign matter Dissolve about 20 g of the substance being
examined, accurately weighed, in 20 ml of petroleum ether
(boiling range 60-90ºC), in a conical flask. Filter through a
dry and weighed sintered-glass filter (if necessary, add more
volume of petroleum ether to facilita te filtration). Wash the
residue and filter with petroleum ether, dry to constant
weight at 105ºC and weigh accurately. The weight increased
represents the content of foreign matter in the substance
being examined.
Water and volatile matter Weigh accurately about 5 g of the
substance being examined in a weighting bottle dried to
constant weight previously, weigh accurately, dry at 105ºC
for 40 min, allow to cool in desiccator and weigh accurately.
Repeat the drying at l05ºC for further 20 min, cool and
weigh again until two consecutive weighings do not differ by
more than O. 001 g. The loss on drying is the content of
water and volatile matter in the substance being examined.
NOTE Preparation of iodine bromide solution Dissolve
completely 13. O g of powdered iodine in 1000 ml of glacial
acetic acid by warming in a dry glass flask with a ground-
glass stopper. Add promptly 2. 5 ml (or 7. 8 g) of bromine,
stopper the flask and mix well. In order to check the quantity
of bromine added, two titrations may be performed.
(1) Before the addition of bromine, pipette 20. O ml of the
solution, titrate with sodium thiosulfate (O. 1 mol/L) VS,
take the volume (ml) required as A.
(2) After the addition of bromine, pipette 20. O ml of the
solution, add 10 ml of freshly prepared potassium iodide TS,
ti trate with sodium thiosulfate (O. 1 mol/L) VS, take the
volume ( mD required as B. B is slightly less than 2 A.
lodine bromide solution should be kept in a well-closed
container and protected from light.
0721 Vitamin A Assay
The content of Vitamin A in the drug substance or
formulated preparation, expressed in lnternational Units
(IU), is determined by ultraviolet-visible spectrophotometry
( 0401 ) or high performance liquid chromatography
( 0512), 1 IU is equivalent to O. 344 µg of all-trans-retinol
acetate or O. 300 µg of all-trans-retinol.
Carry out the assay as rapidly as possible and avoid exposure
to actinic light.
Method 1 ( Ultraviolet-visible Spectrophotometry)
The absorbance of Vitamin A could not be measured alone
using ultraviolet-visible spectrophotometry due to the
presence of diluting oil and impurities related to Vitamin A.
Under the conditions described below, the absorbance of
nonvitamin A substances was adjusted by using a correction
equation and the proper value was obtained.
The correction equation used involves triple-wavelength
spectrophotometry, the 3 selected wavelength are: the
maximum wavelength, 2 adjacent wavelength on each side of
the maximum. In order to ensure the instrumental accuracy
of the wavelength, the wavelength scale of the spectro-
photometer should be verified before the measurement.
Procedure Dissolve an accurately weighed quantity of the
substance being examined in cyclohexane to produce a
solution containing 9-15 units per ml. Carry out the method
for ultraviolet-visible spectrophotometry ( 0401 ) , det~rmine
the wavelength of maximum absorption, and measure the
absorbance at the specified wavelengths as described in the
following table. Calculate the ratio of the absorbance
measured at the given wavelengths to that measured at 328
nm, as well as the value of El~ at 328 nm.
Wavelength Ratio of Wavelength Ratio of
(nm) absorbance (nm) absorbance
300 0.555 340 o. 811
316 0.907 360 0.299
328 l. 000
If the wavelength of maximum absorption lies between 326
and 329 nm, and the obtained ratios of absorbance are within
±0. 02 of those listed in the table, the content of vitamin A
in units per g can be calculated from the expression:
El~ = (328 nm) X 1900
If the wavelength of maximum absorption lies between 326
and 329 nm, and the obtained ratio of absorbance exceed
±O. 02 of those listed in the table, the absorbance at 328 nm
can be corrected as follows:
A32s (corr.) =3. 52 (2A32s -A316 -A340)
If the difference between corrected absorbance and measured
absorbance at 328 nm is within ± 3. O%, the content of
vitamin A should still be calculated from the measured
absorbance.
If the difference between corrected absorbance and measured
absorbance is within -15 % to -3 %, the content of vitamin
A should be calculated from the corrected absorbance.
If the difference between corrected absorbance and measured
absorbance exceeds - 15 % to - 3 %, or the wavelength of
maximum absorption líes outside the range of 326 and
329 nm, the substance being examined should be assayed as
follows:
Place an accurately weighed quantity ( equivalent to less than
500 units of the total amount of vitamin A, not more than
2 g) of the substance being examined in a saponification
flask. Add 30 ml of ethanol and 3 ml of 50 % ( g/ g)
potassium hydroxide solution. Boil on a water bath for 30
min under a reflux condenser. Allow to cool and rinse the
inner wall of the condenser with 10 ml of water. Transfer the
saponified liquid to a separating funnel ( using glycerin starch
paste as the lubricant for the stopcock of the separating
funneD. Wash the flask with 60-100 ml of water subdivide in
several portions, add the washings to the separating funnel
and extract with 4 quantities (60 ml, 40 ml, 40 ml and 40
mD of peroxide-free ether successively, shaking for 5 min in
each extraction. Combine the ether extracts and wash with
100 ml of water in several portions by swirling gently to
avoid emulsification until the water layer <loes not become red
on the addition of phenolphthalein IS. Filer the ether layer
through a funnel containing anhydrous sodium sulfate and
absorbent cotton wool. Wash the funnel with ether, combine
the washings and the filtrate into a 250 ml volumetric flask,
dilute with ether to volume and mix well. Transfer an
accurately measured quantity of the ethereal solution into an
evaporating dish, and remove the ether by gentle heating.
Dissolve the residue immediately in isopropanol to produce a
solution containing 9-15 Units per ml. Carry out the method
for ultraviolet-visible spectrophotometry ( 0401), determine
the wavelength of maximum absorption and measure the
absorbance of resulting solution at 300 nm, 310 nm, 325
nm and 334 nm. If the wavelength of maximum absorption
lies between 323 nm and 327 nm and the ratio of the
absorbance measured at 300 nm to that measured at the
absorption maximum at 325 nm <loes not exceed O. 73, the
absorbance at 325 nm can be corrected as follows:
A32s (corr.) =6. 815 A32s -2. 555 A310 -4. 260 A334
The content of vitamin A in units per g can be calculated as

follows:
El~ = (325nm, corr.) X 1830
If the corrected absorbance is in the range of 97 %-103% of
measured absorbance, the content of vitamin A should be
calculated from the measured absorbance.
If the wavelength of maximum absorption does not lie
between 323 nm and 327 nm or the ratio of the absorbance
measured at 300 nm to that measured at the absorption
maximum at 325 nm exceed O. 73, it is necessary to measure
accurately another quantity ( equivalent to 300-400 Units of
vitamin A) from the 250 ml of ethereal extract obtained after
saponification, evaporate the ether under reduced pressure to
about 5 ml and continue the evaporation to dryness under a
current of nitrogen. Dissolve the residue immediately in
accurately measured 3 ml of methanol, inject 500 µl of the
resulting solution into a column system for purification
described under method II in Vitamin D Assay ( 0722 ) .
Accurately collect the eluate containing vitamin A and
evaporate to dryness under a current of nitrogen. Continue
the procedure described above beginning at the words
"Dissolve the residue immediately in isopropanol. .. ", and
calculate the content of vitamin A.
Method 2 ( High Performance Liquid Chromatography)
This Method is used to determine the content of the vitamin
A in drug substances and formulated preparations.
Chromatographic conditions and system suitability test U se a
column packed with silica gel anda mixture of n-hexane and
isopropanol ( 997 : 3) as mobile phase, and the detection
wavelength is 325 nm. lnject 10 µl of the system suitability
test solution into the column, the resolution factor between
the peaks due to all-trans-retinol and cis-retinol should be
greater than 3. O. lnject 10 µl of the reference solution into
the column, and the relative standard deviation of the peak
area for all-trans-retinol acetate is not more than 3. O% from
5 replicate injections of the reference solution.
Preparation of system suitability test solution Place an
accurately weighed quantity of vitamin A CRS (equivalent to
about 300 mg of all-trans-retinol acetate) in a beaker, add
O. 2 ml of iodine TS, shake well, allow to stand for 10 min,
transfer quantitatively to a 200 ml volumetric flask, dilute
with n-hexane to volume and shake well. Transfer accurately
measured 1 ml of the resulting solution to a 100 ml
volumetric flask, dilute with n-hexane to volume and shake
well.
Procedure Place an accurately weighed quantity of the
substance being examined (equivalent to about 15 mg of all-
trans-retinol acetate) in a 100 ml volumetric flask, dilute
with n-hexane to volume and shake well. Transfer accurately
measured 5ml of the resulting solution to a 50 ml volumetric
flask, dilute with n-hexane to volume, shake well and use as
the test solution. Prepare the reference solution in the same
manner but using vitamin A CRS instead of the substance
being examined. lnject separately 10 µl of the test solution
and the reference solution into the column and record the
chromatogram. Calculate the content of all-trans-retinol
acetate with respect to the peak area obtained in the
chromatogram by the externa! standard method.
NOTE
(1) Glycerin starch paste To 22 g of glycerin add 9 g of
soluble starch and heat at 140ºC for 30 min with stirring,
allow to cool.
(2) Peroxide-free ether Carry out the test for peroxide as
described under the monograph of Anaesthetic Ether. If the
reagent fails to comply with the requirements, shake it with
5% sodium thiosulfate solution, allow to stand, separate the
0722 Vitamin D Assay l ·····Uf.
ether layer. Wash the ether layer with water and distil,
discard the initial and final aliquots of the distillate, each
about 5 % of the total volume. Examine the remaining
distillate for peroxide, it should comply with the
requirements.
(3) Vitamin A CRS may be used as system suitability test
solution for the evaluation of resolution, if it contains an
appropriate quantity of cis-retinol.
0722 Vitantin D Assay
This method is used for the determination of the content of
vitamin D and pre-vitamin D (including vitamin Dz and D3)
in drug substances, preparations, vitamin AD preparations
or cod liver oil, calculated as the total amount of vitamin Din
International Units ( IU) of activity, by high performance
liquid chromatography ( 0512). Each unit is equivalent to
O. 025 µg of vitamin D.
Protect from light and avoid exposure to air throughout the
procedure.
Method 1 is used for assay of the substance being examined
that has no retinol and impurities which interfere the
determination, otherwise it should be treated according to
Method 2 before assay; if the impurities still interfere the
determination even after the treatment by Method 2, proceed
the assay by Method 3.
Method 1
Preparation of stock reference solution According to the
ingredient stated in vitamin D preparation, place 25 mg of
vitamin Dz or ~ CRS, accurately weighed, in a 100 ml amber
volumetric flask, add 80 ml of iso-octane to dissolve with the
aid of ultra-sonication for 1 minute and protect from heating,
dilute to volume with iso-octane, shake well and use as the
stock solution (1). Transfer accurately measured 5 ml of the
stock solution ( 1) into a 50 ml amber volumetric flask,
dilute to volume with iso-octane, shake well and fill with
nitrogen. Stopper and use as the stock solution ( 2). Store
below OºC and protect from light.
In the Assay of vitamin Dz, prepare the vitamin D3 stock
reference solution in the same manner by using 25 mg of
Vitamin Th CRS, and use as the system suitability test
solution.
Chromatographic conditions and system suitability test Using
a column packed with silica gel and a mixture of n-hexane
and n-amyl alcohol (997 : 3) as the mobile phase. Detector
wavelength is 254 nm. Transfer 5 ml of vitamin D3 stock
solution ( 1) into a glass container with stopper and purge
the solution with nitrogen and stopper. Place the container in
a water bath at 90ºC for 1 hour, allow to cool rapidly, add 5
ml of n-hexane and shake well. Introduce the solution in a
1 cm quartz cell with stopper, and incline the cell at 45º.
Excite under two 8 W ultraviolet lamps with main
wavelength of 254 nm and 365 nm each at a distance of 5-6
cm for 5 minutes to convert sorne vitamin Th to pre-vitamin
D3, trans-vitamin Th, and tachysterol D3. Inject the resulting
solution into the column 5 times and record the
chromatograms. The relative standard deviation for the area
of the peak due to vitamin Th is not more than 2. O%; the
approximate relative retention times with reference to
vitamin ~ are O. 5 for pre-vitamin D3, O. 6 for trans-vitamin
Th and l. 1 for tachysterol D3. The resolution factors between
each pair of adjacent peaks due to pre-vitamin ~ , trans-
vitamin ~, vitamin Th and tachysterol Th are not less than
l. o.
 0731 Determination of Protein Content
Det.ennination of correction factor Transfer accurately measured
5 ml of the stock solution (2) into a 50 ml volumetric flask and
dilute to volume with iso-octane, shake well and use as the
reference solution. lnject 10 µl of the reference solution into the
column and record the chromatogram, calculate the correction
factor f1 of vitamin D from the expression:
f1 = c1/A1
Where: c1 is the concentration, in µg per ml, of the
vitamin Din the reference solution; and A 1 is
the peak area of vitamin D in the chroma-
togram obtained with the reference solution.
Transfer accurately measured 5 ml of the stock solution (2)
into a 50 ml volumetric flask, add a grain of 2, 6-di-
tertbutyl-p-cresol crystal, displace the air by purge the
mixture solution with nitrogen and stopper the flask. Place it
in a water bath of 90ºC for l. 5 hours, allow to cool rapidly,
dilute to volume with n-hexane, shake well and use as the
reference mixture solution. Inject 10 µl of the reference
mixture solution into the column and record the
chromatogram. Calculate the correction factor f 2 for pre-
vitamin D from the expression:
fz=Cc1 -f1A1)/A2
Where: c 1 is the concentration, in µg per ml, of the
vitamin Din the reference solution;
f1 is the correction factor of vitamin D;
A1 is the peak area of vitamin D in the
chromatogram obtained with the reference
mixture solution;
Az is the peak area of pre-vitamin D in the
chromatogram obtained with the reference
mixture solution.
Procedure Using the test solution prepared as described
under Assay in the specified monograph, calculate the total
content (e;) of vitamin D refer to the content of vitamin D
and pre-vitamin D from the expression:
+
e;= f1A;1 fzAi2
Where: A;1 is the peak area of vitamin D, Ai2 is the peak
area of pre-vitamin D.
Method 2
Preparation of test solution A Place an accurately weighed
quantity ( equivalent to not less than 600 Units of total
vitamin D, not more than 2 g) of the substance being
examined in a saponification flask. Add 30 ml of ethanol,
O. 2 g of ascorbic acid, and 3 ml of 50% (g/g) potassium
hydroxide solution ( if the substance being examined is 3 g,
add 4 mD. &il on a water bath for 30 minutes under reflux.
Allow to cool and wash the inner wall of the condenser with
10 ml of water. Transfer the saponification solution into a
separating funnel. Wash the flask with 60-100 ml of water in
portions, add the washings to the separating funnel and
extract with 3 quantities ( 60, 40, 40 mD of peroxide-free
ether. Combine the ether extracts and wash with several
portions of 100 ml of water cautiously to avoid emulsification
until the water layer <loes not become red on the addition of
phenolphthalein IS. Allow to stand until the layers are
separated completely. To the ether layer add several pieces of
dry filter paper strip and shake to remove the residual water
in the ether extract. Wash the separating funnel and filter
paper with a small volume of ether, combine washings and
ether extracts into a round-bottomed flask with stopper, and
evaporate the solvent to about 5 ml on a water bath at low
temperature. Remove the residual ether to dryness with a
stream of nitrogen and immediately add accurately measured
3 ml of methanol. Stopper and dissolve the residue with the
aid of ultra-sonication. Transfer the resulting solution into a
centrifuge-tube, centrifuge and use the clear supernatant
liquid as the test solution A
Fractionating collection of Vitanlin D by cleanup chroma-
tographic column Inject accurately measured 500 µl of the
test solution A into the column packed with octadecylsilane
bonded silica gel, using a mixture of methanol-acetonitrile-
water ( 50 : 50 : 2) as the mobile phase and the detector
wavelength is 254 nm. Record the chromatogram. The peak
of vitamin D is overlapped with the peak dueto pre-vitamin D
and they are separated from the peaks due to vitamin A and
related impurities. Accurately collect the total fractions of
mobile phase containing the total amount of vitamin D and
pre-vitamin D into a round-bottomed flask, evaporate the
mobile phase rapidly with a stream of nitrogen. Add an
accurately measured volume of the n-hexane solution to the
flask to produce a solution containing 50-140 Units of vitamin
D, stopper and place in an ultra-sonication bath to dissolve
residue and use as the test solution B.
Procedure Carry out the procedure as described under Method
l. Inject 100-200 µl of the test solution B into the column.
Method 3
Preparation of test solution To the test solution A prepared
as described under Assay in specified preparation, treat the
test solution A as described under Fractionating collection of
Vitamin D by cleanup chromatographic column in Method 2,
beginning with the words "evaporate the mobile phase rapidly
with a stream of nitrogen... ", immediately add accurately
measured 2 ml of iso-octane to dissolve, replace the air by
purge with nitrogen, stopper and place it in a water bath at
90ºC for l. 5 hours, evaporate the solvent with nitrogen
stream in 2 minutes, add accurately 2 ml of n-hexane to
dissolve the residue and use as the test solution C.
Preparation of reference solution Dilute an accurately
measured volume of the stock solution ( 1) with iso-octane
to produce a solution containing about 50 Units of vitamin D
per ml, transfer accurately measured 2 ml of the solution
into a round-bottomed flask, carry out the method as
described under Preparation of the test solution beginning
with the words "replace the air by purge with nitrogen", and
use the resulting solution as the reference solution.
Procedure Using the chromatographic conditions as
described under Method 1, inject separately 200 µl of the
reference solution and the test solution C into the column,
record the chromatogram, calculate the content of vitamin D
with respect to the peak area in the chromatogram by the
externa! standard method.
0731 Determination of Protein Content
Amino acid is the basic unit of protein. Peptide chains are
formed between amino acids by dehydration and conden-
sation. Protein is biological macromolecule composed of one
or more polypeptide chains. Suitable analytic methods are
applied to different test of proteins depending on protein' s
characteristics, and the methods must be validated. Use a
protein reference material that is the same as or similar to the
test proteins in composition as far as possible.
Method 1 Kjeldahl nitrogen assay
This method is based on nitrogen analysis as a means of
protein determination. Protein is digested when heated with
sulfuric acid, copper sulfate or potassium sulfate. The
ammonia produced by the decomposition is combined with
sulfuric acid to form ammonium sulfate, then ammonia is
released by distillation in alkaline solution. After absorbed in
boric acid solution, the distilled ammonia is titrated with
sulf uric acid VS. The protein content can be determined from

the nitrogen content in the test solution, calculated according
to the volume of sulfuric acid consumed, by multiplying with
the conversion factor.
This method has lower sensitivity and can be applied to
determine about O. 2 to 2. O mg of nitrogen in the proteins.
The conversion factor by which nitrogen is converted into
protein may be slightly different due to the different
compositions of amino acids in the proteins.
Preparation of Test Solution Prepare the solution as described in
the monograph. For biologicals, carry out the following
steps.
Accurately measure a suitable volume of substance being
examined ( for lyophilized products or solid powders,
measure after reconstitution) , dilute to volume with O. 9 %
sodium chloride solution to quantitatively produce a solution
containing about 1 mg of nitrogen per ml. Use 1 ml of the
solution, accurately measured, as the test solution for
determination of total nitrogen. Prepare the test solution of
non-protein nitrogen, unless otherwise specified, in
accordance with the tungstic acid precipitation method under
Annotation.
Procedure Unless otherwise specified, procedure ( 1) is to
be used. For biologicals, carry out the procedure (2).
( 1) This procedure is applied to the test samples which do
not contain inorganic nitrogen and non-protein organic
nitrogen. Transfer a volume of the test solution, accurately
measured, as specified in the monograph, to a Kjeldahl
flask. Determine the nitrogen content in the test solution
according to Determination of Nitrogen <0704 method 2 or
method 3). Unless otherwise specified, the conversion factor
by which nitrogen is converted into protein is 6. 25.
(2) This procedure is applied to the test samples which
contain inorganic nitrogen and non-protein organic nitrogen.
Unless otherwise specified, transfer respectively a quantity of
total nitrogen content of the test solution and non-protein
nitrogen content of the test solution, accurately measured as
specified in the monograph, to two Kjeldahl flasks. Carry out
the Determination of Nitrogen <0704 method 2 or method 3 ) .
Calculate the content of nitrogen in the test solution by
subtracting the content of non-protein nitrogen from the content
of total nitrogen. Unless otherwise specified, the conversion
factor by which nitrogen is converted into protein is 6. 25.
Annotation: Common methods for preparation of non-protein
nitrogen test solution
Tungstic acid precipitation method Transfer a quantity of
the substance being examined ( lyophilized preparation
should be measured after reconstitution or solid powder
after dissolution) ( the protein content is not more than
O. 2 g), accurately measured, to a 20 ml volumetric flask.
Add 1 O ml of water, 2 ml of 1 O% sodium tungstate
solution, 2 ml of O. 33 mol/L sulfuric acid solution, and
dilute to volume with water and mix well. Or introduce
2. O ml of the substance being examined in a volumetric
flask, add 14. O ml of water, 2. O ml of 10% sodium
tungstate solution, 2. O ml of O. 33 mol/L sulfuric acid
solution, and mix well. Allow to stand for 30 min, filter,
discard primary filtrate and use the successive filtrate.
( Adjust the volume of 1 O% sodium tungstate solution and
O. 3 3 mol/L sulfuric acid solution according to the protein
concentration, to maintain the final concentration of
tungstic acid in the solution at 1 %) .
Trichloroacetic acid precipitation method To a quantity of
the substance being examined ( lyophilized preparation
should be measured after reconstitution or solid powder
after dissolution) ( the protein content is about 6-12 mg) ,
0731 Determination of Protein Content
accu-rately measured, add equal volume of 1O%
Trichloroacetic acid solution and mix well. Allow to stand
for 30 min, filter, discard primary filtrate, and use the
success1ve filtra te. ( Adjust the volume of 1O%
trichloroacetic acid solution according to the protein
concentration, to maintain the final concentration of
trichloroacetic acid in the solution at 5 %) .
Method 2 Folin phenol method ( Lowry method)
This method is based on the following principie: Chelation of
cupric (Cuz+) ions with peptide bond of proteins in alkaline
solution forms a protein-copper complex, and the complex
makes phosphomolybdic acid in Folin phenol reagent
reducing to form a blue compound. At the same time Folin
phenol reagent in alkaline condition can be easily reduced by
tyrosine, tryptophan, cysteine in protein to develop blue colour.
The intensity of colour obtained is proportional to the
concentration of protein within a suitable range. The content of
protein in test samples can be determined by calibration curve of
protein reference solution according to the colourimetric method.
This method has high sensitivity and may usually reach the
measuring range of 20 to 250 µg of protein. Many substances
can interfere with this method. The ions which interfered
with biuret reaction can easily interfere with Folin-phenol
reaction, and the interfering effects are even more
significant. Reducing substances, phenols, citric acid,
ammonium sulfate, trometamol BS, glycine, sugar, glycerol
etc. can all interfere with this method.
Unless otherwise specified, carry out Method 2. l. If any
interfering substance exists, unless otherwise specified,
carry out Method 2. 2 and methodological verification 1s
needed.
Method 2.1
Reagent Alkaline Copper TS Dissolve 10 g of sodium
hydroxide and 50 g of sodium carbonate in 400 ml of v1ater as
solution A. Dissolve respectively O. 5 g of potassium tartrate
in 50 ml of water and O. 25 g of copper sulfate in 30 ml of
water, mix the two solutions as solution B. Mix solution A
and solution B before using, and dilute to 500 ml with water.
Preparation of reference solution Unless otherwise specified,
dissolve bovine serum albumin RS or national standard for
determination of protein content in water to produce a
solution of O. 2 mg per ml.
Preparation of test solution Prepared as described in the
monograph ( the concentration of the protein should be the
same as that of the reference solution).
Procedure Transfer respectively O. O ml, O. 2 ml, O. 4 ml,
O. 6 ml, O. 8 ml, l. O ml (The volume of reference solution
used can be adjusted within the test range specified in this
method) of reference solution into six test tubes with
stoppers, dilute each to l. O ml with water, then add l. O ml
of alkaline copper reagent to each tu be, mix well, place in
room temperature for 10 min. Add 4. O ml of Folin phenol
reagent [ dilute 1 volume of the Folin phenol stock solution
( the concentration of acid is 2 mol/L) to 16 volumes with
water] to each tube, mix immediately. Place in room
temperature for 30 min. Measure the absorbance at 650 nm
<0701 ) , using O. O ml test tube as a blank. Calculate the
linear regression equation from the absorbances obtained
versus the concentrations of the reference solutions. Repeat
the operation, using the substance being examined,
accurately measured, instead of the reference solution, and
calculate the concentration of the protein in the test solution
by multiplying the quantity of protein obtained from the
equation by the dilution factor.
 0731 Determination of Protein Content
Method 2. 2
Befare testing, add deoxycholate-trichloroacetic acid in to the
sample solution to remove interfering substances by
precipitation of proteins. This method also can be used to
concentrate proteins from a dilute solution.
Reagents Solution A Mix 200 ml of 1 % sodium hydroxide
solution and 200 ml of 5 % sodium carbonate solution, then
dilute to 500 ml with water.
Solution B Mix 100 ml of 2. 98 % disodium tartrate
dihydrate solution and 100 ml of l. 25 % copper sulfate
solution, then dilute to 250 ml with water. Prepare this
reagent freshly befare using.
Solution C Mix solution A and solution B in 50 : l. Prepare
this reagent freshly befare using.
Folio' s phenol solution Dilute the stock solution of Folin' s
solution Cacid concentration of 2 mol/L) in 1 - 2 CThe
prepared Folin' s phenol solution meets the fallowing
requirement: Mix lml of test solution, 5 ml of solution C
and O. 5 ml of the prepared Folin' s phenol solution. The pH
of the mixed solution should be 10. 3±0. 3. If the pH is out
of the range, appropriately adjust the dilution factor of Folin' s
phenol solution. )
Sodium deoxycholate solution Dissolve a suitable quantity of
sodium deoxycholate in water to produce a solution of l. 5mg
per ml.
Preparation of reference solution Unless otherwise specified,
dissolve a suitable quantity of bovine serum albumin RS or
nationai standard for determination of protein content in
water to produce solutions of O. OOmg, O. Olmg, O. 02mg,
O. 03mg, O. 04mg, and O. 05mg per ml, respectively CThe
concentration of reference solution can be appropriately
adjusted within the range defined in this Method).
Preparation of test solution Carry out the preparation
method specified in the monograph CThe concentration of
protein is consistent with that of the reference solution).
Procedure Accurately measure l. O ml of each reference
solution to a glass test tube respectively, add O. 1 ml of
sodium deoxycholate reagent, mix well by vortex, allow to
stand at room temperature far 10 min, then add O. 1 ml of
72 % trichloroacetic acid solution, mix well by vortex,
centrifuge at 3000 g, for 30 min, gently pour the super-
natant, and remove the remaining liquid by pipettes.
Reconstitute the protein precipitate with 1 ml of solution C,
then add 5 ml of solution c, mix well, allow to stand at
room temperature for 10 min, then add O. 5 ml of Folin' s
phenol TS, immediately mix, allow to stand at room
temperature far 30 min, then carry out the method far UV-
visible spectrophotometry ( 0401 ) to measure the
absorbance at 750 nm. In parallel, perfarm a blank test using
O. 00 mg tube. Calculate a linear regression equation by the
concentrations of reference solutions and the corresponding
absorbances. Accurately measure another l. O ml of the test
solution, detect using the same method. Calculate the
concentration of protein in the test solution from the linear
regression equation, and then multiply with the dilution
factor.
Method 3 Biuret method
This method is based on the fallowing principle: protein with
more than two peptide bonds reacts with cupric CCuz+ ) ion
farming a purple complex in alkaline solution. The colour
intensity of the complex is proportional to the protein
concentration within a suitable range. The protein content in
test samples can be determined by calibration curve of protein
reference solution according to the colourimetric method.
This method is fast, but has low sensitivity, and may usually
reach the measuring range of 1 to 10 mg of protein. The
interfering substances in this method are ammonium sulfate,
trometamol BS, and certain amino acids.
Reagent Biuret TS Dissolve l. 5 g of cupric sulfate, 6. O g
of potassium sodium tartrate and 5. O g potassium iodide in
500 ml of water. While stirring, add 300 ml of 10% sodium
hydroxide, dilute to 1000 ml with water, mix well.
Preparation of reference solution Unless otherwise specified
in the monograph, dissolve serum albumin Cbovine) RS or
national standard far determination of protein content in
water to produce a solution of 10 mg per ml.
Preparation of test solution Prepared as described in the
e
monograph the concentration of the protein should be the
same as that of the reference solution).
Procedure Transfer respectively O. O ml, O. 2 ml, O. 4 ml,
O. 6 ml, O. 8 ml, l. O ml of CThe volume of reference
solution to be used can be appropriately adjusted within the
range defined in this Method) reference solution into six test
tubes with stoppers, dilute each to l. O ml with water, then
add 4. O ml of biuret reagent to each tube, mix immediately.
Place each tube at room temperature far 30 min. Measure the
absorbance at 540 nm ( 0401 ) , using O. O ml test tube as a
blank. Calculate the linear regression equation from the
absorbances obtained versus the concentrations of the
reference solutions. Repeat the operation, using the subs-
tance being examined, accurately measured, instead of the
reference soiutions, and caicuiate the concentration of the
protein in the test solution by multiplying the quantity of
protein obtained from the equation by the dilution factor.
Method 4 Bicinchoninic acid method ( BCA method)
This method is based on reduction of cupric CCuz+ ) ion to
cuprous CCu+ ) ion by protein in alkaline solution. The BCA
reagent bonds with cuprous ecu+) ion to form a purple
complex. The colour intensity of the complex is proportional
to the protein concentration within a suitable range. The
protein content in test samples can be determined by
calibration curve of protein reference solution according to the
colourimetric method.
This method has high sensitivity and may usually reach the
measuring range of 80 to 400 µg of protein. Reducing agents and
copper chelates in test samples will interfer the determination.
Reagent Coppe~OCA TS Dissolve 1 g of disodium bicincho-
ninate, 2 g of Anhydrous sodium carbonate, O. 16 g of
Sodium tartrate, O. 4 g of sodium hydroxide and O. 95 g of
sodium hydrogen carbonate in 100 ml of water, adjust pH to
11. 25 as solution A. Use 4 % Copper sulfate solution as
solution B. To 100 ml of solution A add 2 ml of solution B
befo re use, and mix well.
Preparation of reference solution Unless otherwise specified,
dissolve serum albumin (bovine) RS or national standard far
determination of protein content in water to produce a
solution of O. 8 mg per ml.
Preparation of test solution Pre pared as descri bed in the
monograph ethe concentration of the protein should be the
same as that of the reference solution).
Procedure Transfer respectively O. O ml, O. 1 ml, O. 2 ml,
O. 3 ml, O. 4 ml, O. 5 ml (The volume of reference solution
to be used can be appropriately adjusted within the range
defined in this Method) of reference solution into six test
tubes with stoppers, dilute each to O. 5 ml with water, then
add 10. O ml of copper-BCA reagent to each tube, mix

immediately. Place each tube in water bath at 37°C far 30
minutes, and cool. Measure promptly the absorbance at 562
nm ( 0401), using O. O ml test tube as a blank. Calculate the
linear regression equation from the absorbances obtained
versus the concentrations of the reference solutions. Repeat
the operation, using the substance being examined,
accurately measured, instead of the ref erence solutions, and
calculate the concentration of the protein in the test solution
by multiplying the quantity of protein obtained from the
equation by the dilution factor.
Method 5 ~ie brilliant bine method ( Bradford method)
This method is based on the fallowing principie: Coomassie
brilliant blue G250 bonds with basic amino acids (e. g.
arginine) and aromatic amino acids in proteins to farm a blue
complex in acid solution. The colour intensity of the complex
is proportional to the protein concentration within a suitable
range. The protein content in test samples can be determined
by calibration curve of protein reference solution according to
the colourimetric method.
This method has high sensitivity and may usually reach the
measuring range of 1 to 200 µg of protein. The main
interfering substances are detergent, Tri ton X-100, sodium
laurylsulfate ( SDS) etc. Sometimes highly alkaline buffer
also interferes with the colour development.
Reagent Acid Staining reagent Dissolve O. 1 g of
Coomassie brilliant blue G250 in 50 ml of ethanol, add 100
ml of phosphoric acid, diluted to 1000 ml with water, and
mix well. Filter and use the successive filtrate. The solution
should be kept in an amber bottle. Filter again befare use
when precipitate is farmed.
Preparation of reference solution Unless otherwise
specified, dissolve serum albumin ( bovine) RS or national
standard far determination of protein content in water to
produce a solution of 1 mg per ml.
Preparation of test solution Prepared as described in the
monograph ( the concentration of the protein should be
almost the same as that of the reference solution).
Procedure Transfer respectively O. 00 ml, O. 01 ml,
O. 02 ml, O. 04 ml, O. 06 ml, O. 08 ml, O. 10 ml ( The
volume of reference solution to be used can be appropriately
0800 Limit Tests
0801 Limit Test for Chlorides
Test solution Unless otherwise specified, weigh a quantity
of the substance to be examined as prescribed in the
individual monographs and dissolve it in about 25 ml of water
( if the solution is alkaline, neutralize with nitric acid
dropwise). Add 10 ml of dilute nitric acid and filter if
necessary, transfer the solution to a 50 ml Nessler cylinder,
add water to produce 40 ml and mix well.
Reference solution Transfer a volume of sodium chloride
standard solution as prescribed in the individual monographs
to a 50 ml Nessler cylinder. Add 10 ml of dilute nitric acid
and sufficient water to produce 40 ml, mix well.
Procedure To each of the Nessler cylinders described above
0801 Limit Test far Chlorides
adjusted within the range defined in this Method) of
reference solution into seven test tubes with stoppers, dilute
each to O. 1 ml with water, then add 5. O ml of acid staining
reagent to each tube, mix immediately. Measure promptly
the absorbances at 595 nm ( 0401), using O. 00 ml tube as
the blank. Calculate the linear regression equation from the
absorbances obtained versus the concentrations of the
reference solutions. Repeat the operation, using the
substance being examined, accurately measured, instead of
the reference solutions, and calculate the concentration of the
protein in the test solution by multiplying the quantity of protein
obtained from the linear regression equation by the dilution
factor.
Annotation Do not use quartz ( silica) spectrophotometer
cells because the dye binds to this material. The spectro-
photometer cells made of glass or other suitable materials are
recommended.
Method 6 Ultraviolet-visible spectrophotometry method
This method is based on the fallowing principie: The
Proteins containing tyrosine, tryptophan and other aromatic
amino acids with conjugated double bonds. have maximum
absorbance at 280 nm. The absorbance obtained is
proportional to the protein concentration within a suitable
range.
This method is easy and fast, and can be applied far the trace
determination of purified proteins which normally have a
concentration of O. 2 to 2 mg per ml. This method is not
accurate and may be interfered by many substances.
Procedure ( 2) is suitable far the determination of protein
content when nucleic acids are present in test solution.
Preparation of reference solution and test solution As
described in the monograph.
Procedure ( 1) Use a quantity of test solution, measure
absorbance at 280 nm ( 0401 ) , calculate the protein content
of test sample by absorption coefficient method or reference
substance comparison method.
(2) Use a quantity of test solution, measure absorbances at
280 nm and 260 nm ( 0401 ) , calculate the protein content of
test sample according to the fallowing equation:
Proteincontent (mg/ml) =l. 45XA2so-O. 74XA 260
add l. O ml of silver nitrate TS, dilute with water to 50 ml
and mix well. Allow to stand in the dark far 5 minutes, and
compare the opalescence produced by viewing clown the
vertical axis of the cylinders against a black background. Any
opalescence in the test solution is not more intense than that
in the ref erence solution.
If the test solution is coloured, unless otherwise specified,
place two aliquots of the test solution in Nessler cylinders
separately, to one cylinder add l. O ml of silver nitrate TS,
mix and allow to stand far 1O minutes, filter the content of
the cylinder repeatedly until the filtrate is perfectly clear,
then add the prescribed volume of sodium chloride standard
solution to the filtrate, use it as the reference solution. To
the other cylinder add l. O ml of silver nitrate TS, use it as
the test solution. Dilute the test solution and the reference
solution with water to 50 ml, mix well and allow to stand in
the dark far 5 min. Compare the opalescence as described above.
 0802 Limit Test for Sulfates
Sodium chloride standard solution Dissolve O. 165 g of
sodium chloride in water in a 1000 ml volumetric flask.
Dilute to volume with water and mix well (stock solution).
lmmediately before use, transfer 10 ml of the stock solution,
accurately measured, into a 100 ml volumetric flask, dilute
to volume with water and mix well ( each ml of sodium
chloride standard solution is equivalent to 10 f.lg of chlorine).
NOTE The filter paper used in this test, if necessary,
should be previously washed with water containing nitric acid
until the washing is free from chlorides.
0802 Limit Test for Sulfates
Test solution Unless otherwise specified, weigh a quantity
of the substance to be examined as prescribed in the indivi-
dual monographs, dissolve it in about 40 ml of water,
neutralize the solution with hydrochloric acid and filter if
necessary. Transfer the solution to a 50 ml Nessler cylinder,
add 2 ml of dilute hydrochloric acid and mix well.
Reference solution Transfer a volume of potassium sulfate
standard solution as prescribed in the individual monographs
to a 50 ml Nessler cylinder, dilute with water to about
40 ml, add 2 ml of dilute hydrochloric acid and mix well.
Procedure To each of the Nessler cylinders described above
add 5 ml of 25% barium chloride solution, dilute with water
to 50 ml and mix well. Allow to stand for 10 minutes and
compare the opalescence produced by viewing clown the
vertical axis of the cylinders against a black background. Any
opalescence in the test solution is not more intense than that
in the reference solution.
If the test solution is coloured, unless otherwise specified,
place two aliquots of the test solution in Nessler cylinders
separately, to one cylinder add 5 ml of 25 % barium chloride
solution and mix well. Allow to stand for 10 minutes, filter
the content of the cylinder repeatedly until the filtrate is
perfectly clear, then add the prescribed volume of potassium
sulfate standard solution to the filtrate and use it as the
reference solution. To the other cylinder add 5 ml of 25 %
barium chloride solution, use it as the test solution. Dilute
the test solution and the reference solution with water to
50 ml and mix well. Allow to stand for 10 minutes and
compare the opalescence as described above.
Potassium sulfate standard solution Dissolve O. 181 g of
potassium sulfate in water in a 1000 ml volumetric flask.
Dilute to volume with water and mix well ( each ml of
potassium sulfate standard solution is equivalent to 100 f.lg of
sulfate cso4) ).
0803 Limit Test for Sulfides
Apparatus Use the same apparatus as shown in method 1
under Limit test for arsenic <0822), except that there is no
lead acetate cotton intube Canda lead acetate paper is placed
between D and E instead of mercuric bromide paper.
Sodium sulfide standard solution Dissolve l. O g of sodium
sulfide in 200 ml of water, transfer 50 ml of the solution,
accurately measured, into an iodine flask, add 25. O ml of
iodine (O. 05 mol/L) VS and 2 ml of hydrochloric acid, mix
well. Ti trate with sodium thiosulfate (O. 1 mol/L) VS until
the end point is nearly reached, add 2 ml of starch IS and
continue the titration until the blue colour disappears. Carry
out a blank determination and make any necessary correction.
Each ml of iodine ( O. 05 mol/L ) VS is equivalent to
l. 603 mg of sulfur. Dilute an accurately measured volume of
the sodium sulfide solution with water to produce a solution
containing 5 /lg of sulfur per ml. This standard solution
should be freshly prepared.
Standard sulfide stain Transfer 1 ml of sodium sulfide stan-
dard solution, accurately measured, into flask A, add 10 ml
of water and 10 ml of dilute hydrochloric acid. Fit tube Cinto
the mouth of flask A, with the lead acetate paper on its top.
Immerse the flask in a water bath at 80-90ºC for 10 minutes.
A stain is produced on the test paper.
Procedure Unless otherwise specified, place a quantity of
the substance to be examined as prescribed in the individual
monographs in flask A, add 10 ml of water ( or ethanol for
oily substances) and 10 ml of dilute hydrochloric acid, and
then proceed as described in "standard sulfide stain". Any
stain produced is not more intense than the standard sulfide stain.
0804 Limit Test for Selenium
Selenium standard solution Dissolve an accurately weighed
quantity of sodium selenite of known purity in nitric acid
solution 0-30) to produce a solution of l. 00 mg Se per
ml. Transfer 5 ml of the solution into a 250 ml volumetric
flask, dilute to volume with water and mix well. Transfer
5 ml of this solution into a 100 ml volumetric flask, dilute to
volume with water and mix well ( each ml is equivalent to
1 f.lg of seleniuro).
Reference solution Transfer 5 ml of the selenium standard
solution, accurately measured, into a 100 ml beaker, add
25 ml of nitric acid solution 0-30) and 10 ml of water,
and mix well.
Test solution Unless otherwise specified, weigh a quantity
of the substance to be examined as prescribed in the indivi-
dual monographs. Carry out the method for oxygen flask
combustion < 0703 ) in a 1000 ml combustion flask using
25 ml of nitric acid solution 0-30) as the absorbing liquid.
After the combustion of organic matter is completed,
transfer the absorbing liquid and washings to a 100 ml
beaker. Rinse combustion flask and platinum wire with 15 ml
of water, combine the washing liquids with the absorbing
liquid.
Procedure Treat the reference solution and the test solution
concomitantly as follows. Adjust pH to 2. O ± O. 2 with
ammonia TS, and transfer to a separator. Wash the beaker
with successive small quantities of water and add the
washings to the same separator, dilute with water to 60 ml.
Add 1 ml of hydroxylamine hydrochloride solution ( 1- 2)
and 5. O ml of diaminonaphthalene TS, mix well and allow to
stand at room temperature for 100 minutes. Add 5. O ml of
cyclohexane, shake vigorously for 2 minutes, allow to
separate and discard the aqueous layer. Remove any trace of
water in the cyclohexane layer with anhydrous sodium sulfate
and measure its absorbance at 378 nm < 0401 ) . The
absorbance of the test solution is not greater than that of the
reference solution.
NOTE Determination of Se in sodium selenite Place about
O. 1 g of sodium selenite, accurately weighed, in an iodine
flask. Dissolve it in 50 ml of water, add 3 g of potassium
iodide and 10 ml of hydrochloric acid solution 0-2), insert
the stopper and allow to stand for 5 minutes. Add 50 ml of
water and ti trate with sodium thiosulfate (O. 1 mol/L) VS
until the colour changes from reddish brown to orange red.
Add 2 ml of starch IS and continue the titration until the

colour changes from blue to violet-red. Each ml of sodium
thiosulfate (O. 1 mol/L) VS is equivalent to 4. 324 mg of
sodium selenite or l. 97 4 mg of selenium.
0805 Limit Test for Fluorides
Fluoride standard solution Transfer 22. 1 mg of sodium
fluoride previously dried for 1 hour at 105ºC, accurately
weighed, into a 100 ml volumetric flask, dissolve and dilute
to volume with water. Dilute 20 ml, accurately measured, of
the solution with water in another 100 ml volumetric flask to
volume and mix well ( each ml of fluoride standard solution
is equivalent to 20 µg of fluorine).
Test solution Carry out the method for oxygen flask com-
bustion <0703 >, using a quantity of the substance to be
examined equivalent to about 2. O mg of fluorine, accurately
weighed, and 20 ml of water as the absorbing liquid. Shake
for further 2-3 minutes after combustion and absorption are
complete, rinse the stopper, platinum wire and gauze with
water. Combine the washing liquids with the absorbing liquid
in a 100 ml volumetric flask, dilute to volume with water and
mix well.
Procedure Measure accurately 2 ml of the fluoride standard
solution and the test solution into separate 50 ml volumetric
flasks. To each flask, add 10 ml of alizarin fluorine blue TS,
and mix well. Add 3. O ml of a solution of 12 % sodium
acetate in dilute acetic acid and 10 ml of cerous nitrate TS
respectively, dilute to volume with water and mix well.
Allow the solutions to stand in the dark for 1 hour. Carry out
the method for ultraviolet-visible spectrophotometry <0401 >
using absorption cells, measure the absorbances at 610 nm
and calculate the content of fluorine.
0806 Determination of Cyanide
Method 1
Apparatus Use the same apparatus as shown in method 1
under Limit test for arsenic <0822 >, except that there is no
lead acetate cotton in tube c and an alkaline ferrous sulfate
paper (freshly prepared by moistening a piece of filter paper
with 1 drop each of ferrous sulfate TS and sodium hydroxide
TS) is placed between D and E instead of mercuric bromide
paper.
Procedure Unless otherwise specified, place a quantity of
the substance to be examined as prescribed in the individual
monographs in flask A, add 10 ml of water and 3 ml of 10%
tartaric acid solution. Fit tube C immediately into the mouth
of flask A with the alkaline ferrous sulfate paper on its top
and shake well. fuil gently for 1 minute, remove the test
paper, add 1 drop each of ferric chloride TS and hydrochloric
acid, no green or blue colour is developed within 15 minutes.
Method 2
Apparatus See Fig. , A is a 200 ml glass-stoppered conical
flask, Bis a 5 ml beaker which can be placed into the flask
A.
Po~iurn cyanide standard solution Weigh accurately 25 mg
of potassium cyanide into a 100 ml volumetric flask, dissolve
and dilute to volume with water, and mix well. Measure
accurately 5 ml of this solution to a 250 ml volumetric flask,
dilute to volume with water and mix well ( each ml of
potassium cyamide standard sdution is equivalent to 2 µg of
cyano).
0806 Determination of Cyanide
lithiuam trinitrophenol
TS(l ml)
B
test or reference
preparation(5 ml)
Fig. Apparatus of method 2
This standard solution should be freshly prepared.
Procedure Unless otherwise specified, place a quantity of
the substance to be examined as prescribed in the individual
monographs in flask A, add sufficient water to produce 5 ml
and mix well. Place beaker B containing l. O ml of lithium
trinitrophend TS into the flask, stopper the flask and allow
to stand ovemight in a dark place. Remove the beaker B, add
2. O ml of water to the solution in the beaker B and mix well.
Carry out the mothod for ultraviolet-visible spectropho-
tometry <0401 >. Measure the absorbance at 500 nm. The
absorbance does not exceed that of the solution obtained in
the same manner with a volume of potassium cyanide
standard solution as prescribed in the individual monographs.
Method 3
The method is based on the colour reaction of cyanogen
bromide with pyridine benzidine under acid condition. The
content of cyanogen bromide in Hib polysaccharide derivative
is determined by UV-visible spectrophotometry.
Reagent ( 1 ) 60 % Pyridine soiution Measure 30 mi of
pyridine, add 20 ml of water and mix well.
(2) 2% Hydrochloric acid Measure O. 5 ml of hydrochloric
acid, add 9. 5 ml of water and mix well.
(3) Pyridine benzidine solution Weigh accurately O. 5 g of
benzidine and dissolve in 50 ml of 60 % pyridine solution, add
1O ml of 2 % hydrochloric acid and mix well. Prepare the
solution just before use.
Reference solution
(1) O. 1 mg/ml Cyanogen bromide reference stock solution
Weigh accurately 10 mg of cyanogen bromide, dissolve with
a quantity of acetonitrile and dilute to 100 ml with water,
mix well. Prepare the stock solution just before use.
( 2) Cyanogen bromide working solution ( 500 ng/ mD
Measure accurately 1 ml of cyanogen bromide reference stock
solution, dilute to 200 ml with water and mix well.
Test solution
Weigh a quantity of polysaccharide derivative and prepare a
solution with a concentration of 10 mg/ml for determination.
Procedure Measure 2. O ml of pyridine benzidine solution,
add 2. O ml of water and mix well. Allow to stand ata tem-
perature below 20ºC in a dark place for 15 minutes, then
determine the absorbance ata wavelength of 520 nm and use
as a blank control.
Measure 2. O ml of the test solution and 2. O ml of pyridine
benzidine solution, mix well, allow to stand ata temperature
below 20ºC in a dark place for 15 minutes, and then
determine the absorbance ata wavelength of 520 nm.
Transfer O. 1, O. 2, O. 4, O. 6, O. 8 and l. O ml of working
solution of cyanogen bromide to separate test tubes, and add
l. 9, l. 8, l. 6, l. 4, l. 2 and l. O ml of water respectively,
 i,ji1-:11::< ,, ,,: 0807 Limit Test for lron
~~;• >' ,.~ ,•,;'N~ ·~· .,
then add 2. O ml of pyridine benzidine solution and mix well.
Allow to stand at a temperature below 20ªC in a dark place
for 15 minutes and determine the absorbance ata wavelength
of 520 nm.
Result calculation A linear regression equation is obtained
by regressing the content of cyanogen bromide of the working
solution with the corresponding absorbance. The content of
cyanogen bromide B ( ng/ mD in the test solution is obtained
by inserting the absorbance of the test solution into the equa-
tion.
The content of cyanogen bromide in the substance being
examined (ng/mD =B/20
Where: B is the content of cyanogen bromide in the test
solution (ng/mD;
20 mg/ ml is the content of polysaccharide deriva-
tive in the test solution (mg/mD.
0807 Limit Test for Iron
Unless otherwise specified, dissolve a quantity of the
substance to be examined as prescribed in the individual
monographs in water to 25 ml. Transfer the solution to a
50 ml Nessler cylinder, add 4 ml of dilute hydrochloric acid
and 50 mg of ammonium persulfate. Dilute with water to
about 35 ml, add 3 ml of 30% ammonium thiocyanate
solution and sufficient water to produce 50 ml, mix well.
Prepare a reference solution in the same manner, using a
volume of iron standard solution as prescribed in the
individual monographs. Any colour in the test solution is not
more intense than that in the reference solution.
If the colour of the test solution is of a different shade as
compared with that of the reference solution, transfer each
solution to a separator and extract with 20 ml of n-butanol.
Transfer each of the n-butanol layers to a 50 ml Nessler
cylinder, add n-butanol to 25 ml and compare the colour of
the two solutions.
Iron standard solution Dissolve O. 863 g of ferric ammonium
sulfate [FeNH 4 CS04 )2 • 12H20], accurately weighed, in
water in a 1000 ml volumetric flask, add 2. 5 ml of sulfuric
acid, dilute with water to volume and mix well. This is the
stock solution.
Transfer 10 ml of the stock solution, accurately measured,
into a 100 ml volumetric flask immediately before use, add
water to volume and mix well ( each ml of iron standard
solution is equivalent to 10 µg of iron).
0808 Limit Test for Ammonium
Unless otherwise specified, place a quantity of the substance
to be examined as prescribed in the individual monographs in
a distillation flask, add 200 ml of ammonia-free distilled
water and 1 g of magnesium oxide, heat to distill, introduce
the distillate to a 50 ml Nessler cylinder containing 1 drop of
dilute hydrochloric acid TS and 5 ml of ammonia-free distilled
water. When the volume of the distillate is about 40 ml, stop
distillation, add 2 ml of alkaline mercuric potassium iodide
TS, mix well, and allow to stand for 15 minutes. Compare
the colour produced with that of a reference solution
containing 2 ml of ammonium chloride standard solution
treated in the same manner.
Ammoniurn chloride standard solution Place 29. 7 mg of
ammonium chloride, accurately weighed, in a 1000 ml
volumetric flask, dissolve and dilute to volume with water,
and mix well ( each ml of ammonium standard solution is
equivalent to 10 µg of ammonium CNH4) ).
0821 Limit Test for Heavy Metals
The term "heavy metals" refers to those metals that react
with thioacetamide or sodium sulfide under the specified
conditions to produce a coloured compound.
Lead standard solution Dissolve O. 1599 g of lead nitrate in
5 ml of nitric acid and 50 ml of water in a 1000 ml volumetric
flask, dilute to volume with water, mix well as the stock
solution.
Transfer 10 ml of the stock solution, accurately measured,
to a 100 ml volumetric flask, dilute with water to volume
and mix well ( each ml is equivalent to 1Oµg of lead). This
solution should be prepared on the day of use.
All glassware used for the preparation and preservation of the
lead standard solution should be free from lead.
Method 1
Unless otherwise specified, use three 25 ml Nessler
cylinders. To cylinder A add the specified volume of lead
standard solution and 2 ml of acetate BS ( pH 3. 5) , dilute
with water or other solvent as specified in the individual
monographs to 25 ml. To cylinder B add 25 ml of the test
solution containing a quantity of the substance being
examined as specified in the individual monographs. To
cylinder C add 25 ml of another solution containing the same
quantity of the substance being examined as cylinder B
dissolved in the solvent as specified in the individual
monographs, the same volume of lead standard solution as
cylinder A, and 2 ml of acetate BS (pH 3. 5).
If the original test solution is colured, it may be matched by
the addition of a few drops of dilute caramel solution or other
suitable solution to cylinder A
To each cylinder add 2 ml of thioacetamide TS and mix well,
allow to stand for 2 minutes, compare the colour produced by
viewing clown the vertical axis of the three cylinders against a
white background. The colour produced in cylinder B is not
more intense than that produced in cylinder A and the colour
produced in cylinder e is equal to or more intense than that
produced in cylinder A lf the colour produced in cylinder C is
lighter than that produced in cylinder A, use Method 2
instead of Method 1 for the substance being examined.
If the colour cannot be matched by the addition of caramel
solution or other suitable solution, use Method 2 instead of
Method 1 for the substance being examined.
If the substance being examined contains a ferric salt which
interferes with the test, the same quantity of O. 5-1. O g of
ascorbic acid should be added to each cylinder.
Unless otherwise specified, evaporate the same quantity of
the same reagents to dryness in a porcelain dish. Dissolve the
residue in 2 ml of acetate BS (pH 3. 5) and 15 ml of water.
Transfer the solution to a Nessler cylinder, add the specified
quantity of lead standard solution and water or other solvent
as specified under individual monographs to 25 ml. The
solution is used as the reference solution for the test solution
which is prepared by using more than 1 ml of hydrochloric
acid, 2 ml of ammonia TS or by treating with other reagents.
Methed 2
Unless otherwise specified, use Method 2 instead of Method
1 for a quantity of the substance being examined or use
immediately Method 2, using the residue obtained in the test
for Residue on ignition ( 0841 ) . If the substance being
examined is a liquid, evaporate a volume of the substance
 '· ... ' ... ,.

0822 Limit Test for Arsenic I···· lit'··

being examined as specified in the individual monographs to E
dryness, using the residue obtained in the test far Residue on D--1;1~-+-...u---~
ignition, add O. 5 ml of nitric acid, evaporate to dryness,
heat until nitrous oxide fumes are no longer evolved ( or
alternatively, ignite a quantity of the substance being e
examined in a crucible until thoroughly charred, cool,
o00
moisten the residue with O. 5-1 ml of sulfuric acid, ignite at a
low temperature until sulfurous acid fumes are no longer -
evolved, add O. 5 ml of nitric acid, evaporate to dryness,
heat until nitrous oxide fumes are no longer evolved and
ignite at 500-600ºC until the incineration is complete). Cool,
add 2 ml of hydrochloric acid, evaporate to dryness on a
water bath, add 15 ml of water, fallowed by ammonia TS
dropwise until the solution is slight pink to phenolphthalein
IS, then add 2 ml of acetate BS (pH 3. 5) and warm to effect
dissolution. Transfer the resulting solution to Nessler
cylinder B, dilute with water to 25 ml. Place the same
quantity of the same reagents used far the preparation of test mm
solution in a porcelain dish and evaporate to dryness, heat
Fig. 1 Apparatus of method 1
gently to dissolve in 2 ml of acetate BS (pH 3. 5) and 15 ml
of water, transfer to the Nessler cylinder A and add the
specified volume of lead standard solution, dilute with water potassium iodide TS and 5 drops of acid stannous chloride
to 25 ml. To each cylinder add 2 mi of thioacetamide TS and TS, allow to stand at room temperature far 10 minutes and
mix well, allow to stand far 2 minutes, compare the colour add 2 g of granulated zinc. Insert the stopper B and conduit C
produced by viewing clown the vertical axis of the two into the mouth of flask A and immerse the flask in a water
cylinders against a white background. The colour produced in bath at 25-40ºC for 45 minutes. Remove the mercuric
cylinder B is not more intense than that produced in cylinder bromide paper.
A Use arsenic standard solution instead of the substance being
examined and treat it in the same manner described in the
Method 3 individual monographs. Then prepare the arsenic standard
Unless otherwise specified, dissolve a quantity of the stain as described above, if the substance being examined
substance being examined in 5 ml of sodium hydroxide TS needs to be destroyed organically befare carrying out the limit
and 20 ml of water. Transfer the solution to a Nessler
test far arsenic.
cylinder, add 5 drops of sodium sulfide TS and mix well, the
colour produced is not more intense than that of a reference Procedure Transfer the test solution prepared as described
preparation containing the specified volume of lead standard in the individual monographs to flask A and proceed as
solution and treated in the same manner. described under arsenic standard stain, beginning with the
words "Then add 5 ml of potassium iodide TS... ". Any stain
produced is not more intense than the standard stain.
0822 Limit Test for Arsenic
Method 2 (Silver diethyldithiocarbamate method)
Apparatus ( as Fig. 2 ) A is a 100 ml conical flask wi th
Arsenic standard solution Dissolve O. 132 g of arsenic
standard ground joint; B is a standard hollow ground glass
trioxide in 5 ml of 20 % sodium hydroxide solution in a
stopper connected to glass conduit C ( at one end, the
1000 ml volumetric flask, neutralize with dilute sulfuric acid
and add 10 ml in excess. Dilute with water to volume and mix 90
well, as a stock solution.
Transfer 10 ml of the stock solution, accurately measured,
to a 1000 ml volumetric flask immediately befare use, add
e interior
10 ml of dilute sulfuric acid, dilute with water to volume and diameter 6
mix well ( each ml is equivalent to 1 µg of arsenic). exterior
diameter 8
Method 1 ( Gutzeit' s method)
exterior
Apparatus (as Fig. 1 ). A is a 100 ml conical flask with diameter4
standard ground joint; B is a standard hollow ground glass interior
stopper connected to glass conduit C ( external diametre diameter 1.6
8. O mm, internal diametre 6. O mm), the total length of B
and C is about 180 mm; D is a plastic screw, the upper part o o00
00
of which has an aperture 6. O mm in diametre and the lower
part of which has an aperture 8. O mm in diametre; E is a
plastic screw cap which has an aperture 6. O mm in diametre. 5.0 ml graduation
A wad of lead acetate cotton weighing about 60 mg is packed o
D N
into tube C to a depth of about 60-80 mm. A clise of mercuric
bromide paper is placed between the contacting surfaces of D
and E.
10
Arsenic standard stain Place 2 ml of standard arsenic
solution, accurately measured, in flask A Add 5 ml of (mm)
hydrochloric acid and 21 ml of water. Then add 5 ml of
Fig. 2 Apparatus of Method 2
 0831 Determination of Loss on Drying
externa! diametre is 8. O mm and the interna! diametre is 6. O
mm; the other end is in length of 180 mm, in externa!
diameter of 4 mm and in interna! diametre of l. 6 mm, the
interna! diametre of sharp end is 1 mm). D is a glass tube
with flat bottom Oength 180 mm, intemal diametre 10 mm,
and with a graduation at 5. O ml). A wad of lead aceta te
cotton weighing about 60 mg is packed into conduit C to a
depth of about 80 mm, and measure accurately 5 ml of silver
diethyldithio-car-bamate TS in tube D.
Arsenic reference solution Transfer 2 ml of arsenic standard
solution as described under Method 1 to flask A, accurately
measured. Add 5 ml of hydrochloric acid and 21 ml of water.
Then add 5 ml of potassium iodide TS and 5 drops of acid
stannous chloride TS, allow to stand at room temperature for
10 minutes and add 2 g of granulated zinc. Connect conduit e
into flask A immediately, and allow the evolved arsine to
enter tube D. lmmerse the flask A in a water bath at 25-40ºC
for 45 minutes. Remove tube D, add chloroform to the
graduation, mix well.
Use arsenic standard solution instead of the substance being
examined and treat in the same manner in the individual
monographs. Then prepare the arsenic reference solution as
described above, if the substance being examined needs to be
destroyed organically befare carrying out the limit test for
arsemc.
Procedure Transfer the test solution prepared as described
in the individual monographs to flask A and proceed as
described under arsenic reference solution, beginning with
the words "Then add 5 ml of potassium iodide TS... ".
Compare the above tv10 solutions against a \vhite background.
Any colour produced by the test preparation is not more
intense than that produced by the standard arsenic reference
solution. lf necessary, determine the absorbance at the
wavelength of 510 nm, using silver diethyldi-thiocarbamate
TS as the blank ( 0401 ) .
Notes (1) Make sure that a blank test produces no arsenic
stain or only a barely visible stain.
(2) The preparation of standard stain and test stain must be
carried out simultaneously.
(3) The granulated zinc should be arsenic free and the size is
such that they will pass through a No. 1 sieve. The quantity
used should be increased and the reaction time should be
extended up to 1 hour, if the granules are of larger size.
(4) Lead acetate cotton is prepared by immersing l. O g of
absorbent cotton in 12 ml of a mixture of equal volumes of
lead acetate TS and water. Drain off excess liquid and make
the cotton fluffy, allow it to dry at a temperature below
lOOºC and preserve in a well closed glass container.
0831 Determination of Loss on Drying
Mix the substance being examined thoroughly. lf it is in the
form of large crystals, reduce them to a size of about 2 mm
by quickly crushing. Place about 1 g or the amount specified
in the individual monographs of the substance being
examined in a tared, shallow weighing bottle, previously
dried to constant weight at 105ºC, unless otherwise specified.
The substance being examined should be evenly distributed to
forma layer of not more than 5 mm in the thickness, or not
more than 10 mm in the case of bulky material. When the
loaded bottle is placed in the drying chamber or desiccator,
remove the stopper and put it beside the bottle, or leave it on
the bottle in half open position. Upon the opening of the
drying chamber or desiccator, the bottle should be closed
promptly. If the substance is dried by heating, allow it to
cool to room temperature in a desiccator befare weighing.
lf the substance melts at a lower temperature than the
specified drying temperature, unless otherwise specified,
maintain the bottle with its content at about 5- lOºC below the
melting point until most of the water is removed, and then
dry it under the specified conditions. For biological products,
dry it under the specified conditions after most of the water
of the substance being examined was removed at a lower
temperature.
lf a vacuum desiccator or constant temperature vacuum
desiccator is to be used (Set the temperature as specified in
the monograph. For biological products, set the temperature
at 60ºC unless otherwise specified.) a pressure of 2. 67 kPa
( 20 mmHg) or less should be maintained unless otherwise
directed. The desiccants used in a desiccator are usually
phosphorus pentoxide, anhydrous calcium chloride or silica
gel. Phosphorus pentoxide is often used in a constant
temperature vacuum desiccator. The desiccants should be
replaced in time.
0832 Determination of Water
Method 1 ( Karl Fischer' s Method)
l. Volumetric titration
This method is based on the quantitative reaction of water
with a solution of sulfur dioxide and iodine in pyridine and
methanol. The apparatus used should be dry and moisture
proof. The determination should be carried out in a dry place.
Preparation and standardization of Karl Fischer reagent
( 1 ) Preparation of Karl Fischer reagent Place 11 O g of
iodine, previously dried in a desiccator over sulfuric acid for
more than 48 hours, in a dry conical flask with stopper. Add
160 ml of anhydrous pyridine, cool, and stir constantly until
the iodine is completely dissolved. Add 300 ml of anhydrous
methanol and weigh. Keep the conical flask cold in an ice
bath and keep the atmospheric moisture excluded from the
system. Continue inleting the sulfur dioxide until the weight
of the solution achieve to 72 g, and then add anhydrous
methanol to give a total volume of 1000 ml. Press stopper
tightly, mix well, and allow to stand for 24 hours protected
from light.
Commercially available Karl Fischer reagents containing basic
compounds other than pyridine or alcohols other than
methanol may be used. These reagents may be made of single
solution or two discrete solutions.
This reagent should be preserved in an airtight container,
protected from light and stored in a cool and dry place, then
standardized befare use.
( 2 ) Standardization of Karl Fischer reagent W eigh 10-
30 mg of purified water accurately, and standardize with a
water determination apparatus directly or use the following
procedure: place about 10-30 mg of purified water, accu-
rately weighed, into a dry conical flask with stopper. Unless
otherwise spelified, add a quantity of anhydrous methanol.
Titrate with Karl Fischer reagent until the colour changes
from pale yellow to reddish brown and keep the atmospheric
moisture excluded from the system. The endpoint may also
be determined electrometrically by dead-stop titration
( 0701 ) . Perform a blank titration and calculate the water
equivalent of the reagent in mg per ml by the formula:
w
F= (A-B)
Where: F is the weight of water equivalent of the reagent
in mg per ml.

W is the weight of purified water in mg.
A is the volume of the reagent consumed m the
titration of water in ml.
B is the volume of the reagent consumed m the
blank titration in ml.
(3) Procedure Weigh a quantity of the substance being
examined accurately ( which is estimated to consume 1-5 ml
of Karl Fischer reagent) and determine with a water
determination apparatus directly. The solvent is anhydrous
methanol unless otherwise specified. Or accurately weigh a
quantity of the substance being examined to a dry conical
flask with stopper, add a quantity of solvent and titrate with
Karl Fischer reagent until the colour changes from pale
yellow to reddish brown, while shaking or stirring continu-
ously. The endpoint may also be determined electrometrically
by dead-stop titration ( 0701 ) . Perform a blank titration and
calculate the water equivalent of the reagent in mg per ml by
the formula:
Water content of the substance (%) =(A-B)F/WX100%
Where: A is the volume of the reagent consumed in the
titration of water in ml.
B is the volume of the reagent consumed in the
blank titration in ml.
F is the weight of water equivalent of the reagent
in mg per ml.
W is the weight of the substance being examined
inmg.
If the substance is hygroscopic, it can be placed in a dry
sealed container ( for example a glove box in an atmosphere
of dry inert gas) and weighed accurately. Use a drying
syringe to add an appropriate volume of anhydrous methanol
or other suitable solvents accurately weighed, shake to
dissolve the substance, and determine the water content of
the solution. Clean and dry the sealed container and weigh it
accurately. Determine the water content of the solvent in the
same way. Calculate using the formula:
Water content of the substance ( % )
(W1 -W3)C1 -(W1 -W2)C2 XlOO%
Wz-W3
Where W1 is the weight of the substance, solvent and dry
sealed container in g.
W 2 is the weight of the substance and sealed
container in g.
w3 is the weight of the sealed container in g.
C1 is the water content of the substance in g per
g.
Cz is the water content of the solvent in g per g.
For the thermostable substance, an apparatus, combined
with a commercially available Karl Fischer type dry oven can
be used to determine the water content. If an oven is used, an
appropriate amount of the sample is introduced into the oven
and heated, then the water is evaporated, carried into the
apparatus and determined.
2. Coulometric titration
This method is also based on the Karl Fischer reaction and
using dead-stop titration ( 0701) to determine the content of
water. However, compared with volumetric titration, iodine
is not added in the form of a volumetric solution from the
titration tube but is produced electrochemically in the
reaction cell by the oxidation of iodide. When all the water
has been consumed, an excess of iodine occurs, which can
make the platinum electrodes polarized, thus indicating the
endpoint. According to Faraday' s law, the amount of iodine
produced is directly proportional to the electric quantity
passed through, so the total amount of water can be
determined by measuring the total amount of electric quantity
0832 Determination of Water
consumed. This method is predominantly used for substances
with a very low water content (0. 0001% to O. 1%). It is
particularly suited to chemically inert substances like
hydrocarbons, alcohols, and esters. All the apparatus used
must be dried and atmosphere moisture is excluded from the
system. The determination should be carried out in a dry
place.
Karl Fischer reagent Prepare or purchase according to the
requirements of Karl Fischer' s coulometric titrator.
Standardization is not necessary.
Procedure Add an appropriate volume of Karl Fischer
reagent to the titration tube. Moisture is eliminated from the
reagent and the system by pre-electrolysis. Then transfer
quickly a quantity of the test specimen estimated to contain
O. 5-5 mg of water, accurately measured, into the titration.
Perform dead-stop titration ( 0701 ) to the electrometric
endpoint. Read the water content of the specimen directly
from the instrument output, and calculate the percentage
content of water in the substance. 1 mg of water is equivalent
to 10. 72 Coulomb of electric quantity.
Method 2 ( drying in oven method)
Procedure Place 2-5 g of the substance being examined,
accurately weighed, in a flat weighing bottle previously dried
to constant weight to form a smooth layer not exceeding
5 mm in thickness, or not exceeding 1 O mm in thickness for
substance of loose texture. Dry in an oven at l00-105ºC for 5
hours with the stopper of the bottle removed. Upon opening
the oven, close the bottle promptly and allow it to cool in a
desiccator for 30 minutes. Weigh accurately and dry it again
under similar condition for 1 hour, cool and weigh. Repeat
the operation until the difference between two successive
weighings is not more than 5 mg. Calculate the percentage
content of water in the substance being examined according
to the weight loss on drying.
The method is used for the determination of water in crude
drugs with little or no volatile constituents.
Method 3 ( Drying under reduced pres.sure method)
Vacuum desiccator Distribute O. 5-1 cm depth of phospho-
rous pentoxide into a Petri dish of 12 cm in diametre and put
the dish in a vacuum desiccator of 30 cm in diametre.
Procedure Mix thoroughly 2-4 g of the substance being
examined. Weigh accurately O. 5-1 g in a weighing bottle,
previously dried to constant weight under the conditions
prescribed for the substance being examined. Place the loaded
bottle in the vacuum desiccator, remove the stopper of the
bottle and leave it also in the desiccator. Reduce the pressure
of the desiccator by suction to 2. 67 kPa ( 20 mmHg) and
last for 30 minutes. Allow to stand at room temperature for
24 hours. Connect an anhydrous calcium chloride tube to the
air inlet and open the desiccator plunger. Upon opening the
desiccator, close the bottle promptly and weigh accurately.
Calculate the percentage content of water in the substance
being examined according to the weight loss on drying.
The method is used for the determination of water invaluable
drugs containing volatile constituents. The substance of
Chinese Medicine for determination should be pulverized to
pass through a No. 2 sieve.
Method 4 ( Toluene distillation method)
Apparatus (As Fig. 2) The apparatus consists of a 500 ml
round bottom flask (A), a graduated receiving tube ( B)
and a reflux condenser ( C) approximately 40 cm in length.
All parts of the apparatus should be cleaned and dried in an
oven.
 0841 Determination of Residue on Ignition
Fig. Apparatus of method 4
Procedure Place a quantity of the substance being examined
which is anticipated to yield about 1-4 ml of water,
accurately weighed, in the flask A Add about 200 ml of
toluene and dry clean unglazed porcelain ora few glass beads
if necessary. Assemble the apparatus and fill the receiving
tube B with toluene through the condenser. Heat the flask
gently, when toluene begins to boíl, adjust the temperature
and allow to distill ata rate of 2 drops per second. When the
volume of water in the receiving tube does not increase any
more, rinse the inside of condenser with toluene and push
clown the toluene adhering to the wall with a brush or other
suitable tools. Continue the distillation for 5 minutes, cool to
room temperature and disconnect the apparatus. Push clown
any droplet of water adhering to the wall of the receiving tube
with a copper wire wetted with toluene. Allow to stand until
water is completely separated from toluene in the receiving
tu be (a small amount of methylene blue may be added to
facilita te observation). Record the volume of water distilled
and calculate the percentage content of water in the substance
being examined.
N01E
(1) The toluene to be used should be prepared as follows.
Add a small quantity of water. Shake thoroughly and allow to
stand for a while. Discard the water layer and distill the
remainder.
(2) The substance of Chinese medicine for determination
should be broken into granules or pieces with no more than
3 mm in diametre. The substances which have less than
3 mm in length and diametre, can be examined without
further breaking.
Method 5 (Gas chromatography)
Chromatographic system and system suitability Carry out the
method for gas chromatography, using porous polymer beads
of divinylbenzene cross-linked ethylvinylbenzene with O. 18-
0. 25 mm in diametre as the support, or capillary column
with similar polarity. Maintain the column temperature at a
range of 140-150ºC, determine by conductivity detector.
Inject anhydrous ethanol and carry out the method for gas
chromatography ( 0521 ) , the performance of the instrument
should comply with the following requirements.
(1) The number of theoretical plates for the column
calculated with respect to the peak of water should be more
than 1000, while the number of theoretical plates for the
column calculated with respect to the peak of ethanol should
be more than 150.
(2) The resolution between the peaks of water and ethanol is
more than 2.
(3) Carry out 5 replicate injections of anhydrous ethanol,
the RSD of the peak areas of water should not be more than
3.0%.
Reference solution Accurately weigh about O. 2 g of purified
water in a 25 ml volumetric flask, dilute to volume with
anhydrous ethanol and mix well.
Test solution Place the pulverized test sample ( containing
about O. 2 g water), accurately weighed, in a conical flask
with stopper. Add anhydrous ethanol 50 ml accurately and
mix well. Ultrasonicate for 20 minutes, allow to stand for 12
hours. Ultrasonicate for another 20 minutes and allow to
stand. After the solution becomes clarified, use the clear
supernatant solution as the test solution.
Procedure Inject 1-5 ,ul of anhydrous ethanol, reference
solution and test solution respectively into the column.
Determine and calculate the content of water.
U se fresh opened anhydrous ethan from the same bottle
when preparing the reference solution and the test solution.
Calculate the water content by the external standard method.
The water contained in anhydrous ethanol should be deducted
as follows:
Peak area of water practically added into the reference
solution = the total peak area of water in the reference
solution-KX peak area of ethanol in the reference solution.
Peak area of water in test sample = the total peak area of
water in test solution-K X peak area of ethanol in test
solution.
K = peak area of water in anhydrous ethanol/Peak area of
ethanol in anhydrous ethanol.
0841 Determination of
Residue on lgnition
Place l. 0-2. O g or the quantity specified in the individual
monographs of the substance to be examined, accurately
weighed, in a suitable crucible ( If the substance to be
examined contains alkali metals or fluorine element, the
platinum crucible should be used). Previously ignited to
constant weight. Heat gently until it is thoroughly charred,
cool and moistens the residue with O. 5-1 ml of sulfuric acid,
unless otherwise directed. Heat gently until white fumes are
no longer evolved and then ignite at 700-800ºC until the
incineration is complete. Cool in a desiccator and weigh
accurately, ignite again at 700-800ºC to constant weight.
If the residue is to be used in the limit test for heavy metals,
the ignition temperature should be controlled at 500-600ºC.
0842 Limit Test for Readily
Carbonizable Substances
Use two Nessler cylinders with same inner diametre, A and
B. To cylinder A, add 5 ml of the reference solution specified
in the individual monographs. To cylinder B, add 5 ml of
sulfuric acid (94.5%-95.5%) (g/g) and then add the
specified quantity of the substance being examined in small
portions. Shake the mixture thoroughly until the substance is
complete dissolved. Allow to stand for 15 minutes and
compare the colour of the two solutions by viewing
horizontally against a white background, unless otherwise
specified. The colour produced in cylinder B is not more
intense than that of the solution in cylinder A
Solid substances should be finely powdered before the test.
When heating is necessary to effect dissolution of the
substance being examined, mix it with sulfuric acid in a test
 0861
tube, heat to dissolve, allow to cool, transfer the solution to
a Nessler cylinder and compare the colour as described above.
0861 Determination of
Residual Solvents
Residual solvents in pharmaceutical products are defined as
organic volatile chemicals that are used or produced in the
manufacture of drug substances or excipients, or in the
preparation of drug products, but not completely removed by
practica! manufacturing techniques. The general residual
solvents in pharmaceutical products and the limits are listed
in table l. Unless otherwise specified, the residual content of
class 1, class 2 and class 3 residual solvents should comply
with the requirement in table 1; for other solvents, according
to the manufacture technology, relevant limits should be
regulated in order to meet product specifications, good
manufacturing practises ( GMP) or other quality-based
requirements.
Carry out the method for gas chromatography ( 0521 ) .
Column
l. Capillary column
Unless otherwise specified, column with the same polarity
can be substituted each other.
(1) Non-polar column U se 100 % dimethylpolysiloxane as
the stationary phase.
(2) Polar column Use polyethylene glycol (PEG-20M) as
the stationary phase.
(3) Moderate polar column Use (35%) diphenyl- (65%)
methylpolysiloxane, ( 50 % ) diphenyl- ( 50 % ) dimethyl-
polysiloxane, ( 35 % ) diphenyl- ( 65 % ) dimethylpolysilo-
xane, 04%) cyanopropylphenyl- (86%) dimethylpolysilo-
xane, (6%) cyanopropylphenyl- (94%) dimethylpolysilo-
xane as the stationary phase.
(4) Lower polar column Use ( 5%) Phenyl- ( 95%)
methylpolysiloxane, ( 5 % ) diphenyl- ( 95 % ) dimethylpo-
lysiloxane as the stationary phase.
2. Packed column
Use divinylbenzene-ethylvinylbenzene copolymer porous
beads with a diametre of O. 25-0. 18 mm, or other suitable
packing materials as the stationary phase.
System suitability
(1) Calculated with reference to the peak of the substance
being examined, the number of theoretical plates for the
capillary column is not less than 5000; the number of
theoretical plates for the packed column is not less than 1000.
(2) In the chromatogram, the resolution between the peaks
of the substance being examined and its neighbor substance is
greater than l. 5.
(3) When the interna! standard method is used, carry out 5
replicate injections for each of the standard solutions. The
relative standard deviation (RSD) of the ratio of the analyte
peak area to the internal standard peak area should not exceed
5%. When externa! standard method is used, the RSD of the
peak areas of the substance being examined should not exceed
10%.
Test solution
l. Head-space injection
Unless otherwise specified, dissolve O. 1-1 g, accurately
weighed, of the substance being examined in water. For the
hydropholic pharmaceutical products, dissolve the substance
being examined in N, N-dimethylformamide, dimethyl
sulfoxide or other appropriate solvents. According to the
Determination of Residual Solvents
solubility of the substance being examined and the solvents
being examined, select the appropriate solvent that not
interfere the determination of the substance being examined.
Prepare the test solution based on the limit of residual
solvent specified in the monograph. The concentration should
meet the requirement of quantitative examination.
2. Direct injection of solution
Weigh accurately a quantity of the substance being examined
and dissolve in water or appropriate organic solvent. Prepare
the test solution based on the limit of residual solvent
specified in the monograph. The concentration should meet
the requirement of quantitative examination.
Reference solution
Weigh accurately a quantity of the organic solvent in the
individual monograph. Use the same method and solvent of
the test solution to prepare the reference solution. When
using water as the solvent, the organic solvent being
examined should be dissolved in 50 % dimethyl sulfoxide or
N, N-dimethylformamide first, and then gradually diluted
with water. The concentration of the reference solution is
based on the limits of residual solvents in the limit test. For
ensuring the veracity of quantitative result, the concentration
of the reference solution is based on the actual content of
residual solvent in the quantitative test. In general, the peak
area of the reference solution should not be more than twice
of the peak area of the test solution. If necessary, the
concentration of the test solution or reference solution should
be readjusted.
Procedure
Method 1 The method of isothermal temperature capillary
column head-space injection
When the number of organic solvents being examined and the
discrepancy of polarity are small, this method can be used.
Chromatographic conditions The column temperature is
usually 40-lOOºC. The carrier gas is nitrogen ata flow rate of
l. 0-2. O ml per minute. When using water as the solvent, the
head-space oven equilibration temperature is 70-85ºC. The
equilibration time is 30-60 minutes, The injector temperature
is 200ºC. The temperature of the flame-ionization detector
(FID) is 250ºC.
Procedure Inject the reference solution and the test solution
not less than two times respectively, and then measure the
peak area of the solvent being examined.
If there are unknown organic solvents in the chromatogram,
use table 2 to prescreen residual solvents.
Method 2 The method of programmed temperature capillary
column head-space injection
When the number of organic solvents being examined is large
and the discrepancy of polari ty is big, this method can be
used.
Chromatographic system In general the column temperature
is maintained at 40ºC for 8 min, then raised at a rate of 8ºC
per minute to 120ºC, and maintained at 120ºC for 10 min.
The carrier gas is nitrogen at a flow rate of 2. O ml per
minute. When using water as the solvent, the head-space
oven equilibration temperature is 70-85ºC. The equilibration
time is 30-60 minutes. The injector temperature is 200ºC.
The temperature of the flame-ionization detector ( FID) is
250ºC.
When examining the residual solvent of a certain substance,
the temperature program can be adjusted based on the
residual solvents specified in the monograph.
Procedure lnject the reference solution and the test solu-
 0861 Determination of Residual Solvents
tion, notless than two times respectively, then measure the
peak area of the solvent being examined.
If there are unknown organic solvents in the chromatogram,
use table 3 to prescreen residual solvents.
Method 3 Direct injection of solution
Use a packed column, or a capillary column of appropriate
polarity.
Procedure lnject the reference solution and the test solution
two or three times respectively, then measure the peak area
of the solvent being examined.
Calculation
( 1) Limit test Unless otherwise specified, determine the
residual solvents in the test solution as specified in the
monograph. When interna! standard method is used, the
ratio of the peak area of the residual solvent in the test
solution to that of the internal standard is not more than the
corresponding ratio in the reference solution. When the
external standard method is used, the peak area of residual
solvent being examined in the test solution is not more than
the corresponding peak area in the reference solution.
( 2) Quantitative test The content of residual solvents can
be calculated by interna! standard method or external
standard method.
NOTE
(1) Unless otherwise specified, the options of head-space
conditions should be as follows
CD The choice of head-space oven equilibration temperature
depends on the boiling point of the residual solvents of the
substance being examined. For the residual solvents of high
boiling point, the head-space oven equilibration temperature
should also be high. Meanwhile the thermal degradation
ability of the substance to be examined should be paid more
attention in arder to avoid the interference of thermal
degradation products.
®The equilibration time of head-space vials is usually 30-
45 minutes to ensure that the time is enough for the test
solution to reach gas-liquid equilibrium. The equilibration
time should not be too long. The hermeticity of the head-
space vials may get worse and decreases the quantitative
accuracy, if the equilibration time excess of 60 minutes.
@The reference solution and the test solution should follow
the same head-space conditions.
(2) Validation of the quantitative method
When using head-space injection, if the substance being
examined and the reference substance are in the matrices
which are not completely uniform, the matrix effect should
be taken into account ( The influence on the gas-liquid
equilibrium in head-space vials by the different composition
of the test and reference solution). As a standard addition
test can eliminate the matrix effect, it is usually adopted to
validate the accuracy of the quantitative method; if the
results of standard addition test and other quantitative
methods a have discrepancy, the result of standard addition
test is correct.
(3) Elimination of the interference peak
The unknown impurities or the thermal degradation products
in the substance being examined can interfere with the
examination of residual solvents. There are two kinds of
interference effects. The first is the unknown impurity or the
thermal degradation products in the substance being
examined which have the same retention time with the
residual solvent being examined ( co-elution). The second is
the thermal degradation product which has the same
structure with the residual solvent being examined (e. g.
compounds with methyoxyl group may produce methanol
when the thermal degradation occurs). When the residual
solvent exceeds the limit but it is not confirmed if there is
interference effects, a further test should be done to
eliminate the interference. For the first kind of interference
effect, the general adopted method is to examine the same
substance being examined using a chromatographic system
with different polarity, then compare the results of the two
different chromatographic systems. If the results are the
same, then there is no co-elution interference. If the results
are different, there is co-elution interference. For the second
kind of interference effect, the general adopted method is to
examine the same substance without the same residual
sol ven t.
( 4) Determination of nitrogenous alkaline substances The
stainless steel transferline and injection liner of ordinary gas
chromatography instrument have strong sorption to nitroge-
nous alkalescence substances such as amines. lt will decrease
the sensitivity. Therefore, the inert silicon steel or nickel
steel line be used. When using the method of direct injection
of solution, the test solution should not be acidic, because
the residual solvent being examined can react with acids to
generate compounds which are hard to boil away.
Usually use weak polar column or columns with the packing
materials dealt with alkali to examine nitrogenous alkale-
scence substances. C-r0od result may be obtained 'Nhen using
the column for amines.
For the nitrogenous alkalescence substance which is not
readily detected by gas chromatography, such as N-methyl-
pyrrolidone, other appropriate procedures can be used, such
as ion chromatography.
( 5) Selection of detector
Electron capture detector (ECD) has high sensitivity for the
residual solvents which have halogen, such as chloroform.
(6) The different laboratories may use different methods to
examine the same substance. If the results are at the edge of
eligible or not eligible, the results of interna! standard test
and standard addition test are correct.
( 7) The equilibration temperature of head-space vial is
usually at least lOºC lower than the boiling point of the
solvent. The solvents with high boiling point, such as
formamide, 2-methoxyethanol, 2-ethoxyethanol, ethylene
glycol and N-methylpyrrolidone, are not readily detected by
the head-space injection conditions.
(8) The most common method for identification in gas
chromatography is using the retention value. The variation
of the flow rate of carrier gas, the temperature of carrier
gas, and the column temperature could change the retention
value and the result of identification. The relative adjusted
retention time ( RART) is a reliable parametre for
identification as it is only influenced by the column
temperature and the property of the stationary phase.
Methane is usually used to determine the dead volume (to )
of the chromatographic system.
RART=t~-to
tR -to
Where: t R is the retention time of the compound.
t~ is the retention time of the reference substance.
 0861 Determination of Residual Solvents

Table 1 General residual solvents in pharmaceutical products and lirnits

Limit Limit Limit Limit
Solvents Solvents Solvents Solvents
<%> <%) <%> <%>
Class 3 solvents Class 3 solvents
Class 1 solvents Class 2 solvents (solvents that should (solvents that shoulc
( solvents that should (solvents that be limited by GMP be limited by GMP
be avoied) should be limited) or other quality or other quality
based requirements) based requirements)
Benzene 0.0002 2-Ethoxyethanol 0.016 Acetic acid 0.5 Methylethyl ketone 0.5

Methylisobutyl
0.5
ketone

Carbon tetrachloride 0.0004 Ethylene glycol 0.062 Acetone 0.5 2-Methyl-l-propanol 0.5
Pentane 0.5
1,2-])ichloroethane 0.0005 Formamide 0.022 Anisole 0.5 1-Pentanol 0.5
1, 1-Dichloroethene 0.0008 Hexane 0.029 1-Butanol 0.5 1-Propanol 0.5
1 , 1, 1-T richloroethane o. 15 Methanol 0.3 2-Butanol 0.5 2-Propanol 0.5
Propyl acetate 0.5
Class 2 solvents 2-Methoxyethanol 0.005 Butyl acetate 0.5 Class 4 solvents

Tert-Butylmethyl 0.5 (solvents for which

( solvents that should ether no adequate
be limited) Cumene 0.5 toxicological
Dimethyl sulfoxide 0.5 date was found)

1, 1-
Acetonitrile 0.041 Methylbutyl ketone 0.005 Ethanol 0.5
])iethoxypropane

1, 1-
])imethoxynethane

2,2-
Chlorebenzene 0.036 Methyleyclohexane o. 118 Ethyl acetate 0.5 Dimethoxypropane
Isoocatne

Trimethylpetane
Chloroform 0.006 N-Methylpyrrolidone 0.053 Ethyl ether 0.5
Isopropyl ether

Methylisopropyl
ketone

Methyltetrahy-
Cyclohexane 0.388 Nitromethane 0.005 Ethyl formate 0.5
drofuran

1,2-])ichloroethene o. 187 Pyridine 0.02 Formic acid 0.5 Petroleum ether

T riehloroacetic acid
])ichloromethane 0.06 Sulfolane 0.016 Heptane 0.5 Trifluoroacetic acid
1, 2-])imethoxyethane 0.01 Tetralin 0.01 Isobutyl acetate 0.5

N,N-
])imethylacetamide
o. 109 T etrahydrofuran o. 072 Isopropyl acetate 0.5

N,N-
0.088 Toluene 0.089 Methyl acetate 0.5
])imethylformamide

1, 1,2-
])ioxano 0.038 0.008 3-Methyl-1-butanol 0.5
T richloroethene

Xylene 0.217
O)usually 60% m-xylene, 14% p-xylene, 9% o-xylene with 17% ethyl benzene.
@Manufacturers should supply justification for residual levels of these solvents in pharmaceutical products.
 0861 Determination of Residual Solvents

Table 2 The relative adjusted retention time (RART) reference value of the residual
solvents relative to methyl ethyl ketone under isothermal conditions
Non-polar column Polar column
Organic solvent tR/min RART Organic solvent tR/min RART
Column temperature 40ºC Column temperature 40ºC
Methanol l. 828 o. 126 Pentane 1.682 0.032
Ethanol 2.090 0.268 Hexane l. 787 0.075
Acetonitrile 2. 179 0.315 Ethyl ether l. 842 0.097
Acetone 2.276 0.368 Isooctane l. 926 o. 131
2-Propanol 2.356 o. 411 Isopropyl ether l. 943 o. 138
Pentane 2.487 0.481 tert-Butylmethyl ether 2.005 o. 163
Ethyl ether 2.489 0.482 Heptane 2.021 o. 169
Ethyl formate 2.522 0.501 cyclohexane 2. 159 0.225
1, 1-Dimethoxymethane 2.584 0.534 1, 1-Dichloroethene 2.209 0.245
1, 1-Dichloroethene 2.609 0.547 1, 1-Dimethoxymethane 2.243 0.259
Methyl acetate 2.635 0.561 Methylcyclohexane 2.405 0.342
Dichloromethane 2.655 0.572 Acetone 2.876 0.515
Nitromethane 2.807 0.654 Ethyl formate 2.967 0.551
1-Propanol 2.982 o. 748 Methyl acetate 3.000 o. 564
1, 2-Dichloroethene 3.109 0.817 1, 2-Dichloroethene 3.347 0.705
tert-Butylmethyl ether 3.252 0.894 Tetrahydrofuran 3.403 o. 727
Methylethyl ketone 3.449 1.000 Methyl tetrahydrofuran 3.481 0.758
2-Butanol 3.666 l. 117 Carbon tetrachloride 3.635 0.821
Hexane 3.898 l. 242 1, 1, 1-Trichloroethane 3.653 0.828
Isopropyl ether 3.908 l. 247 Ethyl acetate 3.810 0.891
Ethyl acetate 3.913 l. 250 Isopropyl acetate 3.980 0.960
Chloroform 3.954 l. 272 Methanol 4.062 0.993
Tetrahydrofuran 4.264 l. 439 Methylethyl ketone 4.079 l. 000
2-Methy1-1-propanol 4.264 l. 440 1, 2-Dimethoxyethane 4.604 l. 212
1, 2-Dicloroethane 4.517 l. 576 Methylisopropyl ketone 4. 716 l. 257
1 , 1, 1-T richloroethane 4.808 l. 733 Dichloromethane 4. 758 l. 274
Methylisopropyl ketone 4.976 l. 823 2-Propanol 4.822 l. 300
1, 2-Dimethoxyethane 4.985 l. 828 Ethanol 4.975 l. 362
Benzene 5.281 l. 988 Benzene 4.977 l. 362
Isopropyl acetate 5. 311 2.004 Propyl acetate 6.020 l. 784
1-Butanol 5.340 2.019 T richloroethene 6.643 2.035
Carbon tetrachloride 5.470 2.089 Methylisobutyl ketone 7.202 2.261
Cyclohexane 5.583 2. 150 Acetonitrile 7.368 2.328
Methyl tetrahydrofuran 5.676 2.201 Isobutyl acetate 7.497 2.380
Trichloroethene 6.760 2. 785 Chloroform 7.985 2.577
Dioxane 6.823 2.819 2-Butanol 8.390 2.740
lsooctane 6.957 2.891 Toluene 8. 746 2.884
Heptane 7.434 3. 148 1-Propanol 9.238 3.083
Propyi acetate 7.478 3. 172 1,4-Dioxane 10.335 3.526
Methylcyclohexane 8.628 3. 792 1, 2-Dicloroethane 10.827 3.724
Methylisobuty1 ketone 8. 738 3.851 Butyl acetate 11. 012 3. 799
3-Methyl-1-butanol 8.870 3.922 Methylbutyl ketone 11. 486 3.990
Pyridine 9.283 4. 145 Methane l. 602
Toluene 11. 180 5. 168 Column temperature 80ºC
1-Pentanol 11. 382 5.276 2-Methyl-1-propanol 3.577 3.045
Methane l. 594 1-Butanol 4.460 4.334
Column temperature 80ºC Ni tromethane 4.885 4.948
lsobutyl acetate 3. 611 2.099 Cumene 5.288 5.543
 0861 Determination of Residual Solvents

continued
Non-polar column Polar column
Organic solvent tR/min RART Organic solvent tR/min RART
Methylbutyl ketone 3.859 2.345 Pyridine 5.625 6.035
Butyl acetate 4.299 2. 778 3-Methyl-1-butanol 5.934 6.486
Chlorobenzene 5.253 3. 726 Chlorobenzene 6.439 7.223
Anisole 7.436 5.890 1-Pentanol 7.332 8.527
Cumene 8. 148 6.589 Methylethyl ketone 2. 176 1.000
Methylethyl ketone 2.502 l. 000 Methane l. 491
Methane l. 493 Column temperature 120ºC
Column temperature 120ºC Anisole 3.837 9.890
Tetralin 8.067 29.609 Tetralin 7.427 24.484
Methylethyl ketone l. 630 l. 000 Methylethyl ketone l. 650 l. 000
Methane 1.405 Methane l. 404

Table 3 The relative adjusted retention time( RART} reference value of the
residual solvents relative to methyl ethyl ketone under prograrnmed temperature conditions
Non-polar column Polar column
Organic solvent tR/min RART Organic solvent tR/min RART
Methanol l. 846 o. 127 Pentane l. 691 0.033
Ethanol 2. 121 0.272 Hexane l. 807 0.076
Acetonitrile 2.201 0.314 Ethyl ether l. 856 0.094
Acetone 2.303 0.367 Isooctane l. 957 o. 131
2-Propanol 2.401 0.419 Isopropyl ether l. 966 o. 135
Pentane 2.512 0.477 tert-Butylmethyl ether 2.053 o. 167
Ethyl ether 2.519 0.481 Heptane 2.063 o. 171
Ethyl formate 2.544 0.494 Cyclohexane 2.217 0.228
1,1-Dimethoxymethane 2. 611 0.529 1, 1-Dichloroethene 2.267 0.246
1 , 1-Dichloroethene 2. 623 0.535 1, 1-Dimethoxymethane 2.303 0.260
Methyl acetate 2.665 0.558 Methylcyclohexane 2.488 0.328
Dichloromethane 2.674 0.562 Aceto ne 2.988 0.513
Nitromethane 2.839 0.649 Ethyl formate 3.094 0.552
1-Propanol 3.051 o. 760 Methyl acetate 3. 126 0.564
1, 2-Dichloroethene 3. 128 0.801 1, 2-Dichloroethene 3. 511 o. 707
tert-Butylmethyl ether 3.302 0.892 Tetrahydrofuran 3.561 o. 725
Methylethyl ketone 3.507 l. 000 Methyl tetrahydrofuran 3.653 o. 759
2-Butanol 3. 756 l. 131 Carbon tetrachloride 3.821 0.822
Hexane 3.966 l. 241 1 , 1 , 1-T richloroethane 3.833 0.826
Isopropyl ether 3. 971 1.244 Ethyl acetate 4.017 0.894
Ethyl acetate 3.981 l. 249 Isopropyl acetate 4.207 0.964
Chloroform 4.005 l. 262 Methanol 4.295 0.997
T etrahydrofuran 4.387 l. 462 Methylethyl ketone 4.303 l. 000
2-Methyl-1-propanol 4.397 l. 468 1, 2-Dimethoxyethane 4.875 l. 212
1, 2-Dicloroethane 4.612 l. 581 Methylisobutyl ketone 5.005 l. 260
1, 1 , 1-T richloroethane 4.843 l. 702 Dichloromethane 5.041 l. 273
Methylisopropyl ketone 5.087 l. 830 2-Propanol 5.069 l. 284
1, 2-Dimethoxyethane 5.099 l. 837 Ethanol 5.275 l. 360
Benzene 5.380 l. 984 Benzene 5.275 l. 360
Isopropyl acetate 5.398 l. 994 Propyl acetate 6.437 l. 790
1-Butanol 5.402 l. 996 1, 1, 2-Trichloroethene 7. 108 2.039
Carbon tetrachloride 5.501 2.048 Methylbutyl ketone 7.735 2.271
Cyclohexane 5.649 2. 126 Acetonitrile 7.892 2.329
 , ... , . ,. ~·

·~=, ·,

0871 Determination of Methanol

continued
Non-polar column Polar column
Organic solvent tR/min RART Organic solvent tRlmin RART
Methyl tetrahydrof uran 5.739 2. 173 Isobutyl acetate 8.068 2.394
1, 1, 2-Trichloroethene 6.815 2. 738 Chloroform 8.533 2.566
Isooctane 6.928 2. 798 2-Butanol 8.848 2.683
1, 4-Dioxane 6.928 2. 798 Toluene 9. 156 2. 797
Heptane 7.563 3. 131 1-Propmnol 9.461 2.910
Propyl acetate 7.583 3. 142 1 , 4-Dioxane 10. 183 3. 177
Methylcyclohe:xane 8.581 3.666 1, 2-Dicloroethane 10.446 3.274
Methylisobutyl ketone 8.830 3. 797 Butyl acetate 10.543 3.310
3-Methyl-1-butanol 8.968 3.870 Methylbutyl ketone 10.801 3.406
Pyridine 9. 178 3.980 2-Methyl-l-propanol 11. 606 3. 704
Toluene 10.259 4.548 1-Butanol 13.046 4.237
1-Pentanol 10.448 4.647 Cumene 13.258 4.315
Isobutyl acetate 10.638 4.747 Nitromethane 13.396 4.367
Methylbutyl ketone 11. 025 4.951 Pyridine 13.949 4.571
Butyl acetate 12. 175 5.555 3-Methyl-1-butanol 14.519 4. 782
Chlorobenzene 13. 166 6.076 Chlorobenzene 14.562 4. 798
Aniso le 15.270 7. 181 1-Pentanol 15.516 5. 151
Cumene 15. 724 7.420 Aniso le 17.447 5.866
Tetralin 22.409 10.933 Tetralin 21. 708 7.444
Methane l. 604 Methane l. 602
Note: The data in Table 2 and Table 3 are the results of non-polar SPB-1 colum (30 mX O. 32 mm, l. O µm) and polar HP-
INNOWAX column (30 mXO. 32 mm, O. 5 µm).

solution into separate 10 ml head-space vials and seal the

0871 Determination of Methanol
Gas chromatography ( 0521) is used for the determination of
methanol in preparations containing ethanol such as
Medicinal Wines and Tinctures. Unless otherwise specified,
the following methods can be used.
Method 1 (Capillary Columns)
Chromatographic system and system suitability The gas
chromatograph is equipped with a flame-ionization detector
(FID), anda fused-silica capillary column bonded with a film
of 6 % cyanopropylphenyl siloxane 94 % polydimethyl-
silo:xane. Maintain the temperature of the column at 40ºC for
2 minutes, then raise the temperature at a rate of 3ºC per
minute to 65ºC, and then raise the temperature at a rate of
25ºC per minute to 200ºC and maintain it at 200ºC for
10 minutes. Maintain the temperature of the injection port at
200ºC and that of the detector at 220ºC. The split ratio is 1 :
l. The head-space sampler parameters are set as follows: the
equilibration temperature is 85ºC and the equilibration time is
20 minutes. The number of theoretical plates for the column
is not less than 10000 calculated with reference to the peak of
methanol, and the resolution of the peaks of methanol and
other compounds is more than l. 5.
Procedure Use the solution to be examined as the test
solution. Measure accurately 1 ml of methanol into a 100 ml
volumetric flask, dilute to volume with water and mix well.
Measure accurately 5 ml of this solution into a 100 ml
volumetric flask, dilute to volume with water and mix well
as the reference solution.
Transfer accurately 3 ml of the reference solution and the test
vials, respectively. Determine and calculate the content of
methanol with respect to the peak area by the externa!
standard method
Method 2 ( Packed Columns)
Chromatographic system and system suitability Carry out the
method for gas chromatography, using a column packed with
porous polymer beads (O. 18-0. 25 mm) of ethylvinylbenzene
cross-linked with divinylbenzene. The temperature of the
column is maintained at 125ºC. The number of theoretical
plates for the column is not less than 1500 calculated with
reference to the peak of methanol. The resolution of the
peaks of methanol, ethanol, the internal standard and other
compounds shall comply with the requirements.
Conection factor Measure accurately 1 ml of n-propanol into
a 100 ml volumetric flask, dilute to volume with water and
mix well, as the interna! standard solution. Measure
accurately 1 ml of methanol into a 100 ml volumetric flask,
dilute to volume with water and mix well. Measure accurately
10 ml of this solution and 10 ml of the interna! standard
solution into a 100 ml volumetric flask, dilute to volume with
water and mix well, as the reference solution. Inject 1 µl of
the reference solution for 3-5 times successively and calculate
the correction factor according to the peak area of each
injection.
Procedure Measure accurately 1 ml of the internal standard
solution into a 10 ml volumetric flask, dilute to volume with
the solution to be examined and mix well, as the test
solution. Inject lµl of the test solution and calculate the
content of methanol.
Unless otherwise specified, the content of methanol in the
solution to be e:xamined is not more than O. 05% (ml/mD.
 0873
NOTE
(1) When using Method 2, if any peak corresponding to the
location of the internal standard appears in the chroma-
togram, the assay method can be replaced by the external
standard method.
(2) lt is recommended to use a column with wide internal
diametre and thick film (30 mX O. 53 mm X 3. 00 µm).
0872 Acetic Acid in Synthetic Peptides
The amount of acetic acid or acetate in synthetic peptides is
determined by high performance liquid chromatography
( 0512 >. The following procedure is to be used, unless
otherwise specified.
Reference solution Dissolve a quantity of glacial acetic acid,
accurately weighed, in a mixture of 5 volumes of mobile
phase B and 95 volumes of mobile phase A to prepare the
reference solution containing about O. 1 mg of glacial acetic
acid per ml (The concentration can be adjusted depending on
the amount of acetic acid in the substance being examined).
Test solution Proceed as specified in the monograph. ( The
amount of sample may be adapted depending on the expected
amount of acetic acid in the substance being examined.)
Chromatographic system and system suitability test Carry out
the method for high performance liquid chromatography
( 0512 >, using a column packed with octadecylsilane hended
silica gel ( 250 mm X 4. 6 mm, 5 µm). Mobile phase A is
phosphoric acid solution ( add O. 7 mi of phosphoric acid to
1000 ml of water, and adjust to pH 3. O with O. 42% sodium
hydroxide TS) , and mobile phase B is methanol. The flow
rate is l. 2 ml per minutes and the detection wavelength is
210 nm. Perform the gradient elution described below. The
number of theoretical plates for the column is not less than
2000, calculated with reference to the peak of acetic acid.
The retention time of acetic acid is approximately 3-
4 minutes.
Time Mobile phase A Mobile phase B
(min) C% V/V) C% V/V)
0-5 95 5
5-10 50 50
10-20 50 50
20-22 95 5
22-30 95 5 Percentage content of 2-ethylhexanoic acid ( % )
Procedure Inject separately 10 µI of the reference solution
and the test solution into the column. Record the chroma-
tograms. Calculate the content of acetic acid in the peptide
with respect to the peak area obtained in the chromatograms
by the external standard method.
0873 Determination of
2-Ethylhexanoic Acid
Gas chromatography ( 0521 > is used for the determination of
2-ethylhexanoic acid in ~-lactam drugs.
Chromatographic system and system suitability U se a
capillary column coated with polyethylene glycol ( PEG-
Determination of 2-Ethylhexanoic Acid
20M) or equivalent polar column. The column temperature
is 150ºC. Maintain the temperature of the injection port at
200ºC and that of the detector at 300ºC. The number of
theoretical plates for the column is not less than 5000
calculated with reference to the peak of 2-ethylhexanoic acid,
and the resolution between the peaks should be greater than
2. O. lnject the reference solution for five times. The relative
standard deviation CRSD) of the ratio of the peak area of 2-
ethylhexanoic acid to that of the interna! standard is not more
than 5%.
Intemal standard solution Transfer 100 mg of 3-cyclohexy-
lpropionic acid to a 100 ml volumetric flask, dissolve and
dilute to volume with cyclohexane, and mix well.
Test solution Dissolve about O. 3 g of the substance to be
examined, accurately weighed, in 4. O ml of 33%
hydrochloric acid, add 1 ml of the internal standard solution,
accurately measured, and shake vigorously for 1 minute.
Allow the mixture to stand and the layers to separate
( centrifuge if necessary). The upper layer solution is used as
the test solution. If necessary, proceed the secondary
extraction as follows. To the lower layer solution add 1 ml of
the internal standard solution, accurately measured, and
shake vigorously for 1 minute. Allow the mixture to stand
and the layers to separa te ( centrifuge if necessary) , discard
the lower layer solution, and combine the upper layer
solutions as the test solution.
Reference solution Transfer about 75 mg of 2-ethylhexanoic
acid CRS, accurately weighed, to a 50 ml volumetric flask,
dissolve and dilute to volume with internal standard solution,
and mix well. To 1 ml of the above solution, accurately
measured, add 4. O ml of 33% hydrochloric acid and shake
vigorously for 1 minute. Allow the mixture to stand and the
layers to separate ( centrifuge if necessary) , and the upper
solution is used as reference solution. If the secondary
extraction is proceeded for the test solution, the same
procedure is needed for the reference solution. To the lower
layer solution add 1 ml of the interna! standard solution,
accurately measured, and shake vigorously for 1 minute.
Allow the mixture to stand and the layers to separate
(centrifuge if necessary), discard the lower layer solution,
and combine the upper layer solutions as the reference
solution.
Procedure lnject respectively 1 µl of the test solution and
the reference solution into the column and record the
chromatogram. Calculate the percentage content of 2-
ethylhexanoic acid from the expression:
_ATXIRXMRX0.02 %
- AR XhXMT XlOO o
Where: AT is the peak area of 2-ethylhexanoic acid in the
chromatogram obtained with the test solution;
AR is the peak area of 2-ethylhexanoic acid in the
chromatogram obtained with the reference
solution;
IT is the peak area of internal standard in the
chromatogram obtained with the test solution;
IR is the peak area of internal standard in the
chromatogram obtained with the reference
solution;
MT is the weight of the substance being examined,
g;
MR is the weight of 2-ethylhexanoic acid CRS, g.
 0901 Colour of Solution

0900 Tests f or Special Properties or Other Indicators

5 drops of xylenol orange IS. Titrate with disodium edetate

0901 Colour of Solution
Colour of Solution is a method for testing the colour of a
solution by comparing the colour of solution of the drug with
that of the reference solutions specified, or, measuring the
absorbance at the specified wavelength.
A solution which is termed " colourless" specified in an
individual monograph means that the colour of solution of the
drug is the same as that of water or the solvent being used
for the preparation of the solution, and a solution which is
termed "almost colourless" means that the colour of solution
(O. 05 mol/L) VS until the solution turns to yellow. Each ml
of disodium edetate (O. 05 mol/L) VS is equivalent to 11. 90
mg of CoClz • 6H 2O. Dilute the rest of cobaltous chloride
solution with hydrochloric acid solution ( 1-40) to contain
59. 5 mg of CoClz •6H20, based on the above assay result.
Preparation of stock reference solutions Prepare the
following stock Reference Solutions, by mixing the above-
mentionded Soluions, and water as described in following
Table l.
Table 1 Preparation of stock reference solutions
(exp~inml)

of the drug is not more intense than that of a reference Cobaltous Potassium Cupric
solution No. O. 5 of the corresponding tint. chloride dichromate sulfate
Colour Water
standard standard standard
Method 1
solution solution solution
Unless otherwise specified, dissolve a quantity of the subs-
tance being examined specified in an individual monograph greenish yellow
27 15 58
with water in a 25 ml Nessler cylinder, dilute with water to (GY)
10 ml. Place in another Nessler cylinder 10 ml of reference yellowish green
l. 2 22.8 7. 2 68.8
solution of specified tint and colour. View clown vertically the (YG)
'7'> '7
cylinders against a white background, or view horizontally yellow (Y) A f\
'±•V
')<)
[.,.J,.J
<) {\
V /[.,, I

against a white background in diffused light. The colour in orange yellow

the cylinder containing the test solution is not more intense 10. 6 19.0 4.0 66.4
(OY)
than that in the cylinder containing the reference solution. If orange red
the colour in the cylinder containing the test solution is, or is (OR)
12.0 20.0 o 68.0
almost, the same as that in the cylinder containing the
brownish red
reference solution, or the tint between cylinders is not 22. 5 12.5 20.0 45.0
(BR)
consistent, so that it is unable to estimate the result by naked
eye, Method 3 (Colour difference metre method) should be
Preparation of reference solutions Prepare the ma tching
used, and the result should be accorded for justifying.
reference solutions by mixing the stock reference solutions
Potassium dichromate standard solution as reagents Dis- and water as described in following table 2.
solve O. 4000 g of reference potassium dichromate, previously
Table 2 Preparation of reference solutions
dried to constant weight at 120ºC and accurately weighed, in
{exp~inml)
water in a 500 ml volumetric flask and dilute with water to
volume, shake thoroughly. Each ml of the solution contains Number of
O. 800 mg of Kz Cr2 01. reference 0.5 1 2 3 4 5 6 7 8 9 10
solution
Cupric sulfate standard solution as reagents Dissolve about
32. 5 g of cupric sulfate in a quantity of hydrochloric acid
Stock
solution 0-40) to produce 500 ml. Transfer 10 ml of the reference 0.25 o. 5 l. o l. 5 2.0 2.5 3. o 4.5 6.0 7. 5 10.0
solution, accurately measured, toan iodine flask, add 50 ml solution
of water, 4 ml of acetic acid and 2 g of potassium iodide.
Titrate with sodium thiosulfate (0. 1 mol/L) VS, add 2 ml Water 9.75 9. 5 9.0 8.5 8.0 7.5 7.0 5. 5 4.0 2. 5 o
of starch IS towards the end point of the titration and
continue to titrate until the blue colour disappears. Each ml Method 2
of sodium thiosulfate ( O. 1 mol/L) VS is equivalent to Unless otherwise specified, dissolve a quantity of the
24. 97 mg of CuS0 4 • 5H 2O. Dilute the rest of cupric sulfate substance being examined specified in an individual
solution with hydrochloric acid solution ( 1-40) to contain monograph with water to produce a solution of 10 ml, filter
62. 4 mg of CuS04 •5H 20 per ml, based on the above assay if necessary. Measure the absorbance at the wavelength
result. specified ( 0401 ) , it gives not more than the required value.
Cobaltous chloride standard solution as reagents Dissolve Method 3 ( Colour difference metre method)
about 32. 5 g of cobaltous chloride in a quantity of hydro- This is a method describing and analyzing the colour of a
chloric acid solution 0 -40) to produce 500 ml. Transfer solution quantitatively by direct measurement of its tristi-
2 ml of the solution, accurately measured, to a flask, add mulus values of transmittance using colour difference metre.
200 ml of water, mix well. Add ammonia TS until the Using this method for determination recognition when it is
solution turns from pink to green colour, add 10 ml of acetic difficult to identify the colour difference between a test
acid-sodium acetate BS (pH 6. O), heat to 60ºC and then add specimen and a reference solution visually.

Colour difference between a test specimen and a reference
solution is achieved by comparing their tristimulus values
directly or by comparing them with those of water respectively.
According to the modern colour vis ion theory, there are three
kinds of colour-sensitive pyramidal cells in retina of human
eyes, which are sensitive to red, green and blue respectively.
The colour vision procedure can be divided into two steps.
Firstly, the three kinds of independent colour-sensitive
pyramidal substances in retina absorb light radiation of
various wavelengths of spectrum selectively and at the same
time each by itself produces the reaction of white in the
strong light or of black without stimulation outside. Then
these three reactions combine again in the course of nerve
stimulation transmission from the pyramidal receptors to the
vision centre, resulting in three pairs of opposite neural
reactions, i. e., red or green, yellow or blue, and white or
black reaction. At last various senses of colours are formed in
the vision centre of the pallium.
All colours in nature can be composed of a mixture of proper
ratio of red, green and blue, the three primary colours,
which are suitably chosen to excite the three kinds of receptor
cells in human eyes. So a new concept, the tristimulus
values, (X, Y, and Z), can be defined as three primary
source stimulation values to match the colour being examined
in the given three-colour system. Through extensive colour-
matching experiments with human subjects having normal
colour vision ( who are called standard observers or standard
eyes) , distribution coefficients (X O.) , y O.) and z (il))
have been measured for each visible wavelength ( 400-760
nm) which stimulates each pyramidal receptor to give the
relative data. The curve of the combined distribution
coefficients is called the spectral tristimulus values curve of
the CIE 1931 standard colourimetric observer (as Fig. D.
Q.)
:::1 1.5
ta
:>
rJl
:::1
=
·s
1.0
·;;
·¡::
E-t 0.5
o
400 500 600 700
W avelength/nm
Fig. 1 The spectral tristimulus values curve of CIE 1931
standard colourimetric observer
( the visual field being 10°)
The relationships between the distribution coefficient and the
tristimulus values are given in following equations:
f
X = K S (il) P (il) x (il) d(il)
Y= K f S(.il)P(.il) y(.il)d(.il)
Z =Kf S(.il)P(.il) z(.il)d(.il)
Where: K is the normalization coefficient;
s (il) is the relative spectral power distribution of
the illuminant;
P (il) is either the spectral reflectance or spectral
transmittance produced by the colour object;
x (il), y (il) and z
(il) are the distribution
coefficients of standard observers;
D (.il) is the wavelength interval, usually of 10 nm
or 5 nm.
0901 Colour of Solution
Once the tristimulus values of a colour have been
determined, they may be used to calculate the coordinates of
the colour in an idealized three-dimensional colour space.
Many sets of colour equations named as the colour express
system have been developed to define the space, such as the
CIE 1931-XYZ colour express system, the CIE 1964
supplemental standard chroma system, the CIE 1976
L *a* b * colour space or the CIE Lab uniform colour space,
the Hunter colour express system, etc.
In order to understand and compare conveniently, the CIE
Lab uniform colour space is often used to describe colour and
colour difference. It is constructed by a right-angle coordinate
labelled by L * a* b * respectively. Every point in the three-
dimensional-colour coordinate represents a colour and the
geometrical distance between the point and the reference
point illustrates the difference between the two colours (as
Fig. 2 and Fig. 3). The same distance means the same colour
difference. When the instrumental method is applied to
compare the colour of a test preparation with that of a
standard, the parameter needed to be compared is the
difference, in the visually uniform colour space, between the
colour of the blank and that of the test specimen or standard.
+b*
yellow
-a
green +a
red
-b*
blue
Fig. 2 The L *a* b * chromaticity diagram
white
black
Fig. 3 The L * a * b * colour space and
the colour difference tlE *
In the CIE Lab uniform colour space, the relationships of
among colour coordina tes L * , a* and b * , the tristimulus
values X, Y and Z and the colour difference tlE * are
defined by
Luminosity lndex L * = 116 X (Y/Yn) 113 -16
Chromaticity lndex a* = 500 X [(X/ X n) 113 - (Y/Yn) 113 ]
Chromaticity lndex b * = 200 X [(Y/Yn) 113 - (Z / Z n) 113 ]
Colour Difference ll,E* = j(t::,.L * ) 2 +(~ * ) 2 +(M* )2
Where X, Y and Z are the tristimulus values of the test
specimen, Xn, Yn and Zn are the tristimulus values, of the
absolutely diffuse reflection material, and X/ X n , Y /Yn and
Z / Z n should be more than O. 008856. tlE * , t::.L * , both ~ *
and M * are the colour difference, the luminosity index
 0902 Clarity of Solution
difference, and the chromaticity index differences of the test
specimen and the standard respectively.
The colour of the test specimen is brighter than that of the
standard when óL * is positive, and deeper or more saturated
when &l * and t::.b * are both positive.
Briefly, the operating principie of the colour difference metre
is to simulate the sense of sight system of human eyes and
turn the tristimulus values of spectral data into the mathe-
matical expression of the colour through its inner analogy
integrating optical system, then calcula te L * , a* , b * and
t::.E *. Instrumental methods can provide accurate and precise
measurements of colour and colour differences with the same
spectral power distribution of the standard light source as
that of the light source used in common colour observation
(e. g. , in daylight) and with the same photo-electrical
response receptor as the colour vision of the observers (e. g. ,
the visual field is 10°). The provided data are more objective
that do not drift with time, place and individuals.
l. General requirements of the instrument The instrument is
commonly a photo-electric integrating colour difference
metre. The illumination and observation condition is o/d or
d/o, the light source is D65, and the visual field is 10º. It
can detect the tristimulus values X, Y and Z and calculate
L * , a * , b * , t::.E * , the hue and the hue number of the
test solution directly.
Far the measurement of the spectral transmittance of clear
liquids, the colour of solutions changes with the thickness of
the layer measured. Unless otherwise specified, a cell of
1 cm should be used.
To ensure the reliability of the measurement, the instrument
should be calibrated at regular intervals. Colourless materials
such as water or air should be used as a standard and
assigned a transmittance of l. 000 at all wavelengths during
the calibration.
The tristimulus values of water or air are X= 94. 81, Y=
100. 00, and Z = 107. 32 under the conditions of the light
source being D65 and the visual field being 10º at room
temperature.
2. Determination Unless otherwise specified, the colour
difference metre should be calibrated with water firstly.
Then detect the solutions of the test specimen and the
standard which are prepared as directed in the individual
monograph. The colour difference t::.E * between the
solutions of the test specimen and water should not be more
than that between the solutions of the standard and water.
If there are two tints specified in the individual monograph,
and the actual tint of the solution of the test specimen is
between the two standard tints specified, and difficult to
judge, the colour difference t::.E * between the solutions of
the test specimen and water should not be more than the
mean value of the colour differences between the solution of
the two standard tin ts and water [t::.E * <, ( t::.E ,*1 + t::.E ;2) / 2].
0902 Clarity of Solution
Clarity of Solution is a method far testing of clarity or
opalescence of a solution by comparing the opalescence of
solution of the drug with that of the reference suspension
specified. Unless otherwise specified, Method 1 (visual
method) should be adopted.
The solution which is termed "clear" means that the clarity
of solution of the substance being examined is the same as
that of the solvent being used far the preparation of the
solution, or its opalescence is not more pronounced than that
of reference suspension No. O. 5. The term of "almost clear"
far a solution means that the opalescence of the solution of
the substance being examined is as pronounced as that of the
reference suspension between No. O. 5 and No. l.
Method 1 (Visual method)
Unless otherwise specified, dilute the solution being
examined specified in individual monograph with water to
definite concentration at room temperature, place the
solution and reference suspension of appropriate concen-
tration into separate matched, flat-bottomed test tubes, 15-
16 mm in diametre, of colourless, transparent, neutral hard
glass, accurately measured. Compare the contents of the test
tubes after 5 minutes in preparation of the reference
suspension against a black background by viewing under
diff used light clown the vertical axes of the tubes. Or other-
wise place the tubes under the light source of the illuminating
shade chamber of 1000 lx, view horizontally through the
tu bes.
If it' s hard to identify the clarity differences between the test
solution and the reference suspension accurately, method 2
(Turbidimetre method) should be used, and the result
should be accorded far justifying.
Preparation of opalescence reference standard stock solution
Dissolve l. 00 g of hydrazine sulfate, previously dried to
constant weight at 105ºC, in 100 ml volumetric flask with
water, warm in a water bath at 40ºC if necessary, and dilute
with water to volume, shake thoroughly and allow to stand
far 4-6 hours. Mix the solution with equal volume of 10 %
urotropine solution, shake thoroughly and allow to stand far
24 hours at 25ºC, protected from light. The suspension is
stable far 2 months in a cool place, protected from light.
Shake thoroughly befare use.
Preparation of opalescence reference standard solution Dilute
15. O ml of opalescence reference standard stock solution with
water in 1000 ml volumetric flask and make up to volume,
shake thoroughly. Transfer a quantity to a 1 cm cell,
measure the absorbance at 550 nm ( 0401), the absorbance is
O. 12-0. 15. The solution should be used within 48 hours of
preparation. Shake thoroughly befare use.
Preparation of reference suspension Prepare the fallowing
reference suspensions using the opalescence ref erence
standard solution and water. The reference suspension should
be freshly prepared. Shake thoroughly befare use.
Reference
0.5 1 2 3 4
suspension No.
Opalescence
reference
standard
2.50 5. o 10.0 30.0 50.0
solution/ ml
Water/mi 97.50 95.0 90.0 70.0 50.0
Method 2 ( Turbidimeter method)
The turbidity of the test solution can also be examined by
turbidimeter. Particles with different sizes or different
characteristics including coloured substances in solution
might cause light to be scattered. The degree of opalescence
can be determined by the measurement of the transmitted or
scattered light. Three determination modes including trans-
mitted light mode, scattered light mode and transmitted-
scattered light mode (ratio turbidimetry mode) are usually
u sed.
l. General requirements of the instrument
Using the turbidimeter with scattered light mode, the
maximum of light sources should around 860 nm; the
 0903
measuring range should cover O. 01-100 NTU. The resolution
should be O. 01 NTU within the range of 0-10 NTU, and O. 1
NTU within the range of 10-100 NTU.
2. Application range and principie
For measurements of slightly to moderate opalescent suspen-
sions of colourless solution ( the turbidity is less than
100 NTU), the turbidimeter with scattered light mode is
suitable. Since the high turbidity of the solution might cause
multiple scattering, make the scattered light intensity
decrease rapidly, and the turbidity of the solution might not
be properly detected. The turbidity range of reference
suspensions No. O. 5 to reference suspensions No. 4 is about
0-10 NTU.
Using the turbidimeter with scattered light mode, the angle
of incident light and the measured scattered light are 90º, the
relationship between incident light intensity and scattered
light intensity is:
I=K'Tio
Where: I is scattered light intensity, the unit is cd;
lo is incident light intensity, the unit is cd;
K' is the scattering coefficient;
T is turbidity value of the test solution, the unit is
NTU ( NTU is the scattering turbidity unit,
based on the turbidity of formazin turbidity
reference suspension. formazin turbidity refe-
rence suspension is the opalescence reference
standard stock solution mentioned in method 1).
The scattered light intensity I is directly correlated to
turbidity, when incident light intensity I o is constant.
Therefore, the turbidity measurement can be transformed to
the measurement of scattered light intensity.
3. System suitability test
The instrument should be regularly ( generally once a
month) evaluated for the linearity and repeatability of the
reference suspensions. The results of measurement of the
reference suspensions No. O. 5-No. 4 should show a linear
relationship between the concentrations and measured NTU
values. The correlation coefficient of the linear equation
should be no less than O. 999; Measure reference suspensions
No. O. 5-No. 4 for 5 times, the relative standard deviation of
turbidity should be no more than 5% for reference
suspensions No. O. 5 and No. 1, and 2 % for reference sus-
pensions No. 2-No. 4.
4. Determination
The instrument should be calibrated with specified suspen-
sions according to the manual. Liquid preparations can be
determined directly, drug substances or other preparations
should be freshly prepared specified in the individual
monograph. Detect the solutions of the test specimen and the
corresponding ref erence suspension separately, shaking
thoroughly befare use, and avoid bubbles, then read their
turbidity values. The turbidity value of the solution of the
test specimen should not be more than that of the corres-
ponding reference suspension.
0903 Test f or Particulate
Matter in lnjections
The test for particulate matter is applied to determine the
size and number of particulate matter in injections for
intravenous use ( include solution for injection, sterile
powders for injection, concentrated solutions for injection) ,
and sterile raw materials far intravenous injection.
This test consists of light obscuration and microscopic test.
Test for Particulate Matter in Injections
If the test result using light obscuration test <loes not comply
with the requirements or this test is not applicable for the
sample, microscopic test should be followed to reach a
conclusion on conformance to requirements.
Light obscuration test is not suitable for the preparations
having increased viscosity, or the preparation which is apt to
crystallization or the injections which will produce air bubbles
when drawn into the sensor. If the viscosity of the
preparation to be tested is sufficiently high so as to preclude
its examination directly by either test method, a quantitative
dilution with an appropriate diluent may be made to decrease
viscosity, as necessary to allow the analysis to be perfarmed.
Test environment Perform the test in an environment that
<loes not contribute any significant amount of particulate
matter. Test preparation should be carried in a laminar flow
cabinet. The glassware and other required equipment should
be clean and particle free. The water or other suitable solvent
used in the test should be filtered through a filter with a
porosity of not more than l. Oµm.
Place a 50 ml volume of water or other suitable solvents for
the test of particulate matter in a sample container,
determined as directed under test procedure. Far light
obscuration test it should meet the requirements that not
more than 10 particles of equal to or greater than 10 µm in
size and not more than 2 particles of equal to or greater than
25 µm in size are observed in the combined 10 ml of solvent.
For microscopic test it should meet the requirements that not
more than 20 particles of equal to or greater than 10 µm in
size and not more than 5 particles of equal to or greater than
25 µm in size are observed in the combined 50 ml of solvent.
Method 1 (Light obscuration particle count test)
Measurement principie When particles in solution pass
through a narrow testing area, the incident light at right
angle with direction of fluid is weakened by obscuring of
insoluble particles, therefore the output signal of sensor is
reduced. The variations of signal are related to the cross-
sectional areas of particles.
Test apparatus Usually consists of sample-feeding device,
sensor and data processor.
The range of particular size determination is from 2 to
100 µm and the concentration of particles far measurement is
0-10 000 particulates per ml.
Instrument calibration The instrument should be calibrated
every 6 months.
( 1) Sample volume Transfer a volume of water far the
test of particulate matter that is greater than the sample
volume to a container after the instrument equilibrated, and
weigh. Withdraw through the sample-feeding device a certain
volume of the water, and weigh the container again. Obtain
the sample volume by calculating the diff erence of the two
weights. Test far 3 times consecutively. Each volume
obtained should be within ± 5 % of the volume specified, and
the mean value should be within ± 3% of the volume
specified. The results should comply with the requirements
mentioned above if it is calibrated by the other suitable
methods.
( 2 ) Particle counting Prepare a suspension contammg
1000-1500 particles of standard particles having average
diametres of 10 µm or per ml and the relative standard
deviation of the size distribution of the standard particles
used is not more than 5 % . Degas by allowing to stand for
2 minutes. Turn on the stirrer and agitate the sample gently
to disperse the particles evenly ( avoid introducing air
bubbles). Withdraw and obtain the particle counts for three
consecutive samples. Record the cumulative count of 5 µm
 0903 Test for Particulate Matter in lnjections
channel disregarding the first count. The average of second
and third counts should be within ± 20 % of the limits of
number of particles.
( 3 ) Sensor resolution Prepare a suspension containing
1000-1500 particles per ml of standard particles having
average diametre of 10 µm (standard deviation of mean
diametre is not more than 1 µm) and the relative standard
deviation of the size distribution of the standard particles
used is not more than 5 %. Degas by allowing to stand for
2 minutes. Turn on the stirrer and agitate the sample gently
to homogenize it ( taking care not to introduce air bubbles) ,
determine the particles at 8 µm, 10 µm and 12 µm channel.
Calculate two count differences of the count at 8 µm with that
at 10 µm channel, and the count at 10 µm with that at 12 µm
channel, respectively. The ratio of the two count differences
of two channels and the accumulative count of 10 µm channel
is not less than 68 %, respectively. If the instrument
calibration <loes not comply with the requirements, repeat the
calibration steps after adjusting the instrument until it is well
done.
Note Self-test can be conducted if the instrument is
attached by the self-test software.
Procedure
( 1) Intravenous injection or concentrated solutions for
injection with a labelled volume of 25 ml or larger Unless
otherwise specified, not less than 4 containers of the injection
being examined should be tested by the following steps.
Wash the outside walls of the container of the injection being
examined with water. Mix the contents by inverting the
container 20 times and carefuUy open the container imme-
diately. Pour a potion of specimen to wash the bottleneck and
sample container, transfer the specimen in the sample
container, <legas by allowing to stand for 2 minutes or an
appropriate time, then place it on the sample-feeding device
( or directly place the specimen on the sample-feeding device
without stirring after <legas). Turn on the stirrer or swirl
gently by hand to disperse evenly ( avoid introducing air
bubbles) , withdraw a minimum of three aliquots for each
container, each not less than 5 ml in volume, record the data.
Discard the data from the first portian of each specimen,
calculate the average of consecutive counts.
( 2) Intravenous injection or concentrated solutions for
injection with a labelled volume of less than 25 ml Unless
otherwise specified, not less than 4 containers of the injection
being examined should be tested by the following steps.
Wash the outside walls of the container of the injection being
examined with water. Mix the contents by inverting the
container 20 times. Degas by allowing to stand for 2 minutes
or an appropriate time. Carefully open the container then
place it on the sample-feeding device. Turn on the stirrer or
swirl gently by hand to disperse evenly avoid introducing air
bubbles). Withdraw directly a suitable volume without
introducing air bubbles, record the data. Discard the data
from the first specimen, calculate the average of consecutive
specimens.
If the viscosity of concentrated solutions for injection to be
tested under procedure ( 1 ) or ( 2) above is too high to
determine directly, determination may be carried out after a
quantitative dilution with an appropriate diluent.
An appropriate method also can be used to combine the
contents of 4 or more units to obtain a volume of not less
than 25 ml into the sample container in the laminar flow
cabinet. Degas by allowing to stand for 2 minutes or an
appropriate time, then place it on the sample-feeding device.
Turn on the stirrer or swirl gently by hand to disperse evenly
(avoid introducing air bubbles), withdraw a minimum of
four aliquots, each not less than 5 ml in volume into the light
obscuration counter sensor, record the data. Discard the data
with the first portian, calculate the average of consecutive
counts as the result. Calculate the number of particles in each
container with reference to the volume being examined and
the lablled volume of each container.
( 3 ) Sterile powder for intravenous injection Unless
otherwise specified, wash the outside walls of the container
of the injection being examined with water. Carefully remove
the cover, accurately add a quantity of water for particulate
matter or appropriate solvent, replace the closure carefully
and gently agitate the container to ensure the contents
dissolved. Degas by allowing to stand for 2 minutes or an
appropriate time. Carefully open the container then place it
on the sample-feeding device, turn on the stirrer or swirl
gently by hand to disperse evenly ( awoid introducing air
bubbles). Withdraw directly a suitable volume without
introducing air bubbles, record the data Determine a
minimum of another three specimens as the same way.
Discard the data from the first specimen, calculate the
average of consecutive specimens.
An appropriate method also can be used, wash the outside
walls of 3 or more containers of the injection being examined
with water in the laminar flow cabinet. Carefully remove the
cover, accurately add a quantity of water for particulate
matter or appropriate solvent respectively, gently agitate the
container to ensure the contents dissolved. Combine the
solution of each container to obtain a total volume of not less
than 25 ml into the sample container. Degas by allowing to
stand for 2 minutes oran appropriate time, then place it on
the sample-feeding device. Turn on the stirrer or swirl gently
by hand to homogenize the sample ( avoid introducing air
bubbles), withdraw a minimum of four aliquots, each not
less than 5 ml in volume, record the data Discard the data
from the first specimen, calculate the number of particles in
each container by average the consecutive counts.
( 4) Sterile raw material for injection Prepare the test
specimens as directed under the individual monograph.
Transfer an accurately weighted quantity of powder
(equivalent to the maximal strength of its preparations) into
the sample container or an appropriate container, proceed as
directed for procedure ( 3) above, beginning at the words
"accurately add a quantity of water for particulate matter or
appropriate solvent, gently agitate the container to ensure the
contents dissolved... ", carry out the method, record the
data Determine a minimum of another three specimens as
the same way. Discard the data from the first specimen,
calculate the average of consecutive specimens.
Evaluation
(1) Intravenous injection with a labelled volume of 100 ml
or larger Unless otherwise specified, each ml of the
injection contains not more than 25 particles that are equal to
or greater than 10 µm and not more than 3 particles that are
equal to or greater than 25 µm.
(2) Intravenous injection with a labelled volume of less
than 100 ml, steril powder for intravenous injection,
concentrated solution for injection and sterile raw material
for injection Unless otherwise specified, each container of
the specimen contains not more than 6000 particles that are
equal to or greater than 10 µm and not more than 600
particles that are equal to or greater than 25 µm.
Method 2 ( Microscopic particulate count test)
Test apparatus Usually consists of microscope, micro-
porous membrane filter, filtration apparatus and Petri dish.
Laminar flow cabinet HEPA-filter having a pore of
O. 45 µm, the direction of airflow is from inside to outside.
Microscope Use a suitable binocular microscope with big

visual field, equipped with an objective micrometer with a
calibrated ocular micro meter ( 5-1 O µm per grid ) . The
coordinate axis can move up and clown, left and right, move
scope should larger than 30 mm. The microscope is equipped
with an illuminator which the incident angle of light and the
strength of light can be adjusted. It can give a magnification
of 100 X during examination.
Microporous membrane The membrane is 25 mm or
13 mm in diametre with pores of O. 45 µm in effective
diametre, it is white in colour and grids with partition of
3 mm has been printed on one side. It should be washed with
water for particular matter until free from particular matter
of an effective diametre greater than 25 µm, and not more
than 5 particles greater than 10 µm are left on the membrane.
Preparation be/ore examination Rinse the filter under the
cabinet with water for particulate matter or other suitable
solvent Rinse the membrane with water for particulate
matter or other suitable solvent, then place it on the filter
holder with a blunt forceps, invert the fixed filter and wash
the inner wall of the filter repeatedly with water for
particulate matter or other suitable solvent. Allow to drain
and place the filter on a suction flask.
Procedure
( 1) Intravenous injection or concentrated solutions for
injection with a labelled volume of 25 ml or larger Unless
otherwise specified, not less than 3 containers of the injection
being examined should be tested by the following steps.
Wash the outside walls of the container of the injection being
examined with water. Under the cabinet mix the contents by
inverting the container 20 times and carefully open the
container immediately. Transfer 25 ml of specimen tq the
pretreated filter (25 mm in diametre of membrane) gentlly
along the inner wall of the filter, allow to stand for 1 minute
and filter under gentle suction. Release the vacuum and wash
the inner walls of the filter with 25 ml of water for
particulate matter, remove the washings by suction. Release
the vaccum and remove the membrane with the forceps. Place
the membrane on a Petri slide, using a very thin layer of
glycerine, if necessary, to hold the membrane flat and in its
place. Allow the membrane to dry and place the covered slide
on the micrometer stage of the microscope. Examine the
membrane under 100 X magnification with the incident light
at a suitable angle and adjust the microscope to see the grid
clearly. Count the number of particles that are equal to or
greater than 10 µm, and those equal to or greater than 25 µm
in effective diametre. Calculate the average of three
determined data
(2) Intravenous injection or concentrated solutions for injection
with a labelled volume of less than 25 ml Unless otherwise
specified, not less than 3 containers of the injection being
examined should be tested by the following steps. Wash the
outside walls of the container of the injection being examined
with water. Mix the contents by inverting the container 20
times carefully under the cabinet. Carefully open the
container immediately. Withdraw all solutions in each
container directly with an appropriate method, Transfer the
specimen to the pretreated filter ( 13 mm in diametre of
membrane) gently along the inner wall of the filter, proceed
as directed for procedure ( 1) above. ·
(3) Sterile powder for intravenous injection or sterile raw
material for injection Unless otherwise specified, prepare
specimen as directed under procedure ( 3) or ( 4) in light
obscuration particle count test, proceed as directed for
procedure (1) above.
Evaluation
(1) Intravenous injection with a labelled volume of 100 ml
0904 Test for Visible Particles
or larger Unless otherwise specified, each ml of the
injection contains not more than 12 particles that are equal to
or greater than 10 µm and not more than 2 particles that are
equal to or greater than 25 µm.
(2) Intravenous injection with a labelled volume of less
than 100 ml, sterile powder for intravenous injection,
concentrated solution for injection and sterile raw material
for injection Unless otherwise specified, each container of
the specimen contains not more than 3000 particles that are
equal to or greater than 10 µm and not more than 300
particles that are equal or greater than 25 µm.
0904 Test for Visible Particles
Visible particles are defined as insoluble substance that
present in injections, ophthalmic liquid preparations or
sterilized drug substances and can be observed under the
required conditions by visual test. Their sizes or lengths are
usually more than 50 µm.
Injections or ophthalmic liquid preparations should be
produced according to the Good Manufacture Practise
( GMP). All of the products should be detected respectively
by suitable methods befare released, and the unacceptable
products are eliminated synchronously. It should not be used
if the visible particles were found in the product by visual
cheque under natural daylight ( non-direct sunshine) befare
used
There are two methods for the test for visible particles-lamp
test and light scattering method. Lamp test is commonly
used, light scattering method also may be used especially for
sorne products that are unsuitable for lamp test, such as the
containers are colored transparent glass or the colour of the
solution being examined is too dark to be observed (Usually
more intense than that of corresponding reference solution
No. 7). Emulsions and suspensions for injections and eye
drops are unsuitable for light scattering method.
When the test is performed in laboratory, special attention
should be paid to prevent the introduction of foreign visible
particles. If the containers of the test solution prevents direct
test by its opacity or irregular shape etc. the test solution
should be transfered to suitable containers under a B class
environment or a B class laminal-air-flow cabinet
Samples used in this test must be sampled according to the
regulation.
Method 1 ( lamp test)
This test is carried out in a darkroom.
Apparatus See the following figure:
Fig. Apparatus for lamp test
A-a light source of daylight lamp with panel far obstructing from
light: The intensity of illumination is adjustable between 1000-4000
lx;
B-a non-glare black background;
C-a non-glare white background and the bottom (supply with test
far coloured particles) ;
D-a glare white background (inboard of light baffle).
 0904 Test far Visible Particles

Requests for inspector Inspector' s visions of distant and near with the method as described in the package insert of the
distance should be 4. 9 or better ( Corrected visions should be drug products. If other assistant conditions besides shaking
5. O or better) ; inspector should not be colour blindness. needed, that should be described in the monograph.
Procedure Ophthalmic liquid preparations Unless otherwise specified,
Take the specified containers according to the following take 20 containers being examined randomly. Then conduct
requirements of preparation types far the test. Remove any the test as the above procedures. For the ophthalmic
adherent labels from the containers, clean the outer surface. preparations that prepared befare use with special solvents,
If necessary, transfer the solution being examined into the solvents should meet the requirements of the test as the
another clean and diaphanous appropriative glass container. above procedures befare dissolving the contents.
Hold the bottle-neck of the container at the edge of light Evaluation
baffle, gently swirl and invert the container in order to force The obviously visible foreign matters such as broken bits of
the visible particles in the solution being examined floating metal or glass, fibres with length of more than 2 mm or
(ensuring that air bubbles are not introduced), meanwhile block with size dimeusion of more than 2 mm, the smoky
observe any insoluble substances in the container and keep precipitate composed by particles formed by gently inverting
appropriate distance from inspectors' eyes to the container the container after a period of stationary standing, the cluster
Ot' s usually 25 cm to inspect clearly) with visual method of particles which are difficult to count, the precipitate not
respectively in front of black background and white dispersed on shaking, and the protein flocculus which are
background. Repeat several times within 20 seconds. Hold 2 difficult to count within the specified time should not be
ampoules ( vials) far each observation if the volume of found.
solution being examined is not more than 10 ml per ampoule Unless otherwise specified, the numbers of container which
(vial). For the injections with a filling volume of not less found out the tiny visible particles such as tiny insoluble
than 50 ml, they shall be examined by viewing vertically, substance, short fibres or block that length or size less than 2
horizontally and invertedly, respectively. If a large number of mm and the translucent protein flocculus or protein particles
air bubbles are introduced, the solution should allow to stand of less than 1 mm in length far biochemical medicine or
far a sufficient time befare examination until bubbles disap- biologics, comply with the requirements listed in the tables
peare. below.
At the edge of light baffle, the intensity of illumination
should be 1000-1500 lx far the colourless solution being Table 1 Evaluation for biological injections and eye drops
examined with colourless and diaphanous container. The Tiny visible particles limits
intensity of illumination should be 2000-3000 lx far the
Category 40 containers
coloured solution being examined, and the solution with
20 containers in primary test in primary and
brown glass or transparent plastic container. The intensity of
repeat test
illumination should be about 4000 lx far the suspensions or
emulsions. No more than 3 tiny visible par- The number
ticles per container with a filling of containers
lnjections Unless otherwise specified, take 20 containers
volume of 50 ml. beyond the
being examined randomly. Then conduct the test as the above lnjec-- No more than 5 tiny visible test limits as
procedures. tions particles per container with a listed shall be
Sterilized preparations for injection Unless otherwise filling volume of more than 50 ml. not more than
specified, take 5 containers being examined, dissolve the The number of containers beyond 2.
contents completely with appropriate solvent and available ------< the above test limits shall be not ,___ _ _ __
method, then conduct the test as solutions, emulsions and more than l. The number
suspensions. For the sterilized powders far injection with If 2 containers are found beyond of containers
special solvents, the solvents should meet the requirements Eye the test limits, the test shall be beyond the
of injection befare dissolving the contents. For test sample repeated. test limits as
drops
under vacuum, in order to facilitate the contents dissolving, The number of containers beyond listed shall be
the vacuum should be released with suitable method if the test limits shall be not more not more than
necessary. The cold storage test sample should be balanced to than 2. 3.
room temperatures befare being dissolved and examined.
Table 2 Evaluation for non-biological
Sterilized drug substances Unless otherwise specified, injections and eye drops
weight 5 portions according to the sampling requirement, the
weight of each portian is equal to the maximum strength of Tiny visible particles limits
the preparation, transfer them into appropriate clean and 40 containers
Category 20 containers in
diaphanous glass containers respectively, dissolve the powder in primary and
completely with appropriate solvent and available method, primary test
repeat test
then conduct the test as solutions, emulsions and suspen-
sions. If tiny visible particles The
The solvent selected far sterilized powders far injection and number
have been found in 1
sterilized drug substances should be free from visible of containers
container, repeat the
particles. The solvent of water soluble sample is usually the in which tiny
procedure.
water far the test far Particulate matter in injections ( 0903). lnjec-- For visible
. The number of contai-
If other solvents used far preparation, it should be prescribed tions mtravenous h. h . particles have
ners m w ic tmy
in the monograph. The volume of solvent should be enough been found
visible particles have
to dissolve the powders completely. shall be not
been found shall be
The available dissolving method far sterilized powders far more than l.
not more than l.
injection and sterilized drug substances should be consistent
 0904 Test for Visible Particles

continued scattering energy is related to the size of insoluble substance.

This method is intended to· measure the light scattering
Tiny visible particles limits
energies caused by insoluble substance in solutions and then
Category 40 containers compare the energies with liminal value which preseted to
20 containers in cheque out the visible particles.
in primary and
primary test The light scattering energies caused by insoluble substance
repeat test
can be analyzed base on the series of the images collected
If tiny visible particles
The number Suppose the light scattering energies is E, it is processed
have been found in 1
of containers opto- electrical signal change over, became a corresponding
or 2 containers,
in which tiny stereopicture with the height of pyramid is H and the
F repeat the procedure.
lnjec- . or non- The number of contai- visible diametre is D and a pick-up camera can catches it. The light
tions mtravenous . h' h . particles have scattering energies E is a monotonous function for D and H,
ners m w 1c tmy
been found i. e. E= f(D, H). Meanwhile, suppose the intensity of the
visible particles have
shall be not light scattering energies is q, the exposure time of take a
been found shall be
more than 2. photograph is T, there is furthermore E = g ( q , T ) .
not more than 2.
Reasoning can be obtained from the above that the
If tiny visible particles The relationship between D with q and D with T is D = w (q,
number
have been found in 2 T) , remain the relationship of monotonous function. After D
of containers
or 3 containers, repeat has been determined, the light scattering energies can be
in which tiny
the procedure. visible calculated according to the curve of the function.
Eye drops The number of contai-
particles have Apparatus The apparatus consists of the rotator far sample
ners m which tiny
been found bottle, laser photosource, image collecting equipment, data
visible particles have shall be not
processing system and terminal monitoring system. It also
been found shall be
more than 3. includes the automatic send and deliver devices for the bottle.
not more than 3.
The samples are transferred to the rotator by the send device.
Injections used both for intravenous and non-intraven- ous The rotator must rotate the bottle rounding the perpendicular
must comply with the requirement for Injections for line in high speed for a while and then stopped sharply. The
intravenous. For suspensions and emulsions, any obviously homogeneous laser beam from the laser photosource should
visible foreign matters should not be found in 20 containers. evenly illuminates through the solutions being examined. As
soon as the turbulent flow in the solutions disappears, but
Sterilized preparations for injection If tiny visible particles
the solution is still rotating due to inertia, the image colle-
have been found in 5 containers, the numbers of tiny visible
cting equipment takes images continuously at specific angle
particles within each container should comply with the
for light scattering energies that resulting from insoluble
requirements listed in the table 3. If one container doesn' t
substances in the rotating solution. The numbers of image
meet the requirement, repeat the procedure 'Nith another 10
should be not less than 75. Data processing system then
containers, all of them should comply with the requirements
processes the series of the images. It will assess automa-
listed in the table 3.
tically the existing insoluble substance more than a given size
Table 3 Evaluation for sterilized or not according to the predefined criteria, or it will display
preparations for injection the images on the monitor in arder to making decision
Tiny visible particles manually. The results will be recorded at the same time.
Category Then the deliver device will be instructed to diff erentiate the
limits per container
qualified and unqualified samples automatically.
With a reconstitution
volume of not more ~3
Standardization for Instrument The instrument should have
than 50 ml the automatic calibrating function. The calibration should be
Biologics carried out using the reference particles befare the test.
With a reconstitution Unless otherwise specified, calibrate the instrument using
volume of more ~5 the reference particles with diameters 40 µm and 60 µm
than 50 ml respectively. The curve equation resulting from the calibra-
Freeze-dried preparations ~3 tion then is used to calculate the corresponding pinel
Non- parameter of the reference particle with 50 µm diametre far
biologics Non-freeze-dried test criterion.
~5
preparations After the pinel parameter is set according to the 50 µm
reference particle, detect the solutions containing the 60 µm
Sterilize drug substances Any obviously visible fareign reference particle three times, each result should be positive.
matters should not be found in 5 portions. If tiny visible Procedure
particles have been found, the numbers of tiny visible
particles within each portian should comply with the Solutions for injection Unless otherwise specified, take 20
requirements as sterilized preparations for injection. If one containers being examined randomly. Remove any adherent
portian doesn' t meet the requirement, repeat the procedure labels from the containers, clean the outer surface, put the
with another 10 portions, all of them should comply with the containers onto the send device, select the corresponding test
requirements. parameters according to the instrument manual, start the
instrument, test 3 times and record the results. If one result
Method 2 (Light scattering method) fails the test, change the method to the lamp test for further
Principie When a beam of monochromatic colour laser pass confirmation except the containers are dark transparent
through the solution being examined, the insoluble vessel, or the colour of the solution being examined is too
substances in the solution cause the light scattering. The dark to observe by visual test.
 0921 Determination of Disintegration
Sterilized powders for injection Unless otherwise speci-
fied, take 5 containers being examined, dissolve the contents
totally with appropriate solvent and available method, then
examine them in same way as Solutions for injection.
Sterilize drug substances Unless otherwise specified,
weight 5 portions according to the sampling requirement, the
weight of each portian is equal to the maximum strength of
the preparation, transfer them into appropriate clean and
diaphanous glass containers respectively, dissolve the powder
totally with appropriate solvent and available method, then
examine them in same way as Solutions for injection.
In general conditions, the left and right lines of sampling
window should overlap with the edges of the container, the
upper line should be a secant line with the curve surface of
the solution in the container. Rotating time parameter should
be set longer than the time that the whirlpool in the solution
reaches to the bottom of the bottle in arder to force the salid
substances floating and air bubble broken. Holding time
parameter should be set as short as possible but not shorter
than the time to the whirlpool calming clown in arder to avoid
the interference of air bubbles meanwhile retain the salid
substances in moving state when image collecting equipment
start to work. The tightness parameter for holding bottle
prefer to same with the diameter of the bottom of the bottle
(mm), and it is adjustable based on the qualities of the
bottle. If the bottle is not smooth and straight enough to
avoid the vibration during rotating, air bubbles would be
introduced. In this case the tightness parameter should be
increased to decrease the vibration, meanwhile, the rotating
time parameter should be increased so ensuring that the
whirlpool in the solution reaches to the bottom of the bottle.
Evaluation
See lamp test.
0921 Determination of Disintegration
Disintegration is provided to determine whether the oral salid
preparations disintegrate or disperse into fragments or
particles within a prescribed time when placed in a liquid
medium under the prescribed experimental conditions.
Disintegration is considered to be achieved when no residue,
except fragments of undissolved tablet coating or of capsule
shell, remains on the screen of the test apparatus.
Disintegration can be also considered to be achieved when the
residue remained consists of a soft mass having no palpably,
unmoistened core, or floats because of light weight.
Unless otherwise specified, disintegration is not required for
the preparations which comply with the requirements for
Dissolution, Drug release, or Dispersible uniformity.
l. Tablets
Apparatus The apparatus mainly consists of a basket-rack
assembly with disks and a metallic device capable of raising
and lowering the basket in the liquid medium at a constant
rate between 30 and 32 cycles per minute through a distance
of 55 mm± 2 mm.
Basket-rack assembly The basket-rack assembly consists of
six glass tubes, each 77. 5 mm±2. 5 mm long, 21. 5 mm in
interna! diametre wi th a wall of 2 mm in thickness. The tubes
are held in a vertical position by two transparent plastic
plates, each about 90 mm in diametre and 6 mm in
thickness, with six hales, each about 26 mm in diametre. On
the top of the upper plastic plate is a stainless-steel plate
about 90 mm in diametre and 1 mm in thickness with six
holes each about 22 mm in diametre. Attached to the under
surface of the lower plate is a disk of stainless-steel wire
gauze about 90 mm in diametre, with apertures of 2. O mm
in interna! diametre. A stainless-steel central shaf t about
80 mm long is fixed with the upper plastic plate and
stainless-steel plate. The stainless-steel plate, the two
plastic plates and the wire gauze are fixed together by three
screws (as Fig. 1).
(mm) (mm)
Fig.1
Disks The disk is made of a smooth transparent plastic
material having a specific gravity of l. 18-1. 20 with 20. 7 ±
O. 15 mm in diametre, and 9. 5 mm±O. 15 mm in thickness.
It has five hales with 2 mm in diametre, one of the holes is
located in the centre and the others at an equal distance of
6 mm from the central hole. Equally spaced on the sides of
the disk are four V-shaped notches. The dimensions oí each
notch are such that the upper openings are 9. 5 mm wide and
2. 55 mm deep and the lower openings are l. 6 mm wide and
l. 6 mm deep (as Fig. 2).
~
2~55 ~
1.6
20.7
Fig. 2
Procedure The basket is suspended in a water bath pre-
ferably using a 1000 ml of beaker, maintained at 37°C±lºC,
the volume of the fluid in the vessel is adjusted appropriately
so that at the highest point of the upward stroke the wire
mesh remains at least 15 mm below the surface of the fluid
and descends to a distance not less than 25 mm from the
bottom of the vessel on the downward stroke. The top of the
upper plate should not be immersed in water.
Unless otherwise specified, place 1 tablet in each of the six
tubes of the basket and operate the apparatus. Ali of the
tablets should disintegrate completely within 15 minutes. If 1
tablet fails to disintegrate completely, repeat the test on 6
additional tablets. All of the tablets should comply with the
requirement.
Tabtets made from traditional Chinese medicine extract and
Tablets made Jrom traditional Chinese medicine powder
Carry out the test as described above. Add a disk to each
tube. All of the extract tablets should disintegrate within
60 minutes and the powder tablets should disintegrate within

30 minutes. If the tablets adhere to the disks, repeat the test
on additional six tablets without disks. If any one of the
tablets has not disintegrated, repeat the test on additional six
tablets. All the tablets should comply with the test.
Film-coated tablets Carry out the test as described above.
Replacing the water in the beaker with hydrochloric acid (9-
1000). All of the Chemical medicine tablets should disinte-
grate within 30 minutes. For Chinese traditional medicine,
add a disk to each tube, all of the tablets should disintegrate
within 60 minutes. If the tablets adhere to disks, repeat the
test on additional six tablets without disks. If any one of the
tablets has not disintegrated, repeat the test on additional six
tablets, all the tablets should comply with the test.
Sugar-coated tablets Carry out the test as described above.
All of the chemical medicine tablets should disintegrate
within 60 minutes. For Chinese traditional medicine, add a
disk to each tube, all of the tablets should disintegrate within
60 minutes. If the tablets adhere to disks, repeat the test on
additional six tablets without disks. If any one of the tablets
has not disintegrated, repeat the test on additional six
tablets; all the tablets should comply with the test.
Enteric-coated tablets Entric-coated tablets are tested in the
same way as described above using hydrochloric acid solution
( 9 - 1000) as the immersion fluid and the tablets should
show no evidence of cracking, disintegrating or softening in 2
hours. Remove the basket, wash the tablets with a small
quantity of water, add a disk to each tube. Repeat the
operation using phosphate BS ( pH 6. 8) as the immersion
fluid, all tablets should disintegrate completely within
1 hour. If 1 tablet fails to comply with the test, repeat the
operation with additional 6 tablets. All the tablets should
comply with the test.
Buccal tablets Unless otherwise specified, buccal tablets
are tested in the same way as described above. All of the
tablets should not disintegrate or dissolve within 10 minutes.
If any one of the tablets fails to comply with the test, repeat
the test on additional six tablets. All the tablets should
comply with the test.
Sublingual tablets Unless otherwise specified, sublingual
tablets are tested in the same way as described above. All of
the tablets should disintegrate and dissolve within 5 minutes.
If any one of the tablets has not disintegrated, repeat the test
on additional six tablets. All the tablets should comply with
the test.
Soluble tablets Unless otherwise specified, soluble tablets
are tested in the same way as described above maintained the
temperature at 15-25ºC. All of the tablets should disintegrate
and dissolve within 3 minutes If any one of the tablets has not
disintegrated, repeat the test on additional six tablets. All the
tablets should comply with the test.
Colon-located enteric-coated tablets Unless otherwise
specified, colon-located enteric-coated tablets are tested in
the same apparatus as described under the individual
monograph. All of the tablets should not release and
disintegrate in the sulfuric acid solution (9-1000) and the
phosphoric acid buffer solution with pH value less than 6. 8,
but release and disintegrate in the phosphoric acid buffer
solution with pH value between 7. 5 and 8. O within 1 hour,
and the tablet core also disintegrates. If any one of the tablets
has not disintegrated, repeat the test on additional six
tablets. All the tablets should comply with the test.
Effervescent tablets Effervescent tablets are tested in the
following way. Place one tablet in a 250 ml beaker containing
200 ml of water maintained at 15-25ºC, numerous gas
0921 Determination of Disintegration
bubbles are evolved. When the evolution of gas around the
tablet or its fragments has ceased the tablets disintegrate,
dissolve or disperse in the water so that no agglomerates of
particles remain. Unless otherwise specified, the preparation
being examined complies with the test if 6 tablets used in the
test disintegrate in the manner prescribed within 5 minutes.
If any one of the tablets has not disintegrate, repeat the test
on additional six tablets. All the tablets should comply with
the test.
Orally Disintegrating Tablets
Unless otherwise specified, orally disintegrating tablets are
tested in the same apparatus as described under the individual
monograph.
Apparatus The apparatus mainly consists of a stainless-steel
tube with a wire gauze anda metallic device capable of raising
and lowering the stainless tube in the liquid medium at a
constant rate of 30 cycles per minute through a distance of 10
mm±lmm.
Stainless-steel tube The stainless-steel tube is 30 mm long
and 13. O mm in diametre. Attached to the bottom of the tube
is the stainless-steel wire gauze with apertures of 710 µm in
internal diametre. (as Fig. 3)
2 13. o
t"" - -------
~ti ti 1 11
Fig. 3
Procedure The stainless-steel tube is suspended in a water
bath using a 1000 ml of beaker, maintained at 37°C ± 1 ºC,
the volume of water in the beaker should be about 900 ml and
adjusted appropriately so that at the lowest point of the
downward stroke the wire gauze remains 15 mm below the
surface of water. Unless otherwise specified, place 1 tablet in
the stainless-steel tube and operate the apparatus. The tablet
should disintegrate completely and through the wire gauze
thoroughly within 60 seconds. It is acceptable if traces of
powders are floating on the water or sticking to the wire
gauze. A total of 6 tablets should be tested. If 1 tablet fails to
disintegrate completely, repeat the test on 6 additional
tablets. All of the tablets should comply with the requirement.
2. Capsules
Unless otherwise specified, hard capsules or soft capsules
tested comply with the test as described above. If the
capsules float on the surface of the water, a disk may be
added. For Chinese traditional medicine, a disk should also
be added. The hard capsules should disintegrate within
30 minutes and the soft capsules disintegrate within 1 hour. If
one of the capsules has not disintegrated, repeat the test on
further six capsules, all the capsules should comply with the
 0922 Disintegration Test far Suppositories and Vaginal Tablets

test. Far soft capsules, replacing the water in the beaker with three equally-spaced hooks.
simulated gastric fluid.
,-....
Enteric capsules Unless otherwise specified, 6 enteric cap- -,---1
'7- I J
sules to be examed are tested in the same way as described ~
','
above using hydrochloric acid solution ( 9 - 1000) as the I ,_
immersion fluid and the capsule shells show no evidence of o
.,...
cracking, disintegrating in 2 hours. Remove the basket, wash ~
the capsules with a small quantity of water, add a disk to
_,__
each tube. Repeat the operation using simulated intestinal
fluid as the immersion fluid, all capsules should disintegrate ~

completely within 1 hour. If 1 capsule fails to comply with

the test, repeat the operation with another 6 capsules. All the
capsules should comply with the test.
Unless otherwise specified, colon-enteric capsules are tested
in the same way as described above using hydrochloric acid
solution (9-1000) as the immersion fluid and the capsule
shells show no evidence of cracking, disintegration in
2 hours. Remove the basket, wash the capsules with a small
quantity of water, repeat the operation using phosphate BS
(pH 6. 8) as immersion fluid and the capsule shells show no
evidence of cracking. Remove the basket, wash the capsules
with a small quantity of water, add a disk to each tube.
Repeat the operation using phosphate BS ( pH 7. 8) as
immersion fluid, all capsules should disintegrate completely
within 1 hours. If 1 capsule fails to comply with the test,
repeat the operation with another 6 capsules. All the capsules
should comply with the test.
3. Dripping pills
Carry out the test with the same apparatus as described as
tablets using the stainless steel wire gauze with apertures of
O. 42 mm in internal diametre. Carry out the test as described
above using 6 dripping pills, unless otherwise specified. All
of the dripping pills disperse completely within 30 minutes,
and the coated-dripping pills should disperse completely
within 60 minutes. If any one of the dripping pills has not
dispersed completely, repeat the test on further six dripping
pills, all the dripping pills should comply with the test.
Dripping pills made of gelatine matrix may be tested in
simulated gastric fluid.
Annotation Simulated gastric fluid Add about 800 ml of
water and 10 g of pepsin to 16. 4 ml of dilute hydrochloric
acid, mix well, dilute with water to produce 1000 ml.
Simulated intestinal fluid See phosphate-pancreatin BS
(pH 6. 8) (8004 >.
50
52
(mm}
(mm}
Fig. l Fig. 2
Procedure Take 3 suppositories being examined and allow to
stand at room temperature far 1 hour. Place them on the
lower clises of 3 metal devices respectively, then insert the
devices into separate sleeves and fix them by means of the
hooks. Unless otherwise specified, place each piece of the
above apparatus in 3 vessels separately, each containing not
less than 4 litres of water at 37. OºC ±O. 5ºC and fitted with a
rotating device and a means of holding the apparatus
vertically 90 mm below the surface of water. After each
10 minutes invert each apparatus without allowing it to
emerge from the liquid.
Interpretation Unless otherwise specified, all of the fat-
based suppositories should disintegrate, soften or have no
salid core offering resistance to pressure within 30 minutes;
all of the water-soluble based suppositories should completely
dissolve within 60 minutes. If any one of the suppositories
fails to comply with the requirements, repeat the test on 3
additional suppositories and all of them should comply with
the requirements.
2. Vaginal tablets
Apparatus The apparatus is the same as that used far
suppositories except that set the hook-end upside clown in the
vessel (as Fig. 3).

0922 Disintegration Test f or

Suppositories and Vaginal Tablets I
1 ~

The test far disintegration of suppositories and vaginal tab- Fig. 3

lets determines whether a salid dosage farm such as supposi-
tories and vaginal tablets can disintegrate, soften, dissolve or
disperse under the specified experimental conditions.
l. Suppositories
Apparatus The apparatus is composed of a transparent
sleeve and a metal device (as Fig. 1).
Transparent sleeve The transparent sleeve is made of glass
or suitable plastic, with a height of 60 mm, an internal
diametre of 52 mm andan appropriate wall thickness.
Metal device The metal device consists of two stainless steel
clises and three metal hooks. Each of the clise has a diametre
of 50 mm and contains 39 hales each 4 mm in diameter (as
Fig. 2). The clises are separated by a distance of 30 mm. The
metal device is attached to the outer sleeve by means of the
1-Vaginal tablet; 2-Glass plate;
3-Water surface
Procedure Adjust the water surface level until the hales of
the upper metal clise are just covered by a unifarm layer of
water. Put 3 vaginal tablets being examined on separate
upper clises and cover the apparatus with a glass plate to
maintain appropriate humidity.
Interpretation Unless otherwise specified, all of the vaginal
tablets should dissolve or disintegrate into fragments and
pass through the perfarated plates or only remain soft masses
which have no salid core within 30 minutes. If any one of the
tablets fails to comply with the requirements, repeat the test
on 3 additional tablets and all of them should comply with the
requirements.
 0931
0923 Test f or Tablet Friability
This chapter provides guidelines for the friability deter-
mination of compressed, uncoated tablets. The test proce-
dure presented in this chapter is generally applicable to most
compressed tablets and supplements other physical strength
measurements, such as tablet crushing strength.
Apparatus Use a drum, with an internal diametre of about
286 mm and a depth of 39 mm, made of a transparent
synthetic polymer with polished internal surfaces, and not
subject to static build-up (as Fig. ). One side of the drum is
removable. The tablets are tumbled at each turn of the drum
by a curved projection with an inside radius of 80 mm± 1 mm
that extends from the middle of the drum to the outer wall.
The drum is attached to the horizontal axis of a device that
rotates at 25± 1 rpm. Thus, at each turn the tablets roll or
slide and fall onto the drum wall or onto each other.
inner-diameter
286±0.2
thickness 6 ± 0.1 diamv eter
Fig. Tablet friability apparatus
Procedure For tablets weighing up to O. 65 g each, take a
6. 5 g of sample; for tablets weighing over O. 65 g each, a 10-
tablet sample is sufficient. Befare the test, remove any loose
dust with the aid of air pressure or a soft brush. Accurately
weigh the tablet sample, and place the tablets in the drum.
Rotate the drum 100 times, and remove the tablets. Remove
any loose dust from the tablets as befare and weigh.
Generally, the test is run once. If the results are doubtful or
if the weight loss is greater than 1 % , the test should be
repeated twice and determine the mean of the three tests. A
maximum weight loss of not more than 1 % of the weight of
the tablets being tested is considered acceptable and any
tablets broken, chipped and smashed are not picked up.
If tablet size or shape causes irregular tumbling, adjust the
drum base so that the base forms a 10º angle with the
horizontal and the tablets no longer bind together when lying
next to each other, which prevents them from falling freely.
If tablet size or shape causes severe irregular tumbling in the
drum, or tablets are produced by special procedure, this
method is not suitable and may not be used to test the
tablets.
For hygroscopic tablets, conduct the procedure cautiously to
avoid moisture ( Generally under relative humidity less than
40%).
0931 Dissolution and Drug
Release Test
Dissolution test is used to determine the dissolution rate and
Dissolution and Drug Release Test
degree of the active pharmaceutical ingredients ( APis) from
conventional-release dosage forms such as tablets, capsules
or granules etc. under the specified conditions. Referred to
the dissolution of sustained-release dosage forros, controlled-
release dosage forms, enteric-coated dosage forms and trans-
dermal patches etc. , the test is also named drug release test.
Apparatus
Method 1 (Basket)
( 1) The basket assembly consists of a basket and a shaft
made of stainless steel or other inert material. The
specifications are shown in Fig. l. The basket (A) is made
of stainless steel woven cloth with a nominal aperture of O. 40
mm ± O. 04 mm; the stainless steel wire is O. 28 mm ±
O. 03 mm in diametre. The basket is cylindrical, 20. 2 mm±
l. O mm in internal diametre, with a flanged rim at each end.
The shaft (B) is 9. 75 mm± O. 35 mm in diametre and is
connected to the lid of the basket. The lid has a vent hole,
2. O mm±O. 5 mm in diametre and 3 retention springs with 3
tangs on 120º centres. The upper part of the lid has a
diametre equal to the outside diameter of the basket and the
lower part has a diameter equal to the inside diameter of the
basket.
( 2) The vessel is cylindrical, made of hard glass or other
inert materials, with a hemispherical bottom and a nominal
capacity of 1000 ml. The internal diametre is 102 mm±4 mm
( the difference between maximum and mínimum in interna!
diametre is not more than O. 5mm) and the height is 185 mm
±25 mm. A fitted cover may be used to retard evaporation,
the cover has a number of holes, the central one is used for
the shaft to pass through, and the others are used for the
insertion of thermometer and withdrawal of specimens. The
vessel is placed in a constant temperature water bath or other
heating device.
( 3) The shaft is connected with a motor with a speed
regulator capable of maintaining the speed of rotation of the
basket within±4% of that specified in the monograph. When
the motor is in operation, no part of the assembly, including
the environment in which the assembly is placed, contributes
any significant motion or vibration beyond that due to the
smoothly rotating element. The shaft is positioned so that its
axis is not more than 2 mm at any point from the vertical axis
of the vessel and maximum allowable runout at "A" is ±
l. O mm when the part is rotated on centre line axis with
basket mounted.
( 4) The apparatus is equipped with at least six sets of
assembly described above.
9.75±0.35
(mm) (mm)
Fig. 1 Basket Fig. 2 Paddle stirring
stirring element element
 0931 Dissolution and Drug Release Test

Method 2 ( Paddle)
Use the same assembly described in method 1, except
Vesscl - - - + - - - Paddle
replacing the basket with the paddle. The paddle blade and
the lower part of shaft may be coated with a suitable inert 25(distance between the
material such as Teflon. The specifications are shown in bottom of the paddle blade and disk)
Fig. 2. The symmetry of shaft ( i. e. the difference between
the length from the left of shaft to the left edge of blade and
Disk assembly
the length from the right of shaft to the right edge of blade)
is not more than O. 5 mm, the verticality between shaft and
(mm)
blade is 90°±0. 2º. The shaft is positioned so that its axis is
not more than 2 mm at any point from the vertical axis of the Fig. 5 Paddle over disk ( Method 1)
vessel. A and B dimensions are not to vary more than
O. 5 mm when part is rotated on centre line axis.
2(Hole diameter) 0.9(Hole diameter mash 2x2)

ilH
Method 3 ( Small Vessel) ( Fig. 3)
(1) The paddle conforms to the specifications shown in
Fig. 3. The upper part of the shaft is 9. 75 mm±O. 35 mm in
diametre and the lower part is 6. O mm ± O. 2 mm in
~ 111111111111 JOfiickn=l

diametre. The symmetry of shaft (i. e. the difference of the 80

86
length from the left of shaft to the left edge of blade and the 92
length from the right of shaft to the right edge of blade) is
Aupperdisk
not more than O. 5 mm, the verticality between shaft and
blade is 90°±0. 2º. The shaft is positioned so that its axis is
l.5(11ole diameter) ~ire diameter)
not more than 2 mm at any point from the vertical axis of the
vessel. A and B dimensions are not to vary more than
O. 5 mm when part is rotated on center line axis.
( 2) The vessel is cylindrical, made of hard glass or other
~rjglllllllllllJ~t 3nni0kn=>
Mash 2x2 - -....8....
inert materials, with a hemispherical bottom and a nominal 0- - -
1• 86 •I
capacity of 250 ml. The specifications are shown in Fig. 4. I• 92 •I
The internal diametre is 62 mm ± 3 mm ( the difference
between the maximum and minimum in interna! diametre is B Downdisk
not more than O. 5 mm) and the height is 126 mm± 6 mm. (mm)
The others used are the same assembly described in method 1 Fig. 6 Disk assembly (Method 1)
(2).
(3) The motor is connected with the shaft. The speed of the
paddle is maintained within ± 4 % of that specified in the
monograph. The others used are the same assembly described
Vessel
in method 2.
+----+- Paddle
25 ± 2 Distance between the
bottom ofthe paddle blade
'---'-+-.- and disk

AJ --li_;-IA
6 Patch Disk assembly

~ fük """"bly
41.2
Fig. 7 Paddle over disk ( Method 2)
62±3

Method 5 ( Rotating cylinder)

Use the same vessel described in method 2, except replacing
the paddle with a stainless steel rotating cylinder. The
stirring element of rod and cylinder are both made of stain-
(mm) (mm) less steel. The specifications are shown in Fig. 8.
Fig. 3 Paddle assembly Fig. 4 V essel Procedure
of small vessel assembly of small vessel
Method 1 and Method 2
Method 4 ( Paddle over disk) Conventional-release dosage fonns Before the test, adjust the
apparatus so that the distance between the inside bottom of
Method ( 1) Use the paddle and vessel assembly described in
the vessel and the bottom of the basket or paddle blade is
method 2, with the addition of a stainless steel disk assembly
25 mm ± 2 mm. Place the stated volume of dissolution
designed for holding the transdermal system at the bottom of medium ( ± 1 % ) in each of vessels, equilibrate the
the vessel ( Fig. 5 ) . The disk assembly conforms to the temperature of dissolution medium to 37°C ±O. 5ºC. When
specifications shown in Fig. 6. method 1 is used, place 1 tablet (capsule or packet) in each
Method ( 2) Use the same assembly described in method of 6 dry baskets, lower the baskets into the vessels. When
(1), except replacing the disk assembly in method (1) with method 2 is used, place 1 tablet (capsule or packet) in each
that in Fig. 7. of 6 vessels ( when a sinker device is specified in the
 0931 Dissolution and Drug Release Test

Sustained-rel8R cbage forms or rontrolled-rel8R cbage forms

Proceed as described far conventional-release dosage farms
with generally 3 sampling times. Within the time interval
specified, or at each of the times stated, withdraw a
specimen at the specified site and replace the aliquots
dia, equally spaced on withdrawn far analysis with equal volumes of fresh
25.40±0.2dia.b.c at 63.5° dissolution medium at 37ºC ±O. 5ºC immediately, filter the
± 0.5 angle to surface. :--i2.22 - lnterference fit
63.4º _, : i-_ _ _ _ _ _ _ _.....- specimen. Complete sampling and filtering the specimen
~--~ within 30 seconds. Proceed as described in the monograph
9.4--0.l¡Ii~.
. . using the successive filtrate and calculate the amount of
1
: : 1
-.: . f
1112 1 406.40
active ingredient released from each tablet ( or capsule).
. . ·. .. . ~ 50.79 1 Enteri~coated dosage fonns Proceed as method ( 1 ) or
Maximun radius3.00:±0.127 ; 36j67 J
method (2).
1-44.5*0.2 --;1 42.7-43.0
Tolerances:±0.127-1 ~Jl¡ · ~ Method ( 1) The amount released in acid Unless otherwise
specified, place 750 mi of O. 1 mol/L hydrochloric acid
42.69-42.70- ~ solution in each of 6 vessels, within a tolerance of ± 1 %.
Degrease before ! 37 .70 .s Allow the dissolution medium to equilibrate to a temperature
final assembly : §
of 37°C ±O. 5ºC. Place 1 tablet ( or capsule) in each of 6
of rod and cylinder ¡ ~1 ·~ baskets or vessels ( when a sinker device is specified in the
: 93.83 t monograph, the dosage unit is allowed to sink to the bottom
Material: ~ ,~ 1 ,., 1 g. of the vessel befare rotation of the blade is started. When a
stainless steel- _ ~~ :
sinker device is not specified in the monograph, a small,
1.78 wall t-- 445.2±0.2-t ~
loase piece of nonreactive material, such as not more than a
few turns of wire helix, may be attached to the capsule that
(mm)
would otherwise float). Exclude air bubbles from the surface
Fig. 8 Cylinder stirring element of the dosage unit, immediately operate the apparatus at the
specified rate in the monograph. Withdraw a specimen at the
monograph, the dosage unit is allowed to sink to the bottom specified site in 2 hours, and filter immediately. Complete
of the vessel befare rotation of the blade is started. When a sampling and filtering the specimen within 30 seconds.
sinker device is not specified in the monograph, a small, Proceed as described in the monograph using the successive
loase piece of nonreactive material, such as not more than a filtrate and calculate the amount of active ingredient released
few turns of wire helix, may be attached to the capsule that from each tablet ( or capsule) in acid.
would otherwise float. An alternative sinker is shown in The rest of the procedure is the same as that far
Fig. 9). Exclude air bubbles from the surface of the dosage conventional-release dosage farms under method 1 and
unit, immediately operate the apparatus at the specified rate method 2.
in the monograph and start the counting of time. Within the The amount released in buffer solution To the acid solution
time interval specified, or at each of the times stated ( within above add 250 ml of O. 2 mol/L sodium phosphate solution
a tolerance of ± 2%), withdraw a specimen from a zone (adjust to pH 6. 8 with 2 mol/L hydrochloric acid solution or
midway between the surface of the dissolution medium and 2 mol/L sodium hydroxide solution, if necessary) at 37°C ±
the top of the rotating basket or blade, 10 mm from the O. 5ºC. Continue to operate the apparatus far 45 minutes, or
vessel wall ( Where multiple sampling times are specified, far the specified time given in the monograph, withdraw an
the sum of the volume of the samples withdrawn should be aliquot of the fluid at the specified site in a specified time,
within 1 % of the volume of the dissolution medium. If the and filter immediately. Complete sampling and filtering the
sum is greater than 1 %, replace the aliquots withdrawn far specimen within 30 seconds. Proceed as described in the
analysis with equal volumes of fresh dissolution medium at monograph using the successive filtrate and calculate the
37°C immediately, or correct far the volume change in the amount of active ingredient released from each tablet ( or
calculation ). Filter immediately through a suitable capsule) in buffer solution.
membrane. Complete sampling and filtering the specimen
within 30 seconds. Proceed as described in the monograph Method ( 2) The amount released in acid U nless otherwise
using the clear successive filtrate and calculate the amount of specified, place 900 mi of O. 1 mol/L hydrochloric acid
active ingredient dissolved from each tablet ( capsule or solution in each of 6 vessels, proceed as directed under
packet). method ( 1) to measure the amount released in acid.

3r¡~'\.. 3.0-3.5 ++ The amount released in buffer solution Drain the acid from
each of 6 vessels, and immediately add 900 mi of pH 6. 8

3.5~.o+@ • D+3.5~.o
B B
phosphate buffer at 37°C ± O. 5ºC to each of 6 vessels,
prepared by mixing O. 1 mol/L hydrochloric acid solution
with O. 2 mol/L sodium phosphate solution ( 3 : 1 ) , and
adjusting with 2 mol/L hydrochloric acid solution or 2 mol/L
sodium hydroxide solution to pH 6. 8, if necessary. This may
/ 1---2s-26 --1 l~J also be accomplished by transferring each of 6 tablets ( or
A
(mm)
~ capsules) to 6 other vessels containing 900 ml of phosphate
A. Acid-resistan wire clasp buffer ( pH 6. 8 ) respectively, proceed as directed under
B. Acid-resistant wire suppport method (1) to measure the amount released in buffer solution
Fig. 9 Alternative sinker Method 3
Conventional-release dosage fonns Befare test, adjust the
 0931 Dissolution and Drug Release Test
apparatus so that the distance between the lower edge of the
blade and the inside bottom of the vessel is 15 mm± 2 mm.
Place 150-250 ml dissolution medium in each of 6 vessels,
within a tolerance of ± 1 % ( when a sinker device is specified
in the monograph, the dosage unit is allowed to sink to the
bottom of the vessel before rotation of the blade is started
When a sinker device is not specified in the monograph, a
small, loose piece of nonreactive material, such as not more
than a few turns of wire helix, may be attached to the
capsule that would otherwise float ). The rest of the
procedure is the same as described in method 2. Withdraw a
specimen from a zone midway between the surface of the
dissolution medium and the top of the blade, 6 mm from the
vessel wall.
Sustained-release dosage fonns or controlled-release dosage
fonns
Proceed as described for conventional-release dosage forms
under method 3, other requirements consist with those for
sustained-release dosage forms or controlled-release dosage
forms under method 1 and method 2.
Method 4
Transdermal patches Place the stated volume of the
dissolution medium in each of vessels, within a tolerance of
±1%. Equilibrate the dissolution medium to 32ºC ±0. 5ºC.
Apply the patch between two disks (method 1) or above the
disk (method 2), ensuring that the release surface of the
patch is as flat as possible and is facing up. Place the disk
assembly flat at the bottom of the vessel with the release
surface parallel to the edge of the paddle blade. A distance of
25 mm±2 mm between the bottom edge of the paddle blade
and the surface of the disk assembly is maintained during the
test. lmmediately rotate the paddle at the specified rate in the
monograph. Within the time interval specified, or at each of
the time stated, withdraw a specimen and replace with equal
volumes of fresh dissolution medium at 32ºC ± O. 5ºC
immediately.
The rest of the procedure is the same as that for sustained-
release dosage forms or controlled-release dosage forms
under method 1 and method 2.
Method 5
Transdermal patches Place the stated volume of the
dissolution medium in each of vessels, within a tolerance
of ±1%. Equilibrate the dissolution medium to 32ºC ±
O. 5ºC. Unless otherwise specified, prepare the patch as
follows. Remove the protective liner from the patch, and
place the adhesive side on a piece of cuprophanCD that is at
least 1 cm larger on all sides than the patch. Place the patch
on a clean surface with the cuprophan in contact with this
surface, and apply a suitable adhesive to the exposed
cuprophan borders. If necessary, apply adhesive to the back
of the patch. Dry for 1 minute, carefully apply the adhesive
side of the patch to the external wall of the cylinder, so that
the long axis of the patch fits around the circumference of the
cylinder. Press the cuprophan covering to remove trapped air
bubbles. Place the cylinder in the apparatus and make sure
that the distance between the inside bottom of the vessel and
the cylinder is maintained at 25 mm± 2 mm during the test.
Immediately rotate the cylinder at the specified rate in the
monograph. Within the time interval specified, or at each of
the time stated, withdraw a specimen and replace with equal
volumes of fresh dissolution medium at 32ºC ±O. 5ºC immE,-
diately. Repeat the test with additional transdermal patches.
The rest of the procedure is the same as that for sustained-
release dosage forms or controlled-release dosage forms
under method 1 and method 2.
When in-situ optical fibre real time measurement is used in
the five methods above, interference of excipients could be
neglected, or eliminated by setting reference wavelength.
lnsitu optical fibre real time measurement is mainly suitable
for dissolution curve determination and drug release of
sustained-release dosage forms.
Interpretation
Conventional-release dosage fonns
The requirements are met if the quantities of active ingredient
dissolved from tablets (capsules or packets) conform to one
of the following states.
(1) The amount of active ingredient dissolved from each of 6
tablets (capsules or packets) is not less than the specified
quantity ( Q) calculated on the labelled content.
(2) If 1-2 of 6 tablets (capsules or packets) fail the
requirement, but not less than Q-10% and the average
amount of active ingredient dissolved is not less than Q.
( 3) If 1-2 of 6 tablets ( capsules or packets) fail the
requirement, and only 1 tablet (capsules or packets) is less
than Q-10 % but not less than Q-20 % , and the average
amount of active ingredient dissolved is not less than Q,
repeat the test on 6 additional tablets (capsules or packets);
if 1-3 of the total of 12 tablets (capsules or packets) are less
than Q, and only 1 of tablets (capsules or packets) is less
than Q-10 % , but not less than Q-20 % , and the average
amount of active ingredient dissolved is not less than Q.
The 10 % and 20 % values stated in the above interpretation
are expressed as percentages of the labelled content.
Sustained-release dosage forms or ccmtrolled-release dosage
fonns
Unless otherwise specified, the requirements are met if the
quantities of active ingredient released from tablets ( or
capsules) conform to one of following states.
(1) The amount of active ingredient released from each of 6
tablets ( or capsules) lies in the specified range, calculated
on the labelled content at each test time.
(2) If 1-2 of tablets (or capsules) fail the requirement, but
does not exceed 10% and the average amount of active
ingredient released lies in the specified range at each test
time.
(3) If 1-2 tablets (or capsules) fail the requirement, and
only 1 tablet ( or capsule) exceed 10 % , but not exceed
20 % , and the average amount of active ingredient released
lies in the specified range at each test time, repeat the test on
6 additional tablets ( or capsules); if 1-3 of tablets ( or
capsules) of the total of 12 tablets (or capsules) exceed the
specified range, and only 1 tablet ( or capsule) exceeds
10 % , but does not exceed 20 % , and the average amount of
active ingredient released lies in the specified range at each
test time.
The 10 % and 20 % values stated in the above interpretation
are expressed as percentages of the labelled content.
Exceeding the specified range 10 % means that the amount of
active ingredient released from each tablet is not less than
10 % of the lower limit of the specified range, and not more
than 10 % of the upper limit of the specified range at each
time. The amount of active ingredient released at each time
includes the amount of active ingredient released at terminal
time.
Enteric-coated dosage fonns
Unless otherwise specified, the requirements are met if the
quantities of active ingredient released from tablets ( or
CD 11 µm±O. 5 µm thick inert porous cellulose membrane

capsules) conform to one of the following states.
The amount released in acid C1 ) The amount of active
ingredient released from each of 6 tablets Cor capsules) is
not more than 10 % , calculated on the labelled content.
C2) If 1-2 of 6 tablets Cor capsules) are more than 10%,
but the average amount of active ingredient released is not
more than 10%.
The amount released in buffer solution C1) The amount of
active ingredient released from each of 6 tablets Cor
capsules) is not less than the specified quantity CQ) of the
labelled content. Unless otherwise specified, the value of Q is
70 % of the labelled content.
C2) If 1-2 of 6 tablets Cor capsules) is less than Q, but not
less than Q-10 % , and the average amount of active
ingredient released is not less than Q.
C3) If 1-2 of 6 tablets Cor capsules) is less than Q, only 1
of tablets Cor capsules) is less than Q-10 % , but not less
than Q-20 % , and the average amount of active ingredient
released is not less than Q, repeat the test on 6 additional
tablets (capsules) ; if 1-3 of tablets Cor capsules) of the
total of 12 tablets Cor capsules) are less than Q, and only 1
of tablets Cor capsules) is less than Q-1 O% , but not less
than Q-20 % , and the average amount of active ingredient
released is not less than Q.
The 10% and 20% values stated above interpretation are
expressed as percentages of the labelled content.
Transdermal patches Unless otherwise specified, the
requirements consist with those far sustained-release dosage
forms or controlled-release dosage forms.
Dissolution conditions and attentions
C1) Apparatus suitability and performance verification
test Besides every mechanical property of the apparatus
should comply with the requirements above, the suitablilitv
far the individual apparatus is also demonstrated by th~
performance verification test. Test the tablets RS far
dissolution according to the operating conditions specified.
The apparatus is suitable if the results obtained are within
the acceptable range stated in the certificate far the tablets
RS in the performance verification test.
C2) Dissolution medium Use the dissolution medium specified
in the monograph. Unless otherwise specified, the volume of
medium is 900 ml at room temperature, and the medium is
freshly prepared and degassed befare use. If the dissolution
medium is a buffered solution, adjust the solution so that its
pH is within O. 05 unit of the specified pH given in the
monograph
C3) Sampling time Withdraw a specimen within the time
interval specified in the monograph, complete taking speci-
mens from 6 vessels within 1 minute.
C4) Unless otherwise specified, granules and dry suspen-
sions should be scattered rather than being placed in one area
on the surface of dissolution medium.
C5) If capsule shells interfere with analysis, take at least 6
capsules and remove completely the contents, place a shell in
one vessel. Determine the value of each shell according to the
specified method in the monograph and make any necessary
correction. The test is invalid if the value of correction is
greater than 25 % of the labelled content. The interference is
negligible if the value of correction is not more than 2 % of
the labelled content.
0941 Test for Content Uniformity
The test far content uniformity is based on the contents of
0941 Test far Content Uniformity
single-clase preparations of salid, semi-salid, and
inhomogeneous liquid to determine whether the individual
contents are within limits set with reference to the labelled
amo unt.
Unless otherwise specified, the test far content uniformity is
required far the following dosage forms: tablets, hard
capsules, granules or powders containing less than 25 mg or
active pharmaceutical ingredient CAPD comprising less than
25 % of a single-clase unit by weight; sterilized powders for
injection prepared by mixing APis or API with excipient;
soft capsules filled with inhomogeneous solution; and oral
single-clase suspensions, transdermal patches, suppositories.
Above dosage forms in general requirements far preparations
should meet the requirements for content uniformity. For
compound preparations, the test for content uniformity is
only required far the ingredient which complies with the
above conditions. The test far content uniformity is not
required far multivitamin and trace-element preparations.
When the test far content uniformity is specified, the test for
weight variation may not be required. The test for weight
variation may not be required for compound preparations too,
when the test far content uniformity is presented for all
APis.
Unless otherwise specified, take 10 dosage units of the
substance being examined, carry out the method as specified
in the monograph and determine separately the relative
content x; of each dosage unit which is expressed 100 as the
l
labelled amount. Calculate the mean value X, the standard
deviation 5 [s ~ t, (x; - X)' and the absolute value
n-1
A CA= 1 100- X 1 ) which is the difference between the
labelled amount and the mean value.
If Á + 2. 25 ~ L, the substance being examined compiies
with the requirements far content uniformity;
If A+ 5 > L , the substance being examined fails to comply
with the requirements for content uniformity;
If A+ 2. 25 > L and A + 5 ~L, then repeat the test using
another 20 dosage units.
According to the results of the first and the repeated tests of
30 dosage units, calculate the mean values X, the standard
deviation S and the absolute value A which is the difference
between the labelled amount and the mean value. Then
calculate and judge as follows.
WhenA~O. 25L, if A 2 +5 2 ~0. 25L2, the substance being
examined complies with the requirements for content
uniformity; If A 2 + 5 2 > O. 25L 2 , the substance being
examined fails to comply with the requirements for content
uniformity.
When A > O. 25L , if A + l. 7 5 ~ L , the substance being
examined complies with the requirements far content
uniformity; If A+ l. 75 > L, the substance being examined
fails to comply with the requirements far content uniformity.
L in the above formulas is specified. Unless otherwise
specified, L is 15. O; Far oral single-dose suspensions, soft
capsules filled with inhomogeneous solution, powders far
spray administration supplied in capsules or blisters, eye, ear
and nasal single-dose suspensions, salid or semi-salid
preparations, L is 20. O; For transdermal patches and
suppositories, L is 25. O.
If the limit of content uniformity is ± 20 % or other
percentage as specified in the monograph, L is 20. O or other
corresponding value.
When the mean value CT) of upper and lower limit of
contents in the monograph is more than 100. O C% ) , if X<
 0942 Minimum Fill

100.0, A=lOO-X; If 100.0~X~T, A=O; If X>T, as the percentage of the labelled quantity with three
significant figures, and then identify the results.
A= X - T. Calculate as described above and judge the
results. When T is less than 100. O ( %) , the calculation Injection, liquid Oral or external
method of A should be specified in the monograph. concentrates for solids, semisolids,
If the method used for content uniformity is different to the injection liquids; Viscous liquids
Labelled
assay method, and the individual content can not be obtained
quantity Quantity Quantity
from the response value, take 10 dosage units of the Average Average
substance being examined, carry out the method of content in each in each
quantity quantity
uniformity as specified in the monograph. Determine container container
separately the response value Y; ( absorbance, peak area Not less
Less than Not less than
etc), calculate the mean value Y. Determine the content XA than the
which is expressed 100 as the labelled amount by the assay
20 g / / labelled
93% of the
(ml) labelledquantity
method, divide X A by the mean value Y to get the propor- quantity
tional coefficient K CK = XA /Y). Every response value Y; Not less
multiply by K to calculate the relative content C%) x; Not less than
20-50 g than the
Cx; = KY;) of each dosage units which is expressed 100 as (ml) / / labelled
95% of the
labelled quantity
the labelled amount. Calculate X, S and A as described quantity
above, then judge the results. If necessary to repeat the test,
take another 20 dosage units, carry out the method as Not less
Not less Not less
than 97% Not less than
described above. Calculate the mean value Y of 30 dosage More than than the than the
of the 97% of the
units, the proportional coefficient K, the relative content 50 g (ml) labelled labelled
labelled labelled quantity
( %) X;, the standard deviation S and A, then judge the quantity
quantity
quantity
results.

0942 Minimum Fill 0951 Preparations For Inhalation:

The following tests and specifications apply to solid,
semisolid, and liquid dosage forms packed in containers,
except those preparations for which the tests for Weight
Variation of Contents and Volume in Container are otherwise
specified or radioactive pharmaceuticals.
Procedure
Gravimetric method (for containers labelled by weight)
Unless otherwise specified, select 5 containers ( or 3
containers if the labelled quantity is more than 50 g), remove
the cover and label. Thoroughly clean and dry the outside of
the containers by suitable means, and weigh individually and
accurately. Accurately remove the contents from each
container, wash the container with a suitable solvent. Dry,
and again weigh each empty container. Determine the net
weight of contents in each container and the average net
weight of contents. The results should comply with the requi-
rements. If any one container fails the requírements, a further 5
containers ( or 3 containers if the labelled quantity is more
than 50 g) may be tested individually and all must comply.
Volumetric method (for containers labelled by volume).
Unless otherwise specified, select 5 containers ( or 3
containers if the labelled quantity is more than 50 ml). Open
the containers with caution to avoid any loss of the contents.
Pour individually the contents of each container into a
calibrated, dry cylinder (graduated to contain rather than to
deliver the desighnated volumes) of such size that the
volume to be measured occupies at least 40 % of its graduated
volume. For viscous liquids, keep the containers upside clown
for 15 minutes to pour the contents as completely as possible.
If the labelled quantity is not more than 2 ml, take up
individually the contents of each container into a dry
previously calibrated syringe. Measure the volume of the
contents in each container, calculate the average volume of
contents. The results should comply with the requirements.
If any one container fails the requirements, a further 5
containers ( or 3 containers if the labelled quantity is more
than 50 ml) may be tested and all must comply.
Calculate the average quantity and quantity in each container
Aerodynamic Assessment of Fine Particles
rr'\1 . • 1 1 1 . • 1 • . ·1 . • r· . ~ · 1
1 ne paruc1e or arop1et s1ze mstnounon ano1 nne parnc1e 1
aose
in the spray discharged from inhalation preparations are
important parameters for evaluating the quality of inhalation
preparations. While particle size measurement by micro-
scopy, light obscuration, light scattering or light diffraction
methods can be used to evaluate the number of particles in
the emissions of inhalations in the manufacturing process,
the particle or droplet size distribution of the finished
products should be expressed by the aerodynamic size
distribution of the drug aerosol leaving the inhalers
Unless otherwise specified under individual monograph,
Apparatus 1 should be used to assess aerodynaminc charac-
teristic of fine particles
APPARATUS 1 (Twin impinger)
APPARATUS The apparatus is shown in figure 1, and the
component units are described as follows.
The dimension tolerance of the glassware is ± 1 mm.
Connect all the component units according to the figure and
operate in a fume hood at a temperature of 20-25ºC.
Introduce 7 ml of solvent specified in the individual mono-
graph as the absorbent in the first impingement chamber D,
and 30 ml of solvent specified in the individual monograph as
the entrapping liquid in the second impingement chamber H.
Connect all the component units and ensure that jet spacer
peg of the jet assembly G, just touches the bottom of the
second impingement chamber. Clip the second impingement
chamber and make all the component units tightly collected.
Ensure that the assembly is vertical and adequately supported
and that components C and E parallel each other. Connect a
pump to the outlet F. Switch on the pump, adjust the air
flow through the apparatus, as measured at the inlet to the
throat, to 60±5 L/min.
Procedure
( 1 ) Inhalation aerosols
Select one aerosol container and allow it to stand at 22ºC ±
2ºC for at least 1 hour. Shake thoroughly and fire several
 0951 Preparations For Inhalation: Aerodynamic Assessment of Fine Particles
95
Fig. 1 Appartusl: Twin impinger
A: Mouthpiece adapter, connected to the mouthpiece of inhalation.
B: Throat, a modified 50 ml round-bottomed flask with 29/32
ground-glass inlet socket and 24/29 ground-glass outlet cone.
C: Neck.
D: First impingement chamber, a modified 100 ml round-bottomed
flask with 24/29 ground-glass inlet socket and 14/23 ground-glass
outlet socket.
E: Coupling tube, a glass wall tube with 14 ground-glass outlet
connected to D.
F: Outlet of three-way tube, a side outlet is 14 ground-glass outlet
cone, the upper end connect with a plastic screw cap (containing
a ring) to make E and F sealed, the outlet of the lower is 14
ground-glass outlet.
G: Jet assembly, a modified polypropylene filter holder with 4 jets
(l. 85 mm± O. 125 mm in diametre) arranged on a circular clise
and with an integral jet spacer peg with 2 mm protrusion ( 2 mm
in diametre).
H: Second impingement chamber, a 250 ml conical flask with 24
ground-glass inlet socket.
discharges to waste. Insert the actuator into rubber adapter,
switch on the pump, shake the canister for 5 seconds,
replace the canister in its actuator, discharge once
immediately. Remove the canister from its actuator, shake
for 5 seconds, relocate the canister in its actuator and
discharge again. Unless otherwise prescribed, repeat the
discharging for 10 times. After the last discharging, remove
the container, wait for 5 seconds and then switch off the
pump and dismantle the apparatus.
( 2) Powder inhalers
1 ) Powder inhalers supplied in capsules. Select one
capsule, place it in the dry powder inhaler, unless otherwise
specified, squeeze the buttons on each side of the inhaler to
pierce the two sides of the capsule, switch on the pump.
lnsert assembled inhaler with a suitable rubber adapter into
the throat and ensure that the inhaler is tightly connected to
the throat and lines up along the horizontal axis of the throat
B. Af ter 1O seconds remo ve the inhaler, place another
capsule in the inhaler. Conduct the determination for 10
capsules. After the last discharging, switch off the pump and
dismantle the apparatus.
2) Powder inhalers supplied in powder reservoir. Load the
inhaler with a <lose by turning the grip or squeezing the
inhaler. Switch on the pump. Insert the assembled inhaler
with a suitable adapter into the throat and ensure that the
inhaler is tightly connected to the throat and line up along the
horizontal axis of the throat B. After 10 seconds remove the
inhaler. Repeat the discharging for 10 times, unless other
prescribed. After the last discharging, switch off the pump
and dismantle the apparatus.
( 3) Inhalation nebulizers
1) Inhalation nebulizers for nebulizer machine Unless other
justified, select one container, put it into the devices. lnsert
the assembled inhaler with a suitable adapter into the throat
and ensure that the inhaler is tightly connected to the throat
and lines up along the horizontal axis of the throat B. Switch
on the pump of the apparatus ( with filter of suitable
diametre) and after 10 seconds actuate the nebulizer. After
60 seconds, stop nebulizing, wait for about 5 seconds and
then switch off the pump of the apparatus and dismantle the
apparatus.
2 ) Multiple-doses metre inhalation nebulizers Select one
container, and put it into the devices. Insert the assembled
inhaler with a suitable rubber adapter into the throat and
ensure that the inhaler is tightly connected to the throat and
lines up along the horizontal axis of the throat B. Unless
other justified, prepare sample according to patients
instructions, actuate the nebulizer to discharge a <lose, wait
for 5 seconds and actuate the nebulizer again. Unless
otherwise justified, repeat the discharge for 10 times.
EVALUATION
Wash F interface, the inner of the inlet tubes to the lower
conical flask and its out surface where it projects into the
flask and assembly jet using the blank entrapping liquid.
Combine the washings into the second impingement chamber
H and dilute with the same solvent to a volume. Determine
the quantity of the active ingredient in this solution using the
method described under the individual monograph, and
calculate the fine particle <lose by dividing the result with the
sampling frequency.
For inhalation nebulizers for nebulizer machine, wash the
inner surface of the upper impingement chamber with blank
entrapping liquid, combine the washing with the entrapping
liquid in upper impingement, dilute to a volume. Wash the
filter preceding the pump and its connection to the lower
impingement chamber, the inner surface of the lower
impingement chamber with the blank entrapping liquid,
combine the washings with the entrapping liquid in the lower
impingement chamber, dilute to a volume. Determine the
amount of active substance collected in each of both flasks.
Express the results for each of the 2 parts of the apparatus as
a percentage of the total amount of active substance.
APPARATUS 2
APPARATUS Each component of apparatus 2 was shown in
fig. 2-5.
Drill, counter-hore and tap
for an M-4 cap screw
Note:use minimun cllearance 19.0
for screw in the lower part to aid
in precise alignment.
10° ± lº (mm)
Fig. 2 Critica! dimensions for the L induction port.
 0951 Preparations For Inhalation: Aerodynamic Assessment of Fine Particles

Apparatus 2 ( The Andersen cascade impactar, ACI)

consists of 8 stages together with a final filter. Material of
construction may be aluminium, stainless steel or other
D suitable material. The stages are clamped together and sealed
Vacuum
with 0-rings (Table. 1 ). In the configuration used for
pump pressurised inhalers the entry cone of the impactar is
Two-way Flow connected to the L induction port. A suitable mouthpiece
Solenoid Control
Valve C
adapter is used to provide an airtight seal between the inhaler
Valve F
and the L induction port. In the configuration for powder
Connector Vacuum Tubing inhalers, a pre-separator is placed above the top stage to
A B collect large masses of non-respirable powder. To accom-
Fig. 3 Experimental set-up for testing powder inhalers. modate high flow rates through the impactar, the outlet
nipple, used to connect the impactar to the vacuum system is
enlarged to have an internal diametre of greater than or equal
to 8 mm. To ensure efficient particle capture, coat each plate
with glycerol, silicone oil or similar high viscosity liquid,
typically deposited from a volatile solvent.
Table 1 Critica) dimensions for nozzles
Description Number Dimension (mm)
Stage O nozzle diametre 96 2. 55±0. 025
Stage 1 nozzle diametre 96 l. 89±0. 025
Stage 2 nozzle diametre 400 o. 914±0. 0127
Stage 3 nozzle diametre 400 o. 711 ±o. 0121
Stage 4 nozzle diametre 400 o. 533±0. 0127
Stage 5 nozzle diametre 400 o. 343±0. 0127
Stage 6 nozzle diametre 400 o. 254±0. 0127
Stage 7 nozzle diametre 201 o. 254±0. 0127
Procedure
{ 1 ) Pres.surised inhalers
Put a suitable filter in the last stage, assemble Apparatus 2
step by step, ensure that the system is airtight. Place a
Fig.4 Apparatus 2 used for pressurised inhalers
suitable mouthpiece adapter in position at the end of the L
44 induction port so that the mouthpiece end of the actuator,
Cross-section
38.~ when inserted, lines up along the horizontal axis of the L
19 ---+'
15 -1
14.4_,
¿;PT=o:::N1r--R15 induction port and the inhaler unit is positioned in the same
Rl5.87~05 orientation as the intended use. Connect a suitable pump to the
1~ :::::¡ R14.4 outlet of the apparatus and adjust the air flow through the
Top view ' Groove for 0-ring
R19 apparatus, as measured at the inlet to the L induction port, to
R44 28. 3 L/rnin (±5%). Switch off the pump.
Unless otherwise prescribed, conduct according to the patient
instructions. Locate the mouthpiece end of the actuator in the
adapter, switch on the pump to the apparatus, shake the
container for 5 seconds, replace the container in its actuator,
discharge once immediately. Remove the container from its
actuator, shake for 5 seconds, relocate the container in its
R6.'iU±0.03 actuator and discharge again Repeat the procedure until
10º± lº required discharges have been finished. After the final
Do not break edge discharge, remove the assembled inhaler from the adapter,
45º±3ºV. Material: Aluminum, stainless steel wait for 5 s and then switch off the pump. The number of
or other suitahle material. discharges should be minimised and typically would not be
Except where noted, all the edges to greater than 1 O. The number of discharges should be
Sideview be broken and burrs removed.
Smface to be clean machine- sufficient to ensure an accurate and precise determination of
tooled finish. the fine particle <lose.
Inner bore smface oughness Ra Dismantle the apparatus. Carefully remove the filter. Remove
./27 ahout 0.4 mm.
_ 22 0-rings: nominal dimensioning: the mouthpiece adapter and L induction port from the
-14 ID29mm, apparatus and extract the active substance with the solvent
OD 32 mm,with 1.8 mm. described under the individual monograph and dilute to a
----0
~Do not break edge suitable volume. Extract the active substance from the inner
walls and the collection plate of each of the stages of the
Fig. 5 Connection of the L induction port to the apparatus with the solvent described under the individual
preseparator of the apparatus 2 (Dimensions in monograph and dilute to a suitable volume.
millimetres unless otherwise stated). Determine the quantity of active substance contained in each
 0951 Preparations Far Inhalation: Aerodynamic Assessment of Fine Particles

solution with the method described under the individual
monograph.
( 2) Powder inhalers
The aerodynamic cut-off diametres of each stage at 28. 3 L/ min
far apparatus 2 are provided in table 2.
Put a suitable filter in the last stage, assemble Apparatus 2,
ensure that the system is airtight. Depending on the product
characteristics, the pre-separator may be omitted, where
justified and authorised. Stages 6 and 7 may also be omitted
at high flow rates, if justified. The pre-separator may be
coated in the same way as the plates or may contain 10 ml of
a suitable solvent. Unless otherwise defined, conduct the test
at the flow rate, Oout, at the inlet to the L induction port.
Oout is used in the test far unifarmity of delivered <lose
drawing 4 L of air from the mouthpiece of the inhaler and
through the apparatus. Unless otherwise prescribed, conduct
according to the patient instructions. With the pump running
and the 2-way solenoid valve closed, locate the mouthpiece of
the inhaler in the mouthpiece adapter and ensure horizontal.
Discharge the powder into the apparatus by opening the valve
far the required time, T ( ± 5 per cent) (T is used in the
test far uniformity of delivered <lose). Repeat the discharge
sequence until required discharges have been finished. After
the final discharge, wait far T sand then switch off the
pump. The number of discharges should be minimised and
typically would not be greater than 10. The number of
discharges should be sufficient to ensure an accurate and
precise determination of fine particle <lose.
Dismantle the apparatus. Carefully remove the filter. Wash
the mouthpiece adapter, L induction port and pre-separator
with the solvent described under the individual monograph
and dilute to a suitable volume. Extract the active substance
from the inner walls and the collection plate of each of the
stages of the apparatus with the solvent and dilute to a
suitable volume.
Determine the quantity of active substance contained in each
solution with the method described under the individual
monograph.
EVALUATION
From the analysis results of the solutions, calculate the mass
of active substance deposited on each stage per discharge and
the mass of active substance per discharge deposited in the L
induction port, mouthpiece adapter and when used, the pre-
separator.
Starting at the final collection si te ( filter), calcula te the
cumulative mass from specified stage, which is the fine
particle <lose.

Table 2 Calculations for Apparatus 2 when used ata flow rate of 28. 3 L/min
Cut-off diametre Mass of active substance deposited Cumulative mass of active substance Cumulative fraction of active
(.um) per discharge deposited per discharge substance( per cent)
d1 =O. 4 mass from stage 8, ms e1=ms f7 = (e 1 /e) X 100

d6 =O. 7 mass from stage 7, m1 e6 =e1+m1 f6=(e6/e)X100

ds =l. 1 mass from stage 6 ,m6 es =e6+m6 fs =(es/e) X 100
d4 =2. 1 mass from stage 5 ,ms e4 =es+ms f 4=(ede)X100
mass from stage 4, m4 f 3 = (e 3 /e) X 100
dz=4. 7 mass from stage 3,m3 ez =e3+m3 fz=(ez/e)XlOO
di=5.8 mass from stage 2 ,mz ei =e2+m2 fi = (e 1/e) X 100

do=9. O mass from stage 1, m 1 eo=e1 +m1 fo = (e o/e) X 100

mass from stage O,mo e=eo+mo 100

APPARATIJS 3 lnterstage passageway

APPARATIJS Each component of Apparatus 3 was shown Removable Micro-orífice
collecit(MOC)
in figure 6-10.
+- Lid witb seal
lndyction port body attacbed
Tbe entrance ---+

--......."'li=':~~=""""/ .........__Bottom frame witb

cup tray in place

Fig. 7 Apparatus 3 showing component parts.

Stage 2 Stage 4 Stage 6

6 boles 52 boles 396 boles

Clamping mecbanism
Stage 1 Stage 3 Stage 5 Stage 7
Fig. 6 Apparatus 3 (shown with the 1 boles 24 boles 152 boles 630 boles
pre-separator in place). Fig. 8 Apparatus 3: nozzle configuration.
 0951 Preparations For lnhalation: Aerodynamic Assessment of Fine Particles

lnterstag~ passage Interstage passage continued

to next stage from previous stage
Dimension
Description
(mm)
;:::;:.=~ ,-seal body Stage 3 nozzle to seal body distance • • -
,-Cup tray 8.445 ± 0.410
dimension c
Stage 4 nozzle to seal body distance • • -
, -Bottom frame
dimension c
11.379 ± 0.237

Stage 5 nozzle to seal body distance • • -

dimension c
13.176 ± 0.341
Fig. 9 Apparatus 3: configuration of
interstage passageways. Stage 6 nozzle to seal body distance * * -
dimension c
13.999 ± 0.071

Stage 7 nozzle to seal body distance * • -

dimension c
14.000 ± 0.071

MOC nozzle to seal body distance * * -

Nozzie diameter. 14.429 to 14.571
dimensiona
dimension c
* See Fig. 8
* * See Fig. 9

In routine operation, the seal body and lid are held together
as a single assembly. The impaction cups are accessible when
this assembly is opened at the end of an inhaler test. The
cups are held in a support tray, so that all cups can be
removed from the impactar simultaneously by lif ting out the
Pre-separator insert tray.
Connect the L induction port to the impactar inlet. For
powder inhalers, a pre-separator should be added between
Fig. 10 Apparatus 3: pre-separator configuration. the L induction port and the impactar in the general. A
suitable mouthpiece adapter is used to provide an air tight
Apparatus 3 (Next Generation Impactar, NGD is a cascade seal bet\veen the inhaler and the induction port.
impactor with 7 stages and a micro-orifice collector (MOC). Apparatus 3 contains a terminal Micro-Orifice Collector
Over the flow rate range of 30 L/min to 100 L/min the 50 (MOC) which for most formulations will eliminate the need
per centefficiency cut-off diametres ( Dso values ) range for a final filter by method valida tion. The MOC is an
between O. 24 µm to 11. 7 µm. In this flow range, there are impactar plate with nominally 4032 holes, each approxi-
always at least 5 stages with Dso values between O. 5 µm and mately 40 µm in diametre. Most particles not captured on
6. 5 µm. stage 7 of the impactar will be captured on the cup surface
Material of construction may be aluminium, stainless steel or below the MOC. For impactors operated at 60 L/min, the
other suitable material. MOC is capable of collecting 80 percent of O. 14 µm particles.
The impactar configuration has removable impaction cups For formulations with a significant fraction of particles not
with all the cups in one plane. There are 3 main sections to captured by the MOC, there is an optional filter holder that
the impactar; the bottom frame that holds the impaction can replace the MOC or be placed downstream of the MOC
(a glass fibre filter is suitable). To ensure efficient particle
cups, the seal body that holds the jets and the lid that
capture, coat each plate with glycerol, silicone oil or similar
contains the inter stage passageways (Figures 6-7). Multiple
high viscosity liquid, typically deposited from a volatile
nozzles are used in all but the first stage (Figure 8). The
solvent (unless validation result shows that is not necessary).
flow passes through the impactar in a saw-tooth pattem
(Critical dimensions are shown in table 3). Procedure
Table 3 Critical dimensions for apparatus 3 ( 1) ~urized inhalers
Place cups into the apertures in the cup tray. lnsert the cup
Dimension
Description tray into the bottom frame, and lower into place. Close the
(mm)
impactar lid with the seal body attached and operate the
Pre-separator (dimension a-see Fig. 10) 12.80 ± 0.05 handle to lock the impactar together so that the system is
Stage 1 • Nozzle diametre 14.30 ± 0.05 airtight. Connect the L induction port to the impactar inlet.
Stage 2 * Nozzle diametre 4.88 ± 0.04 Place a suitable mouthpiece adapter in position at the end of
Stage 3 * Nozzle diametre 2.185 ± 0.02 the L induction port so that the mouthpiece end of the
Stage 4 • Nozzle diametre 1.207 ± 0.01 actuator, when inserted, lines up along the horizontal axis of
Stage 5 • Nozzle diametre 0.608 ± 0.01 the L induction port. The front face of the inhaler mouthpiece
Stage 6 • Nozzle diametre 0.323 ± 0.01 must be flush with the front face of the L induction port.
Stage 7 • Nozzle diametre 0.206 ± 0.01 When attached to the mouthpiece adapter, the inhaler is
MOC* approx. 0.070 positioned in the same orientation as intended for use.
Cup depth ( dimension b-see Fig. 9) 14.625 ± 0.10 Connect a suitable pump to the outlet of the apparatus and
Collection cup surface roughness (Ra) 0.5-2 µm adjust the air flow through the apparatus, as measured at the
inlet to the L induction port, to 30 L/min ( ± 5 per cent).
Stage 1 nozzle to seal body distance • • -
dimension c
o± 1.18 Switch off the pump.
Unless otherwise prescribed, conduct according to the patient
Stage 2 nozzle to seal body distance • * - instructions. Locate the mouthpiece end of the actuator in the
dimension c
5.236 ± o. 736
adapter, switch on the pump to the apparatus, shake the
 0951 Preparations Far Inhalation: Aerodynamic Assessment of Fine Particles
canister far 5 s, insert the canister in to the actutor,
discharge one delivery into the apparatus immediately;
remove the canister, shake the canister far 5 s, reinsert the
canister into the actutor, discharge a second delivery of the
inhaler into the apparatus. Unless otherwise prescribed,
repeat the procedure until specific discharges have been done.
After the final discharge, remove the assembled inhaler from
the adapter, wait far 5 s and then switch off the pump. The
number of discharges should be minimised and typically
would not be greater than 10. The number of discharges is
sufficient to ensure an accurate and precise determination of
the fine particle <lose.
Dismantle the apparatus. Remove the L induction port and
mouthpiece adapter from the apparatus, unless otherwise
prescribed, extract the active substance with the solvent
described under the individual monograph and dilute to a
suitable volume. Open the impactar by releasing the handle
and lifting the lid. Remove the cup tray, with the collection
cups, and recover the active substance in each cup with the
same solvent and dilute to a suitable volume.
Determine the quantity of active substance contained in each
solution with the method described under the individual
monograph
( 2) Powder inhalers
Assemble the apparatus with the pre-separator. Without
inter-stage loss increasing ( > 5 %) or re-entrainment, the
preseparator may be omitted, where justified.
Place cups into the apertures in the cup tray. lnsert the cup
tray into the bottom frame, and lower into place. Clase the
impactar lid with the seal body attached and operate the
handle to lock the impactar together so that the system is
airtight. Assemble the pre-separator insert into the pre-
separator base, fit the pre-separator base to the impactar
inlet. Add 15 ml of the solvent used far sample recovery to
the central cup of the pre-separator insert. Place the pre-
separator body on top of this assembly and clase the latches.
Connect the L induction port to the impactar inlet or pre-
separator inlet. Place a suitable mouthpiece adapter in
position at the end of the induction port so that the
mouthpiece end of the inhaler, when inserted, lines up along
the horizontal axis of the L induction port. The front face of
the inhaler mouthpiece must be flush with the front face of
the L induction port. When attached to the mouthpiece
adapter, the inhaler is positioned in the same orientation as
intended far use. Connect the apparatus to a flow system.
Unless otherwise prescribed, conduct the test at the flow
rate, Qaut, at the inlet to the L induction port. Q 0u1 is used in
the test far unifarmity of delivered <lose. Switch on the
pump, connect a flowmeter to the L induction port. Adjust
the flow control valve to achieve steady flow through the
system at the required rate, Q0 u1 ( ± 5 per cent). With the
inhaler in place and the test flow rate established, measure
the absolute pressure on both sides of the control valve. A
ratio P 3 / P 2 of less than or equal to O. 5 indicates critical flow.
Switch to a more powerful pump and re-measure the test
flow rate if critical flow is not indicated. Switch off the pump.
Unless otherwise prescribed, operate on the basis of the
patient instructions. With the pump running and the 2-way
solenoid valve closed, locate the mouthpiece of the inhaler in
the mouthpiece adapter. Discharge the powder into the
apparatus by opening the val ve far the required time, T ( ±
5 per cent) ( T is used in the test far unifarmity of delivered
<lose). Repeat the discharge sequence until required charges
have been finished. The number of discharges should be
minimised and typically would not be greater than 10. The
number of discharges is sufficient to ensure an accurate and
precise determination of fine particle <lose.
Dismantle the apparatus. Remove the mouthpiece adapter and
L induction port from the apparatus. Unless otherwise
prescribed, extract the active substance with the solvent
described under the individual monograph and dilute to a
suitable volume. When used, remove the pre-separator
carefully from the impactar. Transfer the solution in the pre-
separator into a suitable flask, wash the pre-separation with
solvent, combine the washing and dilute to the mark with
solvent. Open the impactar by releasing the handle and lifting
the lid. Remove the cup tray, with the collection cups, and
recover the active substance in each cup and dilute to suitable
volume.
Determine the quantity of active substance contained in each
solution with the method described under the individual
monograph.
( 3) Preparations for nebulisation
A back-up filter in addition to the micro-orífice collector
(MOC) must be used to ensure quantitative recovery of
active substance from the nebulised aerosol at the specified
flow rate of 15 L/min. The filter is located below the MOC
( internal filter option) or a filter in holder, external to the
impactar, is used to capture any fine droplets that pass
beyond the last size fractionating stage. A pre-separator is
not needed far testing nebuliser-generated aerosols.
Pre-cool the assembled impactar and induction port in a
refrigerator (set at about 5ºC) far not less than 90 min and
start the determination within about 5 min of removal of the
impactar from the refrigerator.
Set up the nebuliser with a supply of driving gas ( usually air
or oxygen), or use a compressor, at the pressure and flow
rate specified by the manufacturer of the nebuliser. Take
precautions to ensure that the gas supply line <loes not
become detached from the nebuliser when under pressure.
Fill the nebuliscr VJ"ith the volume of the medicinal product as
specified in the patient instructions.
Remove the impdctor from the refrigerator. Attach the L
induction port to the impactar, and connect the outlet of the
impactar/ external fil ter to a vacuum source. Turn on the
flow through the impactar. Connect a flow metre to the L
induction port. Adjust the flow control valve located between
the impactar and the vacuum source to achieve a steady flow
through the system at 15 L/min ( ±5 per cent). Remove the
flow metre.
Make sure the nebuliser is positioned in the same orientation
as intended far use then attach the mouthpiece of the
nebuliser to the L induction port, using a mouthpiece adapter
if required, ensuring that connections are airtight. Switch on
the flow/compressor far the nebuliser. Sample far a
predetermined time (To ) . Switch off the driving gas flow /
compressor to the nebuliser, remove the nebuliser from the L
induction port and switch off the flow from the vacuum
source to the impactar. Dismantle the appartus. Unless other
prescribed, determine the quantity of active substance
collected in the induction port, on each stage and on the
back-up filter/ external filter with the method described under
individual monograph.
EVALUATION
From the analysis of the solutions, calculate the mass of
active substance deposited on each stage per discharge and
the mass of active substance per discharge deposited in the L
induction port, mouthpiece adapter and when used, the pre-
separator.
Starting at the final collection site (filter or MOC), calculate
the cumulative mass from specified stage, which is the fine
particle <lose.
 0952 Determination of Adhesion

continued
0952 Detennination of Adhesion The number Diametre/ mm Weight/kg
35 28.575 95.5
This method is to measure the adhesion of cataplasms and 36 30. 163 112. 8
patches applied to the surface of skin. Four indexes of 37 31. 750 131. 9
measurement are tack, shear adhesion, peel strength and
38 33.338 152
binding power. Tack is an adhesion force between patches or
cataplasms and skin surface under mild touch, i. e. , a 39 34.925 175
peeling resistance when pressed mildly. Shear adhesion can 40 36.513 198. 1
reflect the resistance to persistent extemal force causing
41 38. 100 227.3
deformation or fracture. Peel strength is a peeling resistance
between patches cataplasms and skin. Binding power is a kind 42 41. 275 287.57
of force that draw the cataplasms together with skin. 43 42.863 320.4
Method 1 ( Tack test) 44 44.450 361
Tack is measured by Bowl incline stopping. A series of 45 47.625 439.5
fitting friction halls under the table roll past the adhesive
46 50.800 538.8
surface of the inclined plate separately. The tack is evaluated
by the biggest friction hall the adhesive surface could cling. ( 1 ) Apparatus
Table The number of friction halls and the specification The tack tester consists of inclined plate, base frame,
stainless steel hall and hall meeting box ( as Fig 1 ) . A
The number Diametre/ mm Weight/kg
stainless steel plate with about 2 mm thickness is used as the
1 0.794 0.002 inclined plate (angle of inclination is 15º, 30º or 45º), two
2 l. 588 0.016 horizontal lines with the interval scale of 10 mm were drawn
3 2.381 0.055 on it. The top one is the mark of the halls zero position and
the bottom line is the mark of samples stationary position.
4 3. 175 o. 132 The base frame could be adjusted and keep horizon of the
5 3.969 0.257 tester. The hall meeting box is used to meet the hall rolling
6 4. 763 0.440 clown the plate, the inwall of the box is lined with soft
abrasives. The number and specification of the friction halls
7 5.556 0.702
should comply with the description in the table above.
8 5.953 0.86
9 6.350 l. 03
10 7. 144 l. 50
11 7.938 2.06
12 8.731 2.66
13 9.525 3. 55
14 10.319 4.43
15 11. 113 5.64 Ball meeting box.
16 11. 509 6.20
Fig. 1 Apparatus for tack testing
17 11. 906 6.93
18 12.303 7.5
( 2) Procedure
19 12. 700 8.42 Place the cataplasms or patches ( including packing material)
20 13.494 10. 1 under an environment of 18-25ºC and 40%-70% relative
21 14.288 12.0 humidity for more than 2 hours avoiding overlap before the
test. Scrub the test board and loading board with anhydrous
22 15.081 14. 1 ethanol dipping ambulant, use clean gauze to wipe dry.
23 15.875 16.5 Repeat the operation above over 3 times to make sure that
24 16.669 19. 1 the working plane is clean.
Place three pieces of the sample between the two lines of the
25 17.463 21. 9
tiltboard ( the tilt angle as specified in an individual
26 18.256 25.0 monograph) with the plaster side upward. The lower end of
27 19.050 28.4 the sample should be placed on the second horizontal line and
28 19.844 ali the pieces of the sample should be flattened on the
32.4
tiltboard. Roll the halls specified in an individual monograph
29 20.638 36.2 from the top of the inclined plane freely.
30 22.225 45.2 ( 3) lnterpretation
31 23.019 so.o Among the numbers of the friction hall which is stuck to 3
32 23.8131 55.5 samples, if 3 all are the same biggest one, or if 2 of them are
the biggest one and the other is only the neighboring smaller
33 25.400 57.4
one, the samples comply with the requirement; if one of
34 26.988 80.8 them is the biggest one and the other two are only the

neighboring smaller one, then repeat the test on additional
3 samples, all the samples are the same biggest one.
Method 2 ( Shear adhesion test)
Apply the cataplasms or patches to the plate surface. Keep
the plate verticality, hanging a poise with a standard mass
along the length direction of the patches, record the time
period the patches slip from starting to dropping out or the
distance it slips clown in a certain period of time.
( 1) Apparatus
1) Test frame lt consists of a base frame, which could be
adjusted to keep horizontal, and a bracket, which is used to
hang, and fix the test plate. Test frame keep the acting
surface of the test plate to be hanged vertically.
2) Test plate Test plate is l. 5-2. O mm in thick, 125 mm
in wide, 125 mm in long. The test plate is made of stainless
steel. with the surface rugosity less than O. 4 µm.
3) Press stick Press stick is a steel axis coated by rubber
2000 g in weight.
4) Loading board The requirements for the material, dim-
ension and surface are same as test plate.
(2) Procedure Before the test, Place the cataplasms and
patches (including packing material) under an environment
of 18-25ºC and 40%-70% relative humidity for more than
2 hours avoiding overlap before test.
Dip the cleaning material in the detergent and scrub the test
plate and loading board, then towel off carefully with a neat
gauze. Repeat this operation more than 3 times, until the
acting surface of the plate is clean by visual cheque. Do not
touch the acting surface of the plate with your finger or other
things af ter cleaning. Stick the sample to the middle between
the test plate and loading board and the sample is parallel
with the end of the plate. Roll the sample with press stick.
When the sample sticks to the plate, hold at room
temperature for 20 minutes, fix it to the test frame. Record
the starting time of the test.
Remove weight at the schedule time, measure the displace-
ment of the sample downslide, or record the time the sample
dropping out the plate.
( 3) Interpretation The displacement or the dropping time
should comply with the requirements specified in individual
monograph.
The test result is the arithmetic mean of a set of samples'
displacement or dropping time.
Method 3 ( Peel strength test)
Conduct this test by 180ºpeel strength test method.
( 1 ) Apparatus
1) Testing machine Sample' s breakdown load should be
15 %-85 % of lacerating machine' s full load. The indication
error of force should be less than 1 % . The machine peels
continuously with a lowering speed of 300 ± 10 mm/min,
usually accompanied by a plotting device record peeling load
automatically.
2) Test plate The stainless steel test plate is l. 5-2. O mm
thick, 50±1 mm in wide, 125±1 mm in long.
3) Melinex The PET film is O. 025 mm thick, about
110 mm in long, 20 mm in wide. The melinex should comply
with the requirements of JB1256-77 (6020 PET).
(2) Procedure Before the test, Place the cataplasms and
patches (including packing material) under an environment
of 18-25ºC and 40%-70% relative humidity for more than
2 hours avoiding overlap before test.
Fix the patches' backface to test plate with double faced
adhesive tape and fix the sample to the incline plate with
adhesive tape, fix sample with adhesive tape along its up side
0952 Determination of Adhesion ¡ ,11 .f,~{
and clown side if necessary, let the test sample be stuck on
the plate flat.
Stick sample' s adhesive layer to clean melinex, rolling on the
sample back and forth with press stick to ensure adhesion,
and no air blister at the joining part. Before the test, keep the
sample under the room temperature for 20-40 minutes after
sticking Fold the free end of the melinex 180º, grasp
melinex' s free end and the test plate to up side and clown side
separately of the testing machine. Keep the peeling surface
and testing machine in line. The testing machine peels conti-
nuously with a lowering speed of 300 ± 10 mm/min, the
peeling curve is drawn by an automatic recording device.
(3) Interpretation Peel strength should comply with the
requirements specified in individual monograph.
The 180ºpeel strength a CkN/m) of the patch is calculated
according to the following formula:
s
a=LB•c
In the formula: S is the area under the curve m the
numeric area, mm 2 ;
L is the length of the curve m the
numenc area, mm;
B is the actual width of the sample,
mm;
e is the load of record sheet' s unit
height, kN/m.
The test result is the arithmetic mean of the peel strength.
Method 4 ( Binding power test)
This method is suitable for the cataplasms or patches with
the size of no less than 35 mm X 60 mm.
( 1) Apparatus
Consists of pressing roller, pull rod, bracket, fixture,
sensor, gearing device and electrical appliances, etc (Fig. 2).
All of them are made of stainless steel. Pressing roller takes
the cylindrical shape, with the external diametre of 50 mm
and a roller through it. Both end of the pressing roller are
fixed with bearing axis. The weight that the pressing roller
and bracket act on the specimen is 2000 ± 20 g. The pull rod
is connected with the bracket with the angel of 90º. There are
two joggles at each side of the bracket, which are used for
supporting these rollers. Under the joggles are two
supporting wheels, and they are used to control the upper
and lower movement of the bracket. When the supporting
wheel keeps upright, the centre of the pressing roller to the
bottom of the supporting wheel is 30±30 mm; when tipped,
it should not be lower than the bottom of the roller.
electrical appliance
f
cylindrical
body
b~rcketjoggle
pul! rod .
!H
( roIler supportmg
wheels
bearing axis
Fig. 2 Apparatus for the determination of binding power
 0981 Crystallinity
The fixture includes the bottom plate and the pressing plate
(Fig. 3). The bottom plate takes the shape of 'I', with two
raised parallel rectangular bars in the middle. The bottom
plate is inward together with the middle of the rectangular
bars, which ensure two 12 mm-in-width bars can make
parallel shift during the loading process. The pressing plate is
also of the 'I' shape and with two parallel empty slot in the
middle. The bottom plate is a perfect match to the pressing
plate, yet leaves a gap for loading the cataplasms and
patches. Tighten the screw lightly after the loading and make
sure the fixture is in the same plane. The fixture is of
different size to match the cataplasms and patches with
different thickness.
pressing plate
o o
rn
o o
o o
Fig. 3 The fixture and the loading structure in the
determination of binding power
( 2) Procedures
Place the cataplasms and patches ( including packing
material) under an environment of 18-25ºC and 40%-70%
relative humidity for more than 2 hours avoiding overlap
before test.
The instrument is initialized and balanced for more than
30 minutes before use. Tension calibration must be conducted
when the instrument is newly used, continuous use after
prolonged withdrawal, replacement of important parts
(fixture, pull rod, bracket and sensor) or data abnormity.
Except for otherwise, the calibration should be referred to
the instrument, which is measuring the signal values of the
pressing roller bearing different balancing weights respec-
tively ( 5 points above generally). The instrument replaces
the balancing weights with the adhesive power, and the
measured signal is the longitudinal coordinate. Then the
standard curve of adhesive power and signal is drawn
automatically. Correlation coefficient should be greater than
O. 99 and the instrument is ready to use.
Choose the fixture according to the thickness of the
cataplasms and patches. Scrub the test board and loading
board with anhydrous ethanol dipping abluent, use clean
gauze to wipe dry. Repeat the operation over 3 times to make
sure that the working plane is clean.
Take three pieces of specimen and cut into the size of
50 mmX70 mm (no less than 60 mm in length and 35 mm in
width). If there is elasticity, the elastic side or the more
elastic side should be with the same direction with the long
side of the loading instrument ( Fig. 3 ) . The specimen is
fixed on the loading instrument and aligned the appropriate
scale, with the sticky side upward. Tear both sides of the
specimen a little, expose two sticky sides, and press both of
the sticky sides with pressing bars. The loading instrument is
placed in the middle of the bottom plate. Make sure that the
tablets of the specimen should be flatted on the bottom plate.
Horizontal press the pressing plate, fix the bottom plate and
the press plate with bolts on both sides. The sticky side on
the rectangular bars should be in uniform tension and placed
on the device. After fixation, a suitable model for determi-
nation should be selected.
The forward and backward movement of the pressing roller is
modeled in the original settings, which is suitable for the
pressing roller homing and testing process. Except for special
circumstances, the forward speed of the pressing roller is
600 mm/min, backward speed is 21 mm/min. The necessary
basis is needed for setting other speed.
( 3) Interpretation
The binding power tested should be in accordance with the
specified individual monograph. Two principles: indepen-
dently attached to the skin during the medication and is
inacceptable range to body feeling. Generally the binding
power for rubber pastes is suggested to be 3000-6000 mN
while 1000-2000 mN for gel paste.
The binding power of all three pieces should be within the
specified limits. If one piece fails, repeat the test on three
other pieces. The specimen complies with the requirement if
the second test succeed, and if not, the specimen fails to
comply. Under all circumstances, the specimen fails to
comply if one piece has the paste removed or wire drawing.
0981 Crystallinity
Solid substance may be classified as two forms: crystalline
and noncrystalline. The following methods are used to test its
crystallinity.
Method 1 ( polarizing microscope method ) Generally
crystals have optical anisotropism except isometric crystal
system. The transparent crystals exhibit the phenomena of
birefringence when the light passes through them.
Mount a few particles of the specimen on a clean glass slide,
add a quantity of liquid paraffin to immerse the particles.
Examine the mixture under a polarizing microscope: the
particles exhibit the phenomena of birefringence, extinction
positions and optical crystalline characteristics specified in the
individual monograph when the microscope stage is revolved.
Method 2 (X-ray powder diffraction) Crystalline materials
present characteristic diffraction decorative patterns ( sharp
diffraction pattern), while noncrystalline materials, exhibit
broad dispersion diffraction patterns. The test method is
described under X-ray Diffraction ( 0451 ) .
0982 Determination of Particle Size and
Particle Size Distribution
The following procedures are used for the determination of
particle size or particle size distribution. The method 1 and
method 2 are used for the determination of particle size or
size limit in pharmaceutical preparation. The method 3 is
used for the determination of particle size distribution in drug
substance or pharmaceutical preparation.
Method 1 ( microscopy)
The particle size determined by this method is expressed as
the length of particles observed under a microscope.
Standardization of ocular micrometer Standardization of
ocular micrometer is carried out the method is described
under Microscopical identification ( 2001 ) .
Procedure Thoroughly mix the substance being examined.
 0982 Determination of Particle Size and Particle Size Distribution
For the substance with high viscosity, a suitable quantity of
glycerine solution ( 1 - 2 ) specified in the individual
monograph may be added. Introduce 1-2 drops of the
substance being examined onto a slide as specified in the
appendix of the dosage form or in the individual monograph,
cover with cover glass, and press gently to make the particles
spread uniformly. Prevent formation of bubbles. For the
substance being examined which is semisolid, it can be
spread directly onto a slide. Immediately examine the slide
under a microscope, in the whole field of view, using 50-
100 X magnification. No agglomeration occurs and none of the
particles has a dimension of 50 µm or greater than specified
in the individual monograph. Examine the slide again under a
microscope, in the field of view specified in the appendix of
the dosage form or in the individual monograph, using 200-
500 X magnification. Record the total number of particles
observed and the number of particles having the specified
dimension. Calculate the percentage.
Method 2 ( sieving)
Sieving generally falls into three main types: manual sieving,
mechanical sieving and air-jet sieving. Manual sieving and
mechanical sieving are most suitable where the majority of
the particles are larger than about 75 µm. For particles
smaller than 75 µm, air-jet sieving or other means of
agitation may be more appropriate.
Mechanical sieving methods using mechanical agitation or
electromagnetic agitation, and that can induce either a
vertical oscillation or a horizontal circular motion, or
tapping, or a combination of both tapping and horizontal
circular motion is available. Entrainment of the particles in an
air stream may be used in air-jet sieving.
The relative humidity of the environment in which the sieving
is carried out must be controlled to prevent moisture uptake
or loss by the sample. If the substance being examined is
known to deveiop an electrostatic charge, an antistatic agent,
such as colloidal silicon dioxide and/or aluminum oxide, may
be added at a O. 5 per cent ( m/m) level to minimize this
effect.
l. Manual sieving
( 1) Single sieve Place a quantity of the substance being
examined, as specified in the individual monograph, upan the
specified No. sieve with a clase-fitting collecting pan and
cover. Shake the sieve in a rotary horizontal direction for not
less than 3 minutes, and gently tap on the sieve frequently in
vertical direction. Weigh accurately the amount in the
collecting pan, and calculate the percentage.
( 2) Two sieves Place the accurately weighed content of
5 single-clase units or of 1 multiple-dose unit upan the upper
sieve ( coarser) specified in the appendix of the dosage form
or in the individual monograph. Shake the sieve in a lef t-to-
right horizontal direction by gently tapping on the sieve
for 3 minutes. Weigh accurately the amount remaining on
the coarser sieve and passing through the finer sieve.
Calculate the percentage of the fraction.
2. Mechanical sieving
Unless otherwise specified, weigh accurately the test sieves
having a 200 mm diameter and the collecting pan. Place 25-
100 g of the substance being examined, accurately weighed,
depending on the bulk density of the material, upan the top
sieve ( coarsest) ( The bottom sieve is assembled with a
clase-fitting collecting pan) and cover. Set the mode and the
frequency of agitation, and agitate the nest of sieves for
5 minutes, then weigh accurately each sieve and the
collecting pan and determine the mass of substance on each
sieve. Calculate the percentage. Repeat these steps until the
mass on any of the test sieve <lose not change by more than
5 % or O. 1 g of the previous mass on that sieve. If less than
5 % of the total sample mass is present on a given sieve, the
endpoint for that sieve is increased to a mass change of not
more than 20 % of the previous mass on that sieve.
3. Air-jet sieving
A single sieve is used at a time. It requires sequential
analyses on individual sieves starting with the finest sieve to
obtain a particle size distribution. Unless otherwise specified,
weigh accurately the test sieve having a 200 mm diametre.
Place 25-100 g of the substance being examined, accurately
weighed, depending on the bulk density of the material,
upan the sieve with a cover. Set the pressure and air-jet for
5 minutes, then weigh accurately the sieve and determine the
mass of substance on it. Calculate the percentage. Repeat
these steps until the mass on the test sieve <lose not change
by more than 5 % or O. 1 g of the previous mass on that sieve.
If less than 5 % of the total sample mass is present on a given
sieve, the endpoint for that sieve is increased to a mass
change of not more than 20 % of the previous mass on that
s1eve.
Method 3 (Laser Diffraction)
Laser diffraction particle size analysis is based on the
scientific phenomenon that particles in a laser beam scatter
laser light at angles that inversely proportional to the size of
the particles. There is a direct relationship between the
distribution of the scattered light energy and the particle size
distribution. Mie theory or Fraunhofer approximation theory
is used to obtain the size distribution of the particles. The size
range of this method is O. 02-3500 µm.
1. Instrument
Laser diffraction analyzer: The intensity of the laser source
should be stable and the electronic background and optical
background should be automatically eliminated.
"Standard reference material" with certain distribution and
eigenvalues [d (0. 1), d (O. 5), d (O. 9) ] can be used
for verification of the analyzer. Relative Standard Deviation
CRSD) is generally used to express the size distribution of
"standard reference material". Far samples where the
coefficient of variation of the particle size distribution is less
than 50 % ( or ratio of diametre of largest to smallest particle
about 10 : 1), five repeatable measurements should be
performed. The deviation of d ( O. 5 ) of the " standard
reference material" from the eigenvalue should be less than
3 %, and the RSD of the five measurements should not
exceed 3%. The deviation of the d (0. 1) and d (0. 9) of
the "standard reference material" from the eigenvalue should
be smaller than 5 %, and the RSD of the measurements
should not exceed 5 %. As for the " standard reference
material" with size below 10 µm, the deviation of the
measured d (O. 5) from the eigenvalue should be less than
6 %, and the RSD of the paralleled tests should not exceed
6 %; the deviation of the d (O. 1) and d (O. 9) from the
eigenvalue should be less than 10%, and the RSD of the
paralleled tests should not exceed 10%.
2. Procedure
Either wet dispersion unit or dry dispersion unit can be
chosen according to the properties and solubility of the
material for test. Wet method can be used for measurement
of the suspended materials or the materials that are not
soluble in the dispersion media. Dry method is used for
measuring water-soluble materials or solid materials without
any suitable dispersion media.
Wet method The lower size limit is usually 20 nm.
The suitable dispersion method should be chosen according to
 0983 Measurement of Consistency by Penetrometry
the properties of the material for test so that sample can be
dispersed in to stable suspended solution. Generally, such
physical dispersion methods as ultrasonication and stirring
can be used. When necessary, a certain amount of chemical
additive or surfactant can be added, so that the sample can be
dispersed in a stable system. Thus, the sample can evenly
and stably pass through the cell window, so as to get the
accurate results.
Only when the electrical double layer potential ( Zeta
Potential) is in a certain range, can the dispersion system be
in a stable state. Therefore, when the dispersion system of
the material for test is prepared, attention should be paid to
the measurement of Zeta Potential of the system, so as to
assure the repeatability of the measurement.
Normally the amount of sample for test is displayed as
obscuration. The optimal obscuration is between 8% and
20%. For the most advanced analyzer the lower limit can be
clown to O. 2%.
Dry method The lower size limit is usually 200 nm.
Generally, sample path should be obturated to prevent the
sample from absorbing moisture. The velocity biasing should
be eliminated from selecting dry powder feeder and sample
cell. According to how easily the material can be dispersed,
the air pressure applied for sample dispersion should be able
to be adjusted, so that particles with different sizes can pass
through the cell window evenly and stably at the same speed,
thus assuring the accuracy of results.
For chemical drug substance, the ejective dispersion unit
shall be used. A proper amount of metal halls can be added
into the unit. The vibration feeding rate, the air pressure
( usually 0-0. 4 MPa) , and the gap of sample tray should be
adjusted to control the dispersion and the amount of the
sample that passes through the analyzer.
The amount of sample for dry measurement should reach
O. 5 %-5 % in obscuration.
Annotations
( 1) The setting of optical parameters is related to the size
distribution of the sample. The refractive index and
absorption index affect slightly those particles with sizes
above 10 µm, and they affect considerably to those particles
with size below 10 µm. When measuring the size distribution
of different drug substances and products, currently there is
no mature theory available for guiding the setting of the
optical parameters of the instrument, so that the setting can
be determined by experiment. "Standard reference material"
can be used to verify the instrument.
(2) For measurement of coloured materials, emulsions, and
partid es smaller than 1O µm, the Mie theory should be the
best general solution so as to reduce measuring errors.
Fraunhofer approximation theory should not be used in this
regard.
(3) When measuring the broad size distribution samples, it
is recommended to use the whole size range analyzer with
wide dynamic detector instead of analyzer with separate size
range to reduce the measurement errors.
0983 Measurement of Consistency
by Penetrometry
The following tests and specifications apply to semi-solid
substances, such as ointments, eye ointments and their usual
matrix (vaseline, wool fat and beeswax etc.). It is necessary
to control the hardness and viscidity of those substances as
they could influence the spreading ductility of the products.
Consistency measured by penetrometry is described as the
depth of penetration which is examined at 25 ºC when
penetrating object with specified mass is released from
penetrometer and held free for 5 s.
Apparatus
The penetrometer should automatically release the
penetrating object, and show the real-time depth of
penetration when the penetrating object is released and held
free for 5 s, and should has a device to ensure that the shaft
is vertical, should have a centre locating device to make sure
that cone tip of the penetrating object is consistent with the
centre of the container, should ha ve a lifting regulator to
accurately adjust cone tip of the penetrating object. There
should be no obvious friction between shaft and joint when
penetrating object is released, and measurement range of the
penetrometer should exceed 65 mm.
(1) Test frame It consists of a horizontal base, vertical
kingpost, vertical shaft to maintain and guide the penetrating
object, fixed handle, coarse adjustment screw, readjust
handle and penetrometry display device etc.
(2) Penetrating object and Shaft The penetrating object,
made of a suitable material, has a smooth surface, and is
classified by its shape and mass. There are three kinds of
penetrating objects suitable to be selected: penetrating object
l possesses a mass of 102. 5 ±O. 05 g and a matched shaft
weights 47. 5±0. 05 g, penetrating objectll possesses a mass
of 22. 5±0. 025 g anda matched shaft weights 15±0. 025 g,
the total weight of penetrating object m
and matched shaft is
9. 38 ±O. 025 g. The shape and dimensions are shown in
Fig. 1- Fip ~
l(')
d
+t
O'I
N
N </>0.4
d
+t
!!:l </>8.4
(mm)
Fig. 1 Penetrating object I
(3) Sample container It is a flat-bottomed cylinder, anda
different type of sample container supporting the use of a
different type of penetrating object. The shape and
dimensions of the matched sample containers of penetrating
object l -ID are shown in Fig. 4-Fig. 6.
The type of penetrating object should be selected according to
the amount of sample being examined. Penetrating object 11
is recommended for the study and determination of
penetrometry.
Procedure Adjust the equipment befare measurement, so
that cone tip of the penetrating object fall right on the centre
hole of the corrected apparatus.
Prepare the test samples by one of the following procedures
and store the samples at 25 ±O. 5ºC for 24 hours, unless
 0983 Measurement of Consistency by Penetrometry

-
00

(m~)
Fig. 6 Sample container of penetrating object ID

(mm) otherwise specified.

Fig. 2 Penetrating object 11 (1) Carefully and completely fill container, for about 2 mm
higher than the top edge, without forming air bubbles. Shake
for 5 minutes on a flat table to remove the bubbles that may
be mixed in.
( 2) According to the method described under the mono-
graph, melt sample and carefully fill container 2 mm higher
than the top edge, without forming air bubbles.
At 25ºC ±O. 5ºC, place the test sample on the base of the
penetrometer. Verify that its surface is perpendicular to the
vertical axis of the penetrating object. Zero, release the
penetrating object (in O. 1 s) and hold it free for 5 s.
Measure the depth of penetration, expressed in tenths of
millimetre. In order to guarantee the comparison of the
</JO.l results determined by three different penetrating object, the
measured value of penetrating object 11 and penetrating
<P2 object m should be converted to speculated value of
penetrating object I , based on formulas.
Evaluation
( 1 ) The use of penetrating object I
(mm)
Repeat for 3 times and calculate the arithmetic mean of the
Fig. 3 Penetrating object ID and shaft three measurements. If any of the individual results differ
from the mean by more than 3. O%, repeat the test and
express the results of the six measurements as the mean and
the relative standard deviation (RSD), the relative standard
deviation should less than 5. 0%.
h
n
l
( 2) The use of penetrating object
Repeat for 3 times, and convert the measured value of
penetrating object II to the predictive value of penetrating
object I according to the following formula:
p=2r+5
Fig. 4 Sample container of penetrating object I Where: p is the predictive value of penetrating object l ;
(d=75 mm or 102 mm, h~62 mm) r is the measured value of penetrating object II.
Calculate the arithmetic mean of the three measurements. If
any of the individual results differ from the mean by more
than 3. O% , repeat the test and express the results of the six
measurements as the mean and the relative standard deviation
(RSD), the relative standard deviation should less than 5. 0%.
When penetrating object I is specified for samples in the
individual monograph, the penetrometry value can be deter-
mined by the use of penetrating object l , and converted to
the predictive value of penetrating object I. If the predictive
value exceeds the limits of sample standard regulation,
penetrating object I should be used to determine again to
judge whether the testing samples comply with requirement.
(mm) ( 3) The use of penetrating object I[
Fig. 5 Sample container of penetrating object 11 Repeat for 3 times, and convert the measured value of
penetrating object m to the predictive value of penetrating
 1101 Sterility Tests
object I according to the following formula:
p=3. 75s+24
Where: p is the predictive value of penetrating object I ;
s is the measured value of penetrating object III.
Calculate the arithmetic mean of the three measurements. If
1100 Biological Tests
1101 Sterility Tests
Test for sterility is a method to detect whether medicines,
biologics, medical devices, raw materials, excipients or other
articles, which are required to be sterile according to the
Pharmacopoeia, are sterile. However, a result conforming
with the requirements only indicates that no contaminating
micro-organism has been found in the sample examined under
the conditions of the test.
Test for sterility should be carried out in aseptic conditions.
Test environment must meet the requirements of sterility
testing. The whole process should be performed under
strictly aseptic conditions to avoid any microbial contami-
nation. The precautions taken to avoid contamination are
such that they do not affect any microorganisms that are to
be revealed in the test. The cleanliness of working areas
including laminar flow cabinet, working bench, background
room should be monitored regularly according to the current
national standard such as the detecting method for airborne
particles, airborne microbe and settling microbe in the clean
room ( area) of the pharmaceutical industry. The isolator
should be validated regularly according to the relevant
requirements, and the cleanliness of inner isolator space
should also comply with aseptic requirements. The working
conditions in which the tests are performed are monitored at
routine inspection.
Culture Media
Fluid thioglycollate medium is primarily intended for the
culture of anaerobic bacteria; however, it will also detect
aerobic bacteria. Soya-bean casein digest medium is suitable
for the culture of both fungí and aerobic bacteria.
Media preparation and incubation condition
Media for the test may be prepared as described in the below
formulations, and dehydrated media or prepared media may
be used provided that they have the same ingredients and
comply with the requirements. Media should be sterilized
using a validated process. Store ata temperature between 2ºC
and 25ºC and protect from light. If the prepared media are
stored in unsealed containers, they should be used within 3
weeks. If stored in suitable sealed containers, the media
should be used within 1 year.
l. Fluid thioglycollate medium
Pancreatic digest of casein 15.0 g
Y east extract 5.0 g
Glucose anhydrous 5.0 g
Sodium chloride 2.5 g
L-Cystine 0.5 g
O. 1% Resazurin sodium solution (freshly prepared) l. O ml
Sodium thioglycollate o. 5 g
or (Thioglycollic acid) co. 3 mD
Agar o. 75 g
any of the individual results differ from the mean by more
than 5. O%, repeat the test and express the results of the six
measurements as the mean and the relative standard deviation
( RSD) , the relative standard deviation should less than
10. 0%.
Water 1000 ml
Mix the above ingredients except glucose and resazurin
solution with water to dissolve by warming gently, adjust the
pH to slightly alkaline, boíl and filter, add glucose and
resazurin solution, mix, adjust the pH so that after
sterilization the solution will have a pH of 7. 1±0. 2 at 25ºC.
Dispense the medium in suitable containers which provide a
ratio of surface to depth of medium such that not more than
the upper half of the medium has undergone a colour change
( pink colour) indicati ve of oxygen uptake at the end of the
incubation period. Sterilize using a validated process. When
ready for use, not more than the upper one-fifth of the
medium has acquired a pink colour, otherwise, the medium
can be restored only once by heating the containers in a
water-bath until the pink colour disappears ( not more than
20 minutes), cooled quickly, taken care to prevent the
introduction of non-sterile air into the container.
Unless otherwise specified, Fluid Thioglycollate Mediwn is
to be incubated at 30-35ºC.
2. Soya-bean casein digest medium
17.0 g
Pancreatic digest of casein
Sodium chloride 5.0 g
Papaic digest of soya-bean meal 3.0 g
Dipotassium hydrogen phosphate 2.5 g
Glucose monohydrate/ anhydrous 2.5 g/2.3 g
Water 1000 ml
Mix the above ingredients except glucose to dissolve by
warming gently, filter, adjust the pH so that after
sterilization the solution will have a pH of 7. 3±0. 2 at 25ºC,
add glucose, dispense and sterilize.
Soya-bean casein digest medium is to be incubated at 10-25ºC.
3. Neutralizing or inactivating medium
Before sterilization or inoculation, add proper quantity of
suitable neutralizer or inactivator or surface-active substance
to each of the media described above. The quantity of
neutralizer or inactivator or surface-active substance adopted
in sterility test should be the same as that used in validation
test.
4. O. S %glucose broth medium
( used in the test of antibiotic such as Streptomycin sulfate)
Peptone 10.0 g
Glucose 5. O g
Beef powder 3. O g
Sodium chloride 5. O g
Water 1000 ml
Mix the above ingredients except glucose with water to
dissolve by warming gently, adjust the pH to slightly
alkaline, boíl, add glucose, mix well, filter, adjust the pH
so that after sterilization the solution will have a pH of 7. 2±
O. 2 at 25ºC, dispense and sterilize.
S. Soya-bean casein digest agar medium
Pancreatic digest of casein 15.0 g
Sodium chloride 5.0 g

Papaic digest of soya-bean meal 5. O g
Agar 15. O g
Water 1000 ml
Mix the above ingredients except agar to tepidly dissolve,
adjust the pH so that after sterilization the solution will have
a pH of 7. 3±0. 2 at 25ºC, add agar, heat to melting, shake
well, dispense, and sterilize.
6. Sabouraud dextrose broth medium
Mixture of equal amounts of animal tissue pepsin
10.0 g
hydrolyzate and Tryptone
Dextrose 20.0 g
Water 1000 ml
Mix above ingredients except glucose to tepidly dissolved,
adjust the pH so that pH value after sterilization at 25ºC is
5. 6±0. 2, add dextrose, shake well, dispense and sterilize.
7. Sabouraud dextrose agar medium
Mixture of equal amounts of animal tissue pepsin
hydrolyzate and Tryptone 10.0 g
Dextros e 40.0 g
Agar 15.0 g
Water 1000 ml
Mix above ingredients except dextrose and agar to tepidly
dissolve, adjust the pH so that pH value after sterilization at
25ºC is 5. 6 ±O. 2, add agar, heat to melting, then add
dextrose, shake well, dispense and sterilize.
Suitability tests of medium
The fluid thioglycollate medium, soya-bean casein digest
medium and the other media used in the test for sterility, as
described above, should comply with the following sterility
and sensitivity tests, carried out before or in parallel with the
test on the product to be examined.
Sterility tests lncubate not less than 5 vessels of each batch
of sterilized medium at the temperature required for each
medium for 14 days. Growth of microorganisms shouid not
occur.
Test for sensitivity of medium
Test strains
The viable microorganisms used in the test must not be more
than five generations ( the dried strains obtained from
Culture Collection Centre are defined as Generation O). The
suitable seed-stock technique should be used so that the
microor-ganism characters can be maintained.
Staphylococcus aureus [CMCG (B) 26003]
Pseudomonas aeruginosa [CMCC (B) 10104]
Bacillus subtilis [CMCC (B) 63501]
Clostridium sporogelles [CMCC (B) 64941]
Candida albicans [CMCC (F) 98001]
Aspergillus niger [CMCC (F) 98003]
Preparation of inoculum lnoculate freshly cultured
Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus
subtilis into soya-bean casein digest medium or soya-bean
casein digest agar medium and freshly cultured Clostridium
sporogelles into fluid thioglycollate medium, incubate at 30-
35ºC for 18-24 hours. Inoculate freshly cultured Candida
albicans into sabouraud dextrose broth medium or sabouraud
dextrose agar medium, incubate at 20-25ºC for 24-48 hours.
Dilute the above cultures to produce a suspension, containing
less than 100 CFU microorganisms per ml, with pH 7. O
sterile sodium chloride-peptone buffer or O. 9 % sterile
sodium chloride solution. lnoculate freshly cultured Asper-
gillus niger into sabouraud dextrose agar medium, incubate
at 20-25ºC for 5-7 days until good sporulation is obtained.
Wash the Aspergillus niger spore culture with 3-5 ml of pH
7. O sterile sodium chloride-peptone buffer or O. 9 % sterile
1101 Sterility Tests
sodium chloride solution containing O. 05 % ( ml/ ml) of
polysorbate 80 and transfer it to sterile tube. Prepare spore
suspension containing less than 100 CFU per ml with pH 7. O
sterile sodium chloride-peptone buffer or O. 9 % sterile
sodium chloride solution containing O. 05% ( ml/ml) of
polysorbate 80.
Use the above suspensions within 2 hours if stored at ambient
temperature, or within 24 hours if stored between 2-8ºC. The
stable spore suspension of A. niger may be maintained at 2-
8ºC for a validated period of time.
Inoculation of medium Take 7 containers of fluid thiogly-
collate medium ( containing 12 ml medium per container),
inoculate 2 containers of the medium with less than 100 CFU
test microorganisms of Staphylococcus aureus, Pseudomonas
aeruginosa , and Clostridium sporogenes respectively, and
the remainmg uninoculated culture medium ( one container)
is used as blank control, then incubate for 3 days. Take
7 containers of soya-bean casein digest medium (containing 9
ml medium per container), inocula te 2 containers of the
medium with less than 100 CFU test microorganisms of
Bacillus subtilis, Candida albicans and Aspergillus niger,
respectively, and use the remaining uninoculated culture
medium ( one container) as blank control, incubate for
5 days. Observe the test containers for growth of the
microorganisms every day during the incubation.
Intepretation of Results No growth of microorganisms shall
be observed in the blank control. If a clearly visible growth of
the microorganisms occurs in an inoculated culture medium,
the medium meets the requirement of the test for sensitivity
of medium.
Diluents, Rinsing Fluids and
Their Preparation Methods
Prepare diluents and rinsing fluids and sterilize using a
validated process.
l. O. 1 % sterile peptone solution
Dissolve tepidly l. O g of peptone in 1000 ml of water, filter,
adjust the pH to 7. 1±0. 2, dispense and sterilize.
2. pH 7. O sterile sodium chloride-peptone buffer
Mix 3. 56 g of potassium dihydrogen phosphate, 5. 77 g of
anhydrous disodium hydrogen phosphate, 4. 30 g of sodium
chloride and l. 00 g of peptone with 1000 ml of water to
tepidly dissolve, filter, dispense and sterilize.
According to the nature of the product being examined, other
suitable solutions which are validated may also be adopted
(e. g. , O. 9 % sterile sodium chloride solution).
If necessary, add suitable neutralizer or surface-active
substance to each of the diluents or rinsing fluids described
above before or after sterilization
Method Suitability Test
In the course of sterility testing method for product to be
examined, the method must be verified to ensure that the
adopted method is suitable for sterility test of the product.
Whenever there is a change of test procedure or drug product
which might impact the test result, the testing method must
be revalidated.
Method suitability test is to be conducted as described below
under Test for sterility of the product being examined using
exactly the same methods and the following instructions. The
tests should be performed separately for each of the
microorganism tested.
Tested strains and preparation of inoculum Except for
Escherichia coli [ CMCC ( B) 44102], preparation of the
strains and the inoculums of Staphylococcus aureus, Baci-
 1101 Sterility Tests
llus suhtilis, Clostridium sporogenes, Candida albicans,
Aspergillus niger is the same as described in the test for
sensitivity of medium. Preparation of E. coli inoculums is the
same as Staphylococcus aureus.
Membrane filtration Filter the specified quantity of the test
specimen with membrane filtration apparatus, rinse the
membrane, add test microorganism with less than 100 CFU
to the final portien of rinsing fluids, filter again. Add fluid
thioglycollate medium or soya-bean casein digest medium to
the filtration apparatus. Use another container containing the
same volume of the medium, then add the same amount of
test microorganisms as control. lncubate the containers at
specified temperature for not more than 5 days. Repeat the
procedure for each of the test microorganism tested.
Direct inoculation Take 6 containers containing certain
required volume of fluid thioglycollate medium for direct
inoculation, inoculate 2 containers of medium with less than
100 CFU test microorganisms of Staphylococcus aureus,
Escherichia coli, and Clostridium sporogenes separately.
Take 6 containers of soya-bean casein digest medium
complying with the required volume for direct inoculation,
inoculate 2 containers of the medium with less than 100 CFU
test microorganisms of Bacillus suhtilis , Candida albicans
and Aspergillus niger separately. Add specified quantity of
the product being tested to one of the inoculated containers of
each test micro--organism, the other inoculated container is
for control. lncubate the bacterial containers at specified
temperture for not more than 5 days.
lnterpretation of Results Compare with the control
container, if clearly visible growth of each test microor-
ganisms is obtained in the test containers containing the
product to be tested, visually either the product possesses no
antimicrobial activity under the conditions of the test, or
such activity has been satisfactorily eliminated. The test for
sterility of the product being examined may then be carried
out using the same method and conditions of the test. If the
growth of test microorganisms is not obtained, or poor, or
slow in any tested container, visually comparable to that in
the control container without product, then the product with
the specified quantity possesses antimicrobial activity under
the conditions of the Test. In such a case, modify the
conditions in order to eliminate the antimicrobial activity by
using a large amount of rinsing fluid, or increasing the
volume of medium, or using suitable neutralizer or
inactivator, or replacing the type of the membrane filter used
etc. and repeat the validation test.
The method suitability test may also be performed
simultaneously with the Test for sterility of the product
being examined.
Test for Sterility of the Product Being Examined
The test for sterility is carried out by the membrane filtration
method or direct inoculation method. The membrane
filtration method is used whenever the nature of the product
permits. The same method and experimental conditions
should be adopted in sterility test as that validated in method
suitability test.
If surface-active substances, neutralizers or inactivators,
etc., are used during the test, their efficacy and their absence
of toxicity for microorganisms must be demonstrated.
Number of products to be tested It is the number of the
mínimum package of the product to be examined for sterility
test once. Unless otherwise specified, the number of the
products to be tested is specified in Table l. The number of
final product to be tested for post-marketing surveillance is
specified in Table 2. The number of the products for positive
control test is not included in table 1 and 2.
Quantity of product to be tested lt is the mínimum quantity
( g or ml) of products tested each mínimum packaging
inoculated into each medium for sterility test once. Unless
otherwise specified, the mínimum quantity of each container
of the product for each medium are defined in Table 3. If the
contents in a single container ( bottle) are sufficient to
inoculate two containers of medium, they should be trans-
ferred to fluid thioglycollate medium and soya-bean casein
digest medium respectively. When using the technique of
membrane filtration, whenever permitted, the whole
contents of each container should be filtered for testing.
Positive control The microorganisms for positive control
should be selected according to the nature of the product
being examined. Staphylococcus aureus is used for the
product possessing no antimicrobial activity or mainly anti-
gram-positive-bacteria activity. Encherichia coli is used for
the product mainly possessing anti-gram-negative-bacteria
activity. Clostridium sporogenes is used for the product
possessing anti-anaerobic bacteria activity. Candida albicans
is used for the product possessing fungistatic activity.
Preparation of the inoculums for positive control is the same
as described under the method suitability test, the number of
test microorganism added should be less than 100 CFU, the
quantity of the product used is the same as described under
the Test for sterility of the product being examined for each
container of medium. lncubate the positive control container
for 72 hours, the growth of the test microorganisms should
be well.
Negative control In the course of the sterility test for
examined product, a negative control should be performed
with the same solvent or diluents or rinsing fluids as used for
the test. There must be no growth of microorganisms.
Processing of test and medium inoculation
Before opening the product containers to be tested, the
exterior surfaces of containers must be thoroughly cleansed
with a suitable disinfectant. If the containers are packaged
under vacuum, sterile air should be transmitted into the
container aseptically with suitable equipments (e. g., needles
with sterilizing fil ter) before the container is opened to
release the contents.
Unless otherwise specified, processing of test and medium
inoculation should be carried out as describled below.
1. Membrane filtration method
Sealed sterility testing system should be used in the technique
of membrane filtration. The membrane used in the test for
sterility has a nominal pore size which is not greater than
O. 45 µm and a diametre about 50 mm. Selection of filtration
membrane type should be performed according to the
characters of product to be tested and solvent used. lntegrity
of the membrane in the apparatus should be kept during the
process filtration.
When filtering aqueous solution, the membrane in the filter
should be pre-wetted with a small quantity of rinsing fluids.
When the samples tested are oily preparations, the filtration
apparatus and membrane must be thoroughly dried before
use. The level of the sample solution and rinsing fluid should
always cover the entire surface of the membrane throughout
the operation for maximal filtration efficiency. If necessary,
wash the membrane by filtering through it 100 ml of rinsing
fluid each time, and the total volume used should not exceed
1000 ml for one single membrane to avoid any damage to the
microorganisms held by the membrane, after filtering the
sample solution.

Aqueous solutions Transfer the specified quantity of samples
tested directly into the membrane or membranes, or mix the
samples into an aseptic vessels containing not less than
100 ml suitable quantity of the sterile diluent and transfer the
mixture to the membrane or membranes. Filter immediately.
If the product being examined contain preservatives, the
membrane should be washed not less than three times by
filtering through it a suitable quantity of the rinsing fluid
same as described under the method suitability test. Except
far biological products, after washing, transfer 100 ml of
fluid thioglycollate medium into one filter, and soya-bean
casein digest medium into another filter. For biological
products, after washing, transfer 100 ml of fluid thiogly-
collate medium into 2 filter, and soya-bean casein digest
medium into one other filter.
Solid soluble in aqueous vehicle Transfer the specified
quantity of specimens tested, dissolve with suitable sterile
diluent or as directed in the label. Proceed with the test as
directed above far aqueous solutions.
Water insoluble substances Transfer the specified quantity of
specimens and filter directly, or mix in a sterile diluent
containing polysorbate 80 or other suitable emulsifying
agent, and transfer it into the membrane or membranes.
Filter immediately. Wash the membrane at least 3 times with
rinsing fluid containing O. 1 %-1 % polysorbate 80. The mem-
branes are inoculated to medium with or without containing
polysorbate 80. Inoculate medium as directed above far
aqueous solutions.
Ointments and viscous oils soluble in isopropyl myristate Mix
specified quantity of specimens tested in suitable quantity of
sterile isopropyl myristatel shake vigorously to completely
dissolve the sample. If necessary, heat the mixture to not
more than 44 ºC , and filter as rapidly as possible befare the
mixture cools clown. In exceptional cases when the mixture is
unable to be filtered, more than 100 ml of diluents should be
added to the above mixture, shake sufficiently and extract
the solution, then transfer the aqueous phase under layer into
the membrane or membranes and filter immediately. Proceed
washing membrane and inoculating medium as described
above far water insoluble substances.
Sterile aerosol ( or nebulization) products Transfer the
specified quantity of product containers tested to a freezer,
and freeze at-20ºC or other suitable temperture far about
1 hour. Aseptically drill quickly a hole in the top end of each
container and release the propellant befare aseptically opening
the container, then transfer the contents in to a sterile vessel.
Proceed as directed above far aqueous solutions or water
insoluble substances.
Prefilled syringes with drug Transfer the specified quantity
of specimens tested and filter directly (if necessary, dissolve
in suitable sterile diluent or in solvent specified by product
label) , or transfer the contents of each container of the
products to be tested into a sterile vessel containing suitable
sterile diluent, proceed with the test as directed above far
aqueous solutions or water insoluble substances. Needles
included in the product package should also be tested far
sterility using appropriate method.
Devices ( e. g. , transfusion bag) with pathways labeled sterile
Take the specified quantity of specimens tested, flush each
package inner wall with 50-100 ml of flushing fluid. Collect
the fluids in an appropriate sterile vessel, proceed with the
test as directed above far aqueous solutions. Needles included
in the product package should also be tested far sterility
using direct inoculation method.
1101 Sterility Tests
11
2. Direct inoculation method
Direct inoculation method is only used when the nature of the
product does not permit far membrane filtration method.
Directly transfer the specified quantity of product tested
equally into fluid thioglycollate medium and soya-bean casein
digest medium respectively. Except biological products,
inoculate the products for sterility testing with the same
amounts of fluid thioglycollate medium and soya-bean casein
digest medium. For biological products, the proportion of
fluid thioglycollate medium and soya-bean casein digest
medium inoculation bottles is 2 : 1 in sterility test. Unless
otherwise specified, in each container incubated, the volume
of samples tested is not more than 10 % of the volume of the
medium, and the volume of fluid thioglycollate medium
should not be less than 15 ml, the volume of soya-bean
casein digest medium should not be less than 10 ml. Use the
same medium volume and height in the container as in the
method suitablity test.
Turbid aqueous solution Transfer the specified quantity of
specimens tested equally into each culture medium.
Solid articles Transfer the specified quantity of specimens
tested directly and equally into each culture medium. Or
prepare a suspension of the product with suitable sterile
solvent or as directed in the label, then transfer the specified
quantity of specimens equally into each culture medium.
Water insoluble substances Transfer the specified quantity of
specimens tested to a sterile container, add the diluent
containing suitable quantity of polysorbate 80 or other
emulsifying agent to the container and mix to emulsify the
specimens. lnoculate the suspension prepared above equally
into each culture medium. Alternatively, transfer directly the
specified quantity of specimens equally into each culture
medium containing polysorbate 80 or other emulsifying
agent.
Dressing Use the specified number of product as prescribed,
open the sealed package with aseptic precautions, and remove
about 100 mg or 1 cm X 3 cm from different parts of
specimens, transfer the test parts equally into each sufficient
culture medium to cover adequately the material to be tested.
Catgut and suture etc. Use the specified number of smallest
package of catgut, suture and other disposables medical
materials as prescribed, open the sealed package with aseptic
precautions, inocubate equally into each sufficient culture
medium to cover adequately the material to be tested.
Sterile medica) devices Use the specified number of product
as prescribed. If necessary, articles can be disassembled or
cutted into small sections, and transfered equally into each
sufficient culture medium to cover adequately the material to
be tested.
Radiopharmaceutical Use one bottle of product, respe-
ctively transfer O. 2 ml of product into 7. 5 ml of fluid
thioglycollate medium and soya-bean casein digest medium.
Incubation and observation
lncubate the medium containers at specified temperature as
prescribed of each medium far 14 days. For biological
products, incubate half of fluid thioglycollate medium
containers above at 30-35ºC, half of fluid thioglycollate
medium containers above at 20-25ºC far 14 days. Observe
and record each medium container far evidence of microbial
growth every day during incubation period. If the product
being tested renders the medium turbid so that the presence
or absence of microbial growth cannot be readily determined
by visual examination, 14 days after the beginning of
 1101 Sterility Tests

incubation, transfer suitable portions of the medium to fresh examined. The test may be considered invalid only when one
containers with the same kind of medium, and incubate the or more of the fallowing conditions are fulfilled.
containers 3 days, then examine whether the containers are (1) The result of the microbiological monitoring of the
turbid far evidence of microbial growth, or smear medium on sterility testing facility and environment shows that it does
microslide, stain and examine with microscope far the not meet the requirements of the test far sterility.
suspected microbial growth. (2) A review of the testing procedure used during the test in
question reveals a fault of microbial pollution.
lnterpretation of Results (3) After determination of the identification of the
Microbial growth should be evident in the positive control microorganisms isolated from the test, the growth of this
container, and must be not faund in the negative control species (or these species) may be ascribed unequivocally to
container. Otherwise, the test may be considered invalid. faults in respect with the material or the technique used in
Except the positive control container, if all of the test conducting the sterility test procedure.
containers described above are clear or turbid but no evidence If the test is validated to be invalid, it should be repeated
of microbial growth, the product to be examined complies with the same number of the samples and procedure as in the
with the test far sterility; if anyone of the test containers is original test. If no evidence of microbial growth is faund in
turbid, the product to be examined does not comply with the the repeated test, the product examined complies with the
test far sterility, unless it can be clearly demonstrated that test far sterility; if microbial growth is faund, the product
the test was invalid far causes unrelated to the product to be examined does not comply with the test far sterility.
Table 1 Minimum number of products and final bulk or bulk of biologics to be tested from each batch
Mínimum number of items to be tested
Product to be tested Number of items in the batch(N)
far each medium
N is not more than 100 10% or 4 containers(whichever is greater)
N is more than 100 but not more than 500 10 containers
Parenteral preparations
2 % or 20 containers( whichever is less)
N is more than 500
20 containers(biologics)
Far large-volume parenteral 2 % or 1O containers( whichever is less)
preparations(more than 100 mD 20 containers(biologics)
N is not more than 200 in each
5 containers
more than freeze-drying cabinet
5 ml N is more than 200 in each
Freeze-dried 10 containers
freeze-drying cabinet
blood products
N is not more than 100 5 containers
not more
N is more thanlOO but not more than 500 10 containers
than 5 ml
N is more than 500 20 containers
Ophthalmic and other non- N is not more than 200 5 % or 2 containers( whichever is greater)
injectable preparations N is more than 200 10 containers
N is not more than 4 Each container
Barreled sterile salid
N is more than 4 but not more than 50 20% or 4 containers(whichever is greater)
raw materials
N is more than 50 2% or 10 containers(whichever is greater)
Antibiotic salid raw
6 containers
material medicine(~5 g)
Each container ( the mínimum quantity to be
tested should be O. 1 % of the whole quantity
Final bulk and bulk of biologics in each container or not less than 1O ml. As
described above at each opening of the
container.)
Diagnostic products and
Each batch(not less than 3 ml)
semi-finished products in vitro
N is not more than 100 10% or 4containers(whichever is greater)
Medical devices Nis more than 100, but not more than 500 10 articles
N is more than 500 2 % or 20 articles( whichever is less)
Note: If the contents of one container are not enough to inoculate the two media, the mínimum number of items to be tested
should be increased to the corresponding ratio.

Table 2 Minimum number of product to be tested for post-marketing surveillance

Product to be tested Mínimum number of items to be tested(bottle or count)

Liquids 10
 1105 Microbiological E:xamination of Non-sterile Products: Microbial Enumeration Tests

continued

Product to be tested Minimum number of items to be tested(bottle or count)

Solids 10
less than 50 ml 6
Blood products
50 ml or more 2
Medica! devices 10
Note: l. If the contents of one container are not enough to inocula te the two media, the mínimum number of items to be tested
should be increased to the corresponding ratio.
2. Mínimum number of antibiotic powder (~5 g) and antibiotic raw material medicine (~5 g) is 6 bottles Cor counts).
Minimum number of barreled. sterile solid raw materials is 4 packages.

Table 3 Minimum quantity of product to be tested

Product to be tested Quantity per container Mínimum inoculum quantity per container
Not more than 1 ml The whole contents of each container
Aqueous More than 1 ml, but not more than 40 ml Half the content, but no less than 1 ml
solutions More than 40 ml, but not more than 100 ml 20 ml
More than 100 ml 10%, but no less than 20 ml
Less than 50 mg The whole contents of each container
Not less than 50 mg and less than 300 mg Half the Contents of each container
Solid product Not less than 300 mg and less than 5 g 150 mg
500 mg
5 g or more
Half the contents of each container(biologics)
Bulk or final
Half the contents of each container
bulk of biologics
Surgical dressing/ cotton/ ga uze( in packages) 100 mg or 1 cmX3 cm

Sutures and other individually

The whole devicem
packaged single-use material
Medical devices The disposable medica! apparatus
Half of internal surface area
tu be( infusion bag)
The whole devicem , cut into pieces or
Other medical devices
disassembled
CD:If the volume of medical devices is too large, more than 2000 ml of medium should be used to immerse the devices
completely.

Unidirectional air flow area, work surfaces, and the environ-

1105 Microbiological Examination of
Non-sterile Products: Microbial
Enumeration Tests
Microbial enumeration tests described hereafter will allow
quantitative enumeration of mesophilic bacteria and fungí
which may grow under aerobic conditions.
The tests are designed primarily to determine whether non-
sterile pharmaceutical products including raw materials and
excipients comply with an established specification for
microbiological quality. When used for such purposes, follow
the instructions given below, including the number of
samples to be taken and interpret the results as stated below.
The methods are not applicable to products containing viable
microorganisms as active ingredients unless otherwise specified.
The conditions of the microbial enumeration test should
satisfy the requirement of microbiological examination.
Strictly sterile operation could avoid extrinsic microbial
contamination of the product. The precautions taken to avoid
contamination must be such that they do not affect any
microorganisms which are to be revealed in the test.
ment should be monitored regularly.
If the product to be examined has antimicrobial activity, this
is insofar as possible removed or neutralized. If neutralizers
or inactivators are used, their efficacy and their absence of
toxicity for microorganisms must be demonstrated.
If surface-active substances are used for sample preparation,
their absence of toxicity for microorganisms and their
compatibility with any neutralizers or inactivators used must
be demonstrated.
Enumeration Methods
Plate method, membrane filtration method and the most-
probable-number(MPN) method can be all used for microbial
enumeration test. MPN method is generally the least accurate
method for microbial counts; however, for certain product
groups with very low bioburden, it may be the most appro-
priate method.
The choice of a method is based on factors such as the nature
of the product and the required limit of microorganisms. The
method chosen must allow testing of a sufficient sample size
to judge compliance with the specification. The suitability of
the chosen method must be established.
 l105 Microbiological Examination of Non-sterile Products: Microbial Enumeration Tests

microbial counting method to confirm the method used is

Growth Promotion of the Counting Media and
suitable for the microbial count in the product to be
Suitability of the Counting Method examined.
The growth promotion of the media used in enumeration Suitability must be reconfirmed if a change in testing
tests must be confirmed. performance~ or the product, which may affect the outcome
Method suitability tests should be carried out for the of the test is introduced.
Table 1 Preparation and use of test microorganisms
Growth promotion Suitability of counting method
Preparation of
Microorganism Total aerobic Total molds
test strain Total aerobic Total molds
microbial count and yeasts count microbial count and yeasts count

Soya bean casein digest

Soya bean casein diges
Soya bean casein digest agar or casein soya
Staphylococcus agar or soya bean
agar or soya bean bean casein digest
aureus casein digest broth
[CMCCCB)
casein digest broth
<100 CFU
/ broth(MPN) /
30-35ºC <100 CFU
26003)] 30-35ºC
18-24 h 30-35ºC
< 3 days
< 3 days
Soya bean casein Soya bean casein
Soya bean casein digest agar or soya digest agar or soya
Pseudomonas
digest agar or soya bean casein digest bean casein digest
aeruginosa
[CMCCCB)
bean casein digest broth broth / broth(MPN) /
30-35ºC <100 CFU <100 CFU
10104]
18-24 h 30-35ºC 30-35ºC
< 3 days < 3 days
Soya bean casein Soya bean casein
Soya bean casein digest agar or soya digest agar or soya
Bacillus h~n~ ~"~~:~ ....1:~~~+
digest agar or soya bean casein digest uc:a.u. \...a,::n:u1 u15c:.:')L
suhtilis
[CMCCCB)
bean casein digest broth broth / broth(MPN) /
30-35ºC <100 CFU <100 CFU
63501]
18-24 h 30-35ºC 30-35ºC
< 3 days < 3 days
Soya bean casein
Sabouraud-dextrose Soya bean Sabouraud- Sabouraud-
Candida digest agar(MPN:
agar or sabouraud- casein digest agar dextrose agar dextrose agar
albicans Not applicable)
dextrose broth <100 CFU <100 CFU <100 CFU
[CMCC(F) <100 CFU
20-25ºC 30-35ºC 20-25ºC 20-25ºC
98001] 30-35ºC
2-3 days < 5 days < 5 days < 5 days
<5 days
Sabouraud-dextrose
Soya bean casein
agar or patato- Soya bean casein Sabouraud- Sabouraud-
Aspergillus digest agar(MPN:
dextrose agar digest agar dextrose agar dextrose agar
niger Not applicable)
20-25ºC <100 CFU <100 CFU <100 CFU
[CMCC(F) <100 CFU
5-7 days, or until 30-35ºC 20-25ºC 20-25ºC
98003] 30-35ºC
good sporulation < 5 days < 5 days < 5 days
< 5 days
is achieved
Note: When rose bengal agar is needed for the total number of molds and yeasts test, growth promotion of the media must be
confirmed according to sabouraud-dextrose agar.

Preparation of test strains and suspensions test strains The
viable microorganisms used for inoculation are not more than
5 generations ( the dried strains obtained from Type Culture
Collection are defined as Generation O). Use appropriate
culture collection techniques to preserve the test strains to
ensure their biological characteristics. Test strains used in
medium growth promotion test and counting method
suitability test are summarized in Table l.
Preparation of microbial suspension Inocula te the test strains
as the procedures described in Table l. Suspend fresh
cultures of Staphylococcus aureus, Pseudomonas aerugi-
nosa , Bacillus suhtilis , and Candida albicans in sterile
buffered sodium chloride-peptone solution pH 7. O or O. 9 %
sterile sodium chloride solution to make test suspensions of
suitable concentrations. Add 3-5 ml sterile buffered sodium
chloride-peptone solution pH 7. O contammg O. 05 % ( ml/
mD polysorbate 80 or O. 9 % sterile sodium chloride solution
in the fresh cultures of Aspergillus niger to elute the spores.
Then, suck out the spore suspension by appropriate method
and place into a sterile test tube. Prepare suitable concen-
trations of spore suspensions of Aspergillus niger with
sterile buffered sodium chloride-peptone solution containing
O. 05% (ml/mD polysorbate 80, pH 7. O, or O. 9% sterile
sodium chloride solution.
Use the suspensions within 2 h if stored at ambient
temperature; Use within 24 h if stored at 2-8ºC. The stable
spore suspension of Aspergillus niger may be maintained at
2-8ºC and used within the validated period of time.
Negative control
To verify testing conditions, a negative control should be
 1105 Microbiological Examination of Non-sterile Products: Microbial Enumeration Tests
performed. There must be no growth of microorganisms. A
failed negative control requires a deviation investigation.
Growth promotion of the media
The media used for microbial counting, such as ready-
prepared medium, the media prepared from dehydrated
culture media or prepared according to certain formulations,
should be validated for growth promotion.
lnoculate tubes/plates of soya bean casein digest broth and
soya bean casein digest agar or inoculate plates of sabouraud
dextrose agar with not more than 100 CFU of the microor-
ganisms indicated in Table 1 and incubate under the
conditions described in Table l. Use 2 tubes or 2 plates for
each test strain in parallel. At the same time, perform the
tests using corresponding reference media instead of the
media to be tested.
The ratio between average numbers of colonies on the solid
test media and reference media ranges in O. 5-2, and the
shape and size of colonies on test media should be consistent
with the reference media; the test microorganisms in the
tubes of liquid test media should grow well compared to the
microorganisms in the tubes of reference media.
Suitability of the counting method
1. Preparation of the sample
The method far sample preparation depends on the physical
and biological characteristics of the product to be tested. If a
heating procedure is required during preparation, the sample
solution should be heated evenly. The temperature should not
exceed 45ºC. The time interval between preparation and
addition into the test medium should be not more than 1 h.
The common methods used for sample solution preparation
are shown as follows. If none of the procedures described
below can be demonstrated to be satisfactory, a suitable
alternative procedure must be developed.
( 1) Water-soluble products Dissolve or dilute the product to
be examined in sterile buffered sodium chloride-peptone
solution pH 7. O, phosphate buffer solution pH 7. 2, or soya
bean casein digest broth to prepare a 1 : 10 dilution. If
necessary, adjust to pH 6-8. Further serial 10-fold dilutions,
where necessary, are prepared with the same diluent. The
stock solution of water-soluble liquid preparation can also be
used as the sample solution.
( 2 ) Non-fatty products insoluble in water Suspend the
product to be examined in buffered sodium chloride-peptone
solution pH 7. O, phosphate buffer solution pH 7. 2, or soya
bean casein digest broth to prepare a 1 : 10 dilution. A
surface active agent such as O. 1 % polysorbate 80 may be
added to assist the suspension of poorly dispersed substances.
If necessary, adjust to pH 6-8. Further serial 10-fald
dilutions, where necessary, are prepared with the same
diluent.
(3) Fatty products Dissolve the product to be examined in
sterile isopropyl myristate, or mix the product to be
examined with the mínimum necessary quantity of sterile
polysorbate 80 or other noninhibitory sterile surface-active
reagent. Temperature of the surface-active agent should be
not more than 40ºC or in exceptional cases, not more than
45ºC. Mix carefully and if necessary maintain the
temperature in a water-bath. Add sufficient of the pre-
warmed chosen diluent to make a 1 : 10 dilution of the
original product. Mix carefully, while maintaining the
temperature for the shortest time necessary for the formation
of an emulsion. Further serial 10-fold dilutions may be
prepared using the chosen diluent containing sterile surface-
active reagent with the same concentration.
( 4) Products required special preparation methods
Film products Cut the product to be examined in to pieces,
add sterile buffered sodium chloride-peptone solution pH
7. O, phosphate buffer solution pH 7. 2, or soya bean casein
digest broth, soak, shake to prepare a 1 : 10 dilution. If
necessary, adjust to pH 6-8. Further serial 10-fald dilutions,
where necessary, are prepared with the same diluent.
Enteric and colonic-coated products Place the product to be
examined in sterile buffered phosphate solution pH 6. 8 ( for
enteric-coated products ) or sterile buffered phosphate
solution pH 7. 6 (far colonic-coated products) , maintain in
45ºC water-bath, and shake to dissolve, to prepare a 1 : 10
dilution. Further serial 10-fald dilutions, where necessary,
are prepared with the same diluents.
Fluids or solids in aerosol form Froze the product to be
examined at - 20ºC or other suitable temperature far 1 h.
Take out the product and disinfect the position to be opened
rapidly. Drill a hale in the disinfected position with a sterile
steel cone. Place until the product recovers back to ambient
temperature, and turn the container gently to release all the
propellant slowly. Other suitable methods can be used also
far sampling. Aseptically transfer all the solution from each
container by sterile syringes into a sterile container, mix and
take samples for examination.
Transdermal patches Remove the protective cover sheets of
the transdermal patches and place them, adhesive side
upwards, on sterile glass or plastic trays. Cover the adhesive
surface with a suitable sterile porous material (e. g., sterile
gauze) to prevent the patches from sticking together, and
transfer the patches to a suitable volume of the chosen diluent
containing surfactants, such as polysorbate 80 or lecithin.
Shake the preparation vigorously for at least 30 minutes.
Further serial 10-fold dilutions, where necessary, are
prepared with the same diíuent.
2. Inoculation and dilution
Inoculate and dilute the sample prepared according to
fallowing requirements. Prepare samples for microbial
recovery test. The volume of the microbial suspension should
not exceed 1 % of the volume of diluted product. To
demonstrate acceptable microbial recovery from the product,
the lowest possible dilution factor of the prepared sample
must be used far the test.
( 1 ) Test group Add the microbial suspension into the
sample prepared as described above and mix. The quantity of
microorganisms in 1 ml sample or solution filtered by each
membrane filter should be not more than 100 CFU.
( 2) Test control group Perform the same examination as
the test group using diluents of prepared sample instead of
the microbial suspension.
( 3 ) Control group of the microbial suspension Add the
microbial suspension into corresponding diluents without
neutralizer, inactivater and surfactants instead of the sample
and perfarm the same examination as the test group and
microbiological recovery test.
If it is impossible to choose the sample of the lowest diluted
level suitability test due to antimicrobial activity or poor
solubility of the sample, further appropriate protocols must
be developed to treat the sample. If inhibition of growth by
the sample cannot be avoided by other methods, the aliquot
of the microbial suspension may be added after
neutralization, dilution, or membrane filtration.
3. Neutralization or removal of antimicrobial activity
After inoculation, obtain the microbial count following the
 1105 Microbiological Examination of Non-sterile Products: Microbial Enumeration Tests
procedure described in Recovery of Microorganism. If the
number of colonies of the test group minus that of the test
control group is less than 50 % of the number of colonies of
the control group of the microbial suspension, the following
procedures should be carried out to remove the antimicrobial
activity of samples.
( 1) An increase in the volume of the diluents or culture medium;
(2) Incorporation of an appropriate neutralizing or inactive--
ting agents.
Neutralizing or inactivating agents may be used to neutralize
or remove the activity of antimicrobial agents (see Table 2).
They may be added to the chosen diluents or the medium prefera-
bly before sterilization. If used, their efficacy and their absence of
toxicity for microorganisms must be demonstrated by carrying
out a blank with neutralizer or inactivator and without product
The ratio of the number of colonies of the neutralizer or
inactivator group and the number of colonies of the microbial
suspension group should fall in the range of O. 5-2.
Table 2 Common neutralizing or inactivating
agents/methods for interfering substances
Potential neutralizing or
Interfering substance
inactivating agents/ method
Glutaraldehyde, mercurials Sodium hydrogen sulfite
Phenolics, alcohol, Dilution
aldehydes, sorba te
Aldehydes Glycine
Quaternary ammonium Lecithin
compounds ( QCAl) ,
parahydroxybenzoa tes
(parabens), bisbiguanides
QACs, iodine, parabens Polysorbate
Mercurials Thioglycollate
Mercurials, mercuric Thiosulfate
compounds, aldehydes
EDTA, Quinolones Mg or Ca ions
Sulfonamides Para-aminobenzoates
beta-lactam antibiotics beta-lactamase
(3) Membrane filtration.
( 4) A combination of the above measures.
If no suitable neutralizing method can be found, the failure of
special test strains recovery suggested the antimicrobial
activity of the product. This information serves to indicate
that the article is not likely to be contaminated with the given
species of the microorganism. However, it is possible that
the product inhibits only sorne of the microorganisms
specified herein, but does not inhibit others not included
among the test strains or those for which the latter are not
representative. Therefore, the test should be performed with
a higher dilution factor in accordance with microbial growth
and the specific acceptance criterion, provided they had no
interfere with the interpretation of test results. If the
suitability of the method meets the requirements, this
dilution factor should serve as the lowest dilution factor to
perform the tests of the product.
4. Recovery of microorganism in the product
For each of the microorganisms listed in Table 1, separate
tests are performed. The plate method, membrane filtration
method, or the MPN method can be used in the recovery of
microorganisms.
( 1) Plate method The plate methods consist of the pour-
plate method and the surface--spread method. For each of the
microorganisms listed in Table 1, perform plate methods at
least in duplicate for each medium and calculate the mean
count of each group as the result.
Pour-plate method For sterile petri dishes 90 mm in
diametre, add into the dish 1 ml of the sample prepared as
described under Preparation of the Sample, Inoculation
and Dilution, and Neutralization/Removal of Antimi-
crobial Activity and 15-20 ml of soya bean casein digest agar
or sabouraud-dextrose agar. Both media maintained at not
more than 45ºC, mix, solidify, and incuba te upside clown. If
larger petri dishes are used, the amount of agar medium is
increased accordingly. Incubate the plates as indicated in
Table 1 and perform the counting. The numbers of
microorganisms in test control group and the control group of
the microbial suspension should be determined with the same
method. Calculate the mean count of each group.
Surfac~spread method For sterile petri dishes 90 mm in
diametre, add 15-20 ml of soya bean casein digest agar or
sabouraud-dextrose agar at not more than 45ºC to each pétri
dish and allow to solidify to form plates. Dry the surface of
medium with appropriate methods. If larger petri dishes are
used, the amount of agar medium is increased accordingly.
Spread a measured volume of not less than O. 1 ml of the
sample, prepared as directed under Preparation of the
Sample, Inoculation and Dilution, and Neutralization/
Removal of Antimicrobial Activity over the surface of the
plate. Incubate the plates and perform the counting as
indicated in Table l. The numbers of microorganisms in test
control group and the control group of the microbial
suspension should be determined by the same method
Calculate the mean count of each group.
(2) Membrane filtration Use membrane filters having a
nominal pore size not greater than O. 45 µm, and generally
50 mm in diametre. If membrane fílters of different
diametres are used, the volume of rinse should be adjusted
accordingly. The type of filter material is chosen in such a
way that the bacteria-retaining efficiency is not affected by
the components of the sample to be investigated or the
solvents. Filters and membranes should be sterilized by
appropriate methods before use. When used, the integrity of
the membrane should be ensured before and after filtration.
Before the filtration of water-soluble products, filter a small
amount of rinsing fluid to wet the membrane. For fatty
products, the membrane and filter should be dried thorou-
ghly before use. For the maximum filtration efficiency of the
membrane, attention should be paid to maintaining the entire
surface of the membrane covered by the sample solution and
the rinsing liquid. After membrane filtration, if the
membrane needed to be rinsed, the volume of rinsing liquid
is 100 ml per membrane for each time. The total volume
should not exceed 1000 ml to avoid the microorganisms on
the membrane being injured.
Add a suitable quantity of the sample prepared as described
under Preparation of the Sample, Inoculation and
Dilution, and Neutralization/Removal of Antimicrobial
Activity (preferably representing 1 g, 1 ml or 10 cm2 of the
product, or less if large numbers of microorganisms are
expected) into an appropriate volume of diluents, mix,
filter, and rinse the membrane with rinsing liquid.
For the determination of total aerobic microbial count
CTAMC) , transfer the membrane fil ter and spread on plates
of soya bean casein digest agar with the surface containing
microorganisms upside; For the determination of total
combined yeasts/molds count CTYMC) transfer the mem-
 1105 Microbiological Examination of Non-sterile Products: Microbial Enumeration Tests

brane filter and spread on plates of sabouraud-dextrose agar continued

with the surface containing microorganisms upside. lncubate Number of g or ml
the plates as indicated in Table l. Perform the counting. For of product per tube MPN/g Lower Upper
each of the test strains and media, at least one membrane or ml limit limit
filter is used for each medium. The numbers of microor- o. 1 0.01 0.001
ganisms in test control group and the control group of the 3 o o 23 5 94
microbial suspension should be determined with the same 3 o 1 38 9 104
method. 3 o 2 64 16 181
( 3) MPN Method The precision and accuracy of the MPN
3 1 o 43 9 181
3 1 1 75 17 199
Method is less than that of the Membrane Filtration method
3 1 2 120 30 360
or the Plate Method. Far these reasons, the MPN Method is
3 1 3 160 30 380
only reserved for the enumeration of sample T AMC in
situations where no other method is available and it is not
3 2 o 93 18 360
3 2 1 150 30 380
applicable to the enumeration of molds. If the use of the
3 2 2 210 30 400
method is justified, proceed as follows.
3 2 3 290 90 990
Prepare a series of at least 3 serial 10-fold dilutions of the
product as described under Preparation of the Sample,
3 3 o 240 40 990
3 3 1 460 90 1980
Inoculation and Dilution, and Neutralization/Removal of
3 3 2 1100 200 4000
Antimicrobial Activity. From each level of dilution,
3 3 3 >1100
3 aliquots of 1 ml are used to inoculate 3 tubes with 9-10 ml
of soya bean casein digest broth. The control group of the Note: When the amounts listed in the table switch to 1 g (or
microbial suspension should be determined with the same mD, O. 1 g (or mD, and O. 01 g (or mD, the figures in this
method. If necessary, a surfactant, a neutralizer or an table should be correspondingly reduced by 10 times; when
switch to O. 01 g ( or mD, O. 001 g ( or mD, and O. 0001 g
inactivator may be added into the medium.
lncubate all tubes at 30-35ºC for 3 days. Observe the growth (or mD, the number should be multiplied by 10 times. Other
situation could be done by analogy.
of microorganisms in each tube daily. If reading of the results
is difficult or uncertain owing to the nature of the product to S. lnterpretation of results
be examined, subculture in the same broth, or soya bean When verifying the suitability of the membrane filtration
casein digest agar, for 1-2 days at the same conditions and method or the plate method, the ratio of the count differences
observe the growth of microorganisms. Determine the most between test group and the test control group to the control
probable number of T AMC per gram or millilitre of the group count should be not out of the range of O. 5-2. When
product to be examined from Table 3. verifying the suitability of the MPN method, the calculated
Table 3 Most-probable-number values of microorganisrm value from the inoculum must be within 95 % confidence
limits of the results obtained with the control. If the recovery
Numbers of tubes MPNof 95 % Confidence of each test strain meets the requirement, the count method
showing growth TAMC limits could be used to examine the total aerobic microbial count
Number of g or ml CTAMC) and total yeasts and molds count CTYMC).
of product per tube MPN/g Lower Upper If one or more of test organism cannot be recovered with any
or ml limit limit of the methods described above, choose the method and test
o. 1 0.01 0.001 conditions with the closest recovery to the criteria for test the
o o o o 9.4 product examination.
<3
o o 1 3 o. 1 9. 5 Testing of Products
o 1 o 3 o. 1 10
o 1 1 6. 1 l. 2 17 Test amount
o 2 o 6.2 l. 2 17 The amount used for the test refers to the quantity (g, ml or
o 3 o 9.4 3. 5 35 cm2 ) of the product to be examined for one test. Generally
1 o o 3. 6 0.2 17 the product to be examined should be taken no less than
1 o 1 7. 2 l. 2 17 2 mínimum packing units randomly. Then mix and take
1 o 2 11 4 35 specified amount of sample to test.
1 1 o 7.4 l. 3 20 Unless otherwise specified, the common amount used for the
1 1 1 11 4 35 test is 10 g or 10 ml of the product to be examined. For film
1 2 o 11 4 35 form, sample 100 cm2 • The amount to be tested may be
1 2 1 15 5 38 reduced for expensive medicines and micro-packaging drugs.
1 3 o 16 5 38 Samples should be taken from more than 2 mínimum packing
2 o o 9. 2 l. 5 35 units. For big honey pill, samples should not be less than
2 o 1 14 4 35 4 pills. Far film, sample should not be less than 4 patches.
2 o 2 20 5 38 Examination of the product
2 1 o 15 4 38 Examine the total aerobic microbial count ( T AMC) and
2 1 1 20 5 38 total yeast and mold counts ( TYMC) according to the
2 1 2 27 9 94 counting methods confirmed in Suitability of the counting
2 2 o 21 5 40 method.
2 2 1 28 9 94 Soya bean casein digest agar or soya bean casein digest broth
2 2 2 35 9 94 is used for the total aerobic microbial count ( T AMC).
2 3 o 29 9 94 Sabouraud-dextrose agar is used for the total yeasts and
2 3 1 36 9 94 molds count CTYMC).
 1106 Microbiological Examination of Non-sterile Products: Tests far Specified Microorganisms
Negative Control Test
Negative control test is conducted by replacing the sample
preparation with diluents according to the confirmed exami-
nation method There should be no growing bacteria.
Otherwise, deviation investigation should be perfarmed.
l. Plate methods
The plate methods consist of the pour-plate method and the
surface-spread method. Unless otherwise specified, take
specified amount of sample, prepare the sample and count
the organisms in the product according to a method that has
been confirmed during suitability of the counting method.
Prepare far each medium at least 2 petri dishes far each level
of dilution.
Incubation and enumeration Unless otherwise specified,
incubate the plates of soya bean casein digest agar at 30-35ºC
far 3-5 days and the plates of sabouraud-dextrose agar at 20-
25 ºC far 5-7 days. Observe the growth of microorganisms
daily. Count all the growing colonies and report. Far plates
with colonies spreading into pieces should not be counted.
After counting, calculate the mean count far each level
dilution and report in accordance with the reporting criteria of
microbial counts. If both counts at the same level of dilution
are not less than 15, the difference between the two petri
dishes should be not more than 1-fald
Reporting criteria of microbial counts
Far the total aerobic microbial count ( T AMC) choose a
dilution less than 300 CFU. Far total yeasts/molds count
(TYMC), choose a dilution less than 100 CFU. Both levels
of dilution described above are the basis of microbial counts
of reporting. Calculate the number of microorganisms
contained in 1 g, 1 mi or 10 cm2 of products based on the
highest mean colony count. Declare microbial counts of
reporting to 2 significant figures.
If no colony grows in the petri dishes of each dilution, or
only the lowest level of dilution grows less than 1 colony in
average, the colony count should be reported as <
1 multiplied by the lowest dilution factor.
2. Membrane filtration
Unless otherwise specified, prepare the sample using a
method that has been confirmed during Suitability of the
Counting Method. Transfer the appropriate amount of
sample equivalent to 1 g, 1 mi or 10 cm2 of the product to be
examined to the appropriate amount of diluents in accordance
with the confirmed method in accordance with Suitability of
the Counting Method, filter immediately and rinse. Far
samples containing larger numbers of microorganisms,
choose the suitable dilutions to filter. After rinsing, spread
the membrane on plates of soya bean casein digest agar or
sabouraud-dextrose agar with the surface containing microor-
ganisms upside.
lncubation and enumeration Far incubation and enum-
eration, proceed as plate method. The number of colonies per
membrane should not be more than 100 CFU.
Reporting criteria of microbial counts Report the number of
microorganisms equivalent to 1 g, 1 mi or 10 cm2 of the
product. If no colony grows on the membrane, report the
number of colony as <1 (1 g, 1 mi or 10 cm2 of the product
filtered by one piece of membrane) , or report the count as <
1 multiplied by the lowest dilution factor.
3. MPN method
Prepare and dilute the specified amount of sample using a
method that has been confirmed during Suitability of the
Counting Method. lncubate all tubes far 3-5 days at 30-35ºC.
Confirm microbial growth if necessary, using the procedure
shown under Suitability of the Counting Method. Record far
each level of dilution the number of tubes showing microbial
growth. Determine the most probable number of microor-
ganisms per gram or millilitre of the product to be examined
from Table 3.
Interpretation of the Results
The total aerobic microbial count (TAMC) is considered to
be equal to the total number of colonies faund in soya bean
casein digest agar ( including fungí). The total combined
yeast/ mold counts ( TYMC) are considered to be equal to
the number of colonies faund in sabouraud-dextrose agar
(including bacteria). When TYMC exceed the acceptance
criterion due to the bacteria} growth, sabouraud-dextrose
agar containing antibiotics ( such as chloramphenicol,
gentamicin) or other selective media ( such as rose bengal
agar) may be used. When a selective medium is used,
suitability of the medium should be tested. If MPN method is
adopted, it is far TAMC.
Microbial contamination acceptance limits far different
preparations is interpreted as fallows:
10 1 CFU: maximum acceptable count= 20;
10 2 CFU: maximum acceptable count= 200;
10 3 CFU: maximum acceptable count= 2000: and so farth.
If results of the total aerobic microbial count and the total
combined yeasts/mold count meet the specified requirements
far the product being examined, the product is qualified. If
one of them is out of the range, the product is unqualified
Diluents, Rinsing fluids, Medium
See Microbiological Examination of Non-sterile Products:
Tests far S pecified Microorganisms ( 1106 ) .
1106 Microbiological Examination of
Non-sterile Products: Tests f or
Specified Microorganisms
The tests far specified microorganisms described hereaf ter
will allow determination of the absence of, or limited
occurrence of, specified microorganisms that may be detected
in products.
The tests are designed primarily to determine whether non-
sterile pharmaceutical products, including raw materials and
excipients, comply with Microbial contamination limit of
pharmaceutical preparations. When used far such purposes,
fallow the instructions given below, including test amount
and result interpretation.
If specified microorganisms or other pathogens are detected
in products once, the product is unqualified. No retest should
be taken.
The sample preparation and experimental environmental
requirements are in accordance with that described in
Microbiological Examination of Non-sterile Products:
Aficrobial Enumeration Tests <1105).
If the product to be examined has antimicrobial activity, this
is insofar as possible removed or neutralized. If neutralizers
or inactivators are used in tests, their efficacy and their
absence of toxicity far microorganisms must be demonstrated.
If surfactants are used far sample preparation, their absence
of toxicity far microorganisms and their compatibility with
any neutralizers or inactivators used must be demonstrated.
Growth Promoting and Inhibitory Properties of
the Media and Suitability of Test Method
The suitability of the media used in the Tests for Specified
 1106 Microbiological Examination of Non-sterile Products: Tests for Specified Microorganisms

Microorganisms must be tested. lnoculate Clostridium sporogenes in reinforced medium for

Suitability of the test method should be carried out to clostridia under anaerobic conditions at 30-35ºC for 24-
guarantee that the method used for the tests of specified 48 hours or fluid thioglycollate medium at 30-35ºC for 18-
microorganisms in the product is suitable. 24 hours. Suspend fresh cultures of test strains with sterile
Suitability of the test method must be reconfirmed if there is buffered sodium chloride-peptone solution pH 7. O or O. 9 %
a change in testing procedure or the product, which may sterile sodium chloride solution to suitable concentrations.
affect the outcome of the test. Use the suspensions within 2 h if stored at ambient
Preparation of test strains and suspensions temperature. Use within 24 h if stored at 2-8ºC. Suspension
Test strains The viable microorganisms used for inoculation of spores of Clostridium sporogenes can replace a fresh
are not more than 5 Generations ( the dried strains obtained suspension. The spore suspension may be maintained at 2-
from Culture Collection Centre are defined as Generation 0). 8ºC within the validated period of time.
Use appropriate culture collection techniques to preserve the Negative control
test strains to ensure their biological characteristics. Negative control is performed to verifying testing conditions.
Staphylococcus aureus [CMCC (B) 26003] There should be no growth of microorganisms in a negative
Pseudomonas aeruginosa [CMCC (B) 10104] control. A deviation investigation should be carried out if
Escherichia coli [CMCC (B) 44102] there are microorganisms in negative control.
Salmonella paratyphi B [CMCC (B) 50094] Growth promoting and inhibitory properties of tbe media
CAndida albicans [CMCC (F) 98001] Ready-prepared medium, the dehydrated culture media or
Clostridium sporogenes [CMCC (B) 64941] media prepared according to formulations can all be used for
Preparation of microbial suspension lnoculate each of specified microorganisms examination. The growth promo-
Staphylococcus aureus, Pseudomonas aeruginosa, tion and inhibitory properties of the media should be
Escherichia coli , and Salmonella paratyphi B separately in confirmed.
containers with soya bean casein digest broth or soya bean The suitability test of the media used in Tests for Specified
casein digest agar at 30-35ºC for 18-24 hours. lnoculate Microorganisms consists of growth promoting properties,
CAndida albicans separately on sabouraud-dextrose agar or inhibitory properties and indicative properties. Test items and
m sabouraud-dextrose broth at 20-25ºC for 2-3 days. test strains of each medium are summarized in Table l.
Table 1 Growth Promoting, Inhibitory, and lndicative Properties of Media used in Tests for Specified Microorganisrm
Test Medium Property Test Strains
Bile-tolerant gram-negative Enterobacteria enrichment Growth promoting E. coli
bacteria broth mossel P. aeruginosa
Inhibitory S. aureus
"t7'._LL ___ J L'.1- _!_ _____ ----- Growth promoting E. coli
v iu1eL reu uue g1ucu::;e agar
+ Indicative P. aeruginosa
Escherichia coli MacConkey broth Growth promoting E. coli
lnhibitory S. aureus
Growth promoting E. coli
MacConkey agar
+ lndicative

Salmonella Rappaport vassiliadis salmonella Growth promoting Salmonella paratyphi B

enrichment broth
lnhibitory S. aureus
Xylose lysine deoxycholate agar Growth promoting Salmonella paratyphi B
+ lndicative
Triple sugar iron agar Indica ti ve Salmonella paratyphi B
Pseudomonas aeruginosa Cetrimide agar Growth promoting P. aeruginosa
lnhibitory E. coli
Staphylococcus aureus Mannitol salt agar Growth promoting S. aureus
+ lndicative
lnhibitory E. coli
Clostridia Reinforced medium for clostridia Growth promoting Cl. sporogenes
Columbia agar Growth promoting Cl. sporogenes
Candida albicans Sabouraud dextrose broth Growth promoting C. albicans
Sabouraud dextrose agar Growth promoting C. albicans
+ lndicative
Candida chromogenic agar Growth promoting C. albicans
+ Indicative
lnhibitory E. coli
 1106 Microbiological Examination of Non-sterile Products: Tests for Specified Microorganisms
Test for growth promoting properties, liquid media Inoculate
not more than 100 CFU of the appropriate microorganism
(see Table 1) separately into test media and reference media.
lncubate at the specified temperature for not more than the
shortest period of time specified in the test. The test media is
approved if clearly visible growth of the microorganisms in
the test media occurs compared with the reference media
Test for growth promoting properties, solid media Perform
inoculation with Surface-Spread Method. lnoculate not
more than 100 CFU of the appropriate microorganism ( see
Table 1) separately onto the surface of test media and
reference media. lncubate at the specified temperature for not
more than the shortest period of time specified in the test.
The test media is approved if the sizes and morphological
feature of the colonies on test media consist with the
reference media.
Test for inhibitory properties Inocula te not less than
100 CFU of the appropriate microorganism ( see Table 1)
separately to test media and reference media. lncubate at the
specified temperature for not less than the longest period of
time specified in the test. No growth of the test microor-
ganism occurs.
Test for indicative properties Perform inoculation with
Surface-Spread Method. Inoculate not more than 100 CFU
of the appropria te microorganism ( see Ta ble 1) separately
onto the surface of test media and reference media. lncubate
at the specified temperature for not more than the shortest
period of time specified in the test. The test media is
approved if the sizes, morphological and indicative feature of
colonies on test media consist with the reference media.
Suitability of the test method
Preparation of the sample Sample solutions are prepared as
described under the Testing of Products.
Test strains Select the appropriate test strains according to
Microbial contamination limit of pharmaceutical prepara-
tions. In the verification tests for bile-tolerant gram-negative
bacteria, Escherichia coli and Pseudomonas aeruginosa are
all used as test organisms.
Suitability test lnoculate appropriate amount of sample
solution and not more than 100 CFU of test strains according
to tests for specified microorganisms in the appropriate
media. For membrane filtration, filter specified volume of
sample solution and rinse. Add the test strains in the last
rinsing solution and filter. Add appropriate media specified or
place the filter membrane onto specified media. lncubate at
appropriate temperature for the shortest period according to
the corresponding methods in Tests for Specified Microor-
ganisms. After incubation, test stains must be detected with
characteristics typical of a postive test.
lnterpretation If the test described above detected test
strains, the products could be examined according to the
sample solution preparation and the test method for specified
microorganisms. If the test described above failed to detect
test strains, the antimicrobial activity of samples should be
removed [ (see Microbiological examination of non-sterile
products: Microbial enumeration tests ( 1105), Neutrali-
zation/removal of antimicrobial activity] and perform the
method suitability test again.
If the antimicrobial activity of a given product cannot be
neutralized after suitability verification, it is assumed that the
inhibited microorganism cannot be present in the product.
Select methods with thoroughly elimination of the antimi-
crobial activity to perform the test.
Testing of Products
The methods for test of specified microorganisms should be
adopted after suitability test.
Positive control tests The positive control tests are in
accordance with tests methods for specified microorganisms.
lnoculate not more than 100 CFU test strains. Test strains
should be detected in positive control tests.
Negative Control Tests Negative control test is conducted by
replacing the sample preparation with diluents according to
the confirmed examination method. There should be no
growing bacteria. Otherwise, deviation investigation should
be performed.
Bile-Tolerant Gram-Negative Bacteria
Sample preparation and pre-incubation Prepare a sample
using soya bean casein digest broth as described in
Microbiological examination of non-sterile products:
Microbial enumeration tests ( 1105) to make 1 : 10 dilu-
tion, mix and incubate at 20-25ºC for a time sufficient to
resuscitate the bacteria but not sufficient to encourage
multiplication of the organisms ( usually about 2 hours).
Test for absence
Unless otherwise prescribed, transfer the appropriate amount
of sample equivalent to 1gor1 ml of the product prepared in
pre-incubation broth into enterobacteria enrichment broth-
mossel. lncubate at 30-35ºC for 24-48 hours. Subculture on
violet red hile glucose agar and incubate at 30-35ºC for 18-
24 hours. No bile-tolerant Gram-negative bacteria detected if
there is no growth of colonies on the plates.
Quantitative test
Selection and subculture lnoculate suitable quantities of
enterobacteria enrichment broth-mossel with the preparation
as described above and/ or dilutions of it contammg
respectively O. 1 g, O. 01 g, and O. 001 g ( or O. 1 ml,
O. 01 ml, and O. 001 mD of the product to be examined.
lncubate at 30-35ºC for 24-48 hours. Subculture each of the
cultures on a plate of violet red hile glucose agar and incubate
at 30-35ºC for 18-24 hours.
Interpretation Growth of colonies on violet red hile glucose
agar constitutes a positive result. Otherwise, the
corresponding tube shows a negative result. Determine from
Table 2 the most-probable-number of bile-tolerant Gram-
negative bacteria contained in the product based on the test
results of each tube.
Table 2 The Most Probable Number of Bile-tolerant
Gram-Negative Bacteria ( N)
Results for each quantity
Probable number of
of product
bacteria ( N) per g (or
O. 1 g or O. 01 g or O. 001 g or mD of product
0.1 ml O. 01 ml O. 001 ml
+ + + N>l0 3
+ + - 2
l0 <N<l0 3
+ - - lO<N<l0 2
- - - N< 10
Note: ( 1) + represents there are colonies growth on the
plate of violet red hile glucose agar; - represents no colonies
growth on the plate of violet red hile glucose agar.
(2) If the amount of the sample decreased by 10 times
(such as O. 01 g or O. 01 ml, O. 001 g or O. 001 ml, O. 0001 g
or O. 0001 mD, the probable number of bacteria (N) per g
( or ml) of product should mcrease by 10 times
 1106 Microbiological Examination of Non-sterile Products: Tests for Specified Microorganisms
correspondingly.
Escherichia coli
Sample preparation and pre-incubation According to the
method described in Microbiological examination of non-
sterile products : Microbial enumeration tests ( 1105 ) to
make a 1 : 10 dilution of the product. lnoculate sample
equivalent to 1 g or 1 ml of the product to be examined to a
suitable amount ( determined as described under Suitability
of the test method) of soya bean casein digest broth, mix
and incubate at 30-35ºC for 18-24 hours.
Selection and subculture Transfer 1 ml pre-incubation broth
to 100 ml macconkey broth and incuba te at 42-44 ºC for 24-48
hours. Subculture a small amount of macconkey broth onto
macconkey agar and incubate at 30-35ºC for 18-72 hours.
Interpretation Growth of colonies on macconkey agar
indicates the possible presence of E. coli. This is confirmed
by isolation, purification and appropriate identification tests.
The product is qualified if no colonies are present or if the
identification tests are negative.
Salmonella
Sample preparation and pre-incubation lnoculate 10 g or
10 ml of sample to a suitable amount ( determined as
described under Suitability of the test method) of soya bean
casein digest broth, mix and incubate at 30-35ºC for 18-
24 hours.
Selection and subculture Transfer O. 1 ml pre-incubation
culture to 10 ml rappaport vassiliadis salmonella enrichment
broth and incubate at 30-35ºC for 18-24 hours. Subculture a
small amount of the rappaport vassiliadis salmonella enrich-
ment broth on xylose-lysine-deoxycholate agar and incubate
at 30-35ºC for 18-48 hours.
The Salmanella is indicated by the growth of well-developed,
erythroic, colourless, transparent or semitransparent colonies,
with or without black centres. Select suspected colonies with an
inoculating needle and puncture into the triple sugar iron
agar, then incubate at 30-35ºC for 18-24 hours. Or use other
appropriate method for further identification.
Interpretation If there are suspected colonies grow on xylose
lysine deoxycholate agar, and the triple sugar iron agar with
red in the slant, yellow in the bottom, or yellow in the slant,
black in the bottom, appropriate identification should be
performed to confirm if they are Salmonella. The product is
qualified if no colonies grow on the plates, or the
identification tests are negative, or the triple sugar iron agar
shows no red/yellow in the slant, no yellow/black in the
bottom.
Pseudomonas aeruginosa
Sample preparation and pre-incubation According to the
method described in Microbiological examination of non-
sterile products : Microbial enumeration tests ( 1105 ) to
make a 1 : 10 dilution of the product. lnoculate sample
equivalent to 1 g or 1 ml of the product to be examined to a
suitable amount ( determined as described under Suitability
of the test method) of soya bean casein digest broth, mix
and incubate at 30-35ºC for 18-24 hours.
Selection and subculture Subculture the pre-incubation broth
described above on cetrimide agar and incubate at 30-35ºC for
18-72 hours.
Select the colonies on cetrimide agar for oxidizing enzyme
test, or perform further identification with other appropriate
methods.
Oxidizing enzyme test Place clean filter paper in the petri
dish. Transfer the colonies on the plate to the filter paper by
glass rod, and add drops of 1 % of freshly dimethyl-p-
phenylene diamine dihydrochloride test solution. The result is
positive if the cultures turn pink in 30 seconds and gradually
becomes purple, otherwise negative.
Interpretation If there are colonies on cetrimide agar, and
the oxidizing enzyme test is positive, appropriate identifi-
cation should be performed to confirm if they are Pseudo--
monas aeruginosa. The product is qualified if there no colony
grows on the plates, or the identification tests of colonies are
negative, or the oxidizing enzyme test is negative.
Staphylococcus aureus
Sample preparation and pre-incubation According to the
method described in Microbiological examination of non-
sterile products : Microbial enumeration tests ( 1105 ) to
make a 1 : 10 dilution of the product. lnoculate sample
equivalent to 1 g or 1 ml of the product to be examined to a
suitable amount ( determined as described under Suitability
of the test method) of soya bean casein digest broth, mix
and incubate at 30-35ºC for 18-24 hours.
Selection and subculture Subculture the pre-incubation
culture described above on mannitol salt agar and incubate at
30-35ºC for 18-72 hours.
Interpretation Growth of colonies on mannitol salt agar with
yellow/white colonies surrounded by a yellow zone indicates
the possible presence of S. aureus. This is confirmed by
isolation, purification and appropriate identification tests.
The product is qualified if no colony is similar with the
feature described above, or if the identification of colonies is
negative.
Clostridia
Sample preparation and heat treatment According to the
method described in Microbiological examination of non-
sterile products : Microbial enumeration tests ( 1105 ) to
make a 1 : 10 dilution of the product. Divide the sample
solution equivalent to 2 g or 2 ml of the product to be
examined into 2 portions. Heat 1 portion at 80ºC for 10 min
and cool rapidly.
Incubation, selection and subculture Inoculate two portions
mentioned above separately into a suitable amount of
reinforced clostridium medium ( determined as described
under Suitability of the test method ). lncubate under
anaerobic conditions at 30-35ºC for 48 hours. After
incubation, subculture on Columbia agar and incubate under
anaerobic conditions at 30-35ºC for 48-72 hours.
Catalase test Transfer the colonies on columbia agar to clean
glass slides, add drops of 3 % hydrogen peroxide test
solution. If bubbles are produced on the surface of the
colonies, the catalase test is positive, otherwise negative.
Interpretation Appropriate identification test should be
carried out for further confirmation of Clostridia if there is
growth of anaerobic bacteria with or without spores on
columbia agar, and the catalase test is negative. The product
is qualified if there is no anaerobic bacteria colonies, or the
identification tests are negative, or the catalase test is
positive.
Candida albicans
Sample preparation and pre-incubation Prepare the product
to be examined as described in Microbiological examination
of non-sterile products: Microbial enumeration tests
( 1105 ) to make a 1 : 10 dilution of the product. Inoculate
sample equivalent to 1 g or 1 ml of the product to be
 1106 Microbiological Examination of Non-sterile Products: Tests for Specified Microorganisms

examined to a suitable amount ( determined as described sterilize.

under Suitability of the test method ) of sabouraud- 4. Rose bengal sodium salt agar
dextrose broth and mix. lncubate at 30-35ºC for 3-5 days. Peptone 5. O g
Selection and subculture Subculture the pre-incubation broth Rose bengal sodium salt O. 0133 g
on sabouraud-dextrose agar and incubate at 30-35ºC for 24- Glucose 10. O g
48 hours. Agar 14. O g
Candida albicans in sabouraud dextrose agar develop creamy Potassium dihydrogen phosphate l. O g
Water 1000 ml
white colonies, occasionally show pale yellow, smooth
surface, with the smell of concentrated yeast. The longer Magnesium sulfate O. 5 g
incubation period is, the larger colonies become with darker Mix the above ingredients except glucose and rose bengal
colour, harder texture or wrinkled. Subculture suspected sodium salt, tepid dissolve. Add glucose and rose bengal
colonies onto candida chromogenic agar and incubate for 24- sodium salt, shake well, dispense and sterilize.
48 hours ( extend to 72 hours if necessary) , or use other 5. Fluid thioglycollate
appropriate methods for further identification. Conducted according to Sterility Tests ( 1101 ).
lnterpretation Appropriate identification test should be 6. Enterobacteria enrichment broth ossel
carried out for further confirmation of Candida albicans if Pancreatic digest of gelatine 10. O g
there are suspected colonies on sabouraud dextrose agar and Disodium hydrogen phosphate dehydrate 8. O g
the test of suspected colonies on candida chromogenic agar is Dehydrated ox hile 20. O g
positive. The product is qualified if there no colony on Brilliant green 15 mg
sabouraud dextrose agar, or if the identification tests are Glucose 5.0 g
negative, or if the test of suspected microorganism on Water 1000 ml
candida chromogenic agar is negative. Potassium dihydrogen phosphate 2. O g
Mix the above ingredients except glucose and brilliant green,
Dilutions
tepid dissolve. Adjust the pH so that after heating it is 7. 2±
Dilutions should be sterilized using validated procedures for O. 2 at 25ºC. Add glucose and brilliant green, heat to lOOºC
sterilization after they are prepared. for 30 minutes, cool immediately.
l. Sterile buffered sodium chloride-peptone solution pH 7. O 7. Violet red hile glucose agar
Prepared according to the methods specified in Sterility Y east extract 3.0 g
Tests (1101) . Neutrai red 30 mg
2. Sterile buffered phosphate solution pH 6. 8 , sterile buffered Pancreatic digest of gelatine 7.0 g
phosphate solution pH 7. 2, and sterile buffered phosphate Crystal violet 2 mg
solution pH 7. 6 Prepared according to the methods specified Sodium deoxycholate l. 5 g
in Buffer (8804). Filter, dispense, and sterilize. Agar 15.0 g
Surfactants or neutralizers can be added into the above Glucose monohydrate 10. o g
dilutions prior to sterilization or after sterilization, if necessary. Water 1000 ml
Sodium chloride 5.0 g
3. Sterile sodium chloride solution O. 9%
Dissolve 9. O g of Mix the above ingredients except glucose, neutral red,
sodium chloride in 1000 ml water, filter, dispense, and crystal violet and agar, tepidly dissolve. Adjust the pH so
sterilize. that after heating it is 7. 4 ± O. 2 at 25ºC. Add glucose,
Culture Media and the Methods of Preparation neutral red, crystal violet, and agar, heat to boiling (Do not
autoclave).
Media can be prepared according to the following formula-
tions, or prepared from the certified dehydrated media that 8. Macconkey broth
manufactured in accordance with the following formulations. Pancreatic digest of gelatine 20. O g
Sterilize in an autoclave using a validated cycle after preparation Bromocresol purple 1O mg
Lactase 10. O g
l. Soya-bean casein digest broth medium ( 1SB), soya-bean Water 1000 ml
casein digest agar medium ( TSA) , sabouraud dextrose Dehydrated ox hile 5. O g
broth medium ( SDB) , should be prepared according to Mix the above ingredients except lactase and bromocresol
Sterility Tests (1101>. purple, tepidly dissolve. Adjust the pH so that after
2. Sabouraud-dextrose agar ( SDA) sterilization it is 7. 3 ± O. 2 at 25ºC. Add Lactase and
Prepared according to Sterility Tests ( 1101 ) . If antibiotics bromocresol purple, dispense and sterilize.
used in sabouraud-dextrose agar, the amount of antibiotics 9. Macconkey agar
added in culture medium should be confirmed. The antibiotics Pancreatic digest of gelatine 17. O g
should not affect the growth of mold and yeast. Neutral red 30. O mg
3. Potato dextrose agar ( PDA) Peptones 3. O g
Potato (peeled) 200 g Crystal violet 1 mg
Dextrose 20. O g Lactase 10. O g
Agar 14. O g Agar 13. 5 g
Water 1000 ml Sodium deoxycholate l. 5 g
Cut the potatoes into small tablets and add 1000 ml water, Water 1000 ml
boil for 20-30 min and filter with bandages of 6-8 layers. Sodium chloride 5. O g
Collect the filtrate and add water to 1000 ml. Adjust the pH Mix the above ingredients except lactase, neutral red, crystal
so that after sterilization it is 5. 6 ±O. 2 at 25ºC. Add agar violet and agar, tepidly dissolve. Adjust the pH so that after
and melt it. Then add glucose, shake well, dispense and sterilization it is 7. 1 ±0. 2 at 25ºC. Add lactase, neutral red,
 1107 Microbiological Acceptance Criteria of Non-sterile Pharmaceutical Products

crystal violet, and agar, heat to boiling for 1 minute, Peptic digest of animal tissue 5. O g
shaking regularly, dispense and sterilize. Phenol red 25 mg
10. Rappaport vassiliadis salmonella Beef extract l. O g
Soya peptone 4. 5 g Agar 15. O g
Magnesium chloride hexahydrate 29. O g D-mannitol 10. O g
Sodium chloride 8. O g Water 1000 ml
Malachite green 36 mg Mix the above ingredients except D-mannitol, phenol red,
Dipotassium hydrogen phosphate O. 4 g and agar, dissolve, warming gently. Adjust the pH so that
Water 1000 ml after sterilization it is 7. 4±0. 2 at 25ºC, heat and shake. Add
Potassium dihydrogen phosphate O. 6 g D-mannitol, phpenol red, and agar, heat to boiling for
Mix the above ingredients except malachite green, tepidly 1 minute, dispense and sterilize.
dissolve. Adjust the pH so that after sterilization it is 5. 2± 15. Reinforced medium for clostridia
O. 2 at 25ºC. Add Malachite green, dispense and sterilize ata Peptone 10.0 g
temperature not exceeding 115ºC. Cysteine hydrochloride o. 5 g
11. Xylose lysine deoxycholate agar Beef extract 10.0 g
Yeast extract 3.0 g Sodium acetate 3.0 g
Sodium chloride 5.0 g Y east extract 3.0 g
L-Lysine 5.0 g Sodium chloride 5.0 g
Sodium thiosulfate 6.8 g Soluble starch l. o g
Xylose 3.5 g Agar 0.5 g
Ammonium iron CID) citrate 0.8 g Glucose 5.0 g
Lactase 7.5 g Water 1000 ml
Phenol red 80 mg Mix the above ingredients except glucose, heat to boiling to
Sucrose 7.5 g dissolve, stirring regularly. Adjust the pH so that after
Agar 13.5 g sterilization it is 6. 8 ±O. 2 at 25ºC, if necessary. Add the
Sodium deoxycholate 2.5 g glucose, mix, pack and sterilize.
Water 1000 ml 16. Columbia agar
Mix the above ingredients except the three saccharide, Pancreatic digest of casein 10.0 g
phenol red and agar, tepidly dissolve. Adjust the pH so that Maize starch l. o g
after heating it is 7. 4 ± O. 2 at 25ºC. Add the three Meat peptic digest 5.0 g
saccharides, phenol red, and agar, heat to boiling (Do not Sodium chloride 5.0 g
autoclave), cool to 50ºC and pour into petri dishes. Heart pancreatic digest 3.0 g
12. Triple sugar iron agar ( TSI) Agar (according to gelling power) 10. O g to 15. O g
Peptone 20.0 g Y east extract 5.0 g
Sulfuric acid f errous 0.2 g Water 1000 ml
Beef leaching powder 5.0 g Mix the above ingredients except agar, heat to boiling to
Sodium thiosulfate 0.2 g dissolve, stirring regularly. Adjust the pH so that after
Lactase 10.0 g sterilization it is 7. 3 ±O. 2 at 25ºC, if nessesary. Add the
O. 2%Phenol methyl phthalate indicator solution 12. 5 ml agar, heat to melt, dispense and sterilize. Cool to 45-50ºC
Sucrose 10. O g after sterilization, add appropriate sterile gentamicin sulfate
Agar 12. O g equivalent to 20 mg of gentamicin, if necessary, mix, and
Glucose l. O g pour into plates.
Water 1000 ml
17. Chromogenic candida agar
Sodium chloride 5. O g
Peptone 10. 2 g
Mix the above ingredients except the three sugars, O. 2 %
Agar 15 g ·
phenol methyl phthalate indicator solution and agar, tepidly
Hydrogen poppy O. 5 g
dissolve. Adjust the pH so that after sterilization it is 7. 3 ±
Water 1000 mi
O. 2 at 25ºC. Add the agar and melt, then add remaining
Chromogenic substrates 22. O g
ingredients, shake well, dispense and sterilize, to make a
Mix the above ingredients except agar, dissolve, warming
short slant with higher and lower lays (2-3 cm).
gently. Adjust the pH so that after heating it is 6. 3±0. 2 at
13. Cetrimide agar 25ºC. Filter, add agar, heat to boiling to dissolve, stirring
Pancreatic digest of gelatine 20.0 g regularly until agar melted. Pour into plates.
Glycerol 10. O ml
Magnesium chloride l. 4 g
Agar 13.6 g
1107 Microbiological Acceptance
Dipotassium sulfate 10.0 g Criteria of Non-sterile
Cetrimide 0.3 g Pharmaceutical Products
Water 1000 ml
Mix the above ingredients except agar, tepidly dissolve, and The microbial acceptance criteria of non-sterile preparations
filter. Adjust the pH so that after sterilization it is 7. 4±0. 2 are drawn with full consideration of the administration routes,
at 25ºC. Add the agar, heat to boiling for 1 minute, dispense potential harm to the patients and the particularity of drugs.
and sterilize. Unless otherwise prescribed, microbiological examination
14. Mannitol sodium chloride agar criteria for pharmaceuticals should be based on the criteria
Pancreatic digest of casein 5.0 g followed in many fields, such as examination during manu-
Sodium chloride 75.0 g facture, storage or market, examination of raw material,
 1107 Microbiological Acceptance Criteria of Non-sterile Pharmaceutical Products

excipients or Chinese medicine extract, establishment of new 2. Topical administration preparations for surgery, bum or
preparations standards, evaluation of imported preparations, serimas injury
quality control and arbitration of preparations. Comply with Sterility Tests.
l. Sterile preparations specified in general charpter or mono- 3. Microbiological acceptance criteria of nonsterile chemical
graphs or preparations, raw materials and excipients labeled preparation, biological products preparation and Chinese
sterile medicine preparations without crude power are listed in Table
Comply with Sterility Tests. l.
Table 1 Microbiological acceptance criteria for nonsterile preparations
( chemical, biological products and Chinese medicine without crude powder}
TAMC TYMC
Adminístrate Route (CFU/g, CFU/ml CCFU/g, CFU/ml Specified microorganisms
or CFU/10 cm2) or CFU/10 cm2)

Oral useCD Absence of Escherichia coli ( 1 g or 1 mD

non-aqueous 103 102 Absence of Salrrwnella m preparations contain organ
aqueous 102 101 extracts (10 g or 10 mD

Oromcosal use
Absence of Escherichia coli, Staphylococcus aureus,
Gingival use 102 101
Pseudorrwnas aeruginosa (1 g, 1 ml or 10 cm2)
Nasal use

Auricular use Absence of Staphylococcus aureus, Pseudorrwnas aerugi-

102 101
Cutaneous use nosa (1 g, 1 ml or 10 cm2)

Absence of Escherichia coli, Sta ph ylococcus aureus,

lnhalation use 10 2 101 Pseudorrwnas aeruginosa , Bile-tolerant Gram-negative
bacteria ( 1 g or 1 mD

Absence of Staphylococcus aureus, Pseudorrwnas aerugi-

Vaginal use 2 nosa, Candida albicans (1 g, 1 ml or 10 cm2)
10 '1v-
"1
Urethra use Absence of Clostridium in Chinese medicine preparations
(1 g, 1 ml or 1O cm2)

Rectal use
102 Absence of Staphylococcus aureus, Pseudorrwnas aerugi-
non-aqueous 103
nosa (1gor1 mD
aqueous 102 102

Absence of Staphylococcus aureus, Pseudorrwnas aerugi-

Other topical use 10 2 102
nosa (1 g, 1 ml or 10 cm2)
Note: CD Salrrwnella should be absent in 10 g (10 mD if chemical/biological product preparation contain natural (botanical,
animal ingredients or minerals) origin without extraction.
T AMC-Total Aerobic Microbial Count; TYMC-Total combined Yeast and Mold Counts

4. Microbiological acceptance criteria of non-sterile Chinese medicine preparations with crude powder are listed in Table 2.
Table 2 Microbiological acceptance criteria for non-sterile Cbinese medicine preparations with crude powder
TAMC TYMC
Adminístrate Route (CFU/g, CFU/ml (CFU/g, CFU/ml Specified microorganisms
or CFU/10 cm2) or CFU/10 cm2)

Non-aqueous oral use

without fermented crude powder 104 10 2 Absence of Escherichia coli ( 1 g)
from bean, medicated leaven or other (pill 3X10 4) Absence of Salrrwnella (10 g)
Bile-tolerant Gram-negative bacteria are not
with crude powder above 105 sx10 2 more than 102 (1 g)

Aqueous oral use

Absence of Escherichia coli (1 mD
without fermented crude powder
Absence of Salrrwnella (10 mi)
from bean, medicated leaven or other sx10 2 102
Bile-tolerant Gram-negative bacteria are not
more than 101 Cl mD
with crude powder above 10 3 10 2

Absence of Staphylococcus aureus, Pseudo-

Non-aqueous topical use rrwnas aeruginosa (1 g or 10 cm2)
103 102
for wound epidermal or mucosal Absence of Candida albicans, Clostridium
for intact epidermal or mucosal m vagina/ urethra preparation ( 1 g or
104 102
10 cm2)
 1121 Antimicrobial Effectiveness Testing

continued
TAMC TYMC
Administrate Route (CFU/g, CFU/ml (CFU/g, CFU/ml Specified microorganisms
or CFU/10 cm2 ) or CFU/10 cm2 )

Absence of Staphylococcus aureus, Pseudo-

Aqueous topical use
102 102 monas aeruginosa ( 1 ml)
for wound epidermal or mucosal
Absence of Candida albicans , Clostridium
for intact epidermal or mucosal
102 102 in vagina/urethra preparations (1 mD
Note: TAMC-Total Aerobic Microbial Count; TYMC-Total combined Yeasts and Molds Count

S. Microbiological acceptance criteria of non-sterile raw materi- potential hazardsous microbes depending on nature, usage,
al and excipients are Usted in Table 3. manufacturing process and so on.
In addition to the specified microorganisms required above,
Table 3 Microbiological acceptance criteria for
the significance of other microorganisms recovered should be
non-sterile raw material and excipients
evaluated in terms of the following:
TAMC TYMC Specified -administration route: hazards vary according to the route of
(CFU/g (CFU/g microorga- administration,
or CFU/ml) or CFU/ml) msms -nature of the product: whether the product supports growth
or whether it has adequate antimicrobial preservation,
raw material -the usage of the product,
10 3 102 *
and excipients -the intended recipient: risk may differ for neonates, infants
and the debilitated,
Note: * -There is no unified requirement.
-use of immunosuppressive agents, corticosteroids,
TAMC-Total Aerobic Microbial Count; TYMC-Total com-
-the presence of disease, wounds, organ damage, and so on.
bined Y east and Mold Counts.
When a risk-assessment basis on these factors is necessary,
6. Microbiological acceptance criteria of Chinese medicine ex- the process should be conducted by personnel with specialized
tract and prepared pieces are Usted in Table 4. training in microbiology and in the interpretation of
microbiological data. For raw material and excipients, the
Table 4 Microbiological acceptance criteria of assessment takes account of the processing to which the
Chinese medicine extract and prepared pieces product is subjected, the current technology of testing, and
TAMC TYMC the availability of materials of the desired quality.
Specified
(CFU/g or (CFU/g or
microorganisms
CFU/mD CFU/mD 1121 AntimicrOOial Effectiveness Testing
Chinese medicine 3 2
10 10 *
extract
Antimicrobial preservatives refer to the chemical substances
Absence of Sal- inhibiting the growth of microorganisms, sometimes called
Prepared pieces monella ( 1O g) , preservatives. Antimicrobial effectiveness testing is applied to
for oral precious Bile-tolerant determine the effectiveness of antimicrobial preservatives in
powder for oral * * Gram-negative the sterile and non-sterile pharmaceutical preparations, in
directly, after bacteria are not order to evaluate the antimicrobial effectiveness of the final
soak more than 104 preparations. lt can also be applied to help the manufacturers
(1 g) to determine the concentration of antimicrobial preservatives
Note: * -There is no unified requirement. during development of the preparations.
TAMC-Total Aerobic Microbial Count; TYMC-Total If a pharmaceutical preparation does not itself have adequate
combined Yeast and Mold Counts antimicrobial activity, antimicrobial preservatives may be
added, according to the characteristics of the preparations
7. Preparations for more than one administration routes. (particularly to aqueous preparations), to prevent prolife-
Comply with the requirements of each administration route. ration or to limit microbial contamination which, during
Total aerobic microbial count (TAMC) and total combined normal conditions of storage and use, particularly for multi-
yeast and mold counts (TYMC) of non-sterile products are dose containers, could occur in a product and presenta hazard to
tested according to the method in "Microbiological Exami- the patient from infection and spoilage of the preparation.
nation of Non-sterile products: Microbial Enumeration Antimicrobial preservatives should not be used as a substitute
Tests" ( 1105 ). Specified microorganism examinations in for good manufactüring practises or solely to reduce the
non-sterile products are performed according to the method viable microbial population of a non-sterile product or control
in "Microbiological Examination of Non-sterile products: the presterilization bioburden of multi-dose formulations
Tests for Specified Microorganisms" ( 1106). during manufacturing. All useful antimicrobial agents are
When an acceptance criterion for microbial enumeration is toxic substances, and the amount of added preservative
specified, it is interpreted as follows: should be minimumal effective. For maximum protection of
10 1 CFU: The maximum acceptable count is 20; patients, the concentration of the preservative shown to be
102 CFU: The maximum acceptable count is 200; effective in the final packaged product and should be below a
103 CFU: The maximum acceptable count is 2000; and so forth level that may be toxic to human beings.
The specified microorganisms listed in tables above are not During storage, antimicrobial effectiveness of the preserva-
necessarily exhaustive. Thus, raw material, excipients and tive may be changed by the active ingredients of the
sorne special preparations may be necessary to control other formulation or by the container and so on. Thus it must be
 1121 Antimicrobial Effectiveness Testing

validated that antimicrobial effectiveness in the final package
will not be reduced by the storage conditions befare the
expiry date.
The tests and criteria for effectiveness apply to a product in
the original, unopened container.
Culture Media
Media preparation
The media prepared according to Sterility Tests ( 1101 >.
The media are soy bean casein digest medium, soy bean
casein digest agar medium, sabouraud dextrose agar medium
or sabouraud dextrose broth medium.
Suitability test for the media
Carry out suitability test for the media which will be used for
antimicrobial effectiveness testing, including the finished
product of the medium and the media which are prepared
from dehydrated media or the formula.
Test strains The viable microorganisms used in the test
must not be more than five passages removed from the
original master seed lot ( the freeze dried microorganism
obtained from the microorganism preservation centre is
generation 0). The suitable seed-stock technique shall be used
so that the microorganism biological characters can be
maintained. The microorganisms and freshly cultured
bacteria for the suitability test of culture medium shall be
cultured as specified in Table l.

Table 1 Culture conditions for microorganisms for suitability test of culture medium,
operational suitability test, and antimicrobial effectiveness testing
lncubation lncubation
Organisms Suitable medium
temperature time

Staphylococcus aureus Soy bean casein digest agar medium or

30-35ºC 18-24 h
[CMCC (B) 26 003] soy bean casein digest medium

Pseudomonas aeruginosa Soy bean casein digest agar medium or

30-35ºC 18-24 h
[CMCC (B) 10 104] soy bean casein digest medium

Escherichia coli Soy bean casein digest agar medium or

30-35ºC 18-24 h
[CMCC (B) 44 102] soy bean casein digest medium

Candida albicans Sabouraud dextrose agar medium or

20-25ºC 24-48 h
[CMCC CF) 98 001] sabouraud dextrose broth medium

Aspergillus niger Sabouraud dextrose agar medium or 5-7 days or till there are
20-25ºC
[CMCC CF) 98 003] sabouraud dextrose broth medium abundant spores

Preparation of inoculum Take freshly cultured Escherichia
coli or Staphylococcus aureus or Pseudomonas aeruginosa as
described in Table 1 and prepare them with pH 7. O sterile
sodium chloride-peptone buffer solution or O. 9 % W/V
sterile sodium chloride solution to produce a suspension with
appropriate concentration. Take the freshly cultured
aspergillus niger as described in Table 2 and wash the spore
with 3 to 5 ml of pH 7. O sterile sodium chloride-peptone
buffer solution containing O. 05 % ( ml/ mD polysorbate 80
or with 3 to 5 ml O. 9 % W/V sterile sodium chloride
solution. Transfer the spore suspension to a sterile test tube
with proper method. Dilute the above suspension with sterile
O. 9 % W/V sodium chloride solution containing O. 05 %
( ml/ml) polysorbate 80 to produce a suspension with
appropriate concentration.
Prepared microorganism suspensions should be used within
2 hours if stored at room temperature, or within 24 hours if
stored at 2 to 8°C. Aspergillus niger spore suspensions
should be stored at 2 to 8ºC and used befare the expiry date
which has been validated.
Suitability test lnoculate not more than 100 CFU of
Escherichia coli or Staphylococcus aureus or Pseudomonas
aeruginosa into TSA respectively, and use two petri dishes
for each test strain. Mix well, allow the contents to solidify,
incubate at 30 to 35ºC for not more than 3 days, and count
the plate. lnoculate not more than 100 CFU of Aspergillus
niger or Candida albicans into sabouraud dextrose agar
medium respectively, and use two petri dishes for each test
strain. Mix well, allow the contents to solidify, incubate at
20 to 25ºC for not more than 5 days, and count the plate.
Meanwhile, carry out the above method, using the reference
medium instead of the medium being examined.
Evaluation of results If average colony numbers of the
medium being examined is not less than 70 % of average
colony numbers of the reference medium and the form and
size of colonies are the same as that of the reference medium,
the medium being examined meet requirements of the
suitability test.
Determination of Antimicrobial Effectiveness
Test Strains Use the strains described in Table 1 for deter-
mination of antimicrobial effectiveness. Normal contami-
nating microorganisms in the pharmaceutical preparations
could be used as test strains if needed.
Preparation of inoculum Culture of freshly cultured bacteria
is shown in Table l. In the case of agar culture for
Pseudomonas aeruginosa or Staphylococcus aureus,
Escherichia coli or Candida albicans, wash the culture on
the agar surface with suitable volume of sterile O. 9 % W /V
sodium chloride solution. Transfer the suspension to a sterile
test tube. Dilute the above suspension with sterile O. 9 %
W /V sodium chloride solution to produce a suspension of
108 CFU per ml. In the case of liquid culture, harvest the
bacteria by centrifugation, wash them with sterile O. 9 %
W /V sodium chloride solution to produce a suspension of
108 CFU per ml. lnoculate freshly cultured Aspergillus niger
and wash the spore culture with 3 to 5 ml of sterile O. 9 %
W /V sodium chloride solution containing O. 05 % ( ml/ ml)
polysorbate 80. Transfer the spore suspension to a sterile test
tube using suitable method. Dilute the above suspension with
sterile O. 9 % W /V sodium chloride solution containing
 1141 Test for Abnormal Toxicity

o. 05% e ml/ml) polysorbate 80 to produce a spore per ml or g of the test preparation. In the table, the "A"
suspension of 108 CFU per ml. Determine the microor- criteria express the recommended efficacy to be achieved In
ganisms counting per ml of the suspension. justified cased where the A criteria cannot be attained, far
The prepared inoculum shall be used within 2 hours if stored example for reasons of an increased risk of adverse reactions,
at room temperature, or within 24 hours if stored at 2 to the "B" criteria must be satisfied.
8ºC. Aspergillus niger spore suspension should be stored at 2
Table 2-1 Criteria for antimicrobial effective~ of
to 8ºC and used within one week.
injections, ophthalmic preparations, preparations
Inoculation of test product Features of a container that will for breast and endometrial
influence antimicrobial effectiveness include the material from
lg reduction
which it is made, its shape and size evolume) and the type
6h 24 h 7d 14 d 28 d
of closure, etc.. The test can be conducted in original
containers if sufficient volume of the product is available in Bacteria A 2 3 NR
each container and the prepared microorganisms' suspension B 1 3 NI
can be inoculated, mixed and sampled in the product Fungi A 2 NI
container by sterile operation. If the product should be
B 1 NI
transferred to sorne other sterile container because of the
characteristics of the product or size of the original product Note: NR means no recovery of growth of the test
container, the material from which the container is made microorganisms.
should not affect the characteristics of the product efor NI means no increase in number of viable microorganisms of
example, by sorption of ingredients). Particular attention not more than O. 5 lg growth compared to the previous
should be paid to possible changes in product pH since these reading.
can markedly affect preservative activity. A container with a
Table 2-2 Criteria for antimicrobial effectiven~ of
suitable wide neck that will allow easy of transferring and
aural preparations, nose drops, skin preparations,
mixing is recommended.
inhalation preparations
lnoculate directly the prepared microorganisms, suspension
into each of at least five completely packaged products, or lg reduction
transf er sufficient volume of products to each of five sterile 2d 7d 14 d 28 d
containers separately eif the number of the test strains Bacteria A 2 3 NI
exceeds five, the containers to which the products are
B 3 NI
transferred should be increased). Inoculate each container
with one of the prepared microorganisms' suspension, the Fungi A 2 NI
concentration of test microorganisms that is added to the B 1 NI
product is 105 -10 6 CFU perg or per ml, and the volume of Note: NI means no increase in number of viable microor-
the prepared microorganisms' suspension used should be not ganisms of not more than O. 5 ig growth compared to the
more than 1% of the volume of the product, and mix well, previous reading.
in order to achieve homogeneous distribution of the inoculum.
Maintain the inoculated product at 20-25ºC, protected from Table 2-3 Criteria for antimicrobial effectiven~ of oral
light. preparations, oral mucosa preparations, rectal preparations
Viable microorganisms counting Sample the test preparation lg reduction
1 ml e g ) from each of the containers above at the 14 d 28 d
appropriate intervals specified in Table 2-1, Table 2-2 and Bacteria 3 NI
Table 2-3 according to product categories. Determine the
Fungi 1 NI
number of CFU present in each test preparation, using soy
bean casein digest agar medium far bacteria test and Note: NI means no increase in number of viable microor-
sabouraud dextrose agar medium for fungi test. Viable micr- ganisms of not more than O. 5 lg growth compared to the
oorganisms counting method and the operational suitability previous reading.
test according to Microbiological Examination Of
Nonsterile Products: Microbial Enumeration Tests (1105).
Strains for the operational suitability test are specified in
1141 Test for Abnonnal Toxicity
Table 1, and preparation of inoculum is the same as that in
the suitability test of culture media. Recovery rate of test Abnormal toxicity is different from the toxicity of the drug,
microorganisms for operational suitability test shall not be and it is introduced by manufacture process or other reasons.
lower than 70%. The test is determined by observing the abnormal reaction or
According to the results of the microorganisms counting, death of animals within a period of time after having received
calculate the concentration of CFU per ml or g of each test a dose of the substance being examined. It is perfarmed to
preparations at the start of the test and at the applicable test find out any possible contamination caused by exogenous
intervals, and express the concentration in terms of lg values. agents and those unexpected factors that may create safety
The test results should be rounded off to the first decimal problems.
place by the roles on rounding off significant figures. Preparation of test solution
Evaluation of results The criteria for evaluation of antimi- The test solution shall be prepared to concentration pres-
crobial effectiveness of the test preparation are given in Table cribed in the monograph and brought to a room temperature
2-1, Table 2-2 and Table 2-3, in which "the lg reductions" befare use.
refers to the lg values of the concentration at the applicable Animals
test intervals deducted by the lg values of the concentration Select the healthy animals not previously used far testing,
''·h· ..{.>>;·
fll' 1142 Test far Pyrogens
···•• I
and maintain on an adequate balanced diet befare the test and
throughout the observation period.
Test for non-biological products
Unless otherwise specified, inject intravenously O. 5 ml of the
test solution into each of the five mice, weighing 18 g to
22 g. Inject the test solution at a unifarm rate, the injection
occupying 4-5 seconds usually. Inject the test solution about
30 seconds, as is specified to inject slowly in the monograph.
Unless otherwise specified, the substance passes the test if
none of the mice die within 48 hours. If any mouse dies,
repeat the test using respectively 10 mice, each weighing
between 19 g and 21 g. The substance passes the test if none
of the mice in the second group die within 48 hours.
Test for biological products
Far abnormal toxicity test, both mice and guinea pigs shall
be used, unless otherwise specified. The blank control group
of animals from the same batch of the animals used in the
test shall be established. The test result is valid if all the
animals of the blank control group remain healthy and
survive the observation period, without any abnormal reac-
tions, and with increase of body weight by the end of obser-
vation period. Inject the test solution slowly to the body of
the animal according to the specified route of administration.
( 1 ) Test in mice
Five mice are used far the test, unless otherwise specified.
Weigh each mouse befare test. The body weight of each
mouse shall be 18-22 g. Each mouse shall be injected i. p.
with O. 5 ml of test solution and observed far 7 days. The
product passes the test if all the mice remain healthy and
survive the observation period, without any abnormal
reactions, and with increase of body weight by the end of
observation period. If the product fails to pass the test, the
test may be repeated once by using another ten mice weighing
19-21 g, and the result shall be judged as described above.
( 2) Test in guinea pigs
Two guinea pigs are used far the test, unless otherwise
specified. Weigh each guinea pig befare test. The body weight
of each guinea pig shall be 250-350 g. Each guinea pig shall
be injected i. p. with 5. O ml of test solution and observed far
7 days. The product passes the test if all the guinea pigs
remain healthy and survive the observation period, without
any abnormal reactions, and with increase of body weight by
the end of observation period. If the product fails to pass the
test, the test may be repeated once by using another faur
guinea pigs, and the result shall be judged as described above.
1142 Test for Pyrogens
The test far pyrogens is carried out in rabbits by measuring
the rise of body temperature fallowing the intravenous injec-
tion of a specified dose of the preparation being examined.
Test animal U sed healthy, matured rabbits of male or non-
pregnant females, weighing not less than l. 7 kg ( rabbits
weighing l. 7-3. O kg far biological products ). Keep the
rabbits under observation and fed with a complete and
balanced diet far 7 days befare the selection test, loss of
weight, loss of appetite or other abnormal signs should not
appear. During 7 days proceeding the pyrogen test, rabbits,
which have not previously been used in a pyrogen test, or
which have been given a substance which maximum rise of
temperature in this group is up to O. 6ºC while the test
substance passes the pyrogen test, or which were not been
used in pyrogen test far three weeks, should be subjected to
a selection test as prescribed under the procedure except
injection. Record the body temperature of each rabbit 8 times
successively at 30 min intervals. Any animal, showing body
temperature out of the range of 38. 0-39. 6ºC or a
temperature variation greater than O. 4 ºC, is not used in the
pyrogen test. Do not use a rabbit far pyrogen testing more,
frequently than once every 48 hours, or prior to 2 weeks
fallowing a maximum temperature rise of O. 6ºC, or more,
while being subjected to the pyrogen test. All rabbits in
which the preparation being examined was adjudged to be
pyrogenic should not be used again. Rabbits used in pyrogen
test far blood products, antitoxins and other test substance
with same antigenicity only can be used again far the same
product during 5 days.
Test conditions 1-2 days befare the test. House the rabbits
in a quiet area with a unifarm temperature between 17-25ºC.
Carry out the test in a lab where there is no risk of
disturbance exciting the animals and in which the room
temperature is within 3ºC of that of the rabbits' living area.
Withhold faod from the rabbits and place the animals in
suitable fitting boxes at least 1 hour befare the test, until the
test is completed. Measure the body temperature with a
suitable temperature-sensing device with a precision of
±0. l°C. lnsert the thermometer or temperature-sensing
probe into the rectum of the rabbit to a unifarm depth
( about 6 cm) far not less than l. 5 minutes. Record the body
temperature of each rabbit once at an interval of 30 minutes
befare the injection of product to be examined, usually
measures twice. The difference between the two readings
taken immediately befare injection should not be greater than
O. 2ºC, use the mean value of these two readings as the
normal body temperature of each rabbit. In any one test, the
normal body temperature of each rabbit must be within 38. 0-
39. 6ºC and must not differ from one another by more than
1 ºC on a group.
Render the syringes, needles and glassware free from
pyrogens by heating at 250ºC far more than 30 minutes or by
other suitable method.
Procedure Warm the preparation being examined to about
38. OºC. Within 15 minutes after the determination of normal
body temperature, inject a dose as specified in the mono-
graph into the ear vein of each of 3 rabbits slowly. Record the
temperature of each rabbit at 30-minutes intervals, measure
6 times after injection. The temperature rises of each rabbit is
calculated by subtracting its normal temperature from the
highest temperature recorded. If one of the 3 rabbits shows a
temperature rise of O. 6ºC or more, or the sum of the
3 temperatures rise is l. 3ºC or more, continue the test using
5 other rabbits under the same procedure.
Evaluation of the results If none of the 3 rabbits in the first
test shows a temperature rise of O. 6°C or more, and the sum
of the 3 tempera ture rises does not exceed l. 3 ºC , or if only
one of the 5 rabbits in the second test shows a temperature
rise of O. 6ºC or more, and the sum of the 8 temperature
rises does not exceed 3. 5ºC, the preparation being examined
passes the test.
If more than one rabbit in either test show a temperature rise
of O. 6ºC or more; or the sum of the 8 temperature rises
exceed 3. 5ºC, the preparation being examined is considered
to be pyrogenic.
When the temperature rise is negative, the result is counted
as a zero response.

1143 Test for Bacterial Endotoxin
The test for bacteria! endotoxins is used to detector quantify
endotoxins of gram-negative bacteria! origin using amoe-
bocyte lysate from horseshoe crab CTachypleus tridentatus
or Limulus polyphemus). It is used to determine the limit
concentration of bacteria! endotoxin in a preparation being
examined.
Two methods are used for this test: the gel-clot method and
the photometric method. The latter includes a turbidimetric
method and a chromogenic method. Proceed by any one of
these two methods. In the event of doubt or dispute, the final
decision is made based on the gel-clot limit method, unless
otherwise indicated in the monograph.
Avoid microbial contamination during the test.
Endotoxin is expressed in Endotoxin Units CEU ). One
Endotoxin Unit CEU) is equal to one lnternational Unit
CIU) of endotoxin.
The National Standard Endotoxin CNSE) is prepared and
purified from Escherichia coli. lt is used for calibration of the
working standard endotoxin CWSE). It also is used for
calibration, verifying and arbitrating the sensitivity of lysate
reagents. NSE can be used for bacteria! endotoxin test as
positive control Csolution B and solution C), for interference
test, for sensitivity test of lysate reagent, and for verification
of criteria for the standard curve in photometric method.
The working standard endotoxin CWSE) has been calibrated
against the NSE. It is used for bacteria! endotoxin test as
positive control Csolution B and solution C), for interference
test, for sensitivity test of lysate reagent and for verification
of criteria for the standard curve in photometric.
Water for bacteria! endotoxins test (water for BET) should
meet the standard of sterile water for injection, and must be
free of interfering factors. The quantities of bacteria!
endotoxin of water used in the gel-clot method are less than
O. 015 EU/ml, while the quantities are less than O. 005 EU/
ml in the photometric method.
Depyrogenate all glassware and other heat-stable apparatus
by heating in a hot-air oven at 250ºC for more than
30 minutes Cor by any other validated suitable method) to
eliminate extraneous endotoxin that may present. If
employing plastic apparatus, such as microtitre plates and
pipette tips for automatic pipetters, use apparatus shown to
be free of detectable endotoxin and of interfering effects for
the test.
Preparation of the test solutions Prepare the test solutions
by dissolving or diluting active substances using water for
BET. If necessary, adjust the pH of the test solution Cor
dilution thereof) so that the pH of the mixture of the lysate
reagents and test solution falls within the pH range of 6. O to
8. O. The pH may be adjusted by the use of acid, base or a
suitable buffer. Acids and bases may be prepared from
concentrates or solids with water for BET in containers free
of detectable endotoxin. Buffers must be validated to be free
of detectable endotoxin and interfering factors.
Establishment of endotoxin limits The endotoxin limit CL)
for drugs or biological products is usually defined as follows;
L=K/M
Where: L is the endotoxin limit for active substances
administered parenterally, which is specified in
units such as EU/ml, EU/mg, or EU/Unit of
biological activity;
1143 Test for Bacteria! Endotoxin
1
K is the threshold human pyrogenic <lose of
endotoxin per kg of body mass in a single hour
period, which is expressed as EU / Ckg • h).
Far injections, K = 5 EU/ Ckg • h); for
injections of radio-pharmaceuticals, K =
2. 5 EU/ C kg • h ) ; and for intrathecal
injections, K=O. 2 EU/ Ckg•h);
M is the maximum recommended human <lose of
product per kg of body mass in a single hour
period, which is specified in units such as ml/
Ckg•h), mg/ Ckg•h), or U/ Ckg•h). Here
the human average body weight is 60 kg;
human body surface area is calculated accor-
ding to l. 62 m 2 , the injection period is
calculated as 1 hour when the injection is
completed within 1 hour. The <lose of test
product per square metre of body surface area
multiplied by O. 027 can be converted to <lose of
product per kg of body mass CM).
The endotoxin limit calculated by human <lose maybe
adjusted according to the situation of manufacture and clinical
use if necessary. In that case, appropriate reasons for
adjustment have to be submitted.
Determination of the maximmn valid dilution ( MVD) The
maximum valid dilution CMVD) is the maximum allowable
dilution of a sample at which the endotoxin limit can be
determined. Determine the MVD using the following formulae;
MVD=cL/J¡
Where: L is the endotoxin limit of the sample being
examined;
e is the concentration of the test solution, when
L is expressed as EU/mg or EU/U, the unit of
e is mg/ ml or U/ ml; When L is expressed as
EU/ ml, e is l. O ml/ ml. Mínimum valid
dilution concentration Cthe concentration of
substance for injection when MVD) , can be
calcula ted using the form ulae, e = }¡ / L ;
A is the labelled lysate reagent sensitivity of in the
gel-clot method CEU/ ml) or the lowest point
used in the standard curve of the photometric
method.
Gel-clot Method ( Method 1)
The gel-clot method detects or qualifies endotoxins based on
clotting of the lysate reagent in the presence of endotoxins.
Test for confinnation of labelled lysate reagent sensitivity
The labelled lysate reagent sensitivity CEU/mD is defined as
the lowest concentration of endotoxin that is needed to cause
the lysate reagent to clot under the conditions specified. The
test for confirmation of the labelled lysate reagent sensitivity
is carried out when each new batch of lysate reagent is used
or when there is any change in the experimental conditions
which may aff ect the outcome of the test.
Dissolve NSE or WSE using water for BET, mix
continuously for 15 minutes using a vortex mixer. Prepare
standard solutions of 4 concentrations equivalent to 2A.,
l. OA., O. 5A. and O. 25A. by diluting the NSE or WSE with
water for BET, mix each dilution for 30 seconds using a
mixer. Use 18 tubes of 10 mm X 75 mm in size containing of
O. 1 ml of lysate solution or use 18 original ampoules of
O. 1 ml of dissolving lysate. Add O. 1 ml endotoxin standard
solutions to each of 16 tubes, every standard concentration in
four replicates, and O. 1 ml of water for BET to each of
2 tubes as negative control. Mix gently after each addition,
cover the tubes tightly and incubate the tubes vertically at
37±1 ºC for 60±2 minutes avoid vibration. Take each tube in
 1143 Test for Bacteria! Endotoxin

turn directly from the incubator and invert it through Where: X is the log endpoint concentration. The endpoint
approximately 180º in one smooth motion to test the integrity concentration is the last positive result in a
of the gel. If a firm gel has formed that remains in place upon series of decreasing concentrations of endotoxin;
inversion, record the resultas positive. A result is negative if n is the number of replicates.
an intact gel is not formed. Handle the tubes with care to If the Ac is not less than O. 5A. and not more than 2A., the
avoid vibration, or false negative may result. The test is not labelled sensitivity (A.) is confirmed and is used in tests
valid unless the highest concentration (2. OA.) of the standard performed with this lysate reagent.
solution shows positive results in all replicate tubes, the Test for interfering factors Prepare solutions A, B, C and
lowest concentration (O. 25A.) shows negative results in ali D as shown in Table 1, and use the test solutions at a
replicate tubes and the duplicated tubes of negative control dilution less than the MVD, not containing any detectable
show negative results. Calculate the geometric mean endpoint endotoxins, operating as described under the "Test for
concentration, i. e. the measured sensitivity of the lysate confirmation of labelled lysate reagent sensitivity''.
reagent (..\e ) , using the following expression:
Ac=antilg CL:X/n)
Table 1 Preparation of solutions in the test for interfering factors by using gel-clot method
Endotoxin concentration/Solution Initial endotoxin Number of
Solu-tion Diluent Dilution factor
to which endotoxin is added concentration replica tes
A None/Test solution - - - 2
1 2A. 4
2 1A 4
B 2A./Test solution Test solution
4 0.5A. 4
8 0.25A. 4
1 2A. 2
2 1A 2
e ZA/Water for BET Water for BET
4 0.5A. 2
8 0.25A. 2
D None/Water for BET - - - 2
Solution A: Solution of the preparation being examined that is free of detectable endotoxins.
Solution B: test for interference
Solution C: control of the labelled lysate reagent sensitivity
Solution D: negative control (water for BET)

The test is not valid unless all replicates of solutions A and D was completed.
show negative results and the result of solution C confirms Table 2 Preparation of solutions in gel-clot limit test
the labelled lysate reagent sensitivity.
If the result of solution B confirms the labeled lysate reagent Endotoxin Concentration/Solution Number of
Solution
sensitivity, the test solution does not contain interfering to which endotoxin is added replica tes
factors under the experimental conditions used. Otherwise, A None/Diluted test solution 2
the solution interferes with the test. If the preparation being
examined interferes with the test at a dilution less than the B 2A/Diluted test solution 2
MVD, repeat the test for interfering factors using a greater
dilution, not exceeding the MVD. e 2A./Water for BET 2
Interference may be overcome by suitable treatment, such as D None/Water for BET 2
filtration, neutralization, dialysis or heat treatment. To
establish that the treatment chosen effectively eliminates Solution A: solution of the preparation being examined
interference without loss of endotoxins, repeat the test for Solution B: positive product control
interfering factors using the preparation being examined to Solution C: positive control
Solution D: negative control (water for BET)
which the standard endotoxin has been added and which has
then been submitted to the chosen treatment.
Invalidation of results The test is not valid unless both
When establishing a method of bacteria! endotoxin test for a replicates of the two positive control solutions B and C are
new drug, the test for interfering factors should be carried positive and those of negative control solution D are negative.
out. When the source of the lysate reagent, or the formula or The preparation being examined complies with the test when
the manufacture process of the substance being examined are a negative result is found for both replicates of solution A
changed, or there is any change in the experimental The preparation being examined does not comply with the
conditions that are likely to influence the result of the test, test when a positive result is found for both replicates of
the test for interfering factors should be performed again. solution A Repeat the test by 4 replicates of solution A if a
Procedure positive result is found for one replicate of solution A and a
( 1 ) Gel-clot limit test negative result is found for the other. The preparation being
Prepare solutions A, B, C and D as shown in Table 2, and examined complies with the test if a negative result is found
perform the test following the procedure in the "Test for for ali replicates of solution A in the repeat test. Otherwise,
confirmation of labelled lysate reagent sensitivity''. the preparation being examined does not comply with the
Prepare solution A and solution B using a dilution not greater test.
than the MVD, with which the Test for interfering factors When a positive result is found for both replicates of solution

A, and the preparation being examined is diluted to a dilution
less than the MVD, the test should be repeated at a dilution
of MVD.
( 2) Gel-clot semi-quantitative test
The test quantifies bacteria! endotoxins in the test solution by
titration toan endpoint. Prepare solutions A, B, C and Das
shown in Table 3, and perform the test following the
procedure in the "Test for confirmation of labelled lysate
reagent sensitivity''.
Invalidation of results The test is not valid unless the follo-
wing 3 conditions are met: (1) both replicates of solution D
( negative control) are negative; ( 2) both replica tes of
solution B (positive product control) are positive; and (3)
the geometric mean endpoint concentration of solution C is in
the range of O. 5A. to 2A..
To determine the endotoxin concentration of solution A,
calculate the endpoint concentration for each replicate series
of dilutions by multiplying each endpoint dilution factor by /....
Table 3 Preparation of solution in the gel-clot semiquantitative test
Endotoxin concentration/Solution
Solution Diluent
to which endotoxin is added
A None/Test solution Water for BET
B ZA/Test solution
e ZA/Water for BET Water for BET
~- ....
D None/Water for BEI
Solution A: test solution at the dilution, not exceeding the MVD, with which the test for interfering factors was carried out,
Subsequent dilution of the test solution must not exceed the MVD. Use water for BET to make two dilution series of 1, 1/2,
1/ 4, and 1/8, relative to the dilution with which the test for interfering factors was carried out.
Solution B: solution A containing standard endotoxin ata concentration of 2A. (positive product control).
Solution C: series of water for BET containing the standard endotoxin at concentrations of ZA., A., O. 5A., and O. 25A., respectively
Solution D: negative control (water for BET)
Photometric Method ( Method 2)
The photometric method includes a turbidimetric method and
a chromogenic method. The turbidimetric method measures
the endotoxin concentrations of test solutions based on the
measurement of the increase in turbidity during the gel
formation of the lysate reagent. Depending on the test
principie used, this method is classified as being the
endpoint-turbidimetric test or the kinetic-turbidimetric test.
The endpoint-turbidimetric test is based on the quantitative
relationship between the endotoxin concentration and the
turbidity ( absorbance or transmission) of the reaction
mixture at the end of an incubation period. The kinetic-
turibidimetric test in a method to measure either the onset
time needed for the reaction mixture to reach a predetermined
absorbance, or the rate of turbidity development.
The chromogenic method measures the chromophore released
from a suitable chromogenic peptide by the reaction of
endotoxins with the lysate reagent. Depending on the test
principle employed, this method is classified as being the
endpoint-chromogenic test or the kinetic-chromogenic test.
The endpoint-chromogenic test is based on the quantitative
relationship between the endotoxin concentration and the
quantity of chromophore released at the end of an incubation
period. The kinetic-chromogenic test is a method to measure
1143 Test far Bacteria! Endotoxin 1;
If the test is conducted with a diluted test solution, calculate
the concentration of endotoxin in the original solution (e) by
multiplying the result by the dilution factor.
The preparation being examined meets the requirements of
the test if the endotoxin concentration is less than that
specified in the individual monograph. The endotoxin concen-
tration in the test solution is the geometric mean endpoint
concentration of the replica tes [CE= antilg( I;c /2) J. If none
of the dilutions of the test solution is positive in a valid test,
record the endotoxin concentration as less than A. (or, if a
diluted sample was tested, as less than A.X the lowest dilution
factor of the sample).
The preparation being examined <loes not meet the require-
ments of the test, if the endotoxin concentration is not less
than that specified in the individual monograph. If all
dilutions are positive, the endotoxin concentration is recorded
as equal to or greater than the greatest dilution factor
multiplied by A..
lni tial endotoxin Number of
Dilution factor
concentra tion replica tes
1 - 2
2 - 2
4 - 2
8 - 2
1 2A. 2
1 2A. 2
2 1A. 2
4 O. 5A. 2
8 0.25A. 2
-
either the onset time needed for the reaction mixture to reach
a predetermined absorbance or light transmittance, or the
rate of colour development.
All photometric tests are usually carried out by special
instrumentation at the incubation temperature of 37±1 ºC.
The volume ratios of the substance being examined and the
lysate reagent, incubation time, etc. employed in the test are
decided according to the related instructions of instruments
and the lysate reagents.
To assure the precision or validity of the turbidimetric and
chromogenic tests, preparatory tests are conducted to assure
that the criteria for the standard curve are satisfied and that
the test solution <loes not interfere with the test.
Assurance of criteria for the standard curve The test for
Assurance of criteria for the standard curve must be carried
out when each new batch of lysate reagent is used or any
changes are made to the experimental conditions that are
likely to influence the result of the test. Using the standard
endotoxin solution, prepare at least three endotoxin
concentrations ( the dilution factor of adjacent concentrations
is not greater than 10) to generate the standard curve within
the range of endotoxin concentrations indicated by the lysate
reagent manufacturer. The mixing time for every dilution is
 1144 Test far Vasopressor Substance
the same as that of gel-clot method. Perfarm the test using at
least three replicates of each standard endotoxin solution, and
duplicate of negative control solution at the same time. When
absorbances or light transmittances of the duplicate of
negative control solution are less than that of the lowest
concentration, or both reaction times of the duplicate of
negative control solution are greater than that of the lowest
concentration, perform statistical analysis of linear regression
far all the data.
The test is not valid unless the absolute value of the
correlation coefficient, 1 r 1 , is greater than or equal to
O. 980, otherwise repeat the test.
Test for interfering factors Select an endotoxin concentration
O.m) at or near the middle of the endotoxin standard curve.
Prepare solutions A, B, C and Das shown in Table 4.
Table 4 Preparation of solutions in the test for
interfering factors by using photometric method
Endotoxin Solution to which Number of
Solution
concentration endotoxin is added replica tes
Not less
A None Test solution
than 2
Middle
concentration Not less
B Test solution
(A.m) of the than 2
standard curve
At least 3
Each
concentra tions
concentration
e Oowest Water far BET
not less
concentration
than 2
is designated A.)
Not less
D None Water far BET
than 2
Solution A: test solution that may be diluted not to exceed
the MVD.
Solution B: preparation to be examined at the same dilution
as Solution A, containing added endotoxin ata concentration
equal to or near the middle of the standard curve.
Solution C: standard endotoxin solution at the concentrations
used in the validation of the method described in assurance of
criteria far the standard curve.
Solution D: negative control (water for BET.)
Calculate the content of endotoxin contained in the solution A
( C) and solution B (C.) , respectively, and calcula te the
recovery of the endotoxin added to solution B as follows;
R =[(Cs-C)/A.m]XlOO%
If under the conditions of the test, the recovery of the
endotoxin added to solution B is within 50 % to 200 %, the
test solution is considered free of interfering factors. When
the endotoxin recovery is out of the specified ranges, the
interfering factors must be removed as described in the Test
far interfering factors under Gel-clot Method 1. The
efficiency of the treatment is verified by repeating the test far
interfering factors.
When the source of the lysate reagent, or the origin, formula
and the manufacture process of the substance being examined
are changed, or there is any change in the experimental
conditions that are likely to influence the result of the test,
the Test far interfering factors should be perfarmed again.
Procedure Follow the procedure described in the "Test for
interfering factors" und~r Photometric Method. Calculate the
endotoxin concentration of each replicate of solution A using
the standard curve generated by the senes of positive
controls, solution C.
The test is not valid unless the fallowing 3 requirements are
met;
(1) the results obtained with the series of positive controls,
solution e, comply with the requirements for validation
defined in the "Assurance of criteria for the standard curve"
under Photometric Method ;
(2) the endotoxin recovery, calculated from the endotoxin
concentration found in solution B after subtracting the
endotoxin concentration found in solution A, is within the
range of 50 % to 200 %;
(3) the result obtained with solution D (negative control) is
less than the lowest concentration of the standard curve, or
the reaction time of solution D (negative control) are greater
than that of the lowest concentration of the endotoxin
standard curve.
Invalidation of Results The preparation being examined
complies with the test if the mean endotoxin concentration of
the replicates of solution A, after correction for dilution and
concentration, is less than the endotoxin limit for the
product. Otherwise, the preparation being examined <loes not
meet the requirements of the test.
Note: In this chapter, the term "tube" includes all types of
receptacles, for example, microtiter plate wells.
1144 Test for Vasopressor Substance
The vasopressor substance of the preparation being examined
(T) is determined by comparing its vasopressor activity on
anesthetized rat with that of standard preparation of lysine
vasopressin (S) under the condition of the fallowing method.
Preparation of standard solution Dissolve the lysine
vasopressin standard in sodium chloride injection to produce a
standard solution of O. 1 Unit per ml immediately befare the
test.
Preparation of test solution Dissolve the preparation being
examined in water for injection ( or sodium chloride
injection) to produce a solution of suitable concentration
specified in the monograph. Adjust the concentration of the
solution so that of the volume of the solution being injected
will be equal to that of the standard dilution.
Procedure Carry out the test on a healthy male rat
weighting over 300 g which anaesthetized with suitable
anesthetics (such as i. p.1 g urethane per kg body weight).
Tie the rat on it' s back to a operating table and maintain the
body temperature of the rat during the test. Dissect the
trachea for cannulation when necessary. lnsert the femoral
(or carotid) vein a cannula filled with sodium chloride
injection for intravenous injection. Inject through the venous
cannula 50-100 Units of heparin per 100 g body weight.
Insert another cannula filled with a heparinised sodium
chloride injection into the common carotid artery and connect
it to a device capable of giving a continuous record of the
blood pressure.
At this stage, adjust the pressure of the device to a level
corresponding to the normal blood pressure of the rat, and
remove the artery clamp. Inject slowly a a-adrenoceptor
blocking agent ( such as phentolamine mesylate O. 1 mg per
100 g body weight) and repeat the same <lose of injection
after 5-10 minutes. Start the test only when the blood
pressure of the rat remains stable. Injection should be made
at a uniform rate and at a regular interval of 5-10 minutes
depending on the time at which the blood pressure returns to

its original level. Each injection is followed by about O. 5 ml
of sodium chloride injection.
lnject intravenously in turn two doses of standard dilution
(ml) with the dose ratio of about 1 : O. 6, and repeat the
injection for 2-3 times. If the elevation response of blood
pressure to the lower doses is corresponding to l. 33-
3. 33 kPa, and the mean response caused by higher doses is
clearly greater than that of the mean response caused by
lower doses, the sensitivity of the rat is suitable for the test.
Let ds, represent a dose of standard dilution ( mD which
lies between the range of lower and higher doses used in the
sensitivity test, and dT the dose of test preparation specified
in the monograph, inject a series of four doses in the order of
ds, dT, dT, d 5 , compare the elevation response of blood
pressure caused by the third dose (dT) to that caused by the
first dose ( d s ) , and the response caused the second dose
(dT) to that caused by the fourth dose ( ds ). The
preparation complies with the test if the two responses
caused by d T are not greater than one half of that caused by
ds. If not, repeat another series of four doses of ds, and dT
and compare the responses in the same way. The preparation
complies with the test if, in the two series of doses, none of
the response of dT is greater than that of d 5 • The
preparation fails the test if all the responses caused by d T are
greater than that of d 5 • If it is not the case, repeat the test
with another rat. The test preparation fails the test if in the
repeated test the responses caused by d T is greater than that
caused by ds.
1145 Test for Depressor Substances
The depressor substance of a preparation being examined
(T) is determined by comparing its depressor effect on
anesthetized cat v1ith that of the histamine reference standard
(RS) under the condition of the following method.
Preparation of standard solution Dissolve an accurately
weighed quantity of the histamine phosphate RS in water to
produce a solution of 1 mg histamine base per ml. Store the
solution ata temperature of 4-8ºC. The solution may be used
overa period of not exceeding three months if it is validated
to meet the activity test requirement.
Preparation of standard dilution Dilute the standard solution
with sodium chloride injection to produce a solution of O. 5 µg
of histamine base per ml immediately befare the test.
Preparation of test solution Prepare a solution of the
substance preparation being tested to produce a solution of
suitable concentration specified in the monograph so that the
volume of the solution being injected will be equal to that of
the standard dilution.
Procedure A healthy cat weighting over 2 kg, male or non-
pregnant female, is anaesthetized with a suitable anesthetic
such as one of the barbiturates. Fix the cat to an operating
table and maintain the body temperature of the cat during the
test. Dissect the trachea and introduce a short glass cannula
into the trachea so as to be ready for artificial respiration
when necessary. lnsert a cannula filled with suitable
anticoagulant solution into one of the common carotid artery
and connect the cannula to a suitable pressure measuring
device for making a continuous record of blood pressure.
Cannulate the femoral vein for intravenous injection. Adjust
the pressure of the pressure measuring device to a level
corresponding to the normal blood pressure of the animal
( usually 13. 3-20. O kPa) and remove the artery clamp. The
doses can be given only when the blood pressure of the
1146 Test for Histamine
animal is constant. lnjections should be made at a uniform
rate and at regular intervals depending on the speed at which
the blood pressure returns to a constant level. Each injection
is followed by a fixed volume of sodium chloride injection.
lnject intravenously in turn 3 doses of standard solution
corresponding to O. 05 µg, O. 1 µg and O. 15 µg of histamine
base per kg of the body weight. Repeat the injections for 2-
3 times, if the depressor responses to the injections of
O. 1 µg/kg are approximately the same and correspond to a
fall of blood pressure not less than 2. 67 kPa and the
responses to the graded doses are clearly discriminated, the
sensitivity of the animal is suitable for the test.
Let d s represent the dose of O. 1 µg of the histamine base
per kg of the cat' s weight and d T represent the dose of the
test preparation specified in individual monographs, inject a
series of doses in the order of d s , d T , d T , d s , compare the
depressor response caused by the third dose with that caused
by the first dose, and the response caused by the second dose
with that caused by the fourth dose. The preparation com-
plies with the test if none of the depressor responses caused
by dT is greater than 1/2 of that caused by ds. If one
response caused by dT is greater than 1/2 of that caused by
ds, inject another series of ds, dT, dT, ds and compare
their responses in the same way. The test preparation
complies with the test if none of the depressor responses
caused by dT is greater than that caused by d 5 • The test
preparation fails the test if all responses caused by dT are
greater than that caused by ds. If it is not the case, repeat
the test with another cat. The test preparation fails the test if
in the repeated test one depressor response caused by d T is
greater than that caused by ds.
The animal can further be used for testing of depressor
substances if the sensitivity of the animal is still suitable for
the test.
1146 Test for Histamine
Histamine substance being examined (T) is determined by
comparing its contraction effect on isolated guinea-pig
intestine with that of the histamine reference standard (RS)
under the condition of the following method.
Preparation of standard solution Dissolve an accurately
weighed quantity of the histamine phosphate RS in water to
produce a solution of 1 mg histamine base per ml. Store the
solution ata temperature of 4-8ºC. The solution may be used
over a period of not exceeding three months if it is validated
to meet the activity test requirement.
Preparation of standard dilution On the day of the assay,
take a quantity of histamine reference standard diluted with
sodium chloride injection to produce two dilutions of ( S)
(ds 2 , ds 1 ). Adjust 2 dose levels so that the higher dose
does not produce a maximum response and the lower dose
produces approximately half as great as the higher dose.
Make sure the contraction is reproducible. The final
concentration of histamine reference in organ bath may be
about 10- 1 -10- 9 g/ml with the injection volume O. 2 -0. 5 ml
generally, and the ratio ( r) of the two dose is about 1 :
o. 5.
Preparation of test solution Prepare in the same way as
described under Preparation of standard solution with
suitable concentration specified in the monograph. The
volume of the solution being injected will be equal to that of
the standard solution.
Preparation of lock solution for isolated intestine Solution A:
 ·1a4.: 1147 Test for Allergen
on the day of assay, prepare the solution to 700 ml of
aqueous solution contammg the following ingredients:
sodium chloride 160. O g, potassium chloride 4. O g, calcium
chloride ( anhydrous) 2. O g, magnesium chloride ( anhy-
drous) l. O g, disodium hydrogen phosphate dodecahydrate
O. 10 g, and then add water for injections to 1000 ml.
Solution B: Dissolve O. 5 mg of atropine sulfate, l. O g of
sodium hydrogen carbonate, O. 5 g of glucose monohydrate
in sufficient water for injections, mix with 50 ml of solution
A, and then add water for injections to 1000 ml, adjust to
pH 7. 2-7. 4. Solution B should be freshly prepared.
Assay Sacrifice a guinea-pig, male or nonpregnant female,
weighing 250 g to 350 g that has been deprived of food for the
preceding 24 h. Remove a portian of the distal small intestine
2 cm in length and empty the isolated part by rinsing
carefully with solution B using a syringe. Attach a fine thread
to each end and make a small transverse incision in the
middle of the piece of intestine. Place it in an organ bath with
a capacity of 10 ml to 30 ml, containing solution B main-
tained at a constant temperature ( 34 ºC to 36ºC) and pass
through the solution a current of a mixture of 95 % of oxygen
and 5 % of carbon dioxide. Attach one of the threads near to
the bottom of the organ bath and the other thread to an
isotonic myograph and record the contractions of the organ
on a kymograph or other suitable means of giving a
permanent record. If a lever is used, its length is such that
the movements of the organ are amplified about 20 times.
The tension on the intestine should be about 1 g and it should
be adjusted to the sensitivity of the organ. Flush out the
organ bath with solution B. Allow it to stand for 15 -30 miP_
Flush 2 or 3 times with solution B before each addition of
histamine reference standard or test solution. The successive
additions should be made at regular intervals allowing a
complete relaxation between additions (about 2 min).
Let ds representa dose of standard dilution (ml) which lies
between the range of lower and higher doses used as indicated
above, and dT represents the dose of test preparation
specified in the monograph, add into the bath the six dose in
the order of ds 2 , ds 1 , dy, dy, ds 1 , ds 2 , adjust the
dilution so that the contraction due to the lower dose of
histamine is smaller than that of the higher dose. Determine
whether the contraction, if any, is reproducible and that the
responses to the higher and lower doses of histamine are
unchanged. Compare the contraction response caused by the
second dose (d 51 ) to that caused by the fourth dose (dy),
and the response caused by the fifth dose ( d 51 ) to that
caused by the third dose ( d T ) • The test preparation
complies with the test if the two responses caused by d T are
not greater than that caused by d 51 , while it fails if both of
the responses caused by dT are greater than that of d 51 • If
one of the responses caused by d T is greater than that of
ds 1 , repeat the test with another guinea-pig and compare the
responses in the same way. The test preparation complies
with the test, if in the two series of doses, none of the
response of dy is greater than that of d 51 • The preparation
fails the test if one of the responses caused by d T is greater
than that of d 51 • If the solution to be examined does not
produce a contraction, prepare a fresh test solution with a
quantity of histamine reference standard corresponding to
that specified in the monograph, by adding higher and lower
doses of histamine reference standard. Add a series of fourth
doses in the order of ds 2 , ds 1 +T, ds 2 +T, ds 1 , and repeat it.
The test preparation complies with the test if the contractions
caused by the test solution with histamine reference standard
correspond to that caused by the higher and lower doses of
histamine reference standard. If this is not the case, as
subsequent responses to test solution with higher and lower
doses of histamine are diminished or showed no contraction,
or if not reproducible, the results of the tests are invalid and
repeat the test with another guinea-pig. If the results of test
for histamine are invalid, the test for depressor substances
should be carried out.
1147 Test for Allergen
The general allergic reactions of a substance being examined
are determined by injecting adose of test solution into guinea
pigs, after a fixed interval challenging intravenously into each
sensitized animal a solution of the substance being examined
and observing allergic reactions of each challenged animal.
Use healthy guinea pigs weighing 250-350 g, male or non-
pregnant female. Breed in normal condition before and during
test. Guinea pigs can only be used once for the test.
Preparation of test solution Unless otherwise prescribed,
prepare an solution of a substance being examined with
suitable concentration specified in the monograph.
Procedure Unless otherwise prescribed, inject intraperito-
neally or an appropriate route of administration O. 5 ml of the
test solution into each of 6 guinea pigs for 3 times
successively, once every other day. Examine the guinea pigs
every day and measure the weight of each animal before
sensitization and challenge. Then distribute sensitized animals
into two groups with 3 guinea pigs in each group. Challenge
intravenously into each guinea pig of one group 1 ml of the
test solution on the fourteenth day and each of the other
group on the twenty-first day after the first injection.
Observe if allergic reactions happen within 30 minutes after
challenge.
Evaluation of the results The guinea should have no allergic
reactions within 30 minutes after challenge. If one of the
challenged guinea pigs shows two or more types of allergic
reactions, su ch as piloerection, shivering, retching,
continuous sneezing for three times, continuous cough for
three times, purpura and short of breathing; or one of the
following allergic reactions: incontinence, instability of gait
or fall clown steps, twitching, shock or dying, the substance
being examined does not meet the requirements of the test.
1148 Test for Hemolysis and
Agglomeration
The test is to observe the effect of test solution on
erythrocytes by mixing and incubating with test solution and
2 percent rabbit erythrocyte suspension for certain time.
Preparation of 2 percent erythrocyte suspension Introduce a
few millilitres of freshly rabbit blood into a flask containing
glass pearls, horizontally rotate the flask about 10 minutes,
or stir the blood with a glass rod to remove fibrin. Fill the
flask about 10 times more than that of the volume of blood
with O. 9 % NaCl solution, stir and centrifuge at a speed of
1000-1500 rpm for 15 minutes, remove the supernatant and
wash the precipitated erythrocyte with O. 9 % NaCl solution
2-3 times by above means until the colour of supernatant is
not red. Prepare the 2 percent suspension of erythrocyte with
O. 9 % NaCl solution.
Preparation of test solution Unless specified otherwise, the
test solution should be prepared with suitable concentration
specified in the monograph.
 1201
Procedure Use 5 clean and labelled glass tubes, No. 1 and
Z tubes are used for test solution, No. 3 tube for negative
control, No. 4 tube for positive control and No. 5 tube for
test solution control. As shown in the below sheet, add in
turn to the tubes with 2 percent erythrocyte suspension,
O. 9 % NaCl solution or purified water, invert the mixed
solutions gently and immediately incubate at 37°C ±O. 5ºC
incubator for 3 hours. Observe the haemolysis and agglo-
meration 3 hours later.
Tu bes 1, 2 3 4 5 ration occurred in negative tube meanwhile hemolysis
2 % erythrocyte suspension/ ml z. 5 Z.5 2.5
O. 9% NaCl/ml 2. 2 2. 5 4. 7
Purified water/ ml 2.5
Test solution/ml 0.3 0.3
If the mixed solution is red and clear with no or a little
erythrocyte stayed in the bottom of the tube, heamolysis has
occurred. If all of the erythrocytes sank and the supernatant
is colourless and clear, or the supernatant is colour and
clear, meanwhile no obvious diff erence among the No. 1, 2
1200 Biological Activity Assays
1201 Microbiological Assay of Antibiotics
The activity ( potency) of antibiotics may be demonstrated
under suitable conditions by their inhibitory effect on
microorganisms. The assay is designed on the basis of the
principle of parallel line model.
Two general methods are employed, the agar diffusion
method and the turbidimetric method.
If the calculated potency is less than 90 % or more than 110 %
of the estimated potency, the assay should be repeated by
changing the estimated potency.
Unless otherwise specified, the average percentage of fiducial
limits is not more than 5%.
Method 1 Agar Diffusion Method
The agar diff usion method is a method to determine the
potency of an antibiotic to be tested, and is based on
comparing the dose response of the preparation being
examined that inhibits the growth of a suitable susceptible
microorganism and that of the standard preparation of that
antibiotic are in the same degree of inhibition expressed by
the sizes of the corresponding inhibition zones.
Preparation of inoculum
Bacillus subtilis suspension Transfer the growth of Bacillus
subtilis [ CMCC ( B) 63501 J from the nutrient agar slant
onto a large slant surface, incubate at 35-37°C for 7 days,
examine the growth stained by Grams method
microscopically, it contains not less than 85 % of spores.
Wash off the spores with sterile water and heat at 65ºC for
30 minutes.
Bacillus pumilus suspension T ransfer the growth of
Bacillus pumilus [ CMCC ( B) 63202] from the nutrient
agar slant onto a large slant surface, prepare the suspension
as described under Bacillus subtilis suspension.
Staphylococcus aureus suspension Transfer the growth of
Microbiological Assay of Antibiotics
and 5 tubes can be obtained from gross appearance, no
haemolysis has occurred.
If red brown or brown red flocculated precipitation has
appeared, which can not be dispersed by turning over gently
for 3 times, agglomeration might has occurred. Then the
flocculated precipitation should be further observed under
microscopy, if erythrocyte aggregation is observed, the agg-
lomeration is true.
Evaluation of the results When no haemolysis and agglome-
occurred in the positive tube, and if no hemolysis and
agglom-eration occurred in all test solution in 3 hours, the
substance being tested complies with the requirements,
otherwise, it does not comply with the requirements. If
hemolysis and/ or agglomeration are observed in one of two
test solution tubes in 3 hours, four test solution tubes should
be examined in same procedure. Hemolysis and/ or
agglomeration are not expected in each of four test solution
tubes, or else the substance being examined does not meet
the requirements of the test.
Staphylococcus aureus [ CMCC ( B) 26003 J from the
nutrient agar slant onto a fresh slant surface, incubate at 35-
37ºC for 20-22 hours. Wash off the growth with sterile water
or sterile solution of O. 9 % sodium chloride immediately
before use.
Micrococcus lutea suspension Transfer the growth of
Micrococcus lutea [ CMCC ( B) 28001] from the nutrient
agar slant onto a large slant surface, incubate at 26-27°C for
24 hours. Wash off the growth with medium fil or sterile
solution of O. 9 % sodium chloride immediately before use.
Escherichia coli suspension Transfer the growth of
Escherichia coli [CMCC (B) 44103] from the nutrient agar
slant onto a fresh slant surface, incubate at 35-37°C for 20-
22 hours. Wash off the growth with sterile water
immediately before use.
Saccharomyces cerevisiae suspension Transfer the growth of
Saccharomyces cerevisiae ( ATCC9763) from medium V
slant onto medium N slant, incubate at 32-35ºC for
24 hours. Wash off the growth with sterile water into a tube
containing sterile glass beads, shake well.
Klebosiella pneumoniae suspension Transfer the growth of
Klebosiella pneumoniae [ CMCC ( B) 46117 J from the
nutrient agar slant onto a fresh slant surface, incubate at 35-
37°C for 20-22 hours. Wash off the growth with sterile water
immediately befare use.
Bordetella bronchiseptica suspension Transfer the growth of
Bordetella bronchiseptica [ CMCC ( B) 58403 J from the
nutrient agar slant onto a fresh slant surface, incubate at 32-
35ºC for 24 hours. Wash off the growth with sterile water
immediately befare use.
Reference preparation The reference substances should be
handled as directed in the package inserts. Solutions of the
ref erence substance are pre pared using sterile buffers
described in Table 1 and diluted immediately befare use.
The reference substances, molecular formula and theoretical
potency are shown in Table 2.
 1201 Microbiological Assay of Antibiotics

Test preparation Dissolve an accurately weighed quantity of individual monographs and dilute to approximately the same
the substance being examined in the solvent prescribed under concentration as that of the reference preparation.
Table 1 The Experimental Design of the Agar Diffusion ~y of Antibiotics
Medium Sterile buffer Concentration lncubate condition
Antibiotics Test organism
No. pH pH Unit/ml temp./°C time/h
Streptomycin Bacillus subtilis I 7. 8-8. o 7.8 o. 6-1. 6 35-37 14-16
Kanamycin [CMCC(B)63501] I 7. 8-8. o 7. 8 o. 9-4. 5 35-37 14-16
Amikacin I 7. 8-8. o 7.8 o. 9-4. 5 35-37 14-16
Paromomycin I 7. 8-8. o 7.8 o. 9-4. 5 35-37 14-16
Ribostamycin I 7. 8-8. o 7.8 2. 0-12. o 35-37 14-16
Capreomycin I 7. 8-8. o 7.8 10. 0-40. o 35-37 14-16
Sul benicillin I 6. 5-6. 6 6.0 5. 0-10. o 35-37 14-16
Norvancomycin '111 6.0 6.0 9. 0-43. 7 35-37 14-16
AcetylspiramycintD 11 8. 0-8. 2 7.8 5-40 35-37 14-16
Tobramycin I 7. 8-8. o 7.8 1-4 35-37 14-16
Roxithromycin 11 7. 8-8. o 7.8 5-10 35-37 16-18
Kitasamycin 11@ 8. 0-8. 2 7.8 20-40 35-37 16-18
nutrient agar
Meleumycin 8. 0-8. 2 7. 8 5-40 35-37 16-18
medium
Micronomycin I 7. 8-8. o 7.8 o. 5-2. o 35-37 14-16
Josamycin 11 7. 8-8. o 7.8 7. 5-30 35-37 14-16
Josamycin 11 7. 8-8. o 7.8 20-80 36-37 14-16
pro piona te
Teicoplanin Il 6. 5-6. 6 6.0 20-40 36-37 14-16
Vancomycin '111 6.0 6.0 2. 5-12. 5 35-37 14-16
Gentamycin Bacillus pumilus I 7. 8-8. o 7.8 2. 0-12. o 35-37 14-16
Erythromycin [ CMCC(B) 63202] I 7. 8-8. o 7.8 5. 0-20. o 35-37 14-16
Netilmicin I 7. 8-8. o 7.8 5-20 35-37 14-16
Sisomicin I 7. 8-8. o 7.8 5-20 35-37 14-16
Azithromycin I 7. 8-8. o 7.8 o. 5-20 35-37 16-18
Clarithromycin I 7. 8-8. o 7.8 2. 0-8. o 35-37 14-16
Staphylococcus aureus
Neomycin 11 7. 8-8. o 7. 8® 4. 0-25. o 35-37 14-16
[CMCC(B)26003]
T etracycline Micrococcus lutea 11 6. 5-6. 6 6.0 10. 0-40. o 35-37 14-16
Oxytetracycline [CMCC(B)28001] 11 6. 5-6. 6 6.0 10. 0-40. o 35-37 16-18
Chlortetracycline 11 6. 5-6. 6 6.0 4. 0-25. o 35-37 16-18
Chloramphenicol 11 6. 5-6. 6 6.0 30. 0-80. o 35-37 16-18
Bacitracin 11 6. 5-6. 6 6.0 2. 0-12. o 35-37 16-18
Fosfomycin 11 7. 8-8. o 7.8 5-20 35-37 18-24
VI 7. 2-7. 4 6.0 614-2344 35-37 16-18
Colistin Escherichia coli
Polymyxn B [CMCCCB)44103] nutrient agar
6. 5-6. 6 6.0 1000-4000 35-27 16-18
medium

Saccharomyces
Amphotericin B® cerev1s1ae N 6. 0-6. 2 10.5 o. 5-2. o 35-37 24-36
(ATCC 9763)

Klebosiella
Spectinomycin Pneumoniae 11 7. 8-8. o 7.0 50-200 35-37 16-18
[CMCCCB)46117]
CDUse 15 ml Saccharomyces cerevisiae inoculated seed layer instead of base and seed layers for the preparation of inoculated
plate.
@Adjust the pH of medium 11 to 8. 0-8. 2 after it is sterilized.
®Containing 3% sodium chloride.
@Add O. 3 % glucose to medium 11
 1201 Microbiological Assay of Antibiotics

Table 2 The Antibiotic Reference Standards and Theoretical Potency

Calculated Calculated
Antibiotic Antibiotic
Molecular formula theoretical Molecular formula theoretical
reference reference
or name potency or name potency
standards standards
Unit/mg Unit/mg
Streptomycin CC21 839 N1012 )z •382S04 798.3 Erythromycin C318s1NÜ13 1000
Kanamycin C1s 83s N4 011 • 82 S04 831. 6 Chloramphenicol Cu 812 C12 Nz Os 1000
Amikacin Cz2 843 Ns Ü13•n 82 S04 (n =l. 8 or 2) Bacitracin Baci tracin Zinc
Ribostamycin C11 834 N4 010•n 82 S04 Cn<2) Colistin Colistin Sulfate
Neomycin Neomycin sulfate Norvancomycin Css 8n C12 N9 024 • 8Cl 975.2
Gentamycin Gentamycin sulfate Capreomycin Capreomycin Sulfate
Sulbenicillin C16 816 Nz Na2 01 S 904.0 Amphotericin B C418nN011 1000
T etracyclline Cz2 824 Nz Os• 8Cl 1000 Paromomycin Cz3 84s Ns Ü14•n82S04
Oxytetracycline Cz2 824 Nz Ü9• 282 O 927 Netilmicin CC21 841Ns01 )z •582S04 660. 1
Sisomicin (C19 831Ns01 )z •582S04 646.3 Azithromycin C3s 812N2Ü13 1000
Fosfomycin C38sCa04P•820 711. 5 Tobramycin C1s831NsÜ9 1000
Acetylspiramycin Acetylspiramycin Roxithromycin C41 81s Nz Ü1s 1000
Clarithromycin C3s8s9NÜ13 1000 Kitasamycim Kitasamycin
Spectinomycin C14824N201 •28Cl•5820 670.9 Meleumycin Meleumycin
Micronomycin Czo841Ns01 •5/282S04 654.3 Josamycin C428s9NÜ1s 1000
Josamycin
Polymyxin B Polymyxin B sulfate C4s8nN016 937
propionate
Chlortetracvcline Cz2 822 CIN2 Os• 8Cl 1000 Teicoplanin C12-s9 Hss-99 C}z Ns-9 Ü2s-33 1000

Preparation of inoculated plates fill Petri dishes with flat
bottom, 90 mm diametre, 16-17 mm high with 20 mi of
molten medium listed in the table l. Place the dishes on a
horizontal platform to produce layers of hardened culture
medium in uniform thickness. Then add to each plate 5 mi of
the same medium which has previously been inoculated at 48-
50ºC ( or 60ºC for spores) with an inoculum of the test
organism listed in the table l. The concentration of the
inoculum should be so selected that the sharpest zones of
inhibition are obtained. The diametres of the inhibition zones
produced by the high <lose of the reference preparation are
18-22 mm for 2-dose assay, and 15-18 mm by middle dose
for 3-dose assay respectively. Spread the medium evenly over
the entire surface and allow it cool on a horizontal platform.
Place 4 ( 2-dose assay) or 6 ( 3-dose assay) stainless steel
cylinders ( 6. O mm± O. 1 mm in interna} diametre, 7. 8 ±
O. 1 mm in externa} diametre and 10. 0±0. 1 mm in height)
on the surface of each plate at equal distance, then cover
them with circular clay lids.
~y
conditions prescribed in the table 1 measure the diametre ( or
area) of the inhibition zones. Carry out the statistical
analysis of variance ( 1431, design 3. 3) and calculate the
potency of the substance being examined.
Method 2 Turbidimetric Metbod
The turbidimetric method is a method to determine the
potency of an antibiotic, depends upon the preparation being
examined that inhibits the growth of a microbial culture in a
fluid medium and that of the standard preparation of that
antibiotic are in the same degree of inhibition expressed by
the turbidity of the microbial culture which can be measured
photometrically.
Preparation of inoculum
Staphylococcus aureus suspension Transfer the growth of
Staphylococcus aureus [ CMCC ( B) 26003] from the
nutrient agar slant onto a fresh slant surface, incubate at 35-
37ºC for 20-22 hours. Wash off the growth with sterile water
or sterile solution of O. 9 % sodium chloride immediately
before use.

2-dose assay Use not less than 4 inoculated plates prepared
as above, fill two of the diagonal cylinders on each plate with
the high and low doses of the reference preparation, fill the
remaining cylinders with the high and low doses of the test
preparation. The <lose levels should be in the ratio of 2 : 1 or
4 : l. Incubate the plate under the conditions prescribed in
the table 1 measure the diametre ( or area) of the inhibition
zones. Carry out the statistical analysis of variance ( 1431 ,
design 2. 2) and calculate the potency of the substance being
examined
3-dose assay Use not less than 6 inoculated plates prepared
as above, fill three of the alternate cylinders on each plate
with the high, middle and low doses of the reference
preparation, fill the remaining cylinders with the high,
middle and low doses of the test preparation. The <lose levels
should be in the ratio of 1 : O. 8. Incubate the plates under the
Escherichia coli suspension T ransfer the growth of Escheri-
chia coli [CMCC (B) 44103] from the nutrient agar slant
onto a fresh slant surface, incubate at 35-37ºC for 20-
22 hours. Wash off the growth with sterile water immediately
before use.
Candida albicans suspension Transfer the growth of
Candida albicans [ CMCC ( F) 98001] from the Martin
agar modified medium slant onto 10 mi of medium IX,
incubate at 35-37°C for 8 hours. Dilute to a suitable concen-
tration with medium IX.
Reference preparation The reference substances should be
handled as directed in the package inserts. Solutions of the
reference substance are prepared using sterile buffers
described in Table 3 and diluted immediately before use.
The reference substances, molecular formula and theoretical
potency are shown in Table 2.
 1201 Microbiological Assay of Antibiotics

Test preparation Dissolve an accurately weighed quantity of individual monographs and dilute to approximately the same
the substance being examined in the solvent prescribed under concentration as that of the reference preparation.
Table 3 The Experimental Design of the Turbidimetric Assay of Antibiotics
Medium Sterile buffer Caneen tration lncubate condition
Antibiotics Test orgnism
pH pH Unit/ml Temp./ºC
No.
Gentamycin III 7. 0-7. 2 7.8 0.15-1. o 35-37
Steptomycin III 7. 0-7. 2 7. 8 2.4-10.8 35-37
Amikacin III 7. 0-7. 2 7. 8 o. 8-2. o 35-37
Erythromycin III 7. 0-7. 2 7. 8 o. 1-0. 85 35-37
Neomycin III 7. 0-7. 2 7. 8 o. 92-1. 50 35-37
T etracycline III 7. 0-7. 2 6.0 o. 05-0. 33 35-37
Chloramphenicol III 7. 0-7. 2 7.0 5.5-13.3 35-37
Neltimicin III 7. 0-7. 2 7.8 0.1-2.5 35-37
Si© somicin Staphylococcus III 7. 0-7. 2 7.8 o. 1-0. 25 35-37
Azithromycin aureus III 7. 0-7. 2 7. 8 l. 0-5. o 35-37
Acetylspiramycin [ CMCCCB) 26003] III 7. 0-7. 2 7. 8 5. 0-16. o 35-37
Tobramycin III 7. 0-7. 2 7.8 o. 3-1. 1 35-37
Kitasamycin III 7. 0-7. 2 7.8 o. 8-2. 4 35-37
Meleumycin III 7. 0-7. 2 7.8 l. 2-3. 2 35-37
Micronomycin III 7. 0-7. 2 7.8 o. 5-1. 2 35-37
Bacitracin III 7. 0-7. 2 6.0 o. 06-0. 30 35-37
Josamycin III 7. 0-7. 2 5. 6 l. 0-4. o 35-37
Josamycin propionate III 7. 0-7. 2 7. 8 o. 8-4. 8 35-37
Fosfamycin sodium III 7. 0-7. 2 7.0 12-42 35-37
Fosfamycin calcium Escherichia coli III 7. 0-7. 2 7.0 12. 0-31. o 35-37
Fosfamycin trometamol [CMCC(B)44103] III 7. 0-7. 2 7.0 12. 0-31. o 35-37
Spectinomycin III 7. 0-7. 2 7.0 30-72 35-37

Preparation of inoculated fluid medium Add suitable
quantity of inoculum of the test organism listed in the Table
3 to the fluid medium immediately befare use, and swirling
to attain a homogeneous suspension. The quantity of the
inoculum should be so selected that the microbial culture
should have absorbance between O. 3 to O. 7 after an
incubation period of 3-4 hours at 35-37°C. The difference of
absorbances of the microbial culture inhibited between
adjacent doses of the reference preparation is at least O. 1 for
the dose levels is in the ratio of 2 : l. It is preferable to obtain
the optimum <lose-response relationship and measurable
opacity occurs in the conditions of the test.
Use the inoculated medium immediately after its prepara-
tions.
Assay
Standard curve method (1-dose assay) Use glass or plastic
test tubes that are relatively uniform in length, diametre,
and thickness, sterilized befare use. Unless otherwise
specified, it may be necessary to select the median concen-
tration from defined <lose-response linearity as the median
test level. Prepare dilutions representing 5 test levels which
include one corresponding to the median concentration of the
standard. The <lose levels should be in the ratio of 1 : l. 25 or
less. Prepare a solution of a single median test level of the
antibiotic to be examined according to its estimated potency
or labelled concentration. Add each dose to 3 replicate tubes
at least. Distribute l. O ml of each solutions of standard or the
antibiotic to be examined into identical test tube and add to
each tube 9. O ml of inoculated medium, mix immediately.
The tubes randomized block arrangement, place them under
the appropriate incubation temperature and identical incuba-
tion time (incubate usually for 4 hours) in order to obtain a
readily measurable opacity under in the conditions of the test.
Measure the opacity online using suitable apparatus, or after
incubation, stop the growth of the microorganisms by adding
O. 5 ml of farmaldehyde ( 1-3) to each tube and read its
absorbance in a suitable spectrophotometer fitted with a 530
nm or 580 nm filter. Prepare at the same time 2 control tubes
both containing l. O ml of the test diluents and 9. O ml of the
inoculated medium but no antibiotic. One incubates at the
same condition as the test tubes to observe the growth of
microorganisms, the other adds immediately O. 5 ml of dilute
farmaldehyde Cl-3) to be the blank of absorbance.
Carry out the statistical analysis ( Calculation and Statis-
tical Analysis of Standard Curve Method) and calculate the
potency of the substance being examined.
Calculation and Statistical Analysis of Standard Curve Method
l. Calculation of Standard Curve
Logarithm Clg) of each <lose of Standard and corresponding
absorbance are shown in Table 4.
Table 4 Logarithm of Each Dose of Standard and
Corresponding Absorbance
Group of Units lg-dose of Standard Absorbance
1
2 Xz Yz
3
 1201 Microbiological Assay of Antibiotics

continued Percentage of fiducial limits

- upper limit of X 0 - lower limit of X 0
Group of Units lg-dose of Standard Absorbance FL %- 2Xo (9)

4 Where: Xo is the <lose of antibiotic.

Unless otherwise specified, the percentage of fiducial limits
is not more than 5 %.
n Xn Yn
( 4) Estimation of potency The potency of antibiotic are
Mean X y
obtained by multiplying the concentration ( lg-dose
transformed to concentration) by dilution factor of antibiotic.
Regression coefficient ( slope) b , intercept a and linear
regression equation of standard curve are calculated as 2-dose assay or 3-dose assay Use glass or plastic test tubes
follows: that are relatively uniform in length, diametre, and thick-
Regression coefficient: ness, sterilized befare use. Unless otherwise specified, it
b= I;(x; --X)(y; -y) I;x;Y; --XI;y; may be necessary to select the optimum high, ( middle,)
(1)
I;(x;-x) 2 I;x7-xI;x; low doses of the reference preparation. Distribute l. O ml of
each solutions of standard or the antibiotic to be examined
lntercept a=y-bx (2)
into identical test tube. For 2-dose assay the <lose levels
Linear regression equation Y=bX+a (3) should be in the ratio of 2 : 1 or 4 : 1, and for 3-dose assay
2. Significance tests of regression coefficient the <lose levels should be in the ratio of 1 : O. 8. With the
It used t-test to judge if the linear regression equation is same operation as standard curve method ( 1-does assay)
established, if the relation between X and Y can be repre- test, add each <lose to 4 replicate tubes at least. Assign tubes
sented by a straight line. at random block design and place them under the appropriate
Assume Ho: b = O, calculate the value of t with the incubation conditions. Carry out the statistical analysis of
following equations: variance < 1431 , design 2. 2 and 3. 3 ) and calculate the
Estimated standard deviation: potency of the substance being examined.
-JI;(y; -Y)2 Culture Media
Sy,x- (4)
n- 2
Mediuml:
Standard error of regression coefficient:
Peptone 5 g
SY,X
Sb (5) Beef extract 3 g
./2: (x;-x) 2 Dipotassium hydrogen phosphate 3 g
b-0 Agar 15-20 g
t=-- (6)
sb Water 1000 ml
Where: y; is the true absorbance of standard;
Mix the above ingredients with the exception of agar, adjust
y is the estimated absorbance may be obtained
the pH value to O. 2-0. 4 higher than the final pH of the
from linear regression equation;
solution. Add agar, heat to dissolution and filter, adjust the
y is the mean true absorbance of standard;
pH of the solution so that it is 7. 8-8. O or 6. 5-6. 6 after
X; is the logarithm of <lose of standard;
sterilization. Sterilize the medium at 115ºC for 30 minutes.
X is the mean logarithm of <lose of standard.
lf the calculated value of t is larger than the tabulated value Medium Il:
of to. os12<n-2> corresponding to p =O. 05 and 2n-4 degrees of Peptone 6 g
freedom, the regression is highly significant, the relation Beef extract l. 5 g
between X and Y can be represented by a straight line. Y east extract 3 g
Glucose 1 g
3. Estimation of potency and fiducial limits
Agar 15-20 g
( 1 ) Estimation of logarithm of dose of antibiotic When the Water 1000 ml
regression is highly significant, lg-dose of antibiotic may be Mix the above ingredients with the exception of agar and
estimated by the following equations: glucose, adjust the pH value to O. 2-0. 4 higher than the final
Logarithm of <lose of antibiotic X 0 =Yo;ª ( 7) pH of the solution. Add agar, heat to dissolution and filter,
then mix thoroughly with glucose and adjust the pH of the
( 2 ) Estimation of fiducial limits of logarithm of dose of solution so that it is 7. 8-8. O or 6. 5-6. 6 after sterilization.
antibiotic Fiducial limits of lg-dose of antibiotic for p = Sterilize the medium at 115ºC for 30 minutes.
O. 05 are calculated as follows: Medium ][:
Fiducial limits of Xo Peptone 5 g
2
SY·xJl 1 CXo-x) Beef extract l. 5 g
FL=Xo ±to.OS(n-2) 0
b ¡ • -+-+ "G 2 _ - " G
- ,
(8)
m n L.iX; X L.iX; Y east extract 3 g
Where: n is the number of <lose multiplying the number of Sodium chloride 3.5 g
responses to S; Dipotassium hydrogen phosphate 3.68 g
m is the number of responses to T; Potassium dihydrogen phosphate l. 32 g
Xo is the logarithm of <lose of T which is obtained Glucose 1 g
from Linear regression equation; Water 1000 ml
Yo is the mean absorbance of antibiotic. Mix the above ingredients with the exception of glucose,
( 3) Estimation of percentage of fiducial limits Percentage of heat to dissolution and filter, then mix well with glucose and
fiducial limits of lg-dose of antibiotic are calculated as adjust the pH of the solution so that it is 7. 0-7. 2 after
follows: sterilization. Sterilize the medium at l 15ºC for 30 minutes.
 1201 Microbiological Assay of Antibiotics

Medium N: solution. Add agar, heat to dissolution and filter, then mix
Peptone 10 g thoroughly with glucose and adjust the pH of the solution to
Sodium chloride 10 g 6. 5-6. 6 after sterilization. Sterilize the medium at 115ºC far
Sodium citrate 10 g 30 minutes.
Glucose 10 g Medium 1X:
Agar 20-30 g Peptone 7. 5 g
Water 1000 ml Beef extract l. O g
Mix the above ingredients with the exception of agar and Yeast extract 2. O g
glucose, adjust the pH value to O. 2-0. 4 higher than the final Sodium chloride 5. O g
pH of the solution. Heat it with agar at 109ºC far 15 minutes Glucose 10. O g
and allow to stand at above 70ºC far one hour, filter. Mix Water 1000 ml
thoroughly with glucose and adjust the pH of the solution so Mix the above ingredients with the exception of glucose, heat
that it is 6. 0-6. 2 after sterilization. Sterilize the medium at to dissolution and filter, then mix thoroughly with glucose
115ºC far 30 minutes. and adjust the pH of the solution to 6. 5 after sterilization.
Sterilize the medium at 115ºC far 30 minutes.
Medium V:
Peptone 10 g Nutrient broth medium:
Maltose 40 g Peptone 10 g
Agar 15-20 g Sodium chloride 5 g
Water 1000 ml Beef infusionCD 1000 ml
Mix peptone with water, adjust the pH value to O. 2-0. 4 Dissolve peptone and sodium chloride in the beef infusion,
higher than the final pH of the solution. Add agar, heat to warming slighting until the substances are dissolved, adjust
dissolution and filter, then mix well with maltose and adjust the pH value to slightly alkaline, heat to boíl, filter if
the pH of the solution to 7. 2-7. 4 af ter sterilization. Dispense necessary, adjust the pH of the solution to 7. 2 ±O. 2 after
sterilization. Sterilize the medium at 115ºC far 30 minutes.
the solution into suitable vessels and sterilize the medium at
115ºC far 30 minutes. Allow it to set in a sloped farm with Nutrient agar medium:
butt on cooling. Peptone 10 g
Sodium chloride 5 g
Medium VI:
Agar 15-20 g
Peptone 8 g
Beef infusionm 1000 ml
Beef extract 3 g
Mix the above ingredients with the exception of agar, adjust
Yeast extract 5 g
the pH value to O. 2-0. 4 higher than the final pH of the
Sodium chloride 45 g
solution. Add agar, heat to dissolution and filter, adjust the
Dipotassium hydrogen phosphate 3. 3 g
pH of the solution to 7. 2-7. 4 after sterilization. Dispense the
Potassium dihydrogen phosphate 1 g
solution into suitable vessels and sterilize the medium at
Glucose 2. 5 g
115ºC far 30 minutes. Allow it to set in a sloped farm with
Agar 15-20 g
butt on cooling.
Water 1000 ml
Mix the above ingredients with the exception of agar and Modified Martín medium:
glucose, adjust the pH value to O. 2-0. 4 higher than the final Peptone 5.0 g
pH of the solution. Add agar, heat to dissolution and filter, Y east extract 2.0 g
then mix thoroughly with glucose and adjust the pH of the Glucose 20.0 g
solution so that it is 7. 2-7. 4 after sterilization. Sterilize the Dipotassium hydrogen phosphate l. o g
medium at 115ºC far 30 minutes. Magnesium sulfate 0.5 g
Agar 15-20 g
Medium "W: Water 1000 ml
Peptone 5 g Mix the above ingredients in water except agar and glucose,
Beef extract 3 g warming slightly until the substances are dissolved, adjust
Dipotassium hydrogen phosphate 7g pH value to about 6. 8. Add agar, heat to boíl and filter,
Potassium dihydrogen phosphate 3 g then mix thoroughly with glucose and adjust the pH of the
Sodium citrate 2. 5 g solution to 6. 4±0. 2 after sterilization. Dispense the solution
Agar 15-20 g into suitable vessels and sterilize the medium at 115ºC far 30
Water 1000 ml minutes. Allow it to set in a sloped farm with butt on
Mix the above ingredients with the exception of agar, adjust cooling.
the pH value to O. 2-0. 4 higher than the final pH of the
solution. Add agar, heat to dissolution and filter, then mix Medium for Polymyxin B:
thoroughly with glucose and adjust the pH of the solution so Peptone 6.0 g
that it is 6. 5-6. 6 after sterilization. Sterilize the medium at Beef extract l. 5 g
115ºC far 30 minutes. Y east extract 3.0 g
Glucose l. o g
Medium ": Papaic digest of soybean 4.0 g
Y east extract 1 g Agar 15-20 g
Ammonium sulfate 1 g Water 1000 ml
Glucose 5 g Mix the above ingredients with the exception of agar, adjust
Agar 15-20 g
Phosphate BS (pH 6. O) 1000 ml
Mix the above ingredients with the exception of agar, adj ust CD Beef infusion can also be prepared by dissolvíng 3 g of beef extract
the pH value to O. 2-0. 4 higher than the final pH of the powder in 1000 mi of water.

the pH value to O. 2-0. 4 higher than the final pH of the
solution. Add agar, heat to dissolution and filter, adjust the
pH of the solution to 6. 5-6. 7 after sterilization. Sterilize the
medium at 115ºC for 30 minutes.
The culture medium can be replaced by the dry culture
medium containing the same ingredients, preparation and
sterilization is in accordance with the manufacture' s instruction.
Sterile Buffer Solutions
Phmphate BS ( pH 6. O) Dissolve 9. 07 g of potassium
dihydrogen phosphate in water to produce 1000 ml. Adjust
with 1 mol/L sodium hydroxide to pH 5. 6, filter, sterilize at
115 ºC for 30 minutes.
Phmphate BS ( pH 6. O) Dissolve 2 g of dipotassium
hydrogen phosphate and 8 g of potassium dihydrogen
phosphate in water to produce 1000 ml, filter, sterilize at
115ºC for 30 minutes.
Phmphate BS ( pH 7. O) Dissolve 9. 39 g of disodium
hydrogen phosphate and 3. 5 g of potassium dihydrogen
phosphate in water to produce 1000 ml, filter, sterilize at
115ºC for 30 minutes.
Phmphate BS ( pH 7. 8) Dissolve 5. 59 g of dipotassium
hydrogen phosphate and O. 41 g of potassium dihydrogen
phosphate in water to produce 1000 ml, filter, sterilize at
115 ºC for 30 minutes.
Phmphate BS ( pH 10. 5 ) Dissolve 35 g of dipotassium
hydrogen phosphate in water, add 2 ml of 10 mol/L
potassium hydroxide solution and sufficient water to produce
1000 ml, filter, sterilize at 115ºC for 30 minutes.
1202 Preparation of Penicillinase and
Determination of lts Activity
Culture medium
Peptone 15 g
Sodium chloride 4 g
Sodium citrate 5. 88 g
Dipotassium hydrogen phosphate 4 g
Glycerine 50 g
O. 1 %Ferrous sulfate CFeS0 4 • 7H 2 0) solution O. 5 ml
20% Magnesium sulfate (Mg S04 •7Hz0) solution 1 ml
Beef infusion 1000 ml
Mix the above ingredients, adjust the pH to 7. 0-7. 2 after
sterilization. Dispense in 500 ml conical flasks, 80 ml for
each flask, sterilize at 115ºC for 30 minutes.
Preparation of penicillinase solution
Inoculate one slant culture of Bacillus cereus [CMCC (B)
63301] to one of the culture media flasks, incubate on a
rotatory shaker at 25ºC for 18 hours. Inoculate 10 ml of this
culture to each of the remaining culture media flasks and add
4500 Units of sterile penicillin solution, incubate at 25ºC for
24 hours, again add 20000 Units of sterile penicillin solution
and incubate for 24 hours, and once more add 20 000 Units
of sterile penicillin solution and further incubate for another
24 hours. Centrifuge, sterilize the supernatant liquid by
filtration through a suitable filter medium after adjusting to
pH about 8. 5. Adjust the pH of the filtrate to neutrality
using aseptic technique, dispense the sterile filtrate to
suitable containers and stored below lOºC.
Determination of the activity of penicillinase
Preparation of penicillin solution Dissolve an accurately
weighed quantity of benzylpenicillin sodium ( or potassium)
in phosphate BS (pH 7. O) to produce a solution containing
1205 Biological Assay of Vasopressin
10000 penicillin units per ml.
Preparation of dilute penicillinase solution Dilute the
penicillinase solution with phosphate BS ( pH 7. O) to
produce a solution of about 8000 to 12000 penicillinase units
per ml, warm to 3 7°C befo re use.
Procedure Measure accurately 50 ml of penicillin solution to
a 100 ml volumetric flask, warm to 37°C, add accurately
25 ml of dilute penicillinase solution, previously warmed to
37°C, mix promptly and allow to stand at 37°C for exactly
1 hour. Add 3 ml, accurately measured, to 25 ml, accurately
measured, of iodine ( O. 005 mol/L) VS immediately
[measure accurately 10 ml of iodine (O. 05 mol/L) VS to
100 ml volumetric flask, dilute to volume with sodium
aceta te BS ( pH 4. 5) ] , allow to stand in a dark place at
room temperature for 15 minutes, titrate with sodium
thiosulfate ( O. 01 mol/L ) VS, using starch IS added
towards the end of the titration and continue the titration to
the disappearance of the blue colour.
Blank titration Allow 2 ml, measured accurately, of
preheated penicillin solution to stand at 37°C for 1 hour, add
accurately 25 ml of iodine (O. 005 mol/L) VS, and then
1 ml of dilute penicillinase solution. Allow to stand in dark
place at room temperature for 15 minutes, titrate with
sodium thiosulfate (0. 01 mol/L) VS, calculate the enzyme
activity as follows:
E= (B-A) XMX F X D X 100
Where: E is enzyme activity of penicillinase, Units/
(ml •hour);
B is volume of sodium thiosulfate VS consumed
in blank titration, ml;
A is volume of sodium thiosulfate VS consumed
in the titration of penicillinase, ml;
M is concentration of sodium thiosulfate VS,
rnol/L;
F is the penicillin units, equivalent to each ml of
iodi:rJ.e ( O. 005 mol/L ) VS, under the
experimental condition described above;
D is dilution factor of penicillinase solution.
[Annotation] Phosphate BS (pH 7. 0) To dipotassium
hydrogen phosphate 7. 36 g and potassium dihydrogen
phosphate 3. 14 g, add sufficient water to produce 1000 ml.
Sodium acetate BS ( pH 4. 5) To glacial acetic acid
13. 86 ml, add sufficient water to produce 250 ml; to
crystalline sodium aceta te 27. 30 g, add sufficient water to
produce 200 ml. Mix the two solutions.
1205 Biological Assay of Vasopressin
The potency of the preparation of vasopressin being examined
(T) is estimated by comparing its blood pressor activity on
the rat with that of the Lysine Vasopressine Standard (S)
under the condition of following method of assay.
Preparation of standard solution On the day of the assay,
dissolve Lysine Vasopressine Standard in sodium chloride
injection to produce two dilutions of standand solution ( S).
The ratio ( r) of the concentration between two dilutions
should not be greater than 1 : O. 6. Choose 2 doses of the
standard preparation such that the elevation of the blood
pressure is significant for the lower <lose and not maximum
for the higher <lose.
Preparation of test solution According to the sample' s
potency (AT) of vasopressin, which labelled or assumed.
Dilute two doses of the preparation being tested with the
 1206 Determination of Cytochrome C Activity
same inter-dose ratio as that of standard, matching the
effects of the dose of the standard preparation as closely as
possible.
~y Anaesthetize a healthy male rat weighing over 300 g
by suitable anaesthetics (such as i. p. 1 g of urethane per kg
body weight). Tie the rat on its back to an operating table
and maintain the body temperature of the rat during the test.
Dissect the trachea and introduce a cannula into it if
necessary. Insert in to the carotid vein ( or femoral vein) a
cannula filled with sodium chloride injection, through which
can be injected the solutions of standard preparation and the
test preparation to be examined. Give through this venous
cannula 50-100 Units of heparin per 100 g body weight.
Insert another cannula filled with 200-400 Units of
heparinised sodium chloride injection into the common
carotid artery and connect it to a device capable of giving a
continuous record of the blood pressure.
At this stage, adjust the pressure of the device to a level
corresponding to the normal blood pressure of the rat, and
remove artery clamp. Inject slowly a a-adrenoceptor blocking
agent (such as O. 1 mg of phentolamine mesylate per 100 g
body weight) and repeat the same dose of injection after 5-
10 minutes. Administration only when the blood pressure of
the rat remains stable. Injections should be made ata uniform
rate and at a regular interval of 5-10 minutes depending on
the time at which the blood pressure returns to its original
level. Each injection is followed by about O. 5 mi of sodium
chloride injection. The two doses of standard preparation and
two doses of the preparation being tested Cds1 , ds-¿ , dT1 ,
1 '\1 _ 1 1 1 • • 1 r1 11 • 1
a Tz J snoum oe g1ven m a ranaom oraer 1011owmg ranaom
block design, and repeat 4-6 replicate of four doses.
Record the maximum rise in blood pressure in response to
each dose and calculate the result of the assay by Statistical
Method for Biological Assays; Parallel Line Assay
(1431 ). The percentage of fiducial limits of error (FL%)
should not be greater than 20 %.
1206 Determination of
Cytochrome C Activity
Reagents ( 1) Phosphate BS (O. 2 mol/L) Dissolve 71. 64 g
of disodium hydrogen phosphate in water to produce 1000 ml
as solution A; dissolve 27. 60 g of sodium dihydrogen
phosphate in water to produce 1000 ml as solution B. Mix 81
ml of solution A with 19 ml of solution B, adjust to pH 7. 3.
(2) Phosphate BS (O. 1 mol/L) Dilute 500 mi of phosphate
BS (O. 2 mol/L) with water to produce 1000 ml, adjust to
pH 7. 3.
( 3 ) Phosphate BS ( O. 02 mol/L ) Dilute 100 ml of
phosphate BS (0. 2 mol/L) with water to produce 1000 mi,
adjust to pH 7. 3.
( 4) Succinate solution Dissolve 4. 72 g each of succinic acid
and potassium hydroxide in water to produce 100 ml, adjust
to pH 7. 3.
(5) Potassium cyanide solution Dissolve O. 65 g of potas-
sium cyanide in water to produce 100 ml, adjust to pH 7. 3
with dilute sulfuric acid.
(6) Cytochrome C-free heart suspension Remove the fat
and connective tissue of two fresh pig or bovine hearts, strip
and mince with a mincing machine. Transfer to a gauze bag,
rinse with tap water for about two hours, stir frequently and
squeeze to remove the haemochrome, squeeze dry, wash
with water in portions, squeeze dry. Soak it in phosphate BS
(O. 1 mol/L) for about 1 hour, squeeze dry. Repeat the
soaking process. Wash, with water in portions, squeeze dry.
Transfer to a homogenizer, immerse in a quantity of
phosphate BS (0. 02 mol/L) and homogenize toan uniform
paste. Centrifuge for 10 minutes. To the supernatant
suspension add sorne ice, adjust pH to about 5. 5 with dilute
acetic acid rapidly and centrifuge for 15 minutes immediately.
To the precipitate add an equal volume of phosphate BS
(O. 1 mol/L), triturate with a glass homogenizer to form a
homogenate, store in a refrigerator. Dilute l. O ml with
phosphate BS (O. 1 mol/L) to produce 10 ml befare use.
Preparation of test solution Prepare a solution in water
containing about 3 mg of cytochrome e per ml.
Procedure Transfer 5 ml of phosphate BS (O. 2 mol/L),
l. O ml of succinate solution and O. 5 ml of test solution ( if
the test solution is a reducing type, add O. 05 ml of
O. 01 mol/L potassium ferricyanide solution) to a 25 ml
nessler cylinder with stopper, add O. 5 ml of cytochrome
e-free heart suspension and l. o ml of potassium cyanide
solution, dilute with water to produce 10 mi, mix well.
Measure the absorbance using reagent as a blank until the
absorbance value ceases to increase at the maximum
wavelength found at intervals of O. 5 nm in the vicinity of
550 nm ( 0401 ) , this is the absorbance due to enzymatic
reduction. Add about 5 mg of sodium hydrosulfite to each
tube, mix well, allow the solutions to stand for about
10 minutes, measure the absorbance until the value ceases to
increase at the same wavelength. This is the absorbance due
to chemical reduction. Calculate according to the following
expression.
absorbance due to
enzymatic reductionX lOO%
Cytochrome C activity
absorbance due to
chemical reduction
1207 Assay of Hyaluronidase
Reagents ( 1) Acetic acid-potassium acetate BS Dissolve
14 g of potassium acetate and 20. 5 ml of glacial acetic acid in
water to produce 1000 ml.
( 2) Phosphate BS Dissolve 2. 5 g of sodium dihydrogen
phosphate, l. O g of anhydrous disodium hydrogen
phosphate and 8. 2 g of sodium chloride in water to produce
1000 ml.
(3) Hydrolysed gelatine Dissolve 50 g of gelatin in 1000 ml
of water, heat at 121 ºC for 90 minutes and freeze dry.
(4) Hydrolysed gelatine solution To a mixture of 250 ml
of phosphate BS and 250 ml of water add 330 mg of
hydrolysed gelatine, mix well and store at 0-4ºC. It is usable
if no turbidity appears in the solution.
(5) Serum stock solution Dilute 1 volume of fresh bovine
serum with 9 volumes of acetic acid-potassium acetate BS,
adjust to pH 3. 1 with 4 mol/L hydrochloric acid solution and
allow to stand for 18-24 hours before use. Store at 0-4 ºC and
use within 30 days.
( 6) Dilute serum solution Determine the content of total
serum solids in serum stock solution as follows: Place an
accurately weighed quantity of fresh bovine serum in a
crucible containing clean sand, previously dried to constant
weight at 105ºC, evaporate to dryness on a water bath and
dry to constant weight at 105ºC. Calculate the content of
total solids in the serum stock solution. On the day of the
assay, dilute 1 volume of the serum stock solution containing
about 8 % of total solids with 3 volumes of acetic acid-
potassium acetate BS; or dilute with 2 volumes of acetic acid-
potassium aceta te BS for that containing about 5 % of total
solids.

(7) Potassium hyaluronate stock solution Prepare a stock
solution by dissolving potassium hyaluronate, previously
dried over phosphorous pentoxide under reduced pressure for
48 hours, in water to produce a solution of O. 5 mg per ml.
Store ata temperature below OºC and use within 30 days.
( 8 ) Dilute potassium hyaluronate solution Dilute one
volume of potassium hyaluronate stock solution with one
volume of phosphate BS. Prepare on the day of the assay.
Preparation of standard solution To a quantity of hyaluro-
nidase RS, accurately weighed, add cold hydrolysed gelatine
solution to produce a solution of l. 5 Units per ml. Prepare
on the day of the assay.
Preparation of test solution To a quantity of the substance
being examined, accurately weighed, add cold hydrolysed
gelatine solution to produce a solution of l. 5 Units per ml
calculated on the basis of the labelled or assumed potency.
Prepare on the day of the assay.
Preparation of standard curve To 6 pairs of the same size test-
tu bes in turn add O. 00 ml, O. 10 ml, O. 20 ml, O. 30 ml,
O. 40 ml and O. 50 ml of standard solution per pair, and then
in turn add O. 50 ml, O. 40 ml, O. 30 ml, O. 20 ml, O. 10 ml
and O. 00 ml of hydrolysed gelatine solution per pair, respec-
tively. At intervals of 30 seconds add to each tube succes-
sively O. 50 ml of dilute potassium hyaluronate solution,
making the final volume in each tu be l. 00 ml, mix well and
place each tube in a water-bath maintained at 37 ±O. 5ºC.
After exactly 30 minutes, remove each tube successively
from the water bath at intervals of 30 seconds and immedia-
tely add 4. O ml of dilute serum solution. Shake and allow
them to stand at room temperature for 30 minutes. Mix well
and determine the absorbance of the resulting solutions at
640 nm <0401 ) . Repeat the operation described above, using
a mixture of O. 50 ml of phosphate BS and O. 50 ml of
hydrolysed gelatine solution, beginning at the words "place
each tube in a water bath maintained at 37±0. 5ºC ... ". Plot a
standard curve with absorbance as the ordinate and units of
standard solution as the absicissa.
Procedure To 3 pairs of the same size test-tubes in turn add
O. 20, O. 30 and O. 40 ml of test solution per pair, and then in
turn add O. 30 ml, O. 20 ml and O. 10 ml of hydrolysed
gelatine solution per pair respectively. Carry out the
operation described under the preparation of standard curve
beginning at the words "At intervals of 30 seconds add to
each tube successively O. 50 ml of dilute potassium
hyaluronate solution", read the units from the standard
curve, divide by the corresponding weights ( mg) of
substance being exami-ned. The mean of the six values is the
potency of the sample being examined.
1208 Biological Assay of Heparin
The potency of a preparation of heparin being examined (T)
is estimated by comparing its clot delaying effect on fresh
rabbit blood ( rabbit or pig plasma) with that of Heparin
Standard ( S) under the conditions of the following assay
method.
Preparation of standard solution Dissolve an accurately weig-
hed quantity of the Heparin Standard in sterile water to produce a
solution of 100 Units per ml. Store the solution at a temperature
of 4-8ºC. The solution may be used within 3 months if the
validation of activity complies with the requirement
Preparation of standard dilution On the day of the assay,
dilute an accurately measured portian of the above solution
1208 Biological Assay of Heparin
with sodium chloride injection to produce 3 <lose levels of
standard solution in geometric progression ( ds 3 , dSz ,
d 51 ) , the ratio ( r) of adjacent <lose level should be equal.
Adjust the <lose to make the mean clotting time for the
lowest <lose should be longer than the blank recalcification
time (generally l. 5 times). For an assay using Method 1,
the ratio ( r) is about 1 : O. 7 and the highest <lose may
contain 2-5 Units of heparin per ml. The mean clotting time
for the highest <lose should not be more than 60 minutes. For
an assay using Method 2, the ratio ( r) is about 1 : O. 85
and the highest <lose (may contain O. 5-1. 5 Units of heparin
per ml ) should give a clotting time of not more than
30 minutes. For an assay using Method 3, the ratio ( r) is
about 1 : O. 85 and the highest <lose ( may contain O. 4-1. 7
Units of heparin per ml) should give a clotting time of not
more than 90 seconds.
Preparation of test solution and dilution Prepare 3 <lose levels
of test solution ( dT3 , dT2 , dT1 ) , in the same way as
described under preparation of standard solution and dilution.
The concentration of the test solution is calculated on the
basis of the labelled or assumed potency (AT) of the heparin
being examined. The ratio (r) of adjacent <lose levels should
be the same as that of standard solution. The clotting time
observed with each <lose should be nearly the same as that
observed with the corresponding doses of the standard
solution.
Preparation of plasma Collect fresh blood from rabbit or pig
in a vessel containing 109 mmol/L sodium citrate solution,
ratio of sodium citrate solution and blood volume is 1 : 9,
shake gently while the blood is being collected. When the
necessary amount of blood is collected, centrifuge at 1500
folds of the gravity constant at room temperature for not less
than 15 minutes. Transfer portions of the pooled plasma into
fiasks and aiiow freezing. On the day of the assay, thaw the
plasma by putting the flasks in a constant temperature water
bath at 37±0. 5ºC, filter the thawed plasma through porous
fil ter paper or two layers of gauze. During the assay, keep
the plasma at a temperature of 4-8ºC. Rabbit or pig plasma
can be used in Method 2, and rabbit plasma is used in
Method 3.
Assay
Method 1 ( Rabbit Blood) Use clean, dry test tubes of
uniform size (O. 8 cm X 3. 8 cm or l. O cm X 7. 5 cm). Add
O. 1 ml of one of the 3 doses of standard solution, or one of
the 3 doses of test solution to each tube, for each <lose at
least 3 tubes are used, followed by O. 9 ml of fresh rabbit
blood and mix well, taking careto avoid the formation of air
bubbles. The time elapsed between the addition of blood to
the first and the last tube should be not more than 3 minutes.
Place the tubes in a constant temperature water bath
maintained at 37 ± O. 5ºC and record the time of blood
clotting for each tube.
The percentage of fiducial limits of error (FL %) should not
be grea ter than 1O%.
Method 2 ( Plasma Recalcification) Add to each tube
O. 5 ml Cor O. 8 mD of plasma, place the tubes in a constant
temperature water bath at 37±0. 5ºC for 5-10 minutes. Add
to each tu be O. 4 ml ( or O. 1 ml) of one of the 3 doses of
standard solution or one of the 3 doses of test solution, for
each <lose at least 3 tubes are used, followed by O. 1 ml of
1% calcium chloride solution, mix well, taking careto avoid
the formation of air bubbles. Record the time of blood
clotting for each tube.
The percentage of fiducial limits of error CFL %) should not
 1209 Biological Assay of Chorionic Gonadotrophin
be greater than 5 %.
Method 3 (Activated Partial Thromboplastin Time APTT)
Use the sample cups of blood coagulation analyzer, add to
each tube 50 µl of plasma, 50 µl of one of the 3 doses of
standard dilution or one of the 3 doses of test dilution, 50 µl
of APTT reagent, mix well, taking care to avoid the
formation of air bubbles. Preheat at 37±0. 5ºC far 180 seconds,
add to each tube 50 µl of CaClz reagent, the clotting time
( APTT ) was determined immediately using blood
coagulation analyzer, the number of tubes far each <lose of
standard or test solution should be the same, far each <lose at
least 3 tu bes are used. The amount of the plasma, standard
or test solution, APTT reagent, CaClz reagent and prehea-
ting time can be adjusted according to the instrument or
reagent instruction. The testing arder of the standard and test
dilution should fallow the principie of parallelism, so that the
measurement time of the standard dilution is clase to that of
the same concentration of the test dilution.
Carry out the statistical calculation far quantitative response
based on the linear relationship of the logarithm of clotting
time to the logarithm of heparin concentration ( 1431 ).
The percentage of fiducial limits of error (FL %) should not
be greater than 10 %.
1209 Biological Assay of
Chorionic Gonadotrophin
The potency of a preparation of czhorionic gonadotrophin
being examined (T) is estimated by comparing it' s effect in
increasing the weight of the uteri of immature female mi ce
with that produced by Chorionic Gonadotrophin Standard
(S) under the condition of the fallowing assay method.
Preparation of solvent On the day of the assay dissolve a
quantity of bovine serum albumin in O. 9 % sodium chloride
solution to produce a solution of 1 mg per ml, adjust to pH
7. 2±0. 2 with 1 mol/L sodium hydroxide solution.
Preparation of standard solution On the day of the assay,
dissolve the contents of the sealed container of Chorionic
Gonadotrophin Standard in the above solvent to produce 3
<lose levels of standard solution ( d 53 , d 52 , d 51 ) in
geometric progression, the ratio (r) of adjacent <lose levels
should be equal and not be greater than 1 : O. 5 and the
highest <lose ( may contain O. 14-0. 8 Units of chorionic
gonadotrophin per ml) should not produce a maximum
response of uteri weight gain; the lowest <lose should produce
a response of uteri weight gain significantly. Store the
dilution at 2-lOºC and use it within three days.
Preparation of test solution Prepare three <lose levels of test
solution (dr 3 , dr 2 , dr 1 ) in the same way as described
under preparation of standard solution. The concentration of
the test solution is calculated on the basis of the labelled or
assumed potency (AT) of the chorionic gonadotrophin being
examined. The ratio ( r) of adjacent <lose levels should be
the same as that of standard and the mean value of the
responses given by each <lose level of test solution should be
approximately the same as that of standard solution.
Assay Select healthy female mice of the same strain, 15-
23 days old or weighing 9-13 g, but in an assay the age
difference between individual mouse should not be greater
than 3 days, or the weight difference not be greater than 3 g.
Distribute the mice at random by weight to 6 groups with at
least 10 mice in each group. Inject subcutaneously into each
of 6 groups one of the three <lose levels of standard solution
or test solution with equal volume (O. 2 mD far each mouse
on three successive days at approximately the same time each
day. About 24 hours after the last injection, sacrifice, weight
and dissect the mice, and then remove the uterus of each
mouse by cutting through the cervix. Free the uterus from
extraneous tissue, and severing at the uterus-tubal junction,
gently squeeze out the uterine fluid on an absorbent paper and
weigh immediately ( precision O. 1 mg). Covert the data into
the farm of uterus weight per 10 g body weight. Calculate the
result of the assay by Statistical Method for Biological
Assays; parallel line assay ( 1431).
The percentage of fiducial limits of error (FL %) should not
be grea ter than 25 %.
1210 Biological Assay of Oxytocin
The potency of oxytocin being examined (T) is determined
by comparing the contraction effect produced on isolated rat
uterus with that produced by Synthetic Oxytocin Standard
(S) under the condition of the fallowing assay method.
Preparation of standard solution Synthetic Oxytocin Standard
(S) is diluted to produce a solution of 1 IU per ml with
freshly prepared O. 2% chlorobutanol solution (adjust to pH
3. 5 with 1 mol/L hydrochloric acid). Distribute the solution
into a suitable container, and store it at 4-8ºC. The solution
may be used within 3 months ·if the validation of activity
complies with the requirement.
Preparation of standard dilution On the dav of the assav,
dilute synthetic oxytocin standard soluti~n with O. 9 %
sodium chloride solution to produce two dilutions of ( S).
The higher concentration may contain O. 01-0. 02 Units per
ml, the ratio (r) of the concentration between two dilutions
should not be greater than 1 : O. 7. The concentration of two
dilutions should be chosen as such that, the lower <lose
produces a response of contraction ( generally about 20-
50 mm) , the higher <lose should not produce maximum
contraction ( generally about 50-85 mm ) and the contr-
actions produced by higher and lower doses should be clearly
discrimina ted.
Preparation of test solution and dilution On the day of the
assay, prepare the solution and dilutions of oxytocin injection
being examined by diluting the injection with O. 9 % sodium
chloride solution to produce two dilutions of ( T). The
concentration of the dilution of (T) is calculated on the basis
of the labelled or assumed potency of the preparation being
examined. The ratio ( r) of the concentration between two
dilutions of (T) should be the same as that of (S). The
mean value of contractions produced by each <lose level of
(T) should be approximately the same as that of (S).
Preparation of lock-solution for isolated rat uterus Prepare
the solution by adding slowly 200 ml of O. 25 % sodium
bicarbonate ( dissolve O. 5 g sodium bicarbonate in 200 mi)
to 800 ml of solution containing the following ingredients:
sodium chloride 9 g, potassium chloride O. 42 g, calcium
chloride (anhydrous) O. 06 g, glucose O. 5 g, mix well.
Animal Use healthy female adult rat less than 3 months old
weighing 160-240 g, separating from male adult rat after
weaning. On the day of the assay, confirm by vaginal smear
that the rat is in proestrus. The proestrus stage of the rat
can be produced by treating with sorne oestrogen hormone.
Assay Kill the rat and remove the uterus freeing from
extraneous tissue. Cut the two horns of the uterus in the
bifurcation. Suspend one horn of the uterus in an isolated
 1212 Test far the Prolongation Effect of Protamine Zinc lnsulin
organ bath by fixing the lower end of the uterus horn to the
bottom of the bath and connecting the upper end of the
uterus horn to a recording lever for making a record of the
contraction of the uterus. Fill the organ bath with equal
volume of lock solution ( about 30-50 ml ) , air input
continuously, maintain the bath at a constant temperature
(±O. 5ºC) of about 32-35ºC. 15 minutes later, add in turn
into the bath the two standard dilutions and two test dilutions
in equal volume (0. 3-0. 8 mD. After each addition, when the
contration of uterus reaches its maximum and begin to relax
(approximately 60-90 seconds), change and replace twice the
bath solution with fresh lock solution. The time interval
between two additions should be equal ( about 3-5 minutes).
The second addition can be given only when the uterus is
restored. to its normal condition. Repeat 4-6 replica te of faur
doses ( ds1 , dSz , dT1 , dT2 ) , fallowing random block
design. Measure all the responses and calculate the result of
the assay by the Statistical Method for Biological Assays;
parallel line assay, random block design ( 1431 ) .
The percentage of fiducial limits of error (FL %) should not
be greater than 10%.
1211 Biological Assay of Insulin
The potency of a preparation of insulin being examined (T)
is estimated by comparing the hypoglycaemic effect it
produces on mice with that produced by Insulin Standard (S)
under the condition of the fallowing assay method.
Preparation of standard solution Dissolve an accurately
weighed quantity of lnsulin Standard in O. 9 % sodium
chloride solution ( acidified with hydrochloride acid to pH
2. 5 and containing O. 2 g phenol in 100 mD to produce a
solution of 20 Units of insulin per ml. Store the solution at a
temperature of 4-8ºC. The solution may be used within
5 days.
On the day of the assay, dilute an accurately measured
portian of this solution with O. 9 % sodium chloride solution
(pH 2.5) to produce two dilutions. The ratio (r) of the
concentration of two dilutions is not greater than 1 : O. 5. In
general, the higher concentration may contain O. 06-0. 12
Units of insulin per ml. Adjust the concentration of two
dilutions to such that the smaller <lose produces a positive
depletion of blood sugar and the larger dose does not produce
maximum depletion of blood sugar.
Preparation of test solution Prepare the solution and
dilutions of (T) in the same way as that described under the
preparation of standard solution. The concentration of the
solution of (T) is calculated on the basis of the labelled or
assumed potency CAT) of preparation being examined. The
ratio (r) of the concentration between two dilutions should
be the same as that of two standard dilutions and the mean
value of the blood sugar depletion produced by each
concentration of ( T) should be approximately the same as
that of (S).
Assay Use healthy mice of the same strain, same sex and
approximately the same in age. In one assay, the difference
of weight should not be greater than 3 g. Distribute the mice
at random by weight into four equal groups with at least
10 mice in each group. Mark the mice in each group far
identification. Inject subcutaneously each of the faur prepared
dilutions of (S) (dSz , ds1 ) and (T) (dT2 , dT1 ) into one
group of mice, using the equal volume of O. 2-0. 3 ml far each
mouse. Exactly 40 minutes after each injection, take a
suitable amount of blood sample from the orbital venous
sinus of each mouse. Determine the glucose concentration of
each blood sample by a suitable method such as glucose
oxidase--peroxidase method. Not less than 3 hours later,
inject each of the dilutions into the above faur groups of mice
fallowing the twin cross-over design, and at the same time
interval after injection, determine the blood glucose
concentration of each mouse. Calculate the result of the assay
by the Statistical Method for Biological Assays; parallel line
assay, twin cross-over design ( 1431 ) . The percentage of
fiducial limits of error ( FL %) should not be greater than
25%.
1212 Test for the Prolongation Effect
of Protamine Zinc lnsulin
The prolongation of the hypoglycamia effect of protamine
zinc insulin being examined (T) is tested by comparing the
hypoglycamia it produces on rabbits with that produced by
lnsulin Standard ( S) under the condition of the following
method.
Preparation of standard solution Dissolve an accurately
weighed quantity of lnsulin Standard in O. 9 % sodium
chloride solution (pH 2. 5, acidified with hydrochloric acid)
containing O. 2 % of phenol to produce a solution of the same
concentration of Units per ml as that of the protamine zinc
insulin injection being tested.
Preparation of test solution The protamine zinc insulin
injection is used directly without further dilution.
Procedure Use healthy rabbits weighing 2. 0-3. O kg, male
or nonpregnant female, and house the rabbits singly, 18-
20 hours befare the test, deprive the rabbits of food and feed
with drinking water only. During the day of the test, provide
no food or water to the rabbits and handle them with care to
avoid any excitement. Distribute the rabbits at random by
weight and sex into two equal groups of approximately the
same weight and sex in each group. Take a blood sample of
not more than l. 5 ml from the marginal ear vein and
determine the normal blood sugar level for every rabbit using
a suitable blood sugar determination method.
Inject subcutaneously the rabbits in one group an accurate
dose of about l. 2 Units of the standard solution of insulin
(S) and the rabbit in the other group the same volume of
solution being examined (T) at the same site as that of the
standard group. At 2 and 6 hours after injection of standard
solution and at 6 and 9 hours after injection of test solution,
take blood samples and determine the blood sugar level ( mg
of glucose per 100 ml of blood) far every rabbit at each
bleeding time. Calculate the blood sugar levels as the
percentage of its normal blood sugar level and expressed itas
percentage of blood sugar. If none of the blood sugar
percentage far a rabbit was lowered below 90 %, or if the
rabbit died or convulsed in the course of the test, the data of
that rabbit should be rejected. There should be at least six
rabbits available in each group. Calculate the mean percentage
of blood sugar for ( S) and ( T) group at each bleeding
time.
Evaluation of result In the standard group, if the number of
rabbits died or convulsed is more than 1 in five, or if the
mean percentage of blood sugar at 2 hours bleeding time has
not lowered to 65 % or at 6 hours the bleeding time has not
returned to 95%, adjust the <lose of (S) and repeat the test.
The protamine zinc insulin being examined complies with the
test far prolongation if its mean percentage of blood sugar
has lowered to or below 75 % either at 6 hours or at 9 hours
 1213 Biological Assay of Protamine Sulfate
bleeding time.
1213 Biological Assay of
Protamine Sulfate
The potency of a preparation of protamine sulfate being
examined ( T) is determined by testing its capacity to
neutralize the anticoagulant effect of Heparin Standard (S)
in test tubes containing fresh rabbit blood or plasma under
the condition of the fallowing assay method
Preparation of heparin standard solution Dissolve an
accurately weighed quantity of Heparin Standard in O. 9 %
sodium chloride solution to produce a series of solutions
different in concentration of not more than 5 Uníts per ml in
succession, e.g. 85, 90, 95, 100, 105, 110, 115, 120,
125 Units per ml.
Preparation of test solution Dissolve an accurately weighed
quantity of protamine sulfate being examined (T) in O. 9 %
sodium chloride solution to produce a solution of 1 mg per
ml, calculated on the dried basis or, if it is an injection,
dilute with O. 9 % sodium chloride solution to 1 mg per ml,
calculated with reference to the labelled amount.
Preparation of plasma Carry out the preparation of plasma
as described under the Biological Assay of Heparin
(1208).
Assay Use 8 dry clean test tubes of unifarm size (O. 8 cmX
3. 8 cm). To the first and eighth tube add O. 2 ml each of
O. 9 % sodium chloride solution as the control tube. To each
of the six remaining tubes add O. 1 ml of the solution being
examined fallowed by O. 1 ml of one of the heparin standard
solutions in succession, mix thoroughly. To each of the eight
tubes add O. 8 ml of fresh rabbit blood, mix immediately
after each adding, taking care to avoid the formation of air
bubbles. Record the time of blood clotting far each tube.
Place the tubes in a constant temperature water bath maintai-
ned at 37 ± O. 5ºC. The time elapsed between the blood
collection and placing the tubes in the bath should not be
more than 2 minutes.
If, instead of fresh rabbit blood, plasma is used in an assay,
add O. 7 ml of plasma to each of 8 tubes, mix well and allow
the tubes to stand in the water bath far 5-10 minutes. Then
to each tube add O. 1 ml of 1 % calcium chloride solution, mix
immediately after each addition taking care to avoid the
farmation of air bubbles and record the clotting time for each
tu be.
Evaluation of result The assay is valid if the diff erence of
clotting time between two control tubes is not more than 1 :
l. 35. Take the mean clotting time of the two control tubes as
the normal clotting time.
In the series of six test tubes in arder of increasing
concentration of heparin, the last tube which shows the
clotting time of not more than 150 % of the normal clotting
time is the terminal tube of the test.
Perfarm 5 assays in series, if the heparin concentration of
five terminal tubes does not differ by more than 10 Units,
take the mean value of the 5 assay results as the potency of
protamine sulfate in Units of heparin per mg.
1214 Biological Assay of Digitalis
The potency of a preparation of digitalis being examined (T)
is determined by comparing the mínimum lethal dose it
produces on pigeons with that produced by Digitalis Standard
(S) under the condition of the following assay method.
Preparation of standard solution Transfer a suitable quantity
of the Digitalis Standard, weighed accurately and rapidly to
avoid the absorption of moisture, to a glass stoppered vessel.
To each unit of digitalís, calculated wíth reference to the
stated potency of the standard, add 1 ml of 76 % ethanol.
Stopper the vessel tíghtly and shake continuously far 1 hour,
filter through a dry filter: the solutíon contaíns 1 Unit of
digitalis per ml. Store the solutíon ata temperature of 4-8ºC.
The solution may be used wíthin 1 month íf the validatíon of
activity complies with the requirement.
On the day of the assay, dilute an accurately measured
portion of the standard solution with O. 9 % sodium chloride
solutíon so that the mean lethal dose of the diluted solutíon
wíll be 25-34 ml per kg of pígeon body weight ( usually 1 ml
of the standard solutíon is to be diluted to 30 ml with O. 9 %
sodium chloride solution).
Preparation of test solution Weigh accurately a quantity of
the digitalis being examined (if it is in the farm of tablets,
accurately weigh more than 20 tablets to determine the
average weight, powder the tablets rapidly, weigh a quantity
of the powdered tablet equivalent to not less than 20 tablets)
and prepare the test solution as described under preparation
of standard solution. The concentration of the test solution is
calculated on the basis of labelled or assumed potency (AT)
of digitalis being examined and the lethal dose of test solution
(ml/kg) should be approximately the same as that of the
standard solution.
.Assay Use healthy pigeons v1eighing 250-400 g, but in one
assay the weight difference between individual pigeon should
not be greater than 100 g; 16-24 hours befare assay, deprive
the pigeons of food and give drinking water only. On the day
of the assay, distribute pigeons at random by weight into two
equal groups with at least 6 pigeons in each group, one is the
standard group, another is the test group. Secure the pigeon
on a board pluck a few feathers covering the alar vein and
inserta fine needle connected with a microburette (precision
O. 02 ml) into the exposed vein. lnfuse O. 5 ml of the
standard solution or test solution into the vein, fallowed by a
continuous infusion of O. 2 ml per minute until cardiac arrest
occurs. Pigeons may ha ve tremor, emesis or evacuation of
stool befare dying, but only the dilation of pupil and
cessation of breath can be considered as the critical point of
death. Record the total volume of standard solution or test
solution infused for each pigeon and calculates the mínimum
lethal dose as ml (or Units) per kg of body weight. Multiply
the mínimum lethal <lose by 10 and convert it into logaríthm.
Carry out the statistical calculation far direct assays <1431),
the percentage of fiducial limits of error (FL%) should not
be grea ter than 15 %.
1215 Toxicity Test of Sodium
Stiboglu-conate
The toxlClty of sodium stibogluconate is determined by
comparing the number of mice kílled by injection of the
preparation being tested ( T) with that by Sodium Stibog-
luconate Standard ( S) under the condition of fallowing
method.
Preparation of standard solution Dissolve an accurately
weighed quantity of the standard in water by warming at
about 70ºC for 15 minutes. Cool to room temperature and add
water to make a suitable concentration. Warm the solution at
50ºC far 30 minutes (water evaporation should be avoided) ,
 1217
cool to room temperature. The concentration of the solution,
calculated on pentavalent antimony, should be such that after
intravenous injection into a group of mice, using O. 02 ml/g
body weight, will cause a mortality of approximately 50 %
(20%-80% is suitable).
Preparation of test solution If the sample being tested is a
powdered material, prepare the test solution in the same
manner as that of standard solution. If the sample is sodium
stibogluconate injection, prepare the test solution by diluting
it with water and warm the solution at 50ºC for 30 minutes
(water evaporation should be avoided) , cool to room tempe-
rature. In either case, the concentration of the solution of test
preparation should be 83% as that of standard solution.
Procedure Use 40 or 20 healthy mice weighing 17-25 g with
the difference not exceeding 3 g in one test. Distribute the
mice into two groups at random by weight with 20 or 10 mice
in each group. lnject intravenously one group the standard
solution and the other the test solution using a volume of
O. 02 ml per g body weight. The injection for each mouse
should be completed within 4-5 seconds. Record the number
of mice died within 15 minutes after injection.
Evaluation of result When two groups of twenty mice are
used in a test: the sample complies with the test if the
number of mice killed by the injection of the test solution is
not greater than the number killed by standard solution; the
sample fails the test if the number of mice killed by the test
solution is greater than the number killed by standard solution.
When two groups of ten mice are used in a test: the sample
complies with the test if the number of mice killed by the test
solution is at least two less than the number killed by
standard solution. The sample fails the test if the number of
mice killed by test solution is two or more greater than that
killed by standard solution. If it is not the case, repeat the
test with another two groups of ten mice and evaluate the test
result in the same way as that described above by comparing
the total number of mice killed in the two series of ten mice
of (S) and (T) group.
1216 Biological Assay of Follicle
Stimulating Hormone
The potency of menotrophin being examined ( T) with
respect to its follicle stimulating hormone activity is estima-
ted by comparing its effect in increasing the weight of the
ovaries of immature female rats with that of Menotrophin
Standard ( S) under the condition of following method of
assay.
Preparation of solvent On the <lay of the assay dissolve a
quantity of bovine serum albumin in O. 9 % sodium chloride
solution to produce a solution of 1 mg per ml, adjust to pH
7. 2±0. 2 with 1 mol/L sodium hydroxide solution.
Dissolve chorionic gonadotrophin ( raw material or
preparation for injection), accurately weighed, in above
solution to produce a solution of 20 Units per ml calculated
on the basis of the labelled.
Preparation of standard solution On the <lay of the assay
dissolve the contents of Menotrophin Standard with respect
to its follicle stimulating hormone potency in above solvent to
produce 3 <lose levels of standard solution (ds 3 , ds 2 , ds 1 )
in geometric progression, the ratio ( r) of adjacent <lose
levels should be equal and not be greater than 1 : O. 5 and the
highest <lose ( may contain 2-5 Units of follicle stimulating
hormone per mD should not produce a maximum response of
ovaries weight gain, the lowest <lose should produce a
Biological Assay of Luteinising Hormone
response of significant ovary weight gain. Store the dilution
at 2-lOºC and use it within three days.
Preparation of test solution Prepare three <lose levels of test
solution (dT 3 , dT 2 , dT 1 ) in the same way as described
under preparation of standard solution. The concentration of
the test solution is calculated on the basis of the labeled or
assumed potency (AT) of the follicle stimulating hormone
being examined. The ratio (r) of adjacent <lose levels should
be the same as that of standard and the mean value of the
responses given by each <lose level of test solution should be
approximately the same as that of standard solution.
Assay Select healthy female rats of the same strain, 19-
23 days old, or weighing 36-60 g, but at assay the age
difference between individual rats should not be greater than
3 days, or the weight difference not be greater than 15 g.
Distribute the rats at random by weight to 6 groups of at
least 8 rats in each group. lnject subcutaneously into each of
six groups one of the three <lose levels of the standard
solution or test solution with equal volume (O. 5 ml) for
each rat on three successive days at approximately the same
time each <lay. About 24 hours after the last injection,
sacrifice, weigh, and dissect the rats and remove the ovaries.
Free the ovaries from extraneous tissue and oviduct remove
any extraneous fluid on an absorbent paper and weigh
immediately ( precision O. 1 mg). Covert the data into the
form of ovaries weight per 10 g body weight. Calculate the
result of the assay by Statistical Method far Biological
Assays; parallel line assay, random design <1431 ).
The percentage fiducial limits of error ( FL %) should not
be greater than 45%.
1217 Biological Assay of
Luteinising Hormooe
The potency of menotrophin being examined ( T) with
respect to its luteinising hoqnone activity is estimated by
comparing its effect in increasing the weight of the seminal
vesicles of immature male rats with that of Menotrophin
Standard ( S) under the condition of following method of
assay.
Preparation of solvent On the <lay of the assay dissolve a
quantity of bovine serum albumin in O. 9 % sodium chloride
solution to produce a solution of 1 mg per ml, adjust to pH
7. 2±0. 2 with 1 mol/L sodium hydroxide solution.
Preparation of standard solution On the <lay of the assay
dissolve the contents of Menotrophin Standard with respect
to its luteinising hormone potency in above solvent to produce
3 <lose levels of standard solution ( ds3 , dSz , ds1 ) in
geometric progression, the ratio (r) of adjacent <lose levels
should be equal and not be greater than 1 : O. 5 and the
highest <lose (may contain 8-15 Units of luteinising hormone
per mD should not produce a maximum response of seminal
vesicle weight gain, the lowest <lose should produce a
response of seminal vesicles weight gain significant. Store the
dilution at 2-lOºC and use it within four days.
Preparation of test solution Prepare three <lose levels of test
solution (dT3 , dT2 , dT1 ) in the same way as described
under preparation of standard solution. The concentration of
the test solution is calculated on the basis of the labelled or
assumed potency (AT) of the luteinising hormone being
examined. The ratio ( r) of adjacent <lose levels should be
the same as that of standard and the mean value of the
responses given by each <lose level of test solution ( T)
 1218 Biological Assay of Calcitonin
should be approximately the same as that of (S).
Assay Select healthy male rats of the same strain, 19-23
days old, or weighing 36-60 g, but in an assay the age
difference between individual rats should not be greater than
3 days, or the weight difference not be greater than 15 g.
Distribute the rats at random by weight to 6 groups with at
least 6 rats in each group. Inject subcutaneously into each of
6 groups one of the three dose levels of standard solution or
test solution with equal volume (O. 5 ml) for each rat on
four successive days at approximately the same time each
day. About 24 hours after the last injection, sacrifice, weigh
and dissect the rats and remove the prostate glands. Free the
seminal vesicles from extraneous tissue and anterior lobe of
prostate glands remove any extraneous fluid on an absorbent
paper and weigh immediately (precision O. 1 mg). Covert the
data .into the form of seminal vesicles weight per 10 g body
weight. Calculate the result of the assay by Statistical
Method Jor Biological Assays; parallel line assay ( 1431 ).
The percentage of fidU:cial limits of error (FL%) should not
be grea ter than 35 %.
1218 Biological Assay of Calcitonin
The potency of the preparation of calcitonin being examined
(T) is estimated by comparing the hypocalcemic effect it
produces with that produced by the Calcitonin Standard (S)
under the condition of following method of assay.
Preparation of solvent Dissolve O. 2 g of bovine serum
albumin in 20 ml of water, mix well, warm in a water bath
at 56ºC for 1 hour, cool to room temperature, freeze at-10
to-20ºC. On the day of the assay thaw in a water bath at 36ºC
±0. 5ºC. Transfer to a 200 ml volumetric flask, a solution
containing 2 g of sodium acetate previously added, add
3. 5 ml of hydrochloric acid. Dilute to approximate total
volume with water, adjust to pH 3. 5-4. 5 with a solution of
hydrochloric acid or sodium h~droxide. Dilute to volume with
water. Another •solvent is prepared by using the following
method: A solution containing l. 88 g of sodium chloride and
O. 5 g of crystalline sodium acetate previously added, add O. 5
ml of acetic acid, then dilute to 250 ml with deionized water,
and then dissolve O. 25 g of bovine serum albumin in the
solution, mix it well 20 min later. The solution may be
prepared the day before the assay and store at 4 ºC.
Preparation of standard solution On the day of the assay,
according to labeled, dissolve Calcitonin Standard in above
solvent to produce two doses (S) (d 52 , ds 1 ) , the ratio
( r) of high to low dose should not be greater than 3 : 1 and
the solution of higher dose may contain 50-100 mlU per ml.
Preparation of test solution According to the sample' s
potency (AT) of calcitonin, which labeled or assumed,
dilute two doses of the preparation (dr 2 , dr 1 ) being tested
with the same inter-dose ratio as that of standard, matching
the effects of the dose of the standard preparation as closely
as possible.
Assay Use healthy rats of the same sex and of the same
strain, weighing 200 to 250 g ( subcutaneous injection, the
range of weights in one test should be kept as small as
possible and should, in an case, not exceed 20 g), weighing
70 to 80 g Cintravenous injection, the range of weights in one
test should be kept as small as possible and should, in an
case, not exceed 15 g). Deprive the rats of food 16 hours
befare the test but allow access to unlimited quantities of
water prepared by distilled water or deionized water. On the
day of test assign the rats at random by weight to four
groups of at least five animals, tow groups being allocated to
the higher and lower dose levels of Standard and two to those
of the preparation being examined. Weigh and mark the rats.
lnject subcutaneously into the abdomen or intravenously into
tail vein of each rat of four groups one of the two dose levels
of the standard solution or test solution according to body
weight of each animal with the dose volume O. 4 ml per 100 g
of body weight. Exactly one hour after injection, take a
sample of blood from each rat with ophthalmic venous plexus
bleed in the order of injection. Determine the calcium content
of each sample of plasma by suitable method, such as o-
cresolphthalein complexation method.
Calculate the result of the assay by Statistical Method for
Biological Assays; parallel line assay, random design
<1431 ). The percentage of fiducial limits of error (FL%)
should not be greater than 45%.
1219 Biological Assay of Growth Hormone
Method 1
The potency of the preparation of growth hormone being
examined ( T) is estimated by comparing its effect in
increasing the body mass of immature hypophysectomised rat
with that of Growth Hormone Standard ( S) under the
condition of following assay method.
Preparation of standard solution On the day of the assay
dissolve the contents of Growth Hormone Standard in O. 9 %
sodium chloride solution containing O. 1 % bovine serum
albumin to produce two solutions ( higher and lower dose
levels) of (S). The ratio (r) of the concentration between
two solutions may be 1 : O. 25. The solution of higher dose
may contain O. 1-0. 2 IU per ml and the solution of lower dose
may contain O. 025-0. 05 IU per ml. Store the daily doses in
tightly closed ampoules ata temperature below-15ºC. Thaw
the daily doses before use.
Preparation of test solution Prepare and store two solutions
of the preparation being examined in the same way as
described under preparation of standard solution. The concen-
tration of the test solution is calculated on the basis of the
labelled or assumed potency (AT) of the preparation being
examined.
Assay Select healthy rats of the same sex and of the same
strain 26 days to 28 days old, weighing 60 to 80 g. From 2 to
3 weeks before the test, weigh the rats and carry out
hypophysectomy. After operation, keep the rats at a clean
animal lab and allow them to recovery.
On the first day of the test, weigh the rats again and discard
those which have gained or lost more than 10 per cent of their
body mass. Assign the remaining rats at random by weight
into four equal groups of not fewer than eight. Weigh and
number the rats. lnject subcutaneously into the neck of each
rat of four groups one of the two dose levels of the standard
solution or test solution with equal volume (O. 5 ml) for
each rat on six successive days at approximately the same
time each day. Kill the rats 24 hours after the last injection,
weigh the rats, cleave the sellar region and inspect
macroscopically for traces of pituitary gland if necessary.
Exclude from the test rats showing remnants of the organ.
The difference in mass of each rat between the day of the
first injection and the last weighing is taken as the response.
The mean value of the responses given by each dose level of
(T) should be approximately the same as that of (S) and
the lower dose should produce a response of body weight gain
 1401 The Test of Radiopharmaceutical Preparations
significantly, the higher dose should not produce a maximum
response of body weight gain.
Calculate the result of the assay by Statistical Method for
Biological Assays; parallel line assay, random design
( 1431 >. The percentage of fiducial limits of error (FL%)
should not be greater than 50 %.
Method 2
The potency of the preparation of growth hormone being
examined ( T) is estimated by comparing its effect in
increasing the width of the proximal epiphysis of ~he tibia in
immature hypophysectomised rat with that of Growth
Hormone Standard ( S) under the conditions of following
method of assay.
Preparation of standard solution and preparation of test
solution As described under the method l.
~y Carry out the assay as described under the method
l. Cut out the two tibiae from each of the rats after killing
them and store them in 10 % formaldehyde solution. Cleave
the proximal part of the bone in the middle sagittal plane.
Rinse the bone parts with water for 10 minutes, with acetone
for 10 minutes and again with water for 3 minutes. Transfer
to 2 % silver nitrate solution. Allow to stand for two minutes
and flush with water. Place each of the bone parts in water
and expose them to strong light until the calcified parts have
become dark brown. Fixate them in 10% sodium thiosulfate
solution for 30 seconds, and then put them in 80 % ethanol
solution and ready for measuring. The width of the epiphysis
is measured as the response using 1 mm thickness of slice
under a microscope.
Calculate the result of the assay by Statistical Method for
Biological Assays; parallel line assay, random design
<1431 >. The percentage of fiducial limits of error (FL%)
should not be greater than 50%.
1401 The Test of Radiopharmaceutical
Preparations
Radiopharmaceutical Preparations are the preparations
containing one or more radionuclides for diagnostic and
clinical usage. The production, business, testing and use of
radiopharmaceutical preparations should comply with the
requirements in the Drug Administration Law of the People' s
Republic of China and "The Regulations for the Adminis-
tration of Radiopharmaceutical preparations" promulgated by
the Sta te Council of the People' s Republic of China.
l. Tenninologies and definitions
Nuclide Refers to a species of atom characterised by its
mass number, the number of protons and the nuclear energy
state and its average life is long enough to be observable.
Isotope Refers to nuclides with the same number of protons
but different mass numbers, which occupy the same position
in the periodic table of elements, are called isotopes of a
given element, or each other' s isotopes.
Radioactive and radioactive nuclide Refers to the property
of certain nuclides emitting spontaneously radiation of one or
more particles or y-ray, or emitting of X-ray after the
generation of tracked electron capture or spontaneous fission.
Radionuclide is a nuclide that is radioactive.
Radioactive decay Refers to a transformation process of
radionuclide emitting spontaneously one or more particles or
y-ray and transforming into another nuclide or another energy
state of the same nuclide. Also known as decay or radioactive
disintegration. The main types of radioactive decay may
involve: a decay, ¡r decay, 13+ decay, electron capture, y
transition and isomeric transition.
Radioactive decay law Refers to the law of the radionu-
clides decay, that is, the exponential decay law:
Ni =No e-At
Where: Ni is the number of atoms of nuclide at elapsed
time t;
N 0 is the number of atoms of nuclide when t=O;
A is the decay constant of radionuclide;
is the time elapsed;
e is the base of natural logarithm.
Half-life Refers to the time in which the amount of
radionuclide decays to half of its initial value in the process of
individual radioactive decay. It is usually expressed as T 112.
Each nuclide has its specific half-life which is related to the
decay constant as follows:
T112 =O. 693/A
Radioactivity Refers to the number of nuclear trans-
formation of nuclide per second. The official unit of
radioactivity is Becquerel ( Bq). lBq = one nuclear trans-
formation/second. Other units commonly used are megabec-
querel (MBq), gigabecquerel (GBq), kilobecquerel (kBq).
Specificactivity Refers to the radioactivity per unit mass
of the element or of the compound concerned.
Radioactiveconcentration Refers to the radioactivity in a
unit volume of radiopharmaceutical preparation.
Radionuclidicpurity Refers to the percentage of total radi-
oactivity that is present in the form of radionuclide
concemed.
Radiochemicalpurity Refers to the ratio, expressed as a
percentage, of the radioactivity of the stated radionuclide
present in the stated chemical form, to the total radioactivity
of the radionuclide present in the radiopharmaceutical preparation.
Carrier Refers to the stable nuclide or its compound added
or present in the radionuclide or its compound concemed.
2. Test for identification
Tests for identification of radiopharmaceutical preparations
may be classified into the tests for the nature of the
radionuclide and for identity. The later may be identified by
the method described under Radiochemical purity.
ldentification of the radionuclide depends upon the
characteristic transformation and the nature of the
radionuclide. The basic method employed is the accurate
measurement of the half-life, mass absorption coefficient or
the y-spectrum of the radionuclide.
(1) Gamma-ray spectrum method The gamma-ray energy
spectrum of radionuclide should be identical with the energy
of main photon complies with the requirement specified under
the nuclide concemed. The energy of photoelectric peak
should be identical with the y-ray transition energy given in
the decay scheme of the radionuclide.
The y-ray spectrum of the nuclide in a radiopharmaceutical
preparation is recorded by multiple channel y-spectrometer
with sodium ( thallium) iodide scintillating crystal, or high
purity germanium semiconductor as detector through the use
of the calibration curve of energy and channel by means of a
series of y-ray emission standard specimens or sources of
known energy.
(2) Measurement of half-life A suitable detection apparatus is
chosen based upon the nature of the radionuclide. The
radioactive source is prepared by an appropriate amount of
radiopharmaceutical preparation being examined with
 1401 The Test of Radiopharmaceutical Preparations
reference to the measuring range of the detection apparatus
and the half-life of the radionuclide. Keep the source and the
detector of the apparatus in a fixed geometrical condition and
measure the count rate successively at certain time intervals
and a period of not less than a quarter of the half-lives of the
radionuclide concerned, repeat at least three times. Draw a
curve with time as abscissa and count rate as ordinate on a
semi log paper. The half-life T 1; 2 can be obtained by
calculating the slope which should not differ by more than
5% from the half-life stated for the individual radionuclide.
The following should be considered in the measurement.
CDit is necessary to ensure that the efficiency of the detection
apparatus remains constant;
@The geometrical conditions of the detection assembly are
kept constant;
@Make any necessary correction for the dead time on the
basis of the radioactivity of the radionuclide.
Note geometric condition The validity of relative calibra-
tion and measurement of radionuclides is dependent upon the
reproducibility of the relationship of the source to the
detector and its geometric condition, which must be
coincident in the measurement.
Correction for deadtime The minimum time interval that is
required for the counter to resolve two consecutive signal
pulses is known as the dead time. If the counting rate is high
when measuring, the correction for dead time must be made.
f,=m/n=l-mr (mr<<l)
where f, is the correction factor, r the dead time, m the
counting rate, n the true counting rate.
(3) Mass absorption coefficient method Generally used for
a purified fj-ray radionuclide with a long half-life. Take as an
example of 32 P: Prepare a membrane source with 32 P solu-
tion, mount on a suitable counter ( about 20000 counters/
minute). Carry out count rate determinations individually and
successively using 6 aluminium foils, different in thickness,
chosen from a range of 20-50 mg/ cm2 and one foil of
800 mg/ cm2 at least as the absorber. The sample and
absorbers should be placed as clase as possible to the detector
in arder to minimize the scattering effects. The net fj-ray
count rate is obtained by subtracting the count rate found
with the thickest absorber of 800 mg/ cm2 or more from the
count rates found with various absorbers. Plot the logarithm
of the net [j-ray count rate against the total absorbers
thickness, which is the sum of the thickness of aluminium
absorbers, the thickness of the counter window and the air
equivalent thickness ( the distance, expressed in cm, of the
sample from the counter window multiplied by l. 205 under a
pressure of 76 cm of Hg column and 20ºC). An
approximately straight line is obtained. Select two of the
absorbers of diff erent thickness that are 20 mg/ cm2 or more
apart, their total absorber thickness should fall on the linear
part of the absorption curve. The absorption coefficient is
calculated from the equation:
_ 1 l Nn
µ- t2-t1 n Ne2
Where ti and t2 are the thinner and the thicker absorber
expressed in mg/ cm2 ; N 11 , N 12 represent the net {1 count
rate with t1 and t2 absorbers, respectively.
The error of the calculation result should be not more than
± 10 % in comparison with the mass absorption coefficient of
the same pure nuclide measured under identical conditions.
3. Test for purity
( 1) Determination of radionuclidic purity Radionuclidic
impurities may be present in pharmaceutical preparation. The
requirement for limit of specific radionuclidic impurities must
be considered with regards to the nature of radiation and its
effects to human. Usually, the nuclidic purity may be
specified by the percentage of the radioactivity of
radionuclidic impurities at the time when the measurements
are made or that of total radioactivity present in the form of
labelled radioactivity of the main nuclide.
The determination of nuclidic purity is carried out with a
multiple channel gamma spectrometer, using Germanium
semiconductor as detector. Keep the form and size of the
reference standard source and of the sources of samples being
examined constant and maintain the geometric conditions of
the sources and the detector under identical conditions and a
suitable environmental condition for performing of the
spectrometric measurements. The radionuclidic purity may
be obtained from the curve plot against the accurately
measured energy and the efficiency of the detection based
upon the known nuclidic parameters and the calculated y-
spectrum peak area with reference to a series of y-ray
emission reference sources.
Certain decay products of radionuclides are still radioactive.
These are referred to as mother and daughter radionuclides,
respectively. The daughter radionuclides are usually excluded
from the calculation of radionuclidic purity. The radionuclidic
purity should be stated with the date and hour when the
measurement is made.
(2) Determination of radiochemical purity The radio-
chemical impurities of radiopharmaceutical preparation may
be produced from the decomposition of the preparation itself
or in the process of production. Radiochemical purity
determination includes the separation of different chemical
components and the measurement of the corresponding
radioactivi ti es.
Method 1 Carry out the method of ascending paper chroma-
tography ( 0501 ) or thin-layer chromatography ( 0502),
using a suitable amount of sample or proceed as described in
the individual monograph if necessary. Apply the carrier
solution on the base line, allow it to dry and then apply the
sample solution on the same position. After developing and
removal of the paper or plate, dry it in air, determine the
distribution of radioactivity on the chromatogram with a
suitable apparatus. Mapping and calculate the Rf value and
radiochemical purity by the following expression.
In the identification of individual radiopharmaceuticals, the
word "about" means that the measured Rf value should be
within±l0% of the specified value.
Radiochemical purity ( % ) =
net counts ofradioactivity of
specifiedchemicalidentity
---='------------=---X 100 %
sum of net counts of
radioactivity onthechromatogram
Method 2 Carry out the paper electrophoresis ( wet method)
or electrophoresis on cellulose aceta te membrane ( 0541 ) .
Apply the carrier solution on the base lineas described in the
individual monograph and followed by the sample solution on
the same position if necessary. The base line is l. 5 cm from
the anode or cathode support of the electrophoresis
apparatus. Remove the paper or membrane after the specified
time interval, dry it in air, determine and calculate the
radiochemical purity as described under method l.
Method 3 Carry out the method of ascending paper chroma-
tography ( 0501 ) or thin-layer chromatography < 0502)
described in the individual monograph, using multiple
separating system. After developing and removing of the
paper or plate, dry it in air, determine the distribution of
radioactivity on chromatograms applied in each separating
 1401 The Test of Radiopharmaceutical Preparations
system with a suitable apparatus, and mapping.
In case the radioactive chemical impurities B and C contained
in radiopharmaceutical A, B and (A +C) may be separated
+
in separating system 1; C and (A B) being separated in
system 2; the radiochemical purity of sample A is calculated
by the following expression:
B % net radioactive c?unt~ of B peak X % 1) It is essential to ensure the apparatus being operated
100
sum of net rad1oact1ve counts
of paper tested with system 1
= net radioactive counts of C peak X l %
e%o sumo f net ra d"ioact1ve
. counts o f 00 o
paper tested withs ystem 2
A%=100%-CB+C)%
In addition, other separating analytical methods, which can
separate various radiochemical impurities, can also be used
for measurement of radiochemical purity after being
validated, such as HPLC and column chromatography, etc.
4. Test for particle size
The diametre and distribution of the particulate matter or
particles should be tested for the radiopharmaceutical prepa-
rations which are produced as colloidal solution or suspen-
sion. Usually, particles of nm in diametre are measured with
an electronic microscope and that of µm with a microscope.
Electronic microscopical measurement Mount the solution
being examined or an appropriate amount of dilute solution
on a copper wire gauze of 3 mm (300 pores) which is coated
with film suitable for mounting the specimen. Allow it to
dry, observe directly or by photographing. Select an area
where the particulate matter is distributed uniformly and
measure at random the diametres of more than 100 particles.
Calculate the diameter and distribution of particulate matter
or partid es wi th reference to both electronic and optical
magnifying powers.
Microscopical measurement Mount the solution being
examined or an appropriate amount of dilute solution in a
blood cell counter and place it on the microscope stage.
Observe the homogenicity of the distribution of particulate
matter at first with eye piece X 10, objective lens (X 10),
then observe with objective lens ( X 40) at the selected
representing field or by photographing. Measure at random
diametres of over 100 particles. Calculate the percentage
distribution and the diametres of the particles in the
preparation being examined with reference to optical magni-
f ying powers.
S. Detennination of pH value
The pH value of the solutions of radiopharmaceutical prepa-
rations must be in the definite scope. It is determined by a pH
metre or a precise pH test paper which has been calibrated.
Method of pH test paper Drop a drop of the solutions of
radiopharmaceutical preparations on a precise pH test paper,
compare the colour with the standard colour plate.
Method of pH metre Carry out the Determination of pH
value <0631 ) in a defensive condition.
6. Measurement of radioactivity and radioactive concentration
In the field of pharmaceutical sciences, specific apparatus has
been developed for the measurement of radioactivity, using
<lose calibrator with well-type ionization chamber as the
detector. The apparatus has been calibrated carefully for
release with reference radiation sources comply with the
requirements for such measurement with the total
indeterminacy no greater than ± 5% ( fiducial probability
99. 7 %). The apparatus should be calibrated periodically to
ensure the accuracy of measurement.
(1) Measurement of radioactivity and radioactive concen-
tration of r-ray emitting nuclides using adose calibrator
under normal working conditions. Preheat the apparatus
sufficiently and set it under the conditions required for the
measurement of the nuclide concerned to measure the
background reading or zero point adjustment.
2) Place the preparation being examined, accurately
measured, into the ionization chamber under identical
geometrical conditions as that for calibration.
3) Carry out the measurements for 10 times consecutively,
calculate the average value and subtract from it the back
ground reading correspondingly as the radioactivity A of the
sample.
4) The radioactive concentration C of the preparation being
examined is calculated by the following equation:
C=A/V
Where V is the volume of the preparation being examined
( 2) Measurement o f radioactivity and radioactive concen-
tration of p-ray emitting nuclides using adose calibrator.
The <lose calibrator must be calibrated with a standard
source, using the same condition described under the
measurement of the substance being examined, and then may
be used directly for the measurement The procedure and
calculation of results are similar to that described under y-ray
emitting radio nuclides.
1) The <lose calibrator must comply with the requirements of
the measuring apparatus enforced by the Government and
calibrated and certificated by the National Bureau of
T echnical Supervision.
2) The date and time of measurement shouid be printed and
the results should be within 90. 0%-110. 0%, or the range
specified in the individual monograph of the labelled value of
radioactivity.
3) The <lose calibrator must be stable and reliable, with
detection so urce of long half-life nuclide (e. g. , 137 Cs).
7. Other requirements for the Radiophannaceutical prepara-
tions
( 1) Containers Solutions of radiopharmaceutical prepara-
tions should be stored in vials with rubber closure for
multiple injections. The radiation level on the surface of the
containers complies with the requirement for protection
against ionization radiation.
(2) Expiry date The designated expiry date begins with
the date at which the radioactivity is measured It is not
recommended to use beyond the expiry date or in any case,
an extraordinary condition is observed within the expiry date.
( 3 ) Label and insert The label of radiopharmaceutical
preparations should indicate the name of the preparation, the
name of manufacturer, approved number, batch number and
sign for radiopharmaceutical preparations, etc. The insert
should indicate the name of the preparation, the chemical
state, the date of production, batch number, radioactive
concentration with the date and hour when the measurement
was made, content (mi), total radioactivity, expiry date,
name of manufacturer, sign for radiopharmaceutical prepara-
tions, indications, category, usage, dosage, strength,
package, storage, notice, etc.
 1401 The Test of Radiopharmaceutical Preparations

Physical Characteristics of Radionuclides

Electron emitting Photon emitting
Half-life
Nuclide Emitting Emitting
period Type of decay Energy/MeV Type of decay Energy/MeV
probability/ % probability/%
3H 12. 33 y ~- 0. 019CD 100
ne 20. 39 min ~+ 0.960CD 99.8 y o. 511 199. 5®
13N 9. 965 min ~+ l. 198CD 99.8 y o. 511 199.6®
i4c 5730 y ~- 0. 156CD 100
15Q 122. 2 s ~+ l. 732CD 99.9 y o. 511 199.8®
isF 109. 8 min ~+ 0.633CD 96. 7 y o. 511 193.5®
32p 14. 26 d ~- l. 71 CD 100
s1cr 27. 70 d eA 0.004 66.4 X 0.005 21. 6
y 0.320 9. 9
57Co 271. 8 d eA +ce o. 006-0. 007 176.2 X o. 006-0. 007 55.4
ce 0.014 7. 4 y 0.014 9.2
0.115 l. 8 o. 122 85. 6
o. 129 l. 3 o. 136 10.7
o. 692 o. 15
sseo 70. 82 d eA 0.006 48.8 X o. 006-0. 007 25.8
~+ 0.475CD 14.9 y o. 511 29.8®
o. 811 99.4
0.864 o. 7
l. 675 0.5
60Co 5. 271 y ~- 0. 317CD 99.9 y l. 173 99.9
l. 333 100.0
6szn 244. 3 d eA 0.007 47.5 X o. 008-0. 009 38.3
~+ 0.330CD l. 4 y o. 511 2.8®
l. 116 50.0
66Ga 9. 49 h eA 0.008 20. 1 X o. 009-0. 010 18.5
~+ 0.362CD 1 y o. 511 114®
0. 772CD o. 7 0.834 5. 9
0. 924CD 3. 7 l. 039 37.0
l. 781 CD 0.3 l. 333 l. 2
4. 153CD 51 l. 918 2.0
2. 190 5. 3
2.423 l. 9
2. 752 22. 7
3.229 l. 5
3.381 l. 5
3.791 l. 1
4.086 l. 3
4.295 l. 8
4.806 l. 9
61Ga 3. 261 d eA 0.008 60. 7 X o. 008-0. 010 56. 1
ce o. 082-0. 084 29.3 y o. 091-0. 093 41. 9
o. 090-0. 092 3. 6 o. 185 21. 4
o. 175 0.3 0.209 2. 5
0.300 16.6
0.394 4.6
0.888 o. 15
6sGa 67. 63 min eA 0.008 5. o X o. 009-0. 010 4. 7
~+ 0.822CD l. 2 y o. 511 177.8
l. 899CD 87.7 l. 077 3.2
 1401 The Test of Radiopharmaceutical Preparations

continued
Electron emitting Photon emitting
Half-life
Nuclide Emitting Emitting
period Type of decay Energy/MeV Type of decay Energy/MeV
probability/ % probability/ %
68Ge 270. 8 d eA 0.008 41. 8 X 0.001 l. 5
o. 009-0. 010 43.8
75Se 119. 8 d eA 0.009 41. 5 X 0.001 2. 1
ce 0.013 4.2 o. 011-0. 012 54.9
0.023 0.8 y 0.066 l. 1
0.054 0.3 0.097 3.4
0.085 2. 7 o. 121 17. 2
0.095 0.4 o. 136 58.5
o. 109 0.6 o. 199 l. 5
o. 124 l. 5 0.265 58.9
o. 134 0.2 0.280 25.0
0.253 0.4 0.304 l. 3
0.268 0.2 0.401 11. 4
ssKr 10. 76 y ~- o. 173© 0.43 y 0.514 0.43
0.687© 99.56
sgsr 50. 53 d ~- l. 501© 99.99 y 0.909 0.01
9ºSr 28. 78 y ~- 0.546© 100
9oy 64. 10 h ~- 2.280© 99.99
99Mo 65. 94 h ~- 0.436© 16.4 X o. 018-0. 021 3.3
0.848© l. 2 y 0.041 l. 1
l. 214© 82.2 o. 181 6. 1
0.366 l. 2
o. 740 12.3
o. 778 4.3
99mTc 6. 01 h ce 0.002 99 X o. 018-0. 021 7.4
eA 0.015 2. 1 y o. 141 89
ce o. 119-0. 121 9.4
o. 137-0. 140 l. 3
99Tc 2.111Xl05y ~- 0.294© 100
io3Ru 39. 26 d eA+ce 0.017 11. 4 X 0.003 4.0
ce o. 030-0. 039 86.3 o. 020-0. 023 8. 6
~- o. 113© 6. 5 y 0.497 91. o
0.227© 92.0 o. 610 5.8
m 1n 2. 805 d eA 0.019 15.5 X 0.003 6.8
ce o. 145 8. 1 o. 023-0. 027 82.8
0.219 5.0 y o. 171 90. 7
0.245 94. 1
113m In l. 658 h eA 0.020 4.3 X 0.003 2.24
ce 0.364 28.8 o. 024-0. 028 24.2
0.387 5. 6 y 0.392 64.9
0.391 l. 1
114m In 49. 51 d ce l. 62 40. 1 X o. 024-0. 027 32.8
o. 186-0. 189 38.6 y o. 190 15. 6
~- l. 939© 99.4 0.558 4.4
(1141n: 71. 9 s) o. 725 4.4
123 I 13. 27 h eA 0.023 12.4 X 0.004 9. o
ce o. 127 13.6 o. 027-0. 032 85. 1
o. 154 l. 8 y o. 159 83.3
0.158 0.4 o. 346 o. 1
0.440 0.4
 1401 The Test of Radiopharmaceutical Preparations

continued
Electron emitting Photon emitting
Half-life
Nuclide Emitting Emitting
period Type of decay Energy/MeV Type of decay Energy/MeV
probability/ % probability/%
0.505 0.3
0.529 l. 4
0.538 0.4
1241 4.18 d eA 0.003 63.8 X 0.004 6.0
0.023 8.3 o. 027-0. 032 57
ec o. 571 0.3 y o. 511 45
~+ 0.812 0.3 0.603 62.9
l. 535 11. 7 o. 723 10.4
2. 138 10. 7 l. 326 l. 6
l. 376 l. 8
l. 509 3.2
l. 691 11. 2
1251 59. 41 d eA +ce 0.004 78. 1 X 0.004 14.8
0.023-0.035 33. 1 0.027 112. 7
0.031 23.5
y 0.035 6. 7
1261 13. 11 d eA 0.023 5. 5 X 0.004 4.0
ce 0.354 0.5 o. 027-0. 032 38. 1
0.634 o. 1 y 0.389 35.6
~- 0.378® 3.6 0.491 2.9
0.870® 33.4 0.511 2. o@
l. 258® 10. 3 0.666 32.9
~+ l. 133® 0.8 o. 754 4.2
0.880 0.7
l. 420 0.3
1311 8. 021 d ce 0.046 3.5 X o. 029-0. 030 4.4
0.330 l. 6 y 0.080 2.6
~- 0.248® 2. 1 0.284 6. 1
0.304® 0.6 0.364 81. 5
0.334® 7. 2 0.637 7.2
0.606® 89.6 o. 723 l. 8
0.807® 0.4
133Xe 5. 243 d eA 0.026 5. 7 X 0.004 5.8
ce 0.045 52.8 0.031 38.6
0.075 8.0 o. 035-0. 036 8.3
~- o. 267® l. 4 y 0.081 36.9
0.346® 98.5
137Cs 30. 07 y eA 0.026 0.8 X 0.004 0.9
ce 0.624 7.8 o. 032-0. 037 6. 9
0.656 l. 4 y 0.662 85. 1
~- o. 514<D 94. 7
l. 176® 5.3
ls3Sm 46. 27 h eA 0.005 54.6 X 0.006 11. o
0.034 4.6 o. 041-0. 048 59.0
ce 0.021 21. 2 y 0.070 4. 7
0.055 42.4 o. 103 29.3
0.062 3.45
0.095 6. 3
o. 101 l. 4
~- 0.635 31. 3
 1421 Methods of Sterilization

continued
Electron emitting Photon emitting
Half-life
Nuclide Emitting Emitting
period Type of decay Energy/MeV Type of decay Energy/MeV
probability/ % probability/ %
0.704 49.4
0.808 18.4
19s Au 2. 695 d eA 0.054 o. 1 X 0.010 l. 19
ce 0.329 2. 9 o. 069-0. 082 2. 7
0.397 l. o y 0.412 95.6
0.408 0.3 o. 676 0.8
~- 0.285<D l. o 1.088 0.2
o. 96l<D 99.0
199 Au 3. 139 d eA 0.054 o. 7 X 0.010 6. 9
ce 0.035 3. 21 0.050 0.36
0.075 11. 8 o. 069-0. 082 17. 3
o. 125 6. 6 y o. 158 40.0
o. 155 4.. 8 0.208 8. 7
o. 193 l. 24
~- 0.244 21. 5
0.294 72.0
0.452 6. 5
200Tl 26. 1 h eA 0.054 3.3 X 0.010 31. 8
ce 0.285 3.4 o. 069-0. 071 64.4
0.353 l. 4 o. 080-0. 082 17.6
~+ l. 066<D 0.3 y 0.368 87
0.579 13. 7
0.828 10.8
l. 206 30
l. 226 3. 3
l. 274 3. 3
l. 363 3.4
l. 515 4.0
201Tl 72. 91 h eA 0.054 3.0 X 0.010 30
ce 0.052 7. 2 o. 069-0. 071 59
0.084 15.4 o. 080-0. 082 16
o. 121 l. 2 y o. 135 2. 6
o. 153 2.6 o. 167 10.0
202Tl 12. 23 d eA 0.054 3. 1 X 0.010 29.4
ce 0.356 2.4 o. 069-0. 071 60. 1
o. 080-0. 082 16.4
y 0.440 91. 5
CD Maximum energy of the beta spectrum.
@ Maximum intensity corresponding to a total annihilation in the source.
eA : Auger electrons.
ce: Conversion electrons.
y=year, d=day, h=hour, min=minute, s=second.
Absolute sterility of a product can be neither guaranteed nor
demonstrated by testing to any batch of sterilized product.
1421 Methods of Sterilization The sterility attribute of a batch of article only can be
expressed by the probability of decreasing viable micro-
Sterilization is the process to inactivate or remove any viable organisms in article to certain acceptable level, namely
micro-organisms of a product by physical or chemical means, sterility assurance level. In practise, a product is sterilized to
so that the survival probability of viable organism can decrease the survival probability of micro-organisms to a
decrease to intended SAL. The methods described in this specified level, and is designated as sterility assurance level
chapter may be used for the sterilization of preparations, raw (SAL). The survival probability of micro-organisms of a
materials, percipients and medical devices. product treated by terminal sterilization is not more than
Sterility is the absence of any viable micro-organisms. 10- 6 • The SAL of a process for a given product is establi-
 1421 Methods of Sterilization
shed by appropriate validation studies.
The sterility assurance of a sterile product can' t be guaran-
teed by testing, but depends on the use of the validated
sterilization process, good manufacturing practises ( GMP) ,
and good quality assurance system. It is essential that the
fallowing factors are fully considered in choosing a suitable
sterilization procedure, including nature of the product to be
sterilized, effectiveness and economy of the procedure, and
integrity and stability of the product after sterilization.
It is essential to validate the process befare being applied to
practise. The validated items include:
( 1) Establish the validation protocols and the evaluation
standard.
(2) Ensure that the equipments are suitable and running
effectively.
(3) Demonstrate that the key equipment and instrumenta-
tion are capable of operating within the prescribed parameter
criteria.
(4) Perform replicate cycles by actual or simulated products
by the specified sterilization procedure to verify the confar-
mance of the critica! process parameter and demonstrate the
effectiveness of the process (performance validation).
( 5 ) Summarize the records and complete the documents
above, to farm a validation report.
In manufacturing practise, the process of sterilization should
be monitored, and the key parameters of the procedure
( such as temperature, pressure, duration, humidity,
concentration of the gas and the dose of radiation, etc.)
should be within the validated limits. The validation of the
sterilization process should be repeated at suitable intervals.
Revalidation is carried out whenever major changes in the
procedure, including changes in the load, take place.
The sterility assurance is related to the sterilization proce-
dure, the degree of microbial contamination of the product
and the resistance of the contaminating microorganisms
befare sterilization. Therefare, according to the characteris-
tics of the sterilization procedure, it is essential to establish
the microorganism contamination level and resistance limit
befare sterilization to control and monitor it, and choose
adequate precautions to minimize the contamination within
the prescribed limits in the production process.
It is necessary to avoid re-contamination of the product
during cooling stage. In all cases, the container and closure
are required to maintain the sterility of the product
throughout its shelf-life.
Methods of Sterilization
The frequently used methods include steam, dry heat,
ionization radiation, gas and filtration. One or combinations
of the above methods may be used, depending on the nature
of the product. Wherever possible, the terminal sterilization
should be chosen. If terminal sterilization is not possible,
filtration or aseptic processing can be used. Wherever
possible, appropriate additional treatment (far example,
steam) of a non-final product is applied.
l. Stearn sterilization
In this method, the products are placed in a sterilizing
chamber, and sterilized by saturated steam under pressure or
overheated water flow which leads to denaturation of protein
and nucleic acid to achieve the inactivation of micro-
organisms. Steam is the most effective and most widely used
method, and may be used far pharmaceutical preparations,
containers, culture media, sterile coats, plastic plugs, and
other materials resistant to high temperature and moisture.
Steam sterilization does not guarantee the complete inacti-
vation of bacteria! spores, and is often used as a comple-
mentary sterile technique far thermo-labile products.
Thermal stability, heat penetration, the degree of viable
micro-organisms contamination and other factors should be
considered when choosing the conditions of steam sterili-
zation. The process is usually carried out by the fallowing
conditions: 121 ºC X 15 min, 121 ºC X 30 min, l16°C X 40
min. Other combinations of time and temperature may be
used provided that they could give an SAL of 10- 6 or less
after sterilization. When choosing F 0 concept as the selection
of sterilization programs ( F 0 means the standard sterili-
zation time in the condition of 121 ºC of sterile temperature),
it is necessary to take special measures to ensure that the
products being sterilized to meet the requirements of sterility
assurance. At the same time, apart from the sterilization
validating procedure, it is also necessary to monitor micro-
organisms during manufacturing process to prove that the
contamination is within the prescribed limits. For thermo-
stable products, over-killed method should be prior adopted
to ensure that the products will be sterilized to get adequate
low SAL. For thermo-unstable products, the determination
of sterilization process depends on the contamination degree
and thermo resistance of certain product batches befare being
sterilized at a certain time. So it is necessary to monitor the
product contamination continuously and strictly and adopt
various measures to decrease the level of microbial contami-
nation, especially the heat-resistant bacteria contamination.
The F 0 of thermo-unstable goods is not less than 8 minutes.
For steam sterilization, the products to be sterilized should
be loaded in the sterilizing chamber appropriately, no
tightness, to ensure that they are all effectively and equally
steriiized. Knowiedge of the cooiest part of the chamber is
obtained befare this method is applied. Put the biological
indicator at the coolest patt to ensure that SAL of the
products can be achieved. Spores of Bacillus stearother-
mo philus are usually used as the indicator far this method.
2. Dry heat sterilization
Dry heat sterilization is carried out in an oven or tunnel
equipment far sterilization with farced air circulation, where
micro-organisms and pyrogens are inactivated by high
temperature. This method is suitable far products where
steam sterilization is inappropriate, such as glass utensils,
metal containers, fibre products, solid drugs, and liquid
paraffin wax and so on.
The process is usually carried out by heating the product at
160-17 OºC far 120 minutes or longer, 17 0-180 ºC far
60 minutes or longer, or 250ºC far 45 minutes or longer.
Other combinations of time and temperature may be used
provided that they could give an SAL of 10- 6 or less. It is
unnecessary to control the microbial contamination befare
sterilization of those over-killed products by dry heat. Dry
heat at 250ºC far 45 minutes can also remove the pyrogens of
containers and other equipments.
The products should be loaded appropriately far dry heat
sterilization to ensure that they are all effectively and equally
sterilized.
The distribution of the temperatures in the sterilizing
chamber should comply with the prospective requirements
and the coolest part of the chamber should be monitdred
when this method will be applied. Spores of Bacillus suhtilis
are usually used as the indicator far this method. Endotoxin
inactivation test can verify the effectiveness of depyro-
genation. Generally not less than 1000 EU of endotoxin
which is derived from Escherichia coli may be added into the
items prior to depyrogenation, in this case the reduction of
the endotoxins should be at least 3-log.

3. lonising radiation sterili7.ation
Sterilization by this method is achieved by exposure of the
product to ionizing radiation in the farm of gamma radiation
from a suitable radioisotopic source ( such as cobalt 60) or of
a beam of electrons energized by a suitable electron accele-
rator. This method may be used far medica! devices,
containers, manufacturing equipments, and other raw
materials and preparations resistant to radiation.
The SAL of the product is not more than 10-s after ionising
radiation sterilization. The key parameter of ionising radiation
sterilization is mainly the absorbed radiation <lose. The
radiation <lose absorbed by the material being irradiated
should be identified according to the suitability of the
products and the maximum amount of the contaminated
micro-organisms and the maximum resistance to radiation. lt
should be validated befare the procedure that the safety,
efficiency and stability of a product should not be changed by
the radiation <lose. A reference absorbed <lose is 25 kGy. Use
the lowest possible radiation <lose far the final product, raw
materials and medica! devices. Befare irradiation, test the
number of contaminated microorganisms and the resistance
to the irradiation of the product to evaluate the SAL of the
proce- dure. Far setting dosage, it should be examined
regularly to validate its efficacy.
During the procedure, the radiation absorbed by the product
should be monitored regularly by means of suitable chemical
or physical methods to ensure that the <lose is appropriate. If
dosimetres radiated along with sterilized products are
adopted, place themat specified positions. Calibrate against a
standard source at suitable intervals.
Spores of Bacillus pumilus are usually used as the indicator
far this method.
4. Gas sterili7.ation
In this method, micro-organisms are inactivated in a high-
pressure chamber filled with gas of disinfectors, far
example, ethylene oxide, hydrogen pero:xide, farmaldehyde
and ozone ( 0 3 ) • This method is suitable far the products
which are stable in these gases. lnflammability, teratogenesis
and residue toxicity of the gases should be considered in the
process of sterilization.
The most generally used gas in this method which is carried
out in a high pressure chamber filled with sterilized gas is
ethylene oxide, usually mixed with 80 %-90 % inert gases.
This method is applicable to medica! devices and plastic
products that are not durable by using high temperature
methods. This method is not applicable to products which
contain chlorine or can absorb ethylene oxide.
Temperature, humidity and gas concentration in the
chamber, as well as the duration could affect the effecti-
veness of sterilization by using ethylene oxide. The fallowing
conditions are recommended:
Temperature: 54±10ºC
Relative humidity: 60%±10%
Pressure: 8X 10 5 Pa
Duration: 90 minutes
The process of sterilization is validated befare application.
During the procedure, the chamber is vacuumized first, and
then filled with vapour to the specified humidity and
temperature. After that, the filtered and pre-heated ethylene
oxide is filled in. During the process, the temperature,
humidity, pressure, concentration of ethylene oxide and time
should be seriously monitored. Biological indicators can be
used to monitor the effectiveness of sterilization, if neces-
sary. The process should be supervised by skilled techni-
cians. After sterilization, adequate time should be left to
allow dispersa! of residual ethylene oxide and other volatile
1421 Methods of Sterilization
residues. lt should be monitored that the residues are within
the prescribed limits to prevent toxicity.
Leak test should be carried out to ensure obturation of the
chamber. Package materials and alignment of the products
can be considered because they may affect the gas penetration
and diffusion when validation of sterilization is perfarmed.
Spores of Bacillus suhtilis are usually used as the indicator
far this method.
5. Filt ration
In this method, micro-organisms in the gas or liquid products
are removed by filtering through a certain type of filter
material. lt is usually used far gas, thermo-labile solutions of
drugs and raw materials.
Sterilization filter units use micro porous filter membranes
( hydrophilic or lipophilic ) as the filter material. The
material of filter membranes depends on the manufacturer' s
practise. The pare size is usually less than O. 22 µm in
pharmaceutical manufacture. The definition of pare size of a
filter is adsorption of bacteria on or in the filter matrix but
not average pore size distribution coefficient. Hence, only the
filter that can be manifested by microorganism retention test
can be used at the final sterilization stage. Adsorption of
liquid by filter must not affect the drug quality. Fibre-
shedding filters, particularly those containing asbestos
should be avoided The user of filters should understand the
nature and quantity of precipitates during filtration and
evaluate their toxicity influence. The filter unit and mem-
branes should be sterilized befare use. The filter units are
cleaned first and then new membranes or filter units are used
far a new batch of products.
The sterility assurance level in this method is related to
bioburden of a product prior to sterilization and the lag
reduction value ( LRV) of the filter unit. Calculate LRV
with the fallowing expression:
LRV=lgNo-lgN
Where N 0 represents the number of micro-organisms befare
filtration, and N is the number after filtration.
LRV is used to express the efficiency of filtration. The LRV
per cm2 of active filter surface is not less than 7 far O. 22 µm
pare size membranes. Therefare, the total contamination
amount of filtration products should be controlled within the
prescribed limits far filtration sterilization. There are two
ways to make sure sterilization effect iveness, namely
connecting two sterilization filters together or refiltering
befare filling.
Key factors, such as average pare size, distribution of sizes,
integrity and LRV, are not able to be monitored in the
filtration process normally. Therefare, the integrity test and
effectiveness of the membranes should be perfarmed befare
use and af ter use far every batch, such as a bubble point
test, pressure hold test, or diffusive airflow test. Specifi-
cations of the integrity tests should come from the data of
relevant micro-organism retention test. The duration of
filtration process should be validated, which is normally not
more than one working day.
Pseudomonas diminuta are usually used as the indicator far
this method.
The products sterilized by this method should be processed in
a cleaned area with rigorous monitoring and a sterile area is
recommended. Relative facility, package container, stopper
and other materials should be sterilized by appropriate
:methods, and recontamination should be avoided.
6. Aseptic preparation
Aseptic preparation denotes the manufacturing of sterile
preparations under sterile condition. lt may include aseptic
 1421 Methods of Sterilization
filling of products into containers and aseptic lyophilization.
Filtration is usually used in the latter process.
It is necessary to monitor the environment of aseptic
preparation, and the filtration should be carried out under
sterile conditions. The equípment, containers, plugs and
other materials should be sterilized using suitable sterilization
methods, and recontamination should be avoided.
Sterility assurance of this process is carried out by the
simulating test for sterility by aseptic filling culture media.
Sterility of the circumstance, personnel quality and materials
should be monitored in the whole process.
The aseptic preparation should be validated at regular inter-
vals, including periodic environmental filter examination and
simulating test for sterility by aseptic filling culture media.
Biological Indicators
Biological indicators are preparations of viable m1cro-
organisms used to assess the performance of sterilization
equipments, to validate and monitor the effectiveness of
sterilization process. They usually consist of bacteria! spores.
l. The Requirements for micro-organisms used as biological
indicators
Different biological indicators are used for different sterili-
zation methods. The micro-organisms used as biological
indicators comply with the following requirements:
( 1) The test strain is more resistant than any possible conta-
minating micro-organisms in the product to be examined.
(2) The test strain is non-pathogenic.
(3) The test strain is resistant to mutation and easy to store.
( 1) The test strain is easy to culture. If resting spores are
used, they should be more than 90 % of the indicator.
2. Preparation of biological indicators
The biological indicators are prepared using a specified
procedure. Characteristics of the micro-organisms are deter-
mined before use, for example, D value, which is the
duration (expressed in minutes) with which the number of
viable organisms are reduce to 10 % of the original number at
a definite temperature. Use suitable culture media for the
incubation. Produce the spore suspension in innutrient
liquids, which mainly consist of spores. Store the spores as
suspen-sions in innutrient liquids.
Biological indicators consist of a definite number of one or
more types of spores. They are usually placed on an inert
carder, for example a strip of filter paper, a glass slide,
stainless or plastic materials. Spore suspensions may, be
presented in sealed ampoules. Sorne biological indicators have
medium system. The D value is not only related with
sterilization conditions but also the survival environment of
microorga-nism. Therefore, after completing the preparation
of certain type of biological indicators, the D value and the
number of spores should be determined. Biological indicators
are packed with suitable materials, and an expiry date should
be provided. The carders and package materials protect the
biological indicators from any deterioration or contamination,
while allowing the sterilizing agent to enter into and contact
with the micro-organisms. The packages are designed to
facilitate storage, transportation, sampling and subculture.
Sorne biological indicators may be inoculated directly into a
liquid product to be sterilized or into a liquid substitute which
is similar in chemistry and physics. The equivalence between
the liquid product and the substitute should be demonstrated.
3. Application of biological indicators
In validation of sterilization process, though sorne parameter
can be monitored, the effectiveness of a sterilization process
is most easily assessed by the degree to which biological
indicators are inactivated. Commercial biological indicators,
or spores prepared from resistant micro-organisms isolated
from contaminants are used as biological indicators. The D
value, purity and number of spores are validated for the
biological indicators. The quantity and resistance of spores of
the biological indicators used should be more or greater than
the contamination of the product to ensure the effectiveness
of the process.
Put the biological indicators at different positions in the
chamber for the terminal sterilization. Avoid direct contact of
the indicator with the product to be sterilized. lncubate
indicators after specified sterilization on suitable culture
media to guarantee inactivation of all the spores.
Commercial biological indicators may be used for the
validation test of deep sterilization in which the bioburden of
product is ignored. For susceptible products, suitable strains
and spores can be selected as the biological indicator and the
process is designed depending on the degree that the products
are conta- minated. Sterility assurance of these products are
assessed by monitoring the number and resistance of
contaminating micro-organisms and the validation test.
4. Common biological indicators
( 1 ) Steam sterilization Spores of Bacillus stearother-
mophilus ( for example, NCTC 10007, NCIMB 8157,
ATCC 7953) are recommended as the indicator for steam
sterilization. The D value is l. 5-3. O minutes, the number of
viable spores in each tablet (or ampul) is 5 X 10 5 -5 X 10 6 ,
and all the micro-organisms are inactivated at 121 ºC for
19 minutes. Spores of Clostridium sporogenes (for example,
NCTC 8594, NCIMB 8053, ATCC 7955 ) may also be
used, with D-value of O. 4-0. 8 minutes.
( 2) Dry-heat sterilization Spores of Bacillus suhtilis ( for
example, NCIMB 8058, ATCC 9372) are recommended as
the indicator for dry-heat sterilization. The D-value is more
than l. 5 minutes, the number of viable spores in each tablet
is 5 X 105 -5 X 10 6 • Escherichia coli endoxin for validation of
depyrogenation is used, and the amount added is not less
than 1000 EU.
( 3 ) Ionizing radiation sterilization Spores of Bacillus
pumilus ( for example, NCTC 10327, NCIMB 10692,
ATCC 27142) are recommended as the indicator for ionizing
radiation sterilization. The number of viable spores in each
tablet is 10 7 -10 8 • The D-value is around 3 kGy when a
25 kGy radiation <lose is applied. Contaminated micro-
organisms in the product may be more resistant to radiation
than B. pumilus. Therefore, the latter can only be used to
monitor the process, and cannot be used to establish the
radiation <lose.
( 4) Gas sterilization Spores of Bacillus suhtilis ( for exa-
mple, NCTC 10073, ATCC 9372) are recommended as the
indicator for gas sterilization with ethylene oxide. The
number of viable spores in each tablet is 1X106 -5X10 6 • The
D-value is more than 2. 5 minutes, and the concentration of
ethylene oxide is 600 mg/L. The relative humidity is 60%.
The microorganisms are inactivated at 54 ºC for 60 minutes.
Spores of Bacillus stearothermophilus (for example, NCTC
10007, NCIMB 8157, ATCC 7953) are recommended as the
indicator for gas sterilization with hydrogen peroxide.
(S) Filtration Pseudomonas diminuta (for example ATCC
19146) is recommended as the indicator when O. 22 µm pare
size membranes are used for filtration. Serratia marcescens
(for example, ATCC 14756) is recommended when O. 45 µm
pare size membranes are used.
 1431 Statistical Methods for Biological Assays

1431 Statistical Methods f or Biological Assays

1. Introcluction
Biological assay is a method by utilizing animal in vivo experiments and tissue, organ, cell strain, microorganism in vitro
experiment, etc., used to evaluate the biological activity of the drug. It is based on the drug' s pharmacological effects using
biological statistics as a tool. Specified experiments are designed to compare the special response produced by the test
preparation with that of the standard or the reference standard. The potency and the biological activity of preparation being
examined or the toxicity from the impurity can be determined by calculating the ratio of equally effective doses or the biological
response of limited doses.
This chapter presents mainly the basic principies and general requirements to be observed, the assay design and statistical
method to be adopted in a biological assay. The assay procedure, experimental conditions and sorne other specifications are
described under individual monographs.
Standard preparation ChP standard preparations (S) are available for biological assays, the potency of (S) is expressed in
Unit which is equivalent to the corresponding International Unit.
Test preparation Test preparation ( T) or (U) is a pharmaceutical products whose potency is to be assayed. The
pharmacological properties of (T) or (U) should be essentially similar to those of the standard preparation. AT or Av is the
labelled or assumed potency of (T) or (U).
Ratio of equally effective doses Biological assays are carried out by comparing the responses produced by the standard
preparation (S) and the test preparation (T) when applied as stimuli to living matter at the same time and under the same
condition. From the results of an assay, the doses of (S) and (T) producing equal response-the equally effective doses (ds,
dT) can be calculated and the potency of (T) is estimated from the ratio of equally effective doses (R), i. e.
R is the ratio of ds and dT. R=ds/dT.
Mis the difference between logarithm of ds and dT. M=lg ds-lg dT=xs-xT. R=antilog M.
PT is the estimated potency of (T) obtained by multiplying the value of R or antilog M by assumed potency AT.
P T = AT• R = AT• antilog M. P T is expressed as u/ mg, u/ ml etc.
Control of biological variation Biological assays are subject to random errors due to the inherent variability of biological
responses. In arder to control and minimize the biological variation and reduce the error of an assay, the following conditions
must be noticed.
(1) The living matter used in an assay should be of the same species and same source, bred in an uniform environment or
cultured under uniform condition.
(2) When the totality of individual unit appears to be reasonably homogeneous, allocate the individual unit to different doses by
random process. If the individuals in subgroup, such as body weight, Petri dishes, sex, litter are likely to be more
homogeneous than the totality of individual unit, allocation will be made by arranging the individuals in contiguous subgroups at
random to diff erent doses.
(3) When the assay design requires to segregate the variation between subgroups or blocks such as between litters or Petri
dishes, the member in each subgroup is selected at random for each <lose.
Error variance The residual error of an assay is obtained by subtracting the variations allowed for in the design (e. g.
variations between doses) from the total variation in response. Error variance (5 2 ) is the mean square of residual error which
is obtained by dividing the sum of squares of residual error by its degrees of freedom.
The value of 5 2 will affect the value of standard error of M (SM) and the range of fiducial limits.
Validity test For parallel line assay, it is required that the relation between the logarithm of doses and responses ( or
transformed responses) can be represented by a straight line in a certain range of doses, and the line for the test preparation
must be parallel to that for the standard preparation. If the <lose-response line of (S) or (T) is not significantly deviated from
a straight line and the two lines are not significantly deviated from parallelism, the assay result is said to be statistically valid,
the calculation methods described in this chapter can be used to estimate the potency of the test preparation and its fiducial
limits.
Fiducial limits of error The required precision of a biological assay is expressed in terms of fiducial limits (FL ). The fiducial
limits for M are M ± t • SM, where SM is the standard error of M, t is a value depending on the degrees of freedom (j) of 5 2
(Table 1).
Table 1 Degrees of freedom and the corresponding values of t ( P =O. 95)

f t f t f t f t

3 3. 18 8 2.31 14 2. 15 30 2.04
4 2. 78 9 2.26 16 2. 12 40 2.02
5 2.57 10 2.23 18 2. 10 60 2.00
6 2.45 11 2.20 20 2.09 120 l. 98
7 2.37 12 2. 18 25 2.06 = l. 96
 1431 Statistical Methods for Biological Assays

In ChP, the fiducial limits are calculated with reference to a probability of O. 95, i. e., the true potency would be expected to líe
between the upper and lower fiducial limits CP=O. 95). The precision of an assay is expressed in FL% which is the average
distance between the upper and lower fiducial limits expressed as a percentage of the estimated PT or R. The value of FL% is
prescribed for each assay method in ChP, if the result of an assay <loes not comply with the required FL %, the experiment
should be repeated. FL % may be reduced as muchas practicable by increasing the number of animals or petri dishes; reducing
the weight or age variation of animals; changing the doses etc.
The results of n repeated assays, including the one with a FL% in excess of that specified in the monograph, can be combined
to obtain a single value.
2. Direct Assay
In a direct assay, the threshold <lose of standard or test preparation which produces sorne fixed effect is being measured. The
assay of digitalis with pigeons is an example of such an assay.
In an assay of digitalis, xs and XT are the logarithm of the mínimum lethal <lose of (S) and (T) for each pigeon, their mean
value xs and Xy are taken as the equally effective doses, ns and ny are the number of pigeons treated with standard .and test
preparation respectively.
( 1 ) Calculation of potency
M=xs-xT (1)
R =antilogCXs-xT) =antilogM (2)
Py=R•Ay (3)
( 2) Error variance and fiducial limits

(4)

2 ·ns+
s ---
ny
(5)
ns•ny
FL of R=antilog(M±t •SM) (6)
FL of PT =Ay •antilog(M±t •SM) (7)

The percentage fiducial limits CFL %) of the potency CR of P T) is the percentage of the difference between the upper limit
and the lower limit divided by doubled mean Cor potency).
FL %= upper fiducial limit-lower fiducial limit X lOO % (8)
2 X mean ( or potency)
When two or more test preparations (T, U. .. ) are compared with the standard preparation simultaneously, the combined error
variance (s 2) of (S), (T), (U) ... is calculated as follows:
¿X~ (¿xs)2 +¿x~
ns
52 (9)
ns-1 +ny-l +nu-1 +···
f=ns-l +nT-l +nu-1 + ...
Example 1 Direct assay of digitalis using pigeons (MLD method}
The assay is carried out as described in the Biological Assay of Digitalis ( 1214 ). The substance being examined is digitalis
powder, Ay=lO Units/g. The results of test are shown in table 1-1.
Table 1-1 An assay of digitalis powder
s T

MLDs Cds) Xs MLDT (dy) XT

Units/kg Units/kg
lg Cds X 10) lg (dyXlO)
body wt. body wt.

l. 15 l. 061 1.11 l. 045

l. 01 l. 004 l. 23 l. 090
l. 10 l. 041 l. 06 l. 025
l. 14 l. 057 l. 31 l. 117
l. 06 l. 025 0.94 0.973
0.95 0.978 l. 36 l. 134

¿Xs 6. 166 ¿XT 6.384

- -Xy
Xs l. 028 l. 064

M=l. 028-1. 064=-0. 036

R=antilog(-0. 036)=0. 9204
Py=lOXO. 9204=9. 20 Units/g
 1431 Statistical Methods for Biological Assays

6. 166 2 6. 384 2
l. 061 2+1. 004 2+···+0. 978 2--6-+1. 045 2+1. 090 2+···+1.134 2- - 6 -
s2 0.002373
6+6-2
f=6+6-2=10 t=2. 23

SM =Jo. 002373 X~~~ =O. 02812

FL of PT =10 antilog(-0. 036±2. 23XO. 02812) =7. 97-10. 6 Units/g
FL % of PT-
_10. 6-7. 97
X .
%- %
XlOO o-14. 3 o
2 9 20
3. Parallel Line ~y Based on Quantitative Responses
When the magnitude of response which varies with the amount of <lose is measurable, it is known as a quantitative response.
Parallel line assay can be used only when the relation between the logarithm of <lose and response ( or transformed response)
can be represented by a straight line overa certain range of doses, and the straight line for (T) must be parallel to that for (S).
In a parallel line assay, the <lose structure of (S. T) may be (2. 2) or (3. 3) which are generally termed as (k. k) assay;
the <lose structure of (S. T. U) may be (2. 2. 2) or (3. 3. 3) which are generally termed as (k. k. k) assay. When the
number of doses of (S) is not equal to that of (T), (k. k') assay is resulted. Ordinarily the (k. k) structure is used for
parallel line assay, but when the <lose of (T) or (S) used is very different from the right <lose, it is possible that the response
of the largest or smallest <lose may fall outside the linear zone. In such circumstances, the response to the largest or smallest
<lose should be omitted and (3. 2) or (2. 3) assay is the result. Therefore, in (k. k') assay, k is only one more or one less
than k'.
Either in (k. k) assay, (k. k. k) assay or in (k. k') assay, the preceeding k always represents the number of doses of (S),
the k or k ' behind is the number of doses of ( T) or (U). Therefore K = k + k or K = k + k' or K = k + k + k. In this chapter,
the calculation used for parallel line assays is a simplified method, therefore, the following requirements should be observed:
(1) The ratio of adjacent doses (r) of (S) and (T) in an assay must be constant, usually (1 : O. 8) - (1 : O. 5). Igr=I.
(2) The number of responses (m) to each <lose must be equal.
( 1) ~y design CD Random design In this type of assay design, no restriction on the variability of subgroups is applied.
The test animals or other experimental units are allocated randomly to different doses, only the variation caused by <lose levels
can be segregated from the assay result.
® Random block design In this type of design, the test animals or other experimental units are further divided into blocks,
such as litters of experimental animals or Petri dishes in a microbiological assay. Random block design requires that every <lose
is applied once in every block ( litter or petri dish) and is only suitable when the block is large enough to accommodate all
doses. In this design, the error variance may be reduced by segregating the variation between blocks.
@ Cross-over design This design is useful when the single animal used in an experiment can be tested on two occasions
separated at suitable time interval. The cross over dcsign uscd in (2. 2) parallel line assay is knovn1 as tv~in crossover test, in
which two doses of standard preparation and of a test preparation are used. Animals are divided randomly into four groups with
equal numbers of animal in each group, and each animal in every group is marked for identification. In the first part of the test,
each group receives one of the four doses of (S) and (T). Animals which received one preparation in the first part of the test
receive the other preparation in the second part of the test, and animals receiving small doses in one part of the test receive large
doses in the other. Thus the precision of the experiment can be increased by eliminating the different effect between animals and
the different responses at the two stages of the test. The arrangement of doses for twin cross-over design is shown in the
following table

Group 1 Group 2 Group 3 Group 4

lst test ds1 ds-¿ dT¡ dT 2

2nd test dT 2 dT¡ ds-¿ ds1

( 2) Variance analysis and validity test

For random and random block design
G)Enter the value of the measured responses or their transformations (y) into Table 2 K is the total number of doses and
mis the number of y in each <lose. In randomized block design, the variation between rows (m) represents the variation
between blocks. n is the total number of responses, n=m K.
Table 2 The value of y in different dose groups
Number of doses
(1) (2) (3) ... (K)
Sum

1 Y1 (1) Y1 (2) Y1 (3) ... Y1 (k) ~Y1

2 Y2 (1) Y2 (2) Y2 (3) ... Y2 (k) ~Y2
Between
blocks
3
...
Y3 (1)
...
y3 (2)
...
y3(3)
... ... y3 (k)
...
~Y3
...
...
m Ym (1) Ym (2) Ym (3) Ym (k) ~Ym
Sum ~Ym ~Yc2) ~YC3) ... ~YCk) ~y

®Replacement of missing value and rejection of aberrant value When an accident leads to the loss of one or more
responses, the number of responses (m) in each <lose will not be equal. The balance may be restored by one of the following
 1431 Statistical Methods for Biological Assays

procedures:
For random design, the missing value can be replaced with the arithmetic mean of other responses to the same <lose. If there are
one or two surplus responses in one <lose, reject the surplus response by random selection. For randomized block design, the
missing value can be replaced with a value calculated from equation (10):
KC+mR-G
(10)
y CK-1) Cm-1)
Where: C is the sum of responses in the <lose group containing the missing value;
R is the sum of responses in the row containing the missing value;
G is the sum of all responses recorded in the assay;
K is the total number of doses;
m is the number of blocks used in the assay.
When there are enough blocks Clitters or dishes) used in the design, it is preferable to omit the whole block in which the
missing value líes.
Each replacement of a missing value causes a loss of 1 degree of freedom for error variance, and the number of missing values
should not be more than 5 % of the total number of responses.
When the largest or smallest response in one of the doses is questionable, the following aberrant value test should be applied.
Let Ya be the supposed aberrant value, arrange the responses in the <lose in order of magnitude from Ya to Ym (yª, Y2, y3 . ..
Ym- 2 , Ym-l, Ym). If Ya is the largest value, the series is in descending arder; if Ya is the smallest value, the series is in
ascending arder.
Calculate the value of J with the following equations:
When m=3-7,
Y2 -ya
11 (11)
Ym -ya
When m=8-13,
Y3-Ya
12 (12)
Ym-1 -ya
When m=14-20,
_ Y3 -yª (13)
l 3 -
Ym-2 - Ya
!f the calculated value of] exceeds the critical value shown in Table 3; y. can be judged as an aberrant value and rejected.
Table 3 The value of J for aberrant value test
m 3 4 5 6 7

11 o. 98 0.85 o. 73 0.64 0.59

m 8 9 10 11 12 13

12 o. 78 o. 73 0.68 0.64 o. 61 o. 58
m 14 15 16 17 18 19 20

13 0.60 0.58 0.56 o. 54 0.53 o. 51 0.50

®Variance analy5i5 Far randomized block design, the sum of squares of total variation is divided into components of 'between
doses', 'between blocks' and 'residual error'. The values for sum of squares were obtained using the following equations.
(¿y)2
Total=¿y 2- mK (14)

Í101a1=mK-l
¿ [¿y(k)J2 (¿y)2
Between doses=----- (15)
m mK
J between doses = K - 1
¿(¿ym)2 (¿y)2
Between blocks = K mK (16)

f between blocks = m - 1
Residual error= total- between doses- between blocks (17)
J residual error= (K -1) (m-1)
The mean squares for each component are calculated by dividing the sum of squares of each component by its degrees of
freedom. The mean square for residual error is the error variance (5 2) of the assay.
sum of squares for error
52 (18)
f for error
2
Km¿y -K • ¿ [¿y<kJ -m • ¿(¿ym) +(¿y) 2 2 2
or 52 Km (K-1) (m-1)
(19)
f=(K-1) (m-1)
For random design, it is only necessary to calculate the sum of squares for total, between doses and residual error, using
equations 04), (15) and (20).
Residual error= total- between doses (20)
 1431 Statistical Methods for Biological Assays

Í residual enor = K ( m -1)

and s 2 is obtained from equation (18) or (21).
m¿y2-¿ [¿y<k)J2
52 (21)
Km(m-1)
f=K(m-1)
@ Validity test For (2. 2) and (2. 2. 2) assays, the between doses component can be subdivided into terms of preparations,
regression and parallelism; for ( k. k ) assays other than ( 2. 2) assay and ( k. k. k ) assays other than ( 2. 2. 2) assay,
additional terms of quadratic and difference of quadratics are required.
1
For (k. k) and (k. k ) assays, calculate m • ¿c7 and ¿ [C;• ¿y(k)] for each term, using the equations shown in Table 4.
The sum of squares for each component is obtained from equation (22).
Table 4 Orthogonal coefficient for validity tests of ( k. k } and ( k. k' } ~y

Source of Orthogonal coefficient(C;)

Design m•¿c7 ¿[C;• ¿Y<k)J
variation Si S2 s3 s4 Ti T2 T3 T4

Preparations -1 -1 1 1 4m T2+Ti -S2-Si

(2. 2)
Regression -1 1 -1 1 4m T2-Ti +s2-Si
Parallelism 1 -1 -1 1 4m T2-Ti -S2+Si

Preparations -1 -1 -1 1 1 1 6m T3+T2+Ti -S3-S2-Si

Regression -1 o 1 -1 o 1 4m T3-Ti +s3-Si
(3. 3) Parallelism 1 o -1 -1 o 1 4m T3-Ti -S3+Si
Quadratic 1 -2 1 1 -2 1 12 m T3-2T2+Ti +s3-2S2+Si
Difference of -1 2 -1 1 -2 1 12 m T3-2T2+Ti -S3+2S2-Si
Quadratics

Preparations -1 -1 -1 -1 1 1 1 1 8m T4 +T3+T2+Ti -S4-S3-S2-Si

Regression -3 -1 1 3 -3 -1 1 3 40 m 3T4 +T3-T2-3Ti +3S4 +S3-S2-3Si
(4.4) Parallelism 3 1 -1 -3 -3 -1 1 3 40 m 3T4 +T3-T2-3Ti -3S4 -S3+S2+3Si
Quadratic 1 -1 -1 1 1 -1 -1 1 8m T4 -T3-T2+Ti +S4-S3-S2+Si
Difference of -1 1 1 -1 1 -1 -1 1 8m T4-T3-T2+Ti -S4 +s3+S2-Si
Quadratics

Preparations -2 -2 -2 3 3 30 m 3(T2 +Ti)-2(S3+S2+Si)

(3.2) Regression -2 o 2 -1 1 10 m T2-Ti +2(S3-Si)
Parallelism 1 o -1 -2 2 10 m 2CT2-Ti)-S3+Si
Quadratics 1 -2 1 o o 6m S3-2S2+Si

Preparations -3 -3 -3 -3 4 4 4 84 m 4(T3+T2+Ti)-3(S4 +s3+s2+Si)

Regression -3 -1 1 3 -2 o 2 28 m 2(T3-Ti)+3(S4 -Si )-S2+S3
(4. 3) Parallelism 3 1 -1 -3 -5 o 5 70 m 5(T3 -Ti)-3(S4 -Si )-S3 +S2
Quadratic 3 -3 -3 3 2 -4 2 60 m 2(T3+Ti)-4T2+3(S4 -S3-S2+Si)
Difference of -1 1 1 -1 1 -2 1 10 m T3-2T2+Ti -s4+s3+S2-Si
Quadratics
Notes: For (2. 3) or (3. 4) assay, calculate m • ¿c7 and ¿ [C;• ¿Y<k>] by interchanging the orthogonal coefficient of (S) and (T) in
(3. 2) or (4. 3) assay.
In Table 4 Si, 5 2... Ti, T 2 ... are the total responses for each <lose of (S) and (T) corresponding to the term ¿Y<k>in table 2. The subscript
1, 2, 3... are used to represent the respective <lose level and 1 is always the smallest <lose.

[¿ CC; • ¿y<k)) ] 2
Sum of squares for ech component (22)
m•¿c7
f=l

For (k. k. k) assays, calculate the sum of squares of preparation by equation (23) and (24).
For (2. 2.2) assay
CS2 +Si ) 2+CT2 +Ti ) 2+<U2 +Ui ) 2 (¿y) 2
Sum of squares (preparations) (23)
2 m mK
f=2
For (3.3.3) assay
(Si +s2+S3) 2+CTi +T2+T3) 2+CUi +u2+U3) 2 (¿y) 2
Sum of squares (preparations) (24)
3 m mK
f=2
For calculation the sum of squares for components other than preparations, calculate m • ¿c7 and ¿ [C;• ¿Y<k)J by the
equations shown in Table 5.
 1431 Statistical Methods for Biological Assays

Table 5 Orthogonal coefficient for validity tests of ( k. k. k ) ~y

Source of Orthogonal coefficient (C;)

Design m. ¿;e; ¿:; [C;•L::;y<k)]
variation 51 52 53 Ti T2 T3 U1 U2 u3
Regression -1 1 -1 1 -1 1 6m 52-51 +T2-T1 +U2-U1

(2. 2. 2) 1 -1 -1 1 4m T2-T1 -52+51

Parallelism 1 -1 -1 1 4m U2-U1 -52+51
1 -1 -1 1 4m U2-U1 -T2+T1

Regression -1 o 1 -1 o 1 -1 o 1 6m U3-U1+T3-Ti +53-51

1 o -1 -1 o 1 4m T3-T1 -53+51
Parallelísm 1 o -1 -1 o 1 4m u3-u1 -53+51
1 o -1 -1 o 1 4m U3-U1 -T3+T1
(3. 3. 3)
Quadratic 1 -2 1 1 -2 1 1 -2 1 18 m U3-2U2+u1 +T3-2T2+T1 +53-2..52+51

Dífference -1 2 -1 1 -2 1 12 m T3-2T2+T1 -53+252-51

of -1 2 -1 1 -2 1 12 m U3-2U2+U1 -53+252-51
quadratícs -1 2 -1 1 -2 1 12 m U3-2U2+U1 -T3+2T2-T1

The sum of squares for regression and parallelism is obtained from equation (22). The sum of squares of quadratic and
difference of quadratics is calculated from equation (25).
. 'ff f . 2¿{¿[C;•¿Y<k>]}2 C
Quadrat1c and d1 erence o quadrat1cs ¿ (m. ¿e;) 25)
f=2
The mean square of each term is obtained by dividing the sum of squares of each term by its degrees of freedom.
Table 6 is a model of validity test for (3. 3) assay, randomized block design.
Table 6 Validity test for ( 3. 3) ~y, randomb:ed block design
Source ofvariation Degrees of freedom(f) Sum of squares Mean Square F p

Preparations 1 Equation(22) Sum of squares/ f Mean square/ s 2

Regression 1 Equation(22) Sum of squares/ f Mean square/ s 2
Parallelism 1 Equation(22) Sum of squares/ f Mean square/ s 2
Quadratic 1 Equation(22) Sum of squares/ f Mean square/ s 2
Difference of quadratics 1 Equation(22) Sum of squares/ f Mean square/ s 2

Doses K-1 Equation(l5) Sum of squares/ f

Mean square/ s 2
Blocks m-1 Equation(16) Sum of squares/ f
Mean square/ s 2
Error CK-l)(m-1) Equation( 17) Sum of squares/ f(s 2 )

Total mK-1 Equation(14)

In Table 6, the F ratio is the mean square of each variable expressed as a ratio of s 2 , and the significance of these values are
assessed by use of Table 7.
Table 7 The F distribution table
f i Degrees of freedom for numerator
1 2 3 4 6 12 20 40 =
1 161 200 216 225 234 244 248 251 254
4052 4999 5403 5625 5859 6106 6208 6286 6366
2 18.51 19.00 19. 16 19.25 19.33 19.41 19.44 19.47 19. 50

f2 98.49 99.00 99. 17 99.25 99.33 99.42 99.45 99.48 99.50

Degrees of 3 10. 13 9.55 9.28 9. 12 8.94 8. 74 8.66 8.60 8.53
freedom
for 34.12 30.82 29.46 28. 71 27.91 27.05 26.69 26.41 26. 12
denominator 4 7. 71 6.94 6.59 6.39 6. 16 5. 91 5.80 5. 71 5.63
21. 20 18.00 16.69 15.98 15. 21 14.37 14.02 13. 74 13.46
5 6.61 5. 79 5.41 5. 19 4.95 4.68 4.56 4.46 4.36
16.26 13.27 12.06 11. 39 10.67 9.89 9.55 9.29 9.02
 1431 Statistical Methods for Biological Assays

continued
f 1 Degrees of freedom for numerator
1 2 3 4 6 12 20 40 =
6 5.99 5. 14 4.76 4.53 4.28 4.00 3.87 3. 77 3.67
13.74 10. 92 9. 78 9. 15 8.47 7.72 7.39 7. 14 6.88
7 5.59 4. 74 4.35 4. 12 3.87 3.57 3.44 3.34 3.23
12.25 9.55 8.45 7.85 7. 19 6.47 6. 15 5.90 5.65
8 5.32 4.46 4.07 3.84 3.58 3.28 3. 15 3.05 2.93
11. 26 8.65 7.59 7.01 6.37 5.67 5.36 5.11 4.86
9 5. 12 4.26 3.86 3.63 3.37 3.07 2.93 2.82 2. 71
10.56 8.02 6.99 6.42 5.80 5.11 4.80 4.56 4.31
10 4.96 4. 10 3. 71 3.48 3.22 2.91 2.77 2.67 2.54
f2 10.04 7.56 6.55 5.99 5.39 4.71 4.41 4. 17 3.91
Degrees of 15 4.54 3.68 3.29 3.06 2.79 2.48 2.33 2.21 2.07
freedom
8.68 6.36 5.42 4.89 4.32 3. 67 3.36 3. 12 2.87
for
denominator 20 4.35 3.49 3.10 2.87 2.60 2.28 2.12 l. 99 l. 84
8. 10 5.85 4.94 4.43 3.87 3.23 2.94 2.69 2.42
30 4. 17 3.32 2.92 2.69 2.42 2.09 l. 93 l. 79 l. 62
7.56 5.39 4.51 4.02 3.47 2.84 2.55 2.29 2.01
40 4.08 3.23 2.84 2. 61 2.34 2.00 l. 84 l. 69 l. 51
7. 31 5. 18 4.31 3.83 3.29 2.66 2.37 2. 11 l. 81
60 4.00 3. 15 2. 76 2.52 2.25 l. 92 l. 75 l. 59 l. 39
7.08 4.98 4. 13 3.65 3. 12 2.50 2.20 l. 93 l. 60
= 3.84 2.99 2.60 2.37 2.09 l. 75 l. 57 l. 40 l. 00
6.64 4.60 3. 78 3.32 2.80 2. 18 l. 87 l. 59 l. 00
The upper values:P=O. 05; The lower values:P=O. 01

If the calculated F value is larger than the tabulated value at the level of probability of P=O. 05 or P=O. 01, the value being
tested is said to be signiíicant at the level oí P=O. 05 or highly significant at the level of P=O. 01.
The assay result is said to be valid if the outcome of the test is as follows.
The regression term should be highly significant CP<O. 01).
The deviation from parallelism should not be significant CP>O. 05).
When 3 or more dose levels are used in (k. k) or (k. k. k) assay, the quadratic and difference in quadratics should not be
significant (P>O. 05).
The significant level for the term of preparations is not used for assessing the validity of an assay, but a highly significant F
ratio for preparations may indicate that the choice of assumed potency is nota good one. The estimated potency may serve as a
guide for the choice of assumed potency when the assay is repeated.
For twin c~over design
CD In twin cross-over design Animals are divided into four equal groups at random with a number of animals in each group and
every animal of each group is tested on two separated occasions, therefore 2 X 4 m value of responses are obtained. Enter the
2 X 4 m values into the form of Table 8.
Table 8 The value of y for twin cross-over test
1 st group 2 nd group 3rd group 4th group
Test Test Test Test Test Test Test Test
(1) (2)
¿; (1) (2)
¿; (1) (2) (1) (2)
¿;
(1)+(2) (1)+(2) (1)+(2)
ds 1 dT 2 ds 2 dT¡ dT¡ ds 2 dT 2 ds 1

Ys1 m YT 2<2> Ym +y(2) Ys2m YT¡(2) Ym +Y<2> YT¡ (1) Ys 2<2> Ym +y(2) YT 2m YS¡(2) Ym + Y(2) ¿;
y

51m 51(2) 51
52m 52(2) 52
I;y T1(2) T1m T1
T2<2> T2m T2

@Replacement of missing wlue In twin cross-over test, each animal has two values of measured response, if one value of an
animal is lost, the total two values of that animal will be considered to be missed. Replace the two missing value separately with
the arithmetic mean of the other responses to the same dose of the test or reject the one surplus value of other doses in each test
by random selection.
 1431 Statistical Methods for Biological Assays

Each replacement of a missing value causes a loss of 1 degree of freedom for error variance. The number of missing value should
not be more than 5 % of the total number of responses and the missing value in one <lose should not be more than l.
@Variance analysis For twin cross-over design, the sum of square of total variation of 2 X 4 m responses can be divided into
variation of between animals(inter-animal variation)and variation of intra-animal.
Sum of squares of
(2:;y)2
Total= 2:;y 2 - - - - (26)
2X4 m
Í<TotaD =2X4 m-1

Sum of square of ínter-animal variation is calculated by equation (27) using the value in the third column of each group in Table
8
(2:;y)2
:¿;[y (1) +y (2) ]2
In ter-animal (27)
2 2X4m
f <inter-animaD = 4 m -1
Substract ínter-animal variation from the total variation, the residue is the sum of square of intra-animal variation.
Calculate m • 2:;Cr and 2:; (C;• 2:;y) of each component by multipling the sum of total response for each test of the four groups
in Table 8(S1m ,Szm, Tim, T2m ,S1(2) ,Sz(2), Ti(2), T2(2) )by the orthogonal coefficient shown in Table 9. Sum of square of
. . . [2:;(C,•2:;y)]2
each component in Table 9 is obtained by m. L;C~

Table 9 Orthogonal coefficient for validity test of twin c~over design

Test(l) Test(2)
Source of ¿ce;.¿y)
51(1) 52m T1m T2m 51(2) 52(2) T1(2) T2<2) m• ¿c7
variation
Orthogonal coefficient C;

Preparation * -1 -1 1 1 -1 -1 1 1 8m T2m +T1m-52m-51m +T2(2) +T1(2)-S2(2)-S1(2)

Regression * -1 1 -1 1 -1 1 -1 1 8m T2m-T1m +52m-51m +T2(2)-T1(2) +s2<2)-S1(2)
Parallelism 1 -1 -1 1 1 -1 -1 1 8m T2m-T1m-52m +51m +T2(2)-Tl(2)-S2(2) +S1(2)
'T':~~ ~! +-~~+- ~
.l lHU:: Ui U::~L /\ -1 -1 -1 -1
.l 1 1 1 1 º-
u lit
7'
i 2(2)
-L.7'
1 .L 1(2)
-LC
1 v2(2)
-LC
1 v 1(2)
-7'
l 2(1)
-7'
.Ll(l)
-C
v2(1)
-C
vl(l)

Time X preparation 1 1 -1 -1 -1 -1 1 1 8m T2(2) +T1(2)-S2<2)-51<2)-T2m-T1m +S2m +S1m

Time X regression 1 -1 1 -1 -1 1 -1 1 8m T2(2) -T1(2) +S2<2) -51<2) -T2m + T1m -S2m +S1m
Time X parallelism * -1 1 1 -1 1 -1 -1 1 8m T2(2)-T1(2)-52(2) +51<2)-T2m +T1m +S2m-S1m

The degrees of freedom far each component is l. * mean the four components of intra-animal variation, the remaining three components are
ínter-animal variation.

Sum of square of error variance for intra-animal (error I) and ínter-animal (error II) are calculated by equation (28) and
equation (29).
Error ( l) =Total-Inter-animal-Preparation- Regression-Time-TimeX parallelism (28)
J error( l) = J Total - Íinter-animal - J preparation - J regression - J time - J timeXparallelism = 4(m -1)
Error ( II) = Inter-animal- Parallelism-TimeX Preparation-TimeX regression (29)
J error( U) = J inter-animal - J parallelism - J time X Preparation - J time X regression = 4 ( m -1)
@Validity test The result of validity test for twin cross-over design is shown in Table 10.
Table 10 Validity test for twin c~over design
Source of variation f Sum of square Mean square F p

Parallelism 1 Equation(22) Sum of square/ f Mean square/sli

Time X preparation 1 Equation(22) Sum of square/ f Mean square/sli
Time X regression 1 Equation(22) Sum of square/ f Mean square/sli
Error( II) 4(m-1) Equation(29) Sum of square/ f(s 2u)

Inter-animal 4 m-1 Equation(27) Sum of square/ f Mean square/ s 2

Preparation 1 Equation(22) Sum of square/ f Mean square/ s 2
Regression 1 Equation(22) Sum of square/ f Mean sq uare/ s 2
Time of test 1 Equation(22) Sum of square/ f Mean square/ s 2
Time X parallelism 1 Equation(22) Sum of square/ f Mean square/ s 2
Error( l) 4(m-1) Equation(28) Sum of square/ f(s 2) Mean square/ s 2

Total 2X4 m-1 Equation(26)

In table 10, the mean square for each component is obtained by dividing the sum of square of each component by its degrees of
freedom. F ratio for three ínter-animal components (In the upper part of Table 10) is the mean square for each component
expressed as a ratio of mean square of Error ( II) - (sli) and the F ratio for three intra-animal components (The lower part
of table 10) is the mean square for each component expressed as a ratio of mean square of error ( I ) s 2 • The significance of F
value are assessed in the same way as that for other parallel line assays.
 1431 Statistical Methods for Biological Assays

Assay results are said to be statistically valid if the outcome of these tests is as follows:
The terms of regression, parallelism and preparation are assessed in the same way as that of ( 2. 2) assay. The three
interaction components-time X preparation, time X regression, time X parallelism-indicate the variation of these components
(preparation, parallelism, regression) from the first test to the second test, if the F value of these components obtained are
significantly high, care should be taken in interpretting the results of the assay, and if possible, the assay should be repeated.
(3) Estimation of potency and fiducial limits
For (k. k), (k. k') and (k. k. k) assays, calculate the values of V, W, D, A, B andg by use of the equations listed in
Table 11, and calculate R, SM, FL by use of equations (30) - (33) .
. IV
R=D •anti1og W (30)

SM W 2(:-g) Jms 2[(l-g)AW 2+BV2] (31)

1 ~g ±t •SM J
FL of R=antilog[
1
(32)

FL of PT=AT •antilog[ ~g ±t •SM J

1
1
(33)
Calculate PT by use of equation (3) and its FL% by use of equation (8).
Table 11 Equations used for paralled line assay
Design (5) (T) V w D A B g

1 1 dSz t 2s 2m
2. 2 dsi dSz dTi dT 2 z-CTi +T2-5i -52) z-CT2-Ti +S2-5i) - 1 1
dT2 w2

1 1 ds 3
-
2 -
1 t 2s 2m
--
3. 3 dsi dSzds3 dTi dTzdT3 3CTi +Tz+T3-5i -52-53) 4CT3-Ti +53-5i)
dT 3 3 4 4W2

1 1 22
dsi dSzds 3 dTi dT 2 dT 3 4CTi +T2+T3+T4-5i - [ (T3-T2+53-5z)+ d54 1 1 t s m
4.4 20 - - - - -
ds4 dT 4 dT4 2 10 1owz
52-53-54) 3(T4-Ti +54-5i)]

1 1 1 ds3 1
3. 2 dsi dSz ds3 dTi dTz z-CT2+Ti)-3C5i +52+53) 5[CT2-TiH2(53-5i)] 0

dTz .[r
5 2 2t 2s 2m
- -
6 5 swz
1 1 1 dSz
2. 3 dsi dSz dTi dTz dT 3 3CTi +Tz+T3)-z-C5i +5z) 5[2(T3-Ti H2C52-5i )] -·.Ji
dT 3

1 1 1 ds4 1
dsi dSz ds3 3< Ti+ Tz + T3) - 4 (Si+ 14[2(T3 -Ti)+ (53 -5z) +
dTi dT 2dT 3
4. 3
ds4
52+53+54) 3(54 -5i) J
dT 3 •.rr
7 1 tz szm
- - --
1 12 7 7W 2
1
dTi dT 2 dT 3 4CTi +Tz+T3+T4)- 14[2(53-5i HCT3 -T2H ds3
3.4 dsi dSzds 3
dT4 1
-·.Ji
dT
3(T4-Ti)] 4
3C5i+52+53)

1 dSz
2. 2. 2 dsi dSz dTi dT2 z-CTi +T2-51 -52) 1 -
3CT2 -Ti +U2 -Ui + dTz
2 2t 2 szm
1 -
5z-5i) 3 3W2
1 dSz
dui duz z-CUi +Uz-5i-52) -
duz

1 ds 3
-
3. 3. 3 dsi dSzds3 dTi dT2dT3 3CT1 +T2+T3-51 -52-53)
61 ( T3 - Ti + U3 - U1 + dT 3
2 1 tz szm
53-5i) - - --
3 6 6Wz
1 ds3
du1du2du3 3<Ui +uz+u3-51 -5z-53) -
du3

For twin cross-over design, 2 m values of measured responses for each <lose of (S) and (T) are obtained. Calculate V, W, D,
and g by use of equations far (2. 2) assay in table 11 using the sum of 2 m responses far each <lose (the values of S1, Sz, Ti,
Tz in table 8). Calculate SM by equation (34).
 1431 Statistical Methods for Biological Assays

(34)
s 2 •t 2 •2m
g w2
Example 2 Parallel line ~y of HCG by oteros weight method in mice, random design (3. 3. 3)
5 is the standard preparation of HCG;
T is HCG bulk substance, AT=2500 Units/mg;
U is HCG for injection, Au=500 Units/ampoule.
The assay result is shown in Table 2-1.
Table 2-1 An ~y of HCG
r=l: O. 6, I=lg r=O. 222 (3. 3. 3) assay; K=9, m=l5

Dose ds 1 ds 2 ds3 dT¡ dTz dT3 dm duz dw

Units/mouse 0.135 0.225 0.375 0.135 0.225 0.375 0.144 0.240 0.400

9.31 33.70 15. 10 20.80 25. 70 35. 60 26.20 10.00 55.00

17.50 56.80 47.20 16.40 6.37 48.40 10.00 40.20 41. 70
21. 90 44.60 51. 80 5.66 38.30 41. 90 19.22 22. 30 15.40
14.60 32.30 47.30 9.50 46.80 44. 70 22.00 40.50 53. 60
8.20 16.70 49.90 9.27 43.40 29.80 20. 70 50.90 53. 70
11. 00 6. 17 47.20 7.56 27.80 38.80 23.20 23. 50 33.00
Uterine weight
24.40 41. 50 47. 10 15.40 26.00 37.40 18. 70 19.60 44.30
mg/10 g (body
16.80 36.20 45. 10 20.30 27. 20 33.70 12.60 27. 20 44. 70
weight)
29.90 9.83 46.40 11. 50 27.30 35.40 20.90 30.30 23.00
y
8.95 20.00 52.90 22.20 11. 90 47.90 19. 10 58.80 31. 60
17.80 22.00 32.50 20.60 33.40 14. 60 19.40 55.30 49.20
18.00 60.60 56.40 13.90 29.00 49.80 14.50 40. 70 55.30
13. 70 6.43 39.50 12.60 6.43 14.50 11. 40 35.40 23.80
8.82 26.00 8.08 7.25 27.80 42.00 16.20 15.20 21. 80
17.80 34.80 37. 10 15.80 17. 70 11. 50 20.80 28. 70 36.00

238.68 447.63 623.58 208. 74 395. 10 526.00 274.92 498.60 582. 10 ¿y

¿YCk)
51 52 53 T1 T2 T3 U1 U2 u3 3795.35

CI)5um of squares
K=9; m=15
Total=9. 31 +17. 50 +···+23. 80 2+21. 80 2+36. 00 2
2 2 (3795.35)2 29868.26
9Xl5
f=9X15-1=134
238. 68 2+477. 63 2+···+582. 10 2 (3795. 35) 2
Between doses= X 12336. 55
15 9 15
f=9-l=8
Error=29868.26-12336.55=17531.71
f=l34-8=126
@Variance analysis and validity test
(238.68+447.63+623.58)+(208.74+395. 10+526.00)+
Sum of squares (preparations)
3Xl5
(274. 92+498. 60+582. 10) 2 (3795. 35) 2-633 23
3Xl5 9X15 .
f =2
The results of variance analysis are shown in Table 2-2; the results of validity test are shown in Table 2-3.
Table 2-2 Variance analysis for HCG ~y (3. 3. 3)
L;y(k) Sum of squares

Source of S1 S2 53 T1 T2 T3 U1 U2 u3 m• ¿; [C·
variation 238. 68 447.63 623.58 208. 74 395. 10 526.00 274. 92 498. 60 582. 10 ¿;e; L;y(k)J { L;[C;•L;y (k) ]}2 22:;{2:;[C; •2:;y(k)]}2
m • 2:;C7 L;(m • L;CD
Orthogonal coefficient C;

Regression -1 o 1 -1 o 1 -1 o 1 6Xl5 1009.34 11319. 64

1 o -1 -1 o 1 4X15 -67. 54
119. 08
Parallelism 1 o -1 -1 o 1 4X15 -77. 72
1 o -1 -1 o 1 4X15 -10. 08
 1431 Statistical Methods far Biological Assays

continued
~y(k) Sum of squares

Source of S1 S2 53 T1 T2 T3 U1 U2 1/3 m• ~[C¡•

variation 238.68 447. 63 623. 58 208. 74 395. 10 526.00 274. 92 498. 60 582. 10 ~e; ~YCk)J {~[C;·~y(k)]}2 2~{~[C; • ~Yck)]} 2
m·~C7 ~(m·~CD
Orthogonal coefficient C;

Quadratic 1 -2 1 1 -2 1 1 -2 1 18X15 -228. 64 193.62

Difference -1 2 -1 1 -2 1 12X15 -22. 46
-1 -2 71. o
of 2 -1 1 1 12Xl5 -107.18
quadratics -1 2 -1 1 -2 1 12X15 -87. 72

Table 2-3 Validity test or HCG ~y( 3. 3. 3)

Source of variation Degree of freedom(f) Sum of squares Mean square F p
Preparations 2 633.23 316.6 2.28 >0.05
Regression 1 11319.64 11319.64 81. 35 <0.01
Parallelism 2 119. 08 59.54 <1 >0.05
Quadratic 1 193.62 193.62 l. 39 >0.05
Difference of
quadratics 2 71. 00 35.50 <1 >0.05
Doses 8 12336.55 1542.07 11. 08 <0.01
Error 126 17531. 71 139. 14(s 2)
Total 134 29868.26

Conclusion The regression was highly significant, the deviation from parallelism, the quadratic and difference of quadratics
were not significant. Therefore, the assay result was satisfactory.
® Potency and fiducial limits
r=l : O. 6 I=O. 222
s 2=139.14 f=l26 t=l.98
VT = ~ X (208. 74+395. 10+526. 00-238. 68-447. 63-623. 58) = -60. 017

W= ~ X (526. 00-208. 74+623. 58-238. 68+582.10-274. 92)=168. 223

139. 14X l. 98 2 X15
g 6 X (168. 223) 2 O. 048

RT= 0.375
O.
·1 (-60.017)
•ant1 og _ XO. 222=0. 833
375 168 223
PT=2500XO. 833=2082. 5 Units/mg

SMT
o. 222
168. 2232 X (1-0. 048) X 15X 139. 14X e-o.
Ít 2 2 1
048) X 3X168. 223 +6 X (-60. 017) 2 =O. 05129
J
FL of RT =antilog(i1:_º~.8g:8 ±1. 98XO. 05129) =O. 653-1. 043
FL of PT =2500X (O. 653-1. 043)
=1632. 5,.._,2607. 5 Units/mg
2607. 5-1632. 5 %- %
FL % of PT X . XlOO o-23. 4 o
2 2082 5
Vu = ~ X (274. 92+498. 60+582. 10-238. 68-
447. 63-623. 58)=15. 243
W=168. 223 g=O. 048
o.
R u= o. 375 ·1 ( 15. _243 XO. 222 ) =O. 982
•antl og
400 168 223
Pu=500XO. 982=491. O Units/ampoule
S
Mu
O. 222
168. 223 2 X (1-0. 048 )
X 1 1 Ít 2
15 X 39. 4 ~l -0. 048)3X168. 223
2+ 1
6X15. 243
2]
=O. 05051

0 ~~. ~;8 ) ±l. 98 X O. 05051 J=O. 779-1. 235

1 9
FL of Ru =antilog [
FL of Pu=500X (0. 779-1. 235)=389. 5-617. 5 Units/ampoule
617. 5-389. 5 - %
FL% of Pu X 1. O Xl00-23. 2 o
2 49
 1431 Statistical Methods for Biological Assays

Calculate s 2 from equation (21)

15(9. 31 2 +17. 50 2 +···+21. 80 2 +36. 00 2 ) - (238. 68 2 +447. 63 2 +···+582.10 2 )
s2 9X15(15-1) 139.14
Exarnple 3 Parallel line assay of neomycin, randomized block design (3. 3) assay
5 is the standard preparation of neomycin;
T is neomycin bulk substance, AT =670 Units/mg.
The assay result is shown in Table 3-1.
Table 3-1 An assay of neomycin
Units/ ds1 d52 ds3 dT1 dT 2 dT3
~Ym
ml 8.0 10.0 12.5 8.0 10.0 12.5
16.05 16.20 16.50 15.80 16.35 16.60 97.50
16.20 16.45 16.65 16.20 16.45 16. 70 98. 65
16.00 16.45 16.70 16.05 16.35 16. 70 98. 25
15.95 16.35 16.60 16.00 16.25 16.60 97. 75
y 15. 70 16.25 16.60 15.85 16.25 16. 60 97.25
15. 55 16.20 16.55 15. 70 16.20 16.60 96. 80
15. 65 16.20 16.40 15.80 16. 15 16.40 96. 60
15.90 16. 10 16.45 15.80 16. 10 16.50 96.85
15.60 16.00 16.30 15. 70 15.95 16.30 95.85
142. 60 146.20 148. 75 142.90 146.05 149.00
~Y<k) 875.50
51 52 53 T1 T2 T3

Q)5um of squares
K=6, m=9
875. 52
Total=16. 05 +16. 20 +···+16. 50 2 +16. 30 2 -
2 2
X =5. 4709
9 6
f=9X6-1=53
(142. 60) 2 +046. 20)2 +···+ (146. 05) 2 + (149. 00) 2 (875. 5) 2
Between doses 4. 1926
9 9X6
f=6-l=5
. h (97. 50) 2 +(98. 65) 2 +···+(96. 85) 2 +(95. 85) 2 (875. 5) 2
Be tween d is es= 9X6 l. 0018
6
f=9-l=8
Error=5. 4709-4. 1926-1. 0018=0. 2765
f=53-5-8=40
®Variance analysis and validity test
The results of variance analysis are shown in Table 3-2; the results of validity test are shown in Table 3-3.
Table 3-2 Variance analysis for neomycin in assay ( 3. 3)
~Y<k)
Sum of squares
Source of T2 ~[C;• ~Y<k) J
51 52 53 T1 T3
m·~Cr [~CC·~y(k))] 2
variation 142.60 146.20 148. 75 142.90 146.05 149.00
m·~Cr
Orthogonal coefficient C;
Preparations -1 -1 -1 +1 +1 +1 9X6 0.4000 0.002963
Regression -1 o +1 -1 o +1 9X4 12.25 4. 168
Parallelism +1 o -1 -1 o +1 9X4 0.05000 0.00006944
Quadratic +1 -2 +1 +1 -2 +1 9Xl2 l. 250 0.01447
Difference of -1 +2 -1 +1 -2 +1 9X12 0.8500 0.006690
quadratics

Table 3-3 Validity test for neomycin assay( 3. 3)

Source of variation Degrees of freedom(j) Sum of squares Mean square F p

Preparations 1 0.002963 0.002963 <1 >O. 05

Regression 1 4. 168 4. 168 602.9 >0.01
Parallelism 1 0.00006944 0.00006944 <1 >0.05
Quadratic 1 0.01447 0.01447 2. 1 >0.05
Difference of quadratics 1 0.006690 0.006690 <1 >0.05
Doses 5 4. 1926 0.8385 121. 3 <0.01
Dishes 8 l. 0028 o. 1252 18. 1 <0.01
Error 40 0.2765 0.006912(s 2 )
Total 53 5.4709
 1431 Statistical Methods for Biological Assays

Conclu5ion The regression was highly significant, the deviation from parallelism, the quadratic and difference of quadratics
were not significant. Therefore, the assay result was satisfactory. The difference between dishes was highly significantCP<
O. 01), the precision of the assay would be increased by segregating the variation between dishes from the total.
@Potency and fiducial limit5
r= 1 : O. 8 I =O. 0969 5 2 =O. 006 912 f=40
t=2. 02(P=O. 95)
V= ~ X 042. 90+146. 05+149. 00-142. 6-146. 2-148. 75)=0. 1333

W= ! 049. 0-142. 9+ 148. 75-142. 6) =3. 0625

2. 02 2 XO. 006 912X9
g 4X3. 0625 2 O. OO 7
12. 5 ·1 o. 1333
R=12. °ant1 og . XO. 0969=1. 01
5 3 0625
PT=l. OlOX670 Units/mg=676. 70 Units/mg

SM
o. 0969
3.06252X(l-0.007) X
r,: 2
9X0.006912X~l-0.007)3X3.0625 +-;¡-X0.1333
2 1 2] -0.006469
-
FL of R =antilog[(ll~~.o~g7 ) ±2. 02XO. 006469 J=O. 980-1. 041
FL of PT =670XO. 980-670Xl. 041=656. 60-697. 47 Units/mg
FL% of PT= 697~~76~6~576~ 60Xl00%=3. o%
6X9Cl6. 05 +16. 20 +· .. +16. 50 2+16. 30 2)-6(142. 62+···+149. 02)-9(97. 52+···+95. 85 2)+875. 52
2 2
52 0.006912
6X9(6-l)X(9-1)
f=(6-l) X (9-1)=40
Example 4 Parallel line assay of oxytocin, randomized block design ( 2. 2)
S is the standard preparation of oxytocin;
T is oxytocin injection, AT = 10 Units/ml.
The assay result is shown in Table 4-1.
Table 4-1 An assay of oxytocin injection
ds1 dSz dT1 dT 2
Doses
0.0068 0.009 0.0080 0.0106 ~Ym
Units
(O. 34 mD (O. 45 mi) (O. 40 mD co. 53 mD
39.5 68.0 41. o 71. o 219.5
37.0 62.5 36.0 53.0 188.5
35.0 63.0 37.0 62.0 197.0
y
34.5
31. 5 58.0 60.0 184.0
(15. O)
30.0 50.0 35.0 60.0 175.0

173.0 301. 5 183.5 306.0

~Y<k) 964.0
S1 S2 T1 T2

(J)Di5po5al of an aberrant value

In Table 4-1, the response value of 3rd column (dT1 ) 4th row (15) is rather small, and the aberrant value test is applied.
Arrange the responses to dT 1 in ascending order: 15. O, 35. O, 36. O, 37. O, 41. O.
m=5 Ya =15 Y2 =35 Ym =41
Ji =Y2-Ya =35-15=0. 77
Ym-Ya 41-15
In Table 3, when m=5, ]1 =O. 73, smaller than the calculated J1 (O. 77). Therefore, ya can be rejected and the missing
value replaced by a value calculated as follows:
C=149 R=l49. 5 G=929. 5
K=4 m=5
4Xl49+5Xl49. 5-929. 5
y ( 4-1) ( 5-1) 34. 5
@Sum of 5quare5
964. 0 2
Total =39. 52+37. 02+···+60. 02+60. 02- X =3600. 20
5 4
f=5X4-1=19
173. 02+301. 52+183. 52 +306. 02 964. 02
Between doses - X =3163.10
5 5 4
f=4-1=3
 1431 Statistical Methods for Biological Assays

219. S2+188. S2+···+184. 02+11s. 02 9

~~ ~
2

Between blocks = 28S. 82

4
f=5-1=4
Error=3600. 20-3163. 10-28S. 82=151. 28
The replacement of a missing value causes a loss of 1 degree of freedom, therefore,
f=l9-3-4-1=11, s 2 =1Sl. 28/11=13. 75
@Variance analysis and validity test
The results of variance analysis are shown in Table 4-2; the results of validity test are shown in Table 4-3.
Table 4-2 Variance analysis for an assay of oxytocin injection
¿::;Y<k)

Source of 51 S2 T1 T2 [2:::(C;• L:Y<k) )] 2

173.0 301. s 183.5 306.0 m•L:Cr I:[C;• L:Y<k>]
variation m•L:Cr
Orthogoral coefficient C¡

Preparations -1 -1 1 1 SX4 lS.O 11. 2S

Regression -1 1 -1 1 SX4 2Sl. o 311SO. os
Parallelism 1 -1 -1 1 SX4 -6.00 l. 80

Table 4-3 Validity test for an as&iy of oxytocin injection

Source of variation f Sum of squares Mean square F p

Preparations 1 11. 2S 11. 2S <1 >0.05

Regression 1 31S0.05 31SO.OS 229.06 <0.01
Parallelism 1 l. 80 l. 80 <1 >O.OS

__.,-n,
Doses 3 3163. 10 1054.37 76.67 /'\"1
.....__V, V.I.

Blocks 4 28S.82 71. 46 S.20 <O.OS

Error 11 lSl. 27 13. 75(s 2)

Total 19 3600.20

Conclusion The regression was highly significant, the deviation from parallelism was not significant. Therefore, the assay
result was satisfactory.
@Potency and fiducial limits
r=l: O. 7S I=O. 12S s 2 =13. 7S f=ll t=2. 20
1
V=2(183. S+306. 0-173. 0-301. 5)=7. S

W= ~ (306. 0-183. S+301. S-173. 0)=12S. S

13. 7SX2. 20 2 XS
g 12S. S2 o. o21
o. 009 ·1 7. s
R= O. 0106 ant1og 12 S. S XO. 12S=O. 864
PT=lOXO. 864=8. 64 Units/ml

~·/1 ~0. 021) X /SX13. 75X[(l-O. 021)12S. S2+7. S2] =O. 008 362
2
SM 125. s2

FL of R =antilog [
0 ~~. i;l) ±2. 2oxo. 008 362J=O. 826-0. 899
1 8

FL of PT=lOX (0. 826-0. 899)=8. 26-8. 99 Units/ml

8.99-8.26 %- %
FL % of p T 2 X 8. 64 X 100 o-4. 2 o
Example 5 Parallel line assay of insulin---Blood sugar depres.sion method in mice, twin cros.s-over design
S is the standard preparation of insulin
T is the test preparation of insulin, AT=27 Units/mg.
ds 1 : 2S mu/ml, O. 25 ml/mouse
dr 1 : 25 mu/ml, O. 25 ml/mouse
ds 2 : SO mu/ml, O. 25 ml/mouse
d Tz : SO mu/ ml, O. 25 ml/ mouse
r=l: O.S I=0.301
y is value of blood glucose (mg%).
The assay result is shown in Table 5-1
 1431 Statistical Methods for Biological Assays

Table 5-1 The data of an ~y of insulin

Group 1 Group 2 Group 3 Group 4

Test(l) Test(2) Test(2) Test(l) Test(2) Test(l) Test(2)

Test Test O) Test Test Test
ds1 dT 2 (1)+(2)
ds.¿ dT¡ m+m dT¡ ds.;,
(1)+(2)
dT2 ds1
(1)+(2)

Ys 1m YT 2<2) Ym+Y<2 Ys 2m YT¡(2) Ym +Y<2 YT¡ (1) Ys 2<2) Ym +Y<2 YT2 (1) Ys 1<2) Ym +Y<2)
103. 99 87.01 191. 00 83.21 110. 13 202.64 116. 54 85.82 202.36 105.37 128. 92 234. 29
113. 21 104. 61 217.82 61. 05 78. 53 137. 58 94. 19 77. 72 171. 91 73.40 126. 95 200. 35
y 106. 94 100.26 207.20 85. 56 139. 40 224. 96 92. 82 100.26 193.08 74. 38 106. 19 180. 57
94. 19 96.10 190.29 76. 54 126. 95 203.49 103. 99 79.89 183.88 72.42 100.26 172. 68
103. 99 74.56 178.55 76. 54 97.49 174.03 113. 21 87.01 200.22 66. 54 90. 77 157. 31
92.82 82.27 175.09 78. 70 130. 90 209.60 101. 05 100.26 201. 31 106. 94 109. 35 216.29
108. 50 87.01 195.51 72. 42 93.34 165. 76 106. 94 122.99 229. 93 98.31 103.22 201. 53
89.09 84. 64 173. 73 77. 52 121. 24 198. 73 92.82 82. 27 175.09 113. 21 132.88 246.09
131. 45 93.34 224. 79 76. 54 110. 93 187. 47 98.31 91. 95 190.26 61. 83 89.58 151. 41
111. 64 88.20 199.84 64. 58 94. 72 159. 30 127.53 106. 19 233. 72 95. 56 110. 93 206.49 Total
1055. 82 1099.05
S1 2154.87
S1m S1<2)
752. 66 934.36
S2 1687.02
S2 m Sz(2)
:L;y¡
1110. 90 1047.40
T1 2158.30
T1<2) T1 m
898.00 867. 96
T2 1765. 96
T2c2) T2m
:L;y 7766. 15

Q)Sum of square
(7766. 15) 2
Total =103. 99 2+113. 21 2+···+89. 58 2 +llo. 93 2 25 865. 8223
2X4Xl0
ÍTotal =2X4X 10-1=79
(7766. 15) 2
inter-animal= 191. 00 2 +217. 82 2+···+151. 41 2+206. 49 2 11 320. 6387
2X4Xl0
Íinter-animal =4X 10-1=39
® Calculatem•¿;c;,¿;cc 1•¿;y) and sum of squares of each component of Table 5-1 by use of equations in Table 9, and
equation(22) (18).
The sum of square of error ( I) and error ( Il) are obtained by equation(28) (29).
Error( l) =25865. 8223-11320. 6387-84. 8102-9249. 0855-1267. 7893-369. 4991 =3573. 9995
f=4X(l0-1)=36
Error( Il) =11320. 6387-71. 2720-215. 7917-137. 8388=110895. 7362
f=4X(l0-1)=36
The result of variance analysis and validity test are shown in Table 5-2 and 5-3.
Table 5-2 Variance analysis for insulin, twin cross-over design
:L;y(l) :L;y(2)
Sum of square
Source of S1m S2m Tw> T2m 51(2) 52(2) T1(2) T2c2> m. :z:;c; [:L; (C;• :L;y) ]2
:L;(C;• :L;y)
variation 1055.82 752. 66 1047.40 867. 96 1099.05 934.36 1110. 90 898.00
m. :z:;c;
(C;• :L;y)
Preparation * -1 -1 1 1 -1 -1 1 1 10X8 82.37 84.8102
Regression * -1 1 -1 1 -1 1 -1 1 10X8 -860. 19 9249.0855
Parallelism 1 -1 -1 1 1 -1 -1 1 10X8 75. 51 71. 2720
Times of test * -1 -1 -1 -1 1 1 1 1 lOX8 318.47 1267. 7893
Time X Preparation 1 1 -1 -1 -1 -1 1 1 10X8 -131. 39 215. 7917
Time X regression 1 -1 1 -1 -1 1 -1 1 lOX8 105.01 137.8388
Time X parallelism * -1 1 1 -1 1 -1 -1 1 lOX8 -171. 93 369.4991
*mean the four components of intra-animal variation, the remaining three components are inter-animal variation.
 1431 Statistical Methods far Biological Assays

Table 5-3 Validity test of insulin

Source of variation f Sum of square Mean square F p
Parellelism 1 71. 2720 71. 2720 <1 >0.05
Time X Preparation 1 215. 7917 215. 7917 <1 >0.05
Time X regression 1 137.8388 137.8388 <1 >0.05
Error( Il) 36 10895.7362 302. 6593(s~)
Inter-animal 39 11320. 6387 290.2728 2.92
Preparation 1 84.8102 84.8102 <1 >0.05
Regression 1 9249.0855 9249.0855 93. 16 <0.01
Time of test 1 1267. 7893 1267.7893 12. 77 <0.01
Time X Prallelism 1 369.4991 369.4991 3.72 >0.05
Error(l) 36 3573.9995 99. 2778(s 2)
Total 79 25865.8223

Conclusion The regression was highly significant, the deviation from parallelism was not significant, the assay result was
satisfactory. The variance between two tests was highly significant, thus the precision of the assay was increased by eliminating
the effects of difference between animals at the two stages of the test.
@Potency and fiducial limits
r=l: O. 5 I=O. 301 s2=99. 2778 f=36 t=2. 03
V= ! X 0765. 96+2158. 30-1687. 02-2154. 87)=41. 185

W= ! X (1765. 96-2158. 30+ 1687. 02-2154. 87) = -430. 095

R = 50 ant1·1og ( _ 41. 185

. XO. 301 ) =O. 936
50 430 095
PT=27XO. 936=25. 27 Units/mg
99. 2778 X 2. 03 2 X 2X10 O.
044
g (-430. 095) 2

c- 430 _095°) 2 ~ 0 _
3 1
sM . ) x /2x 10X99. 2778[0-o. 044) x C-430. 095) 2+41. 185 2] =o. 03204
0 044
FL of R =antilog[ 01~~.9i:4 ) ±2. 03XO. 03204] =O. 803-1. 084
FL of PT=27(0. 803-1. 084)=21. 68-29. 27 Units/mg
o ofp T-(29.27-21.68
FL % - X . XlOO )%-
o-15. O%
o
2 25 27
4. Combination of Potency &timates
When the same test preparation has been assayed n times, it is necessary to combine the n estimated potencies to obtain a mean
potency and its fiducial limits.
In the combination of n estimates, the fallowing points should be observed:
(1) The n estimates were independent and each obtained from a separate assay of the same method, under the same
experimental condition.
(2) The estimated log potency (M) should be corrected by assumed potency (AT) befare they are combined.
Calculated the mean of the combined log PT by the fallowing method.
Homogeneity of n estimates of log potency: The homogeneity of n log potencies is assessed by X2 test.
x2 = L;WM2 - ( :L;WM)2 (35)
L;W
f=n-l
Where Mis the logarithm of the potency estimates and W is the reciproca! of its SM.
1
W= SL (36)
If the calculated value of X2 is smaller than the tabulated value of XZt>o.os corresponding to n-1 degrees of freedom ( shown in
Table 12), the potencies are homogeneous, the weighted mean potency of n estimates and its FL are obtained by equation
(37) - (41), where Mis the weighted mean potency of lg PT.
M_:L;(WM)
(37)
L;W

SM=~ (38)

FL of M=M±t •SM (39)

PT =antilgM (40)
FL of Pr =antilg(M±t •SM) (41)
For equation (39) and (41), the appropriate value of t (in Table 1) is that corresponding to the sum of the numbers off far
the s 2 in the n individual assays.
 1431 Statistical Methods for Biological Assays

If the calculated value of X2 is greater than the tabulated value of XZ¡lo.os in Table 12, the potencies are hetergeneous, The
combined mean potency and the FL of n estimates are obtained by equation (42) (43) and equation (39) - (41).
M=¿M (42)
n
¿Mz_ (¿M)z
__ SM
5 M - ¡;;
J
f=n-1
n (n-1)
n
(43)

Table 12 The value of X2 (P=O. 05)

f xz f xz f xz
1 3.84 11 19. 7 21 32. 7
2 5.99 12 21. o 22 33.9
3 7.82 13 22.4 23 35.2
4 9.49 14 23. 7 24 36.4
5 11. 1 15 25.0 25 37.6
6 12. 6 16 26.3 26 38.9
7 14. 1 17 27.6 27 40. 1
8 15. 5 18 28.9 28 41. 3
9 16. 9 19 30. 1 29 42.6
10 18. 3 20 31. 4 30 43.8

Example 6 Combination of 5 estimates of Heparin

The assay results are shown in Table 6-1.
Table 6-1 The data of 5 ~y results of Heparin

w(s~)
PT WMz
(Units/mg)
M(lg PT) SM WM

189.28 2. 2771 0.0289 1197. 30 2726.37 6208.22

180. 13 2.2556 0.0144 4822.53 10877. 70 24535. 74
189. 72 2.2781 0.0105 9070.29 20663.03 47072.44
185.27 2.2678 0.00633 24957.01 56597.51 128351. 83
181. 25 2.2583 0.0278 1293.93 2922.08 6598.94
¿ 41341. 06 93786.69 212767. 17

93 7 8 6
X2 =212 767. 17 41 3 4 ;_~: =l. 86
f=5-1=4
The calculated X2 (l. 86)is smaller than the tabulated value(XZ4lo.os =9. 49).
M 93 786. 69
2 2686
41 341. 06 .
PT =antilog2. 2686= 185. 61 Units/mg

SA1 41=.J 3 ~1.

06 =O. 004 92
f=5X24=120,t=l. 96
FL of PT =antilog(2. 2686±1. 96XO. 004 92)=181. 53-189. 78 Units/mg
FL%0 = 189. 78-181. 53Xl00%= 2 2 %0
2X185. 61 ° .
Example 7 Combination of 6 estimates of lnsulin
The assay results are shown in Table 7-1.
Table 7-1 The data of 6 ~y results of insulin

PT (Units/mg) M(lg PT) Mz SM w(s~) WM WAfZ

25.91 l. 4135 l. 9980 0.09603 108.44 153.28 216.66

23. 15 l. 3646 l. 8621 0.006202 25997. 79 35476.59 48411. 35
27.48 l. 4390 2.0707 0.02609 1469. 10 2114. 04 3042. 10
28.39 l. 4532 2. 1118 0.03177 990. 75 1439. 76 2092.26
 1431 Statistical Methods for Biological Assays

continued

PT (Units/mg) M(lg PT) M2 SM w(s~) WM WM2

27.56 l. 4403 2.0745 0.03560 789.04 1136.46 1636.84

25. 79 l. 4115 l. 9923 0.03181 988.26 1394.93 1968.95
¿ 8.5221 12. 1094 30343.38 41715.06 57368. 16

x2 =57 368 16 41 715. 062 19.70

. 30 343. 38
f=6-l=5 x~s> 0 . 05 =1i.1
The calculated X2(19. 70)is greater than the tabulated valueCll. 1), the 6 estimates are hetergeneous.
By calculating the rejection of aberrant value by equation (10), there is no aberrant rejection value.
5221 2/6 o. 013 212 06
5 ~ 12. 1094-8.
6-1 6
=O. 001007-0.002 212=-0. 001 205
The value of S~ is negative number, calculate an approximate S~ by omitting the term following the minus sign S~ =O. 001
007. Calculate the variance SK.t, the sum of varianceCSK.t+S~), calibration weight W', ¿w'M of each experiment (in table
7-2).
Table 7-2 The data of as.say results of insulin
PT(Units/mg) M(lgPT) M2 SL SK.t+s~ W' ¿w'M
25.91 l. 4135 l. 9980 0.009222 0.01023 97.75 138. 17
23. 15 l. 3646 l. 8621 0.00003846 0.001045 956.94 1305.84
27.48 l. 4390 2.0707 0.0007236 0.001731 577.70 831. 31
28.39 l. 4532 2. 1118 0.001009 0.002016 496.03 720.83
27.56 l. 4403 2.0745 0.001267 0.002274 439.75 633.37
25. 79 l. 4115 l. 9923 0.001012 0.002019 495.29 699. 10
¿ 8.5221 12. 1094 0.01327206 3063.46 4328.62

M=4328.62/3063.46=1.4130

S"M=J
306 46
!.
=O. 018 07 f=5, t=2. 57
PT=antilogl. 4130=25. 88(Units/mg)
FL of PT =antilog(l. 4130±2. 57 XO. 01807) =23. 26---28. 80 Units/mg
28. 80-23. 26 %- %
FL% x .
2 25 88
X 100 o-10. 70 o

5. Glossary of Symbols
AT The assumed or labelled potency of test preparation(T)
C lncomplete sum of response in a column containing the missing value
C; Orthogonal coefficient used in validity test
ds 1 ,ds 2 ••• Dose levels of the standard preparation(S)
dr 1 ,dr2 ••• Dose levels of the test preparation(T)
F The mean square of each variable expressed as a ratio of s 2
FL Fiducial limits
FL % Percentage of fiducial limits
f Degrees of freedom
G lncomplete sum of response in an assay excluding the missing value
g lndex of regression significance
1 Logarithm of the ratio between adjacent doses(r)
K Total number of doses of S and T
k •k' Number of doses of Sor T
M Logarithm of the potency ratio(R)
m Number of responses to each dose
n Total number of responses to S and T
ns Number of responses to S
nT Number of responses to T
P Probability
PT, Pu Estimated potency of test preparation(T, U)
R Estimated potency ratio befare correction by assumed potency, R=antilog M
R lncomplete sum of response in a row containing the missing value
r Ratio of adjacent doses

s Standard preparation
S1, Sz ... Sum of the responses to each dose of standard preparation
SM Standard error of M
sz Error variance
T Test preparation
T1, Tz ... Sum of the responses of each dose of test preparation
u Another symbol used to represent test preparation
U1, Uz .•. Sum of the responses to each dose of U
X Logarithm of dose
Xs Logarithm of mínimum effective dose of S
XT Logarithm of mínimum effective dose of T
Xs Mean log mínimum effective dose of S
XT Mean log mínimum effective dose of T
y Individual response or transfarmed response
Observed response listed in order of magnitude
Sum of the responses to each dose of S or T
Sum of the responses to each block or row
2000 Special Methods f or Traditional Chinease Medicines
2001 Microscopical ldentification
Microscopical identification is a method with the application
of the microscope to identify the characters of tissues, cells
or cell contents in sections, powders, disintegrated tissues or
surface slides of decoction pieces of crude drugs and dosage
farms including powders of prepared slices of crude drugs.
Representative samples are chosen to be identified and slides
are prepared to meet the requirements of identification far
each drugs. The slides of preparations are made after appropriate
treatment according to their different dosage forms.
l. Microscopical slides of crude ~ or decoction pieces of
crude~
(1) Transverse or Longitudinal Sections Select the part of
the drug, intended to observe cut into slices of 10-20 µ.m in
thickness with a razor blade or using sliding microtome after
softened. Material may be embedded in hard paraffin befare
cutting if necessary. Select flat slices on the glass slide,
according to different objects, treat with glycerol-acetic acid
TS, chloral hydrate TS or other test solutions 1-2 drops, and
then cover the cover glass. If necessary, af ter treat with
chloral hydrate TS, heat until it is transparent, and then
treating with glycerol-ethanol TS or diluent glycerol, cover
the cover glass.
(2) Slides of Pauxler Spread a small amount of the powder,
screen through No. 4 or No. 5 sieve, picking a little on a
slide, examine after treated with glycerol-acetic acid TS,
chloral hydrate TS, or other suitable test solutions, and
covered the cover glass. Heat until it is transparent as the
method above if necessary.
( 3) Slide of Surface Af ter moistening and softening the
materials, cut two copies of about 4 mm 2 of the part intended
to observe, place on the glass slide ( one surface up, the other
surface down) or tear its epidermis, add suitable test solutions
or heat until it is transparent, cover the cover glass.
(4) Slide of Disintegrated Tissue The material should be
cut into small strips of about 5 mm in length, 2 mm in
diametre or pieces of about 1 mm thick befare being
disintegrated. Potassium hydroxide method can be used if
2001 Microscopical ldentification
parenchyma makes most part of the material and with a few
or scattered woody tissues; chromic-nitric acids method or
potassium chlorate method can be used if the material is
hard, with the presence of more woody tissues or the woody
tissues grouped into larger bundles.
CDPotassium Hydroxide Method Place the material in a test
tube, add an adequate quantity of 5 % potassium hydroxide
solution, heat until the material can be disintegrated by
pressing with a glass rod. Decant the alkali solution and
wash the material with water, transfer a little to a slide, tear
with dissecting needle, observe using dilute glycerine as
mountant and cover the cover glass.
@Chromic-Nitric Acids Method Place the material in a test
tube, add an adequate quantity of chromic-nitric acid TS, allow
to stand until the sample can be disintegrated by pressing with a
glass rod. Decant the acid solution, wash with water, prepare a
mount as method l.
@ Potassium Chlorate Method Place the material in a test
tube, add nitric acid solution 0-2) and a little potassium
chlorate, heat gently until effervescence relaxes. Add a small
quantity of potassium chlorate in time to maintain a light
effervescence until the sample can be disintegrated by
pressing with a glass rod. Decant the acid solution, wash
with water and prepare a mount as method l.
(5) Slides of Pollen and Spore Grind pollens, anthers
(or small flowers) or sori (soften the dry material in glacial
acetic acid) with a glass rod and filter by carbasus into a
centrifuga} tube, centrifuge. To the precipitate add 1-3 mi of
a freshly prepared mixture of acetic anhydride-sulfuric acid
(9 : 1), heat on a water bath far 2-3 minutes, centrifuge.
Wash the precipitate with water twice, place a little on the
glass slide, treat with chloral hydrate TS, cover the cover
glass, or add 1-2 drops of 50% glycerine and 1% phenol,
mount in fuchsin-glycerin gelatine [gel 1 g, water 6 mi,
soak till it is colliquative. Add glycerine 7 mi, heat by gently
agitating, until it is mixed well. Add appropriate basic
fuchsin solution ( basic fuchsin O. 1 g, add absolute alcohol
600 mi and camphor wood oil 80 mi, get it dissolved) after
being filtrated onto the culture dish, mix well, and get it
after the solidification].
 2001 Microscopical ldentification
(6) Slides of ground sections The method is proper for
hard drugs of animals or minerals. Place the tested material
of about 1-2 mm in thickness on the coarse grinding stone
(or mat-surface glass), add an adequate quantity of water,
hold or press the material with the index, middle finger, and
abrade it out and hone on the grinding stone until either
surface is smooth with about several micrometers in
thickness. Move the material onto the fine grinding stone,
add water, press it with cork before abrading out and hone
until it is transparent. Rinse with water, treat with ethanol
and then prepare a mount with glycerol-ethanol TS.
2. Microscopical slides of preparations including prepared
slices powder
With reference to different dosage forms of tested items, take
suitable powder directly the powder and capsule ( comminute if
the content is granular); take the tablet of 2-3 pills, water--
paste pill, paste pill, water honeyed pill, pastille (remove
the coating) of several pills or 1-2 troches, pulverize in the
mortar and get suitable powder; for honeyed pill, split it and
select suitable sample, or get a little precipitate after
removing the honey with water. Prepare a mount as methods
for slides of powder ( 1-5 pills).
3. ldentification of Cell Wall
(1) Lignified Cell Wall Add 1-2 drops of phloroglucinol
TS to the specimen on the slide, allow to stand for a
moment, add 1 drop of hydrochloric acid, a red colour or
purplish-red colour is produced depending on the extent of
lignifica tion.
(2) Suberized or Cuticutarized Cell Wall Add Sudan IlI
TS as above, aiiow to stand far a moment or warm gentiy,
an orange-red or red colour is produced.
(3) Cellulose Cell Wall Add zinc chloride-iodine TS, or at
first add iodine TS to moisten it, allow to stand for a
moment, then add sulfuric acid solution (33-50), a blue or
purple colour is produced.
( 4) Siliceous Cell Wall No change takes place on adding
sulfuric acid.
4. ldentification of Cell Contents
(1) starch
Q)Add iodine TS, a blue or purple colour is produced.
® Mount in glycerol-acetic acid TS, examine under a
polarizing microscope, starch granules show crossed
polarized light which disappears in gelatinized granules.
( 2) Aleurone
Q) Add iodine TS, a brown or yellowish-brown colour is
produced.
CZ)Add mercuric nitrate TS, a brick red colour is produced. If
the material is oily, it should be de-fatted with ether or
petroleum ether before being tested.
(3) Fatty Gil, Volatile Gil or Resin
Q)Add Sudan TS, an orange-red, red or purplish-red colour
is produced.
®In 90% ethanol, fatty and resin oil is insoluble, except
castor oil and croton oil, while volatile oil is soluble.
( 4) Inulin Add a 10 % solution of a-naphthol in ethanol
and then add sulfuric acid, the crystals of inulin turn
purplish-red and dissolve rapidly.
( 5) Mucilage Add ruthenium red TS, a red colour is
produced.
( 6) Calcium Oxalate Crystals
Q) Insoluble in dilute acetic acid, soluble in dilute hydro-
chloric acid without effervescenece.
® Dissolve gradually in sulfuric acid solution ( 1 - 2) , but
needle crystals of calcium sulfate appear on standing for a
moment.
( 7) Calcium Carbonate crystals ( stalactile) Soluble m
dilute hydrochloric acid with effervescence.
(8) Silicum Insoluble in sulfuric acid.
5. Microscopical measure
It refers to measure the size of cells and cell contents in the
microscope with ocular micrometer.
(1) Ocular micrometer A scaleplate in the eyepiece sleeve
of a round glass of 18-20 mm in diameter. In the centre,
there is precise parallel-line scale of equal appearing interval,
and 50 or 100 panels as usual. (as Fig. 1).
(2) Stage micrometer A round glass with fine scale pasted
in the centre of specially made glass slide. As usual, divide 1
mm (or 2 mm) precisely into 100 (or 200) panels, and each
panel is 10 µm in length to mark the ocular micrometer. (as
Fig. 2).
o 10 20 30 40 50 60 70 80 90 100
nj11 njn nin mln +
mln mjn 111/111 nl11111l11
Fig. 1 Ocular micrometer
~
8
micro meter
divide into 200
panels one
panel equals to
1/100 mm
Fig. 2 Stage micrometer
(3) Mark of ocular micrometer Determine the length
represented by each panel of ocular micrometer when
applying the identical microscope, object glass, ocular glass
of definite multiple and body tube of definite length.
Place stage micrometer onto the microscope carrier, and
move the scale of it to the centre of visual field in the high-
power objective ( or low-power objective). Lay up ocular
micrometer ( the obverse upward) into eyepiece sleeve, turn
the ocular micrometer and move the stage micrometer to
ensure that the graduation line "O" of ocular micrometer is in
coincidence with the certain graduation line of stage
micrometer. Find the second coincident graduation line to
calculate the corresponding length (µm) represented by each
panel of ocular micrometer in the object glass on the basis of
the number of the panels of the two micrometers between the
two line in coincidence. As shown in Fig. 3 , 77 panels ( 0-
77) of ocular micrometer equal to 30 panels (O. 7-1. O) of
stage micrometer, and the length of each panel of stage
micrometer is 1O µm. The length of each panel of ocular
micrometer: 10 µmX 30777=3. 8 µm.
Mark respectively during the application of microscopes of
different multiples.
( 4) Measurements Lay up the microscopic slide of
measured object on the microscope carrier, measure the
panel number of the object with ocular micrometer, and

0.8 0.9
10 20 30 40 50 60 70 80 90 1
1111¡1111J111111111J1111¡1111¡1111¡1111J1111¡1111ll11111111¡1111l1111¡11111111¡111111111¡111111111!
Fig. 3 Coincident graduation lines
multiply the number of µm of each panel as above. Measure
by the high power lens as usual except for the measure of
longer objects such as fibre, tube, nonglandular hair by the
low power lens. Note the maximum and mínimum (µm),
anda few values higher or lower than the defined values, are
permitted.
2101 Determination of Swelling Capacity
Swelling capacity is an index indicating the swelling property
of drugs. It is defined as the swelled volume (ml) of 1 g of
dried drug in water or in other specified solvent at a definite
time and temperature. It is used mainly for the natural drugs
containing mucilage, gelatin and semicellulose.
Determination Accurately weigh a quantity of the test drug,
pulverize it if necessary, place it into a assay tube ( 160 mm
in total length, 16 mm in inner diameter, 125 mm long in
the graduated portian, graduated in O. 2 mD, add 25 ml of
water or specified solvent at 20-25ºC, stopper tightly, shake
and allow to stand. Shake once every ten minutes in the first
1 hour, than allow to stand for 4 hours, read the volume of
the sample; read again after placed for 1 hour, until the
volume difference between two successive readings is no
more than O. 1 ml. Detect three samples for each drug, and
use the results of the last reading of each determination to
calculate the average value according to the following
formula:
S= V
w 2201
in which S is the swelling capacity; V is the volume (ml) of
a drug af ter swelling and W is the weight ( g) of the drug
calculated on the dried basis.
2102 Determination of
Plaster Softening Point
This method refers to determining the temperature of plaster
when softened by heating under the prescribed conditions. By
determining the temperature at which the plaster descends by
25 mm using the method below, it examines the degree of
the hardness and reflects the viscosity indirectly.
Apparatus Softening point radio meter. The specimen ring
(A) is a turbinate brass ring, with 6. 35 mm high,
17. 46 mm in upper pore diameter and 15. 88 mm in lower
pore diameter. The steel hall localizer (B), with 20. 60 mm
in interna! diameter, is located in the center of the steel hall.
The steel hall (C) is 9. 53 mm in diameter and 3. 50±0. 05 g in
weight. The upper of the bracket (D) is a deplanate brass
board with two horizontal toroidal rings. It supports two
2201 Determination of Extractives
specimen rings. The lower bracket is a deplanate lubricous
brass board. The distance, from the bottom of the turbinate
brass ring on the upper bracket to the supine surface of the
lower bracket is 25 mm. The superficies infería of the lower
1.0 bracket is 16 ± 3 mm over the bottom of the beaker. The
combined apparatus is shown as the following figure.
Detennination Place the sample being examined in a baking
oven and soften it by heating. Take out and scrape the
plaster. Fill each of the two specimen rings with l. 8 g of the
plaster. Place the specimen ring inverted on a tin foil which
is smeared by sorne glycerine and tiled on a glass board.
Heat the sample in a calorstat oven at 75 ± 2ºC until the
surface is flat. Place it at room temperature for 1 hour, and
then put it in the specimen rings of the bracket. Fix the steel
ball localizer. Place the steel hall localizer and the steel halls
in a beaker filled with water. Heat in a water bath at
constant temperature of 37 ± 1 ºC. After 20 minutes for
balance, place the steel halls in the localizer and heat the
bottom of the beaker with the temperature rising l. 0-1. 5ºC
per minute. Read the temperature on the thermometer
(calibrated) when the steel halls touch the surface of the
lower bracket. Take the mean value of the temperature as
the softening point. The temperature deviation between the
two halls should not be more than l. OºC.
D The braeket
C The steel hall
B The steel ball loealizer
A The turbinat brass ring
Fig. Plaster softening point radiometer
Determination of Extractives
l. Determination of Water-soluble Extractives Pulverize the
material and make sure the powder can pass through No. 2
sieve, and mix well.
Cold maceration method Accurately weigh 4 g of the
powdered material in a 250-300 ml stopper conical flask, add
accurately 100 ml of water, and stopper it. Macerate the
material with shaking for 6 hours and allow to stand for
18 hours . Filter rapidly through a dry filter, transfer
accurately 20 ml of the filtrate to an evaporating dish which
is previously dried to constant weight, and evaporate to
dryness on water bath at 105 ºC for 3 hours. Cool for 30
minutes in a desiccator. Weigh rapidly and accurately. Unless
otherwise specified, calculate the content ( %) of water-soluble
extractives with reference to the dried substance.
Hot extraction method Accurately weigh 2-4 g of the
powdered material in an 100-250 ml stopper conical flask,
add accurately 50-100 ml of water, stopper it, weigh and
allow to stand for 1 hour. Heat to reflux and keep boiling
slightly for 1 hour. Allow to cool, remove the flask, stopper
 2202 Determination of Tannins
it, weigh, add water to restore its original weight, shake
well and filter through a dry filter. Accurately measure 25
ml of the filtrate and transfer into an evaporating dish which
is previously dried to constant weight. Evaporate to dryness
on water bath at 105 ºC for 3 hours. Allow to cool for 30
minutes in a desiccator. Weigh rapidly and accurately. Unless
otherwise specified, calculate the content ( % ) of water-soluble
extractives with reference to the dried substance.
2. Detennination of Ethanol-soluble Extractives
Proceed as described under "Determination of Water-soluble
Extractives", unless otherwise specified, using ethanol with
the concentration specified in individual monograph as the
solvent instead of water.
3. Detennination of Volatile Ether Extractives
Accurately weigh 2-5 g of the powdered material ( through
No. 4 sieve), dried for 12 hours in a desiccator with
phosphorus pentoxide. Place it in to a Soxhlet type extractor,
and add a quantity of ether. Unless otherwise specified, heat
under reflux for 8 hours. Transfer ether solution into an
evaporating dish which is previously dried to constant
weight, and evaporate to dryness. Dry the residue for 18
hours in a desiccator with phosphorus pentoxide. Accurately
weigh, slowly heat it to 105ºC, and dry at 105ºC to constant
weight. The weight loss represents the weight of volatile
ether extractives.
2202 Determination of Tannins
This method is provided to determine the content of tanníns
in Traditional Chinese Medicines. All the extraction and
dilution should be operated at the condition of protecting
from light.
Preparation of reference solution Accurately weigh 50 mg of
gallic acid and place it into an 100 ml brown volumetric
flask. Dissolve and dilute it with water to the scale. Take
5 ml of the dilution solution into a 50 ml brown volumetric
flask and dilute it to the scale with water to prepare into a
reference solution with gallic acid content of O. 05 mg/ml.
Preparation of standard curve Take accurately O. 5 ml,
l. O ml, 2. O ml, 3. O ml, 4. O ml and 5. O ml of reference
solution respectively and place them into defferent 25 ml
brown volumetric flask. Add 1 ml of phosphomolybdium
tungstic acid respectively. Add 11. 5ml, 11 ml, 10 ml,
9 ml, 8 ml, 7 ml water respectively. Dilute them with 29%
sodium carbonate to the scale volume and shake up evenly to
prepare solutions with corresponding content. Measure the
absorbance at 760 nm according to the Ultraviolet-visible
Spectrophotometry ( 0401 ) with the corresponding solvent
solution as the blank. Establish a standard curve with the
absorbances as the ordinate and the corresponding
concentrations as the abscissa.
Preparation of test solution Accurately weigh the powdered
material ( according to the prescription under the individual
monograph) and transfer it into a 250 ml brown volumetric
flask. Dilute it with 150 ml water and macerate it overnight.
Ultrasonicate the mixture for 10 minutes and then cool. Add
water to the scale and mix well. Keep the volumetric flask
standing to generate precipitates. Filter the mixture and
discard the initial 50 ml of filtrate. Accurately take 20 ml of
the subsequent filtrate and add it into an 100 ml brown
volumetric flask, and dilute it to the scale with water.
Detennination
Total polyphenols Accurately take 2 ml of the test solution.
Add it into a 25 ml brown volumetric flask. Carry out the
procedure as described under "preparation of standard curve",
beginning at the words "add 1 ml of phosphomolybdium
tungstic acid ". Add 10 ml of water, determine the
absorbance and calculate the content of gallic acid ( mg) in
the test solution from the standard curve.
Polyphenols not adsorbed by hide powder Accurately take
25 ml of the test solution toan 100 ml stoppered conical flask
with O. 6 g casein, and plug with stopper. Heat in water bath
at 30ºC for 1 hour and then cool off; filter the mixture and
discard the initial filtrate. Accurately take 2 ml of the
subsequent filtrate and add it into a 25 ml brown volumetric
flask. Carry out the procedure as described under
"preparation of standard curve", beginning at the words "add
1 ml of phosphomolybdium tungstic acid ". Add 10 ml of
water, determine the absorbance and calculate the content of
gallic acid (mg) in the test solution from the standard curve.
Use the following formula to calculate the content of tannins
in the test solution.
Total tannins = (Total polyphenols) - ( Polyphenols not
adsorbed by hide pov.xler)
Note: Carry out the casein adsorption blank test. Subtract
the blank value when calculate the tannins content.
2203 Determination of Cineol
Carry out the gas chromatography method ( 0521 ) .
Chromatographic system and system suitability Take
polyethylene glycol CPEG-20M) and silicone COV-17) as the
stationary phase. The coating concentration is 10% and 2%
respectively. Pack a column with the corresponding coated
carrier in 7 : 3 (g/g) (PEG at the end of the injection).
Maintain the column temperature at 110±5ºC. The number
of theoretical plates of the column is not less than 2500,
calculated with reference to cineol. The resolution of the
peaks of cineol and its neighboring impurities should meet the
requirements.
Determination of the correction factor Accurately weigh a
quantity of cyclohexanone, dissolved with n-hexane to
prepare the internal standard solution ( 50 mg per ml).
Accurately weigh 100 mg of cineol and place it into a 10 ml
volumetric flask, add accurately 2 ml of the internal standard
solution, and dilute with n-hexane to volume. Shake well.
Inject 1 µl of the solution to the column for 3-5 times
continuously, record the peak areas and calcula te the
correction factor.
Determination Accurately weigh 100 mg of the sample and
place it into a 10 ml volumetric flask, add accurately 2 ml of
internal standard solution, dissolve and dilute with n-hexane
to volume. Shake well to prepare the test solution. Inject
1 µl of the test solution to the column and calculate the
content of cineol.
2204 Determination of Volatile Oil
Unless otherwise specified, pulverize the substance to be
examined through No. 2 or No. 3 sieves and then mix well.
Apparatus The apparatus consists of three parts as shown
in the figure (as Fig. ).
A is an 1000 ml round-bottom flask made of hard glass
(500 ml or 2000 ml flask is also permitted for use). B is a
volatile oil determination tube. C is a reflux condenser. All
parts are connected via ground glass joints. The measuring

e~
1~ ~J
ºY
B
A spread it out in a thin layer. Examine for foreign matter by
(cm)
Fig. Apparatus for Determination of Volatile Oil
tube in B is graduated into divisions of O. 1 ml. Clean the
glass apparatus thoroughly and tightly connect the ground
galss joints in order to avoid the spiii of voiatiie oii.
Determination
Method 1 This method is used for the determination of
volatile oils of which the relative density is less than l. O.
Weigh a quantity of the sample to be examined accurate to
O. 01 g (equivalent to O. 5-1. O ml of volatile oiD into flask A.
Add 300-500 ml of water anda few glass beads, followed by
shaking to mix it well. Connect flask A to volatile oil
determination tube B and then connect B to reflux condenser
C. Add water through the top of reflux condenser C until the
graduated tube of B is filled and overflows to flask A. Heat
the flask gently to boiling in an electric heating jacket or
other suitable means, and continue heating for about 5 hours
until the volume of oil does not increase. Stop heating, allow
to stand for a few minutes, open the stopcock at the lower
part of B, and run off the water layer slowly until the oily
layer is 5 mm above the zero mark. Allow to stand for at
least 1 hour, open the stopcock again, and run off the
remaining water layer carefully until the oily layer is just on
the zero mark. Read the volume of the oil in the graduated
portian of the tube and calculate the content of volatile oil in
the sample to be examined, expressed as percentage (%).
Method 2 This method is used for the determination of
volatile oils of which the relative density is more than l. O.
Add 300 ml of water and a few glass beads to flask A.
Connect flask A to volatile oil determination assembly R
Add water through the top of B until the graduated tube of B
is filled and the water overflows to flask A. Add 1 ml of
xylene with a pipette and then connect the reflux condenser C
to R Heat the flask to boiling and keep distilling at a rate
that will keep the middle part of the condenser cold. Stop
2302 Determination of Ash
heating after 30 minutes, and allow to stand for at least 15
minutes. Read the volume of xylene in the graduated portian
of the tube. Carry out the procedure described under Method
1, beginning at the words "Weigh a quantity of the sample to
be examined accurate to O. 01 g" . Substract the volume of
xylene previously observed from the volume of the oily layer,
and the remainder is taken to be the volume of volatile oil.
Calculate the content of volatile oil in the sample to be
examined, expressed as percentage ( %).
Note The volatile oil determination tube B must be set
vertically, while the connecting point between the side tube
and the graduated measuring tube is ata horizontal level.
2301 Determination of Foreign Matter
Foreign matter is material consisting of any or all of the
following:
l. Matter coming from the source plant but not defined as
the drug.
2. Matter not coming from the source plant.
3. lnorganic impurities such as stones, sand, lumps of soil,
etc.
Determination
(1) Weigh a quantity of the substance to be examined and
inspection with the unaided eye or by use of a lens ( 5 X -
10 X). To separate the foreign matter, a suitable sieve can be
used if necessary.
( 2) Weigh separately each kind of foreign matter and
calculate its percentage content.
Note
( 1) When the foreign matters dosel y resemble the official
drug and difficult to distinguish them, it may be necessary to
use either microscopical, chemical or physical method to
identify the foreign matters.
(2) Cut the big crude drugs and examine for the occurrence
of insects, moulds and deterioration.
(3) Unless otherwise specified, the quantity of substance to
be examined for the determination of foreign matter is
accordant with requirement of sampling of crude drugs.
2302 Determination of Ash
l. Total ash Pulverize the sample to be examined through a
No. 2 sieve, and mix well. Weigh 2-3 g ( 3-5 g for the
determination of acid-insoluble ash) of the powdered drug in
a tared crucible accurate to O. 01 g. lgnite the sample slowly
till it is completely carbonized. To avoid burning, raise the
temperature gradually to 500-600ºC. lncinerate the sample
until free from carbon and dry to constant weight. Calculate
the percentage of ash with reference to the sample.
If a carbon-free ash cannot be obtained in this way, cool the
crucible and moisten the charred mass with hot water or 2 ml
of 10% ammonium nitrate solution. Evaporate to dryness on
a water bath, and incinerate the residue as described above
until carbon-free ash is obtained.
2. Acid-insoluble ash To the crucible containing the residue
from the determination of total ash, add 10 ml of dilute
hydrochloric acid with great care, cover with a watch glass,
and heat on a water bath for 10 minutes. Wash the watch
glass with 5 ml of hot water and add the washings to the
crucible, filter with an ashless filter paper, then transfer the
residue to the filter paper with water, and wash till the
 2303 Limit Tests of Rancidity
filtrate yields no reactions of chlorides. Transfer the filter
paper together with the residue to the original crucible, dry
and ignite to constant weight. Calculate the percentage of
acid-insoluble ash with reference to the sample.
2303 Limit Tests of Rancidity
Rancidity means that the complex chemical reactions occur in
fatty oils or seed medicinal herbs that contain fatty oils
during the storage process, producing the decomposed
components such as free fatty acids, peroxides, low
molecular aldehydes and ketones, turning up distinctive smell
and influencing the sense properties as well as the interior
quality of the crude drugs and the processed pieces.
This method check the rancidity of crude drugs and the
processed pieces through the determination of acid value,
carbonyl value and peroxide value.
l. Extraction of f atty oils
Unless otherwise specified, pulverize 30-50 g of the test sample
( varying in accordance with the content of fatty oil) to coarse
powder. Extract the coarse powder with 100-150 ml of
n-hexane in a Soxhlet type extractor under reflux for
2 hours. After allowing to cool, filter the mixture through a
No. 3 sintered-glass funnel. Recover the solvent to dryness in
vacuum, and the fatty oil will be obtained.
2. Determination of rancidity
Determination of acid value Carry out the method for the
Tests of Fats and Fatty Oils (0713).
Determination of carbonyl value The carbonyl value is the
number that expresses in millimoles of carbonyl compounds
in 1000 g of fatty oils.
Unless otherwise specified, weigh accurately O. 025-0. 5 g of
the fatty oil in a 25 ml volumetric flask. Dissolve and dilute
to the scale volume with toluene and mix well. Take
accurately 5 ml in a 25 ml stoppered tube, add 3 ml of a
4. 3 % solution of trichloroacetic acid in toluene and 5 ml of a
O. 05 % solution of 2, 4-dinitrophenylhydrazine in toluene,
mix well and heat in a 60ºC water bath for 30 minutes. Stand
for cool, add slowly 10 ml of a 4% solution of potassium
hydroxide in ethanol along the tube wall, then add ethanol to
25 ml, stopper well, shake thoroughly for 1 minute and
stand for 10 minutes. Carry out the method for Ultraviolet-
visible Spectrophotometry ( 0401 >, Measure the absorbance
at 453 nm, with the corresponding solvent solution as the
blank. Calculate according to the following formula:
A X 125
Carbonyl value = X W X 1000
854
Where A=absorbance of the substance being examined;
W=weight of the fatty oil (g);
854 = average value of the molar absorption
coefficient of 2, 4-dinitrophenylhydrazine derivatives
of carbonyl compounds.
Determination of peroxide value The peroxide value is the
percentage of free iodine produced by the reaction of peroxide
compounds in fatty oil with potassium iodide.
Unless otherwise specified, weigh accurately 2-3 g of the
fatty oíl in a 250 ml dried iodine flask. Add 30 ml of a
mixture of chloroform and glacial acetic acid ( 1 : 1 ) to
dissolve the sample completely. Add accurately 1 ml of a
freshly prepared saturated solution of potassium iodide,
stopper well, shake gently for 30 seconds, stand in a dark
place for 3 minutes, and then add 100 ml of water. Titrate
with sodium thiosulfate VS (O. 01 mol/L) until the colour
changes to yellow, add 1 ml of starch IS, and continue
titrating until the blue colour disappear. Perform a blank
determination. Calculate the peroxide value according to the
following formula:
P eroxi'de va lue = CA - B) W
X O. 001269 X lOO
Where A= volume of sodium thiosulfate VS ( mD ;
B = volume of sodium thiosulfate VS for blank
determination (ml);
W=weight of the fatty oil (g);
O. 001269=weight (g) of iodine equivalent to 1 ml
of sodium thiosulfate VS (0. 01 mol/L).
2321 Determination of Lead ( Pb) ,
Cadmium ( Cd) , Arsenic (As) ,
Mercury ( Hg) and Copper (Cu)
l. Atomic absorption spectrophotometry
This method is provided to determine the lead, cadmium,
arsenic, mercury and copper in tradi tional chinese medicinal
substances using atomic absorption spectrophotometry. The
instrument used should meet the relevant requirements in
( 0406 ) . The following procedures are performed unless
otherwise specified.
( 1 ) Determination of lead ( graphite fumace method)
Determination conditions Reference condition: wavelength
283. 3 nm; drying temperature: l00-120ºC, maintain
20 seconds; ashing temperature: 400-750ºC, maintain 20-
25 seconds; atomization temperature: l 700-2100ºC, maintain
4-5 seconds.
Preparation of lead standard stock solution Measure
accurately a quantity of lead single-element standard
solution, diluted with 2% nitric acid to prepare the lead stock
solution (1 µg Pb per mD. Store at 0-5ºC.
Preparation of calibration curve Measure accurately a
quantity of lead standard stock solutions, diluted with 2%
nitric acid to the concentration of O ng, 5 ng, 20 ng, 40 ng,
60 ng, 80 ng per ml respectively. Measure accurately 1 ml
the above solution, add O. 5 ml of a mixed solution of 1 %
ammonium dihydrogen phosphate and O. 2 % magnesium
nitrate respectively, mix well, pipette accurately 20 µl into
the atomic generator of graphite furnace and determine their
absorbances. Establish a standard curve with the absorbances
as the ordinate and the corresponding concentrations as the
abscissa.
Pre paration of test solution
Method A W eigh accurately O. 5 g of the coarse powder of
the substance being examined, transfer into a teflon digestion
vessel, add 3-5 ml of nitric acid, mix well, soak ovemight,
cover the inner lid, screw clown the outer coating, and put
the vessel into a suitable microwave slaking oven to slake
( operate according to the instrumental specific slaking
procedure). Then put the digestion vessel on the electric hot
plate, heat slowly until brown fumes are no longer evolved,
and slowly concentrate to 2-3 ml. Allow to cool. Transfer
the solution to a 25 ml flask and dilute to volume with water.
Shake well. Prepare synchronously the reagent blank
solution according to the above method.
Method B Weigh accurately 1 g of the coarse powder of the
substance being examined, transfer into a Kjeldahl flask, add
5-10 ml of the mixed solution of nitric acid and perchloric acid
(4 : 1), add a small hopper on the flask-top, and macerate
ovemight. Heat on the electric hot plate, and keep boiling
slightly. If a brownish-black colour is obtained, add again a
quantity of the above mixture and heat until the solution becomes
 2321 Determination of Lead (Pb), Cadmium (Cd), Arsenic (As), Mercury (Hg) and Copper (Cu)
clear and transparent. Then raise temperature to emit thick
smoke. Heat until white smoke disperses. The slaked solution
becomes transparent, colourless or a little yellow. Allow to
cool. Transfer it into a 50 ml volumetric flask. Wash the
container with 2% nitric acid. Add the washing solution into
the same volumetric flask, dilute with the same solvent to
volume, and shake well. Prepare synchronously the reagent
blank solution according to the above procedure.
Method C Weigh accurately O. 5 g of the coarse powder of
the substance being examined, transfer into a porcelain
crucible, and char at low temperature on the electric hot
plate to smokeless. Transfer into the high temperature oven,
and ignite far 5-6 hours at 500ºC (if incomplete incineration
observed, add proper nitric acid and heat at low temperature
on the electric hot plate. Repeat the procedure till free from
carbon). Remove and allow to cool. Dissolve the ash with
5 ml of 10 % nitric acid. Transfer the solution into a 25 ml
volumetric flask. Wash the container with water. Add the
washing solution into the same volumetric flask, dilute to the
volume, and shake well. Prepare synchronously the reagent
blank solution according to the above method.
Determination Measure accurately 1 ml of the test solution
and its corresponding reagent blank solution respectively,
add O. 5 ml of a mixed solution of 1 % ammonium dihydrogen
phosphate and O. 2% magnesium nitrate, shake well, pipette
accurately 10-20 µl to determine their absorbances according
to the above method of "Preparation of calibration curve",
and calcula te the content of lead ( Pb) in the test solution
from the calibration curve.
( 2) Detennination of cadmium (Cd) ( graphite fumace method)
Determination conditions Reference condition: wavelength
228. 8 nm; drying temperature: 100-120ºC, maintain
20 seconds; ashing temperature: 300-500ºC, maintain 20-
¿5 seconds; atomization temperature: 1500-19üüºC,
maintain 4-5 seconds.
Preparation of cadmium standard stock solution Measure
accurately a quantity of cadmium single-element standard
solution, diluted with 2% nitric acid to prepare the cadmium
stock solution ( 1 µg Cd per mD. Store at 0-5 ºC.
Preparation of calibration curve Measure accurately a
quantity of cadmium standard stock solution, diluted with
2% nitric acid to the concentration of O ng, O. 8 ng, 2. O ng,
4. O ng, 6. O ng, 8. O ng per ml, respectively. Pipette
accurately 10 µl the above solutions into the graphite
furnace, determine their absorbances, and then establish a
standard curve with the absorbances as the ordinate and the
corresponding concentrations as the abscissa.
Preparation of test solution Refer to "Preparation of test
solution" of lead.
Determination Pipette accurately 10-20 µl of the test solution
and its corresponding reagent blank solution respectively,
determine their absorbance according to the above method of
"Preparation of calibration curve" ( if interference occurs,
pipette 1 ml of the standard solution, blank solution and test
solution respectively, add O. 5 ml of a mixed solution of 1 %
ammonium dihydrogen phosphate and O. 2 % magnesium
nitrate, shake well, determine their absorbances according to
the method above), and calculate the content of cadmium
(Cd) in the test solution from the calibration curve.
( 3) Detennination of arsenic (As) ( hydride method)
Determination conditions Apparatus: suitable hydride generator
device; reducing agent: a mixed solution of 1 % sodium
borohydride and O. 3 % sodium hydroxide ( prepared prior to
use) ; carrier liquid: 1 % hydrochloric acid; carner gas:
nitrogen; wavelength: 193. 7 nm.
Preparation of arsenic standard stock solution Measure
accurately a quantity of arsenic single-element standard
solution, diluted with 2% nitric acid to prepare the arsenic
stock solution (1 µg As per mD. Store at 0-5ºC.
Preparation of calibration curve Measure accurately proper
quantities of arsenic standard stock solution, diluted with 2%
nitric acid to the concentration of O ng, 5 ng, 10 ng, 20 ng,
30 ng, 40 ng per ml respectively. Accurately pipette 10 ml of
each solution into 25 ml volumetric flask respectively, add
1 ml of 25 % potassium iodide solution ( prepared prior to
use) , and shake well. Add 1 ml of 10 % ascorbic acid
solution (prepared prior to use), and shake well. Dilute with
hydrochloric acid ( 20 - 100) to the scale, shake well,
stopper and immerse the flask in a water bath at 80ºC far
3 minutes. Allow to cool. Transfer proper quantities of each
solution respectively into the hydride generator device,
determine the absorbance, and establish a standard curve
with the peak areas as the ordinate and the corresponding
concentrations as the abscissa.
Preparation of test solution Refer to A or B method of
"Preparation of the test solution" of lead.
Determination Pipette accurately 10 ml of the test solution
and its corresponding reagent blank solution respectively,
determined as described under "Preparation of calibration
curve" beginning at the words "add 1 ml of 25 % potassium
iodide solution" . Calculate the content of arsenic (As) in
the test solution from the calibration curve.
( 4) Detennination of Mercury ( Hg) ( cold absorption method)
Determination conditions Apparatus: suitable hydride
generator device; reducing agent: a mixed solution of O. 5%
sodium horohvdride and O. 1 % sodium hydroxide ( prepared
prior to use); carrier liquid: 1 % hydrochloric acid; carrier
gas: nitrogen; wavelength: 253. 6 nm.
Preparation of mercury standard stock solution Measure
accurately a proper quantity of mercury single-element
standard solution, diluted with 2% nitric acid to prepare the
mercury stock solution (1 µg Hg per mD. Store at 0-5ºC.
Preparation of calibration curve Measure accurately O ml,
O. 1 ml, O. 3 ml, O. 5 ml, O. 7 ml and O. 9 ml of mercury
standard stock solution, transfer into a 50 ml volumetric
flask respectively, add 10 ml of 20% sulfuric acid and O. 5 ml
of 5% potassium permanganate solution, and shake well.
Add dropwise 5 % hydroxylamine hydrochloride solution
until the violet red colour just disappears, dilute with water
to volume, and shake well. Inject a quantity of each solution
to the hydride generator device, determine the absorbance,
and then establish a standard curve with the peak areas (or
absorbance ) as the ordinate and the corresponding
concentrations as the abscissa.
Preparation of test solution
Method A Accurately weigh O. 5 g of the coarse powder of
the substance being examined, transfer into a teflon digestion
vessel, add 3-5 ml of nitric acid, mix well, and macera te
overnight. Cover the inner lid, screw firmly the outer
coating, and put the vessel into the proper microwave slaking
oven to slake (opera te according to the instrumental specific
slaking procedure). Then put the digestion vessel on the
electric hot plate, heat slowly until brown fumes are no
longer evolved, and concentrate slowly to 2-3 ml. Allow to
cool. Add 2 ml of 20 % sulf uric acid and O. 5 ml of 5 %
potassium permanganate solution, and shake well. Drop 5 %
 2322 Determination of Mercury and Arsenic Speciation and Their Valence States
hydroxylamine hydrochloride solution until the violet red
colour just disappears. Transfer it into a 10 ml volumetric
flask, dilute with water to volume, and shake well.
Centrifugate if necessary. The supernatant is used as the test
solution. Prepare synchronously the reagent blank solution
following the above procedure.
Method B Accurately weigh 1 g of the coarse powder of the
substance being examined, transfer into a Kjeldahl flask, add
5-10 ml of a mixed solution of nitric acid and perchloric acid
(4 : 1), and mix well. Add a small hopper on the flask-top,
and immerse overnight. Heat to complete slaking on the
electric hot plate at 120-140ºC for 4-8 hours. Allow to cool.
Add 5 ml of 20 % sulfuric acid and O. 5 ml of 5 % potassium
permanganate solution. Shake well. Drop 5 % hydroxylamine
hydrochloride solution until the violet red colour just
disappears. Transfer it into a 25 ml volumetric flask, dilute
with water to volume, and shake well. Centrifugate if
necessary. The supernatant is used as the test solution.
Prepare synchronously the reagent blank solute based on the
same procedure.
Determination Pipette accurately a quantity of the test
solution and its corresponding reagent blank solution
respectively, determined as described under "Preparation of
calibration curve" . Calcula te the content of mercury ( Hg)
in the test solution from the calibration curve.
( 5) Determination of Copper (Cu) ( flame method)
Determination conditions wavelength: 324. 7 nm; flame:
air-acetylene flame. If necessary, the result is calibrated with
the absorbance of the background.
Preparation of copper standard stock solution Measure
accurately a proper quantity of copper single--element
standard solution, diluted with 2% nitric acid to prepare the
copper stock solution (1 µg Cu per mD. Store at 0-5ºC.
Preparation of calibration curve Measure accurately a
quantity of copper standard stock solution, diluted with 2 %
nitric acid solution to the concentration of O µg, O. 05 µg,
O. 2 µg, O. 4 µg, O. 6 µg and O. 8 µg per ml respectively.
Inject each standard solution into the flame, determine the
absorbances respectively, and establish a standard curve with
the absorbances as the ordinate and the corresponding
concentrations as the abscissa.
Preparation of the test solution Refer to "Preparation of
the test solution" of lead.
Determination Pipette accurately a quantity of the test
solution and its corresponding reagent blank solution
respectively, determined as described under "Preparation of
calibration curve" . Calcula te the content of copper (Cu) m
the test solution from the calibration curve.
2. lnductively coupled plasma-mass spectrometry
This method is provided to determine arsenic, cadmium,
lead, mercury and copper in traditional chinese medicinal
substances using inductively coupled plasma-mass
spectrometer. The instrument used should meet the relevant
requirements in ( 0412).
Preparation of reference stock solution Measure a quantity
of arsenic, cadmium, lead and mercury single--element
standard solution respectively, diluted with 10 % ni trie acid
to prepare corresponding stock solutions (1 µg Pb per ml,
O. 5 µg As per ml, 1 µg Cd per ml, 1 µg Hg per ml and
10 µg Cu per mD.
Preparation of reference solution Measure accurately a
quantity of reference stock solutions of lead, arsenic,
cadmium and copper, diluted with 10% nitric acid to prepare
a mixed reference solution ( the concentration of lead and
arsenic is O ng, 1 ng, 5 ng, 10 ng and 20 ng per ml; the
concentration of cadmium is O ng, O. 5 ng, 2. 5 ng, 5 ng and
10 ng per ml; the concentration of copper is O ng, 50ng,
100 ng, 200 ng, 500 ng per ml). Measure accurately a
quantity of mercury reference stock solution, diluted with
10% nitric acid to the concentration of O ng, O. 2 ng, O. 5 ng,
1 ng, 2 ng and 5 ng per ml. This solution should be prepared
prior to use.
Preparation of internal standard solution Measure
precisely a quantity of single standard solution of germanium
(Ge), indium Cln) and bismuth (Bi), diluted with water to
prepare the internal standard solution (1 µg Ge per ml, 1 µg
In per ml, and 1 µg Bi per mD.
Preparation of test solution Substances being examined are
dried for 2 hours at 60ºC, ground to coarse powder. Weigh
accurately O. 5 g and put it into a suitable high-pressure--
resistant and high-temperature-resistant digestion vessel.
Add 5-1 O ml of nitric acid ( if violent reaction occurs, lay
aside till the reaction stops ) . Close tightly and slake
according to the instrumental requirements and relative
procedures. After slaking completely, cool clown the
temperature of slaked solution to lower than 60ºC, take out
the digestion vessel, cool, and transfer the slaked solution
into a 50 ml volumetric flask. Wash the digestion vessel with
a small amount of water three times and add them into the
same flask. Add 200 µl of 1 µg/ ml aurum single--element
standard solution, dilute to volume, and shake well ( if
precipitates are generated, get the supernatant after
centrifugation).
Prepare the reagent blank solution according to the same
procedure except adding aurum single--element standard
solution.
Determination The isotopes to be detected are 63 Cu, 75 As,
114
Cd,202
Hg and 208 Pb. Select 72 Ge as the interna! standard
of 63 Cu and 75 As, 115 ln as the internal standard of 114 Cd,
209
Bi as the internal standard of 202 Hg and 208 Pb. Calibra te
the detected elements by choosing the suitable correction
equation according to the requirements of different
instruments.
lnsert the interna! standard sampling pipe of instrument in
the internal standard solution during the entire analyzing
process, while inserting the sampling pipe in turn into the
different concentrations of reference solutions and detect.
Establish a calibration curve with the detected values (mean
value of three readings) as the ordinate and the corresponding
concentrations as the abscissa. lnsert the sampling line into
the test solution, detect and get the average value of three
readings. Calculate the corresponding concentrations from
the calibration curve, subtract the concentrations of the
corresponding blank solutions, and then obtain the content of
the above elements respectively.
2322 Determination of Mercury and
Arsenic Speciation and Their
Valence States
This method is provided for analysis of mercury or arsenic
speciation and its valence state in test samples using high
performance liquid chromatography/ tandem inductively
coupled plasma-mass spectrometry ( HPLC/ICP-MS).
Preparation of test solution, if necessary, should be specified
in the monograph, because pretreatments of elemental
speciation analysis differ greatly in different samples.
 2322 Determination of Mercury and Arsenic Speciation and Their Valence States
l. Determination of mercury speciation and valence state
Carry out the method for HPLC/ICP-MS (0412).
Chromatographic/mass spectrum system and system
suitability Use octadecylsilane bonded silica gel as the
stationary phase; use a mixture of methanol, O. 01 mol/L
ammonium acetate aqueous solution containing O. 12% L-
cysteine (adjust the pH value to 7. 5 by ammonia water)
(8 : 92 V /V) as the mobile phase; the flow rate is lml/min.
Monitor the isotope 202 Hg with inductively coupled plasma-
mass spectrometry equipped with a coaxial nebulizer and a
reaction cell. Choose normal mode or collision cell model
according to the interference. The resolution of three
different mercury speciation should be greater than l. 5.
'] !j
2 requirements. The resolution of arsenocholine, arsenobetaine
2.0
!
4.0 6.0
min
Fig. Chromatogram of mercury speciation
l. mercury ( 11) chloride; 2. methyl mercury; 3. ethyl mercury
Preparation of standard stock solution Weigh accurately a
quantity of mercury ( II) chloride, methyl mercury and ethyl
mercury, diluted respectively with 8% methanol solution to
prepare corresponding standard stock solutions ( 100 ng Hg
per mD.
Preparation of calibration curve Measure accurately a
quantity of standard stock solutions, diluted respectively
with 8% methanol solution to the concentration of O. 5 ng,
1 ng, 5 ng, 1O ng, 20 ng per ml ( calculated as mercury).
Preparation oj test sotution Uniess otherwise specified,
weigh accurately a quantity of test sample, diluted with
artificial gastric JUICe or artificial intestinal JUICe.
Ultrasonicate in water bath at 37 ºC for proper time. Mix
well, take proper amount and allow to stand for 24 hours.
Pipette the middle layer solution, and filter by microfiltration
membrane ( 10 µm ). Measure accurately a quantity of
subsequent filtrate, diluted with O. 125 mol/L hydrochloric
acid to prepare the test solution. Prepare synchronously the
reagent blank solution according to the above method.
Determination Inject 20-100 µl of each calibration curve
solution and test solution into the column, respectively.
Measure the peak areas of different mercury speciations and
establish a standard curve with the peak areas as the ordinate
and the corresponding concentrations as the abscissa.
Calculate the amounts of different mercury speciations in test
solution from the calibration curve.
2. Determination of arsenic speciation and valence state
Carry out the method for HPLC/ICP-MS ( 0412).
Ouomatograplúc/mass spectrum system and system suitability
Use polystyren~divinylbenzene copolymers bonded trimethyl
ammonium anion exchange material or equivalent material as
the stationary phase ( 250 mm X 4. 1 mm; 10 µm); use
O. 025 mol/L ammonium dihydrogen phosphate aqueous
solution (adjust the pH value to 8. O by ammonia water) as
mobile phase A, water as mobile phase B; elute in gradient
ata flow rate of lml/min as the following:
Time mobile phase mobile phase
(min) A C% V/V) B C% V/V)
0-15 0-100 100-0
15-20 100-0 0-100
20-25 o 100
Monitor the isotope 75 As with inductively coupled plasma-
mass spectrometry equipped with a coaxial nebulizer and a
reaction cell. Choose collision cell model or calibrate by
choosing the suitable correction equation according to the
requirements of different instruments. The resolution of six
different arsenic speciation should meet the relevant
and arsenous acid should be no less than l. O.
A
7.0 14.0 21.0
min
Fig. Chromatogram of arsenic speciation and valence state
l. arsenocholine; 2. arsenobetaine;
3. arsenious acid; 4. dimethyl arsenic acid;
5. monomethyl arsenic acid; 6. arsenic acid
Preparation of standard stock solution Weigh accurately a
quantity of arsenious acid, arsenic acid, monomethyl arsenic
acid, dimethyl arsenic acid, arsenocholine and
arsenobetaine, diluted respectively with water to prepare
corresponding standard stock solutions (2. O µgas per mD.
Preparation of calibration curve Measure accurately a
quantity of standard stock solutions, diluted respectively
with O. 02 mol/L disodium ethylene diamine tetraacetic acid
to the concentration of 1 ng, 5 ng, 20 ng, 50 ng, 100 ng,
200 ng, 500 ng per ml (calculated as arsenic) respectively.
Preparation of test solution Unless otherwise specified,
weigh accurately a quantity of test sample, diluted with
artificial gastric juice or artificial intestinal juice. Ultrasonicate in
water bath at 37 ºC for proper time. Mix well, take proper
amount and allow to stand for 24 hours. Pipette the middle
layer solution and filter by microfiltration membrane ( 10
µm). Measure accurately a quantity of subsequent filtrate,
diluted with O. 02 mol/L disodium ethylene diamine
tetraacetic acid to prepare the test solution. Prepare
synchronously the reagent blank solution according to the
above method.
Determination Inject accurately 20-100 µl of each calibration
curve solution and test solution into the column, respectively.
Measure the peak areas of different arsenic speciations and
establish a standard curve with the peak areas as the ordinate
and the corresponding concentrations as the abscissa.
Calculate the amounts of different arsenic speciations in test
solution from the calibration curve.
Notes
( 1) All glassware in the experiment should be immersed in
the 20 % nitric acid (V /V) for 24 hours or processed with
other proper methods to avoid interference in determination.
( 2) The method above is a general approach to determine
arsenic or mercury speciation in test samples. Not all kinds
of arsenic or mercury speciation standard solutions are
necessary in each case. The standard solutions can be
 2331 Determination of Residue of Sulfur Dioxide
prepared according to the requirements.
( 3) End-uncapped silane in stationary phase can reduce column
efficiency significantly in mercury speciation analysis.
Chromatographic column with high silane end-capped ratio is
highly recommended and should be flushed at intervals with
high percentage of organic phase by valve switch technology,
if necessary.
(4) Limitation of arsenic or mercury speciation amounts in
test sample should meet the requirments in each specified
monograph.
2331 Detennination of Residue of Sulfur Dioxide
The methods refer to acid-base titration, gas chromatography
and ion chromatography for determination of sulfur dioxide
residue in medicines or decoction pieces fumigated with sulphur,
respectively as method l , method 11 and method fil. Proper
method may be selected according to specific conditions to
carry out the determination of residue of sulfur dioxide.
Method 1 (Acid-base Titration)
The method refers to that traditional chinese medicines are
treated via distillation. After the sulfite substances in the
sample are converted to sulfur dioxide via acidification, they
are carried together with nitrogen to an absorption bottle
with hydrogen peroxide and oxidized through by hydrogen
peroxide to sulfate ions. Determine and calculate the amount
of sulfur dioxide residue in the medicine or decoction pieces
via acid-base titration.
Apparatus Shown in Fig. 1 A is a 1000 ml double-neck
round-bottom flask; Bis a vertical reflux condensing tube; C
is a separating funnel ( with scale); D is the nitrogen
connecting inlet; E is the sulfur dioxide gas outlet. In
addition, an electric jacket, a nitrogen source anda gas flow
meter are provided.
~ Demostic 19
standard caliber
w
o 250ºC . Temperature programming: keep at initial
(')
3
......
VI
(')
3 cT 50 ml scale
i helium and the flow rate is 2. O ml/min. Headspace sampling
D
Demostic 29
standard caliber
..,__ Demostic 24
standard caliber
Fig l. Apparatus for Determination of
Sulfur Dioxide (Acid-base Titration)
Determination method Place about 10 g of medicine or fine
powder of decoction pieces ( sample amount may be reduced
appropriately if the amount of sulfur dioxide residue is
relatively high, for example more than 1000 mg/kg, but not
less than 5 g) , weighed accurately into a double-neck round-
bottom flask, add 300 to 400 ml of water. Open the reflux
condensing tube and feed in water, connect the upper opening
E of the condensing tube with a rubber gas guide tube to the
bottom of a 100 ml conical flask. Add 50 ml of 3 % hydrogen
peroxide solution into the conical flask as absorption liquid
( end of the rubber gas guide tube shall be below the
absorption liquid leveD. Prior to use, add three drops of
methyl red ethanol solution indicator (2. 5 mg/ml) into the
absorption liquid, and titrate with O. 01 mol/L sodium
hydroxide until the solution turns yellow ( i. e. final point;
the absorption solution in excess of the final point shall be
discarded). Open nitrogen and adjust its flow to about O. 2 L/min
with a flow meter, and open the piston of separating funnel e to
allow 10 ml of hydrochloric acid solution ( 6 mol/L) to flow
into the distilling flask. Heat the solution in the double-neck
flask immediately to boiling and keep at a slight boiling
state. Stop heating after the solution inside the flask is boiled
for l. 5 h; cool the solution naturally, ti trate the absorption
liquid with sodium hydroxide (0. 01 mol/L) VS until yellow
colour does not disappear within 20 s; and correct the
titration results with blank test. Calculate according to the
following formula:
Amount of sulfur dioxide residue in test sample (µg/g) =
CA-B) XCXO. 032X 1000000/W
Where A refers to the volume of titrant sodium hydroxide
consumed by the test sample, ml;
B refers to the volume of titrant sodium hydroxide consumed
by blank test, ml;
C refers to the mol concentration of titrant sodium
hydroxide, mol/L;
O. 032 ref ers to the mass of sulfur dimride equivalent to 1 ml
of titrant sodium hydroxide (1 mol/L), g;
W refers to the weight of test sample, g.
Method ll (Gas Chromatography)
The method refers to determination of the sulfur dioxide
amount of residue in medicines and decoction pieces with gas
chromatography ( 0521 ) .
Chromatographic system and system suitability Use GS-
GasPro bonded silica gel porous layer open tubular
chromatographic column (such as GS-GasPro, column length
30 m, inner diameter O. 32 mm) or equivalent column and
thermal conductivity detector. The detector temperature is
temperature 50 ºC for 2 min, then raised the temperature to
200ºC ata rate of 20ºC/min, and keep at 200ºC for 2 min;
The injection port temperature is 200ºC, the carrier gas is
utilizing gas-tight syringes (syringe temperature 105°C); the
balance temperature of headspace bottle is BOºC; and the
equilibrium time is all 10 min. The system suitability test
shall meet the requirements of gas chromatography.
Preparation of reference solutions Accurately weigh
500 mg of reference substance sodium sulfite and place it into
a 10 ml measuring flask; add in a mixed solution containing
O. 5 % mannitol and O. 1 % disodium ethylene diamine
tetraacetic acid ( disodium EDTA) to dissolve the reference
substance; dilute it to the volume and shake up evenly to
prepare a reference stock solution with sodium sulfite content
of 50. O mg/ ml. Accurately measure O. 1, O. 2, O. 4 and l. 2
ml of reference stock solution respectively and place them
into a 10 ml measuring flask; dilute them with a mixed
solution containing O. 5 % mannitol and O. 1 % disodium
ethylene diamine tetraacetic acid ( disodium EDTA ) to
reference solutions with sodium sulfite contents of O. 5 mg/ rnl,
1 mg/rnl, 2 mg/rnl, 5 mg/rnl and 10 mg/ml respectively.
Accurately weigh 1 g of sodium chloride and 1 g of solid
 2341
paraffin ( fusing point 52 to 56 ºC) and place them into a
20 ml headspace sample bottle respectively. Accurately add
2 ml of 2 mol/L hydrochloric acid solution in to the bottles;
place the bottles into 60 ºC water bath and remove when the
salid paraffin is dissolved completely; cool naturally to room
temperature to cure and seal solid paraffin on the acid liquid
layer (blow off acid mist condensed on bottle wall with air).
Accurately weigh 100 µl of the above reference solutions with
sodium sulfite contents of O. 5 mg/ml, 1 mg/ml, 2 mg/ml, 5
mg/ml and 10 mg/ml and place them above the paraffin layer
respectively; finally seal to obtain the reference solutions.
Preparation of test solution Accurately weigh 1 g of
sodium chloride and 1 g of solid paraffin ( fusing point 52 to
56ºC) and place them into a 20 ml headspace sample bottle
respectively. Accurately add 2 ml of 2 mol/L hydrochloric
acid solution into the bottles; place the bottles into 60 ºC
water bath and remove when the salid paraffin is dissolved
completely; cool naturally to re-cure the salid paraffin; take
and accurately weigh O. 2 g of fine powder of the sample and
place it above the paraffin layer; add 100 µl of a mixed
solution containing O. 5 % mannitol and O. 1 % disodium
ethylene diamine tetraacetic acid ( disodium EDTA) ; finally
seal to obtain the test solution.
Detemúnation method Accurately measure 1 ml of headspace
bottle gases of balanced reference solution and test solution,
inject them into a gas chromatograph respectively, and
record chromatogram. Quantify according to the externa!
standard work curve method, calculate the sulfite content in
sample and multiply the measured result with O. 5079 to
obtain the sulfur dioxide content.
Method ][ (Ion Chromatography)
According to the method, traditional Chinese medicines are
handled with steam distillation, so that sulfite series
substances in the sample which are converted to sulfur
dioxide after acidification are absorbed by hydrogen peroxide
and oxidized into sulfate ions; then such ions are measured
and the amount of sulfur dioxide residue in the medicine or
decoction pieces are calculated with ion chromatography
( 0513 >.
Instrument and devices See Fig. 2 for ion chromatography
steam distillation device. The distillation device needs to be
customized and is provided with an additional electric jacket.
D residues in crude durgs, decoction pieces and preparations by
Fig 2. Apparatus for Determination of
Sulfur Dioxide (Ion chromatography)
A. double-neck bottle; B. absorption bottle;
C. round-bottom bottle; D. glass tube
Chromatographic system and system suitability Use ion
chromatography. The chromatographic column is an anion
exchange column (such as ASll-HC, 250 mmX4 mm) with
alkanol quaternary ammonium as functional group and ethyl
vinyl benzene-divinyl benzene polymer resin as filler, or
Determination of Pesticide Residues
equivalent column; the guard column uses the anion
exchange column (such as AGll-HC, 50 mm X 4 mm) with
the same filler; the elluent is 20 mmol/L potassium
hydroxide solution (produced by automatic elluent generator); if
there is no automatic elluent generator, the elluent adopts a
mixed solution with final concentration of 3. 2 mmol/L
Na2 C03 and l. O mmol/L NaHC03 ; the flow rate is 1 ml/ min;
and the column temperature is 30ºC. Anion suppressor and
electrical conductivity detector. The system suitability test
shall conform to the requirements of ion chromatography.
Preparation of reference solution Take sulfate radical
standard solution, add water and prepare into solutions with
sulfate content of 1 µg/ml, 5 µg/ml, 20 µg/ml, 50 µg/ml,
100 µg/ml and 200 µg/ml respectively; feed 10 µl for each of
the solutions and draw the standard curves.
Preparation of test solution Accurately weigh 5 to 10 g
(not less than 5 g) of rough powder of the test sample; add
bottle A (double-neck flask) and 50 ml of water; shake out
to disperse the powder uniformly; connect the bottle with a
water vapor distillation bottle C. Add 20 ml of 3% hydrogen
peroxide solution to absorption bottle B (100 ml Nessler tube or
measuring flask) as absorption liquid; insert the lower end of
the absorption tube to below the absorption liquid level. Add
5 ml of hydrochloric acid to bottle A along the bottle wall,
and seal the bottle quickly; start distillation; keep bottle e in
boiling state and regulate the heating power of distillation to
maintain the outflow rate of distillate at 2 ml/min. Distill till
the total volume of the solution in bottle B is about 95 ml
(time: around 30 to 40 min) , wash the tail connecting tu be
with water and transfer it to the absorption bottle; dilute the
solution to the volume and shake well; allow to stand for 1
h, filter the solution with a microfiltration membrane to
obtain the product.
Determina.tion metbod Accurately measure 10 µl of corresponr1in..g
reference solution and test solution to determine and calculate
the content of sulfate in the sample. Calculate the content
of sulfur dioxide residue in the sample according to ( S02 /
so¡- =o. 6669).
2341 Determination of Pesticide Residues
This method is provided for the determination of pesticide
gas chromatography ( 0521 > and mass spectrometry ( 0431 >.
Unless otherwise specified, determine as follows.
Method l. Detennination of organochlorine pesticide residues
by chromatography
l. Detennination of 9 organochlorine pesticide residues
Chromatographic system and system suitability Column: a
fused silica capillary column ( 30 m X O. 32 mm) with a film
( O. 25 µm ) of 14 % cyanopropyl phenyl-86 % methyl
polysiloxane or 5% phenyl-95% methyl polysiloxane;
detector: 63 Ni-electron capture detector ( ECD) ; inj ection
port temperature: 230ºC; detector temperature: 300ºC;
injection mode: splitless; column temperature program:
maintain the initial temperature at lOOºC, increase the
temperature ata rate of lOºC per minute to 220ºC, and then
increase the temperature at a rate of 8ºC per minute to
250ºC, hold for 10 minutes. The number of theoretical
plates of the column is not less than 10 6 , calculated with
reference to the peak of a-BHC. The resolution between two
neighboring peaks is more than l. 5.
Preparation of reference stock solution Weigh accurately a
 2341 Determination of Pesticide Residues
quantity of reference substance of benzene hexachloride
(BHC) ( crBHC, ~-BHC, y-BHC, a-BHC ) ,
dichlorodiphenyl trichloroethane (DDT) (p, p'-DDE, p' p'-
DDD, o, p'-DDT, p, p'-DDT ) and
pentachloronitrobenzeen ( PCNB) pesticides, dissolve with
petroleum ether ( 60-90ºC) respectively to prepare solutions
containing 4-5 µg per ml.
Preparation of mixed reference stock solution Measure
accurately O. 5 ml of each of the above reference stock
solutions to a 10 ml volumetric flask, diluted with petroleum
ether (60-90ºC) to volume and mixed well.
Preparation of mixed reference solution Measure accurately
the above mixed reference stock solution, diluted with petroleum
ether ( 60-90ºC) to prepare a series of solutions containing O µg,
1 µg, 5 µg, 10 µg, 50 µg, 100 µg and 250 µg per liter.
Pre paration of test solution ( Crude drug or decoction
pieces ) Weigh accurately 2 g of the substance being
examined (pulverized through the No. 3 sieve), in a 100 ml
conical flask with stopper, macerate with 20 ml of water
overnight. Add accurately 40 ml of acetone, weigh and
ultrasonicate for 30 minutes, allow to cool, weigh, and
replenish the lost weight with acetone. Add about 6 g of
sodium chloride and measure accurately 30 ml of methylene
chloride, weigh and ultrasonicate for 15 minutes, weigh,
replenish the lost weight with methylene chloride and allow
to stand for layer separation. Transfer quickly the organic
layer to a 100 ml stopper conical flask, which contained a
quantity of anhydrous sodium sulfate, stand for 4 hours.
Measure accurateiy 35 mi oí the above solution, concentrate
in vacuum to almost dryness at 40ºC. Add a small quantity of
petroleum ether ( 60-90ºC) to the residue, and repeat the
above procedures to exhaust the solvent of acetone and
methylene chloride. Dissolve the residue with petroleum ether
( 60-90ºC) and transfer to a 10 ml graduated centrifuga! tube
with stopper, dilute with petroleum ether (60-90ºC) to 5 ml.
Add cautiously 1 ml of sulfuric acid, shake for 1 minute, and
centrifuge ( 3000 rpm) for 10 minutes. Measure accurately 2 ml
of the supematant liquid to a graduated concentration flask (as
Fig.), link the rotatory evaporator, concentrate in vacuum
below 40ºC (or concentrate with nitrogen) to a quantity and
dilute accurately to 1 ml.
1.0ml
0.5ml
Fig. Graduated concentration flask
Preparatúm of test solution (Preparations) Weigh accurately a
quantity of substance being examined ( equivalent to 2 g of
crude drug), triturated to fine power (cut into pieces of pills
or measure the liquid preparation directly) , carry out the
method described as the above preparation of test solution.
Determination Inject accurately 1 µl of each of the test
solution and the mixed reference solution with corresponding
concentration, respectively, into the gas chromatograph,
calculate the amount of 9 organochlorine pesticide residues by
extemal standard method.
2. Determination of 22 organochlorine pesticide residues
Chromatographic system and system suitability Column 1:
a fused silica capillary column (30 mXO. 25 mm) with a film
(O. 25 µm) of 50% phenyl and 50% dimethyl polysiloxane;
column 2: a fused silica capillary column (30 mX O. 25 mm)
with a film (O. 25 µm) of 100% dimethylsiloxane polymer;
detector: 63 Ni-electron capture detector ( ECD); injection
port temperature: 240ºC; detector temperature: 300ºC;
injection mode: splitless; flow rate: constant pressure
( initial flow rate is l. 3 ml/ min); column temperature
program: maintain the initial temperature at 70ºC for
1 minute, increase the temperature at a rate of 10ºC per
minute to 180ºC, hold for 5 minutes, then increase the
temperature at a rate of 5ºC per minute to 220ºC, and then
increase the temperature at a rate of lOOºC per minute to
280ºC , hold for 8 minutes. The number of theoretical plates
of the column is not less than 106 , calculated with reference
to the peak of crBHC . The resolution between two
neighboring peaks is more than l. 5.
Preparation of reference stock solution Weigh accurately a
quantity of reference substance, dissolve with isooctane
respectively to prepare solutions of concentration in Table l.
Preparation of mixed reference stock solution Measure
accurately 1 ml of each of the above reference stock solutions
to a 100 ml volumetric flask, diluted with isooctane to
volume. Mix well.
Preparation o/ mixed reference solution Measure
accurately the above mixed reference stock solution, diluted
with isooctane to prepare a series of solutions containing 10
µg, 20 µg, 50 µg, 100 µg, 200 µg and 500 µg per liter ([3-
BHC, Endrin, p, p'-DDD ando, p'-DDT contain 20 µg,
40 µg, 100 µg, 200 µg, 400 µg and 1000 µg per liter).
Preparation of test solution Weigh accurately l. 5 g of the
substance being examined ( pulverized through the No. 3
sieve), place in a 50 ml polystyrene centrifuge tube with
stopper, add 10 ml of water, mix, allow to stand for
2 hours. Add accurately 15 ml of acetonitrile, shake for
1 minute, add about 5 g of a mixture of magnesium sulfate
and sodium chloride ( 4 : 1), shaking for 1 minute,
centrifuge ( 4000 rpm) for 1 minute. Measure accurately
10 ml of the supernatant liquid, concentrate in vacuum to
almost dryness at 40ºC. Dissolve the residue with a mixture
of cyclohexane and ethyl aceta te ( 1 : 1) and transfer to a
10 ml volumetric flask, diluted with a mixture of
cyclohexane and ethyl aceta te ( 1 : 1) to volume and mixed
well. Transfer this solution to a centrifuga! tube containing
1 g of anhydrous sodium sulfate, shake, allow to stand for
1 hour, centrifuge (filter if necessary), clean up the sample
solution by GPC ( 400 mm in length, 25 mm in inside
diameter, BIO-Beads S-X3; mobile phase: cyclohexane-ethyl
acetate mixture (1 : 1); flow rate: 5. O ml/min; injection
volume: 5 mD. Collect the eluted portions of 18-30 minutes,
concentrate in vacuum to almost dryness at 40ºC. Add a
small quantity of hexane twice to place cyclohexane-ethyl
acetate mixture (1 : 1) , dissolve the residue by 1 ml of
hexane and transfer to a Florisil SPE column [ 1000 mg/
6 ml, prewash with 10 ml of a mixture of hexane and acetone
(95: 5) and 10 ml of hexane], rinse the evaporation flask
with 1 ml of hexane three times and transfer to the same SPE
column, elute with the mixture of 10 ml of hexane and
acetone (95 : 5), collect the eluent, blow to almost dryness
with nitrogen gas, dissolve and dilute accurately to 1 ml with
isooctane.
 2341 Determination of Pesticide Residues

Determination Inject accurately 1 µl of each of the test hexachlorobenzene ~ O. 1 mg; sum of heptachlor,
solution and the mixed reference solution into the gas heptachlor-exo-epoxide and heptachlor-endo-epoxide ~ O. 05
chromatograph respectively, calcula te the content of 22 mg; sum of aldrin and dieldrin ~ O. 05 mg; endrin ~O. 05
organochlorine pesticide residues by external standard mg; sum of cis-chlordane, trans-chlordane and oxy-
method. chlordane ~ O. 05 mg; sum of a-endosulfan, ~-endosulfan
Limits Unless otherwise indicated in the monograph, per and endosulfan sulfate~ 3 mg.
kilogram of crude drugs or decoction pieces: total BHCs Notes (1) Confirm the results by another column before
(sum of a-BHC, ~-BHC, y-BHC and a-BHC) ~O. 2 mg; quantitative, and confirm the result by GC/MS if necessary.
total DDTs (the sum of p, p'-DDE, p, p'-DDD, o, p'- (2) Recoveries range from 70% to 120%.
DDT and p, p'-DDT) ~O. 2 mg; quintozene~0.1 mg;
Table 1 Concentration, relative retention time and LOD of 22 organochlorine pesticides residues
Concentration of reference stock Relative Retention Reference Limits of
No. Pesticide
solution (µg/mD Time (Column 1) Detection (mg/kg)

1 Hexachlorobenzene 100 0.574 0.001

2 a-BHC 100 0.601 0.004
3 Quintozene 100 0.645 0.007
4 y-BHC 100 0.667 0.003
5 ~-BHC 200 0.705 0.008
6 Heptachlor 100 0.713 0.007
7 a-BHC 100 0.750 0.003
8 Aldrin 100 0.760 0.006
9 oxy-Chlordane 100 0.816 0.007
10 Heptachlor-exo-epoxide 100 0.833 0.006
11 Heptachlor-endo-epoxide 100 0.844 0.005
12 trans-Chlordane 100 0.854 0.005
13 cis-Chlordane 100 0.867 0.008
14 a-Endosulfan 100 0.872 0.01
15 p, p'-DDE 100 0.892 0.006
16 Dieldrin 100 0.901 0.005
17 Endrin 200 0.932 0.009
18
º' p'-DDT 200 0.938 0.018
19 p, p'-DDD 200 0.944 0.008
20 ~-Endosulfan 100 0.956 0.003
21 p, p'-DDT 100 0.970 0.005
22 Endosulfan sulfate 100 1.000 0.004
Notes: Endosulfan sulfate is the reference substance for calculating the RRTs.

Method 1 . Detennination of organophosphorus pesticide 2, 2-dichlorovinyl dimethyl phosphate ( DDVP ) . The

residues by chromatography
Chromatographic system and system suitability Column: a
fused silica capillary column ( 30 m X O. 32 mm) with a film
(O. 25 µm) of 50% phenyl-50% dimethyl polysiloxane, or
5% phenyl-95% methyl polysiloxane; detector: nitrogen
phosphorous detector (NPD) or flame photometric detector
( FPD ) ; injection port temperature: 220ºC; detector
temperature: 300ºC; injection mode: splitless; column
temperature program: maintain the initial temperature at
120ºC, increase the temperature ata rate of lOºC per minute
to 200ºC, then increase the temperature at a rate of 5ºC per
minute to 240ºC , hold for 2 minutes, and then increase the
temperature at a rate of 20ºC per minute to 270ºC, hold for
O. 5 minutes. The number of theoretical plates of the column is
not less than 6000, calculated with reference to the peak of
resolution between two neighboring peaks is more than l. 5.
Preparation of reference stock solution Weigh accurately a
quantity of reference substance of parathion, parathion-methyl,
rogar, oxidative rogar, acephatemet, monocrotophos, alfatox,
ethion, malathion, methidathion, 2, 2-dichlorovinyl dimethyl
phosphate ( DDVP ) and acephate pesticides respectively,
dissolve with ethyl acetate to prepare solutions containing 100
µg per ml.
Preparation of mixed reference stock solution Measure
accurately l. O ml of each of the above reference stock
solutions to a 20 ml brown volumetric flask, diluted with
ethyl acetate to volume and mix well.
Preparation of mixed reference solution Measure accurately
the above mixed reference stock solution, dilute with ethyl
 2341 Determination of Pesticide Residues

acetate to produce a series of solutions containing O. 1 µg, O. 5 µg, sulfate, then concentrate the filtrate in vacuum to almost
1 µ.g, 2 µ.g, 5 µ.g per ml. dryness at 40-45ºC, operate repeatedly with a small amount
of petroleum ether ( 60-90ºC) to exhaust the solvent of
Preparation of test solution ( Crude drug or decoction
acetone, add proper amounts of petroleum ether ( 60-90ºC)
pieces ) Weigh accurately 5 g of the substance being
examined (pulverized through the No. 3 sieve). Add 5 g of to dissolve the residue, transfer to the small column [it is
anhydrous sodium sulfate and 50-100 ml of ethyl acetate, progressive from up to clown 2 g of anhydrous sodium
sulfate, 4 g of Florisil, 1 g of microcrystalline cellulose,
ultrasonicate for 3 minutes in ice bath. Filter the upper-layer
solution, add 30-50 ml of ethyl acetate into the drugs, 1 g of aluminium oxide and 2 g of anhydrous sodium sulfate,
prewash with 20 ml of a mixture of petroleum ether ( 60-
ultrasonicate for 2 minutes in ice bath, and filter. Combine
90°C) and ether ( 4 : 1) J, elute with the mixture of 90 ml
these two filtrates, wash filter paper and residue with a small
of petroleum ether ( 60-90ºC) and ether ( 4 : 1) , and collect
amount of ethyl acetate, and combine them with the above
the eluent, concentrate the filtrate in vacuum to almost
filtrate. Concentrate the filtrate in vacuum to almost dryness
dryness at 40-45°C. Add 3-4 ml of petroleum ether ( 60-
at 40ºC, transfer into a 5 ml volumetric flask with ethyl
90°C) and concentrate to almost dryness, repeatedly with a
acetate and dilute to volume. Measure accurately 1 ml of the
small amount of petroleum ether ( 60-90ºC) to exhaust the
solution, put on a small column packed with graphitized
solvent of the ether, dissolve with petroleum ether (60-90°C)
carbon (250 mg/3ml, pre-washed with 5 ml of ethyl
and transfer to a 5 ml volumetric flask, dilute to volume.
aceta te) , place in the multifunctional vacuum sample
processor, elute with 5 ml of the mixture of hexane and ethyl Detemúnation Inject accurately 1 µ.l of each of the test
acetate ( 1 : 1), collect the eluent, concentrate to almost solutions and the mixed reference solution with
dryness on the N2-blowing instrument, and add accurately 1 corresponding concentrations, respectively, into the gas
ml of ethyl acetate to dissolve it. chromatograph, calculate the content of three pyrethrin
pesticide residues in the test solution by externa! standard
Determination Inject accurately 1 µ.l of each of the test
method.
solution and the mixed reference solution with corresponding
concentration, respectively, into the gas chromatograph, and Method N • Detennination of 227 pesticide residues by mass
calculate the content of 12 organophosphorus pesticide spectrometry
residues by externa! standard method. l. GC/MS/MS method
Chromatographic system Column: a fused silica capillary
Method ][. Detennination of pyrethrin pesticide residues by
column (30 mXO. 25 mm) with a film (0. 25 µ.m) of 5% phenyl
chromatography
and 95% methyl polysiloxane; injection port temperature:
Chromatographic system and system suitability Column: a 240°C; injection mode: splitless; carrier gas: helium; flow rate:
fused silica capillary column (30 mX O. 32 mm) with a film constant pressure; column temperature program: maintain the
(O. 25 µ.m) of 5% phenyl and 95% methyl polysiloxane; initial temperature at 70ºC for 2 minutes, increase the
detector: 63 Ni-electron capture detector ( ECD) ; injection temperature at a rate of 25°C per minute to 150ºC, then increase
port temperature: 270ºC; detector temperature: 330ºC; the temperature ata rate of 3°C per minute to 200ºC, and finally
injection mode: splitless; column temperature program: increase the temperature at a rate of 8°C per minute to 280ºC ,
maintain the initial temperature at 160ºC for 1 minute, increase hold for 10 minutes.
the temperature ata rate of lüºC per minute to 278ºC, hold for
Mass spectrometry system
O. 5 minute, and then increase the temperature ata rate of 1ºC
Detector: triple-quadrupole mass spectrometer; ion ization
per minute to 290ºC, hold for 5 minutes. The number of
source: El; source temperature: 230ºC; collision gas:
theoretical plates of the column is not less than 105 , calculated
nitrogen or argon; MS transfer line temperature: 280ºC;
with reference to the peak of deltamethrin. The resolution
monitoring mode: multiple reaction monitoring ( MRM).
between two neighboring peaks is more than l. 5.
Monitoring ion pairs, collision energy (CE) and limit of
Preparation of reference stock solution Weigh accurately a detection ( LOD) are shown in table 2. Segmentation
quantity of reference substances of cis-cypermethrin, detection can be used to increase sensitivity.
fenvalerate and deltamethrin pesticides respectively, dissolve
Table 2 Monitoring ion pairs, retention time,
with petroleum ether ( 60-90°C) to produce solutions
CE and WD of 76 pesticides
containing 20-25 µ.g per ml.
Retention Precurs:>r Prcxiuct CE lffi
Preparation of mixed reference stock solution Measure No. Pesticide
Time (min) ion 100 (V) (rrg/kg)
accurately O. 5 ml of each of the above reference stock
solutions to a 10 ml volumetric flask, dilute with petroleum 184.9 93.0 10
ether ( 60-90ºC) to volume and mix well. 1 Dichlorvos 5. 9 0.005
109.0 79.0 5
Preparation of mixed reference solution Measure accurately
the above mixed reference stock solutions, dilute with petroleum 169.0 168.2 15
ether (60-90°C) to produce a series of solutions containing O µg, 2 Diphenylamine 10.5 0.005
169.0 140.0 35
2 µ.g, 8 µ.g, 40 ¡.ig and 200 µ.g of reference substance per
liter. 260.9 203.0 10
3 Tecnazene CTCNB) 10. 2 0.005
Preparation of test solution ( Crude drug or decoction 214. 9 179.0 10
pieces) Weigh accurately 1-2 g of the substance being
examined (pulverized through the No. 3 sieve) and put in a 195.9 181. o 5
100 ml stopper conical flask, add 30 ml of a mixture of 4 Chlordimeform 11. 2 0.025
151. 9 117. 1 10
petroleum ether ( 60-90°C) and acetone ( 4 : 1) , ultrasonicate
for 15 minutes, and filter. The filtrates are combined after 305.9 264.0 5
treated the drugs with the above procedure twice. Dehydrate 5 Trifluralin 11. 6 0.005
the filtrate with proper amounts of anhydrous sodium 264.0 160. l 15
 2341 Determination of Pesticide Residues

continued continued

Retention Precursor Prcxluct CE lffi Retention Precursor Prcxluct CE lffi

No. Pesticide No. Pesticide
Time (min) ion ton (V) <lllYkg) Time (min) ion ton (V) <lllYkg)

216. 9 181. 1 5 Methyl- 296. o 281. O 20

6 crBHC 12. 1 0.005 25 pentachlorophenyl 18. O O. 005
181. 1 145. 1 15 sulfide 296. O 263. O 15
206.1 176. o 10 277. o 109. o 15
7 Dieloran 12.6 0.005 26 Fenitrothion 18.2 0.01
206. o 148. o 20 260. o 125. o 10
283. 8 248. 8 15 223. 9 123. 1 10
8 Hexachlorobenzene 12. 4 0.005 27 Dichlofluanid 18.4 0.01
283. 8 213. 9 30 123. o 77. 1 20
280. o 265. o 12 262. 9 192. 9 35
9 Pentachloranisole 12. 6 0.005 28 Aldrin 18. 5 0.01
280. o 237. o 22 254. 9 220. o 20
205. o 127. o 10 284. o 169. o 15
10
Atrazine-d5
(ethyl-d5)
13. 1 29 ( Fendi~hion-thdl6d 6 )
o, o- me y-
19. O
205. o 105. o 15 284. o 115. o 20
216. 9 181. 1 5 139. o 111. o 15
11 ~-BHC 13.2 0.005 30 Dicofol 19. 2 o. 01
181. o 145. o 15 251. o 139. o 10
216. 9 181. 1 5 313.8 285.8 5
12 y-BHC (Lindane) 13. 4 0.005 31 Chlorpyrifos 19. 3 0.005
181. 1 145. 1 15 313. 8 257. 8 15
295. o 237. o 18 290. 9 109. o 10
13 Quintozene 13. 7 0.005 32 Parathion-ethyl 19. 4 0.01
237. o 143. o 30 290. 9 80. 9 25
230. 9 175. o 10 208. o 181. 1 5
14 Terbufos 13.8 0.005 33 Triadimefon 19.4 0.01
230. 9 129. o 20 208. o 111. o 20
216. 9 181. 1 5 300. 9 223. o 25
15 a-BHC 14.6 0.005 34 Chlorthal-dimethyl 19. 4 0.005
181. l í45. i 15 298. 9 221. o 25
263. 8 229. o 20 330. 8 315. 8 15
16 Chlorothalonil 14. 8 0.025 35 Bromophos-methyl 20. 1 0.005
263. 8 168. o 25 328. 8 313. 8 15
197. o 141. 1 10 266. o 220. 2 10
17 Tefluthrin 15. 1 0.005 36 Butralin 20.2 0.05
177. 1 127. 1 15 266. o 17 4. 2 20
265. o 230. o 15 Heptachlor 354. 8 264. 9 15
18 Pentachloraniline 15. 5 0.005 37 20.7 0.005
265. o 194. o 20 exo-epoxide 352. 8 262. 9 15
212. o 145. o 30 386. 7 262. 7 15
19 Vinclozolin 16. 6 0.005 38 Chlordane-oxy 20. 7 0.005
212. o 109. o 40 184. 9 85. o 30
Chlorpyrifos- 286. o 271.o 15 Heptachlor 354. 8 264. 9 15
20 16. 7 0.005 39 21. o 0.005
methyl 286. o 93. o 20 endo-epoxide 352. 8 262. 9 15
262. 9 109. o 10 251. 8 162. 2 10
21 Parathion-methyl 16. 8 0.01 40 Pendimethalin 21. O 0.01
262. 9 79. o 30 251. 8 161. 1 15
273. 7 238. 9 15 144. 9 112. 1 5
22 Heptachlor 16.8 0.005 41 Dimepiperate 21. 6 0.01
271. 7 236. 9 15 144. 9 69. 1 15

Octachlorodipropyl 129. 9 94. 9 20 128. o o

65. 25
23 17 3 0.005 42 Triadimenol 21. 7 0.01
ether (S421) · 108. 9 83. o 10 168. o 70. o 10
286. o 271. o 15 366. 8 212. 8 35
24 F enchlorphos 17.4 0.005 43 Fipronil 21. 9 0.005
285. o 269. 9 15 350. 8 254. 8 15
 2341 Determination of Pesticide Residues

continued continued
Retention Precursor Pnxluct CE L(]) Retention Precursor Pnxluct CE LCD
No. Pesticide No. Pesticide
Time (min) ion ion (V) (Illdkg) Time (min) ion ion (V) (rrg/kg)

282. 8 96. o 10 181. o 166. o 20

44 Procymidone 22.0 0.01 63 Bifenthrin 28. 9 o. 005
284. 8 96. o 10 181. o 165. o 25
372. 8 265. 8 15 265.0 210.0 8
45 Chlordane-trans 22. O 0.005 64 Fenpropathrin 29. O 0.005
374. 8 265. 8 15 208. o 181. o 5

358. 7 302. 8 15 227. o 212. o 18

46 Bromophos-ethyl 22. 6 0.005 65 Methoxychlor 28. 9 0.005
302. 8 284. 7 15 227. o 169. o 25
271. 9 236. 9 15 273. 8 238. 8 15
47 Chlordane-cis 22.8 0.005 66 Mirex 29.8 0.005
372. 9 265. 9 20 271. 8 236. 8 15
248. o 176. 2 30 29.4 183. o 168. o 12
48 o, p'-DDE 22.5 0.005 67 Phenothrin 0.005
246. o 176. 2 30 29.6 183. o 153. o 12
194. 9 159.0 5 207. 8 181. 1 10
49 a-Endosulfan 22.6 0.01 68 Acrina thrin 30.4 0.005
276. 7 241. 9 15 181. o 152. o 30
143. o 117. o 20 208. o 181. o 5
50 Flumetralin 23.3 0.005 69 Cyhalothrin 30.4 0.005
143. o 107. 1 20 197. o 141. o 10
277. o 241. o 5 31. 4 183. 1 168. 1 10
51 Dieldrin 23.8 0.01 70 Permethrin 0.005
262. 9 193. o 35 31. 6 183. 1 165. 1 10
237. o 165. 2 20 32.3,
52 o, p'-DDD 24.4 0.005 163.0 127.0 5
32.4
235. o 165. 2 20 71 Cyfluthrin 0.025
32. 5,
246. 1 176. 2 30 226. o 206. o 12
53 p, p'-DDE 24.0 0.005 32.6
315. 8 246. o 15
32. 7'
181. o 152. o 10
262. 8 193. o 35 32.9
54 Endrin 24. 7 0.01 72 Cypermethrin 0.025
244. 8 173. o 30 33.0,
181. o 127. o 30
33. 1
202. o 139. 1 20
55 Nitrofen 24.9 0.01 33. 1 198. 9 157. o 10
282. 9 253. o 10 73 Fl ucythrina te o. 025
33.4 156. 9 107. 1 15
246. 9 227. o 15
56 Chlorfenapyr 25.3 0.01 163. o 136. o 10
327. 8 246. 8 15 74 Quizalofop-ethyl 33. O 0.01
371. 8 298. 9 10
237. o 165. 2 20
57 p, p'-DDD 25.7 0.005 34.3 167.0 125. 1 5
235. o 165. 2 20 75 Fenvalerate 0.025
34. 7 225. o 119. o 18
237. o 165. 2 20
58 o, p'-DDT 25.8 0.005 181. o 152. 1 25
235. o 165. 2 20 76 Deltamethrin 36.0 0.025
252. 7 174.0 8
206. 9 172. o 15
59 ~- Endosulfan 25.2 0.01
267. o 196. o 14 Notes: (1) Compound 10 and compound 29 are interna!
standard compounds.
Endosulfan 271. 9 237. o 15 (2) Sorne pesticides have multiple retention time because of
60 26.8 0.01
sulfate 387.0 289.0 4 isomer.
Preparation of reference stock solution Weigh accurately a
237. o 165. 2 20 quantity of pesticides of Table 2 and Table 4, dissolve with
61 p, p'-DDT 27. O 0.005
235. o 165. 2 20 acetonitrile or toluene respectively to prepare solutions of
1000 µg per ml.
341. o 185. o 30
62 Bromopropylate 28. 6 0.005 Preparation of internal standard stock solution Weigh
341. o 183. o 15
 2341 Determination of Pesticide Residues

accurately a quantity of atrazine-d 5 and fenthion-d 6 , collision energy (CE) and limit of detection ( LOD) are
dissolve with acetonitrile to prepare mixed solutions of shown in table 4. Segmentation detection by retention time
1000 µg per ml. can be conducted to increase sensitivity.
Preparation of mixed reference stock solution Measure Table 4 Monitoring ion pairs, retention time,
accurately appropriate amount of each of the above reference CE and WD of 155 pesticides
stock solutions, diluted with O. 05 % acetic acid acetonitrile to
Retention Precursor Prcduce CE L0D
prepare solutions containing 100 µg and 1000 µg per liter. No. Pesticide
Tnres (min) ion ion CV> (rrg/kg)
Preparation of internal standard solution Measure
accurately appropriate amount of internal standard stock 184. o 143. o 13
1 Acephate 2.5 0.05
solution, diluted with acetonitrile to prepare solution 184. o 125. o 24
containing 6 µg per liter.
Preparation of matrix-matched reference solution Weigh 223. 5 126. o 17
2 Acetaniprid 4. 1 0.005
accurately 3 g of blank sample in sextuplicate, prepare in the 223. 5 90. o 43
same manner of test solution till " blow to O. 4 ml with
nitrogen gas at 40ºC", add accurately 50 µl, 100 µl of mixed 270. 1 238. 1 15
reference stock solution ( 100 µg/L) and 50 µl, 100 µl, 3 Alachlor 6. 6 0.005
270. 1 162. 1 26
200 µl, 400 µl of mixed reference stock solution ( 1000 µg/L),
dilute to 1 ml with acetonitrile, vortex and filter (0. 22 µm). 208. 1 116. 1 10
4 Aldicarb 4. 5 0.005
Preparation of test solution ( Crude drug or decoction 208. 1 89. o 22
pieces ) Weigh accurately 3 g of pulverized sample
( through the s1eve of number 3 ) , place m a 50 ml 223. 1 166. 1 8
polystyrene centrifuge tube with stopper, add 15 ml 1% 5 Aldicarb-Sulfone 3.3 0.005
223. 1 148. o 11
acetic acid, vortex, allow to stand for 30 minutes. Add
accurately 15 ml of acetonitrile and 100 µl of intemal 207. 1 132.0 9
standard solution, shake for 5 minutes ( 500 rpm) , add about 6 Aldicarb-Sulfoxide 2.9 0.005
7. 5 g of a mixture of magnesium sulfate and sodium acetate 207. 1 89. o 20
( 4 : 1) , shake immediately, and then shake for 3 minutes 303. 2 135. o 15
(500 rpm), put the samples in an ice bath for 10 min, 7 Allethrin 9. 1 0.25
centrifuge ( 4000 rpm) for 5 minutes. Transfer 9 ml of the 303. 2 169. o 12
supernatant to the d-SPE tu bes ( containing 900 mg anh.
228. 1 186. 1 26
MgS0 4 , 900 mg PSA, 300 mg C18, 300 mg silica and 90 8 Ametryn 5.5 0.005
mg GCB), vortex, and shake for 5 minutes, centrifuge 228. 1 96. 1 34
( 4000 rpm) for 5 minutes. Transfer accurately 5 ml of the
supernatant liquid into the 15 mi poiypropyiene centrifuge 216.1 174.1 23
9 Atrazine 5. 2 0.005
tube, blow to O. 4 ml with nitrogen gas at 40ºC, dilute to 1 216. 1 104. o 38
ml with acetonitrile, vortex and filter (0. 22 µm).
Determination lnject accurately 1 µl of each of the test Atrazine-d s 221. o 178. 8 35
10 5. 1
solution and the matrix-matched reference solution into GC/MS (ethyl-ds) 221. o 101. 1 35
respectively, calculate the amount of 74 pesticides by internal
standard method. 346.0 289.0 8
11 Azinphos-ethyl 6. 7 0.05
2. HPLC/MS/MS method 346. o 261. o 11
Chromatographic system Column: octadecylsilane bonded 318.0 160. 1 9
silica gel column (15 cm in length, 3 mm in inside diameter 12 Azinphos-methyl 5. 8 0.05
and 3. 5 µm in particle size). Gradient elution by table 3 is 318. o 132. o 20
applied with O. 1% formic acid solution (containing 10 mmol/L 404. 1 372. 1 18
ammonium formate) as mobile phase A and acetonitrile as mobile 13 Azoxystrobin 5. 9 0.005
phase B. Chromatographic temperature is 35ºC. Flow rate is 404. 1 344. 1 32
O. 4 ml/min.
326. 2 294. 2 14
Table 3 Mobile phase gradient 14 Benalaxyl 7. 1 0.005
326. 2 208. 1 21
Time (min) Mobile phase A C%) Mobile phase B C%)
301. 1 170. 1 29
0-1 95 5 15 Bifenazate 6.2 0.005
301. 1 198. 1 15
1-4 95-40 5-60
338. 2 269. 2 10
4-14 40-0 60-100 16 Bitertanol 6.4 0.05
338. 2 99. 1 18
14-18 o 100
343. o 307. 1 26
18-26 95 5 17 Boscalid 6. 1 0.005
343. o 140. o 25
Mass spectrometry system Detector: triple-quadrupole 306. 2 201. 1 15
mass spectrometer. Ion Source: ESI in positive ion sean 18 Buprofezin 9.5 0.005
mode. Detection mode: Multiple reaction monitoring 306. 2 116. 1 20
CMRM). Reference retention time, monitoring ion pairs,
 2341 Determination of Pesticide Residues

continued continued
Retention Precursor Pn:x:luce CE lffi Retention Precursor Pn:x:luce CE lffi
Pesticide No. Pesticicle
Tures (rnin) ion ion (V) Cnw'kg) Tures (min) ion ion (V) Cnw'kg)

312. o 238. l 17 276. 1 244. 1 20

19 Butachlor 9. 2 0.005 38 Dimethenamid 6.0 0.005
312. o 162. 2 33 276. 1 168. 1 33
271. o 159. o 21 230. o 199. o 13
20 Cadusafos 7.6 0.005 39 Dimethoate 4.0 0.005
271. o 97. o 51 230. o 125. o 29
202. 1 145. 1 13 326. 1 159. o 42
21 Carbaryl 5.0 0.005 40 Diniconazole 6. 7 0.05
202. 1 127. 1 39 326. 1 70. o 53
192. 1 160. 1 21 275. o 89. o 18
22 Carbendazim 3.4 0.005 41 Disulfoton 8. 1 o. 1
192. 1 132. 1 40 275. o 61. o 49
222. 1 165. 1 16 307. o 261. o 14
23 Carbofuran 4. 9 0.005 42 Disulfoton-Sulfone 5. 5 o. 1
222. 1 123. o 27 307. o 153. o 17

Carbofuran-3- 238. 1 181. 1 14 291. o 213. o 13

24 3. 9 0.005 43 Disulfoton-Sulfoxide 5. O 0.005
Hydroxy 238. 1 163. 1 23 291. o 185. o 20
235. o 207. o 25 311. o 172. 9 25
25 Chinomethionat 7. 9 0.05 44 Edifenphos 6. 9 0.005
2350 163. o 38 311. o 282. 9 16
481. 9 450. 9 23 324. 1 296. o 18
26 Chlorantraniliprole 5. 4 0.005 45 EPN 8. 2 0.005
481. 9 283. 9 20 324. 1 157. o 30
359. o 155. o 16 226. 1 106. 9 21
27 Chlorfenvinphos 6. 9 0.005 46 Ethiofencarb 5. 1 0.005
359. o 127. o 22 226. 1 164. 1 11
360. 1 268. 1 14 385. o 199. o 14
28 Clethodim 8. 6 0.005 47 Ethion 9. 5 0.005
360. 1 164. 1 23 385. o 142. 9 36
363. o 307. o 22 243. 1 215. o 17
29 Coumaphos 7. 6 0.05 48 Ethoprophos 6. 2 0.005
363. o 227. o 35 243. 1 130. 9 29
375. 2 358. 1 10 394. 2 359. 2 15
30 Cyhalofop-Butyl 8. 6 0.05 49 Etofenprox 11. 8 0.05
375. 2 256. 1 22 394. 2 177. 1 21
226. 1 108. 1 35 293. 1 265. o 24
31 Cyprodinil 6. 8 0.005 50 Etrimfos 4.0 0.005
226. 1 93. 1 44 293. 1 125. o 42
259. o 88. 9 20 304. 1 276. 1 19
32 Demeton co+s) 5. 3 0.005 51 Fenamiphos 5. 9 0.005
259. o 60. 9 50 304.1 217. o 31
305. 1 277. 1 19 336. 1 308. 1 21
33 Diazinon 7. 6 0.005 52 Fenamiphos-Sulfone 4. 7 0.005
305. 1 169. 1 29 336. 1 266. o 28
314. 9 258. 9 23 Fenamiphos- 320. 1 292. 1 21
34 Dichlofenthion 9. 3 0.05 53 4. 3 0.05
314. 9 286. 9 17 Sulfoxide 320. 1 233. o 34
238. 1 112. 1 19 331. o 268. 1 32
35 Dicrotophos 3. 5 0.005 54 Fenarimol 6.0 0.05
238. 1 193. o 15 331. o 139. o 48
406. 1 337. o 24 337. 1 125. o 38
36 Difenoconazole 7. 3 0.005 55 Fenbuconazole 6. 3 0.005
406. 1 251. o 36 337. 1 70. o 24
311. o 158. o 21 304. 9 272. 9 30
37 Diflubenzuron 6. 2 0.005 56 Fenchlorphos-oxon 6. O 0.05
311. o 141. o 45 304. 9 109. o 31
 2341 Determination of Pesticide Residues

continued continued
Retention Precursor Praluce CE UD Retention Precursor Praluce CE LCJ)
Pesticide Pesticide
Trrres (min) ion ion (V) (ng/kg) Trrres (min) ion ion (V) (ng/kg)

422. 2 366. 1 24 314. 1 185. o 30

57 Fenpyroximate 9. 9 0.005 76 Hexaconazole 6.4 0.05
422. 2 215. 1 35 314. 1 159. o 41

309. o 281. o 20 253. o 171. 1 25

58 Fensulfothion 5. 2 0.005 77 Hexazinone 4.4 0.005
309. o 253. o 25 253. o 71. 1 45

293. 1 265. o 20 297. 1 255. o 25

59 Fensulfothion-oxon 4. O 0.005 78 lmazalil 5. o 0.005
293. 1 237. o 21 297. 1 159. o 30

F ensulfothion- 309. 1 281. o 15 256. o 209. 1 23

60 4. 5 0.005 79 lmidacloprid 3.9 0.005
oxon-sulfone 309. 1 253. o 23 256. o 175. 1 28

Fensulfothion- 325. o 297. o 16 528. 1 293. o 19

61 5. 7 0.05 80 lndoxacarb 7. 9 0.005
sulfone 325. o 269. o 23 528. 1 249. o 23

279. o 247. o 18 330. o 244. 9 20

62 Fenthion 7.2 0.05 81 lprodione 6. 3 0.005
279. o 169. o 24 332. o 247. o 20

285. 4 249. 9 18 315. o 163. o 22

63 ( Fend~hionh-dl6 d ) 7. 2 82 Isazofos 6. 9 0.005
o , o- 1met y - 6 285. 4 168. 9 24 315. o 120. o 35

263. 1 231. o 22 346. 1 287. 1 8

64 F enthion-oxon 5.2 0.05 83 Isofenphos 8.2 0.005
263. 1 216. o 32 346. 1 245. o 19

F enthion-oxon- 295. o 217.1 26 332. o 273. o 10

65
s ulfone
4. 1 o. 1 84 Isofenphos-methyl 7. 5 0.005
295. o 104. 1 34 332. o 231. o 30

F enthion-oxon- 279. o 264. o 26 194. o 152. o 11

66 3.8 0.005 85 Isoprocarb 5. 3 0.005
suHoxide 279. o 247. o 36 194. o 137. o 13

311. o 279. o 25 290. 9 188. 9 30

67 Fenthion-sulfone 5. 3 o. 1 86 Isoprothiolane 6. 5 0.005
311. o 125. o 28 290. 9 231. o 15

295. o 280. o 26 315. 1 269. o 11

68 Fenthion-sulfoxide 4. 9 0.05 87 Malaoxon 4.8 0.005
295. o 109. o 40 315. 1 127. o 17

384. 1 328. 1 24 331. o 285. o 10

69 Fluazifo¡r-P-butyl 9. O 0.005 88 Malathion 6.4 0.005
384. 1 282. 1 29 331. o 127. o 17

316. 1 247. 1 25 330. 1 227. o 12

70 Flusilazole 6.3 0.005 89 Mecarbam 6.8 0.005
316. 1 165. 1 37 330. 1 199. o 21

324. 1 262. 1 26 270. 1 228. 1 20

71 Flutolanil 6.4 0.005 90 Mepronil 6. 3 0.005
324. 1 242. 1 35 270. 1 119. o 32

247. o 137. o 15 280. 2 248. 1 14

72 Fonofos 7. 7 0.005 91 Metalaxyl 5. 1 0.05
247. o 109. o 25 280. 2 220. 1 19

284. 1 228. o 14 241. o 209. o 12

73 Fosthiazate 5.0 0.005 92 Methacrifos 5.8 o. 1
284. 1 104. o 32 241. o 125. o 26

383. 2 252. 1 17 142. o 125. o 19

74 Furathiocarb 8. 9 0.005 93 Methamidophos l. 8 0.005
383. 2 195. o 25 142. o 94. o 21

376. 1 316. o 25 303. o 145. o 13

75 Haloxyfo¡rmethyl 7. 9 0.005 94 Methidathion 5. 7 0.05
376. 1 288. o 35 303. o 85. o 30
 2341 Determination of Pesticide Residues

continued continued
Retention Precursor Produce CE lffi Retention Precursor Produce CE lffi
No. Pesticide Pesticide
Tirres (min) ion ion (V) (rrg/kg) Tirres (min) ion ion (V) (rrg/kg)

226. 1 169. 1 14 321. o 275. o 8

95 Methiocarb 5. 6 0.005 114 Phenthoate 7. 3 0.005
226. 1 121. 1 26 321.0 247.0 14

163. 1 106. o 13 261. o 75. o 19

96 Methomyl 3.4 0.005 115 Phorate 7. 8 0.005
163. 1 88. o 12 261.0 47.0 49

369. 2 313. 2 10 245.0 245.0 5

97 Methoxyfenozide 6. 2 0.005 116 Phorate-oxon 5. 2 0.005
369. 2 149. 1 24 245. o 75. o 10
285. o 253. o 19 Phorate-oxon- 277. o 249. o 14
98 Metolachlor 6. 6 0.005 117 4.2 0.005
285. o 177. o 33 sulfone 277. o 183. o 16
166. o 109. 1 17 293.0 247.0 9
99 Metolcarb 4.6 0.05 118 Phorate-sulfone 5. 6 o. 1
166. o 94. o 43 293. o 171. o 16
215. 1 187. 1 25 368. o 322. o 14
100 Metribuzin 4.8 0.005 119 Phosalone 7.8 0.05
215. 1 84. 1 28 368. o 182. o 23
225. 1 193. o 11 318. o 160. o 24
101 Mevinphos 3.8 0.005 120 Phosmet 5.9 0.05
225. 1 127. o 22 318. o 133. o 51
188. 1 126. 1 19 300. 1 227. o 19
102 Molinate 6.3 0.05 121 Phosphamidon 4.3 0.005
188. 1 55. 1 34 300. 1 174. 1 19

224. 1 193. o 11 299. 1 153. 1 11

103 Moncrotophos 3.3 0.005 122 Phoxim 7. 7 0.05
224. 1 127. o 22 299. 1 129. o 16
289. 1 125. o 50 Piperonyl 356. 2 177.1 15
104 Myclobutanil 5. 9 o. 005 123
Butoxide
8. 7 o. 005
289. 1 70. o 24 356. 2 119. 1 49

272. 2 199. 1 26 239. 1 182. 1 22

105 Napropamide 6. 3 0.005 124 Pirimicarb 4. 7 0.05
272. 2 171. 1 26 239. 1 137. 1 32

N-desethyl- 278. o 245. 8 24 Pirimiphos- 334. 1 306. 1 23

106 5. 5 0.005 125 9. 6 0.005
pimiphos-methyl 278. o 249. 8 24 ethyl 334. 1 198. 1 21

214. o 183. o 15 Pirimiphos- 306. 1 164. 1 30

107 Omethoate 2. 7 0.05 126 8. 1 0.005
214. o 155. o 21 methyl 306. 1 108. 1 39

345. 1 303. o 19 312. o 252. 1 23

108 Oxadiazon 9.2 0.05 127 Pretilachlor 8.2 0.005
345. 1 220. o 28 312. o 132. 1 63
279. 1 219. 1 16 376. o 308. o 17
109 Oxadixyl 4. 6 0.005 128 Prochloraz 7. o 0.005
279. 1 132. 1 43 376. o 70. o 45
237. 1 220. 1 7 372. 9 344. 9 18
110 Oxamyl 3. 3 0.05 129 Profenofos 8.2 0.005
237. 1 90. 1 12 372. 9 302. 9 26

294. 1 165. o 31 208. 1 109. o 23

111 Paclobutrazol 5.5 0.05 130 Promecarb 5. 8 0.005
294. 1 125. o 52 208. 1 151. o 13
276. o 248. o 14 218. 1 162. 1 21
112 Paraoxon-ethyl 5. 2 0.05 131 Propanil 5. 5 0.005
276. o 220. o 22 218. 1 127. 1 33

248. o 231. o 24 368. 2 231. 2 14

113 Paraoxon-methyl 4. 6 0.05 132 Propargite 9.9 0.005
248. o 202. o 27 368. 2 175. 1 23
 2341 Determination of Pesticide Residues

continued continued

Retention Precursor Produce CE UD Retention Precursor Produce CE UD

Pesticide No. Pesticide
Ttrres(min) 100 100 (V) (rrg/kg) Tlm2S (min) 100 ion (V) (rrg/kg)

282. o 138. o 25 314. 1 178. o 29

133 Propetamphos 6. 6 0.005 152 Triazophos 6.4 0.005
282. o 156. o 19 314. 1 162. 1 25
342. 1 205. o 25 256. 9 109. o 25
134 Propiconazole 6. 8 0.05 153 T richlorfon 3.6 0.05
342. 1 159. o 35 256. 9 221. o 15
210. 1 168. 1 11 190. o 163. o 28
135 Propoxur 4.8 0.005 154 Tricyclazole 4.3 0.005
210. 1 111. o 19 190. o 136. o 34
344. 8 241. o 27 409. 1 206. 1 19
136 Prothiophos 11. o o. 1 155 Trifloxystrobin 8. 1 0.005
344. 8 132. 9 69 409. 1 186. 1 18
388. 1 296. 1 19 Notes: Compound 10 and compound 63 are interna! standard
137 Pyraclostrobin 7. 5 o. 005 compounds.
388. 1 194. 1 17
The preparation of reference stock solution, internal standard
365. o 147. o 31
solution and test solution is the same with the GC/MS/MS
138 Pyridaben 10. 7 0.005
365. o 309. o 19 method.

322. 1 227. 1 21 Determination Inject accurately 1-10 µl ( according to

139 Pyriproxyfen 9. 1 0.005 detection requirement and instrument sensitivity) of each of
322. 1 185. 1 32 the test solution and the matrix-matched reference solution
into HPLC/MS respectively, calculate the amount of 153
299. 1 271. o 19
pesticides by interna! standard method.
140 Quinalphos 7. 1 o. 005
299. 1 163. o 33 [Note appended]
297. o 241. o 8 The preparation of reference solutions and the pesticides
141 RH 5849 5. 2 o. 005 monitored are in accordance with each relevant pesticide
297. o 105. o 25 limitation.
Same pesticides must not be detected m blank matrix
323. o 295. o 14
sample.
142 Sulfotep 7. 6 o. 005
323. o 170. 9 20 The recovery of standard addition must fall in the interval of
70 %-120 % . Under the circumstance of good method
520. 1 208. 1 23 reproducibility, sorne of the recoveries can fall in the interval
143 Tau-fluvalinate 11.5 0.25
520. 1 181. 1 35 of 50%-130%.
Pesticides are confirmed if: the RT chromatographic peaks
308. 1 125. o 55 detected match the standard peaks, ion pairs are both
144 Tebuconazole 6.2 0.005 detected after background correction and the peaks area ratio
308. 1 70. o 27
is the same with the standard peak area ratios ( relative scale
353. 2 297. 2 11 >SO%, ±20% deviation is allowed. Relative scale >20%-
145 Tebufenozide 6. 7 0.05 50%, ± 25% deviation is allowed. Relative scale > 10%-
353. 2 133. 1 25 20%, ± 30% deviation is allowed. Relative scale~ 10%,
332. o 314. o 12 ±50% deviation is allowed. ). If not confirmed, other ion
146 Tetramethrin 8.8 0.05 pairs or instrument can be employed.
332. o 286. o 13 In GC/MS/MS method, Fenthion-d6 is recommended as the
intemal standard, while in HPLC/MS/MS method, Atrazine-d 6
202. o 175. o 35
14 7 Thiabendazole 3.5 0.005 is recommended as the intemal standard.
202. o 131. 1 45 The precursor ion and product ion is only a recommendation
under the given detection condition. Appropriate adjustments
253. o 186. o 20
148 Thiacloprid 4.3 0.05 can be made according to the specific condition of the
253. o 126. o 30 instrument. In case of interference of sample matrix, other
ion pair can be adapted.
292. o 211. 1 18 The proportion of material for purification in SPE can be
149 Thiamethoxam 3. 6 0.005
292. o 181. 1 31 adjusted to particular pesticides and samples only when
methodology study is conducted.
301. o. 269. o 23 In GC/MS/MS method, the solvent of the tested sample can
150 Tolclofos-methyl 7.8 0.05 be replaced with toluene instead of acetonitrile when diluting
301. o 175. o 35
to volume. That is, lml toluene is added after nitrogen
364. o 238. o 21 blowing concentrating.
151 Tolylfluanid 7. 6 0.05
364. o 137. o 38
 2351 Determination of Aflatoxins

Use octadecylsilane bonded silica gel as the stationary phase. Use

10 mmol/L ammomium acetate solution as mobile phase A,
2351 Determination of Aflatoxins methanol as mobile phase B at the rate of O. 3 ml per minute and
the column temperature of 25ºC . Elute with the following
Method I gradient elution program.
High performance liquid chromatography ( 0512) is used for
Time ( min) Mobile phase A ( % ) Mobíle phase B ( % )
the determination of aflatoxins ( calculated as the total amount of
aflatoxin Bi, aflatoxin a, aflatoxin Gr and aflatoxin Gz) in 0-4. 5 65-15 35-85
crude drugs and decoction pieces. Use the following method
unless otherwise direction. 4. 5-6 15-0 85-100
Chromatographic system and system suitability Use 6-6. 5 o-65 100-35
octadecylsilane bonded silica gel as the stationary phase anda
mixture of methanol, acetonitrile and water (40 : 18: 42) as 6. 5-10 65 35
the mobile phase. A post-column derivatizatíon method is
used as the detecting method. (1) Derivatízation with íodine: Detector: triple-quadrupole mass spectrometer. Ion source:
use a O. 05% of iodine solution (díssolve O. 5 g of iodine with ESI. Acquisition mode: positive ion mode. Reference
100 ml of methanol, and dílute with water to 1000 mD as monitoring ion pair and collision energy (CE) are shown in
derivatisation reagent, maintaín the rate of derívatisation the following table.
pump at O. 3 ml per minute and the derivatisation
temperature at 70ºC. (2) Derivatization with photochemical Monitoring ion pair and collision energy ( CE } of Aflatoxins
reactor ( 254nm). Detect with a fluorescence detector, and Bi,~' G1, Gz
set the excitation wavelength at 360 nm (or 365 nm) and
emission wavelength at 450 nm. The resolution between the Precursor Product
No. Aflatoxin CE (V)
neighboring chromatographic peaks should be more than l. 5. 10n ion

Preparation of mixed reference solution Accurately 331. 1 313. 1 33

measure O. 5 ml of the aflatoxin standard solution ( marked 1 Aflatoxin Gz
concentration at l. O µ.g per ml of aflatoxin Bi, O. 3 µ.g per ml 331. 1 245. 1 40
of aflatoxin a , l. o µ.g per ml of aflatoxin Gr and O. 3 µ.g per
329. 1 243. 1 35
mi of aflatoxin G2 ) , add it into a 10 ml volumetric flask, and 2 Aflatoxin G1
dilute it wíth methanol to volume to prepare the stock 329. 1 311. 1 30
solution. Accurately measure 1 ml of the stock solution, add
it into a 25 ml volumetric flask, and dilute it with methanol 315. 1 259. 1 35
to volume.
3 Aflatoxin a
315. 1 287. 1 40
Preparation of test solution Accurately weigh 15 g of the
powder to be examined (pulverized through No. 2 sieve) in a
313. 1 241. o 50
4 Aflatoxin B1
homogeneous flask, add 3 g of sodium chloride, accurately 313. 1 285. 1 40
add 75 ml of 70% methanol, stír for 2 minutes at high speed
(more than 11 000 rpm) , and centrifuge ( 2500 rpm) for Preparation of mixed reference solution series Measure
5 minutes. Accurately measure 15 ml of the supematant accurately proper amount of the aflatoxin standard solution
liquid and add it into a 50 ml volumetric flask, dilute with ( marked concentration at l. O µ.g per ml of aflatoxin Br,
water to volume, mix well and filter through a micropore O. 3 µ.g per ml of aflatoxin a , l. O µ.g per ml of aflatoxin Gr
membrane filter ( O. 45 µ.m ) . Measure 20. O ml of the and O. 3 µg per ml of aflatoxin Gz ) , dilute it with 70 %
subsequent filtrate, apply to an ímmunoaffinity column, methanol to prepare the reference solution series containing
wash the column with 20 ml of water at the rate of 3 ml per aflatoxin a,
Gz of o. 04-3 ng per ml and aflatoxin Bi , Gr of
minute, discard the washing, allow air into the column and O. 12-10 ng per ml respectively. (If necessary, prepare the
squeeze out of water, elute with a quantíty of methanol, matrix reference solution series according to the situation of
collect the eluate in a 2 ml volumetríc flask, dílute with the sample. )
methanol to volume and mix well.
Preparation of test solution the same as method I.
Determination Accurately inject 5 µ.l, 10 µ.l, 15 µ.l, 20 µ.l
Determination Accurately inject 5 µl of the reference
and 25 µ.l of the mixed reference solution into the column solution series into LC-MS respectively and measure the peak
respectively and measure the peak area. Plot the standard
areas. Plot the standard curve, using the peak areas as the
curve, using the peak areas as the ordinate and the
ordinate and the corresponding concentrations as the
corresponding inject amounts as the abscissa. Accurately
abscissa. Accurately inject 5µ.l of the test solution into LC-
inject 20-25 µ.l of the test solution into the column and
MS and measure the peak area. Calculate the concentration
measure the peak area. Calculate the amount of aflatoxin Br,
of aflatoxin Br, aflatoxin Bz, aflatoxin Gi, aflatoxin Gz in
aflatoxin a , aflatoxin Gr and aflatoxin Gz in the test solution the test solution from the standard curve.
from the standard curve.
Notes ( 1) Safety and preventive measures should be
Method ll satisfied during the experiment, and the pollution to
High performance liquid chromatography/ tandem mass enviroment is not allowed.
spectrometry is used for the determination of aflatoxins (2) The glassware containing residue of the waste liquor or
( calculated as the total amount of aflatoxin Bi ' aflatoxin a' leavings should be soaked into solution of 10% sodium
aflatoxin Gr and aflatoxin Gz ) in crude drugs and the decoction hypochlorite in a special storage vessel for over 24 hours, and
pieces. Use the following method unless otherwise direction. then rinse with water.
Oiromatographic/mass spectrwn system and system suitability (3) If the determination results exceed the limit, confirm it
 3102
by method 11 •
2400 Determination of
Materials in lnjections
Materials in injections refer to the residues that need to be
controlled after the chinese medicinal materials having been
extracted, purified and made into injections.
Unless otherwise specified, the content of protein, tannin
and resin should be controlled far injections. Beyond that,
the content of oxalate and potassium ion should be controlled
far intravenous injections. The determination proceeds as
fallows:
Protein Unless otherwise specified, measure 1 ml of the
test injection, add 1 ml of a fresh 30% sulfonic salicylic acid
TS, mix well and allow to stand far 5 minutes. Opalescence
should not be observed. If the injection contains components
which could react with acid to farm precipitation, add 1-
3 drops of tannic acid TS instead. Opalescence should not be
observed.
Tannin Unless otherwise specified, measure 1 mi of the
test injection, add 5 ml of a fresh sodium chloride
physiological solution containing 1 % egg white and allow to
stand for 1O minutes. Opalescence should not be observed. If
it occured, measure 1 ml of the test injection, add 1 drop of
dilute acetic acid, and then add 4-5 drops of sodium chloride
gelatin TS. Opalescence or precipitate should not be observed.
Injections which contain components such as macrogol and
polysorbate will not farm precipitation even if they contain
tannin. For these injections, the semi-finished products
should be determined befare adding additives.
Resin Unless otherwise specified, measure 5 ml of the test
injection, add 1 drop of hydrochloric acid and allow to stand
for 30 minutes. Floccules should not be observed. If it
occured, measure 5 ml of the test injection, add 10 ml of
chloroform instead, shake well, separate the chlorofarm
layer, and evaporate to dryness on a water bath. Add 2 ml of
glacial acetic acid to dissolve the residues, then pour the
3000 Special Mothods f or Biological Products
3100 The Mothods for Content Determination
3101 Determination of Total Solid
The method is based on the evaporation of the liquid
components of test sample at a certain temperature. The
content of total solid is calculated from the residual solid.
Procedure
Method 1 Drying at 105°C
Measure accurately a quantity of test sample and put into a
suitable weighing bottle, which has been dried to constant
weight. Dry to constant weight in an oven at lOSºC.
Method 2 Drying at SOºC
Measure accurately a quantity of test sample anci put into a
Determination of Sialic Acid Content
liquid into a stoppered tube, add 3 ml of water, mix well and
allow to stand far 30 minutes. precipitate should not be
observed.
Oxalate For intravenous injections, unless otherwise
specified, measure 2 ml of the test injection and adjust the
pH value to 1-2 with dilute hydrochloric acid. Filter and
adjust the pH value of the filtrate to 5-6. Add 2-3 drops of
3 % calcium chloride solution and allow to stand for 10 minutes.
No opalescence or precipitate should be observed.
Potassium ion For intravenous injections, unless otherwise
specified, measure 2 ml of the test injection and evaporate to
dryness. lgnite the residues with weak flame till charring
occurs, and then incinerate at a temperature of 500-600ºC
until free from carbon. Add 2ml of dilute acetic acid to
dissolve the ash. Transfer the solution into a 25 ml
volumetric flask, dilute with water to the scale and mix well.
Use the solution as test solution.
Take two 10 ml Nessler cylinders. Measure accurately
O. 8 ml of potassium ion standard solution in cylinder A. Add
O. 6 ml of alkaline formaldehyde ( measure formaldehyde
solution, adjust the pH value to 8. 0-9. O with O. lmol/L
sodium hydroxide), 2 drops of a 3% disodium edetate
solution and O. 5 ml of a 3 % sodium tetraphenylborate
solution. Dilute with water to 10 ml and shake well.
Meanwhile, measure accurately 1 ml of the test solution in
cylinder B, and carry out the operation in exactly the same
manner as for cylinder A. Compare the two Nessler cylinders
by viewing down the vertical axis of the cylinders against a
black background. Opalescence of cylinder B should not be
more pronounced than that of cylinder A.
Note Preparation of potassium ion standard solution.
Grind a defined amount of potassium sulfate into fine
powder, and dry to constant weight at llOºC. Accurately
weigh 2. 330 g into a 1000 mi volumetric flask, add sufficient
water to dissolve the substance and dilute to the scale. Shake
well. Use the solution as stock solution. Befare using,
measure accurately the stock solution 10 ml in a 100 ml
volumetric flask, dilute with water to volume and shake well
Oml of the solution is equivalent to 100 µg of potassium).
suitable weighing bottle, which has been dried to constant
weight. Dry to constant weight in an oven at 50ºC.
Calculate according to the fallowing equation:
Total salid of test sarnple, % (g/ ml) = WX 100 /V
Where: W=Constant weight of test sample after drying, g;
V=Volume of test sample, ml.
3102 Determination of Sialic Acid
Content
(Resorcinol Colourimetry)
The method is based on the principie that conjugated sialic
acid becomes free acid after acid hydrolysis. Free sialic acid
 3103 Determination of Phosphorus Content
reacts with resorcinol to form a coloured compound. The
sialic acid content is determined af ter the coloured compound
is extracted wi th organic a cid.
Preparation of 200 µg/ml sialic acid reference solution
Weigh accurately 10. 52 g of sialic acid CRS ( 1 µg is
equivalent to 3. 24 nmoD and dissolve in a small amount of
water. Transfer totally to a 10 ml volumetric flask and dilute
to volume with water. Mix well to prepare a 1 mg/ml sialic
acid reference stock solution. Dispense the stock solution to
the volume for one test and store at - 70ºC. The validity
period of the dispensed standard is 12 months. It can only be
frozen and thawed once. The validity period is 2 weeks if it is
stored at 4 ºC.
Measure accurately 1 ml of sialic acid reference stock solution
(1 mg/ml) and put into a 5 ml volumetric flask. Dilute to
volume with water. The sialic acid reference solution at a
concentration of 200 µg per ml shall be prepared just before
use.
For the determination of sialic acid content in Group C
meningococcal polysaccharide vaccine, 400 µg/ml sialic acid
reterence stock solution is performed by the above-mentioned
procedure (Weigh accurately 40 mg of sialic acid and transfer
totally to a 100 ml volumetric flask and dilute to volume with
purified water, mix well). Measure accurately 2. O ml of
sialic acid reference stock solution and put into a 10 ml
volumetric flask. Dilute to volume with water. The sialic
acid reference solution at a concentration of 80 µg per ml
shall be prepared just before use.
Procedure
Diiute a quantity of test sampie with water to a protein
concentration of about O. 2-0. 4 mg per ml. To a set of glass
test tubes, add sialic acid reference solution, test sample
solution and water respectively according to the volume listed
in the following table, mix well. To each tube, add 1 ml of
resorcinol-hydrochloric acid solution ( Mix 2. 5 ml of 2%
resorcinol solution and 62. 5 µl of O. 1 mol/L cupric sulfate
solution with 20 ml of 25 % hydrochloric acid. Dilute to 25
ml with water and mix well. This solution is prepared within
4 hours prior to the test). Stopper the tubes and heat in a
boiling water bath for 30 minutes with their liquid surface
level about 2 cm lower than that of water bath. Take the test
tubes out of boiling water bath and cool in an ice bath for 3
minutes with shaking. Then, add 2 ml of butyl acetate-
butanol solution (Mix four volumes of butyl acetate with one
volume of butanol. Store at room temperature and use within
12 hours) to each tu be and mix well. Allow the mixture to
stand at room temperature for 1O minutes. Read the absorbance
at 580 nm by ultraviolet-visible spectrophotometry <0401 >.
Sialic acid CRS tubes Sample
Tube
Blank 2 µg 4 µg 5 µg 6 µg 8 µg tube
Sialic acid
reference 10 20 25 30 40
solution CµD
Water CµD 100 90 80 75 70 60
Test sample
100
solution CµD
For the determination of sialic acid content in meningococcal
polysaccharide vaccine, Measure 2. O ml of test sample
solution (containing about 40 µg sialic acid/ml) and 2 ml of
blank control (water) , Measure O. 1, O. 2, O. 4, O. 8, l. 6
ml of sialic acid reference solution (80 µg/mD and put into a
series of test tubes separately, make up each volume to
2. O ml with water, Add 2 ml of chromogenic reagent (Mix
O. 5 ml of O. 1 mol/L copper sulfate solution, 5 ml of 4 %
resorcinol solution, 80 ml of hydrochloric acid, make up
volume to 100 ml with water. Prepare the stock just before
use.) and mix well. Heat in a boiling water bath for
15 minutes and then cool in an ice bath for 5-10 minutes,
add 4 ml of organic phase (dissolve 15 ml of butanol in butyl
acetate and dilute to 100 ml) and mix well, allow the
mixture to stand at room temperature for 10 minutes. Read
the absorbance at 585 nm by ultraviolet-visible
spectrophotometry, A linear regression equation is obtained
by regressing the concentration of sialic acid reference
solutions with the corresponding absorbance. ( It can be
scaled clown for the volume of test sampie and the reagent)
A linear regression equation is obtained by regressing the
concentrations of sialic acid reference solution with the
corresponding absorbance ( the correlation coefficient shall be
not less than O. 99). The absorbance of sialic acid (5 µg) is
obtained from the linear regression equation. The sialic acid
content of test sample is calculated by the following equation.
Sialic acid content of test sample (mol/mol Protein) =A 2 X
5X3. 24XWXD/ CA1 XPXlOO)
Where: A 1 = Absorbance of sialic acid CRS ( 5 µg) ;
Az =Absorbance of test sample;
D = Dilution factor of test sample;
P = Protein content of test sample, µg/ µl;
W = Amount of 1 nmol erythropoietin is equivalent
to 30. 6 µg.
sialic acid content in Group C meningoc.occ;:il polysaccharide
vaccme ( µg/ ml) =A X n
Where: A= Concentration of sialic acid reference solution
which absorbance equal to the test sample
solution; µg/ml;
n= Dilution factor of test sample.
3103 Determination of Phosphorus
Content
The method is based on the principie that organic phosphorus
is transformed into inorganic phosphorus. The phosphate
radical reacts with ammonium molybdate in acid solution and
forms ammonium phosphomolybdate. In the presence of reducing
agent, phosphomolybdate is reduced to a blue colour substance
called " Molybdenum blue" ( a mixture of molybdenum
trioxide and molybdenum pentoxide). The phosphorus m
test sample is determined by spectrophotometry.
Procedure
Measure accurately a quantity of test sample ( containing
about 4-20 µg of phosphorus) and put into a test tube. Add
four drops ( about O. 08 mD of sulfuric acid and heat until
carbonization occurs. Add two drops Cabout o. 06 mD of
perchloric acid. Digest until the liquid becomes clear and
colourless. Allow to stand for a while and add 2 ml of water
immediately. Add O. 4 ml of O. 04 mol/L ammonium
molybdate solution (Dissolve 5 g of ammonium molybdate in
water and dilute to 100 ml with water) and mix well. Then,
add O. 2 ml of reducing reagent CDissolve 6 g of sodium
bisulfite, l. 2 g of sodium sulfite and O. 1 g of l-amino-2-
naphthol-4-sulfonic acid in water and dilute to 50 ml. Store
in a brown colour bottle and use within a week) and mix well
again. Dilute to 6 ml with water. Allow to stand for 15-20
minutes and read the absorbance at 820 nm by UV-visible
spectrophotometry <0401 ) .
Weigh accurately 439. 3 mg of potassium dihydrogen
 3106 Determination of Aluminium Hydroxide ( or Aluminium Phosphate) Content
phosphate, which has been dried to constant weight, and
dissolve in a small volume of water. Transfer totally to a 100
ml volumetric flask and dilute to volume with water.
Measure accurately 2 ml of the above phosphorus solution
and put into a 100 ml volumetric flask. Dilute to volume with
water to prepare a phosphorus reference solution at a
concentration of 20 µg per ml.
Measure accurately O. 2, O. 4, O. 6, O. 8 and l. O ml of
phosphorus reference solution and put into a series of test
tubes separately. Make up each volume to 1 ml with water.
Carry out the same procedure as that far test sample starting
from the addition of sulfuric acid and read the absorbance of
each tube.
A linear regression equation is obtained by regressing the volume
of phosphorus reference solution with the corresponding
absorbance. The volume of test sample is obtained by inserting
its absorbance into the regression equation.
Phosphorus content of test sample (µg/mD =
VXCR/Vx
Where: V = Volume of phosphorus reference solution
corresponding to the absorbance of the test
sample, ml;
CR = Concentration of phosphorus reference
solution, µg/ml;
Vx=Volume of test sample, ml.
[Note]
( 1 ) If necessary, one to two drops of 30 % hydrogen
peroxide may be added during the digestion with perchloric
acid but hydrogen peroxide must be decomposed completely
at last.
(2) If the mixture after digestion with perchloric acid is
cooled clown, it shall be heated again befare adding water.
( 3) To determine the phosphorus content of group A
mer1Íri..gococcal polysaccharide vaccine; at least three a.rnpoules of
test samples shall be reconstituted and mixed far determination.
3104 Determination of Ammonium
Sulfate Content
The method is based on the principle that ammonium sulfate
may be decomposed by sodium hydroxide and ammonia is
released. The released ammonia is then absorbed by boric
acid to farm ammonium borate, which may be titrated with
acid VS. The ammonium sulfate content of test sample can
be calculated according to the volume of acid VS consumed.
Preparation of test sample solution
The method far deproteinization is the same as that far the
determination of protein content ( 0731 , method 1 ) .
Procedure
Measure accurately 10 ml of deproteinized filtrate and put
into a Kjeldahl distillator. Add 1 ml of 4% sodium hydroxide
solution and a small volume of water. Distill and titrate by
the method far determination of nitrogen ( 0704 ) . Calibrate
the titration result with a blank test.
Calculate according to the fallowing equation:
Ammonium sulfate content C%) = CV1 -Yo) X
C X 14. 01X4. 715X2X100/1000
Where: V1 =Titer of sample, ml;
V 2 =Titer of blank control, ml;
C=Concentration of sulfuric acid VS, mol/L;
The value of 4. 715 is a constant ( 1 g of nitrogen is
equivalent to 4. 715 g of ammonium sulfate);
The value of 14. 01 is the relative atomic mass of nitrogen.
3105 Determination of Sodium
Bisulfite Content
The method is based on the principle that sodium bisulfite
may react with an excess of iodine. The surplus iodine is
then titrated with sodium thiosulfate VS. The sodium
bisulfate content of test sample can be calculated according to
the volume of sodium thiosulfate VS consumed.
Procedure
Measure accurately a quantity of test sample ( containing
about 2. 5 mg of sodium bisulfite) and put into a conical
flask with stopper. Add accurately 20 ml of O. 05 mol/L
iodine solution (Dissolve 36 g of potassium iodide and 13. O g
of iodine in 50 ml of water successively. Add three drops of
hydrochloric acid and dilute to 1000 ml with water. Mix well and
filter with sintered-glass filter) , allow to stand far 5 minutes.
Add 2. O ml of hydrochloric acid ( 5- 1O) along the inside
wall of the bottle and mix well. Titrate with O. 1 mol/L
sodium thiosulfate VS until the end point is nearly reached.
Add O. 5 ml of O. 5 % starch indicator solution and continue
the titration until the disappearance of blue colour. Calibrate
the titration result with a blank test.
Calculate according the fallowing equation:
Sodium bisulfite content ( % ) =
CV0-V1) XCX5. 203Xl00/ CV2 XlOOO)
Where: Va =Titer of blank test, ml;
V1 =Titer of test sample, ml;
Vz =Volume of test sample, ml;
C = Concentration of sodium thiosulfate VS,
mol/L.
[Note]
Preparation and titration of O. 1 moi/L sodium thiosuifate VS
Dissolve 26 g of sodium thiosulfate and O. 20 g of anhydrous
sodium carbonate in freshly boiled and cooled water and dilute to
1000 ml, mix well, allow to stand far one month and filter.
Weigh accurately O. 15 g of potassium dichromate primary
standard, which has been dried to constant weight at 120ºC,
and put into an iodine bottle containing 50 ml of water far
dissolution. Add 2. O g of potassium iodide and dissolve by
shaking gently. Add 40 ml of dilute hydrochloride acid
(5. 7-100). Stopper the bottle tightly and mix well. Allow
the mixture to stand in a dark place far 10 minutes. Add 250
ml of water and titrate with the sodium thiosulfate solution
until the end point is nearly reached. Add 3 ml of starch
indicator solution (Suspend O. 5 g of soluble starch in 5 ml of
water and pour slowly into 100 ml of boiling water with
constantly stirring. Continue the boiling far 2 minutes and
cool clown. Decant and collect the upper clear liquid. The
solution shall be prepared just befare use) and continue the
titration until the blue colour disappears and a brilliant green
colour appears. Calibrate the titration result with a blank
test. One ml of sodium thiosulfate VS is equivalent to 4. 903
g of potassium dichromate. Calculate the concentration of
sodium thiosulfate VS according to the weight of potassium
dichromate taken and the volume of the sodium thiosulfate
solution consumed.
3106 Determination of Aluminium Hydroxide
( or Aluminium Phosphate) Content
The method is based on the principle that aluminium ion
reacts with an excess of sodium ethylenediamine tetraacetate
 3107 Determination of Sodium Chloride Content
and the surplus sodium ethylenediamine tetraacetate is titrated
with zinc VS. The aluminium hydroxide ( or aluminium
phosphate) content in test sample can be calculated
according to the volume of zinc VS consumed.
Procedure
Measure accurately a quantity of test sample ( containing
about 1-10 mg of aluminium) and transfer to a 250 ml
conical flask. Dissolve completely by adding l. 5 ml of
phosphoric acid solution (6-100). Warm in a water bath if
necessary Can additional volume of phosphoric acid solution
may be needed to the test samples which is not easy to
dissolve). Add accurately 10 ml of O. 05 mol/L sodium
ethylenediamine tetraacetate VS. Then, add 10 ml of
ammonium acetate buffer solution (pH 4. 5, dissolve 7. 7 g
of ammonium acetate in 50 ml of water, add 6 ml of glacial
acetic acid and dilute to 100 ml with water). Heat in boiling
water bath far 10 minutes. Take out the flask from the water
bath and cool clown to room temperature. Add 1 ml of
dicresol ( xylenol) orange indicator solution. Titrate with
O. 025 mol/L zinc VS to the end point when the colour of
solution changes from brilliant yellow to orange. Calibrate
the titration result with a blank test.
Calculate according to the following equation:
Aluminium hydroxide content (mg/mD
= CV0-V1) XCX78.0l/V2
Aluminium phosphate content (mg/mD
= CV0-V1) XCX121.95/V2
Aluminium content ( mg/ mD
= CV0-V1) XCX26. 98/Vz
Where: Vo =Titer of blank test, ml;
Y1 =Titer of test sample, ml;
C=Concentration of zinc VS, mol/L;
V2 =Volume of test sample, ml;
The values of 78. 01, 121. 95 and 26. 98 are the
relative molecular ( atomic) masses of aluminium
hydroxide, aluminium phosphate and aluminium
respectively.
[Note]
(1) Preparation and titration of O. 05 mol/L zinc VS
Weigh 15 g of zinc sulfate (equivalent to about 3. 3 g of zinc)
and mix with 10 ml of dilute hydrochloric acid (23. 4-100).
Dissolve in a quantity of water and dilute to 1000 ml with
water. Mix well. Measure accurately 25 ml of the solution.
Add one drop of O. 025 % methyl red ethanol solution. Add
ammonia test solution dropwise until the solution becomes
slightly yellow. Add 25 ml of water, 10 ml of ammonia-
ammonium chloride buffer solution (pH 10. O) and a small
amount of eriochrome black T indicator solution. Titrate
with O. 05 mol/L sodium ethylenediamine tetraacetate VS
until the colour changes from purple to pure blue. Calibrate
the titration result with a blank test. The concentration of
the zinc solution is calculated according to the volume of O. 05
mol/L sodium ethylenediamine tetraacetate VS consumed.
(2) Preparation of O. 025 mol/L zinc VS
Measure accurately 100 ml of O. 05 mol/L zinc VS and dilute
volumetrically to 200 ml with water.
( 3) Preparation and titration of O. 05 mol/L sodium
ethylenediamine tetraacetate VS
Dissolve 19 g of sodium ethylenediamine tetraacetate in water
and dilute to 1000 ml. Mix well. Weigh accurately O. 12 g of
zinc oxide primary standard, which has been ignited to
constant weight at about 800ºC. Add 3 ml of dilute
hydrochloric acid ( 23. 4-100) to dissolve the zinc oxide.
Add 25 ml of water and one drop of O. 025 % methyl red
ethanol solution. Add ammonia test solution dropwise until
the colour of solution becomes slightly yellow. Add 25 ml of
water, 10 ml of ammonia-ammonium chloride buffer
solution ( pH 10. O) and a small amount of eriochrome black
T indicator solution. Titrate with O. 05 mol/L sodium
ethylenediamine tetraacetate VS until the colour of the
solution changes from purple to pure blue. Calibrate the
titration result with a blank test. One ml of O. 05 mol/L
sodium ethylenediamine tetraacetate VS is equivalent
to 4. 069 mg of zinc oxide. Calculate the concentration of the
volumetric solution according to the weight of zinc oxide
taken and the volume of the volumetric solution consumed.
3107 Determination of Sodium
Chloride Content
The method is based on the principie that sodium chloride
reacts with an excess of silver nitrate after the proteins in test
sample are destroyed by nitric acid. Chloride ion reacts
completely with silver nitrate and is precipitated out as sliver
chloride. The surplus silver nitrate is then titrated with
ammonium thiocyanate VS. The sodium chloride content can
be calculated according to the volume of ammonium
thiocyanate VS consumed.
Procedure
Measure accurately l. O ml of test sample and add accurately
5 ml of O. 1 mol/L sil ver nitra te solution (Dissolve 17. O g of
silver nitrate in water and dilute to 1000 mD. If the protein
content of test sample is high, add 2 ml of saturated
potassium permanganate soiution. Mix weii and add 10 mi oí
8. O mol/L nitric acid solution. Digest by heating until the
solution becomes clear. Cool clown. Add 50 ml of water and
1 ml of 8% ferric ammonium sulfate indicator solution.
Titrate with O. 05 mol/L ammonium thiocyanate VS until a
faint brownish red colour appears. The end point is reached
whenever the colour persists after shaking. Calibrate the
titration result with a blank test (digestion may be omitted).
Cá.lculate according to the following equation:
Sodium chloride content (g/L)
CVo-Vx) XCX58. 45
Where: Vo =Titer of blank test, ml;
Vx =Titer of test sample, ml;
C=Concentration of ammonium
thiocyanate VS, mol/L;
The value of 58. 45 is the relative molecular mass of sodium
chloride.
[Note]
(1) Preparation and titration of O. 1 mol/L ammonium
thiocyanate VS
Dissolve 8. O g of ammonium thiocyanate in water and dilute
to 1000 ml. Mix well Measure accurately 25 ml of O. 1 mol/L
silver nitrate VS. Add 50 ml of water, 2 ml of nitric acid and
2 ml of 8% ammonium ferric sulfate indicator solution.
Titrate with the ammonium thiocyanate solution until a faint
brownish red colour appears. The end point is reached
whenever the colour persists after shaking vigorously. The
concentration of the ammonium thiocyanate VS is calculated
according to the volume of the volumetric solution consumed.
(2) Preparation of O. 05 mol/L ammonium thiocyanate VS
Measure accurately 100 ml of O. 1 mol/L ammonium
thiocyanate VS and dilute volumetrically to 200 ml with
water. Mix well.

3108 Determination of Citrate Content
Method 1 Colorimetric Method
Preparation of sodium citrate reference solution
Weigh accurately O. 6 g of sodium citrate (C;Hs NaaCh•2H2Q),
which has been dried to constant weight under reduced pressure,
and dissolve in a small volume of water. Transfer totally to a
100 ml volumetric flask and dilute to volume with water.
Mix well. Measure accurately 5 ml of the solution and put
in to a 50 ml volumetric flask. Dilute to volume with 5 %
trichloroacetic acid and mix well.
Preparation of test sample solution
Measure accurately O. 5 ml of test sample. Add 4. 5 ml of
water and 5 ml of 10% trichloroacetic acid. Mix well and
heat in 60ºC water bath for 5 minutes. Centrifuge at 4000 r/min
for 20 minutes and collect the supernatant for use.
Procedure
Measure accurately 1 ml of the test sample solution and put
into a 25 ml glass test tube with glass stopper. Add
accurately l. 3 ml of pyridine and mix well. Then, add
accurately 5. 7 ml of acetic anhydride. Mix well immediately
and put the tube into 31 ºC ± 1 ºC water bath for exact
35 minutes. Read the absorbance at 425 nm by ultraviolet-
visible spectrophotometry ( 0401 ) .
Measure accurately O. 25, O. 5, O. 75 and l. 00 ml of sodium
citrate reference solution and put separately into a series of
25 ml glass test tubes with stoppers. Add accurately O. 75,
O. 50, O. 25 and O. 00 ml of 5 % trichloroacetic acid
respectively ( The corresponding citrate concentrations of
these mixtures are O. 5, l. O, l. 5 and 2. O mmol/L
respectively). The same procedures as mentioned above are
carried out starting from "Add precisely l. 3 ml of pyridine".
A linear regression equation is obtained by regressing the
concentration of citrate reference solution with the
corresponding absorbance. Calculate the citrate concentration
( mmol/L) of the test sample solution and multiply the
result by dilution factor (20), and the concentration of the
test sample in mmol/L is obtained
Method 2 Ion Chromatographic Method
The citric content is determined by high performance liquid
chromatography ( 0512).
Parameters of chromatography
Ion exchange chromatograph column ( H+ ) at an inner
diameter of 7. 8 mm and a length of 300 mm is used The
column is packed with a co-polymer of styrene and
divinylbenzene ata grain size of 9 µmor 8 µm. The column
temperature is 50ºC. The mobile phase is O. 004 mol/L
sulfuric acid at a flow rate of O. 8 ml per minute and the
detector is differential refractometer.
Procedure
Weighaccurately0.735 g of sodium citrate (C;Hs Na301•
2H 20), which has been dried to constant weight under
reduced pressure, and dissolve in a small volume of water.
Transfer totally to a 100 ml volumetric flask and dilute to
volume with water. Measure accurately 5. O, 10. O and 15. O
ml of the sodium citrate solution and transfer to three 25 ml
volumetric flasks separately. Dilute to volume with water
and mix well to prepare three citrate reference solutions at
concentrations of 5. O, 10. O and 15. O mmol/L respectively.
3108 Determination of Citrate Content
Measure accurately 20 µl of reference solution from each of
the three tubes and inject into the chromatographic column.
Record the chromatograms.
Measure accurately 1 ml of test sample solution and put into
a 15 ml centrifuga! tube. Add accurately 1 ml of l. 5%
sulfosalicylic acid solution and mix well Centrifuge at 2000
r/ min for 10 minutes at room temperature. Determine the
supematant by the same method as above.
A linear equation is obtained by regressing the concentration
of citrate reference solutions with the corresponding peak
areas. Calculate the sodium citrate concentration (mmol/L)
of the test sample solution. The citrate concentration
(mmol/L) of the test sample is obtained by multiplying the
result by the dilution factor (2) of test sample.
[Note]
(1) The concentration of citrate reference solutions may be
adjusted according to the citrate concentration of test sample.
(2) The correlation coefficient of the linear regression shall
be not less than O. 999.
(3) The mobile phase and its flow rate, column temperature
for cation chromatographic column ( H+ ) of different
manufacturers may be different. The operation parameters
can be properly adjusted according to the instructions.
Method 3 High Performance Liquid
Chromatographic (HPLC) Method
The citric content is determined by high performance liquid
chromatography ( 0512).
Parameters of chromatography
The chromatographic column atan inner diameter of 4. 6 mm
and a length of 250 mm, filled with octadecylsilyl silica gel
(grain size 5 µm) , is used. The mobile phase is 18. 2 mmol/L
phosphate buffer and O. 1 % isopropanol solution ( pH2. 0-
2. 5). Column temperature is 40ºC. The flo\v rate is l. O ml
per minute. The temperature of sample cell is room
temperature. The time for operation is 50 minutes. The
wavelength for detection by ultraviolet detector is 210 nm.
Measure 20 µl of 5. O mmol/L citric acid ion solution and
InJect into the chromatographic column. Record the
chromatograms. The tailing factor calculated as the
chromatographic peak of citrate shall be O. 95-1. 40.
Procedure
Weigh precisely O. 735 g of sodium citrate
( C6 Hs Na3 01 • 2 H2 O) , which has been dried to constant
weight under reduced pressure, and dissolve in a small
volume of ultra-pure water. Transfer totally to a 100 ml
volumetric flask and dilute to volume with ultra-pure water.
Measure precisely 5. O, 10. O and 15. O ml of the sodium
citrate solution and transfer to three 25 ml volumetric flasks
separately. Dilute to volume with water and mix well to
prepare three citrate reference solutions at concentrations of
5. O, 10. O and 15. O mmol/L respectively. Measure precisely
20 µl of reference solution from each of the three tubes and
InJect into the chromatographic column. Record the
chromatograms.
Measure precisely 1 ml of test sample solution and put into a
15 ml centrifuga! tube. Add precisely 4 ml of l. 5 %
sulfosalicylic acid solution and mix well, allow to stand at
room temperature for more than 2 hours and centrifuge at 3000
r/ min for 10 minutes. Determine the supernatant by the same
method as above.
A linear equation is obtained by regressing the concentrations
of citrate reference solutions with the corresponding peak
areas. Calculate the citrate concentration ( mmol/L) of the
test sample solution. The citrate concentration ( mmol/L)
 3109 Determination of Potassium Content
of the test sample is obtained by multiplying the result by the
dilution factor (5) of test sample.
[Note]
(1) The concentration of citrate reference solutions may be
adjusted according to the citrate concentration of test sample.
( 2) The amount of sulfosalicylic acid solution can be properly
adjusted according to the protein concentration of test sample.
(3) The correlation coefficient of the linear regression shall
be not less than O. 999.
3109 Determination of Potassium
Content
The potassium content in test sample is determined with
flame photometry.
Procedure
Measure accurately 2 ml of test sample and put into a 50 ml
volumetric flask. Dilute to volume with water to prepare a
test sample solution. Read the intensity of the light emitted
from test sample solution at 769 nm by flame photometry
( 0407). Weigh accurately 56. O mg of potassium chloride,
which has been dried to constant weight at llOºC, and
dissolve with a quantity of water. Transfer totally to a 500
ml volumetric flask and dilute to volume with water.
Measure accurately l. O, 2. O, 3. O, 4. O and 5. O ml of the
above solution and put into a series of 50 ml volumetric flasks
separately. Dilute to volume with water to prepare a series of
potassium reference solutions at concentrations of O. 03,
O. 06, O. 09, O. 12 and O. 15 mmol/L respectively. Carry out
the test with the same procedures as that for test sample
solution.
A linear regression equation is obtained by regressing the
concentration of potassium reference solutions with the
corresponding intensity of emitted light. The potassium
concentration of test sample solution in mmol/L is obtained
by inserting its intensity of emitted light into the linear
regression equation. Calculate the potassium content ( mmol/L)
of test sample by multiplying the result with the dilution
factor (25) of test sample.
3110 Determination of Sodium
Content
The sodium content in test sample is determined with flame
photometry.
Procedure
Measure accurately O. 5 ml of test sample and put into a 50
ml volumetric flask. Dilute to volume with water to prepare
a test sample solution. Read the intensity of light emitted
from test sample solution at 589 nm by flame photometry
(0407>. Weigh accurately O. 293 mg of sodium chloride,
which has been dried to constant weight at l lOºC, and
dissolve in a small volume of water. Transfer totally into a
100 ml volumetric flask and dilute to volume with water.
Measure accurately O. 9, l. 1, l. 3, l. 5 and l. 7 ml of the
above solution and put into a series of 50 ml volumetric flasks
separately. Dilute to volume with water to prepare a series of
sodium reference solutions at concentrations of O. 9, l. 1,
l. 3, l. 5 and l. 7 mmol/L respectively. Carry out the test
with the same procedure as that for test sample solution.
A linear regression equation is obtained by regressing the
concentration of sodium reference solutions with the
corresponding intensity of emitted light. The sodium
concentration of the test sample solution in mmol/L is
obtained by inserting its intensity of emitted light into the
linear regression equation. Calculate the sodium content
( mmol/L) of the test sample by multiplying the result by
the dilution factor 000) of test sample.
3111 Determination of Sodium
Caprylate
The sodium caprylate content in test sample is determined by
gas chromatography ( 0521 ) .
Parameters for chromatography and test for system suitability
Acid denatured polyethylene glycol ( 20M) capillary column
is used. The temperature of column is 160ºC and that of
vaporizer is 230ºC. Flame ionization detector (FID) is used
at the temperature of 23üºC. Flow gas is pure nitrogen at a
flow rate of 35 ml per minute. The separation degree
between peaks of caprylic acid and heptanoic acid shall be
greater than l. 5. The tailing factor for the peak of caprylic
acid shall be O. 95-1. 20. The relative standard deviation
CRSD) for the peak area ratio of caprylic acid to heptanoic
acid shall be not greater than 5 % in five success1ve
determinations of caprylic acid reference solution.
Preparation of intemal standard solution
Dissolve heptanoic acid in trichloromethane to prepare a
solution of 10 mg per ml.
Procedure
Dilute accurately a quantity of test sample with water to a
protein concentration of 40-50 g/L. Measure accurately O. 5
ml of the diluted sample in a test tube. Add 30 µl of interna!
standard solution and O. 2 ml of l. 5 mol/L perchloric acid
solution. Mix for 1 minute on an oscillator. Add 4 ml of
trichloromethane and lid a cover. Mix on an oscillator
vigorously for 2 minutes. Centrifuge at 3000 r/min for 20
minutes. Discard the upper water layer and pour the
trichloromethane layer into a 10 ml test tube carefully.
Evaporate out the trichloromethane until dry. Add 100 µl of
trichloromethane to dissolve the residue. Measure O. 1 µl and
inject into the gas chromatographic apparatus.
Weigh accurately about O. 15 g of caprylic acid and dissolve in
a small volume of trichloromethane. Transfer totally to a 10
ml volumetric flask and dilute to volume with
trichloromethane. Measure accurately 10, 20, 30, 40 and
50 µl of caprylic acid reference solution and put into a set of
test tubes separately. To each tube, add accurately 30 µl of
interna! standard solution. Mix for 1 minute on an oscillator.
Add 4 ml of trichloromethane to each tube and evaporate the
trichloromethane until dry. Add 100 µl of trichloromethane
to each tube to dissolve the residues and proceed with the
same procedures as above.
A regression equation is obtained by regressing the
concentration of caprylic acid reference solution with the
corresponding peak area ratios of caprylic acid CRS to the
internal standard. Calculate the amount of caprylic acid CA)
in test sample solution. The sodium caprylate content in test
sample is calculated by the following equation.
Sodium caprylate content (mmol/g protein) =
AXn/144. 22XBXCX1000
Where: A= Amount of caprylic acid in test sample solution,
µg;
B = Volume of test sample solution taken, i. e.
O. 5 ml;
n = Dilution factor of test sample;
C= Protein content of test sample, g/ml.
 3114
The value 144. 22 is the relative molecular mass of caprylic
acid.
[Note]
( 1) 1 mmol of caprylic acid is equivalent to 1 mmol of
sodium caprylate.
(2) The evaporation speed of sample solution and reference
solution shall be kept in confarmity as far as possible.
( 3) The correlation coefficient of the regression equation
shall be not less than O. 99.
( 4) The temperatures of column, detector and vaporizer as
well as flow rate of flow gas and volume of sample far
injection may be adjusted according to the directions of
manufacturers.
3112 Determination of
Acetyltryptophan
The N-acetyl-DL-tryptophan content in human albumin is
determined by UV-visible spectrophotometry ( absorption
coefficient method).
Procedure
Dilute the test sample of protein with physiological saline to a
concentration of 5 % to prepare the test sample solution.
Measure O. 1 ml of the test sample solution, add O. 3 ml of
physiological saline and 3. 6 ml of O. 3 mol/L perchloric acid
solution and mix well. Meanwhile, measure O. 4 ml of
physiological saline, add 3. 6 ml of O. 3 mol/L perchloric
acid solution, mix well and use as a blank control. Stand the
mixtures at room temperature far 10 minutes, then
centrifuge at 3500 r/min far 20 minutes. Collect the
supernatants and determine the absorbance at a wavelength
of 280 nm. Adjust the zero point with the blank control
solution. The N-acetyl-DL-tryptophan content in test sample
is calcuiated by the foiiowing equation.
N-acetyl-DL-tryptophan content ( mmol/ g)
in test sample= (A280Xn) /5. 25/P
Where: n=Dilution factor of test sample;
5. 25 is the millimolar absorption coefficient of N-
acetyl-DL-tryptophan;
P=Protein content in test sample, g/L.
3113 Detennination of Phenol Content
The method is based on the principle that phenol combines
with bromine, which is produced by the reaction of bromate
and hydrochloric acid, and forros tribromophenol. The
excessive bromine reacts with potassium iodide and iodine is
released. The released iodine is then titrated with sodium
thiosulfate and the phenol content of test sample can be
calculated according to the sodium thiosulfate VS consumed.
Procedure
Transfer accurately 1 ml of test sample to an iodine bottle
and add 50 ml of water. Add accurately 15-25 ml (25 ml far
the test sample in which phenol content is O. 3%-0. 5% and
15 ml far the test sample in which phenol content is less than
O. 3%) of O. 02 mol/L bromine solution (Dissolve O. 56 g of
potassium bromate and 3 g of potassium bromide in water
and dilute to 1000 mD. Then, add 10 ml of 6 mol/L
hydrochloric acid solution along the inner wall of the bottle
and stopper tightly. Mix well, allow to stand for 30 minutes
in a dark place. Add 2 ml of 25 % potassium iodide solution
onto the neck of the iodine bottle. Open the stopper slightly
to let the solution flow into the bottle. Stopper tightly and
Determination of Meta Cresol Content
shake well. Wash the neck of the bottle with a small volume
of water. Titrate with O. 02 mol/L sodium thiosulfate VS
until the end point is nearly reached. Add about O. 5 ml of
starch indicator solution and continue the titration until the
blue colour disappears. The titration result is calibrated with
a blank test.
Calculate the result according to the following equation:
Phenol content of test sample ( % ) =
CVa-V1 ) XCX15. 69Xl00/l000
Where: Va =Titer of blank test, ml;
V1 =Titer of test sample, ml;
C = Concentration of sodium thiosulfate VS,
mol/L.
The value of 15. 69 is the one sixth of the relative molecular
mass of phenol.
[Note]
( 1 ) Preparation and calibration of O. 1 mol/L sodium
thiosulfate VS
Dissolve 26 g of sodium thiosulfate and O. 20 g of anhydrous
sodium carbonate in freshly boiled and cooled water and
dilute to 1000 ml. Mix well. Allow the mixture to stand for
1 month and filter. Weigh accurately O. 15 g of potassium
dichromate primary standard, which has been dried to
constant weight at 120ºC, and put into an iodine bottle. Add
50 ml of water and shake until the salid is dissolved. Add
2. O g of potassium iodide and dissolve by shaking gently.
Add 40 ml of diluted hydrochloric acid (5. 7-100). Stopper
tightly and mix well. Allow the mixture to stand for 10 minutes
in a dark place. Add 250 ml of water and titrate with the
sodium thiosulfate solution until the end point is nearly
reached. Add 3 ml of starch indicator solution (Suspend O. 5
g of soluble starch with 5 ml of water and pour slowly into 100
ml of boiling water with constantly stirring. Continue the
boiling for 2 minutes and cool clown. Decant and collect the
upper clear liquid. The solution shall be prepared just befare
use). Continue the titration until blue colour disappears and
a brilliant green colour appears. Calibrate the titration result
with a blank test. One ml of O. 1 mol/L sodium thiosulfate
solution is equivalent to 4. 903 mg of potassium dichromate.
The concentration of the volumetric solution is calculated
according to the weight of potassium dichromate taken and
the volume of volumetric solution consumed.
(2) Preparation of O. 02 mol/L sodium thiosulfate VS
Measure accurately 100 ml of O. 1 mol/L sodium thiosulfate
titration, dilute volumetrically to 500 ml with water and mix
well.
(3) A limit test may be carried out.
3114 Determination of Meta Cresol
Content
The method is based on the principle that metacresol reacts
with 4-amino antipyrine and potassium ferricyanide under an
alkaline condition and forros a red compound. The
metacresol content in test sample is determined by
spectrophotometry.
Procedure
Measure accurately a volume of test sample, put into a test
tube and dilute 50-fold volumetrically to prepare a test
sample solution. Mix l. O ml of test sample solution with
5. O ml of water thoroughly. Add successively l. O ml of pH
9. 8 buffer solution ( Dissolve 6. 36 g of anhydrous sodium
carbonate and 3. 36 g of sodium bicarbonate in water and
dilute to 800 ml. Adjust pH to 9. 8 with 1 mol/L hydrochloric
 3115 Determination of Thimerosal Content
acid solution and dilute to 1000 mi with water), O. 3% 4-amino
antipyrine solution, l. 2 % potassium ferricyanide solution
and 1 mol/L potassium dihydrogen phosphate solution. Mix
well, allow to stand for 10 minutes at room temperature,
protected from light. Read the absorbance at 510 nm
according to ultraviolet-visible spectrophotometry <0401 ) .
Weigh accurately a quantity of metacresol and dissolve in a
small volume of water. Totally transfer to a suitable
volumetric flask and dilute with water to prepare a
metacresol reference solution at a concentration of 10 µg of
metacresol per ml. Measure accurately l. O, 2. O, 3. O,
4. O, 5. O and 6. O ml of metacresol reference solution and put
into a series of test tubes separately. To each tube, make up
the volume to 6. O ml with water. Carry out the same
procedure as that for test sample solution starting from " add
successively l. O mi of pH 9. 8 buffer solution".
A regression equation is obtained by regressing the
concentration of metacresol reference solutions with the
corresponding absorbance. The metacresol content ( mg/ ml) of
test sample is obtained by inserting its absorbance into the linear
regression equation.
3115 Determination of Thimerosal
Content
Method 1 Titration Method
The method is based on the principie that organic mercury
compound may be digested by strong acid to form inorganic
mercuric ion which reacts with disulfurazone and forms an
orange compound. The thimerosal content of test sample can
be calculated from the disulfurazone VS consumed.
Reagent
(1) Disulfurazone VS
Weigh accurately 50 mg of disulfurazone and dissolve in a
small amount of chloroform. Transfer totally to a 100 ml
volumetric flask and dilute to volume with carbon tetrachloride to
prepare a disulfurazone stock solution.
Measure accurately 2. 5 mi of disulfurazone stock solution,
put into a 100 mi volumetric flask and dilute to volume with
carbon tetrachloride just before use. Mix well to obtain a
disulfurazone VS. Store in a dark cold place.
(2) Mercury reference solution
Weigh accurately O. 135 g of mercuric chloride, which has
been dried to constant weight in a sulfuric acid desiccator,
and dissolve in a small volume of O. 5 mol/L sulfuric acid
solution. Transfer totally to a 100 mi volumetric flask and
dilute to volume with O. 5 mol/L sulfuric acid solution. Mix
well to obtain a mercury reference stock solution.
Upon use, measure accurately a quantity of mercury reference
stock solution and put into a 100 ml volumetric flask. Dilute to
volume with O. 5 mol/L sulfuric acid solution and mix well.
One ml of this reference solution is equivalent to 50 µg of
Hg.
Procedure
( 1) Digestion
Measure accurately a quantity ( containing about 50 µg of
mercury) of test sample and put into a 150 ml round bottom
flask fitted with a 40 cm long reflux condenser through
ground joint. Add 2 ml of sulfuric acid solution and O. 5 ml
of 8. O mol/L nitric acid solution. Mix well and reflux on an
electric heater for 15 minutes (or heat in 85-90ºC water bath
for one hour using a 3 cm X 24 cm test tu be with stopper).
Cool clown. Add 40 ml of water and 5 ml of 20 %
hydroxylamine hydrochloride.
(2) Titration
Wash the digested test sample into a 125 ml separating
funnel for several times with water ata total volume of 40 ml
and titrate with disulfurazone VS. Add about 2 mi of the
volumetric solution each time at the beginning and reduce the
volume to O. 5 ml gradually. At last, the volume of
volumetric solution may be reduced to O. 2 ml. Shake for 10
seconds and discard the car bon tetrachloride af ter the
separation of liquids by standing following each addition.
'Continue the titration procedure until the green colour of
disulfurazone persists, i. e. the end-point.
(3) Standardization of disulfurazone solution
Measure accurately 1 ml of mercury reference solution and
put into a 125 ml separation funnel Add 2 ml of concentrated
sulfuric acid, 80 ml of water and 5 ml of 20 % hydroxylamine
hydrochloride solution. Carry out the same procedure as that
for test sample starting from "titrate with disulfurazone VS".
Calculate according to the following equation:
Thimerosal content ( %) =
V 1 XO. 050X2. 02X 100/ (V2 XV3 X1000)
Where: V1 =Titer of test sample, ml;
V 2 =Titer of mercury reference solution, ml;
V3 =Volume of test sample, mi;
O. 050 = Concentration of mercury reference
solution, mg/ml;
The value of 2. 02 is a constant ( 1 g of mercury is
equivalent to 2. 02 g of thimerosal).
[Note]
(1) A small amount of flocculus, without influence on the
test result, may occur during the titration of test sample of
antitoxins or immunoglobulins by water bath digestion
method.
(2) A limit test may be carried out.
Method 2 Atomic Absorption Spectrophotometry
The method is based on the principie that organic mercury is
digested into inorganic mercury ion under oxidative
condition, which is reduced to mercury atom by stannous
chloride. The mercury content in test sample is determined
by atomic absorption spectrophotometry, based on which
the thimerosal content is calculated.
Reagent
( 1) 20 % Stannous chloride solution
Weigh 20 g of stannous chloride, add 20 ml of hydrochloric
acid and dissolve by heating lightly, then cool clown to room
temperature and dilute to 100 ml with water. Prepare the
solution just before use.
(2) Dilute sulfuric acid
Measure 15 mi of sulfuric acid ( analytically pure), add
15 ml of water and mix well.
(3) 5% Potassium permanganate solution
Weigh 5. O g of potassium permanganate (analytically pure),
dissolve in water and dilute to 100 mi. Boíl the solution for
10 minutes, let it stand overnight then filter.
( 4) 5 % Potassium peroxy-disulfate solution
Weigh 5 g of potassium peroxy-disulfate, dissolve in water
and dilute to 100 ml. Prepare the solution just before use.
(5) 8% Hydroxylamine hydrochloride solution
Weigh 8 g of hydroxylamine hydrochloride, dissolve in water
and dilute to 100 ml.
Preparation of standard mercury solution
Dilute standard mercury stock solution with water precisely
to prepare the standard mercury solutions at five appropriate
concentrations.

Preparation of test sample
Measure a quantity of test sample and dilute with water to a
concentration within the range of standard curve.
Procedure
Measure precisely a quantity of test sample and standard
mercury solution, add 4 ml of dilute sulf uric acid, 1 ml of
nitric acid, 4 ml of 5 % potassium permanganate solution and
mix well. Allow the mixture to stand for 15 minutes, add 2
ml of 5 % potassium peroxy-disulfate solution, heat at about
95ºC for 2 hours and cool clown to room temperature. Add 2
ml of 8 % hydroxylamine hydrochloride solution then add
water to a total volume of 50 ml. Measure a quantity of test
sample after digestion, add the corresponding quantity of
20 % stannous chloride solution, and determine the absorbance at
a wavelength of 253. 7 nm by atomic absorption spectrophotometry
( 0405) at room temperature, using water as a blank control
Result calculation
A linear regression equation is obtained by regressing the
concentration of standard mercury solution with the
corresponding absorbance. The correlation coefficient shall
be not less than O. 99. The mercury content in test sample
solution is obtained by inserting the absorbance of test
sample solution into the equation.
The thimerosal content in test sample is calculated according
to the following equation:
Y=Cttg X2. 02Xn/l000
Where: Y=Thimerosal content in test sample, µg/ml;
Cttg = Mercury content in test sample solution,
ng/ml;
The value of 2. 02 is a constant ( 1 g of mercury is
equivalent to 2. 02 g of thimerosaD ;
n = dilution factor of test sample.
3116 Determination of Methyl
Parahydroxybenzoate and Propyl
p-Hydroxybenzoate Contents
The methyl parahydroxybenzoate and propyl p-
hydroxybenzoate contents in test sample are determined by
gas chromatography.
It complies with the gas chromatography ( 0521 ) .
Parameters for chromatography and test for the suitability of
system
Quartz capillary column spread with 100 % polydimethylsiloxane
CPDMS ) is used. The temperatures of column and
vaporization chamber are 180 and 250ºC respectively.
Hydrogen fire ionizing detector CFID) is used, of which the
temperature is 300ºC. Nitrogen is used as carrier gas, at a
flow rate of 20 mi per minute. The test sample is injected in
a stream splitting mode, ata volume of 1 µl. The separation
degree between the internal standard ep-dihydroxybenzene)
and methyl parahydroxybenzoate or propyl p-hydroxybenzoate
shall be more than l. 5. lnject the reference solutions of methyl
parahydroxybenzoate and propyl p-hydroxybenzoate for
5 consecutive times, and the standard deviations of ratios of
methyl parahydroxybenzoate or propyl p-hydroxybenzoate
peak area to that of p-dihydroxybenzene shall be not more
than 5%.
Preparation of interna) standard
Weigh accurately 50 mg of p-dihydroxybenzene, dissolve in
absolute ethanol and dilute to 50 ml to prepare the internal
standard solution ata concentration of 1 mg/ml.
3117 Determination of 0-Acetyl Content
Preparation of reference solution
Weigh accurately O. 1 g of methyl parahydroxybenzoate and
O. 01 g of propyl p-hydroxybenzoate, dissolve in absolute
ethanol and dilute to 10 ml, in which the former at a
concentration of l. 00% and the latter O. 10%.
Preparation of reference solution for determination of
correction factor
Measure 60 µl of reference solution and 100 µl of interna!
standard solution, add 840 µl of pure water to prepare the
reference solution for determination of correction factor
containing 100 µg/ml interna! standard, about O. 06%
methyl parahydroxybenzoate and about O. 006 % propyl p-
hydroxybenzoate.
Procedure
Measure 840 µl of test sample, add 100 µl of interna!
standard solution and 60 µI of absolute ethanol and mix well.
Measure 1 µl of the mixture and inject into gas chromatograph.
Measure 1 µl of reference solution for determination of
correction factor and proceed with the same procedure as that
for test sample. Calculate the methyl parahydroxybenzoate
and propyl p-hydroxybenzoate contents by corrected interna!
standard method.
3117 Determination of 0-Acetyl
Content
Reagent
(1) 2 mol/L Hydroxylamine hydrochloride solution
Dissolve 13. 9 g of hydroxylamine hydrochloride in water and
dilute to 100 ml. Store in a cold place.
(2) 3. 5 mol/L Sodium hydroxide solution.
Dissolve 14. O g of sodium hydroxide in water and dilute to
100 ml.
(3) 4 mol/L Hydrochioric acid soiution
Dilute 33. 3 ml of hydrochloric acid to 100 ml with water.
C4) O. 37 mol/L Ferric chloride-hydrochloric acid solution
Dissolve 10. O g of ferric chloride CFeCb • 6H 2 O) in O. 1
mol/L hydrochloric acid solution and dilute to 100 ml.
e5) Basic hydroxylamine solution
Mix 2 mol/L hydroxylamine hydrochloride solution with an
equal volume of 3. 5 mol/L sodium hydroxide solution. Use
within 3 hours.
Preparation of reference solution
Weigh accurately 22. 7 mg of acetylcholine chloride Cor 28. 3
mg of acetyl choline bromide) , which has been dried to
constant weight, and dissolve in a small volume of
O. 001 mol/L sodium acetate solution ( pH 4. 5 ). Transfer
totally to a 50 ml volumetric flask and dilute to volume with
O. 001 mol/L sodium acetate solution. Mix well.
Preparation of test sample solution
Dilute the test sample with water to an O -acetyl concentration of
O. 5-2. 5 mmol/L
Procedure
Measure accurately O. 2, O. 4, O. 6, O. 8 and l. O ml of 2. 5
mmol/L acetylcholine chloride ( or acetylcholine bromide)
reference solution and put into a series of test tubes
separately. Make up the volume of each tube to l. O ml with
water and add 2 ml of freshly prepared basic hydroxylamine
solution. Mix well, allow to stand for 4 minutes at room
temperature. To each tube, add 1 ml of 4 mol/L hydrochloric
acid solution to adjust the pH to l. 2 ±O. 2 and mix well.
Then, add 1 ml of O. 37 mol/L ferric chloride-hydrochloric
acid solution and mix well again. Read the absorbance at
540 nm by ultraviolet-visible spectrophotometry ( 0401 ) .
 3118 Determination of Adipic Dihydrazide Content
Corresponding blank controls are prepared by measuring
accurately another set of 2. 5 mmol/L acetylcholine chloride ( or
acetylcholine bromide) reference solution and putting into
another series of test tubes separately. Carry out the blank
control tests with the same procedures starting from "make
up the volume of each tube to l. O ml with water" except that
the order of adding basic hydroxylamine hydrochloride
solution and 4 mol/L hydrochloric acid solution is reversed.
Transfer accurately 1 ml of test sample solution to a test
tube. Carry out the same procedure as that for CRS starting
from "add 2 mi of freshly prepared basic hydroxylamine
solution". Carry out a blank control test for test sample by
transferring 1 mi of test sample solution to another test tube.
Other procedures are the same as that for blank control of
reference solution.
Subtract the absorbance of corresponding blanks from
absorbance of CRS tubes respectively. A linear regression
equation is obtained by regressing the volume of reference
solutions with the corresponding absorbance after blank
correction. Subtract the absorbance of test sample blank
from that of the test sample tube and insert into the
regression equation Calculate the volume of test sample
equivalent to the volume of reference solution (V, ml).
0-acetyl content of test sample solution
(mmol/L) =VX2. 5.
The value of 2. 5 is the acetylcholine concentration of
ref erence solution in mmol/L.
3118 Determination of Adipic
Dihydrazide Content
The method is used for the determination of adipic
dihydrazide ( ADH) content in polysaccharide derivative of
Haemophilus influenzae type h. The amino groups in ADH
can react with trinitrobenzene sulfonate (TNBS) in sodium
tetraborate solution and form a coloured complex. Determine
by ultraviolet-visible spectrophoto metry.
Reagent
(1) Stock solution of ADH reference (1 mg/ml): Weigh
accurately O. 100 g of ADH, dissolve with water and
dilute to 100 ml. Store at -20ºC.
(2) Working solution of ADH reference ( 20 µg/ml):
Measure O. 2 ml of stock solution of ADH referrence, dilute
to 10 ml with water.
(3) 5% Sodium tetraborate solution: Weigh accurately 47. 35 g
of sodium tetraborate (Na2B4Ü1 • lOH20), dissolve with
water and dilute to 500 ml. Store at room temperature.
(4) 3% TNBS solution: Measure 5 mi of TNBS and dilute
to 50 ml with Water. Store at -20ºC.
Procedure
For the blank, measure l. O mi of 5 % sodium tetraborate
solution, add 1 ml of water, mix well, add O. 3 ml of 3%
TNBS solution and mix well, allow to stand at room
temperature for 15 minutes. Read the absorbance at 500 nm.
For the preparation of test sample solution, dilute a quantity
of test sample with water to an ADH concentration of not
more than 20 µg/ ml. Measure l. O ml of test sample
solution, and add l. O ml of 5 % sodium tetraborate solution,
proceed with the same procedure starting from "add O. 3 ml
of 3% TNBS solution".
Measure O. 2 ml, O. 4 ml, O. 6 ml, O. 8 ml and l. O ml of
20 µg/ml ADH referrence solution, put into test tubes
respectively. Add O. 8 ml, O. 6 ml, O. 4 ml, O. 2 ml and
O ml of water, respectively. Add l. O ml of sodium
tetraborate solution, proceed with the same procedure
starting from "add O. 3 ml of 3% TNBS solution".
Calculation
Plot a linear regression curve with the absorbance of ADH
referrence to their concentrations, obtain the linear regression
equation. Calculate ADH content of the test sample solution by
inserting its absorbance into the linear regression equation, and
calculate the ADH content of the test sample according to its
dilution factor.
3119 Determination of Content in
High Molecular Weight Conjugate
The method is to fractionate high molecular weight conjugate,
low molecular weight conjugate and free polysaccharide by
precipitation with different concentrations of ethanol, determine
the phosphorous content in each fraction by ultraviolet-visible
spectrophotometry, and calculate the content of high molecular
weight conjugate.
Reagent
( 1) 5 mol/L Sodium chloride solution: Weigh accurately
29. 22 g of sodium chloride, dissolve with water and dilute to
100 ml. Store at room temperature.
(2) l. 5 mol/L Sulfuric acid: Add 11 volume of water into 1
volume of 98%sulfate acid and mix well.
(3) 2. 5% Ammonium molybdate: Weigh 2. 65 g of ammonium
molybdate [ CNH4)4MÜ1Ü2•4H20], dissolve with water
and dilute to 100 ml.
(4) 10% Ascorbic acid: Weigh 10 g of ascorbic acid,
dissolve with water and dilute to 100 ml.
(5) Mineralization reagent: Prepare by mixing equal volumes
of concentrated sulfuric acid and 70 % Perchloric acid
CHCIQ4).
(6) Chromogenic reagent: Add water, l. 5 mol/L sulfuric acid,
2. 5% ammonium molybdate and 10% ascorbic acid,
according to the volume ratio of 2 : 1 : 1 : 1, and mix well.
( 7) Phosphorous reference stock solution ( 80 µg/ml):
Weigh accurately O. 3665 g of disodium hydrogen phosphate
or O. 3509 g of potassium dihydrogen dried at lOOºC, dissolve
with 500 ml of water and 10 ml of 5 mol/L sulfuric acid and
dilute to 1000 ml with water. Upon use, make a 20-fold
dilution of the reference stock solution of phosphorous to
prepare the reference working solution of phosphorous
(4 µg/mD.
(8) l. O mol/l Sodium hydroxide solution: Weigh 4 g of
sodium hydroxide, dissolve with water and dilute to 100 ml.
Preparation of test sample solution
(1) Fractionational precipitatioin: Measure 3ml of the bulk
conjugate ( pre-diluted with sodium chloride physiological
solution to a polysaccharide concentration of 20-28 µg/ mD or
the final product and serve as test sample solution I, add
O. 75 ml of 5 mol/L sodium chloride solution, mix well and
add 15 ml of absolute ethanol, store at - 20ºC for 72-96
hours. Centrifuge at 4 ºC, 8000 r/min for 90 minutes. Collect
the supernatant to obtain test sample solution Il . Add
O. 5 mi of 50 % ethanol and several glass beads to the
precipitate, mix well and allow to stand at room temperature
for 1 hour, then add l. 5 ml of 50 % ethanol, mix well and
allow to stand at room temperature for 2 hours. Centrifuge at
8ºC, 8000 r/min for 1 hour. Collect l. 8 ml of the supernatant
to obtain test sample solution ill. Add O. 5 ml of l. O mol/L
sodium hydroxide solution to the precipitate, mix well and
allow to stand at room temperature for 1 hour. Add l. 25 ml
of water to prepare test sample solution N.
 3121 Determination of Polymer Content in Human Albumin
(2) Mineralization of test sample solutions: Measure l. O ml
of test sample solution l , l. 5 ml of test sample solution II ,
m
o. 7 ml of test sample solution and o. 5 ml of test sample
solution N, each in duplicate. Add O. 15 ml of mineralization
reagent to each test sample solution, dry at 150ºC for 1 hour,
then raise temperature to 180ºC and dry for 30 minutes, further
raise temperature to 250ºC and dry for 1 hour.
Procedure
To the blank control, add l. 95 ml of water, 50 ml of
mineralization reagent and mix well, add 2. O ml of
chromogenic reagent, mix well and incubate in 37°C water
bath for 2 hours. Read the absorbance at 825 nm.
To the mineralized test sample, add l. 85 ml of water,
2. O ml of chromogenic reagent, mix well and incubate in
37°C water bath for 2 hours. Read the absorbance at 825 nm.
Measure O. 1 ml, O. 2 ml, O. 4 ml, O. 8 ml and l. O ml of the
reference working solution of phosphorous, put in to a series
of test tubes and add l. 85 ml, l. 75 ml, l. 55 ml, l. 15 ml
and O. 95 ml of water, respectively. Add 50 ml of
mineralization reagent and, 2. O ml of chromogenic reagent to
each tube, mix well and incubate in 37"C water bath for
2 hours. Read the absorbance at 825 nm.
Calculation
Plot a linear regression curve with the absorbance of
phosphorous referrence to their concentrations to obtain the
linear regression equation. Calculate phosphorous content of
the test sample solutions by inserting their absorbance into
the linear regression equation.
Calculate according to the following equation:
P1 = CA1 X3) /l. O
P2 = CA2X18. 75) /l. 5
P3 = (A3 X2. 0) /O. 7
P4 = (A4 X2. 0) /O. 5 - (P3 XlO) /100
Validity of the test: 80%~P 1 / CP 2 +P 3 +P4) ~120%
High molecular weight conjugate content of the test sample
C%) =P4/ CP2+P3+P4) XlOO
Free polysaccharide content of the test sample ( % ) = [ 1 - P 4/
CP2+P3+P4) ] XlOO
Where: Pi , P 2 , P 3 and P 4 are total phosphorous contents
in test sample solutions I , II , ID and N, respectively; A1,
Az, A3 and A4 are phosphorous contents in mineralized
samples of test sample solutions I , II , ID and N ,
respectively.
3120 Determination of Saccharide
and Sugar Alcohol Contents in
Human Blood Products
The contents of saccharides and sugar alcohol in human blood
products are determined with ion chromatography ( 0513).
Parameters for chromatography and test for the suitability of
system
Cation chromatographic column ( H+ ) at a diameter of 7. 8
mm and a length of 300 mm filled with co-polymerized
phenylethylene and divinylbenzene matrix with a grain size of
8 µm or 9 µm is used. Column temperature is 50ºC ( 20-
30ºC for sucrose determination) and mobile phase is O. 004
mol/L sulfuric acid solution at a flow rate of O. 8 ml per
minute. The detector is a differential refractometer.
Mix 1 ml of 2 % maltose and 1 ml of l. 5 % sulfosalicylic
acid. Inject 20 µl of the mixture into the chromatographic
column and record the chromatogram. The separation degree
between peaks of maltose and sulfosalicylic acid shall be more
than l. 5 with a tailing factor of O. 95-1. 50 calculated as the
peak of maltose.
Preparation of reference solutions
(1) Maltose reference solution
W eigh accurately l. O, 2. O and 3. O g of maltose, previously
dried to constant weight under reduced pressure, and
transfer totally to three 100 ml volumetric flasks separately.
Dilute each of them to volume with water and mix well.
(2) Glucose reference solution
Weigh accurately O. 5, l. O and l. 5 g of glucose, previously
dried to constant weight under reduced pressure, and
transfer totally to three 100 ml volumetric flasks separately.
Dilute each of them to volume with water and mix well.
(3) Sorbitol reference solution
Weigh accurately O. 5, l. O and l. 5 g of sorbitol, previously
dried to constant weight under reduced pressure, and
transfer totally to three 100 ml volumetric flasks separately.
Dilute each of them to volume with water and mix well.
( 4) Sucrose reference solution
Weigh accurately l. O, 2. O and 3. O g of sucrose, previously
dried to constant weight under reduced pressure, and
transfer totally to three 100 ml volumetric flasks separately.
Dilute each of them to volume with water and mix well.
Preparation of test sample solution
Measure accurately 1 ml of test sample. Add 4. O ml of
l. 5 % sulfosalicylic acid solution and mix well. Allow the
mixture to stand at room temperature for at least 2 hours.
Centrifuge at 3000 r/min for 10 minutes and collect the
supernatant for use.
Procedure
Measure accurately 20 µl of reference solutions and test
sample solutions and inject into the liquid chromatographic
device separately. Record the chromatograms.
Linear regression equations are obtained by regressing the
contents of reference solutions (g/L) of individual saccharide or
sugar alcohol with the corresponding peak areas. Calculate the
content of individual saccharide or sugar alcohol (A) in test
sample solution. The saccharide or sugar alcohol content of the
test sample is calculated by the following equation:
Saccharide or sugar alcohol content ( g/L) =A X n
Where: A = Saccharide or sugar alcohol content in test
sample solution, g/L;
n = Dilution factor of test sample.
[Note]
(1) The volume of CRS and test samples loaded may be
properly adjusted according to the content of saccharide or
sugar alcohol in test sample.
(2) The correlation coefficient of linear regression equation
shall be not less than O. 999.
(3) The mobile phase and its flow rate, column temperature
for cation chromatographic column ( H+ ) obtained from
different suppliers may be different. The chromatographic
parameters may be properly adjusted according to the
instructions.
3121 Determination of Polymer
Content in Human Albumin
The polymer content in human albumin is determined with
molecular exclusion chromatography ( 0514 ).
Parameters for chromatography and test for the suitability of
system
High efficiency volume exclusion chromatographic column
CSEC, exclusion limit 300 kD, grain size 10 µm) at a
 3122 Determination of lgG Monomer and Dimer in Human lmmunoglobulins
diameter of 7. 5 mm and a length of 60 cm filled with
hydrophilic silica gel is used. The mobile phase is O. 2 mol/L
pH7. O phosphate buffer solution containing 1% isopropanol
(Mix 200 ml of O. 5 mol/L sodium dihydrogen phosphate,
420 ml of O. 5 mol/L disodium hydrogen phosphate and 15. 5 ml of
isopropanol with 914. 5 ml of water throughly) ata flow rate of
O. 6 ml per minute. The wavelength for detection is 280 nrn
lnject 20 µl of human albumin solution at a concentration of
12 mg per ml into the chromatographic column and record
the chromatogram. The separation degree between peaks of
monomer and dimmer shall be more than l. 5. The tailing
factor shall be O. 95-1. 40 calculated as peak of human
albumin monomer.
Procedure
Dilute a quantity of test sample with mobile phase to a
protein concentration of about 12 mg per ml. Inject 20 µl into
the chromatographic column. Record the chromatogram for
60 minutes.
Calculate the result using area regression method. Divide the
content ( % ) of total exclusion peak by 2 to obtain the
polymer content of human albumin.
0.20
Monomer
O.IS 26.791
:il0.10
0.05
• ••• • 1
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00
min
Fig. Standard Chromatogram of Human Albumin
3122 Determination of IgG Monomer
and Dimer in Human Immunoglobulins
The sum of IgG monomer and dimer in human immunoglobulins
is determined by molecular exclusion chromatography ( 0514).
Parameters for chromatography and test for the suitability of
system
High efficiency volume exclusion chromatographic column
CSEC, exclusion limit 300 kD, grain size 10 µm), filled
with hydrophilic silica gel, at a diameter of 7. 5 mm and a
length of 60 cm is used. The mobile phase is O. 2 mol/L
phosphate buffer solution ( pH 7. O ) containing 1 %
isopropanol (Mix 200 ml of O. 5 mol/L sodium dihydrogen
phosphate solution, 420 ml of O. 5 mol/L disodium hydrogen
phosphate solution and 15. 5 ml of isopropanol with 914. 5 ml
of water thoroughly) at a flow rate of O. 6 ml per minute.
The wavelength for detection is 280 nm.
Inject 20 µl of human immunoglobulin and human albumin
solutions, each ata concentration of 12 mg per ml, into the
chromatographic column respectively and record the
chromatograms. Both the separation degrees between peaks
of human immunoglobulin reference monomer and lysate and
between peaks of human albumin ref erence monomer and
dimmer shall be more than l. 5. The tailing factor shall be
O. 95-1. 40 calculated as peak of human albumin monomer.
40.00 45.00 50.00 SS.00 60.00
0.35 ~ monomer peak
0.30
0.25 ~
00.20
dimmerpeak
<0.15 polymer peak
0.10
~
0.05 gj clearage peak
o.ooi:;:;:;::;:;:;:;:;:::;:;:;:;::;:~:;::;::;::lk;:;:;:~~;¡¡::::;:;:::;::;:;:;:;:;:::;:;:;:;::;:;::;:;:;:;:;:::;:;:;:;::;:;::;::;
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
Fig. Standard Chromatogram of Human lmmunoglobulins IgG
Procedure
Dilute a quantity of test sample with mobile phase to a
protein concentration of 12 mg per ml and inject 20 µl into
the chromatographic column. Record the chromatogram for
60 minutes. Calculate the result using area regression
method. The content of monomer and dimmer peaks is the
sum of monomer and dimmer representing the percentage of
the total area of the chromatogram. The boundary of peaks
in the chromatogram is the vertical line from the point at the
gorge between two peaks to the base line. The principal peak
corresponds to IgG monomer and there is a peak corresponding to
dimmer with a retention time relative to monomer of about
o. 85.
3123 Determination of Glycine Content
in Human Immunoglobulin
The method is based on the principie that excessive 6-
aminoquinolyl-N-hydroxysuccinimidyl carbamate ( AQC)
forms a stable derivative ( pre-column derivation) with an
amino acid under a certain condition The derivative is determined
by high performance liquid chromatography ( HPLC) , based on
which the glycine content in human immunoglobulin is
calculated.
It complies with the high performance liquid chromatography
( 0512).
Parameters for chromatography and test for the suitability of
system
C1s reversed phase chromatographic column at an internal
diameter of 3. 9 mm and a length of 150 mm, filled with
octadecylsilyl silica gel (grain size 4 µm), is used. Column
temperature is 37°C. The mixture of 140 mmol/L sodium
acetate, 17 mmol/L triethylamine (pH 5. 65) and 1 µg/ml
ethylene diamine tetraacetic acid disodium salt is served as
mobile phase A, 100% acetonitrile as mobile phase B, and
pure water as mobile phase C, each ata flow rate of l. O ml
per minute. The time for gradient elution is 32 minutes ( see
the table) , and the wavelength for detection is 248 nm. The
separation degree between the peak of glycine and its adjacent
peaks shall be more than l. 5. The tailing factor (T) shall be
O. 95-1. 40 (the peaks of glycine and craminobutyric acid).
RSD shall be not more than 2. O% ( the measurement value
of peak area of glycine reference).
Preparation of internal standard solution
Weigh accurately O. 4 g of a--aminobutyric acid reference,
dissolve in ultra-pure water and dilute to 100 ml.
Preparation of reference solution
(1) Weigh accurately 2. 5 g of glycine reference, dissolve in
ultra-pure water and dilute to 100 ml.
(2) Measure accurately l. O ml of solution in section (1),
add 9. O ml of l. 5 % sulfosalicylic acid, mix well and stand
for more than 2 hours, then centrifuge at 3000 r/min for
10 minutes and collect the supernatant for use.
 3125 Deterrnination of Free Histamine Phosphate in Human Histamine Immunoglobulin

(3) Measure accurately O. 4, O. 8, l. O, l. 2 and l. 6 ml of histidine and arginine in blood products, using the corresponding
supernatant in sections ( 2) and transfer into a 10 ml histidine or arginine reference.
volumetric flask separately, then dilute to volume with pure (5) The amounts of reference and test sample taken may be
water. adjusted properly according to the glycine content in test
(4) Measure accurately O. 1 ml of solutions in section (3) sample.
respectively. To each solution, add O. 4 ml of pure water
and O. 02 ml of internal standard solution, and mix well. 3124 Detennination of Protein Content
(5) Measure accurately 10 µl of solutions in section ( 4)
respectively. Transfer each solution to a tube for derivation,
in Recombinant Human Granulocyte
add 70 µl of boric acid buffer ( pH8-10) and mix well by Colony-stimulating Factor
vorticose stirring, then add 20 µl of AQC derivative agent
and mix by vorticose stirring for 15 seconds to prepare a The protein content in recombinant human granulocyte
reference solution. colony-stimulating factor is determined by high performance
liquid chromatography.
Preparation of test sample solution
It complies with the high performance liquid chromatography
(1) Measure accurately l. O ml of test sample solution, add
<0512).
9. O ml of l. 5 % sulfosalicylic acid, mix well and stand for
more than 2 hours, then centrifuge at 3000 r/min for 10 Parameters for chromatography
minutes and collect the supernatant for use. The chromatographic columnata diameter of 4. 6 mm anda
(2) Measure accurately l. O ml of supernatant in section length of 250 mm, filled with octadecylsilyl silica gel (grain
(1), transfer to a 10 ml volumetric flask and dilute to size 5 µm and pore size 30 nm ) , is used. Column
volume with pure water. temperature is 30ºC ± 5ºC. The temperature for storage of
(3) Measure accurately O. 1 ml of solution in section (2), test sample is 2-8ºC. The water solution containing O. 1 %
then add O. 4 ml of pure water and O. 02 ml of internal trifluoroacetic acid is used as mobile phase A, and the
standard solution and mix well. Measure accurately 10 µl of acetonitrile solution containing O. 1 % trifluoroacetic acid as
the mixture and transfer to a tube for derivation, add 70 µl mobile phase B, each at a flow rate of 1 ml per minute. The
wavelength for detection is 214 nm. Gradient elution is
of boric acid buffer and mix well by vorticose stirring, then
performed according to the requirements in the following table.
add 20 µl of AQC derivative agent and mix by vorticose
stirring for 15 seconds to prepare the test sample solution.
Mobile Mobile
Procedure Test Time (min)
phase A (%) phase B (%)
Measure accurately 10 µl of reference and test sample
solutions and inject into liquid chromatograph ic column o 60 40
respectively. Record the chromatogram for 32 minutes. The 2 40 20 80
glycine content is calculated by internal standard method.
3 45 o 100
Gradient 50 60
4 40
Mobile Mobile Mobile 5 60 60 40
Time Flow rate
(min) (ml/min)
phase A phase B phase e Curve
(%) (%) (%) Procedure
Reconstitute one container of standard according to the
o l. o 100 o o directions. Adjust the standard and test sample to an equal
0.5 l. o 99.0 l. o o Transient protein concentration with 20 mmol/L acetic acid-sodium
18.00 l. o 95.0 5.0 o Linear acetate buffer ( pH4. O) , and injected equal volume ( not
less than 10 µl, containing 4-6 µg ) into liquid
19.00 l. o 91. o 9.0 o Linear
chromatographic column separately. Gradient elution is
22.00 l. o 83.0 17.0 o Linear performed according to the requirements in the above-
25.00 l. o o 60.0 40.0 Transient mentioned table. Inject the standard and test sample each for
3 times, record the chromatogram and calculate the peak
28.00 l. o 100 o o Transient
area. The protein content in recombinant human granulocyte
32.00 l. o 100 o o Linear colony-stimulating factor is calculated by the following
equation:
[Note]
(1) The glycine content shall be determined by pre-column Protein content (µg/mD in test sample=
derivation and internal standard methods. Except those CRXAxXnx/ (ARXnR)
required in the appendix, phenyl isothiocyanate and ortho- Where: CR = Protein content in reconstituted standard,
phthaladehyde may also be selected as derivative agents, and µg/ml;
n-valine as internal standard. The grain size of C1s reversed AR =Average peak area of standard solution;
phase chromatographic column may also selected as 5 µm or Ax =Average peak area of test sample;
sub-2 µm. The condition for chromatography may be nR =Dilution factor of standard solution;
adjusted according to the liquid chromatographic system, nx = Dilution factor of test sample.
specification of C1s reversed phase chromatographic column,
derivative agent and internal standard. 3125 Detennination of Free Histamine
(2) The correlation coefficient of linear regression shall be
not less than O. 999. Phosphate in Human Histamine
( 3) The reproducibility in system suitability may be tested Immunoglobulin
by other appropriate methods.
( 4 ) The method is also suitable for the deterrninations of The method is based on the principie that histamine phosphate
 3126 Determination of IgG Content
reacts with o-phthaldialdehyde in alkaline solution to farm a
fluorescence derivative. The free histamine phosphate content
in human histamine immunoglobulin is determined with
fluorometry.
Preparation of histamine phosphate reference solution
Weigh accurately 7 mg of histamine phosphate CRS and
dissolve in a small volume of O. 1 mol/L hydrochloric acid.
Transfer totally to a 25 ml volumetric flask and dilute to
volume with O. 1 mol/L hydrochloric acid Mix well to prepare
a stock histamine phosphate reference solution. Store at
- 20ºC far use. On the date of test, measure accurately
O. 1 ml of stock histamine phosphate reference solution and
put into a 100 ml volumetric flask. Dilute to volume with
O. 1 mol/L hydrochloric acid to prepare a histamine phosphate
reference solution.
Preparation of test sample solution
Mix O. 5 ml of test sample with l. 2 ml of water. Add O. 3 ml
of 25 % trichloroacetic acid and mix well. Centrifuge at 4000
r/min far 10 minutes. The supernatant is taken as test
sample solution.
Procedure
Measure l. 6 ml of test sample solution and put into a test
tube. Add l. 5 g of sodium chloride, 4. O ml of n-butanol
and O. 2 ml of 2. 5 mol/L sodium hydroxide solution. Mix
instantly far 5 minutes. Allow the mixture to stand far the
separation of liquids. Measure 3. 6 ml of the n-butanol phase
and put into a test tube containing l. 2 ml of O. 1 mol/L
hydrochloric acid solution and 2. O ml of n-heptane. Oscillate
for 5 minutes and discard the organic phase. 1'v1ix l. O ml of
the aqueous phase with an equal volume of water. Add
O. 5 ml of O. 4 mol/L sodium hydroxide solution. Mix well
and add quickly O. 1 ml of O. 1 % o-phthaldialdehyde-methane
solution.Mix well instantly. Allow the mixture to stand at
21-22ºC far 10 minutes. Stop the reaction by adding O. 5 ml
of O. 5 mol/L hydrochloric acid solution. Transfer 200 µl of
the reaction mixture to a well of microtiter plate. Measure
the intensity of fluorescence light at an excitation wavelength
of 350 nm and an emission wavelength of 450 nm with a
fluorophotometer.
Measure accurately l. O, O. 8, O. 6, O. 4, O. 2, O. 1, O. 05
and O. 025 ml of histamine phosphate reference solution and
put into a series of test tubes separately. Make up the
volume of each tube to l. O ml with O. 1 mol/L hydrochloric
acid. To each tube, add O. 5 ml of water and O. 1 ml of 25%
trichloroacetic acid solution. Mix well and add l. 5 g of
sodium chloride to each tube. Carry out the same procedure
as that far test sample solution starting from the "add 4. O ml
of n-butanol".
A regression equation is obtained by regressing the
concentrations of histamine phosphate reference solution with
the corresponding fluorescence light intensity. The alkaline
base concentration of test sample solution (G, ng/mD is
obtained by inserting its fluorescence light intensity into the
regression equation. The free histamine content of test
sample is calculated by the fallowing equation.
Free histamine content of test sample (ng/mD
GX2. 76X2. 5
[Note]
The relative molecular mass of histamine phosphate is
307. 148. The concentration of reference solution is calculated
as alkaline base and the relative molecular mass ratio of
alkaline base to histamine phosphate is 1 : 2. 76. The value of
2. 5 is the dilution factor of test sample.
3126 Determination of IgG Content
CUl traviolet-visi ble Spectrophotometry)
The method is based on the principle that IgG may specifically
bind to the corresponding antibody to cause agglutination
and farm antigen-antibody complex at suitable electrolyte
concentration, temperature and pH. The lgG content may
be calculated according to the absorbance of test sample.
Reagent
( 1) Buffer solution: Dissolve 12. 42 g of Tris, 9 g of
sodium chloride, 50 g of polyethylene glycol CPEG 6000),
1 g of bovine serum albumin (BSA) and 1 g of sodium azide
(NaN3 ) in water. Adjust pH to 7. 4 with l. O mol/L
hydrochloric acid solution and dilute to 1000 ml with water.
(2) Anti-human lgG serum: Reconstitute the freeze-dried
anti-human IgG serum fallowing the instructions. According
to the stated potency, dilute a certain amount of anti-human
lgG serum with buffer solution to a titer of 1 : 4 ( far
example, to 2 ml of 1 : 100 anti-human IgG serum, add 48
ml of buffer solution). Mix well and filter with a O. 45 µm
membrane. Store at 4ºC.
Preparation of IgG standard solution
Dilute the standard lgG with physiological saline to make a
series of dilutions ata concentration range of O. 2-6. O mg per
ml. Five dilutions are required in general.
Preparation of test sample solution
Prepare three dilutions (high, medium and low) of the test
sample with physiological saline, in which the IgG content is
within the limits of standard curve.
Procedure
To 10 µl of test sample solution at each dilution, in
duplicate, add 1 ml of antibody solution preheated to 37°C
and mix well. Warm the mixture in 37°C water bath far one
hour. Mix well and read the absorbance at 340 nm by
ultraviolet-visible spectrophotometry ( 0401 ) .
Repeat the above-mentioned procedure using 10 µl of IgG
standard solution instead of test sample solution.
Calculate the mean absorbance of each dilution of standard
and sample respectively. A linear regression equation is
obtained by regressing the logarithm of lgG content of the
standard solutions with the logarithm of corresponding
absorbance. The correlation coefficient of linear regression
equation shall be not less than O. 99. lnsert the logarithm of
absorbance of test sample solutions into the regression
equation and calculate the antilogarithm of corresponding IgG
contents. Multiply the result with the dilution factor to
obtain the lgG content in 1 ml of test sample. The IgG
content of the test sample is the mean of the lgG contents
obtained (g/L).
[Note]
( 1 ) All the measurement of reaction tubes shall be
completed within 10 minutes.
(2) The slit width far ultraviolet-visible spectrophotometry
shall be designed as 2 nm.
(3) The lgG concentration of standard solution may be
properly adjusted according to the lgG content of test sample.
3127 Determination of Molecular
Size Heterogeneities in
Monoclonal Antibodies
This method uses sodium dodecyl sulfate capillary electrophoresis
 3127 Determination of Molecular Size Heterogeneities in Monoclonal Antibodies
( CE-SDS) with UV detection to quantitatively determine the
purity of recombinant monoclonal antibody products based on
the molecular weights under reducing and non-reducing
conditions.
Measurement refers to Capillary Electrophoresis ( 0542).
1. Capillary electrophoresis system
(1) Detector: UV detector, wavelength: 214 nm.
(2) Capillary: Uncoated-fused silica capillary ( the inner
diameter is 50 µm), cut toan overall length of 31 cm andan
effective length of 21 cm.
( 3) Aperture size: 8 ( 100 µm X 800 µm)
2. Reagents
(1) SDS sample buffer: O. 1 M Tris-HCl solution containing
1% SDS, pH 9. O.
(2) SDS gel separation buffer: SDS buffer containing O. 2%
SDS ( pH 8. O), with suitable hydrophilic polymer as the
molecular sieve.
( 3) O. 1 N hydrochloric acid solution
(4) O. 1 N sodium hydroxide solution
( 5) 2-mercaptoethanol
( 6) Alkylation solution: O. 8 M iodoacetamide aqueous solution,
weigh approximately 74 rng of iodoacetamide, add 500 µl of
ultrapure water to dissolve. Prepare fresh and protect from light
(7) The reference solution: The final concentration is 1 rng/ml.
3. Sarnple preparation
( 1) Preparation of test solutions: Dilute the test sample with
SDS sample buffer to 1 mg/ml. Dilute the sample buffer as
the same way as the test sample and use itas blank control.
(2) The preparation of non-reducing sample: Combine 95 µl
test sample solution ( 1 mg/ml) with 5 µl of O. 8 M
iodoacetamide aqueous solution and mix well by vortex. Mix
95 µl blank control with 5 µl of O. 8 M iodoacetamide aqueous
solution by vortex, and use it as the non-reducing blank
control.
(3) The preparation of reducing test solution: Combine 95
µl test sample solution ( 1 mg/ml) with 5 µl of 2-
mercaptoethanol and mix well by vortex. Mix 95 µl of blank
reagent with 5 µl of 2-mercaptoethanol, and use it as the
reduceding blank control.
lncubate the test sample solutions and the blank controls at
68ºC-72ºC. lncubate the non-reducing test solutions far 5 min
and the reducing test solutions far 15 min. After cooling to
room temperature, centrifuge the solutions at 6000 rpm far
1 min. Transfer 75 µl of the solution from each sample tube
into separate vials, and start the running sequence
immediately.
4. Sarnple analysis
The pretreatment of capillary: Rinse the capillary with O. 1 N
sodium hydroxide solution far 3 min ata pressure of 60 psi,
and then rinse with O. 1 N hydrochloric acid solution far 2
min at a pressure of 60 psi, finally rinse with pure water far
1 min at a pressure of 70 psi. These steps should be
conducted prior to each run.
Pre-filling of capillary: Rinse the capillary with SDS gel
separation buffer far 15 min at a pressure of 50 psi. This
operation should be conducted prior to each run.
Sample injection: 10 kV reversed polarity electric injection.
Inject the reducing sample far 30 seconds; inject non-
reducing sample far 40 seconds.
Separation: Conduct at 15 kV far 40 min, under reversed
polarity.
The temperature of sample chamber: 18ºC-22ºC
The temperature of the capillary: 18ºC-22ºC
The injection sequence: Reference, Sample (s), Reference,
Blank control.
S. System suitability
( 1) System suitability requirements far the reducing
candi tions:
Electrophoretogram: The electrophoretogram of the
reference should be consistent with the typical
electrophoretogram provided.
Resolution: Glycosylated heavy chains and non-glycosylated
heavy chains can be clearly distinguished ( the resolution is
set based on the actual measurement data).
The percentage of the non-glycosylated heavy chains of the
reference in the total heavy chains: Calculate the percentage
of the corrected peak area of the non-glycosylated heavy
chains in the corrected peak area of the total heavy chains.
The percentage of the non-glycosylated heavy chains in total
heavy chains in the reference solution should be within the
specified range ( which is set based on the actual
measurement data).
Migration time: The difference of heavy chain migration time
from two injections of reference should be ~l. O min.
The blank control: There should be no interference peak in
the blank solution.
( 2) System suitability requirements far the non-reducing
conditions:
Electrophoretogram: The electrophoretogram of the reference
should be consistent with the typical electrophoretogram
provided.
Resolution: The resolution of lgG main peak and the
fragments is set based on the actual measurement data.
The percentage of the main peak of the reference: Calculate
the percentage of the corrected peak area of the main peak in
the total corrected peak areas. The relative percentage of the
main peak of the system suitability solution should be within
a specified range.
Migration time: The difference of main peak migration time
from two injections of reference should be ~l. O min.
Notes: Depending on the different instruments, the sample
injection conditions and the capillary type can be adjusted to
meet the system suitability requirements.
6. Result analysis
Reducing condition: According to the area normalization
method, the percentage of the heavy chains, non-
glycosylated heavy chains and light chains is respectively
calculated by the percentage of the corrected peak area of the
heavy chains, non-glycosylated heavy chains and light chains
in the sum of all corrected peak areas. The sum of heavy
chains, non-glycosylated heavy chains and light chains is the
purity of the product. [Notes: It is based on the mode of
action of the molecules to determine whether the purity
should include non-glycosylated heavy chains or not.]
Non-reducing condition: According to the area normalization
method, the purity of the main peak is calculated by the
percentage of the corrected peak area of lgG main peak in the
sum of all corrected peak areas.
 3201 Determination of Residual Ethanol Content

3200 Determination of Chemical Residues

blank control test is carried out with the same procedure

3201 Determination of Residual using 1 ml of water instead of test sample solution.
Weigh accurately an appropriate quantity of polyethylene
Ethanol Content glycol (the molecular mass is same as that in test sample)
( Conway Diffusion Method) and dissolve in water to prepare a polyethylene glycol
reference stock solution ata concentration of 100 µg per ml.
The method is based on the principie that ethanol is Prepare the reference solutions at the concentration of 10-50
evaporated from saturated sodium carbonate solution upon µg/ ml according to the following table.
heating. After the evaporation ethanol is absorbed by
potassium dichromate solution, and a yellowish-green to Polyethylene glycol (µg/ml) 10 20 30 40 50
green colour appears. The residual ethanol content in blood
Polyethylene glycol reference
products is determined by spectrophotometry.
stock solution (ml)
0.2 0.4 0.6 0.8 l. o
Procedure
Spread a thin layer of vaseline evenly on the outside rim of a 1 % Protein solution (ml) 0.2 0.2 0.2 o. 2 o. 2
Conway Disk. Measure accurately 2. O ml of potassium
dichromate--sulfuric acid solution ( Dissolve 3. 7 g of
Water (ml) l. 6 l. 4 l. 2 l. o o. 8
potassium dichromate in 150 ml of water. Add 280 ml of
Transfer l. O ml of each of the polyethylene glycol reference
concentrated sulfuric acid slowly. Cool clown and dilute to
solution to a series of test tubes separately. To each tube,
500 ml with water. Mix welD and put into the inner circle of
add 5. O ml of O. 5 mol/L perchloric acid and mix well. Carry
the disk. Measure accurately l. 5 ml of test sample solution
out the same procedure as that for test sample solution starting
and l. 5 mi of saturated sodium carbonate solution [Measure
from " Allow the mixture to stand at room temperature for
a quantity of sodium carbonate (Na2CÜ3• lOH2Ü) and mix
15 minutes".
wi.th the same quantity of water. Mix thoroughly and
A linear regression equation is obtained by regressing the
preserve the supernatant for use] and add into the outer
concentration of polyethylene glycol reference solutions with
circle of the disk. Put on a glass immediately with its rough
the corresponding absorbance. The polyethylene glycol
side downward to seal the disk tightly. Mix well by shaking
concentration F ( µg/ ml) of the test sample solution is
carefully. React at 80ºC for 30 minutes. Determine the
obtained by inserting the absorbance of the test sample
absorbance (A 1 ) of the solution in the inner circle at 650 nm
solution to the linear regression equation.
by ultraviolet-visible spectrophotometry ( 0401 ) .
Calculate the result according to the following equation:
An ethanol reference solution is prepared by measuring
accurately a quantity of absolute ethanol and dilute with Polyethylene glycol concentration of test sample (g/L)
water to a concentration of O. 25 mg per ml. Measure FXnXl0 3 •
accurately l. 5 ml of the ethanol ref erence solution and carry Where: F = Polyethylene glycol content of test sample
out the same procedure as that for test sample solution. The solution, µg/ml;
absorbance obtained is Az. The reading of A1 shall be not n = Dilution factor of test sample.
greater than Az.
[Note]
( 1) The whole course of absorbance reading shall be completed
3202 Determination of Residual within 15-45 minutes after the addition of reagents, otherwise
Polyethylene Glycol Content the result will be influenced.
The sensitivity of this method increases along with the
1be method is based on the principie that !X)lyethylene glycol re.acts increase of relative molecular mass of polyethylene glycol.
with barium and iodine ions and forms a complex (1 : 1). The (2) The protein solution used for the preparation of 1%
!X)lyethylene glycol content is determinei by spectroplntometry. protein solution shall not contain polyethylene glycol.
Procedure
A test sample solution is prepared by diluting the test sample 3203 Determination of Residual
with water to a protein concentration of not more than 1%. Polysorbate 80 Content
Measure accurately l. O ml of the test sample solution and
put into a test tube. Add 5. O ml of O. 5 mol/L perchloric
The method is based on the principie that polyethoxylated
acid and mix well. Allow the mixture to stand at room
radicals in polysorbate 80 react with cobaltic ammonium
temperature for 15 minutes and centrifuge at 4000 r/min for
thiocyanate to form a blue complex, which is soluble in
10 minutes. Mix 4. O ml of the supematant with l. O ml of
dichloromethane. The polysorbate 80 content is determined
5 % barium chloride solution ( Dissolve 5 g of powdered
by spectrophotometry.
barium chloride in water and dilute to 100 mD and O. 5 ml of
O. 1 mol/L iodine solution ( Dissolve 2. O g of potassium Procedure
iodide with a small amount of water. Then, add l. 3 g of Measure l. O ml of test sample and put into a centrifugal
iodine and dilute to 50 ml with water.) Mix well and react at tube. Add 5 ml of saturated sodium chloride ethanol solution
room temperatures for 15 minutes. Read the absorbance at and mix well. Centrifuge at 3000 r/min for 10 minutes and
535 nm by ultraviolet-visible spectrophotometry ( 0401 ) . A collect the supernatant. Wash the wall of tube carefully with
 3205 Determination of Residual Tributyl-phosphate Content
l. O ml of saturated sodium chloride ethanol solution, and
pool the washing and supernatant. Centrifuge at 3000 r/min
for 10 minutes again. Allow the supernatant to stand in 55ºC
water bath and concentrate to a volume of about O. 1-0. 5 ml
with blowing air. Dissolve with 1 ml of water. Add
accurately 2. O ml of dichloromethane and 3. O ml of cobaltic
ammonium thiocyanate solution (Dissolve 6. O g of cobaltic
nitrate and 40. O g of ammonium thiocyanate in water and
dilute to 200 mD. Stopper and mix well, allow to stand at
room temperature for one and a half hours. Shake the
mixture every 15 minutes. Allow the mixture to stand for
another half an hour befare determination. Di.scard the upper
layer. Read the absorbance of the lower layer of dichloromethane
at 620 nm by ultraviolet-visible spectrophotometry( 0401) using
dichloromethane as a blank control
Measure accurately O, 10, 25, 50, 75 and 100 µl of
polysorbate 80 reference solution (Weigh accurately 100 mg
of polysorbate 80 and dissolve in small amount of water.
Transfer the solution totally to a 100 ml volumetric flask and
dilute to volume with water) and put into a series of
centrifugal tubes containing 1 ml of water separately. Mix
welL To each tube, add accurately 2. O ml of dichloromethane
and 3. O ml of cobaltic ammonium thiocyanate solution.
Stopper and mix well. Carry out the same procedure as that
for test sample starting from "allow to stand at room
temperature for one and a half hours".
A linear regression equation is obtained by regressing the
concentrations of polysorbate 80 tubes with the corresponding
absorbance. The correlation coefficient obtained shall be not
less than O. 98. The polysorbate 80 content (µg/mD of the
test sample is obtained by inserting the absorbance of the test
sample into the linear regression equation.
3204
The method is based on the principie that glutaraldehyde
reacts with 2, 4-dinitrophenylhydrazine to form n -pentanal-
dinitrophenylhydrazine. The glutaraldehyde content of test
sample is determined with high performance liquid
chromatography ( 0512).
Parameters for chromatography
The chromatographic column of 4. 6 mm in díameter and 250
mm in length is filled with octadecyl silane of silica gel
(SG-120) of 5 µm grain size. The mobile phase is 70%
acetonitrile solution at a flow rate of l. 2 ml per minute.
Record the chromatogram at 360 nm for 30 minutes.
Procedure
Weigh accurately a quantity of glutaraldehyde and dissolve in
water to prepare a glutaraldehyde reference solution at a
concentration of 10 µg per ml. Measure accurately O. 2,
O. 4, O. 6, O. 8 and l. O ml of glutaraldehyde reference
solution and put into test tubes separately. Make up the
volume of each tube to l. O ml with water. To each tube,
add accurately 1 ml of mobile phase solutíon and O. 1 ml of 2,
4-dinitrophenylhydrazine solution ( Dissolve 2. 4 g of 2, 4-
dinitrophenylhydrazine in 30 % perchloric acid solution to
make a 100 ml solution). Mix by a mixer immediately.
Filter with a O. 45 µm membrane separately. Centrifuge a
quantity of test sample at 3000 r/min for 10 minutes.
Measure accurately 1 ml of the supematant and carry out the
same procedure as that for CRS starting from "add accurately
1 ml of mobile phase solution''. Measure accurately 10 µl of each
of reference solutions and test sample solution, and inject into
the apparatus separately. Record the chromatogram.
A linear regression equation is obtained by regressing the
concentration of glutaraldehyde reference solutions with the
corresponding peak areas. The glutaraldehyde content of test
sample is obtained by inserting its peak area into the
equation.
[Note]
( 1) The purity of glutaraldehyde solution used for the
preparation of reference solution has been determined by
chromatography and the amount of O. 1 g is calculated on the
base of 100% glutaraldehyde.
( 2) The correlation coefficient of the linear regression
equation shall be not less than O. 99.
3205 Detennination of Residual
Tributyl-phosphate Content
The residual tributyl phosphate content in test sample is
determined by gas chromatography ( 0512).
Parameters for chromatography and test for system suitability
Acid denatured polyethylene glycol ( 20M) capillary column
is used. The temperature of column is 140ºC and that of
vaporizer is 190ºC. Flame ionization detector ( FID) or
nitrogen phosphorus detector is used at a temperature of
210ºC. Flow gas is pure nitrogen ata flow rate of 60 ml per
minute. Detection parameters may also be chosen according
to the instrument used. The number of theoretical plates of
column based on the peak of tributyl phosphate shall be not
less than 5000. The separation degree between tributyl
phosphate and tripropyl phosphate shall be not less than
l. 5. The relative standard deviation ( RSD) for the peak
area ratio of tributyl phosphate to tripropyl phosphate shall
be not more than 5 % in five successive determinations of
tributyl phosphate reference solution.
Preparation of internal standard
Weigh a quantity of tripropyl phosphate and dissolve in
n-hexane to prepare volumetrically a solution of 400 µg per mL
Procedure
Measure accurately 3 ml of test sample and put into a glass
centrifugal tube with stopper. Add accurately 50 µl of
tripropyl phosphate internal standard solution and O. 75 ml of
l. 5 mol/L perchloric acid solution. Oscillate for 1 minute
and incubate in 37°C water bath for 10 minutes. Add 4 ml of
n-hexane and oscillate for 2 minutes. Centrifuge at 2000 r/min
for 20 minutes. Pipette out the upper n-hexane layer carefully,
concentrate to about O. 2 ml with blowing air ( heating is not
allowed)and inject O. 1 µl into the gas chromatographic apparatus.
Weigh accurately a quantity of tributyl phosphate CRS and
dissolve in n-hexane to prepare volumetrically a solution of
600 µg per ml. To a set of glass centrifugal tubes with
stoppers, each containing 3 ml of water, add 10, 20, 40,
60 and 80 µl of tributyl phosphate ref erence solutions
(600 µg/mD separately. To each tube, add accurately 50 µl
of tripropyl phosphate internal standard solution. Carry out
the same procedure as that for test sample startíng from
"oscillate for 1 minute".
A regression equation is obtained by regressing the tributyl
phosphate concentration of reference solutions with the
corresponding peak area ratios of tributyl phosphate reference
solutions to the intemal standard. Calculate the tributyl
phosphate content (µg/mD of sample solution.
[Note]
( 1 ) The evaporation speeds of test sample solution and
reference solution shall be kept in conformity as much as
 3206 Determination of Residual Carbodiimide Content
possible. If emulsion fails to disappear completely after
centrifugation, oscillate the tube slightly on the oscillator
and centrifuge once again.
( 2) The correlation coefficient of the linear regression
equation shall be not less than O. 99.
3206 Determination of Residual
Carbodiimide Content
The method is based on the principle that dimethyl barbituric
acid reagent reacts with carbodiimide CEDAC) to farm violet red
complex. The EDAC content is determined by lN-visible
spectrophotometry.
Reagent
( 1) Dimethyl barbituric acid
Weigh 1 g of dimethyl barbituric acid and add into 16 ml of
pyridine, add water to a total volume of 20 ml and mix well.
Prepare the solution just befare use.
(2) Pyridine acetate solution
Mix glacial acetic acid with an equal volume of pyridine well.
Prepare the solution just befare use.
(3) 100 µmol/L EDAC reference stock solution
Weigh O. 0192 g of EDAC, dissolve in water and dilute to
100 ml to prepare 1 mmol/L EDAC solution. Transfer 1 ml
of 1 mmol/L EDAC solution to a 10 volumetric flask and
dilute to volume with water to obtain 100 µmol/L EDAC
reference stock solution. Prepare the solution just befare use.
(4) Working solution of EDAC reference
Dilute 100 µmol/L EDAC reíerence stock solution to prepare
the working solution of EDAC reference at concentrations of
10, 20, 40, 60 and 80 µmol/L, respectively.
Procedure
Measure O. 2 ml of test sample and O. 2 ml of working solution of
EDAC reference and add l. 8 ml of dimethyl barbituric acid
respectively and mix well, using O. 2 ml of water as blank
control. Let the mixtures stand at room temperature in dark
place far 30 minutes, add 2. O ml of pyridine acetate solution
respectively, mix well and determine the absorbance at a
wavelength of 599 nm. If there is interference during the test,
centrifuge the mixtures at 4000 r/ min far 5 minutes befare
determination of absorbance.
Result calculation
A linear regression equation is obtained by regressing the
concentration of EDAC reference solution with the
corresponding absorbance. The EDAC content in test sample
solution is obtained by inserting the absorbance of test
sample solution into the equation, based on which the mean
is calculated.
Residual EDAC content ( µmol/L) in test sample= Mean
concentration of EDAC in test sampleX dilution factor
3207 Determination of Free
Formaldehyde Content
Method 1 Colorimetry
The method is based on the principle that fuchsin-sulfurous
acid can react with farmaldehyde and farm a violet complex.
The farmaldehyde content in test sample is determined by
spectrophotometry.
Preparation of reference solution
Measure accurately a quantity of standardized formaldehyde
solution and put into a 500 ml volumetric flask. Dilute to
volume with water and mix well. A formaldehyde reference
stock solution at a concentration of O. 05 % is prepared.
Measure accurately 10 ml of O. 05 % formaldehyde reference
stock solution and put into a 100 ml volumetric flask. Dilute
to volume with water and mix well. A formaldehyde
reference solution at a concentration of O. 005 % is prepared.
This solution shall be prepared just befare use.
Procedure
A test sample solution is prepared by measuring accurately 1
ml of test sample and dilute to a formaldehyde concentration
of about O. 005 % with water. Transfer accurately 1 ml of the
test sample solution to a 50 ml test tube with stopper. Add
4 ml of water, 10 ml of fuchsin-sulfurous acid solution and
10 ml of mixed acid solution ( Put 783 ml of water into a
beaker. Add 42 ml of hydrochloric acid and 175 ml of
sulfuric acid slowly with gently stirring. Mix well). Mix
well, allow to stand at 25ºC for 3 hours. Read the
absorbance at 590 nm by ultraviolet-visible spectrophotometry
<0401 ).
Measure accurately O, O. 5, l. O, l. 5 and 2. O ml of
O. 005 % formaldehyde reference solution and put into a series
of 50 ml test tubes with stoppers separately. Add water to a
volume of 5 ml. Other procedures are the same as that for
test sample solution starting from adding "... 10 ml of
fuchsin-sulfurous acid solution".
A linear regression equation is obtained by regressing the
concentration of formaldehyde reference solutions with the
corresponding absorbance. The concentration of farmaldehyde in
test sample solution is obtained by inserting its absorbance to
the linear regression equation Calculate the free formaldehyde
content in test sample.
[Note]
( 1 ) Preparation of fuchsin-sulfurous acid solution and
calibration of sulfur dioxide content
Weigh 4. 5 g of basic fuchsin and put into a 3000 ml conical
flask. Add 1500 ml of water. Dissolve the fuchsin
completely by shaking or heating. Cool clown. Add 10 g of
sodium sulfite and mix well. Allow the mixture to stand for
5-1 O minutes. Then, add 40 ml of 3 mol/L sulfuric acid
solution and mix well again. Stopper tightly with a rubber
stopper, allow to stand overnight. If colour appears, add 5-
10 g of bone charcoal and mix rapidly. Filter quickly with a
Buchner funnel. The filtrate is fuchsin-sulfurous acid
solution. The sulfur dioxide content of this solution may be
controlled at a range of 28-48 mol/L. The excess of sulfur
dioxide may be expelled by aeration. If the sulfur dioxide
content is too low, more sulfur dioxide gas may be conducted
into the solution
Determination of sulfur dioxide content
Measure 10 ml of fuchsin-sulfurous acid solution and put into
a conical flask. Add 20 ml of water and 5 ml of starch
indicator solution. Titrate with O. 1 mol/L iodine VS until
light blue colour appears.
Calculate the sulfur dioxide content according to the following
equation:
Sulfur dioxide content (mmol/L) =50XVXC
Where: V= Volume of O. 1 mol/L iodine VS consumed,
ml;
C=Concentration of iodine VS, mol/L.
(2) Calibration of formaldehyde solution
Measure accurately about l. 5 ml of formaldehyde, 37 %
aqueous solution and put into a conical flask. Add 10 ml of
water, 25 ml of hydrogen peroxide solution and two drops of
bromothymol blue indicator solution. Add 1 mol/L sodium
hydroxide VS dropwise until the solution shows a blue
colour. Then, add accurately 25 ml of 1 mol/L sodium
 3208 Determination of Residual Aluminium Content in Human Albumin

hydroxide VS. Put a small funnel on top of the conical flask. [Note]
Heat the flask in a water bath for 15 minutes with shaking For any specific product, quantitative spiking shall be used for
constantly. Cool clown. Wash the funnel with water and let the validation of accuracy and repeatability so as to identify the
the washings flow into the conical flask. Add two drops of interference factors and determine the linear range and suitability
bromothymol blue indicator solution and titrate with 1 mol/L of the method.
hydrochloric acid VS until the solution shows a yellow colour.
Calibrate the result with a blank test One ml of 1 mol/L
3208 Determination of Residual
sodium hydroxide VS is equivalent to 30. 03 mg of formaldehyde.
( 3) Sometimes, the colour developing times of test sample Aluminium Content in Human Albumin
solution and reference solutions are different. In such case,
fuchsin-sulfurous acid solution may be added earlier for those The residual aluminium content in human albumin is
develop slower as required. determined with atomic absorption spectrophotometry.
( 4) If the test sample contains phenol red, the standard Procedure
tubes shall be calibrated correspondingly. Prepare blank control solution, test sample solution, and rnixed
Method 2 Acetylacetone Colorimetry solution of aluminium CRS plus test sample according to the
This method is based on the principie of Hantzsch Reaction requirements in Table l. The test sample and 100 ng/ml
for the determination of trace amount of free formaldehyde. aluminium reference solution (Measure accurately O. 1 ml of
In an approximately neutral mixed solution of acetylacetone 100 µg/ml aluminium reference solution, put into a 100 ml
and ammonium salt, formaldehyde forms a yellow compound volumetric flask and dilute to volume with O. 15 mol/L nitric
[3, 5- diacetyl-1, 4-dihydrolutidine ( DDL ) J. The acid) shall be measured accurately. Carry out the determination
absorbance of the compound at 412 nm increases by atomic absorption ultraviolet-visible spectrophotometry
proportionally to the content of formaldehyde. The content of <0406) at 309. 3 nm using aluminium lamp with a slit of
free formaldehyde is calculated based on the absorbance of O. 7 nm. The temperature-controlling program for the
the test sample. drying, incinerating and atomizing of graphite furnace is
Reagent
listed in Table 2. Measure accurately 30 µl each of blank
Acetylacetone colouring solution: Dissolve 150 g of control solution, test sample solution and mixed solution of
ammonium acetate in a quantity of water, add 3 ml of acetic aluminium CRS and test sample, and inject into the
acid and 2 ml of acetylacetone, mix well, and add water to a instrument separately. Record the readings and calculate by
volume of 1000 ml. Store the solution at room temperature, the following equation:
protected from light. Use the solution within the prescribed Aluminium content of test sample (µg/L)
20X CSo-B) X12.5/ CS-5 0 )
validity period.
Where: B = Reading of blank control solution;
Preparation of fonnaldehyde standard solution
Measure accurately a quantity of previously calibrated So= Reading of test sample solution;
formaldchydc standardsolution, put into a 500 ml volumetric S = Reading of mixed solution of
aluminium CRS and test sample;
flask, dilute to volume with water. Mix well to prepare a
O. 05 % ( W /V) formaldehyde standard stock solution.
The value of 20 is the concentration of aluminium in
Befare use, measure accurately 20 ml of the stock solution, mixed solution of aluminium CRS and test sample,
put into a 100 ml volumetric flask. Dilute to volume with µg/L;
water, and mix well, the formaldehyde standard solution at a The value of 12. 5 is the dilution factor of test
sample.
concentration of 100 µg per ml is preparecl.
Procedure Table 1 Preparation of blank control solution,
Measure accurately a volume of test sample ( containing test sample solution and mixed aluminium
around 50 µg of free formaldehyde) and put into a test tube. reference and test sample solution
Make up to 5 ml with water. Add 5 ml of acetylacetone Aluminium
Test
colouring solution and mix well. lncubate in a water bath at Blank control CRS plus
Solution sample
40 ºC for 40 minutes. Take the test tube out of water bath (ml) test sample
(ml)
and cool to room temperature ( for about 10 minutes). (ml)
Measure the absorbance at 412 nm by ultraviolet-visible Test sample o. 2 0.2
spectrophotometry < 0401 ) . If the mixture is turbid after Aluminium CRS
colour development, centrifuge at 3000 r/min for 15 minutes 0.5
(100 ng/mD
and read the absorbance of supernatant.
O. 15 mol/L HN0 3 2.5 2.3 l. 8
Dilute the formaldehyde standard solution to 100 µg/ml, 75
µg/ml, 50 µg/ml, 25 µg/ml, 5 µg/ml, 1 µg/ml, O. 5
µg/ml, O. 25 µg/ml standard solution. Measure accurately 1 Table 2 Temperature-controlling Program for
ml each of the above standard solution. Next steps of the Graphite Furnace
procedure are the same as that for test sample solution Time ( second)
starting from "··· Make up to 5 ml with water". Temperature
Program Procedure for Climbing+
Carry out a blank control test by measuring accurately 5 ml C°C)
Duration
of water and proceed with the same procedure starting from
1 Pre-heating 80 0+10
"add 5 ml of acetylacetone colouring solution".
A linear regression equation is established between the 2 Drying 220 120+5
concentrations of formaldehyde standard solution and the 3 Incinera ting 1200 10+20
absorbance. The free formaldehyde content of test sample is 4 Atomizing 2600 o+5
obtained by plotting the absorbance value into the regression
5 Clearance 2650 o+5
equation.
 3209 Determination of Residual Hydroxylamine
[Note]
( 1) The amounts of test sample and aluminium CRS taken
may be adjusted according to the function of instrument used
in order to make all the readings fall into the accurate range.
(2) The temperature-controlling program for fumace given
in Table 2 may be properly adjusted according to the function
of instrument used.
(3) The use of glass containers shall be avoided as much as
possible.
3209 Determination of Residual
Hydroxylamine
This method is based on the reaction of hydroxylamine with
iodine to produce nitrous acid in alkaline conditions, followed
by the diazotization reaction with sulfanilic acid, and coupled
with alpha naphthylamine to form coloured azoic compound.
The content of hydroxylamine is determined by UV-visible
spectrophotometry.
Reagents
(1) 6% Sodium acetate solution
Weigh 6 g of anhydrous sodium acetate, add appropriate
amount of water to dissolve and dilute to 100 mi. Mix well.
(2) 1 % Sulfanilic acid solution
Weigh O. 50 g of sulfanilic acid, add appropriate amount of
25 % acetic acid solution to dissolve and dilute to 50 mi.
(3) l. 3% Iodine solution
Weigh O. 65 g of iodine and dissolve with glacial acetic acid,
dilute to 50 ml. Prepare and use in ventilation fume hood.
(4) O. 4 mol/L Sodium thiosulfate solutionWeigh 3. 16 g of
sodium thiosulfate and dissolve with appropriate amount of
water, dilute to 50 mi.
(5) O. 6% anaphthylamine solution
Weigh O. 3 g of naphthylamine and dissolve with 30% acetic
acid. Dilute to 50 ml. Prepare and use in a fume hood.
3300 Microbiological Test
3301 Test for Mycoplasma
Where the test for mycoplasmas is prescribed for a master
cell bank, for a working cell bank, for a virus seed lot, for
control cells or for the cells for therapeutic use, both the
incubation method and the indicator cell culture method
CDNA staining method) are used. Where the test for
mycoplasmas is prescribed for a virus harvest or for a bulk
vaccine, the culture method is used. The indicator cell culture
method may be used, where necessary, for screening of media
Other methods accepted by the NCL may also be used.
Method 1 lncubation Method
Recommended culture media and formulas
C1) Mycoplasma liquid medium
Mycoplasma broth medium
Pig gastric digest 500 ml
Beef extract (1 : 2) 500 ml
Y east extracts 5. o g
Sodium chloride 2. 5 g
Glucose 5. o g
Preparation of Hydroxylamine hydrochloride reference
standard solution
Quantitatively dissolve hydroxylamine hydrochloride reference
standard with water to make stock solution at concentration of
1000 nmol/ml. Accurately measure appropriate amount of stock
solution and dilute with water to make various concentration of
reference standard solutions (150 nmol/ml, 120 nmol/ml, 90
nmol/ml, 60 nmol/ml and 30 nmol/mD. Freshly prepare
befare use.
Test Procedures
Accurately transfer O. 3 ml of test sample into a tube and add
l. 3 ml of 6 % sodium aceta te solution, O. 2 ml of 1 %
sulfanilic acid solution and O. 1 ml of l. 3 % iodine solution in
sequence. Mix well and stand for 10 minutes. Add 50 µl of
O. 4 mol/L sodium thiosulfate solution. Mix well and decolour.
Add 40 µl of O. 6 % a-naphthylamine solution and mix well. Store
the mixture at room temperature for 60 minutes. After
centrifugation at 10 000 rpm for 5min, determine the
absorbance of supernatant at wavelength of 520 nm using
UV-visible spectrophotometry method ( 0401 ) . Accurately
pipette O. 3 ml of each concentration of reference standard
solution into a tube and perform the same procedures
described above from "add l. 3 ml of 6 % sodium aceta te
solution". Accurately pipette O. 3 ml of water into a tube and
perform the same procedures described above from "add l. 3
ml of 6 % sodium aceta te solution" to make a blank control
solution. The concentration of hydroxylamine reference
standard solution is plotted against corresponding absorbance
to generate a linear regression equation. Absorbance of test
sample is substituted into the linear regression equation to
e
calculate concentration nmol/ ml) of residual hydroxylamine.
e
Calculate the content of residual hydroxylamine nmol/ mg
protein) in test sample by the following equation.
Content of residual hydroxylamine in test sample ( nmol/ ml
protein) = Concentration of residual hydroxylamine in test
sample (nmol/ml) /Content of protein in test sample (mg/ml)
Phenol red O. 02 g
pH 7. 6±0. 2. Sterilize at 121 ºC for 15 minutes.
Mycoplasma arginine medium
Pig gastric digest 500 ml
Beef extract (1 : 2) 500 ml
Y east extracts 5. O g
Sodium chloride 2. 5 g
Glucose l. O g
L-arginine 2. O g
Phenol red O. 02 g
pH 7.1±0. 2. Sterilize at 121ºC for 15 minutes.
(2) Mycoplasma semi-solid medium
Prepare the medium according to the formula ( 1). Add 2. 5-
3. O g of agar but no phenol red is added.
C3) Mycoplasma agar medium
Prepare the medium according to the formula C1 ). Add
13. 0-15. O g of agar but no phenol red is added.
Besides the recommended media above, other media which
can support the growth of mycoplasma also can be used, but
the sensitivity of medium must meet the requirements.
Sensitivity test on culture media CColor change unit method)
(1) Strain

Mycoplasma pneumoniae C Strain ATCC 15531 ) and
Mycoplasma orale CStrain ATCC 23714) are distributed by
the NCL.
C2) Procedure
lnoculate the strain in a suitable mycoplasma medium and
incubate at 36ºC ± 1 ºC till the colour of the medium changes.
After two blind passages, inoculate the culture into the
medium to be tested and make a 10-fold serial dilution. For
Mycoplasma pneumoniae, dilute to 10- 7 -10- 9 and then
inoculate into mycoplasma broth medium; for Mycoplasma
orale, dilute to 10- 3 -10- 5 and then inoculate into
mycoplasma arginine broth medium, three tubes of medium
for each dilution. lncubate at 36ºC ± 1 ºC for 7-14 days and
observe the result of colour changing.
C3) Result evaluation
The highest dilution, which makes a colour change in more
than two-thirds of all the inoculated medium tubes, is
regarded as the sensitivity of the culture medium.
Sensitivity of liquid medium: 10-s for Mycoplasma pneumoniae
CStrain JXIU:: 15531) and 10- 4 for Mycoplasma orale CStrain
JXIU:: 23714).
Procedure
CD Test sample may be preserved at 2-8ºC if the test for
mycoplasma is carried out within 24 hours after filling;
otherwise it shall be preserved below -20ºC
C2) Liquid medium and Semi-liquid medium Cor mycoplasma
agar medium) shall be used for the test Before use, boil the
semi-liquid medium Cor agar medium) for 10-15 minutes
and cool clown to about 56ºC. Add inactivated calf serum
Cthe volume ratio of medium to serum is 8 : 2) and a
quantity of penicillin, wherever necessary, and make it even
by shaking. For broth medium the same components as
mentioned above shall be added but boiling is not needed.
lnoculate O. 5-1. O ml of test sample into each of four tubes
containing 10 ml of liquid medium and each of two tubes
containing 10 ml of the corresponding semi-liquid medium
Cwhich have been cooled to 36ºC ± 1 ºC), and incubate at
36ºC ± 1 ºC for 21 days. On the 7th day after inoculation,
take two of four inoculated liquid media tubes respectively
and make a subculture by inoculating each of two tubes into
two fresh tubes containing the corresponding semi-liquid and
liquid media separately and incubate at 36ºC ± 1ºC for 21
days.
C3) Result evaluation
The product to be tested passes the test if growth of
mycoplasmas has not occurred in any of the inoculated media
at the end of incubation. If there is suspected growth of
mycoplasmas, the test may be repeated once using twice the
amounts of test sample. The product complies with the test
if growth of mycoplasma <loes not occur.
[Note]
Quality control department shall examine the sensitivity of
mycoplasma culture media in coordination with medium
preparation department periodically.
Method 2 lndicator Cell Incubation Method ( DNA Staining
Method)
lnoculate indicator cell cultures Cwith Vero cells free from
contamination or other cells accepted by the NCL) with the
test sample and then stain with a specific fluorescent dye. If
the test sample has been contaminated with mycoplasmas,
the DNA of mycoplasmas attached on the cell surface shall be
stained with the fluorescent dye that binds specifically to the
DNA of mycoplasma, which can be easily recognized by
using fluorescent microscopy.
3301 Test for Mycoplasma
Reagent
CD Concentrated solution of bis-benzimide fluorescent dye
CHoechst 33258)
Dissolve 5 mg of bis-benzimide fluorescent dye in 100 ml of
Hank balance solution without phenol red or sodium
bicarbonate. Stir with a magnetic stirrer at room temperature
for 30-40 minutes to make it dissolved completely. Store at
-20ºC rotected from light
C2) Bis-benzimide fluorescent dye working solution
Dilute 1 ml of concentrated solution of bis-benzimide fluorescent
dye with 100 ml of Hank balance solution without phenol red or
sodium bicarbonate. Mix well.
C3) Fixing solution
Mix one volume of acetic acid with three volumes of
methanol.
C4) Sealing solution
Mix 22. 2 ml of O. 1 mol/L citric acid solution and 27. 8 ml of
O. 2 mol/L disodium hydrogen phosphate solution with 50. O ml
of glycerin. Adjust pH to 5. 5.
Culture media and indicator cells
C1) DMEM complete medium
C2) DMEM medium without antibiotic
C3) lndicator cells CVero cell or other cell line which has
been approved to be free from mycoplasma contamination)
Digest the incubated Vero cells and prepare a cell suspension
at a concentration of 105 cells per ml and inoculate O. 5 ml
into each well of a 6-well culture plate or other containers.
To each well, add 3 ml of antibiotic-free culture medium.
lncubate in a 5 % carbon dioxide incubator at 36ºC ± 1 ºC
overnight for use.
Treatment of test sample
C1) Cell cultures
Make not fewer than one subculture of test sample in
antibiotic-free medium. (',ollect the supernatant of the culture
in a three-day-unchanged medium when a confluent cell sheet
is formed.
C2) Viral suspensions
If for viral suspensions the interpretation of results is affected
by marked cytopathic effects, the virus may be neutralized
using a specific antiserum that has no inhibitory effects on
mycoplasmas or a cell culture substrate that <loes not allow
growth of the virus may be used.
C3) Other test samples
The indicator cells used for test shall be those that their
growths are not effected by the test sample.
Procedure
Add 2 ml of test sample Csupernatant of cell culture) or at
least 1 ml of viral suspension or other test sample into the
prepared indicator cell culture plate and incuba te in a 5 %
carbon dioxide incubator at 36ºC ± 1ºC for 3-5 days. Make
not fewer than one subculture of the indicator cell culture and
grow the last subculture in a 6-well culture plate with glass
covers for 3-5 days. Draw out the culture fluid from the
wells, add 5 ml of fixing solution, allow to stand for 5 minutes.
Draw out the fixing solution, add another 5 ml of fixing
solution and fix for 10 minutes. Draw out the fixing solution
again and dry the glass covers in the air. Add 5 ml of
bisbenzimide fluorescent dye working solution Cor other
DNA dyes) and put on covers. Allow the plate to stand at
room temperature for 30 minutes. Draw out the dye solution
and wash each well 3 times with water, 5 ml for each time.
Draw out the water and dry glass covers in the air. Add one
drop of sealing solution on a clean slide and put the glass
cover up side clown on the sealing solution to make sealed
slides. Observe with a fluorescent microscope.
 3302 Test for Adventitious Viruses
Carry out the negative control test with the same procedure
by using 2 ml of antibiotic-free culture medium.
Carry out the positive control test with the same procedure
by using 2 ml of standard strain known as positive.
Result evaluation
( 1) Negative control
Extranuclear fluorescence of the indicator cells shall be
examined which shall be greenish-yellow fluorescent light.
(2) Positive control
Besides the cells, fluorescent particulates with irregular
pattern of fluorescence on the cell surface can be observed
under fluorescent microscope.
The test is valid only when the validity tests of both negative
and positive controls proved qualified.
If the test result is negative, the product complies with the
test. If the test result is positive or suspected, the test shall
be repeated. If the result is positive again, the product fails
to pass the test.
3302 Test f or Adventitious Viruses
During the seed selection and production process of viral
preparations, animal or cell substrates are usually used, so
the preparations may be potentially contaminated with
adventitious agents. Tests for adventitious agents on virus
strain and cells are necessary to ensure the quality of the
prepara ti o ns.
Tests for adventitious agents in virus seed lots
A sufficient quantity of samples shall be taken from the
master or working seed lots for testing of adventitious
agents. In sorne cases, it may be necessary to neutralize the
virus prior to test based on the nature of virus seed. The
virus seed for neutralization is diluted as little as possible. If
the virus seed which cannot be neutralized efficiently by
antibodies need to be diluted, the choose the appropriate
virus dilution which contains the maximum amount of
viruscapable for neutralization and is at least not more than
the dilution multiple for vaccine production use. neutralize
the viruses with specific antibodies from non-human and non-
simian origins, apart from an exceptional case. To reduce the
likelihood, of neutralizing adventitious virus that could be
present in test sample monoclonal antibodies are selected
preferably. The neutralization procedure should not interfere
with detection of adventitious virus The immunizing agents
used to prepare antiserum Cor monoclonal antibody) shall be
produced in cell culture ( or animal) from a species different
from that used for the production of the vaccine and free
from adventitious agents. If the viruses have been propagated
in avian tissues or cells, the antibodies must be of non-avian
origin. If chick embryo is used for the test, it shall be
obtained from a flock free from specified pathogens.
l. Test in animals
(1) Test in adult mice
Inoculate i. c. each of at least ten adult mice, weighing 15-
20 g, with O. 03 ml and inoculate i. p. with O. 5 ml of virus
seed lots or virus suspension after neutralization with
antiserum. Observe the mice for at least 21 days. Carry out
an autopsy of all mice that die after the first 24 h of the test
or that show signs of illness, and examine for the pathological
evidence of viral infection by direct macroscopical observation
The suspensions prepared with the tissues showing pathological
changes are inoculated i. c. and i. p. into at least five
additional mice of 15-20 g which are observed for 21 days.
The test is invalid if more than 20 % of the mice died within
the first 24 hours after inoculation. The test virus seeds
comply with the test if at least 80 % of the initially inoculated
mice and at least 80 % of the mice in blinded-pass group
remain and survive healthy in observation period, and if none
of mice shows evidence of transmissible agents or other virus
infection unrelated to the test virus.
(2) Test in suckling mice
Inoculate i. c. each of at least ten suckling mice, less than
24 h old, with O. 01 ml and inoculate i. p. with at least O. 1
ml of virus seed lots or virus suspension after neutralization
with antiserum. Observe daily for at least 14 days. Carry out
an autopsy of all mi ce that die af ter the first 24 h of the test
or that show signs of illness and examine for the pathological
evidence of viral infection by direct macroscopical
observation The suspensions prepared with the tissues
showing pathological changes as well as with the brain and
spleen are inoculated i. c. and i. p. into at least five
additional suckling mice which shall be observed for 14 days.
The virus seed lot complies with the test if no suckling
mouse shows evidence of viral infection The test is invalid if
more than 20 % of suckling mice died within the first 24
hours after inoculation. The test virus seeds complies with
the test if at least 80 % of the initially inoculated suckling
mice and at least 80 % of the suckling mice in blinded-pass
group remain and survive healthy in observation period and if
none of mice shows evidence of transmissible agents or other
virus infection unrelated to the test virus.
2. Cell culture method
(1) Test for non-haemadsorbing viruses
Inoculate the virus seed lots or neatralized virus suspens1on
neutralizing by antiserum onto the cell cultures of human,
simian origins and of the same cell as that used for the
production of vaccine, respectively. Unless otherwise
specified, take at least 10 ml of virus or neutralize at least 10
ml of virus with antiserum for inoculation onto each of cell
cultures If the virus is grown on human diploid cells or
simian origins cells, the neutralized virus suspension shall be
tested on a separate cultures of the diploid cells or simian
origins cells. At least six flasks of each kind of cell cultures
shall be inoculated, and the volume of virus suspension
inoculated into each flask shall be not less than 25 % of the
total volume of culture medium. Incubate at 36ºC ± 1 ºC and
observe for 14 days. Control flasks without or with virus
inoculation shall be conducted simultaneously for each of cell
cultures. If necessary, feed the cultures with fresh medium
or subculture once, and it shall be not less than 7 days to the
end of observation after the last operation It complies with
the requirements if negative and positive controls are valid
and if no CPE appears in each of cell cultures inoculated with
the test virus sample.
(2) Test for haemadsorbing viruses
Take two flasks of each of above-mentioned cell cultures
inoculated with virus on days 6-8 and 14 respectively and test
for the presence of haemadsorbing viruses. Add the mixed
suspension of O. 2 %-O. 5 % chicken and guinea pig red cells
onto the surface of the cell cultures. incubate one flask at
2-8ºC for 30 minutes and 20-25ºC for 30 minutes. Examine
microscopically for hemadsorption after discarding the excess
red cells. The negative and positive controls shall be valid,
the cells inoculated with the test virus sample shall be no
evidence of hemadsorption
3. Test in chick embryo
The virus seed propagated in avian tissues or cells shall be
tested for avian viruses using embryonated chick eggs.
Unless otherwise specified, take 10 ml of virus seed lots or
neutralize 10 ml of virus seed with antiserum for inoculation

Using O. 5 ml per egg, inocula te a group of at least 10
embryonated SPF eggs, 9-11 days old, by allantonic route
and a second group of at least 10 eggs, 5-7 days old, in to the
yolk sac. Incubate the eggs at 35ºC for 7 days. Observe the
survival of the inoculated eggs. Collect the allantoic fluid and
test forthe presence of haemagglutinating viruses with O. 2-
0. 5 % a mixture of chick and guinea pig red cells. It complies
with the test if at least 80 % of the inoculated eggs survive for
7 days and the result of haemagglutinating test is negative.
Tests for adventitious virus agents in control cells used for
production
l. Test for non-haemadsorbing viruses
( 1) Direct observation on cells
A portion of 5 % (or not less than 500 mD of cell suspension of
each batch of cells used for production shall remain uninoculated
as a control, and cultured in the same cell maintenance medium
under the same conditions as those for production. Observe
the cell cultures for at least 14 days or at the time of the last
viral harvest ( whichever is longer) by microscopic examination for
CPE It complies with the requirements if no CPE appears. The
test is valid if at least 80 % of the cell cultures survive to the end of
observation period.
(2) Test in cell cultures
Pool the supernatant fluid at the end of above -mentioned
observation period. Examine for the presence of adventitious
agents by inoculation of the cell cultures of human and simian
origins. If the vaccine virus is grown in a cell system other
than human or simian, the cells of that species but from a
separate batch are also inoculated. The amount inoculated
shall be not less than the 25 % of total amount of the pooled
supernatant. At least 5 ml of the pooled supernatant shall be
inoculated onto each of indicator cell cultures. The amount
inoculated shall be not less than the 25 % of total amount of
medium in each flask. Incubate the inoculated cultures under
the same condition as that for prod.uction for at least 14 days. lt
complies with the requirements if no evidence of CPE is found
2. Test for haemadsorbing viruses
At least 25 % of cell cultures used for the above direct
observation and cell culture test shall be tested for the
presence of haemadsorbing viruses at the end of observation
period by the same method as that for virus seed lot.
3303 Test for Murine Viruses
Murine monoclonal antibodies may be potentially contaminated
with viruses such as hemorrhagic fever virus, lymphocytic
choriomeningitis virus, respiratory enteric orphan virus type
3, Sendai virus, mouse pox virus, mouse adenovirus,
mouse pneumonia virus and retro virus, etc. The viruses are
classified as groups I and 11 . The first four viruses,
classified as group I, can infect humans and primates. The
viruses in group 11 , including another four viruses, so far
there is no evidence that they can infect humans, but can
replicate in vitro in the cell cultures of human, simian and
monkey origins and may be potentially dangerous to humans,
thus shall be monitored emphatically.
The method is applied to the test for the presence of murine
viruses in hybridoma cell lines and murine monoclonal
antibodies. The live virus antigen and antibody are tested by
in vitro infectivity assay, animal antibody production test,
infectivity assay in embryonated eggs, and so on.
Reagent
(1) O. 01 mol/L pH 7. 4 PBS: Dissolve 2. 9 g of disodium
hydrogen phosphate ( Na2 HP04 • 12H2 O), O. 2 g of sodium
3303 Test for Murine Viruses
dihydrogen phosphate, 8. O g of sodium chloride, O. 2 g of
potassium chloride in water and dilute to 1000 ml.
( 2) pH 9. 6 Coating buffer solution: Dissolve l. 59 g of
sodium carbonate, 2. 93 g of sodium bicarbonate and O. 20 g
of sodium azide in water and dilute to 1000 ml.
(3) O. 01 mol/L pH 7. 4 PBS washing solution: Dissolve 2. 9 g
of disodium hydrogen phosphate ( Na2 HP04 • 12H2 O),
O. 295 g of sodium dihydrogen phosphate, 8. 5 g of sodium
chloride and 5 ml of polysorbate 80 in water and dilute to
1000 ml.
( 4) Substrate buffer solution: Dissolve 12. 9 g of disodium
hydrogen phosphate ( Na2 HP0 4 • 12H2O) and 3. 26 g of
citric acid in water and dilute to 700 ml.
(5) Substrate solution: Dissolve 4 mg of o-phenylenediamine
in 10 ml of substrate buffer solution and add 4 µl of 30 %
hydrogen peroxide.
(6) Washing solution: PBS containing O. 1% Tween 20.
(7) Stopping solution: 1 mol/L sulfuric acid solution.
Preparation of test sample
The test samples include hybridoma cell strain, ascites and
final bulk or final product of monoclonal antibody.
Hybridoma cell strain shall be subjected to in vitro infectivity
assay, animal antibody production test and infectivity assay
in embryonated eggs; however, ascites and final bulk or
final product of monoclonal antibody shall be subjected to
animal antibody production test and infectivity assay in
embryonated eggs.
(1) Test sample for in vitro infectivity assay: Three flasks
of hybridoma cells growing well are subject to freezing and
thawing at - 40ºC for 3 times, pooled aseptically and
dispensed into small tubes, 3 ml per tube. Seal the tubes
with rubber stoppers and store at -40ºC.
( 2) Test sample for animal antibody production test:
Ascites and final bulk or final product of monoclonal antibody
need no treatment and shall be stored at - 20ºC However,
hybridoma cells shall be treated by the following method
before use.
Remove the media in seven flasks in which hybridoma cells
grown well. W ash the cells clown wi th PBS by blowing
slightly and transfer in to small tu bes. W ash the flask once
with PBS to collect the residual cells into the same tube and
centrifuge at 1000 r/min for 10 minutes. Discard the
supernatant and re-suspend the cells with PBS. Repeat the
above procedure twice. Re-suspend the pooled cell pellet in 4
ml of PBS. After 3 times of freezing and thawing,
ultrasonicate the cell suspension and centrifuge at 10 000 r/min
for 30 minutes. Collect the supernatant and centrifuge at
40 000 r/min for 4 hours. Discard the supernatant and
dissolve the pellet in a quantity of PBS to obtain the antigen
for animal antibody production test. Store the antigen at
-40ºC.
Procedure
The methods for detection of murine viruses include in vitro
infectivity assay, animal antibody production test,
infectivity test in embryonated eggs, and so on.
l. In vitro infectivity ~y
Detect the presence of unknown virus antigen in test sample
with known virus antibody.
(1) Cell culture: Select the cells sensitive to the virus to be
tested. Six flasks of each kind of cells growing well shall be
used. Wash the cells twice with O. 01 mol/L pH7. 4 PBS.
Inoculate O. 3 ml of test sample of each batch into each of
four flasks, and O. 3 ml of PBS in to the other two flasks as
negative control. After adsorption at 37°C for one hour,
discard the adsorbed test sample fluid and add a quantity of
 3303 Test for Murine Viruses
1
cell maintenance media into each flask. Observe the
morphology of cells every day under microscope and record
the results. Change the media every 3-4 days. Maintain the
first passage of cells for 10-14 days. After 3 times of freezing
and thawing, pool the two flasks of negative control and faur
flasks of test sample separately. lnoculate the harvested cell
suspensions in test and control groups into the same kind of
cells, and change the medium every 3-4 days.
(2) Smear: After incubation of the pooled cell suspension
far 10-14 days, remove the maintenance media and wash the
cells twice with PBS. Add O. 15 ml of digestion solution into
each flask to detach the cells. Pipette the cell suspension into
a tube and wash twice with PBS. Re-suspend the cell pellet
with a quantity of PBS. Smear the normal cells in negative
control group onto the first row of wells on a glass slide, and
the test sample onto the second row. Dry the slide by
blowing and fix the cells with acetone to obtain the cell slide
of test sample. Store the slide at -40ºC.
lndirect immunofluorescence assay: Prepare a slide of known
virus antigen. Dilute the known specific positive and negative
sera 5-to 20-fald with PBS respectively. Examine the slide of
test sample using the prepared slide of known virus antigen
as a serum control. Add the known positive and negative sera
diluted 10-fald with PBS respectively into different wells on
the slide of test sample and incubate at 37°C far 30 minutes
in a wet box. Wash the slide 3 times, each by immersing
Number of mouse
Mouse
Test group Control group
Suck-ling 10
Aged 3-4 weeks 10 10
Aged 6- 8 weeks 10 10 l.
Serological examination: Bleed the mice injected i. m. and i.
p. with test sample and PBS (control) and separate sera for
detecting antibody by ELISA Coat a 96-well microtitre plate
with virus and normal cell antigens, O. 1 ml per well.
lncubate the plate at 37°C far one hour, allow to stand at
4ºC overnight. Wash the plate sufficiently with washing
solution then pat to dry. Add test sample into one virus
antigen well and one normal cell antigen well respectively and
incubate at 37°C far one hour. Wash the plate sufficiently
with washing solution then pat to dry. Add enzyme-labeled
secondary antibody into each well and incubate at 37°C far
one hour. Wash the plate sufficiently with washing solution
then pat to dry. Add O. 1 ml of substrate solution into each
well and incubate at 37°C far 10-20 minutes. Stop the
reaction by adding O. 1 ml of 1 moVL sulfuric acid solution
into each well when positive serum control wells develop
colour, but negative control wells show no colour. Read the
absorbance of each well.
(3) Result evaluation
If the value of P /N is not less than 2. 1, the result is judged
as positive;
If the value of P / N is l. 5-2. O, the result is judged as doubtful;
with PBS far 5 minutes, and dry. Drop fluorescein-labeled
antibody into each well and incubate at 37°C far 30 minutes.
Wash the slides 3 times each by immersing with PBS for 5
minutes, then wash once with water and dry. Add 50%
glycerin onto the slides and cover with a cover glass.
Examine the slide under microscope.
( 4) Result evaluation
The test is valid if on the slides of known virus antigen,
negative control sera show no fluorescence with normal cell
and virus cell wells, while positive control sera show no
fluorescence with normal cell wells but show fluorescence
with virus cell wells; and if on the slides of test sample,
negative control sera show no fluorescence with normal cell
and test sample wells, while positive control sera show no
fluorescence with normal cell well.
The test is invalid if negative control sera show fluorescence
with normal cell and test sample wells, or positive control
sera show fluorescence with normal cell well.
The result is judged as positive if, on the slide of test
sample, the positive control sera show fluorescence with test
sample well.
2. Animal antibody production test
Preparation of antibody to test sample: Inject each batch of
test sample into 50 SPF BALB/ c or KM mice according to
the requirements in the fallowing table:
Dosage
Ro u te Note
(ml/mouse)
Observe the m1ce far 4
i. m. 0.03 weeks. The survival rate
shall be not less than 80 %.
Observe the mice far 4
l. p. 0.03 weeks. The survival rate
shall be not less than 80 % .
Inject 2 doses at an interval
of 10 days. Bleed the mice
i. m.: 0.1
m. and i. p. 14 days after the second
l. p.: o. 2
inj ection. lnj ect PBS into the
mice in control group.
If the value of P / N is less than l. 5, the result is judged as
negative.
P=Absorbance of conjugate of murine sera immunized with
test sample and virus antigen - Absorbance of conjugate of
murine sera immunized with test sample and normal cell
antigen;
N = Absorbance of conjugate of murine sera immunized with
control and virus antigen - Absorbance of conjugate of murine
sera immunized with control and normal cell antigen.
3. lnfectivity ~y in embryonated ~
Observe the embryonated eggs 24 hours befare inoculation.
Clear blood vessels and dark shadow shall be observed in live
embryonated eggs, and blastokinesis may be observed in
bigger ones. However, if the chick embryo is dead, the
blood vessels are dusky and blurred, and no blastokinesis
shall be observed. Examine the eggs far vitality of chick
embryo again with inspection lamp just before inoculation,
and mark the locations of air chamber and embryo. lnoculate
aseptically the test sample into yolk sac, allantoic cavity and
villous allantoic membrane, incubate the embryonated eggs
far 5 days and observe daily. Harvest the yolk sac, villous

allantoic membrane and allantoic liquid by an aseptic operation.
Grind yolk sac and villous allantoic membrane and centrifuge to
collect the supernatant. Carry out hemagglutination tests on
the supernatant and allantoic fluid with guinea pig or chick
erythrocyte separately.
Take a microtitre plate of eight wells in each row. Add 50 111
of physiological saline into the 2nd to the 8th well. Add 50 111
of test sample treated by above-mentioned procedures into
the lst and the 2nd well respectively. Dilute the test sample
2-fold serially by transferring 50 111 of sample from the 2nd to
the 3rd well with a pipette, then 50 111 from the 3rd well to
the 4th well, and so on, until 50 111 is transferred from the
6th to the 7th well. Pipette out 50 111 of sample from the 7th
well and discard. The 8th well is used as a control. Add
50 111 of 1 % guinea pig erythrocyte suspension into the lst to
the 8th well and mix thoroughly. The test is performed in
duplicate. Allow the two microtitre plates stand at 4 ºC and
room temperature respectively until clear negative result is
observed in control well.
Result evaluation
+ + + +: Erythrocytes are evenly dispersed at the bottom
of well;
+ + + : Erythrocytes are evenly dispersed at the bottom of
well, but their borders are irregular and show a tendency of
decline;
+ + : Erythrocytes form a small loop at the bottom of well,
surrounded by small clots;
+: Erythrocytes form a small block at the bottom of well,
bordered with a small number of clots;
- : Erythrocytes are centralized at the bottom of well and
form red point with a dense border;
If the hemagglutination reaction of "+ +" or more appears,
the result shall be judged as positive.
3304 Test for Nucleotide Sequence of
SV40
The presence of SV40 nucleotide sequence in the test sample
is detected by polymerase chain reaction (PCR) using two
pairs of specific primers designed to amplify two fragments,
i. e. 100 bp (2220-2319) of SV40 VPl and 451 bp (2619-
3070) at C-terminus of major T antigen.
Preparation of test sample and control solutions
Add 25 111 of 2% protease K solution, 50 111 of 10% SDS
solution and 10 111 of O. 05 mol/L EDTA solution (pH8. O)
into 400 111 of the test sample. lncubate at 56ºC for one hour.
Extract with an equal volume of mixture of phenol and
trichloromethane ( 1 : 1), and extract again with an equal
volume of trichloromethane. Then add two volumes of
ethanol. Keep at - 20ºC for 16 hours. Centrifuge at 10000
r/min for 15 minutes. Wash the precipitate with 75%
ethanol and dry. Dissolve in 10 111 of DNase-free and RNase-
free water. Positive control and negative control are treated
by the above-mentioned method simultaneously with test
sample.
Primers
1
Upstream primer for VPl: 2220 5 -ACA CAG CAA CCA
CAG TGG TCC-3'2240
Downstream primer for VPl: 2319 5'-GTA AAC AGC CCA
1
CAA ATG TCA AC-3 2297
1
Upstream primer for C-terminus of T antigen: 3070 5 -GAC
CTG TGG CTG AGT TTG CTC A-3 '3049
Downstream primer for C-terminus of T antigen: 2619 5'-
GCT TTA TTT GTA ACC ATT ATA AG-3'2641
3305 Neurovirulence Test in Monkeys
Procedure
( 1) The amount of primer added to the test sample for
amplification is 30 X 10- 12 mol. A portien of 1 111 of DNA
template is added and the final volume is 50 111. PCR
amplification parameters are as follows: Denature at 94ºC for
3 minutes; a total of 40 cycles of 94ºC for 20 seconds, 50ºC
for 20 seconds and 72ºC for 40 seconds, with final extension
of 72ºC for 3 minutes.
( 2) Detect the amplified products by electrophoresis with
2 % agarose gel containing 1 /lg of ethidium bromide per
millilitre. Perform electrophoresis in 1 X T AE buffer solution
at 100 V for about 40 minutes. The length of the amplified
fragment for VPl is 100 bp, and that for C-terminus of
major T antigen is 451 bp.
(3) Repeat the amplification of VPl fragment with the same
template. Care shall be taken to avoid nonspecific
amplification due to contamination. Alternatively, transfer
the amplified products and the controls from the gel to
Hybond N nylon membrane for Southern blotting with VPl
probe in order to make sure that the amplified fragment is
VPl.
( 4) Sequence the C-terminus coding major T antigen of the
amplified products in the test sample and in the positive
control with automatic sequencer.
Result evaluation
The test is valid if the specific products are obtained in the
positive control and no such products are found in the
negative control. Criteria for evaluation are as follows.
The result is negative if no VPl fragment is amplified.
If the VPl fragment is amplified, the test shall be repeated.
If no VPl amplification is shown in the repeated test, the
result is negative.
If VPl amplification is positive in the repeat test, the
fragment of C-terminus coding major T antigen shall be
further amplified. If the fragment is amplified, its sequence
shall be compared with that amplified from the positive
control. If the two sequences are identical, the result is
judged as positive. If no fragment of C-terminus coding
majar T antigen is amplified, the test shall be repeated
following the steps ( 1) to ( 3). If the assay fails again to
amplify the C-terminus coding major T antigen, the result is
j udged as nega ti ve.
3305 Neurovirulence Test in Monkeys
The method is used for the control tests on live
attenuated poliomyelitis vaccine.
Healthy rhesus monkeys each weighing l. 5 kg or above shall
be used. The monkey serum after 1 : 4 dilution shall be
shown to contain no neutralizing antibody to virus of the
same type. The monkeys shall be selected and quarantined,
and shall have not been used for other tests. The quarantine
period shall be not less than 6 weeks. There shall be no
tuberculosis, B virus infection or other acute infectious
diseases. The serum shall be free from antibody to foamy
virus. The monkeys with serious purulent foci, neoplasm as
well as discernable nephro-and hepatopathy shall not be used
for the test. Intraspinal or intracerebral injection method can
be adopted.
Intraspinal inoculation
(1) Number of monkeys
Reference preparation shall be established. For the evaluation
of vaccines and their respective reference preparations of
types I and II , at least eleven valid monkeys for each type
shall be included. However, for the evaluation of type III,
 3306 Requirements for Virus Nucleic Acid Testing on Source Plasma used for Blood Products
at least eighteen valid monkeys are required. The monkeys
with different body weights and sexes shall be allocated
randomly in every group. One reference preparation can be
tested in parallel with more than one lot of vaccine.
A valid monkey is the one of which neuronal lesions specific
for poliovirus shall be shown in the central nervous system.
I t is permi tted to fill the vacancies if the va lid monkeys are
not enough in the test group, and it shall be necessary to
supplement the same number of monkeys to the reference
group, and vice versa. If the test needs two working days,
the number of monkeys used in both test and reference
groups shall be the same on each day. In order to ensure the
numbers of valid monkeys in the two groups, it is always to
inoculate more monkeys than that required.
(2) Virus titers of test sample and reference preparation
The virus contents of the test sample and the reference
preparation shall be adjusted to be as close as possible. Inject
O. 1 mi ( containing 6. 5-7. 5 lg CCID5o per mD interspinally
between the first and second vertebrates of each monkey.
Only the virus suspension at an appropriate dilution shall be
used for injecting the animals.
( 3) Proced ure
All the inoculated monkeys shall be observed for 17-22 days.
Monkeys that survive the first 24 hours but die afterwards
shall be autopsied to determine whether poliomyelitis is the
cause of death. Those dying due to causes other than
poliomyelitis shall be excluded from the evaluation. The test
is valid if at least 80 % of the monkeys in each group survive
the observation period. Animals that become moribund or
are severely paralyzed shall be killed fer autopsy.
Central nervous system of each monkey shall be subjected to
histopathological examination. Sections shall be made at a
thickness of 10-15 µm and stained with gallocyanine. The
minimum number of sections examined shall be as follows:
12 sections from the whole of the lumbar enlargement;
10 sections from the whole of the cervical enlargement;
2 sections from the medulla oblongata;
1 section each from the pons and cerebellum;
1 section from the mid-brain, and 1 section each from the
left and right of the cerebral cortex and the thalamus.
An evaluation of 4-grade-system of scoring on the severity of the
lesions shall be used. A specially designated, experienced person
shall examine the sections.
Grade 1: Cellular infiltration only ( this is not sufficient to
consider the monkey as valid).
Grade 2: Cellular infiltration with a small number of neuronal
damage.
Grade 3: Cellular infiltration with extensive neuronal damage.
Grade 4: Massive neuronal damage with or without cellular
infiltra tion.
A monkey having neuronal lesions in the section, but no
needle track, shall be regarded as valid. A monkey having
damage due to trauma in the sections, but no specific
pathological lesions, shall not be regarded as valid.
The severity is assessed based on the accumulation of total
seores of readings of the histological sections of lumbar cord
(Le), cervical cord (Ce) and brain (Br). The Lesion Score
(LS) for each valid monkey is calculated as follows:
LS= (¿Lcs/h+ ¿ccs/h+ ¿Brs/h) /3
Note: s/h = score/hemisections
Then calculate the mean LS for each group of valid monkeys.
Only when the mean lesion score of the reference preparation
situates within the range between the upper limit and lower
limit, whether the vaccines being tested are qualified can be
evaluated according to error constants, C1, C2 and C3
values. The criteria for evaluation are as follows.
Compare the mean LS of vaccine tested (test) and that of
reference ( ref).
Qualified: X 1es1 - X re1 < C i
Unqualified: X1es1-Xre1> C2
Retest I : C1 < Xiest - Xre1 < C2 ( retest can only be
conducted once)
Retest JI : If in the same test the difference between the
mean LS of test group and the mean LS of ref erence group is
less than C 1 , while the highest LS of sorne individual
monkey in test group is equal to or more than 2. 5 and,
moreover, 2-fold more than the highest LS of an individual
monkey in the reference group, the test shall be repeated.
Pass in retest: [Xc1es1i+ 1est2) - Xcre1i+re12)] /2<C3
Fail in retest: [Xctestl+ test2) - Xcrefl+ref2) J /2>C3
Intracerebral inoculation
Inocula te O. 5 ml of virus sample ( not less than 7. O
LgCCID50 per ml ) into the thalamus region of each
hemisphere of individual twenty healthy monkeys after being
anesthetized. Ten monkeys receive undiluted virus sample
and the other ten receive 1 : 10 diluted sample. Observe the
inoculated monkeys for 21 days. The test is valid if not less
that 80 % of the animals survive the observation period, and
the number of valid monkeys is not less than 16. Otherwise,
it is necessary to make up the deficiencies. Those die within
48 hours after inoculation or shown nonspecific paralysis
shall be excluded from the evaluation. The CNS tissues of
the animals which die during the observation period and of
those killed at the end of observation period shall be
subjected to histopathologic examination. The criteria of
evaluation are as follows.
Qualified:
The virus preparation shall be judged as qualified if the
examination result accords with one of followings.
CD No histopathologic lesion suggestive to poliomyelitis
infection found in CNS;
® Two monkeys with less than or equal to low-grade
histopathologic lesions;
@ One monkey with less than or equal to mid-grade
histopathologic lesions.
Unqualified:
The virus preparation shall be judged as unqualified if the
examination result accords with one of followings.
CDOne monkey with mid-grade histopathologic lesions and,
at the same time, another monkey with more than or equal
to low-grade histopathologic lesions;
®One monkey with severe histopathologic lesions.
Retest:
If the test result of a pooled lot from several sub-lots of vaccine
does not comply with the requirements, it is permitted to repeat
the test for the sub-lots, separately. The results of retest shall
be evaluated in accordance with the criteria mentioned above.
3306 Requirements for Virus
Nucleic Acid Testing on Source
Plasma used f or Blood Producís
This general chapter applies to test nucleic acid of hepatitis B
virus ( HBV-DNA), hepatitis C virus ( HCV-RNA) and
human immunodeficiency virus type 1 ( HIV-1-RNA) in
source plasma used for blood products.
Direct detection of pathogens by nucleic acid testing (NAT)
is described in this general chapter. NAT is of high
sensitivity, can detect trace of nucleic acid in samples,
significantly shorten the window period compared to ELISA
 3306 Requirements far Virus Nucleic Acid Testing on Source Plasma used far Blood Products
method, and reduce the infection risk of blood transmissible
viruses. Currently, the main methods of NAT used in blood
screening are polymerase chain reaction ( PCR ) and
transcription mediated amplification (TMA) methods.
PCR method is a nucleic acid amplification technology which
simulates the natural replication of DNA in vitro, with the
advantages of high sensitivity, high specificity, rapidity and
simplicity. In principle, PCR is a procedure that allows
specific in vitro amplification of segments of DNA or of RNA
after reverse transcription into cDNA With the template of
DNA or cDNA and the catalysis of DNA polymerase, repeated
cycles of heat denaturation, primer annealing and DNA synthesis
result in an exponential amplification of the target DNA segment
The amplification product can be analyzed by various methods of
high specificity and sensitivity. With the technical improvement,
different derived PCR methods have been set up.
Transcript-based amplification methods include transcription
mediated amplification ( TMA) and nucleic acid sequence-
based amplification ( NASBA) methods. TMA is an isothermal
process using combined action of reverse transcriptase, RNAse
H and RNA polymerase to amplify RNA or DNA With the
primer, reverse transcriptase synthesizes opposite, complementary
DNA copy of the target sequence. RNAse H destroyed the RNA
template from the DNA-RNA compound to form transcription
complex. RNA polymerase recognizes the promoter sequence
in the DNA template and initiates transcription of target
RNA sequences. The newly synthesized RNA amplicon re-
enters the TMA process and serves as a template far a new
cycle of replication leading to an exponential expansion of the
RNA amplicons. The principies of NASBA and TMA are
similar, but nucleic acid extraction and detection method of
amplification product are different.
Materials
Test sample
( 1 ) Measures, such as control of handling time and
temperature, shall be taken to ensure stability of target
sequences during the sample processing.
(2) Anticoagulants that do not interfere with the NAT
process may be used after evaluation. Heparin shall not be
used as anticoagulant when PCR is employed because it' s a
strong inhibitor of Taq enzyme. EDTA and sodium citrate,
etc can be used as anticoagulant.
(3) To ensure the stability of target sequences, the storage
and transportation conditions of samples should be validated.
Sample to be tested in 72 hours can be stored at 2-8 ºC,
otherwise should be stored at -2üºC or below.
Test Kits
The kit shall be the one approved far NAT test of plasma
pool. The storage, transportation and use of the kit should
fallow the instruction of the kit.
Procedures
( 1) Sample pooling
On the basis to ensure the sensitivity of test, according to the
quantity required in the instruction of kit, take equal volume
from each sample and pool them to make the pooled sample
far the test. The preparation process shall ensure the
traceability of each sample and pooled sample.
Measures should be taken to prevent cross contamination and
reduce aerosols as far as possible in the process of pooling,
which may lead to false positive results.
(2) Extraction, amplification and detection
Extraction, amplification and detection shall fallow the
instruction of kit.
(3) Control
To ensure results reliable, controls should be in place according
···.275
to the kit instruction, including internal control, negative control
and positive control
Internal control normal refers to specific nucleic acid sequence
that containing, unless otherwise specified, the primer
binding sites. The internal control is preferably added to the
test sample befare extracting the nucleic acid and therefore
acts as an overall control for the whole process of extraction,
reverse transcription, amplification, and detection.
Negative control is the one resemble as closely as possible to
the test sample, but does not contain target sequences.
Positive control is the one resemble as closely as possible to
the test sample and should contain an appropriate and defined
amount of target sequences.
Only when above mentioned controls comply with the instruction
of kit, shall the results of test be valid
Result Evaluation
(1) Evaluate the results according to the instruction of the
kit.
(2) When test on pooled sample showed negative reaction,
consequently the individual sample of the pool shall be
determined as negative.
( 3) When the test on pooled sample showed positive
reaction, the following procedures shall be followed far
further testing.
Pooled sample is reactive in HBV/HCV/HIV NAT test
Method 1
Test sub-pools using same
Non-reactive
NATmethod
Reactive Every sample of the
sub-pool passed the test
Test each sample of the sub-pool using
same NAT method
The samples shall be rejected The samples passed the test
Quality Control
( 1) Personnel
All personnel involved in the NAT test need pre-post training
and continuous on-job training. Pre-post training should
include at least trainings on nucleic acid amplification
techniques, laboratory management requirements, operation
skills, quality control and biosafety. Operators should master
relevant professional knowledge and skills, can perform test
skillfully, and pass examinations. Continuous on-job training
shall be provided to the operators when necessary, the
training shall include the new progress on relative
technologies, quality control and biosafety. Operators should
be trained befare a new method is implemented.
(2) Laboratory
To ensure the biosafety of operators and environment, as
well as to avoid cross contamination of test samples, NAT
 ::T'>;· ~.·.·. l.~·.·.)~.>: .
··~~T-~U> 3401 Immunoblot
1
laboratory should comply with the requirements of GMP,
National Guideline for Detection of HIV/ AIDS, Laboratories
General Requirements for Biosafety and Medical Waste
Management Regulations. Additionally, it shall meet the
following requirements.
The segregation of NAT laboratory should be in accordance
with the testing system. Generally, pre-amplification and
post-amplification areas should be segregated. Pre-
amplification area, including reagent preparation and sample
preparation, should locate in different rooms or areas; Post-
amplification area, including amplification and detection,
should locate in different rooms or areas. Workflow, allocation of
equipment and facility should be settled reasonably according
to the function of different areas.
The laboratory shall be equipped with corresponding detecting
equipment, cold storage facilities and biosafety protection
facilities. Facilities and equipment in each area should be
specified, and cross-area use shall be avoided. Metrological
devices and critical equipment should be tested, calibrated or
3400 Bioassay Method
3401 Immunoblot
The antigenic specificity of test sample is detected by the
colour developed through enzymological reaction after the
test sample is bound to a specific antibody, which is further
combined with an enzyme-labeled antibody.
Reagent
(1) TG buffer solution: Dissolve 15. 12 g of trishydroxy-
methylaminomethane ( THAM) and 72 g of glyylcine in
water and dilute to 500 ml. Store at 4 ºC.
(2) EBM buffer solution: Dilute 20 ml of TG buffer solution
and 40 ml of methanol to 200 ml with water. Store at 4ºC.
(3) TTBS buffer solution: Dissolve 6. 05 g of THAM and
4. 5 g of sodium chloride in a quantity of water. Add O. 55 ml
of polysorbate 80. Adjust pH to 7. 5 with hydrochloric acid
and dilute to 500 mi with water. Store at 4 "C.
(4) Substrate buffer solution: Dissolve 15 mg of 3, 3'-
diaminobenzidine hydrochloride in 25 ml of TTBS buffer
solution. Add 5 ml of methanol and 15 µl of 30 % hydrogen
peroxide solution. Prepare just before use.
Procedure
Comply with the requirements for SDS -PAGE ( 0541,
Method 5). Both the amounts of test sample and reference
substance loaded shall be more than 100 ng. Take out the gel
and cut off its edge. Immerse the gel into EBM buffer
solution for 30 minutes. Immerse six pieces of thick filter
paper and one piece of nitrocellulose nitrate membrane with
EBM buffer solution sufficiently. Both the sizes of filter
paper and nitrocellulose nitrate membrane are identical to
that the same size of as the gel. Transfer the test sample
onto the nitrocellulose nitrate membrane using semi-dry gel
transfer apparatus according to the following procedures: Put
three pieces of wet filter paper, nitrocellulose nitrate
membrane, electrophoresis gel and the other three pieces of
wet filter paper in turn onto an electrode plate. Cover with
another electrode plate and transfer the test sample for 45
minutes ata constant electric current of O. 8 mA per cm2 of
validated according to regulations.
The NAT system employed should be validated, including
devices/ equipment validation and method validation.
Biosafety protection and treatment of waste and
contaminations should comply with the related national
requirements of biosafety, etc. Emergency treating measures
against virus contamination should be established so that if
contamination occurs, the source of contamination can be
identified and cleaned up in time. NAT test can only be re-
started when the contamination has been cleaned up.
(3) Quality control of laboratory
Regular quality assessment should be carried out, to ensure
the stability of the NAT system and reliability of the test
results.
(4) Data management
All data and information, including sample collection,
sample pooling, nucleic acid extraction, amplification,
detection, and final report, should be recorded and managed.
nitrocellulose nitrate membrane.
Take the nitrocellulose nitrate membrane out and immerse
into blocking solution CTTBS buffer solution containing 10 %
calf serum or other suitable blocking solutions) to block for
60 minutes. Discard the blocking solutíon. Add 10 ml of
TTBS buffer solutíon, and then add a quantity of antibody
( diluted following the instruction) against test sample,
allow to stand overnight at room temperature with shaking.
Rinse the nitrocellulose nitrate membrane once with TTBS
buffer solution by dripping, and then wash 3 times each by
immersing with the buffer solution for 8 minutes. Discard
the buffer solution, add another 10 ml of TTBS buffer
solution, then add a quantity of the biotin-labeled secondary
antibody, allow to stand at room temperature for 40 minutes
with shaking. Rinse the nitrocellulose nitrate membrane once
with TTBS buffer solution by dripping and wash 3 times each
by immersing with the buffer solution for 8 minutes. Discard
the buffer solution, add another 10 ml of TTBS buffer
solution, then add a quantity of avidin solution and biotin-
labeled horseradish peroxidase solution, allow to stand at
room temperature for 60 minutes with shaking. Rinse the
nitrocellulose nitrate membrane once with TTBS buffer
solution by dripping and wash 4 times each by immersing
with the buffer solution for 8 minutes. Discard the buffer
solution, add a quantity of substrate buffer solution, allow
to stand at room temperature for colour development,
protected from light. When a suitable colour appears, stop
the reaction by washing with water.
Result evaluation
The result is judged as positive if obvious color bands appear.
If no colour appears, the result is judged as negative.
3402 Inununodot
The antigenic specificity of test sample is detected by the
colour developed through enzymological reaction af ter the
test sample is bound to a specific antibody, which is further
combined with an enzyme-labeled antibody.

Reagent
( 1) TG buffer solution: Weigh accurately 15. 12 g of
trishydroxymethylaminomethane ( THAM ) and 72 g of
glycine, dissolve in water and dilute to 500 ml. Store at 4ºC.
(2) EBM buffer solution: Dilute 20 ml of TG buffer solution
and 40 ml of methanol to 200 ml with water. Store at 4 ºC.
(3) TTBS buffer solution: Dissolve 6. 05 g of THAM and
4. 5 g of sodium chloride in a quantity of water. Add O. 55 ml
of polysorbate 80. Adjust pH to 7. 5 with hydrochloric acid
and dilute to 500 ml with water. Store at 4 ºC.
( 4) Substrate buffer solution: Dissolve 15 mg of 3, 3-
diaminobenzidine hydrochloride in 25 ml of TTBS buffer
solution. Add 5 ml of methanol and 15 µl of 30 % hydrogen
peroxide solution. Prepare just before use.
Procedure
lmmerse a piece of nitrocellulose nitrate membrane in EBM
buffer solution for 15 minutes. Load test sample, negative
control ( an equal volume of human albumin may be used)
and reference substance onto the membrane by dotting. The
amount of each sample or control loaded shall be more than
10 ng. Dry the nitrocellulose nitrate membrane at room
temperature for 60 minutes and immerse in blocking solution
(TTBS buffer solution containing 10% newborn calf serum
or other suitable blocking solutions) to block for 60 minutes.
Discard the blocking solution. Add 10 ml of TTBS buffer
solution, then add a quantity of antibody ( diluted following
the instructions) against test sample, allow to stand overnight
at room temperature with shaking. Rinse the nitrocellulose
nitrate membrane once with TTBS buffer solution by
dripping and wash 3 times each by immersing with the buffer
solution for 8 minutes. Discard the buffer solution, add
another 10 ml of TTBS buffer solution, then add a quantity
of the biotin-labeled secondary antibody, allow to stand at
room temperature for 40 minutes with shaking. Rinse the
nitrocellulose nitrate membrane once with TTBS buffer
solution by dripping and wash 3 times each by immersing
with the buffer solution for 8 minutes. Discard the buffer
solution, add another 10 ml of TTBS buffer solution, then
add a quantity of avidin solution and biotin-labeled horseradish
peroxidase solution, allow to stand at room temperature for 60
minutes with shaking. Rinse the nitrocellulose nitrate
membrane once with TTBS buffer solution by dripping and
wash 4 times each by immersing with the buffer solution for
8 minutes. Discard the buffer solution, add a quantity of
substrate buffer solution, allow to stand at room
temperature for colour development, protected from light
When a suitable colour appears, stop the reaction by
washing with water.
Result evaluation
The result is judged as positive if obvious colour bands appear. If
no colour appears, the result is judged as negative.
3403 Double Immunodiffusion
The specificity of test sample is examined by the formation of
precipitation lines between antigen and antibody on agarose
gel plate. Antigen and antibody are added to neighboring
wells dug on agarose plate respectively. Precipitation lines of
immuno-complex are formed in a certain length of time when
the antibody is specific to the antigen, and they are at proper
concentrations and with a proper ratio.
Preparation of test sample solution
Dilute test sample with physiological saline to a suitable protein
concentration
3404 Immunoelectrophoresis 1
Reagent
(1) O. 5% Amino-black staining solution: Dissolve O. 5 g of
amino-black lOB in a mixture of 50 ml of methanol, 10 ml of
glacial acetic acid and 40 ml of water.
(2) Destaining solution: Mix 45 ml of ethanol and, 5 ml of
glacial acetic acid with 50 ml of water.
Procedure
Pour the completely swollen l. 5 % agarose solution onto a
level glass plate (O. 19 ml of agarose per cm2 ) • After the
agarose is coagulated, dig several wells each ata diameter of
3 mm in a square array. The distance between neighboring
wells is 3 mm ( see the following figure). The number of
square array is determined according to the actual need. Add
antibody into the central well and the test sample solutions
into the surrounding wells, except one well is reserved for
the corresponding reference serum. The volume of test
sample or control added into each well is 20 µl. Put the
agarose gel plate into a level wet box at 37°C and diffuse for 24
hours. lmmerse the agarose gel plate sufficiently with
physiological saline to remove the unbound protein. Stain the
plate in O. 5 % amino-black solution, then decolourize with
destaining solution until the background is colourless and clear
blue precipitation lines appear. Preserve by a suitable method or
copy the profile.
o
o 00
o Fig. Square array
Result evaluation
The test is valid if the reference substance forms a
corresponding precipitation line. If a precipitation line forms
between the test sample and antibody wells, the test sample
is judged as corresponding to the antibody.
3404 Immunoelectrophoresis
The test sample is separated into antigen bands through
electrophoresis and subjected to double immunodiffusion with
the corresponding antibodies. When the ratio of antigens to
antibodies is appropriate, visible precipitation ares are
formed. The components and properties of test sample are
analyzed by comparing the sites and shapes of the precipitation
ares with those formed by standard antigens and antibodies.
Reagent
(1) Barbital buffer solution (pH 8. 6): Dissolve 4. 14 g of
barbital and 23. 18 g of barbital sodium in a quantity of water
by heating. Cool clown to room temperature. Add O. 15 g of
sodium azide and dilute with water to make 1500 ml.
(2) O. 5 % Amino-black staining solution: Dissolve O. 5 g of
amino-black lOB in a mixture of 50 ml of methanol, 10 ml of
glacial acetic acid and 40 ml of water.
(3) l. 5% Agarose solution: To l. 5 g of agarose, add 50
ml of water and 50 ml of barbital buffer solution. Heat to
swell completely.
 ·218 3405 Peptide Mapping
1
( 4) Destaining solution: Mix 45 ml of ethanol and 5 ml of
glacial acetic acid with 50 ml of water.
(5) Bromphenol blue indicator solution: Dissolve 50 mg of
bromphenol blue in water to make 100 ml.
Reference substance
Normal human serum or other suitable reference substances.
Preparation of test sample solution
Dilute the test sample with physiological saline to a protein
concentration of O. 5%.
Procedure
Pour l. 5 % agarose solution onto a level glass plate to yield a
layer of about 3 mm thick. Coagulate the agarose on standing
to form an even thin layer without bubble. Dig two wells,
each ata diameter of 3 mm and 10-15 mm apart, at the site
about one-third of the full length apart from the negative pole
of the plate. Add 10 µl of test sample and one drop of
bromphenol blue indicator solution into the test well, and
10 µl of normal human serum or human plasma and one drop
of bromphenol blue indicator solution into the control well.
Connect barbital buffer solution ( electrophoresis buffer
solution) to the plate by using three layers of filter paper.
Perform electrophoresis at a constant voltage of 100 V for
about 2 hours ( until the indicator is migrated to the front
edge). Dig a trough at a width of 3 mm between the two
wells. Each end of the trough is 3-5 mm apart from the edge
of the plate. Add antiserum or anti-plasma antibody into the
trough until the trough is full. Put the plate in a wet box and
for diffuse diffusion at 37°C for 24 hours. After diffusion
immcrsc thc platc into physiological salinc to rcmovc thc
unbound protein. Stain the plate with O. 5 % amino--black
staining solution and decolourize with the destaining solution
until the background is basically colourless. Preserve by a
suitable method or copy the profile. Compared with that of
control, the main precipitation line of test sample shall be
the protein to be tested.
Precaution
(1) Cooling system shall be used during electrophoresis,
otherwise the agarose may be cracked due to drying.
(2) The plate shall be immersed sufficiently with physiological
saline, otherwise the background is unclear.
3405 Peptide Mapping
The integrity and accuracy of protein primary structure is
characterized by suitable analytical methods after the protein
has been cleaved by protease or chemical substance.
Method 1 : Trypsin Cleavage-Reverse Phase
HPLC Method
High performance liquid chromatography ( 0512) is applied
for the characterization.
Parameters for chromatography
The column filled with octylsilane or octadecyl silane groups
chemically bonded to porous silica particles for protein or
peptide analysis is used. The column temperature is 30ºC ±
5ºC and the reference solution and test sample is preserved at
4ºC ±O. 5ºC. The solution A of mobile phase is O. 1%
trifluoroacetic acid trifluoroacetic acid in water and solution B
of mobile phase is O. 1 % trifluoroacetic acid in acetonitrile.
Elute with a gradient (solution A from 100% to 30% and
solution B from O% to 70 % ) for 70 minutes at a flow rate of
1 ml per minute and detect at 214 nm.
Procedure
Dialyze the test sample and reference solutions, each at a
concentration of 1 mg per ml, sufficiently against 1%
ammonium bicarbonate solution. The test sample and
reference at low concentrations shall be concentrated
separately to the corresponding concentrations. Add trypsin
solution (Dissolve a quantity of trypsin, pre-treated with L-
1-4' -tosylamino--2-phenylethyl chloromethyl ketone or of pure
grade for sequencing, in 1% ammonium bicarbonate solution
to prepare a solution containing O. 1 mg per ml) to test
sample solution and reference solution separately at a ratio of
1 : 50 ( mg/mg) and incubate at 37°C for 16-24 hours. Add
50% acetic acid solution at a ratio of 1 : 10. Centrifuge at
10 000 r/min for 5 minutes ( or filter with a O. 45 µm
membrane) and collect the supernatant. Measure accurately
100 µl of the supernatant ( or filtra te) and inject into the
HPLC apparatus separately. Perfarm gradient elution and
record the chromatogram. Compare the chromatograms of
test sample solution with those of reference solution.
Method 2: Cyanogen Bromide ( CNBr) Cleavage
Method
Procedure
Dialyze a quantity of test sample and reference solutions,
equivalent to 50 µg of protein, against water for 16 hours
and lyophilize. Dissolve with 20 µl of cyanogen bromide
cleavage solution [Dissolve O. 3 g of cyanogens bromide in
1 ml of formic acid ( 7o- 100) ] , allow to stand a t room
temperature far 24 hours. Add 180 µl of water to the lysate
and lyophilize again. Reconstitute the freeze-dried lysate with
water to an appropriate concentration and perfarm SDS -
PAGE (Appendix N C) (Gel concentration is 20%). Silver
staining is appiied.
Compare the chromatograms of test sample with those of
control reference.
3406 Test f or Losing Rate of Plasmid
The recombinant bacteria in E. coli expression system
contain plasmids for the expression of target protein. In
general, the plasmid carries antibiotic-resistant gene to
facilitate selection. During subculture in the environment
with a certain concentration of antibiotic, such as in seed
culture medium, the recombinant bacterium can not survive
after the plasmid is lost. However, in the fermentation
medium without antibiotic, along with the increasing
number of passage, a part of E. coli may lose the plasmid,
as a result, the antibiotic-resistant gene and the ability to
expressing target protein may lose at the same time. The
stability of plasmid is evaluated by the losing rate of plasmid
determined by comparing the numbers of survival bacteria in
the media with or without antibiotic.
The real-time fermentation liquid during fermentation or
mimic fermentation process, including those at the final
stage of fermentation when the bacteria are subcultured to
the maximum passage, are collected and used for the test in
general. Dilute the fermentation liquid properly, spread to
an antibiotic -free medium and incubate at 37°C overnight.
Select at least 100 colonies on the medium and inoculate onto
the Petric dishes with or without antibiotic respectively.
Incubate at 37°C overnight and compare the numbers of
colonies in the two Petric dishes and calculate the losing rate
of plasmid. The test shall be repeated far more than 2 times
in general. The losing rate of plasmid shall be specified in the
validation of production procedure and shall be within the
permitted range.
 3407
3407 Determination of Residual
Extraneous DNA
For residual extraneous DNA assay, choose one of the
following methods based on the specific circumstance of test
sample.
Method 1 DNA Probe Hybridization
The residual extraneous DNA in test sample is denatured to a
single stranded and adsorbed onto an immobilized membrane.
Under a certain temperature, the single strand binds with a
matching single strand and reassociates to form a double
stranded DNA, which is called as hybridization. A labeled
specified single stranded DNA probe is hybridized with the
single stranded DNA of test sample, which is adsorbed onto
an immobilized membrane. The hybridization result is shown
by a displaying system corresponding to the label. The DNA
content of test sample can be determined by comparing the
result with that of positive DNA reference substance of
known DNA content.
Reagent
( 1) DNA labeling and detection kit
(2) DNA hybridization membrane
Nylon membrane or nitrocellulose membrane
( 3) 2 % Proteinase K solution
Dissolve O. 20 g of proteinase K in 10 ml of sterile water ( the
electrical resistivity of water shall be not less than 18. 2
MQ•cm). Dispense the solution and store at -20ºC for use.
(4) 3% Bovine serum albumin solution
Dissolve O. 30 g of bovine serum albumin in 10 ml of sterile
water ( the electrical resistivity of water shall be not less than
18. 2 M.O.•cm).
(5) 1 mol/L Tris (hydroxymethyl) aminomethane (Tris)
solution (pH 8. 0)
Adjust pH to 8. O with hydrochloric acid.
(6) 5. O mol/L Sodium chloride Solution
( 7 ) O. 5 mol/L Disodium ethylenediamine tetraacetate
solution (pH 8. 0)
Adjust pH to 8. O with 10 mol/L sodium hydroxide solution.
(8) 20% Sodium dodecyl sulfate CSDS) solution
Adjust pH to 7. 2 with hydrochloric acid.
(9) Proteinase buffer solution (pH 8. O)
Mix l. O ml of 1 mol/L Tris solution (pH 8. O), 2. O ml of
5. O mol/L sodium chloride solution, 2. O ml of O. 5 mol/L
disodium ethylenediamine tetraacetate solution ( pH 8. O)
with 2. 5 ml of 20 % SDS solution and dilute to 10 ml with
sterile water ( the electrical resistivity of water shall be not
less than 18. 2 M.O.• cm). If precipitation reaction appears
between test sample and Sodium Chloride Solution, adding
sodium chloride can be avoided.
(10) TE (Tris-EDTA) buffer solution (pH 8. O)
Dilute 10 ml of 1 mol/L Tris solution (pH 8. O) and 2 ml of
O. 5 mol/L disodium ethylenediamine tetraacetate solution
(pH 8. O) to 1000 ml with sterile water ( the electrical
resistivity of water shall be not less than 18. 2 M.O.•cm).
(11) 1% Protamine DNA solution
Weigh accurately O. 10 g of protamine DNA and dissolve in a
small volume of TE buffer solution. Transfer totally to a 10
ml volumetric flask and dilute to volume with TE buffer
solution. Mix well. Shear the DNA into small molecules by
drawing and blowing repeatedly using a syringe fitted with a
No. 7 needle. Dispense the solution and store at -20ºC
(12) DNA dilution solution
Dilute 50 µl of 1% protamine DNA solution to 10 ml with
TE buffer solution.
Determination of Residual Extraneous DNA
Preparation of DNA
DNA used for probe labeling and used as DNA reference
substance is obtained by extraction and purification from
continuous cell line, engineering bacteria or hybridoma cells
where the test sample is produced. The methods of
purification and identification may refer to Molecular
Cloning, A Laboratory Manual , 2002, or Short Protocols
in Molecular Biology, 1998.
Adjust the cell suspension for extraction to a concentration of
about 10 7 cells per ml. If the host cell is bacteria, the
concentration shall be adjusted to about 108 bacteria per ml.
Centrifuge 1 ml of the suspension. Add 400 µ1 of lysis
solution to the precipitate and mix well. React at 37°C for
12-24 hours. Add 450 µl of saturated phenol solution and
mix vigorously. Centrifuge at 10 000 r/min for 10 minutes.
Discard the supernatant and extract with 450 µl of saturated
phenol solution once more. Discard the supernatant, add 450
µl of trichloromethane and mix vigorously. Centrifuge at
10 000 r/min for 10 minutes. Discard the supernatant, add
40 µl of 3 mol/L sodium aceta te ( pH 5. 2) and mix well.
Then, add 1 ml of cold absolute ethanol at a temperature
lower than -20ºC and mix sufficiently. React for 2 hours at
a temperature lower than -20ºC. Centrifuge at 15 000 r/min
for 15 minutes and wash the precipitate once with a quanÚty
of 70% cold ethanol at a temperature lower than - 20ºC.
Centrifuge at 15 000 r/min for 15 minutes and discard the
supernatant. Dry the precipitate by air blowing and dissolve
with a quantity of sterile TE buffer solution. Cleave the
RNA with RNase, extract with phenol/trichloromethane and
purify with molecular sieve chromatography.
ldentify the DNA purity of the reference substance by
electrophoresis on 1 % agarose gel and spectrophotometry:
RNA or oligonucleotide shall not be detected; the ratio of
A26o to A2so shall be in the range of l. 8-2. O ( The test
sample is diluted to A 26 a between O. 2 and l. O for tesiing).
The DNA used for the reference substance and probe labeling
shall be cleaved with enzyme or treated by ultrasonication in
arder to make the size of its fragments suitable for DNA
hybridization and probe labeling.
The DNA concentration of the reference substance is
calculated by the following formula:
DNA concentration (µg/mD = 50XA2 60
The reference substance may be dispensed into suitable
tubes. Store at -20ºC for long-term use.
Probe Iabeling
Carry out according to the instruction of the reagent kit.
Procedure
( 1) Proteinase K pre-treatment
Prepare test sample, reference substance and negative
control according to the following table. lncubate at 37°C for
more than 4 hours to ensure that the reaction of enzyme
cleavage is completed.
Volume of 2% Proteinase
3% BSA Water,
test Proteinase buffer
Tube solution add to,
sample K solution
CµD CµD
CµD CµD CµD
Sample 100 1 20 200
D1 100 1 20 X 200
D2 100 1 20 X 200
D3 100 1 20 X 200
Negative 100 1 20 X 200
 3407 Determination of Residual Extraneous DNA
Precaution
The dilution of test sample: Dilute the test sample ( bulk
preparation) with DNA dilution solution to a concentration
of one human dose per 100 µI; if the maximum dosage of
final product is relatively high and the protein concentration
of the test sample is relatively low, the test sample may be
diluted with DNA dilution to a concentration of 1/10 or
1/100 human dose per 100 µl.
D1, D2, and D3 are diluted DNA reference substances. The
DNA reference substance is diluted with DNA dilution
solution to a concentration of 1000 ng of DNA per mi and
then 10-fold serially diluted to 10 ng/100 µI (Di_), 1 ng/100 µI
(Dz) and 100 pg/100 µI C°-3); if the dosage of final product is
relatively high and the DNA content limit ( 100 pg/ dose) is
strict, the sensitivity of DNA test shall be elevated. Then
the corresponding DNA reference substance shall be diluted
to 100 pg/100 µI CD1), 10 pg/100 µI CDz) and 1 pg/100 µI
(D3 ). The negative control is DNA dilution solution and the
blank control is TE buffer solution which has not been
pretreated with proteinase K.
When 1/100 human dose of test sample is greater than 100 µ!,
the final volume also increases. Usually, the final volume is
around twice the volume of test sample. If the volume
difference between the test sample and final volume is too
small, the activity of proteinase K may be influenced
The volume ratio of 2 % proteinase K solution to proteinase
buffer solution is 1 : 20 and that of proteinase buffer solution
to final volume is 1 : 1O.
Add a quantity of 3 % bovine serum albumin so that the
reference substance and negative control may contain a
certain amount of protein and maintain the same enzyme
cleavage conditions as those for the test sample ( usually
protein in nature). If other substances are used as the test
samples, corresponding substances shall be used
If protein in the pretreated test sample solution interferes
with the test, the DNA in test sample may be extracted by
the above saturated phenol solution extraction method or by
other suitable methods ( extraction shall also be carried out
again for the DNA of reference substance and negative
control in parallel with the test sample). Regardiess of
method to be used for extraction, the limit detection of Vero
cells DNA reference should be 10 pg at least.
( 2) Dot blotting or slot blotting
Immerse the hybridization membrane in TE buffer. Heat the
pre-treated test sample, reference substance and negative
control and blank control in lOOºC water bath far 10 minutes.
Cool clown the tubes in ice bath promptly. Centrifuge at 8000
r/min far 5 seconds. Load the test sample and all controls
onto the hybridization membrane by dot blotting or slot
blotting unit (as protein precipitate exists, the loading
quantity is based on the amount of precipitate and the loading
of protein precipitates should be avoided; the volume far the
test sample, reference substance, negative control and blank
control shall be identical or in the same proportion).
Air-dry the membrane and then use ultraviolet cross-linking
or heat at 80ºC under vacuum for more than one hour.
(3) Hybridization and colour development
Perform according to the instruction of the reagent kit.
Result evaluation
The test is valid if the ref erence substances develop colour
and the intensity of colour corresponds with the DNA
content, and the negative control and blank control do not
develop color or the colour developed is lighter than that of
the reference substance (D3). Compare the test sample with
reference substances and evaluate the residual extraneous
DNA content of test sample according to the colour intensity.
Method 2 Fluorescent Stain ~y
A fluorescent compound could be formed by dsDNA
fluorescent stain specifically binding to double-stranded DNA
(dsDNA). This compound could be activated by 480 nm
excitation light. The fluorescence given off by this compound
could be detected at 520 nm by fluorescence microplate
reader. In a certain range of DNA concentrations and with
excessive fluorescent stain, the fluorescence intensity is
proportional to the DNA content. The residual extraneous
DNA content of test sample could be determined by the
fluorescence intensity of test sample.
Reagent
( 1) 1 mol/L Tris ( hydroxymethyl) aminomethane (Tris)
solution (pH 7. 5) Adjust pH to 7. 5 with hydrochloric acid.
( 2) O. 5 mol/L Disodium ethylenediamine tetraacetate solution
(pH 7. 5)
Adjust pH to 7. 5 with 10 mol/L sodium hydroxide solution.
(3) TE buffer (pH 7. 5)
Mix l. O mi of 1 mol/L Tris solution (pH 7. 5) and O. 2 mi
of O. 5 mol/L disodium ethylenediamine tetraacetate solution
(pH 7. 5) to a final volum of 100 mi with sterile water.
( 4) dsDNA fluorescent stain
Prepare according to the manufacturei.5 instructions.
( 5) DNA standard
The DNA standard is diluted with TE buffer to a
concentration of 50 µg/ml and stored at -20ºC.
The concentration of the DNA standard is calculated by the
following formula:
DNA concentration (µg/ml) 50XA260
Preparation of DNA standard solution
A serial solutions of DNA standard are diluted with TE
buffer to obtain the concentrations of O ng/ml, l. 25 ng/ml,
2. 5 ng/ml, 5. O ng/ml, 10 ng/ml, 20 ng/ml, 40 ng/ml
and 80 ng/ml.
Procedure
Prepare 400 µl of DNA standard solution and test sample
respectively to l. 5 ml tubes, add 400 µl freshly prepared
diluted fluorescent stain, mix thoroughly and incubate far 5
min at room temperature, protected from light. Transfer
250 µl of the above reactions mixture to a 96-well black plate
in triplicate wells. The fluorescence intensity of each reaction
well is obtained by fluorescence microplate reader at
excitation wavelength of 480 nm and emission wavelength of
520 nm, and TE buffer is used as the background control.
A linear regression equation is obtained by regressing the
DNA standard concentration with the fluorescence intensity.
The correlation coefficient of linear regression shall be not
less than O. 99. Calculate the residual extraneous DNA
concentration of the test sample by inserting the fluorescence
intensity of test sample into the linear regression equation.
Precautions
( 1) The fluorescent stain assay shows a good linear detection
with the DNA concentration from l. 25 to 80 ng/ml. So the
residual DNA content of test sample can be determined in
this range. If the residual DNA concentration of test sample
is less than l. 25 ng/ml, reportas less than l. 25 ng/ml.
(2) Methodology validation should be performed, when the
fluorescent stain assay is used for residual DNA detection of
test sample for the first time. The validation includes
precision and recovery tests at least. If the test sample
interferes with the recovery and precision, proper method
should be applied to dilute or purify DNA of test sample
(referred to related contents of the Method 1). If the DNA
of test sample needs to be purified, the recovery test should
be performed at the beginning of purification procedures.
 3409 Determination of Prekallikrein Activator Content
The final result should be corrected by recovery rate.
3408 Determination of Residual
Antibiotics (Culture Method)
Antibiotics may inhibit the growth of microbes on agar
culture medium. The residual ampicillin or tetracycline
content in test sample is determined by comparing the size of
inhibiting rings formed by test sample and control against
corresponding bacteria which grow on culture medium.
Preparation of phosphate buffer solution ( pH 6. O)
Dissolve 8. O g of potassium dihydrogen phosphate and 2. O g
of dipotassium hydrogen phosphate in water and dilute to
1000 ml. Sterilize at 121 ºC far 30 minutes.
Preparation of antibiotic No. 2 medium
Dissolve 6 g of peptone, l. 5 g of beef extract powder and 6 g of
yeast extract powder in a quantity of water. Add 13-14 g of agar
and swell by heating. Filter to remove the insoluble matters.
Dissolve 1 g of glucose in the mixture and dilute to 1000 ml with
water. Adjust pH to a value in the range of 6. 5-6. 6 after
sterilization Dispense the medium in glass tubes or conical
flasks. Sterilize at 115ºC far 30 minutes and store at 4ºC.
Preparation of standard solution
Dissolve a quantity of ampicillin standard with O. 01 moVL
hydrochloric acid and dilute to a concentration of 10 mg of
ampicillin per ml. Measure accurately a quantity of the above
ampicillin solution and dilute with phosphate buffer solution
to a concentration of l. O µg of ampicillin per ml.
Dissolve a quantity of tetracycline standard with physiological
saline and dilute to a concentration of O. 125 µg of tetracycline
per ml.
Preparation of bacterial suspension
( l) Staphylococcus aureus suspension It is used far the
determination of ampicillin
lnoculate the nutrient agar slant culture of Staphylococcus
aureus ( CMCC 26003) strain onto nutrient agar slant and
incubate at 35-37°C far 20-22 hours. Upan using, wash off
the bacteria! lawn with sterilized water or sterile O. 9 %
sodium chloride solution.
( 2 ) Micrococcus luteus suspension It is used far the
determina tion of tetracycline.
lnoculate the nutrient agar slant culture of Micrococcus luteus
CCMCC 28001) strain onto nutrient agar slant and incubate
at 26-27°C far 24 hours. Upan using, wash off the bacteria!
lawn with sterile O. 9 % sodium chloride solution
Procedure
Pour 15-20 ml of melted antibiotic medium No. 2 into a Petri
dish ata diameter of 8 cm or 10 cm and spread evenly on the
dish bottom. Put the dish on a horizontal platfarm and let
the medium solidify to form a bottom layer. Transfer 10-15
ml of antibiotic medium No. 2 to a test tube preheated in
50ºC water bath. Add 300 µl of 0.5%-1.5% (ml/mD of
bacteria! suspension and mix well. Pour a quantity of the
mixture onto the Petric dish spread with a bottom layer. Put
the dish on a horizontal platfarm and cool clown. Place steel
tubes (stainless steel tubes with smooth and flat surface, at
an inner diameter of 6-8 mm, a height of 10-15 mm and a
wall thickness of 1-2 mm) evenly and apart with equal
distances on each Petric dish. Add test sample solution,
negative control solution ( phosphate buffer solution) and
standard solution dropwise into the steel tubes separately.
lncubate the Petri dishes at 37°C far 18-22 hours.
Result evaluation
There is bacterium-inhibiting ring far standard solution but
not far negative control solution. The result is judged as
negative if the diameter of bacterium-inhibiting ring of test
sample solution is smaller than that of standard solution,
otherwise it is judged as positive.
[Note]
The test shall be carried out aseptically. All the glass
apparatus and steel tubes used far the test shall be sterile.
3409 Determination of Prekallikrein
Activator Content
The content of prekallikrein activator CPKA) in test sample
is determined by chromogenic substrate method ( or
chromogenic matrix method).
Reagent
( 1) O. 05 mol/L trishydroxymethylaminomethane--hydrochloric
acid (Tris-HCl) buffer ( containing O. 15 rml/L sodium chloride
solution, TNB): D.ssolve 6. 06 g of trishydro:xymethylamimmethane
(Tris, MW 121. 14) and 8. 77 g of sodium chloride in a
quantity of water, adjust the pH to 8. O with 1 mol/L
hydrochloride acid and add water to a final volume of 1000 ml.
(2) 2 mmol/L Kallikrein chromogenic substrate (S-2302)
solution: Dissolve 12. 5 mg of S-2302 in 10 ml of water.
(3) Prekallikrein CPK): Prepare PK by a suitable method,
dispense in small volumes and store at - 30ºC or below far
use.
Preparation of PKA standard solution
Dilute a quantity of PKA standard with O. 85 % sodium
chloride solution to contain 10. O IU, 20. OIU, 30. OIU,
40. OIU and 50. O IU per ml respectively. According to the
required amount far each test, dispense the standard solution
in small volumes and store at -30ºC. Thaw befare use and
dilute 10-fold with TNR Each tube can only be thawed once.
Preparation of test sample solution
Dilute the test sample 10-fald with TNB.
Procedure
To a 96-well microtitre plate, add 20 µl of test sample
solution and 20-50 µl of PK. Turn on the stopwatch to
ensure that PK are added into every wells at the same time
intervals. Oscillate the microtitre plate far one minute and
cover with a lid, allow to stand at 25-30ºC far 30 minutes.
To each reaction well, add 20 µl of 2 mmol/L S-2302
solution in the same arder at the same time intervals as those
far adding PK. Oscillate the plate far one minute and cover
with a lid, allow to stand at 25-30ºC far 15 minutes. Add 20
µl of 50 % acetic acid solution to each well in the same arder
at the same time interval as those mentioned above and
oscillate far one minute. Read the absorbance of each well at
405 nm by ultraviolet-visible spectrophotometry ( 0401 ) .
At the same time, set up a blank control by using 20-50 µl
of TNB instead of 20-50 µl of PK with the same procedure.
Use 20 µl of PKA standard solution instead of test sample
solution and repeat the procedure. A linear regression
equation is obtained by regressing the logarithm of PKA
activity of standard solutions with the logarithm of their
corresponding absorbance. Calculate the PKA activity of test
sample by the equation.
[Note]
( 1) Three wells shall be set up far each PKA standard and
test sample, among them, two are the test wells, and the
other one is the control well. The diff erence between
absorbance of the two test wells shall be less than O. 020.
(2) According to the PKA content in the test sample, select
 341 O Test for Anticomplement Activity

a suitable dilution range of standard. A= Absorbance of hemolyzed erythrocytes

(3) The correlation coefficient of linear regression shall be before dilution.
not less than O. 99.
Titration of hemolysin
(4) Whenever PK, S-2302 and 50% acetic acid solutions are
added to the wells, the time intervals of addition between Dilute hemolysin according to the parameters in Table
wells, as well as the order of addition, shall be the same so l. Starting from the 1 : 75 dilution of hemolysin, mix l. O
as to allow each well react under the same conditions. ml of hemolysin of each dilution with l. O ml of 5 % sheep
(5) The time for allowing the plate to stand after addition of erythrocyte suspension and incubate at 37°C for 30 minutes.
PK and S2302 solution is recorded starting from addition To O. 2 ml of each incubated mixture, add l. 10 ml of GBB
onto the first well. and O. 2 ml of diluted guinea pig serum (e. g., 1/150
( 6) If the temperature for allowing the plate to stand is
dilution). Incuba te at 37°C for 60 minutes. Centrifuge the
below 25ºC, the plate shall be allowed to stand at 37°C for
incubated mixture at 2000 r/min for 5 minutes. Collect the
10 min within the specified time in two reaction courses.
supernatant and measure the absorbance of each tube at 541
nm by ultraviolet-visible spectrophotometry < 0401 ) . The
3410 lest for Anticomplement Activity above tests shall be done in duplicate. At the same time,
prepare three tubes of non-hemolysis controls by adding O. 1
The method is based on immune hemolytic reaction. ml of 5 % sheep erythrocytes to l. 4 ml of barbital buffer
The anticomplement activity ( ACA) of test sample is solution and another three tubes of complete hemolysis
determined according to the change of hemolysis rate caused controls by adding O. 1 ml of 5 % sheep erythrocytes to l. 4
by the consumption of complement.
ml of water with the same procedure as mentioned above.
Reagent Calculate the hemolysis rate (Y) of each tube according to
(1) Magnesium and calcium stock solution: Dissolve l. 103
the following equation:
g of calcium chloride and 5. 083 g of magnesium chloride
(MgCb• 6H 2 0) in water and dilute to 25 ml. Y As-Acn XlOO%
(2) Barbital buffer stock solution: Dissolve 41. 5 g of sodium Ach-Acn
chloride and 5. 1 g of barbital sodium in 800 ml of water. Adjust Where: Y=Hemolysis rate, % ;
pH to 7. 3 with 1 mol/L hydrochloric acid Add 2. 5 ml of As= Absorbance of each sample tube;
magnesium and calcium stock solution and dilute to 1000 ml with Acn = Absorbance of non-hemolysis control tube;
v:ater. Filter Vv7ith a O. 22 µm membrai1e ar1d store at 4ºC.
Ach = Absorbance of complete hemolysis control
(3) Gelatin-barbital buffer (GBB) solution: Dissolve O. 625
g of gelatin in 30 ml of water by boiling. Add 100 ml of tu be.
barbital buffer stock solution and dilute to 500 ml with water. Plot a curve by using hemolysis rate Y as ordinate and
Prepare fresh solution daily. dilution factor of hemolysin as abscissas to determine the
( 4) Alsever solution: Dissolve O. 5 g of citric acid, 8. O g of optimal dilution of the hemolysin for preparation of sensitized
sodium citrate, 20. 5 g of glucose and 4. 2 g of sodium sheep erythrocyte. Select a dilution such that further increase
chloride in water and dilute to 1000 ml (pH value is about in the amount of hemolysin <loes not cause appreciable change
6. 2). Dispense the solution in blood collecting bottles with a
in the degree of hemolysis, and define this dilution as one
quantity needed for one bleeding of a sheep. Carry out steam
sterilization at l16°C for 10 minutes (after sterilization, the minimal hemolysin dilution unit (1 MHU) in l. O ml. The
steam shall be released as soon as possible). Store at 4 ºC. maximum hemolysis rate shall be within the range of 50%-
( 5) Sheep erythrocytes: Collect sheep blood from j ugular 70 % , otherwise the test is invalid.
vein aseptically and mix with an equal volume of Alsever
Table 1 Preparation of hemolysin dilutions
solution. Dispense the mixture in tubes under an aseptic
condition and store at 4ºC for one week before use. Preparation
(6) Hemolysin: Rabbit anti-sheep erythrocyte serum. Required
dilution of Gelatin-barbita Hemolysin
( 7) Guinea pig serum ( complement) : Dispense the pooled
hemolysin buffer
sera collected from more than ten guinea pigs af ter removing Dilution (1 : x)
solution, ml ml
blood cells by centrifugation at 4 ºC. Store at -70ºC or store
lyophilized. The total complement activity of gumea p1g
serum shall be not less than 100 CH50 /ml. 7.5 0.65 Un-diluted o. 1
10 0.90 Un-diluted o. 1
Preparation of 5 %sheep erythrocyte suspension 75 l. 80 7.5 0.2
Wash a quantity of above-mentioned sheep erythrocytes at 100 l. 80 10 0.2
least 3 times with GBB and suspend in a quantity of the same 150 l. 00 75 l. o
solution. Add O. 2 ml of the erythrocyte suspension to 2. 8 200 l. 00 100 l. o
ml of water. Measure the absorbance at 541 nm by 300 l. 00 150 l. o
ultraviolet-visible spectrophotometry < 0401 ) after the 400 l. 00 200 l. o
complete hemolysis of erythrocytes. Adjust the absorbance 600 l. 00 300 l. o
of the suspension to O. 62 ±O. 01 according to the following
800 l. 00 400 l. o
1200 l. 00 600 l. o
equation After the adjustment, the erythrocyte concentration of
1600 l. 00 800 l. o
the suspension shall be 1 X 109 cells/ ml. 2400 l. 00 1200 l. o
Volume (ml) of erythrocyte suspension after dilution= 3200* l. 00 1600 l. o
V¡XA/O. 62 4800* l. 00 2400 l. o
Where: V¡ = Volume of erythrocyte suspension before
dilution, ml; * Discard 1. O ml of the mixture.
 3411 Determination of Residual Bovine Serum Albumin Content

Preparation of optimal sensitized sheep erythrocytes (EA) by regressing the logarithm of the amount of complement
Add a quantity of 2 MHU/ml hemolysin slowly to an equal used with the logarithm of Y/ ( 1 - Y). Calculate the
volume of 5 % sheep erythrocyte suspension. Allow the intercept (a ) , slope ( b) and correlation coefficient ( r).
mixture to stand at 37°C for 15 minutes. Store at 2-8ºC and The complement activity is calculated by the following
use within 6 hours. equation:
Titration of complement activity in guinea pig serum Complement activity (CHso/ml) = (1/X) XF/5
Dilute the guinea pig serum properly with gelatin barbital Where: (1/X) = Reciprocal of antilog a;
buffer solution and titrate the complement according to the F = Dilution factor of complement
protocol in Table 2. A linear regression equation is obtained
Table 2 Protocol for the titration of complement activity
Tube No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14

GBB CmD l. 2 l. 1 l. o 0.9 0.8 o. 7 0.6 o. 5 0.4 0.3 o. 2 o. 1 l. 3 l. 3

Diluted comple-ment (ml) o. 1 0.2 0.3 0.4 0.5 0.6 o. 7 0.8 0.9 l. o l. 1 l. 2

EA (ml) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 o. 2 0.2 0.2 0.2

Incubate at 37°C for 60 minutes-+- cool in an ice bath-+- centrifuge at 2 000 r/min for 5 minutes-+- measure the absorbance
of supernatant at 541 nm

Determination of anticomplement activity CACA) (3) Only clarified gel solution is permitted to use.
According to the titrated guinea pig serum complement
activity, dilute the complement to 100 CH 50 /ml with gelatin 3411 Determination of Residual
barbital solution. Prepare incubation mixtures according to Bovine Serum Albumin Content
the parameters in Table 3.
The concentration of IVIG in Table 3 is calculated on the The method is used for the assay of the residual bovine
basis of 50 mg/ ml. If the concentration of IVIG is not 50 mg serum albumin (BSA) content in products by ELISA
per ml, the volume of IVIG needed is adjusted by the Preparation of test sample solution Reconstitute the test
following equation: sample in freeze-dried form, as stated on the label and mix
well, stand at room temperature for 30 minutes. Mix again
Volume of IVIG needed (ml) = 10 mg/C
Where~ C=Protein content (mg) in 1 ml of IVIG upon the assay. Liquid test sample can be assayed directly.
Interference test lnterference test is required when the
Table 3 Preparation of incubation mixture
ELISA kit is applied to the test sample at the first time.
Test sample Complement Prepare the following solutions: Solution I ( 2-fold serial
Tube
(ml) control (ml) dilutions of test sample solution) , Solution II (mixture of
test sample and 30 ng/ml internal control at equal amounts),
Test sample (50 mg/mD O. 2
Solution III ( 2-fold serial dilutions of interna! control
Gelatin-barbital buffer (ml) 0.6 0.8 starting from 30 ng/ ml). When BSA content in the test
sample solution is greater than mid-point of detection range
Complement (100 CHso/mD 0.2 0.2 of the kit, make a 2-fold dilution of the test sample solution
prior to preparation of Solution I and Solution 1I.
Then, calculate the quantity of gelatin barbital buffer Solution I and Solution II can be applied by 2-fold serial
solution to be added according to the actual amount of IVIG, dilutions. Solution II1 shall be applied in multiple duplicate
but the total volurne of the mixture shall be maintained at (at least 10 wells), and be distributed evenly on the plate.
O. 8 ml. Allow the mixture to stand at 37°C for 60 minutes. Determine the BSA contents in Solutions I , II and III
To O. 2 ml of the incubated mixture, add 9. 8 ml of gelatin- respectively, according to the instruction of the ELISA kit.
barbital buffer solution ( 50-fold dilution) and determine the The difference between BSA contents of Solutions I and
remaining complement activity. Solution II shall fall into 95 % CI of the BSA content of
The anticomplement activity of test sample is calculated by Solution III, to demonstrate that no interference factor exists
the following equation. The test is valid if the remaining in the test sample during assay.
complement activity of the control is within the range of Procedure Follow the instruction of the ELISA kit, the test
80-120 CHso / ml. sample shall be prepared with the provided diluent. For each
Anticomplement activity of test sample ( %) test sample, at least two dilutions shall be prepared, each
(D-G) /DXlOO dilution shall be applied in duplicate. The assay is valid only
Where: D = Remaining complement activity of control, if the absorbance of standards, the result of interna! control,
CHso/ml; the linear correlation coefficient of standard curve and absorbance
G= Remaining complement activity of test sample, difference between duplicates meet the requirements set forth
CHso/ml. in the kit. Plot a regression curve with the absorbance of
[Note] standards to their concentrations, and calculate BSA content
( 1 ) Leukocytes must be removed during the washing of of the test sample solution by inserting its absorbance into
erythrocytes. the regression equation and multiplying the result by the
(2) Shake the erythrocyte gently during sensitization. dilution factor.
 3412 Determination of Residual Host Bacteria} Protein (E. coli)
[Notes]
(1) For one test sample, if the absorbance of the lower
dilution is lower than that of the higher dilution, HOOK
effect or operation error may occur. The test sample shall be
re-tested, if necessary, adjust the dilution factor upon re-test
(2) Dedicated container and tubes shall be used in the assay
of BSA content to avoid contamination of foreign BSA in the
laboratory.
3412 Determination of Residual Host
Bacteria) Protein (E. coli )
The residual host bacteria! protein content of recombinant
products expressed in E. coli is determined by ELISA
Reagent
(1) Coating solution (Carbonate buffer solution, pH 9. 6)
Dissolve O. 32 g of sodium carbonate and O. 586 g of sodium
bicarbonate in water and dilute to 200 ml.
(2) Phosphate buffer sohition (pH 7. 4)
Dissolve 8 g of sodium chloride, O. 2 g of potassium
chloride, l. 44 g of disodium hydrogen phosphate and O. 24 g
of potassium dihydrogen phosphate in water and dilute to 500
ml. Sterilize at 121 ºC for 15 minutes.
(3) Washing solution (pH 7. 4)
Dilute O. 5 ml of polysorbate 20 to 500 ml with phosphate
buffer solution.
(4) Dilution solution (pH 7. 4)
Dissolve O. 5 g of bovine serum albumin in washing solution
and dilute to 100 ml.
( 5) Concentrated dilution solution
Dissolve l. O g of bovine serum albumin in washing solution
and dilute to 100 ml.
(6) Substrate buffer solution (Citric acid-phosphate buffer
solution, pH 5. O)
Dissolve l. 84 g of disodium hydrogen phosphate (Na2 HP04 •
12H2O) and O. 51 g of citric acid in water and dilute to 100 ml.
(7) Substrate solution
Dissolve 8 mg of o-phenylenediamine in a quantity of
substrate buffer solution. Add 30 µl of 30 % hydrogen
peroxide and dilute to 20 ml with substrate buffer solution.
Prepare just befare use.
(8) Stopping solution
1 mol/L Sulfuric acid.
Preparation of standard solution
Reconstitute the E. coli host cell protein standard with
water according to the instructions and dilute with dilution
solution to 500 ng, 250 ng, 125 ng, 62. 5 ng, 31. 25 ng,
15. 625 ng and 7. 8125 ng of host cell protein per ml
separately.
Preparation of test sample
Dilute a quantity of test sample to a concentration of about
250 µg per ml. If the concentration of the test sample is
lower than 500 µg per ml, dilute with an equal volume of
concentrated dilution solution.
Procedure
Dilute a volume of rabbit anti-E. coli host cell protein
antibody to 10 µg per ml with coating solution. Add 100 µl
of the diluted antibody to each well of a 96-well microtiter
plate. Allow to stand overnight at 4 ºC (16-18 hours). Wash
the microtiter plate 3 times with washing solution. Prepare a
1 % bovine serum albumin solution with washing solution and
add 200 µl of the solution to each well of the 96-well
microtiter plate. lncubate at 37°C for 2 hours. Wash the
blocked microtiter plate 3 times with washing solution. Add
100 µl of the standard and test sample solutions into each
well of the plate separately, in duplicate. Meanwhile, add
dilution solution into other two wells as a blank control.
lncubate at 37°C for 2 hours. Dilute rabbit anti-E. coli host
cell antibody, labeled with horseradish peroxidase ( HRP) ,
with dilution solution ata ratio of 1 : 1000. Add 100 µl of the
diluted solution into each well of the plate. Allow to stand at
37°C for 1 hour. Wash the microtiter plate 10 times with
washing solution. Add 100 µl of substrate solution into each
well of the plate. Allow to stand at 37°C for 40 minutes,
protected from light. Add 50 µl of stopping solution into
each well of the plate to stop the reaction. Read the
absorbance of each well at 492 nm and analyze the data with
computer software or calculate the result manually with
graphic method.
Plot a curve with concentration of standard solution against
the corresponding absorbance. Find out the host cell protein
content on the curve through its absorbance. Calculate the
residual host cell protein content in test sample by the
following equation.
Residual host cell protein content of test sample ( % ) =
CXDX 100%/TX 10 6
Where: C=Residual host cell protein content of test sample
solution, ng/ml;
D=Dilution factor of test sample;
T=Protein content of test sample, mg/ml.
[Note]
Carry out the test according to the package inserts of
validated ELISA kit as well.
3413 Determination of Residual Host
Bacteria) Protein ( Pseudomonas )
The content of residual host bacterial protein in recombinant
products prepared by Pseudomonas expression system is
determined by ELISA
Reagent
(1) Coating solution (carbonate buffer solution, pH9. 6):
Weigh accurately O. 32 g of sodium carbonate and O. 586 g of
sodium hydrogen carbonate, transfer into a 200 ml
volumetric flask, dissolve with water and dilute to the
volume.
(2) Phosphate buffer saline: Transfer 8. O g of sodium
chloride, O. 20 g of potassium chloride, l. 44 g of disodium
hydrogen phosphate and O. 24 g of potassium dihydrogen
phosphate into a 500 ml volumetric flask, dissolve with water
and dilute to the volume. Sterilize at 121 ºC for 15 minutes.
(3) Washing solution: Dilute O. 5 ml of polysorbate 20 to
500 ml with phosphate buffer saline.
( 4) Concentrated diluent: Dissolve l. O g of bovine serum
albumin in washing solution and dilute to 100 ml.
(5) Diluent: Dilute the concentrated diluent with an equal
volume of water.
( 6) Substrate buffer solution (O. 005 mol/L sodium acetate-
citric acid buffer solution): Dissolve O. 68 g of sodium acetate
and l. 05 g of citric acid (C6H 8 01•H2Ü) in water and dilute
to 1000 ml. Adjust pH to 3. 6.
(7) Substrate solution A: Dissolve O. 08 g of 3, 3', 5, 5'-
tetramethylbenzidine CTMB) in 40 ml of dimethyl sulfoxide
(DMSQ), add 60 ml of methanol and mix well. Add 100 ml
of substrate buffer solution, stir in a dark place for 2 hours
 3414 Determination of Residual Host Yeast Protein
until the mixture is dissolved completely. Allow the solution
to stand at room temperature for 4 hours.
(8) Substrate solution B: Dilute 3. 2 ml of l. 5 % hydrogen
peroxide to 1000 ml with substrate buffer solution.
(9) Substrate solution: Mix substrate solutions A and B at
equal volumes just befare use.
(10) Stopping solution: 2 mol/L sulfuric acid solution.
Preparation of reference solution
Dissolve host bacterial protein reference with the diluent
following the instruction of reagent kit, then measure
accurately a quantity of the solution and dilute with diluent to
host bacterial protein concentrations of 20 ng, 10 ng, 5 ng,
2. 5 ng, l. 2 ng, O. 6 ng and O. 3 ng per ml separately.
Preparation of test sample solution
Dilute a quantity of test sample with the diluent to a
concentration of about 100 µ.g of protein per ml. If the
protein concentration is less than 200 µ.g/ ml, the test sample
shall be diluted with an equal volume of concentrated diluent.
Procedure
Dilute the coating antibody to a suitable concentration with
the coating solution. The dilution factor is indicated in the
instructions of the reagent kit. Add 100 µ.l of the diluted
antibody into each well of a 96-well microtitre plate, allow
the plate to stand at 2-8ºC for 16-20 hours. Wash the
microtitre plate 3 times with washing solution. Prepare a 1 %
bovine serum albumin ( BSA ) solution with washing
solution. Add 200 µ.l of BSA solution into each well of the
plate and oscillate (200-300 r/min) at room temperature for
one hour. Wash the plate 3 times with washing solution.
Add the reference and test sample solutions into the wells of
the plate separately, 100 µ.l per well in duplicate.
Meanwhile, add diluent into other two wells as blank
controls. Oscillate ( 200-300 r/ min) the microtitre plate at
room temperature for one hour; and 'x1ash 3 times \11<lith
washing solution. Dilute the first antibody with diluent to a
suitable concentration following the instruction of reagent kit.
Add 100 µ.l of the diluted antibody into each well of the
microtitre plate and oscillate ( 200-300 r/min) at room
temperature for one hour. Wash the microtitre plate 3 times
with washing solution. Dilute horseradish peroxidase
CHRP) -labeled second antibody with diluent to a suitable
concentration following the instructions of reagent kit. Add
100 µ.l of the diluted antibody into each well of the microtitre
plate and oscillate (200-300 r/min) at room temperature for
30 minutes. Wash the microtitre plate 8 times with washing
solution. Add 100 µ.l of substrate solution into each well and
set the microtitre plate at room temperature to react for 10-
15 minutes, protected from light. Add 100 µ.l of stopping
solution into each well to stop the reaction. Read the
absorbance of each well at 450 nm with a microtitre plate
reader and process the data with a computerized reader or
calculate the result manually with graphic method
Plot a curve with the concentrations of the reference solutions
against their absorbance. Find out the host bacteria! protein
content of test sample solution on the curve through its
absorbance. Calculate the residual host bacteria! protein
content in the test sample by the following equation.
Residual host bacteria! protein content C%) in the test sample =
CCXD) / CTX106 ) Xl00%
Where: C= Host bacteria! protein concentration in the test
sample, ng/ml;
D=Dilution factor of the test sample;
T = Total protein content in the test sample,
mg/ml.
[Note]
Carry out the test according to the package inserts of
validated ELISA Kit as well.
3414 Determination of Residual Host
Yeast Protein
The content of residual host yeast protein m recombinant
product prepared by using yeast expression system is
determined by ELISA
Reagent
( 1) Coating solution (carbonate buffer solution, pH9. 6) :
Dissolve O. 32 g of sodium carbonate and O. 586 g of sodium
hydrogen carbonate in water and dilute to 200 ml.
(2) PBS: Dissolve 8. O g of sodium chloride, O. 20 g of
potassium chloride, l. 44 g of disodium hydrogen phosphate
and O. 24 g of potassium dihydrogen phosphate in water and
dilute to 1000 ml. Adjust pH to 7. 4 and sterilize at 121 ºC
for 15 minutes.
(3) Washing solution CPBS-Tween 20): Dilute O. 5 ml of
polysorbate 20 to 1000 ml with PBS.
(4) Diluent: Dissolve O. 5 g of bovine serum albumin with
washing solution and dilute to 100 ml.
(5) Substrate buffer solution (O. 005 mol/L sodium acetate-
citric acid buffer solution) : Dissolve O. 68 g of sodium acetate
and l. 05 g of citric acid CCs Hs 07• H2 0) in water and dilute
to 1000 ml. Adjust pH to 3. 6.
( 6) Substrate solution A: Dissolve O. 08 g of 3, 3' , 5, 5 ' -
tetramethylbenzidine CTMB) in 40 ml of dimethyl sulfoxide
CDMSQ), add 60 ml of methanol and mix well. Add 100 ml
of substrate buffer solution and stir in a dark place for 2
hours until the mixture is dissolved completely. Allow the
solution to stand at room temperature for 4 hours.
(7) Substrate solution B: Dilute 3. 2 ml of l. 5% hydrogen
peroxide to 1000 ml with substrate buffer solution.
(8) Substrate solution: Mix substrate solutions A and B at
equal volumes just befare use.
(9) Stopping solution: 1 mol/L sulfuric acid solution.
Preparation of reference solution
Reconstitute the standard with water, then measure
accurately a quantity of the solution and dilute with diluent to
make 1 ml containing 1000 ng, 500 ng, 250 ng, 125 ng and
62. 5 ng of host yeast protein.
Preparation of test sample solution
Dilute a quantity of test sample with the diluent to a suitable
concentration.
Procedure
Dilute a quantity of guinea pig anti-yeast protein antibody
with coating solution to a suitable concentration. Add 100 µ.l
of the diluted antibody into each well of a 96-well microtitre
plate. Seal the plate with a piece of film, allow to stand at
4 ºC overnight. Wash the plate 3 times with washing
solution. Prepare a 1 % bovine serum albumin ( BSA)
solution with washing solution. Add 200 µ.l of 1 % BSA
solution into each well of the plate, allow to stand at 37°C
for 2 hours. Wash the blocked plate 3 times with washing
solution. Add the reference and test sample solutions into the
wells of the microtitre plate separately, 100 µ.l per well in
duplicate. Meanwhile, add diluent into other two wells as
blank controls. Seal the microtitre plate with a piece of film,
allow to stand at 37°C for one hour. Wash the plate 6 times
with washing solution. Dilute rabbit anti-yeast antibody with
 :··:2s0::. 3415 Test for Blood Group A-Like Substance
diluent to a suitable concentration. Add 100 µl of the diluted
antibody into each well of the microtitre plate. Seal the
microtitre plate with a piece of film, allow to stand at 37°C
for one hour. Wash the plate 6 times with washing solution.
Dilute horseradish peroxidase ( HRP) -labeled sheep anti-
rabbit antibody ( lgG - HRP) solution with the diluent to a
suitable concentration. Add 100 µl of the diluted lgG-HRP
solution into each well. Seal the microtitre plate with a piece
of film, allow to stand at 37°C for one hour. Wash the plate
6 times with washing solution. Add 100 µl of substrate
solution into each well, allow to stand at room temperature
for 5-10 minutes, protected from light. Add 100 µl of
stopping solution into each well to stop the reaction. Read
the absorbance of each well with microtitre plate reader at
450 nm, using 630 nm as a reference wavelength. Process
the data with a computerized reader or calculate the result
manually with graphic method.
Plot a curve with the concentrations of the reference solution
against their absorbance. Find out the host cell protein
content of sample solution on the curve through its
absorbance. Calculate the residual host yeast protein content
of the test sample by the following equation.
Residual host yeast protein content ( % ) =
(CXD) / (TX10 6 ) XlOO%
Where: C= Host yeast protein concentration of test sample,
ng/ml;
D=Dil11tion factor of test sample;
T=Total protein content of test sample, mg/ml.
[Note]
Carry out the test according to the package inserts of
validated ELISA kit as well.
3415
Test for Blood Group
A-Like Substance
(Hemagglutination Inhibition Assay)
The blood group A-like substance content in test sample is
determined by reacting blood group A-like substance
reference and test sample with anti-blood group A serum
respectively and comparing the end-points of the two
reactions.
1 % Blood group A erythrocyte suspension
Mix the blood of group A from six donors at equal volumes, add
a quantity of physiological saline and mix well. Centrifuge at
2000 r/ min for 10 minutes. Decant the supernatant and wash the
precipitate 3 times with physiological saline. Mix 1 ml of the
precipitate with 99 ml of physiological saline to prepare a 1 %
erythrocyte suspension.
Preparation of blood group A-like substance
reference solution
The blood group A-like substance reference is distributed or
accredited by the NCL. The reference solution is made to
contain 1 mg per ml. In a group of ten test tubes, each at a
diameter of 9 mm, carry out a series of 2-fold dilutions for
O. 1 ml of blood group A-like substance reference with
physiological saline starting from 1/100 dilution (0. 01 mg/ml).
Preparation of test sample solution
In a group of ten test tubes, each ata diameter of 9 mm, carry
out a series of 2-fold dilutions for O. 1 ml of test sample with
physiological saline starting from the original concentration.
Detennination of the testing dose of anti-blood group A serum
In a group of ten test tubes, each at a diameter of 9 mm,
carry out a series of 2-fold dilutions for O. 1 ml of anti-blood
group A serum with physiological saline starting from 1/2
dilution. To O. 1 ml of each dilution, add O. 1 ml of 1 %
blood group A human erythrocyte suspension. At the same
time, prepare a negative control by adding O. 1 ml of blood
group A human erythrocyte to O. 1 ml of physiological saline.
Mix well by shaking, allow the mixtures to stand at room
temperature for 15 minutes, centrifuge at 1500 r/min for
one minute and observe the agglutination extent according to
the status of cell sedimentation. The highest dilution of anti-
blood group A serum which still shows complete
agglutination ( + + + +) is served as one testing dose.
Procedure
To O. 1 ml of each dilution of test sample and blood group
A-like substance reference, add O. 1 ml of anti-blood group
A serum containing two testing doses and mix well by
shaking. Allow the mixture to stand at 36. 5-37. 5ºC for 10
minutes and then add O. 1 ml of 1 % human blood group A
erythrocytes in to each tu be. Mix well again by shaking,
allow to stand at 36. 5-37. 5ºC for 15 minutes. Centrifuge at
1500 r/min for one minute and observe the agglutination
extent according to the sedimentation of erythrocytes.
Result evaluation
The content (mg) of blood group A-like substance in 1 ml
of the sample is calculated by multiplying the highest dilution
factor of test sample showing complete hemagglutination
inhibition (end point) by the content of blood group A-like
substance in the highest dilution of control sample showing
the similar extent of hemagglutination inhibition.
3416 Determination of Residual
Murine IgG
The residual murine lgG content in the recombinant product,
after purification by affinity chromatography with monoclonal
antibody, is determined by ELISA
Reagent
( 1 ) Coating solution ( carbonate buffer solution, pH9. 6 ) :
Dissolve O. 32 g of sodium carbonate and O. 586 g of sodium
hydrogen carbonate in water and dilute to 200 ml.
(2) PBS ( pH7. 4) solution: Dissolve 8. O g of sodium
chloride, O. 20 g of potassium chloride, l. 44 g of disodium
hydrogen phosphate and O. 24 g of potassium dihydrogen
phosphate in water and dilute to 1000 ml. Sterilize at 121 ºC
for 15 minutes.
(3) Washing solution (PBS-Tween 20): Dilute O. 5 ml of
polysorbate 20 to 1000 ml with PBS.
Diluent: Dissolve O. 5 g of bovine serum albumin in washing
solution and dilute to 100 ml.
Substrate buffer solution (citric acid-PBS): Dissolve l. 84 g
of disodium hydrogen phosphate (Na2HP04• l2H20) and
O. 51 g of citric acid in water and dilute to 100 ml.
Substrate solution: Dissolve 8 mg of o-phenylenediamine in
20 ml of substrate buffer solution. Add 30 µl of 30 %
hydrogen peroxide. Prepare just befare use.
Preparation of reference solution
Measure precisely a volume of murine lgG reference
substance, reconstituted with a quantity of water according
to the instruction, and dilute with diluent to contain 100,
50, 25, 12. 5, 6. 25 and 3. 13 ng per ml respectively.
Preparation of test sample solution
Dilute a quantity of test sample to make 1 ml containing one
 3418
dose of final product. If specifications of the product are not
defined, the test sample is diluted to a concentration of the
maximum dose of final product per ml.
Procedure
Dilute goat anti-mouse lgG with coating solution to contain
10 µg per ml and add to a 96-well microtitre plate, 100 µl
per well. Allow the plate to stand at 4 ºC overnight (16-18
hours). Wash the plate 3 times with washing solution.
Prepare a 1 % bovine serum albumin solution with washing
solution and add to the plate, 200 µl per well. Block at 37ºC
for 2 hours. Wash the blocked plate 3 times with washing
solution. Add reference and test sample solutions to the plate
separately, 100 µl per well, allow to stand at 37°C for one
hour. Wash the plate 3 times with washing solution. Dilute
horseradish peroxidase-labeled sheep anti-mouse lgG with
diluent following the instructions and add to the plate, 100
µl per well. Allow the plate to stand at 37°C for 30 minutes
and wash 3 times with washing solution. Add substrate
solution to the plate, 50 µl per well, allow to stand at 37°C
for 20 minutes, protected from light. Add 50 µl of stopping
solution (1 mol/L sulfuric acid) into each well to stop the
reaction. Read the absorbance of each well at 492 nm with
microtitre plate reader and analyze the data with computer
software or calculate the result manually with graphic method.
Plot a curve with the concentrations of the reference solutions
against their absorbance. The correlation coefficient of the
curve shall be more than O. 995. Find out the murine lgG
content of test sample solution on the curve through its
absorbance. Calculate the residual murine lgG content of test
sample by the following equation:
Residual murine lgG content (ng) =C XnXF/T
Where: C = Murine lgG content of test sample solution,
ng/ml;
n = Dilution factor of test sample solution;
F = Specification of final product, IU/dose or
µg/dose;
T = Potency or protein content of principal
constituent of the test sample, IU/ml or
µg/ml.
3417ldentity Test for Acellular
Pertussis Vaccines
(Enzyme Linked Immunosorbent Assay)
The antigenic components pertussis toxin ( PT ) and
filamentous hemagglutinin ( FHA) in acellular pertussis
vaccines are identified by this ELISA method.
Reagent
( 1) Coating solution (carbonate buffer solution, pH9. 6) :
Dissolve l. 59 g of sodium carbonate and 2. 93 g of sodium
bicarbonate in water and dilute to 1000 ml.
(2) Phosphate buffer solution (pH 7. 4): Dissolve 8. O g of
sodium chloride, O. 20 g of potassium chloride, l. 44 g of
disodium hydrogen phosphate and O. 24 g of potassium
dihydrogen phosphate in water and dilute to 1000 ml.
Sterilize at 121 ºC for 15 minutes.
(3) Washing solution (PBS-Tween 20): Dilute O. 5 ml of
polysorbate 20 to 1000 ml with PBS.
( 4) Blocking solution: Dissolve l. O g of bovine serum
albumin with washing solution and dilute to 100 ml.
ldentity Test for Antitoxin and Antiserum
(5) Diluent: Dissolve O. 5 g of bovine serum albumin with
washing solution and dilute to 100 ml.
(6) Substrate buffer solution (O. 005 mol/L sodium acetate-
citric acid buffer solution) : Dissolve O. 68 g of sodium acetate
and l. 05 g of citric acid CC6 Ha 01• H 2 0) in water and dilute
to 1000 ml. Adjust pH to 3. 6.
1
(7) Substrate solution A: Dissolve O. 08 g of 3, 3 , 5, 5'-
tetramethylbenzidine CTMB) in 40 ml of dimethyl sulfoxide
CDMSQ), add 60 ml of methanol and mix well. Add 100 ml
of substrate buffer solution and stir in a dark place for 2
hours until the mixture is dissolved completely. Allow the
solution to stand at room temperature for 4 hours.
(8) Substrate solution B: Dilute 3. 2 ml l. 5% hydrogen
peroxide to 1000 ml with substrate buffer solution.
(9) Substrate solution: Mix substrate solution A and B at
equal volumes just befare use.
(10) Stopping solution: 2 mol/L Sulfuric acid solution.
Positive control and negative control
Positive control: Purified PT or FHA reference (2-8 µg/mD.
Negative control: Phosphate buffer solution or other
appropriate solutions.
Preparation of test sample solution
Desorb a quantity of test sample with sodium citrate or other
appropriate reagents.
Procedure
Add 100 µl of coating antibody against PT or FHA at an
appropriate concentration (2-5 µg/mD. Seal the plate with a
piece of film, allow to stand at 2-8ºC for 16-20 hours. Wash
the plate 3 times with washing solution. Add 200 µl of
blocking solution into each well of the plate. Seal the plate
with a piece of film, allow to stand at 37°C for one hour.
Wash the blocked plate 3 times with washing solution. Add
100 µl of positive control PT Cor FHA) reference and the
treated test sample. Seal the plate with a piece of film, allow
to stand at 37°C for one hour. Wash the plate 6 times with
washing solution. Dilute horseradish peroxidase-labelled
mouse anti-PT or FHA antibody at appropriate ratio. Add
100 µl of the diluted antibody into each well of the plate. Seal
the plate with a piece of film, allow to stand at 37°C for one
hour. Wash the plate 6 times with washing solution. Add
100 µl of substrate solution into each well and allow the
microtitre plate to stand at room temperature for 5-15
minutes, protected from light. Add 100 µl of stopping
solution into each stand well to stop the reaction. Read the
absorbance of each well at 450 nm with a microtitre plate
reader.
Result evaluation
The cutoff value is defined as 2. 1 times the highest
absorbance of negative control species (average value of two
wells). If the absorbance of test sample is more than the
cutoff value, the results shall be judged as positive.
3418 Identity Tust for Antitoxin and Antisermn
(Enzyme Linked Immunosorbent Assay)
This ELISA method is an alternative identity test of the
equine protein in the antitoxin and antiserum products
manufactured from horse blood.
Reagent
( 1) Coating solution (carbonate buffer solution, pH9. 6):
Dissolve O. 32 g of sodium carbonate and O. 586 g of sodium
bicarbonate in water and dilute to 200 ml.
 3419 Determination of Molecular Size Distribution for Group A Meningococcal Polysaccharide
(2) Phosphate buffer solution (pH 7. 4): Dissolve 8. O g of
sodium chloride, O. 20 g of potassium chloride, l. 44 g of
disodium hydrogen phosphate and O. 24 g of potassium
dihydrogen phosphate in water and dilute to 1000 ml.
Sterilize at 121 ºC for 15 minutes.
(3) Washing solution (PB&-Tween 20): Dilute O. 5 ml of
polysorbate 20 to 1000 ml with PBS.
( 4) Blocking solution: Dissolve 2. O g of bovine serum
albumin with washing solution and dilute to 100 ml.
(5) Diluent: Dissolve O. 5 g of bovine serum albumin with
washing solution and dilute to 100 ml.
(6) Substrate buffer solution (0. 005 mol/L sodium acetate-
citric acid buffer solution) : Dissolve O. 68 g of sodium acetate
and l. 05 g of citric acid (C6 Hs01•H20) in water and dilute
to 1000 ml. Adjust pH to 3. 6.
1
(7) Substrate solution A: Dissolve O. 08 g of 3, 3 , 5, 5'-
tetramethylbenzidine (TMB) in 40 mi of dimethyl sulfoxide
( DMSO) , add 60 mi of methanol and mix well. Add 100 mi
of substrate buffer solution and stir in a dark place for 2
hours until the mixture is dissolved completely. Allow the
solution to stand at room temperature for 4 hours.
(8) Substrate solution B: Dilute 3. 2 ml l. 5% hydrogen
peroxide to 1000 mi with substrate buffer solution.
(9) Substrate solution: Mix substrate solution A and B at
equal volumes just befare use.
(10) Stopping solution: 2 mol/L Sulfuric acid solution.
Negative control and positive control
Positive control: Horse lgG.
Negative control: Human lgG, bovine lgG, goat lgG and
pig IgG.
Dilute a quantity of positive control solution and negative
control solution with coating solution to a suitable
concentration.
Preparation of test sample solution
Dilute a quantity of test sample with coating solution to 5-10
µg/ml.
Procedure
Add 100 µl of the diluted test sample solution and control
solution into each well of a 96-well microtitre plate
separately, in duplicate. Seal the plate with a piece of film,
allow to stand at 2-8ºC for 16-20 hours. Wash the plate 3
times with washing solution. Add 200 µl of blocking solution
into each well of the plate. Seal the plate with a piece of
film, allow to stand at 37°C for one hour. Wash the blocked
plate 3 times with washing solution. Dilute horseradish
peroxidase-labelled rabbit anti-horse lgG antibody with
diluent at a ratio of 1 : 2000 . Add 100 µl of the diluted
antibody into each well of the plate. Seal the plate with a
piece of film, allow to stand at 37°C for one hour. Wash the
plate 6 times with washing solution. Add 100 µl of substrate
solution into each well and allow the microtitre plate to stand
at room temperature for 5-15 minutes, protected from light.
Add 100 µl of stopping solution into each well to stop the
reaction. Read the absorbance of each well at 450 nm with a
microtitre plate reader.
Result evaluation
The cutoff value is defined as 2. 1 times the highest
absorbance of four negative control (average value of two
wells). If the absorbance is more than the cutoff value, the
result shall be judged as positive.
The test is valid if the absorbance of the positive control is
more than the cutoff value. If the absorbance of test sample
is more than the cutoff value, the result shall be judged as
positive, indicating that the test sample and horse lgG are
homologous.
3419 Detennination of Molecular Size
Distribution for Group
A Meningococcal Polysaccharide
Method 1 Phnsphorus Determination Method
{arbitrary method)
The method is used for the determination of distribution
coefficient ( K o ) of bacterial capsular polysaccharide on
chromatography column and the polysaccharide recovery rate of
the sample eluted before the specified K 0 value is reached.
Reagent
(1) Mobile phase: Dissolve ll. 7 g of sodium chloride and O. 1 g
of sodium azide in water and dilute to 1000 mi. Mix well and
adjust pH to 7. O with O. 1 mol/L sodium hydroxide solution.
(2) Blue dextran 2000 solution: Weigh 20 rng of blue dextran
2000, dissolve in the mobile phase and dilute to 10
mi.
(3) Vitamin Biz solution: Weigh 10 mg of vitamin Biz, dissolve
in the mobile phase and dilute to 10 mi.
Preparation of chromatography oolmnn
Add 400 ml of the mobile phase to about 200 ml of Sepharose 4B
or CL-4B gel and stir sufficiently. Allow it to stand for about one
hour for sediment Discard the suspension containing suspended
particles in the upper layer. Repeat the above procedures for 3-5
times. Add 200 ml of the mobile phase and mix well Degas and
pack the chromatography column (l. 5 cm X 90 cm) to a height
of about 87 cm. Elute with the mobile phase at a flow rate of 15-
20 ml per hour. Two to three times of bed volume of the mobile
phase (about 500 mD is useci to equilibrate the column.
Calibration of chromatography oolmnn
Measure 1 ml of blue dextran 2000 solution, load into the
equilibrated column and elute with the mobile phase at a flow
rate of 15-20 ml per hour. Collect the eluate with a fraction
collector, 3-5 ml far each tube. Read the absorbance of each
tube at 260 nm by ultraviolet-visible spectrophotometry ( 0401 ) .
Plot a graph using elution volume ( ml ) as abscissa and
absorbance as ordinate. The elution volume of the peak at 260
nm is void volume Va.
Measure 1 ml of vitamin Biz solution. Proceed with the same
procedure starting from "load into the equilibrated column''. The
elution volume of the peak at 370 nm is bed volume V;.
Procroure Load about 1 ml of test sample solution ( containing
3-5 rng of polysaccharide antigen. If the test sample is freeze-
dried, it shall be reconstituted with the mobile phase) into the
calibrated chromatography column and elute with the mobile
phase. Collect the eluate with a fraction collector, 5 mi for each
tube. Determine the phosphorus content ( 3103) of each tube.
Plot a graph using elution volume ( ml) of each tube as abscissa
and phosphorus content as ordinate. The elution volume of the
main peak is Ve.
Calculate by the following equation:
_ Ve-Vo
K o-
V; -Vo
Where: K 0 = Distribution coefficient of the test sample
solution;
Ve = Elution volume of the test sample solution,
ml;
Va=Void volume, ml;
V; = Bed volume, mi.
Calculate the polysaccharide recovery rate of the test sample
solution eluted at a K 0 of less than O. 5 by the following
equation:
Rx (%) =1:x100
 3421 Determination of Polysaccharide Content in Haemophilus influenzae Type b Conjugate Vaccine
Where: Rx = Polysaccharide recovery rate of the test sample
solution eluted at a K o of less than O. 5, % ;
Ax= Sum of phosphorus content of eluate in tubes
eluted at a K o of less than O. 5;
At = Sum of phosphorus content of eluate in all
tu bes.
Method 2 lnstrument Method
Reagent and Preparation of chromatography column See
Method l.
Calibration of chromatography column
Mix 1 ml of blue dextran 2000 solution with O. 2 ml of
vitamin Bi 2 and load into the equilibrated column. Elute with
the mobile phase at a flow rate of 15-20 ml per hour and
detect at 206 nm. Collect the eluate with a fraction collector
and record the chromatogram. In the chromatogram, the
first peak is blue dextran 2000, and the elution volume of the
peak is the void volume Vº. The second peak is vitamin Bi2,
and the elution volume of the peak is bed volume V¡.
Procedure
Load about 1 ml of test sample ( containing 3-5 mg of
polysaccharide antigen. If the test sample is freeze-dried, it
shall be reconstituted with the mobile phase) into the
calibrated chromatography column. Elute with the mobile
phase at a flow rate of 15-20 ml per hour and detect at 206
nm. Collect the eluate with a fraction collector, and record
the chromatogram.
Calculate by the fallowing equation
K - Ve-Vo
0
-Vi-Vo
Where: K 0 = Distribution coefficient of the test sample
solution;
Ve = Elution volume of the test sample solution,
ml;
Vo=Void volume, mi;
V¡ =Bed volume, ml.
Calculate the polysaccharide recovery rate of the test sample
solution eluted at a K o of less than O. 5 by the following
equation:
Rx (%) =1: XlOO
Where: R x= Polysaccharide recovery rate of the test sample
solution eluted at a K o of less than O. 5, % ;
Ax = Chromatogram areas of the test sample
solution eluted at a K o of less than O. 5;
At =Sum of total area of chromatogram of the test
sample solution.
[Note] The column chromatography shall be carried out at
10-20ºC
3420 Determination of Molecular
Size Distribution for Typhoid Vi
Polysaccharide
The method is used far the determination of distribution
coefficient ( K 0 ) of bacteria! capsular polysaccharide in
chromatography column and the polysaccharide recovery rate
of the sample eluted befare the specified Ko value is reached.
Reagent, preparation and calibration of chromatography
column
See Method 2 in ( 3419).
Procedure
Load about 1 ml of test sample solution ( containing 3-5 mg of
polysaccharide antigen ) into the calibrated chromatography
column and elute with the mobile phase ata flow rate of 15-20
ml per hour. Collect the eluate with a fraction collector, 3-5
ml per tube. Determine the 0-acetyl content in each tube
according to the method far determination of 0-acetyl
content (3117). The elution volume with the maximum 0-
acetyl content is served as the elution volume (Ve) of
polysaccharide main peak.
Calculate by the following equation:
K _ Ve-Vo
0
-Vi-Vo
Where: K 0 = Distribution coefficient of the test sample
solution;
Ve = Elution volume of the test sample solution,
ml;
Vo=Void volume, ml;
V¡=Bed volume, ml
Calculate the polysaccharide recovery rate of the test sample
solution eluted at a K o of not more than O. 25 by the
following equation:
Rx (%) =~:X 100
Where: R x= Polysaccharide recovery rate of the test sample
solution eluted at a K o of not more than
o. 25%;
Ax = Sum of 0-acetyl content of the test sample
solution eluted in tubes at a K 0 of not more
than O. 25;
A 1 = Sum of 0-acetyl content of the test sample
solution eluted in all tubes.
[Note] The column chromatography shall be carried out at
10-20ºC
3421 Determination of Polysaccharide
Content in Haemophilus influenzae
Type b Conjugate Vaccine
The method is used far the determination of polysaccharide
content based on the following principie: soluble
polysaccharide can be dehydrated by inorganic acid to
generate furfural ( pentose) or furfural derivatives, which
can farm a coloured complex after a condensation reaction
with phenols.
Reagent
(1) O. 1% Ferric chloride hydrochloric acid solution: Weigh
accurately O. 1 g of ferric chloride ( F eCb • 6 H 2O) and put
into a clean bottle, add 100 ml of concentrated HCl, store
at 2-8ºC after dissolvation.
( 2 ) Orcinol ( 3, 5-dihydroxytoluene) ethanol· solution:
W eigh 1 g of orcinol ( C1 H 8 02 • H2 O) and put in to a 1O ml
volumetric flask, add 95 % ethanol and dilute to 10 ml
(prepared just befare use).
(3) 25 µg/ml D-ribose referrence substance solution: Weigh
l. 25 mg of D-ribose and put into a 50 ml volumetric flask,
dissolve with water and dilute to volume.
Procedure
For the blank, measure 1 ml of water, add 5 ml of O. 1%
ferric chloride hydrochloric acid solution and mix well, add
O. 4 ml of orcinol ethanol solution, mix well again. Heat in
water bath far 5 minutes and cool in ice bath. Read the
absorbance at 670 nm.
For the preparation of test sample solution, dilute a quantity
of test sample with water to a ribose concentration of not
more than 25 µg/ml. Measure 1 ml of test sample solution,
 3422 Test for Human Thrombin Activity
proceed with the same procedure starting from "add 5 ml of
O. 1 % ferric chloride hydrochloric acid solution".
Measure O. 1 ml, O. 2 ml, O. 4 ml, O. 6 ml, O. 8 ml and l. O
ml of 25 µg/ml D-ribose standard solution, put into 10 ml
test tubes respectively. Add O. 9 ml, O. 8 mi, O. 6 ml, O. 4
ml, O. 2 ml and O ml of water, respectively. Proceed with
the same procedure starting from "add 5 ml of O. 1 % ferric
chloride hydrochloric acid solution".
Calculation
Plot a linear regression curve with the absorbance of D-ribose
standards to their concentrations to obtain the linear
regression equation. Calculate ribose content of the test
sample solution by inserting its absorbance into the linear
regression equation.
Polysaccharide content of the test sample ( µg/ml) =
aXn/0.41
Where: a = Ribose content of the test sample solution,
µg/ml;
n = Dilution factor of the test sample.
3422 Test for Human Thrombin
Activity
The method is based on the principle that thrombin can
coagulate human fibrinogen. The thrombin activity of test
sample is determined by the formation of coagulum after the
test sample is mixed with human fibrinogen.
Reagent
( 1) O. 5% Fibrinogen solution: Dilute the reconstituted
freeze-dried human fibrinogen solution with physiological
saline to a concentration of 5 mg per ml.
( 2) Human thrombin solution: Dilute the reconstituted
freeze-dried human thrombin with physiological saline to a
concentration of O. 5 IU per ml.
Procedure
To O. 2 ml of test sample, add O. 2 ml of O. 5 % fibrinogen
solution, allow to stand at 37°C for 24 hours. Observe at
least 2 times whether coagulum or fibrin is farmed during the
observation period. Negative and positive control tests shall
be perfarmed in parallel.
(1) Negative control: Repeat the above-mentioned procedure
using O. 2 ml of physiological saline instead of test sample.
(2) Positive control: Repeat the above-mentioned procedure
using O. 2 ml of O. 5 IU/ ml human thrombin solution instead
of test sample.
Result evaluation
The test is valid if coagulum or fibrin is farmed in the
positive control but not in negative control. No coagulum or
fibrin shall be observed visually in the test sample.
[Note]
If the test sample contains heparin, a quantity of protamine
sulfate (10 µg far 1 IU of heparin) shall be added to neutralize
the heparin befare test
3423 Test for Activated Coagulation
Factor Activity
The method is based on the principie that, in the presence of
cephalin, activated coagulation factor can coagulate platelet-
poor human plasma. The presence or absence of activated
coagulation factor in test sample is determined by the
coagulation time after the test sample is mixed with platelet-
poor human plasma and cephalin.
Reagent
( 1) Platelet-poor human plasma: Collect human blood
aseptically and mix well with 3. 8% sodium citrate as an
anticoagulant at a volume ratio of 9 : l. Centrifuge at 1500
r/min, 4 ºC far 30 minutes. Collect the upper two-third of
plasma with a plastic syringe and centrifuge at 3500 r/min,
4 ºC for 30 minutes. Collect the upper two-third of plasma
and fill into plastic tubes, 3 ml per tube. Store at -40ºC.
(2) Tris buffer solution (pH 7. 5): Dissolve 7. 27 g of Tris
and 5. 27 g of sodium chloride in water and dilute to 1000 ml.
Adjust pH to 7. 5 with hydrochloric acid.
(3) Cephalin suspension: Reconstitute freeze-dried cephalin
with water and dilute with a quantity of physiological saline
to a concentration so as to make the blank coagulation time
between 200 and 300 seconds.
(4) O. 025 mol/L Calcium chloride solution: Dissolve 147 g
of calcium chloride (CaClz• 2H 2 0) in 1000 ml of water to
prepare 1 mol/L calcium chloride stock solution. Dilute the
stock solution 40-fald with water just befare use.
( 5 ) Protamine sulfate solution: Dissolve a quantity of
protamine sulfate in Tris buffer solution (pH7. 5) and dilute
to a suitable concentration.
Preparation of test sample solution
Add a quantity of protamine sulfate into the reconstituted
test sample according to the heparin content determined by
the method in ( 3424 ) to neutralize the heparin ( 1 IU of
heparin shall be neutralized with 10 µg of protamine sulfate),
and dilute the mixture 10-fald and 100-fold with Tris buffer
solution (pH7. 5).
Procedure
Mix O. 1 ml of platelet-poor human plasma with O. 1 ml of
cephalin suspension and allow the mixture to stand at 37°C
far one minute. Add O. 1 ml of test sample (dilute 10-fold or
100-fald) and O. 1 ml of O. 025 mol/L calcium chloride
solution preheated to 37°C. Record the coagulation time. A
blank control test is perfarmed by the above-mentioned
procedure using O. 1 ml of Tris buffer solution ( pH7. 5)
instead of test sample.
Result evaluation
The test is valid if the coagulation time of blank control is not
less than 200 seconds. The coagulation times of 1 : 10 and
1 : 100 test sample dilutions shall be not less than 150
seconds.
[Note]
( 1) The apparatus directly contacting blood or plasma shall
be made of plastic or silicified glass. The test shall be
completed within 30 minutes starting from the dilution of
test sample.
(2) Each dilution of the test sample is tested in duplicate.
3424 Determination of Heparin Content
(Coagula tion Method)
The heparin content in test sample is determined based on the
principie that protamine sulfate can neutralize anticoagulant
heparin and influence the coagulation time of plasma
Reagent
( 1) Platelet-poor human plasma: Collect human blood
aseptically and mix well with 3. 8% sodium citrate
anticoagulant at a volume ratio of 9 : l. Centrifuge at 1500
r/min, 4 ºC far 30 minutes. Collect the upper two-third of
the plasma with a plastic syringe and centrifuge at 3500 r/min,
4 ºC for 30 minutes. Collect the upper two -third of the plasma
and fill into plastic tubes, 3 ml per tube. Store at -40ºC.
 3425
(2) Tris buffer solution, pH 7. 5: Dissolve 7. 27 g of Tris
and 5. 27 g of sodium chloride in water and dilute to 1000 ml.
Adjust pH to 7. 5 with hydrochloric acid.
(3) Cephalin suspension: Reconstitute freeze-dried cephalin
with water and dilute with a quantity of physiological saline
to a concentration so as to make the blank coagulation time
between 200 and 300 seconds.
(4) O. 025 mol/L Calcium chloride solution: Dissolve 147 g
of calcium chloride ( CaClz • 2H2 0) in 1000 ml of water to
prepare a 1 mol/L calcium chloride stock solution. Dilute the
stock solution 40-fold with water just before use.
( 5 ) Protamine sulfate solution: Dissolve a quantity of
protamine sulfate in Tris buffer solution (pH7. 5) and dilute
to a concentration of 1-20 mg per ml.
Preparation of test sample solution
Reconstitute the test sample according to the labeled volume.
Transfer O. 5 ml of the reconstituted sample to each of plastic
tubes containing 10 µl of protamine sulfate solution at
different concentrations respectively and mix well.
Procedure
Add O. 1 ml of cephalin suspension into a plastic tube
containing O. 1 ml of platelet-poor human plasma and mix
well. Allow the tube to stand at 37°C for one minute. Add
O. 1 ml of test sample solution and O. 1 ml of O. 025 mol/L
calcium chloride solution preheated to 37°C. Record the
coagulation time. A blank control test is performed by the
above-mentioned procedure using O. 1 ml of Tris buffer
solution (pH7. 5) instead of test sample. The test is valid if
the coagulation time of blank control is not less than 200
seconds. The protamine sulfate content in the tube with the
shortest coagulation time shall be selected for the neutralization
of heparin content in O. 5 rnl of the test sample. On this basis, 1
IU heparin can be neutralized by 10 µg of protarnine sulfate. For
example, if the test sample tube with the shortest
coagulation time contains 30 µg of protamine sulfate, 3 IU of
heparin in O. 5 ml of test sample is then neutralized. So the
heparin content in 1 ml of test sample shall be 6 IU.
[Note]
( 1) The apparatus directly contacting blood or plasma shall
be made of plastic or siliconized glass.
(2) Carry out the test according to the instructions when a
fully-automatic coagulation analyzer is used.
3425 Test for Anti-A and Anti-B
Hemagglutinins
Cindirect Anti-human Globulin Test)
The anti-A and anti-B hemagglutinins in test sample are
determined by indirect anti-human globulin test ( Coombs test).
Reagent
( 1) Erythrocyte suspension: Mix three samples of each group
A, B and RhD-positive group O erythrocytes separately. Wash
3 times with physiological saline. After the last washing,
centrifuge at 2000 r/min for 5 minutes. Measure a quantity of
the deposit and prepare into a 5 % ( rnl/ rnl ) erythrocyte
suspension with physiological saline. The erythrocyte shall be
used within one week after blood collection
( 2) Anti-human globulin serum: Polyvalent antiserum
against human globulin shall be calibrated before use and
diluted to an optima! dilution which is determined according
to the instruction provided by the manufacturer or by the
method described in the Note.
Procedure
Make a quantity of test sample a series of 2-fold dilutions.
Test for Anti-A and Anti-B Hernagglutinins
Set up two rows of small test tubes (75mmX12 mm) for
the test sample of each dilution. Add O. 2 ml of test sample
to each tube. Add O. 2 ml of 5 % group A erythrocyte
suspension to each tu be of the first row and O. 2 ml of 5 %
group B erythrocyte suspension to each tube of the second
row, mix well, allow to stand in 37°C water bath for 30
minutes. Wash the erythrocyte 3 times with a quantity of
physiological saline each by centrifugation at 1000 r/min for
one minute. Add O. 2 ml of anti-human globulin serum into
each tube, mix well and centrifuge at 1000 r/min for one
minute. Observe the results visually. At the same time,
negative, positive and erythrocyte control tests shall be
performed.
Negative control: Add O. 2 ml of 5% erythrocyte suspensions
of groups A and group B, in duplicate, into O. 2 ml of group
AB human serum separately and mix well. Carry out the
same procedure as that for test sample starting from "allow
to stand in 37°C water bath for 30 minutes".
Positive control: Add O. 2 ml of 5 % RhD positive group O
erythrocyte suspension into O. 2 ml of anti-D IgG serum and
mix well. Carry out the same procedure as that for test
sample starting from "allow to stand in 37°C water bath for
30 minutes".
Erythrocyte control: Add O. 2 ml of 5 % erythrocyte
suspension of groups A and B into O. 2ml of physiological
saline separately and mix well. Carry out the same procedure
as that for test sample starting from "allow to stand in 37°C
water bath for 30 minutes".
Result evaluation
The test is valid if the results of negative and erythrocyte
controls were negative and that of positive control is not less
than "+++".
The titers of anti-A and anti-B hemagglutinins are calculated
as the highest dilution of the test sample which produce
agglutination "+", regardless of the volurnes of erythrocyte
suspension and anti-human globulin serum.
[Note]
(1) The test sample of coagulation factor VIII shall be diluted
to 4 IU/ml with physiological saline before test.
(2) Calibration of anti-human globulin serum: Carry out a
series of 2-fold dilutions for O. 2 ml of anti-human globulin
serum and anti-D serum separately with physiological saline.
Add O. 1 ml of 5 % packed erythrocytes suspension of RhD-
positive group O to each dilution of anti-D serum and mix
well. Allow the mixture to stand in 37°C water bath for 30
minutes, wash 3 times separately with physiological saline
and then prepare into a 2 % cell suspensions. Add O. 2 ml of
each dilution of sensitized erythrocytes suspension to a row of
diluted anti-human globulin solutions and mix well.
Centrifuge at 1000 r/min for one minute and read the result
Repeat the same procedure by replacing the sensitized
erythrocytes with an equal volume of un-sensitized human
erythrocytes of RhD positive group O as a negative control.
If the negative control test is valid, the highest dilution of
anti-human globulin serum corresponding to the highest
dilution of anti-D serum showing hemagglutination reaction
( +) is regarded as the optimal dilution.
(3) The criterion for the judgement of haemagglutination is
as follows:
++++: one salid clot;
+ + + : several large clots;
++: medium-sized clots and clear background;
+: small clots and turbid background;
- : no haemagglutination or hemolysis.
( 4) Negative, positive and erythrocyte control tests shall be
performed in parallel.
 3426 Determination of Human Erythrocyte Antibody
3426 Determination of Human
Erythrocyte Antibody
(Microtitre Plate Method)
The method is based on the principie that erythrocytes bind
to the corres¡xmding antibody with the occurrence of coagulation.
The potency of human erythrocyte antibody in test sample is
determined by comparing the hemagglutination reaction end
points of test sample and control.
Reagent
1 % Group O erythrocyte suspension: Mix group O blood
containing an anticoagulant, from three healthy donors or
more, and use within 7 days after blood collection. Wash the
blood 3 times with physiological saline before use. The last
washing is carried out by centrifugation at 2000 r/min for 10
minutes. Dilute the erythrocyte sediment with physiological
saline to 1 % for use.
Procedure
Make the test sample a series of 2-fold dilutions on a
V-shaped 96-well microtitre plate, with an angle of 90º at
the bottom, in duplicate in two rows, 50 µl per well. Add
50 µl of 1 % O group erythrocyte suspension into each well
and mix well by patting the microtitre plate slightly for 30
seconds. Allow the plate to stand at room temperature for 3
hours and observe the result. A negative control test is
performed by the above -mentioned procedure in parallel
using physiological saline instead of test sample.
Result evaluation
Put the microtitre plate on a white background and compare
the results of test sample wells with those of control wells. If
erythrocytes settle at the bottom of the well and form a
regular spot without sticking on the wall, the result shall be
judged as negative. However, if the wall of the well is stuck
with a layer of erythrocytes evenly or only a part of the
erythrocytes settle at the bottom and the rest are stuck on the
wall, the result shall be judged as positive. The highest
dilution of test sample showing positive result is served as
the potency of erythrocyte antibody. If the difference
between results of test sample of the same batch in the two
rows is more than one dilution, the test shall be repeated. If
the difference is one dilution, the highest dilution of test
sample showing positive result in the two rows is served as
the potency of erythrocyte antibody.
3427 Determination of
Human Platelet Antibody
The method is based on the principie that platelet bind to the
corresponding antibody with the occurrence of coagulation.
The potency of human platelet antibody in test sample is
determined by comparing the hemagglutination reaction end
points of test sample and control.
Reagent
(1) 5% Ethylenediaminetetraacetic acid (EDTA) disodium
solution as an anticoagulant: Dissolve O. 365 g of disodium
hydrogen phosphate ( Na2 HP0 4 • 12H2O), O. 875 g of
potassium dihydrogen phosphate, 2. 125 g of sodium chloride
and 12. 5 g of ethylenediaminetetraacetic acid disodium
(EDTA-Na2•2H 20) in water and dilute to 250 ml.
(2) O. 33% EDTA solution: Dissolve O. 73 g of disodium
hydrogen phosphate ( Na2 HP0 4 • 12H2O), l. 75 g of
potassium dihydrogen phosphate, 4. 25 g of sodium chloride
and l. 65 g of ethylenediaminetetraacetic acid disodium
(EDTA-Na2 •2H 20)in water and dilute to 500 ml.
(3) Diluent for platelet: Mix group AB sera from more than
three donors and inactivate at 56ºC for 30 minutes. Add 50 g
of barium sulfate into each 100 ml of group AB serum and
adsorb at 37°C for one hour. Stir the mixture during
adsorption from time to time. Centrifuge the mixture at 3000
r/min for 30 minutes. Discard the precipitate and collect the
supernatant far use.
Dilute one volume of the supernatant with three volumes of
physiological saline to prepare a diluent for platelet on the
date of the test. Care shaU be taken to prevent the existence
of erythrocytes and the occurrence of hemolysis in the group
AB serum.
( 4) Preparation of platelet suspension: Collect 20 ml of
human intravenous blood and mix with 5 % EDT A solution at
a volume ratio of 9 : l. Centrifuge the mixture at 20ºC , 800
r/min far 15 minutes and collect the supernatant (plasma).
Add O. 33 % EDTA solution in to the plasma up to the
original whole blood volume and centrifuge at 20ºC, 1500
r/min for 10 minutes. Discard the supernatant. Wash the
sediment with O. 33 % EDTA solution twice further by the
above-mentioned steps and discard the supernatant. Add O. 5
ml of diluent for platelet into the sediment and mix well.
Count platelet and adjust its concentration to 2. 5X103 -3. 5 X
10 5 platelets/mm3 • (Caution: Allow the platelet suspension
to stand on the counting plate for 2-3 minutes at first and the
counting shall be completed within 10 minutes.)
Preparation of the test sample solution
Make the test sampie a series oí 2-foid diiutions with
physiological saline to a dilution of 1 : 16.
Preparation of positive control solution
Inactivate O. 5 ml of porcine plasma ( or rabbit serum)
immunized with human platelets at 60ºC for 10 minutes and
adsorb with O. 05 g of barium sulfate at 37°C for 15 minutes.
Centrifuge at 3000 r/min for 20 minutes and collect the
supernatant for use.
Negative control solution
Physiological saline and diluent far platelet.
Procedure
To O. 1 ml of test sample of each dilution, add O. 1 ml of
platelet suspension and warm at 37°C far 30 minutes. Drop
the mixture onto a counting plate, allow to stand for 2-3
minutes and count under a microscope with a magnifying
power of 20-40. Negative and positive controls shall be set up
at the same time.
(1) Positive control: To O. 1 ml of positive control solution,
add O. 1 ml of platelet suspension and proceed with the same
procedure as that for test sample.
(2) Negative control: To O. 1 ml of negative control
solution, add O. 1 ml of platelet suspension and proceed with
the same procedure as that for test sample.
Result evaluation
The test is valid if positive control shows "+ +" while
negative control shows " - ". The highest dilution of test
sample when "+" appears is served as the potency of platelet
antibody.
[Note]
(1) - : No coagulum or occasionally two to three platelets
appear in a chain array.
(2) +: Small coagula consisting of three to five platelets
with a few of free platelets appear.
( 3) + +: Large coagula consisting of more than six
platelets with few of free platelets appear.
 3503 Potency Test on Rabies Vaccine for Human Use

3500 Test f or Biological Activity/Potency

dilutions of 1: 2, 1 : 4, 1 : 8, 1 : 16 and 1 : 32 or other

3501 In vitro Test for Relative Potency five appropriate dilutions.
of Recombinant Hepatitis B Vaccine Procedure
Coat the microtiter plate with purified antibody against
(Yeast)
hepatitis A virus, 100 µl per well, allow to stand at 2-8ºC
overnight. Wash the plate and clap to dry, then block with
The HBsAg content in test sample is determined by ELISA, PBS containing 10% bovine serum, 100 µl per well, and
and its relative potency is calculated by double parallel line incubate at 37°C for one hour.
assay against a reference. Add the reference vaccine and test sample at each dilution
Reagent onto the coated microtiter plate, 100 µl per well in
(1) PBS (pH 7. 2): Dissolve 8. 850 g of sodium chloride, triplicate, and incubate at 37°C for one hour or at 2-8ºC
O. 226 g of sodium dihydrogen phosphate ( NaH2 P04 • H2 O) overnight. Wash the plate then add enzyme conjugate, 100
and l. 698 g of disodium hydrogen phosphate ( Na2 HP04 • µl per well, and incubate at 37ºC for one hour.
12H 20) in a quantity of water, adjust pH to 7. 2 and dilute Wash the plate then add colour-developing agent, 100 µl per
wi th water to 1000 ml. well, and incubate at 37°C for 10-15 minutes. Add 50 µl of
(2) Treatment solution for test sample: Mix l. 25 ml of stopping agent and read the absorbance CA).
20% diethylaminoethanol and O. 20 ml of 10% Triton X-100 Result calculation
solution with 8. 55 ml of PBS thoroughly for use. Multiply the mean A values of reference vaccine and test
(3) Diluent for test sample: Dissolve 10. O g of bovine serum sample with 1000 and record in the following table.
albumin in PBS and dilute to 1000 ml.
Preparation of reference and test sample solutions A value of reference A value of test
Dilution
Measure accurately O. 1 ml of reference and test sample, and vaccine X 1000 ( T) sampleX 1000 (S)
transfer to two test tubes containing O. 1 ml of treatment 1: 2 s5 T5
solution for test sample respectively. Lid a cover and mix 1: 4 s4 T4
well, allow to stand at 20-28ºC for 30-35 minutes. Dilute
properly the treated reference and test sample with the 1: 8 s3 T3
diluent for test sample and carry out the tests on the dilutions 1 : 16 Si T2
of 1 : 2000, 1 : 4000, 1 : 8000, 1 : 16 000 and 1 : 32 000 1 : 32 S1 T1
or other series of dilutions, in duplica te. Use the diluent for
test sample as a negative control, in duplicate. No dilution is Calculate by the following equation:
needed for positive or negative control. Antigen content in test sample=
Antigen content in reference vaccineX antilg (V /W X lg2)
Procedure
The test is carried out according to the instructions of reagent Where:
kit. The test is valid if both the mean absorbance of negative V=CT1+T2+T3+TV4+T5) -0.2 CS1+S2+
and positive controls are within the range specified in the s3+s4 +s5)
instructions of the reagent kit. Calculate the relative potency
W=O. l (T5-T1+S5-S1) +o.os (T4-T2+S4-
by <lose-response parallel line assay ( 1431 ) with the data S2)
obtained from the three tests. The geometric mean obtained In vitro relative potency = Antigen content in test sample/
from the three calculations is defined as the in vitro relative Antigen content in ref erence vaccine
potency of the test sample. Using the reference as a
standard, the test result shall be judged as qualified if the 3503 Potency Test on
geometric mean of relative potency of test sample is not less Rabies Vaccine for Human Use
than O. 5.
CNIH Method)
3502 In vitro Test for Relative The immunogenicity of rabies vaccine for human use is
Potency of Hepatitis A Vaccine, determined by the changes of antibody levels in mice
Inactivated immunized with test sample.
Reagent
The hepatitis A virus antigen content in test sample is Diluent (PBS): Dilute 75 ml of O. 9 % potassium dihydrogen
determined by ELISA using reference vaccine as a standard, phosphate solution, 425 ml of 2. 4% disodium hydrogen
based on which the relative potency of test sample is phosphate ( Na2 HP04 • 12H2 O) solution and 500 ml of
calculated. 8. 5% sodium chloride solution to 5000 ml with water.
Adjust pH to 7. 2-8. O.
Preparation of reference vaccine and test sample solutions
Dissociate the reference vaccine and test sample by Preparation of challenging virus strain CVS
appropriate methods then dilute 2-fold serially with the Resuspend the seed virus and dilute to a 10- 2 suspension.
corresponding diluent of test sample, and determine those at lnoculate i. c. O. 03 ml of the suspension into each of not less
 3504 Potency Test on Adsorbed Tetanus Vaccine
than eight mice weighing 11-13 g. Undergo the virus in mice
far two to three passages. Harvest the brain tissues of the
mice with typical rabies signs on the 4th or 5th <lay after
inoculation. Grind the brain tissue to make a 20 % suspension
with 2 % horse or calf serum solution. Centrifuge the
suspension at 1000 r/min far 10 minutes and collect the
supernatant far sterility test and virus titration using ten mice
each weighing 18-20 g. The supernatant qualified in sterility
tests shall be used far challenge.
Dilution of reference vaccine
Dilute the reference vaccine with PBS to make a serial
dilution of 1 : 25, 1 : 125 and 1 : 625, etc.
Preparation of test sample solution
Dilute the test sample 5-fald serially to the dilutions of 1 : 5,
1 : 25, 1 : 125, 1 : 625, etc.
Procedure
lnoculate i. p. O. 5 ml of each dilution of test sample and
reference vaccine into sixteen mice each weighing 12-14 g,
respectively. lnoculate again at an interval of 1 week.
Fourteen days after the first injection, inoculate i. c. each
mouse with O. 03 ml of the challenging virus containing 5-100
LD50 , which was pre-determined. At the same time, dilute
the challenging virus into faur dilutions C10°, 10- 1 , 10- 2
and 10- 3 ) far virulence titration, at least eight mice are
required far each dilution. Observe the animals daily far 14
days starting from the date of challenge, and record the
death. The mice that die or manifest typical signs of
encephalopathy on or after the 5th <lay fallowing challenge
1 11 1 • 1 1 1 •. 1 1 ••
snau oe mc1uaea m 1ne evaiuanon.
Calculate the EDso values of the test sample and reference
vaccine.
The relative potency of vaccine shall be calculated by the
fallowing equation:
P= CT/S) X CdT/ds) XD
Where: P=Potency of test sample, IU/ml
T= Reciproca! of EDso of test sample;
S =Reciproca! of ED50 of reference vaccine;
dT=A single human <lose of test sample, ml;
d s =A single human <lose of reference vaccine, ml;
D= Potency of reference vaccine, IU/ml.
[Note]
CD The vaccine shall be kept in an ice-bath during animal
immunization.
C2 ) All groups of animals shall be fed under the same
condition.
C3) More than 80 % of mice inoculated with original challenging
virus suspension Cdilution 10°) shall die.
3504 Potency Test on
Adsorbed Tetanus Vaccine
The potency of absorbed tetanus vaccine is calculated by
comparing the survival rates of mice Cor guinea pigs) ,
which have been immunized with test sample and standard
and then challenged wi th tetanus toxin.
Preparation of standard and test sample solutions
Prepare three to five dilutions of the test sample and the
standard with physiological saline ata proper ratio separately
Cthe median dilution shall protect about half of animals after
challenge).
Procedure
Inoculate s. c. O. 5 ml Cor 1 ml far guinea pig) of each
dilution of standard and test sample solutions into each of at
least fourteen mice weighing 14-16 g Cor each of at least ten
guinea pigs weighing 250-350 g) respectively. Use ten
unimmunized mice Cor five unimmunized guinea pigs) as
control. Tetanus toxin far challenge was diluted by O. 2%
gelatin phosphate buffer. Four weeks after immunization,
challenge s. c. each of immunized mouse with O. 5 ml of
tetanus toxin containing 50 LD5o Cor l. O ml containing 100
LD50 far guinea pig) , and each of the control mice with O. 5
ml of tetanus toxin containing 1 LDso Cor l. O ml containing
1 LD50 far guinea pig). Record the death of animal daily far 5
days following challenge. The result shall be calculated by
parallel line analysis according to the survival rate on the 5th
<lay after challenge. The 95 % confidence interval shall be not
greater than 50%-200% of the potency, otherwise the lower
95 % fiducial limit shall be greater than the required potency
specification of the corresponding product.
[Note]
The test is valid provided that:
C1) The lowest dilution of test sample protects more than
half of animals;
C2) The highest dilution of test sample protects less than
half of animals;
C3) The <lose-response curves of the test sample and
standard do not deviate significantly in parallelism and
linearity;
C4) Animals in control group die partially but not totally.
3505 Potency Test on
Adsorbed Diphtheria Vaccine
Method l. Toxin Challenge Method in Guinea Pig ( abitration
method)
The potency of adsorbed diphtheria vaccine is determined by
comparing the survival rates of animals, which have been
immunized with test sample and standard and then challenged
with diphtheria toxin.
Preparation of standard and test sample solutions
Prepare three to five dilutions of the test sample and the
standard with physiological saline at an equal proportion
separately, the median dilution shall protect about half of
the animals after challenge.
Procedure
lnoculate each dilution of standard and test sample solutions
in to each of at least ten guinea pigs Csame sex or half gender
each ) weighing 250-350 g respectively. Use five
unimmunized guinea pigs as control.
Four weeks after immunization, challenge s. c. each
immunized guinea pig with l. O ml of diphtheria toxin
containing 100 LDso and each of the control animals with l. O
ml of 100-fold diluted toxin mentioned above. Record the
death of animals daily far 5 days following challenge.
Calculate the potency of test sample by parallel line analysis
according to the survival rate on the 5th <lay after challenge,
using the potency of diphtheria toxoid standard as a standard.
If the 95 % confidence interval is greater than 50 %-200 % of
potency, the lower 95 % fiducial limit of the estímate of
potency shall be greater than the required potency specification of
the corresponding product
[Note]
The test is valid provided that:
C1) The lowest dilution of test sample protects more than
half of the animals;
 3506
(2) The highest dilution of test sample protects less than
half of the animals;
(3) Animals in control group die partially but not totally;
( 4) The <lose-response curves of the test sample and standard do
not deviate significantly in parallelism and linearity.
Method 2. Mouse-Vero Cell Antibody Titration
Method
The potency of adsorbed diphtheria vaccine is determined by
measuring the antitoxin levels in sera of mice immunized with
test sample and standard separately, using Vero cell method.
Reagent
(1) Appropriate medium: Just befare use, add new-born
calf serum, 3% glutamine solution, proper amounts of
penicillin and streptomycin into the medium to final
concentrations of 10%, O. 03%, 100 IU per ml and 100 IU
per ml respectively. Adjust pH to 7. 0-7. 2 with 7 % sodium
bicarbonate solution.
( 2) Calcium and magnesium ions-free buffer solution:
Dissolve 8. O g of sodium chloride, O. 2 g of potassium
chloride and l. 15 g of disodium hydrogen phosphate in water
and dilute to 1000 ml.
(3) O. 25% Trypsin solution: Dissolve 2. 5 g of trypsin and
O. 2 g of disodium ethylene diamine tetraacetate with calcium
and magnesium ions-free buffer solution and dilute to 1000
ml. Adjust pH to 7. O with 7 % sodium bicarbonate solution.
Preparation of Vero cell suspension
Incubate the Vero cell in a 150 cm2 flask. Discard the culture
medium in the upper layer when the cell monolayer is
confluent and about 80%-100% full. Add 10 ml of O. 25%
trypsin solution and digest at 37°C far several minutes.
Discard the trypsin solution and add 10 ml of medium to
disperse the cells. Count the cells and dilute to a
concentration of 2. 5X105 cells per ml with the medium.
Dilution of standard and test sample
Prepare three to five 2-fold serial dilutions of the standard
and test sample of adsorbed diphtheria vaccine with
physiological saline separately.
Procedure
( 1) Immunization and bleeding
Inoculate s. c. O. 5 ml of each dilution of standard and test
sample into each of eight same sex NIH mice weighing 10-14
g respectively. Bleed each mouse 5 weeks after injection,
separate the serum, inactivate at 56ºC far 30 minutes and
store at -20ºC.
(2) Positive control sera
Immunize a number of mice with adsorbed diphtheria vaccine
and bleed 5 weeks later. Separate the sera, inactivate at
56ºC far 30 minutes, dispense it in small tubes, lyophilize
and then store at -20ºC
(3) Determination of test dose of toxin
The concentration of toxin used to determine diphtheria
antibody titer by Vero cell method is 1/10 000 Lcd.
Dilute the toxin 2-fald serially with MEM on a 96-well
microtitre plate, 50 µl per well. Add 50 µl of diphtheria
antitoxin standard (O. 000 1 IU) to each well. Lid a cover
and allow the plate to stand at room temperature far one
hour. Add 50 µl of Vero cell suspension to each well. Lid a
cover, seal the plate with a piece of film, incubate at 37°C
in a 5% carbon dioxide incubator far 6-7 days and obsel"Ve the
result. The highest dilution that causes the death of cells
(red colour) is defined as 1/10 000 Lcd.
The amount of 1/10 000 Lcd of toxin is equivalent to
1X10- 4 Lf and is suitable far the test.
( 4) Antibody titration
The test is carried out on a 96-well microtitre plate.
Determination of Flocculation Unit of Toxoid
CD Add 50 µl of medium to each well except A11 , A1 2 and
H11 , H12. Add 100 µl of medium to wells G11 and G12.
@Add eight serum samples to be tested to wells A 1 to H 1
respectively, 50 µl per well. Carry out a 2-fald serial
dilution horizontally to wells A10 and H10 respectively.
@Dilute the diphtheria antitoxin standard to a concentration
of O. 008 IU per ml. Add 50 µl to each of wells A11 , A1 2 ,
B11 and B12 , then carry out a 2-fald serial dilution vertically
from wells B11 and B12 to wells D11 and D12 respectively.
@Add 50 µl of positive control serum to wells H11 and H 12
respectively.
@Add 50 µl of toxin (Lcd/10 000) to each well except G 11
and G12 and mix well. Lid a cover and allow the plate to
stand at room temperature far one hour.
® Collect and count the Vero cell suspension, and then
dilute to a concentration of 2. 5X10 5 cells per ml.
Cl)Allow the microtitre plate to stand at room temperature
far one hour. Add 50 µl of cell suspension immediately to
each well. Lid a cover, seal the plate with a piece of film and
incubate at 37°C in a 5% carbon dioxide incubator far 6-7
days.
@Take out the plate and record the result according to the
change of colour. Y ellow and red colours represent positive
and negative results respectively. The results can be
examined by microscopy when the change of colour is not
clear. If the cell monolayer is intact, the result shall be
judged as positive. Otherwise it shall be negative. The final
result is expressed as the exponent of 2. That is, the
exponent of highest dilution of serum showing yellow colour
is the end point. Far example, if the highest dilution is 256
( 28 ) falds, the resul t shall be recorded as 8.
The result shall be calculated by parallel line analysis. The
<lose-response curves of the test sample and standard shall
not deviate significantly in parallelism and linearity. The
95 % confidence interval should be within 50 %-200 % of
potency, otherwise the lower 95% fiducial limit shall be
greater than the required potency specification of the
corresponding product.
[Note]
The requirements far the test are as fallows:
( 1) Ali the results of wells Eu , E12 , Fn and F12 , which
represent the amount of toxin and the sensitivity of Vero
cell, shall be negative. If they are positive, both the amount
of toxin and sensitivity of cells are very low, and the test
shall be repeated.
(2) Ali the results of wells Gn and G12 and positive wells
H11 and H12 must be positive. If they are negative, the test
shall be repeated.
(3) If the amount of toxin used is correct 0/10 000 Lcd),
ali the results of wells A11 , A12 , Bn and B12 shall be
positive, and those of wells Cu, C12, D11 and D12 shall be
negative. Otherwise, the test shall be repeated.
( 4) The cell counting shall be accurate.
3506 Determination of
Flocculation Unit of Toxoid
The flocculation unit ( Lf) of toxoid is determined by the
visible reaction of toxoid with the corresponding antitoxin in
test tubes when their contents, ratio, reaction temperature
and reaction time are optimal.
Reagent
Borate buffer solution: Dissolve O. 5 g of sodium borate
(Na2B4 Ü1• lOH2 0), 4. 5 g of boric acid and 8. 5 g of sodium
chloride in 1000 ml of water. The pH of the buffer solution
 3507 Potency Test on Diphtheria Antitoxin
is 7. 0-7. 2.
Preparation of standard solution
Measure the national standard of diphtheria or tetanus
flocculating antitoxin accurately and dilute with borate buffer
solution to contain 100 Lf per ml.
Preparation of test sample solution
Dilute test sample with borate buffer solution to a suitable
limit of flocculation unit.
Procedure
Accurately measure O. 3 ml, O. 4 ml, O. 5 ml, O. 6 ml and
O. 7 ml of 100 Lf per ml standard flocculating antitoxin
( SFA ) and add into five flocculating reaction tubes
respectively. Then accurately measure 1 ml of test sample
solution and add rapidly to each of the above tubes. Mix well
and put the tubes into 45-50°C water bath. Observe
continuously the appearance of flocculating phenomenon in
each tube. Record the SFA volume and time ( kf) of the
tube in which the flocculating phenomenon appears first.
Repeat this test with another five flocculating reaction tubes.
Accurately measure the same SFA volume as that of the tube
in which flocculating phenomenon appears first in the last
test, add into one of the five tubes and put this tube in the
middle. Put the other four tubes on the two sides of this
tube, two for each. To the two tubes at one side, add SFA
volumes increasing by O. 05 ml in turn compared with that of
the tube in the middle, and, to the two tubes at the other
side, add SFA volumes decreasing by O. 05 ml in turn. Add
1 ml of the test sample into each of the five tubes, mix well
and put into 45°C water bath. Observe the appearance of
flocculating phenomenon. Record again the SFA volume and
time (kf) of the tube in which the flocculating phenomenon
appears first. Repeat the test using another five tubes.
However, the interval between the SFA volumes in the five
tubes is O. 02 ml instead. Record again the SFA volume and
time (kf) of the tube in which the flocculating phenomenon
appears first. Repeat this test 2-3 times. The identical result
obtained from two to three tests is defined as the final
determination result.
The flocculation unit of test sample shall be calculated by the
following equation:
Flocculation unit of test sample (Lf/mD =
V X n X 100
Where: V =Volume of 100 Lf/ml SFA in which flocculating
phenomenon appears at first, ml;
n = Dilution factor of test sample.
3507 Potency Test on Diphtheria
Antitoxin
(Rabbit Skin Test)
The potency (IU/mD of diphtheria antitoxin is calculated by
comparing the test sample with standard based on the
principie that toxin can be neutralized by antitoxin.
Reagent
Diluent (borate buffer solution): Dissolve 8. 5 g of sodium
chloride, 4. 5 g of boric acid and O. 5 g of borax (Na2B4 Ü7•
10H2 0) in water, dilute to 1000 ml and filer. After
sterilization, the pH of the solution shall be 7. 0-7. 4.
Preparation of standard solution
The diphtheria antitoxin standard shall be diluted to contain
1/15 IU per ml, that is, each O. 1 ml of the injecting dose
contains 1/300 IU after mixing the standard with an equal
volume of toxin. The volume of stock solution of the
antitoxin standard in one measurement shall be not less than
0.5 ml.
Preparation of test sample solution
Prepare several dilutions of the test sample to contain about
1/15 IU per mL The interval of dilutions is about 5%-10%.
Procedure
The toxin is diluted to contain 20 test doses per ml, that is,
each O. 1 ml of injecting dose contains one test dose (Lr/300)
after mixing the toxin with an equal volume of antitoxin. A
quantity of diluted antitoxin standard and different dilutions
of test sample solution are measured and transferred to test
tubes respectively. Add an equal volume of the diluted toxin
to each tube and mix well. Stopper the test tubes, hold at
37"C for one hour, and inject into rabbits immediately.
Healthy rabbits with white skin, each weighing 2-3 kg, are
used. Remove hair on the back of the rabbits by an
appropriate method one day befare the test. If inflammation
or a large number of spots are found on the skin, the rabbit
shall not be used. Inject each sample solution into two
rabbits, four sample dilutions for each rabbit at the most.
Inject i. d O. 1 ml of each dilution into the rabbit at both
sides of spine. Control test shall be carried out on at least
three different injection si tes ( front, middle and rear) for
each rabbit. Different syringes shall be used for the injection
of standard solution and test sample solution.
Result evaluation
The test rabbits shall be observed 48 and 72 hours after
injection respectively, and the sizes of reaction areas shall be
measured. The final result shall be evaluated by the reactions
observed during 48-72 hours. Generally, mild redness at the
sites of injection with control occurs during 48-72 hours and
their diameters are 10-14 mm. The highest dilution causing
the same reaction intensity as that caused by most controls is
regarded as the potency of test sample. However, the
reaction intensity caused by the test sample shall not exceed
that caused by the control.
The test shall be repeated if one of the following cases
occurs.
( 1 ) The reactions of the control do not conform to the
criteria mentioned above.
(2) The dilutions of test sample are too high or too low.
(3) The reactions are irregular.
[Note]
The toxin is provided by the NCL or prepared by the
manufacturer, but only that with a proper toxicity and has
been stored for more than 12 months shall be used. The
toxin used for testing shall be accurately calibrated for its test
<lose (Lr/300) with the antitoxin standard distributed by the
NCL, and the calibration shall be repeated once 3 months.
The toxin shall be stored at 2-8ºC, protected from light,
and toluene or other appropriate preservatives shall be added.
3508 Potency Test on Tetanus
Antitoxin
(Mouse Bioassay)
The potency (IU/mD of tetanus antitoxin can be calculated
by a comparative test on the test sample and antitoxin
standard based on the principie that toxin can be neutralized
by antitoxin.
Reagent
Borate buffer saline: Dissolve 8. 5 g of sodium chloride, 4. 5
g of boric acid and O. 5 g of borax ( Na2 B.1 01 • 10 Hz O) in
water and dilute to 1000 ml, then filter. After sterilization,
the pH of the solution shall be 7. 0-7. 2.
 3509
Preparation of standard solution
( 1) Dilution of tetanus antitoxin standard: The tetanus
antitoxin standard is diluted with borate buffer saline to
contain O. 5 IU per ml, that is, each O. 4 ml of injecting dose
shall contain 1/10 IU after mixing the standard with an equal
volume of toxin. The volume of stock solution of tetanus
antitoxin standard in one measurement shall be not less than
O. 5 ml.
( 2) Dilution of tetanus toxin: The toxin is diluted with
borate buffer saline to contain 5 test doses 0/lOL+)
per ml, that is, each O. 4 ml of injecting dose contains 1 test
dose (1/lOL +) after mixing the toxin with an equal volume
of antitoxin. The test dose 0/lOL+) of toxin used shall be
accurately calibrated against the antitoxin standard
distributed by the NCL and the calibration shall be repeated
once 3 months.
Preparation of test sample solution
Prepare several dilutions of the test sample with borate buffer
saline to contain about O. 5 IU per ml, that is, each O. 4 ml
of injecting dose contains about 1/10 IU after mixing the
antitoxin with an equal volume of toxin. The interval of
dilutions is about 5 % .
Procedure
A quantity of diluted antitoxin standard and the different
dilutions of test sample solutions shall be measured and
transferred to test tubes respectively. Add an equal volume
of the diluted toxin into each tube and mix well. Stopper the
test tubes. Bind at 37°C far one hour and inject into mice
immediately.
A portian of O. 4 ml of each mixture is injected s. c. into the
abdomen or leg inguinal of each mouse weighing 17-19 g.
Care shall be taken to avoid overflow of the injecting
contents. At least three mice shall be used far each dilution
of test sample and control respectively. Different syringes
shall be used for the injection of control and test sample. The
same syringe may be used far injecting different dilutions of
the same sample in the arder from high dilution to low
dilution. The syringe shall be washed 2-3 times with the next
dilution when changing dilution. The mice shall be observed
at least twice a <lay in the morning and afternoon. Record the
morbidity and death of mice far 5 consecutive days.
Result evaluation
All the mice in control group shall die within 72-120 hours.
The highest dilution of the test sample, which causes the
death or neurotoxic symptoms of mice in test and control
groups at the same time shall be regarded as the potency of
test sample.
The test shall be repeated if one of the fallowing cases
occurs.
(1) The dilutions of test sample are too high or too low.
(2) The mice in control group die within 72 hours or more
than 120 hours after injection.
(3) The death of mice is irregularly, or nonspecific death
occurs in more than two mice injected with the same dilution.
[Note]
The dry toxin used shall be weighed accurately and each
weighing shall be not less than 10 mg. The toxin after being
dissolved shall be used up at one time. The remaining dry
Table Parameters for the potency test of gas-gangrene antitoxins
Cl.
Type of antitoxin
perfringens
Test <lose of toxin 1/5 L+
Potency Test far Gas-gangrene Antitoxins
toxin shall be stored in a sealed vacuum container with
desiccant. The dry toxin may also be made into a liquid farm
by dissolving with physiological saline and mixing with an
equal volume of neutral glycerol ( sterilized at 116ºC far 10
minutes). Each ml of the liquid toxin shall contain at least 20
test doses. The toxin shall be stored at 2-8ºC and protected
from light.
3509 Potency Test f or
Gas-gangrene Antitoxins
(Mouse Bioassay)
The potency of gas-gangrene antitoxins is determined by
comparing the survival and death of mice injected with
different dilutions of test sample and antitoxin standard after
combination with the corresponding toxin, based on the
principle that toxin can be neutralized by antitoxin.
Reagent
Diluent: Dissolve 8. 5 g of sodium chloride, 4. 5 g of boric
acid and O. 5 g of borax ( Naz B4 Ü7 • 1OH2 O) in water and
dilute to 1000 ml, then filter. After sterilization, the pH of
the solution shall be 7. 0-7. 2.
Preparation of gas-gangrene toxin solution: The toxins shall
be provided by the NCL or prepared by the manufacturer.
The test doses of toxins used far testing shall be accurately
calibrated with the antitoxin standard distributed by the NCL
( see the table below) once 3 months. The toxins shall be
diluted with the diluent just befare use to make each ml
contain 5 test doses (20 test doses far Cl. oedematiens ).
Preparation of gas-gangrene antitoxin standard
solution
Gas-gangrene antitoxin standards ( Cl. perfringens, Cl.
oedematiens; Cl. septicum and Cl. histolyticum) shall be
provided by the NCL, stored at 2-8ºC and protected from
light. The antitoxin standards are diluted with the diluent to
make each ml contain the titers as indicated in the table
below. The volume of the stock solution of antitoxin
standards in one measurement shall be not less than O. 5 ml.
Preparation of test sample solution
Prepare several dilutions of the test sample with the diluent
to contain about 5 test doses per ml ( 20 test doses far Cl.
oedematiens). The interval of dilutions is about 5%-10%.
Procedure
Measure accurately O. 8, l. O and l. 2 ml of gas-gangrene
antitoxin standard solutions and transfer to a set of test tubes
separately. Add O. 7, O. 5 and O. 3 ml of the diluent
fallowing the arder of tubes. Measure accurately l. O ml of
each dilution of test sample and transfer to a series of test
tubes separately. Add O. 5 ml of the diluent to each tube.
The diluent may be added befare the addition of antitoxins.
Add l. O ml of gas-gangrene toxin solution (O. 5 ml far Cl.
oedematiens) to each of the above mentioned tubes and mix
well. Stopper the test tubes and bind at 20-25ºC far one
hour. Inject into mice each weighing 17-19 g immediately.
Each dilution shall be injected into faur mice. The dosages
and injection routes are indicated in the table below.
Cl. Cl. Cl.
septicum histol yticum oedematiens
L+ 1/2 L+ 1/50 L+
 29B 3510 Potency Test for Botulinum Antitoxin

continued
Cl. Cl. Cl. Cl.
Type of antitoxin
perf ringens septicum histol yticum oedematiens
Antitoxin
CIU/mD
l. o 5.0 2.5 0.2
Dilution
Toxin testing
5 5 5 20
dose/ml
Antitoxin ( mD l. o l. o l. o l. o
Mixing Toxin (ml) l. o l. o l. o 0.5

Diluent (ml) 0.5 0.5 O. 5 0.5

Dose (ml) 0.5 o. 5 O. 5 0.2

Antitoxin (IU) 1/5 1 1/2 1/50

Injection Test <lose of toxin 1 1 1 1

Animal 4 4 4 4

Ro ute l. v. l. v. l. v. l. m.

Result evaluation
The animals are observed twice a day in the morning and
afternoon. Record the morbidity and death of mice for 3
consecutive days. In antitoxin standard group, at least two
of the four animals injected with the mínimum amount (O. 8
mD of antitoxin shall die within 3 days. Compare the
numbers of death in antitoxin standard and test sample
groups and calculate the potency of test sample.
The test shall be repeated if one of the following cases
occurs.
( 1) All or none of the animals in antitoxin standard group
die within 3 days, or less than two of four mice injected with
the mínimum amount of antitoxins die, or more than two of
four mice injected with the maximum amount of antitoxins
die.
(2) All or none of the animals in test sample group die
within 3 days.
(3) The death of animals occurs so irregularly that the result
can not be evaluated.
( 4 ) Nonspecific death occurs in more than two animals
injected with the same dilution.
[Note]
( 1 ) The bacteria} strains and media used, cultural
conditions and drying methods used by the manufacturer in
toxin preparation shall be consistent with those for the
standards distributed by the NCL.
(2) The dry toxins used for test shall be weighed accurately
and each weighing shall be not less than 1O mg. The toxins
after being dissolved shall be used up at one time. The
remaining dry toxins shall be stored in sealed vacuum
containers with desiccant. The dry toxins may also be made
into a liquid form by dissolving in physiological saline and
mixing with an equal volume of neutral glycerol (sterilized at
116ºC for 10 minutes). Each ml of toxin shall contain at
least 50 test doses. The toxin shall be stored at 2-8ºC and
protected from light.
3510 Potency Test for Botulinum Antitoxin
(1\.1ouse Bioassay)
The potency of botulinum antitoxins is determined by
comparing the survival and death of mice injected with
different dilutions of test sample and antitoxin standard after
combination with the corresponding toxins, based on the
principie that toxin can be neutralized by antitoxin.
Reagent
Diluent: Dissolve O. 7 g of potassium dihydrogen phosphate,
2. 4 g of disodium hydrogen phosphate (Na2 HPQ4• l2H2Ü)
and 6. 8 g of sodium chloride in water for injection and dilute
to 1000 ml. Add 2. O g of gelatin and filter after dissolution.
The pH after sterilization shall be 6. 2-6. 8.
Preparation of botulinum antitoxin standard solution
Dissolve the botulinum antitoxin with physiological saline,
mix with an equal volume of neutral glycerol ( sterilized at
116ºC for 10 minutes) and dilute to a certain concentration.
Store at 2-8ºC and protect from light. Dilute the botulinum
antitoxin standard solution with diluent to make each ml
contain the titers as indicated in the table below just before
use. The volume of stock solution of botulinum antitoxin
standard in one measurement shall be not less than O. 5 ml.
Preparation of botulinum toxin solution
The botulinum toxins shall be provided by the NCL or
prepared by the manufacturer. The test <lose of botulinum
toxins used for testing shall be accurately calibrated every 3
months with the botulinum antitoxin standard distributed by
the NCL as indicated in the table below. The toxin solution
shall be diluted to make each ml contain 5 test doses just
befare use.
Preparation of test sample solution
Prepare several dilutions of the test sample to make each rnl
contain the titers as indicated in the table below. The interval of
dilutions is about 5%-10%.
Procedure
Measure accurately O. 8, l. O and l. 2 ml of botulinum
antitoxin standard solution and transfer to a set of test tubes
separately. Add O. 7, O. 5 and O. 3 rnl of the diluent following
the arder of tubes. Measure accurately l. O rnl of each dilution of
test sample solution, transfer into a set of test tubes separately
and add O. 5 rnl of the diluent to each tube. Add l. O rnl of
botulinum toxin solution to each of the above test tubes and mix
well. Stopper the test tubes and bind at 37°C for 45 minutes.
Inject into mice immediately. Each dilution shall be injected into
four mice each weighing 14-16 g. The dosages and routes of
 3511 Potency Test for Antivenins 1·

injections are indicated in the table below.
Result evaluation
The animals shall be observed twice a day in the morning and
afternoon. Record the morbidity and death of animals for 4
consecutive days. Compare the end point of 50 % animal
protection rate in test sample group with the end point of
50 % animal mortality rate in antitoxin standard group and
calculate the potency of antitoxin.

Table Parameters for the potency test of botulinum antitoxins

Type of antitoxin A B e D E F

Test dos e of toxin 1/5 L+ 1/10 L+ L+ L+ 1/50 L+ 1/20 L+

Antitoxin (IU/ml) l. o o. 5 5.0 5.0 o. 1 0.25

Dilution
Toxin test dose (ml) 5 5 5 5 5 5

Antitoxin (ml) l. o l. o l. o l. o l. o l. o
Mixing Toxin (ml) l. o l. o l. o l. o l. o l. o
Diluent (ml) 0.5 0.5 0.5 0.5 0.5 0.5

Dose (ml) 0.5 0.5 0.5 0.5 0.5 0.5

Antitoxin (IU) 1/5 1/10 1 1 1/50 1/20

Injection Test doses of toxin 1 1 1 1 1

Animal 4 4 4 4 4 4

Route i. p. l. p. i. p. i. p. i. p. i. p.

The test shall be repeated if one of the following cases
occurs.
(1) All or none of animals in antitoxin standard group die,
or the death occurs so irregularly that the end point of 50 %
mortality rate can not be calculated.
(2) All or none of animals in test group die, or the death
occurs so irregularly that the end point of 50 % protection
rate can not be calculated.
(3) Nonspecific death occurs in more than two animals
injected with the same dilution.
[Note]
( 1 ) The bacteria! strains and media used, cultural
conditions and drying methods used by the manufacturer in
toxin preparation shall be consistent with those for the
standards distributed by the NCL.
(2) The dry toxins used for test shall be weighed accurately
and each weighing shall be not less than 1O mg. The toxins
after being dissolved shall be used up at one time. The
remaining dry toxins shall be stored in sealed vacuum
containers with desiccant. The dry toxins may also be made
into a liquid form by dissolving in physiological saline and
mixing with an equal volume of neutral glycerol (sterilized at
116ºC for 10 minutes). Each ml of toxin shall contain at
least 20 test doses. The toxin shall be stored at 2-SºC and
protected from light.
3511 Potency Test for Antivenins
(l\1ouse Bioassay)
The potency of snake antivenins is determined by comparing
the time and number of death of animals injected with
different dilutions of test sample and antivenin standard/
reference after combination with the corresponding snake
venoms, based on the principie that snake venoms can be
neutralized by snake antivenins.
Reagent
Diluent: Dissolve 8. 5 g of sodium chloride, 4. 5 g of boric
acid and O. 5 g of borax ( Naz B4 01 • 1OHz O) in water for
injection, dilute to 1000 ml and filter. The pH of the
solution after sterilization shall be 7. 0-7. 2.
Preparation of snake antivenin standard solution
Dilute snake antivenin standard/reference to make each ml
contain 5 U (Bungarus multicinctus or Agkistrodon halys
antivenin), 5 IU (Naja naja (atra) antivenin) or 10 U
(Agkistrodon acutus antivenin). That is, each O. 4 ml of
injecting dose contains 1 U or 2 U Cor !U) of antivenin after
the standard/reference is mixed with 5 test doses of the
corresponding venom.
Preparation of snake venom solution
The test <lose of snake venom shall be accurately calibrated
by the corresponding snake antivenin standard/reference,
which is distributed by the NCL ( 1 L + for Agkistrodon
halys, Naja naja (atra) and Bungarus multicinctus and
2 L + for Agkistrodon acutus). Dilute the snake venom with
a volume of not more than O. 8 ml containing 5 test doses.
That is, each O. 4 ml of injecting <lose contains one test <lose
of venom after mixing the snake venom with antivenin and
adding diluent to a total volume of 2 ml.
Preparation of test sample solution
Prepare several dilutions of the test sample to make each l. O
ml contain about 5 U ( or IU) of Agkistrodon halys, Naja
(naja) atra or Bungarus multicinctus antivenin, or about
10 U of Agkistrodin acutus antivenin. The interval of
dilutions shall be 5%-10%.
Procedure
Transfer l. O ml of each dilution of test sample to a series of
small test tubes separately. Use 1 ml and l. 2 ml of snake
antivenin standard solutions as controlsCD and @respectively.
To each of above antivenin tubes, add 5 test doses of venom
solution corresponding to the test sample and make up to 2
ml with the diluent, that is, each O. 4 ml of test sample for
injection contains 1 or 2 test doses. Mix well and stopper the
tubes. Bind at 37°C for 45 minutes and inject to mice
immediately.
Inject i. p. O. 4 ml of each dilution of test sample, control
CD and control @ into four mice each weighing 18-20 g
 3512 Potency Test for Rabies Immunoglobulin
separately.
Result evaluation
Observe the test mice once a day for 48-72 hours and record
the morbidity and death. For the test to be valid, not less
than 50% of mice in control group CDshall die, and the mice
in control group C2) shall die later or die less than those in
control group CD, or even no death. The highest dilution of
the test sample which cause the death of mice at the same
time and in the same number as those in control group CD
shall be regarded as the potency of test sample.
The test shall be repeated if the test animals fail to meet the
above-mentioned requirements.
[Note]
(1) The mice shall be injected with an accurate dosage and in
a right injecting site. Meanwhile, the overflow of injecting
contents shall be avoided.
(2) The dry toxins used for test shall be weighed accurately
and each weighing shall be not less than 5 mg. The toxins
after being dissolved shall be stored at 2-8ºC and used up
within 3 days. The remaining dry toxins shall be stored in
sealed vacuum containers with desiccant. They may also be
prepared into a liquid form and mixed each with an equal
volume of neutral glycerol ( sterilized at 116ºC for 10
minutes). One ml of the mixture shall contain at least 50
test doses. The venoms shall be stored at 2-8ºC and
protected form light.
3512 Potency Test for Rabies
Immunoglobulin
Method l. Mouse Neutralization Test
(Arbitration Method)
The potency of rabies immunoglobulin is determined by
comparing the survival and death of mice at specified time,
injected i. c. with different dilutions of test sample and the
standard of rabies immunoglobulin after combination with
rabies virus suspension, based on the principle that rabies
virus can be neutralized by rabies immunoglobulin.
Reagent
( 1 ) Phosphate buffer solution ( PBS) : W eigh O. 24 g of
potassium dihydrogen phosphate, l. 44 g of disodium
hydrogen phosphate ( Na2 HP04 • 12H2 O) and 8. O g of
sodium chloride, dissolve with water and dilute to 1000 ml.
Adjust the pH to 7. 2-8. O with sodium hydroxide.
(2) Phosphate buffer solution containing 2% newborn calf
serum: Add 2 ml of inactivated newborn calf serum to 98 ml
of PBS just befare use.
(3) Phosphate buffer solution containing 20% newborn calf
serum: Add 20 ml of inactivated newborn calf serum into
80 ml of PBS just befare use.
Preparation of virus suspension for neutralization
(1) Preparation of virus suspension
Prepare a 10- 2 virus suspension of CVS strain ( generally
freeze-dried) according to the method described in section
(2) of preparation of virus suspension for neutralization.
Inoculate mice i. c. each weighing 10-12 g with O. 03 ml of
the suspension. Prepare a 10- 2 suspension with the brains of
mice showing signs of illness. Inoculate mice i. c. with the
suspension and undergo for two to three passages in general.
The brains of mice showing signs of illness and paralysis on
the 5th day shall be ground to prepare a 20 % suspension
with skimmed milk. Dispense O. 5 ml of the suspension into
each container, seal under vacuum after lyophilization to
prepare freeze-dried virus for neutralization and store at
-30ºC for use. Alternatively, grind the brain tissues of
mice showing signs of illness and paralysis on the 5th day to
prepare a 10- 1 suspension with phosphate buffer solution
containing 20 % newborn calf serum. After centrifugation at
2000 r/min for 20 minutes, collect the supernatant, mix
well, dispense the mixture into small tubes and store at
-70ºC for use.
( 2) Preliminary determination of virulence for virus suspension
CDPreliminary determination of freeze-dried virus
To the freeze-dried virus containing 20 % mouse brain
suspension, add l. O ml of phosphate buffer solution
containing 2% newborn calf serum and mix well. Then, add
4. O ml of phosphate buffer solution containing 2 % newborn
calf serum and mix well. After centrifugation at 1500 r/min
for 10 minutes, add an equal volume of phosphate buffer
solution containing 2 % newborn calf serum to the
supernatant to prepare a 10- 2 suspension and mix well.
Then, make 10-fold serial dilutions of 10- 3 , 10- 4 , 10-s
and 10- 6 • Transfer O. 5 ml of above dilutions ( from 10- 2 to
10- 6 ) to five small tubes respectively. Add O. 5 ml of
phosphate buffer solution containing 2 % newborn calf serum
to each tube and incubate in 37°C water bath for one hour.
Thirty mice each weighing 10-12 g are divided into five
groups and injected i. c. with O. 03 ml of the incubated ( at
37°C) virus suspensions of 10- 6 , 10-s , 10- 4 , 10- 3 and
10- 2 respectively. Each dilution shall be given to six mice.
Observe the mice daily for 14 days. The deaths within 4 days
after inoculation shall be considered as nonspecific. LDso
shall be calculated statistically based on the mice dying from
the disease beyond 5 days after inoculation.
CZ)Preliminary determination of virus suspension stored at
-70ºC
Thaw the frozen virus suspension containing 10 % of mouse
brain to obtain a 10- 1 suspension. Prepare 10-fold serial
dilutions from 10- 2 to 10- 1 • Transfer O. 5 ml of each of
10- 1 , 10- 6 , 10- 5 , 10- 4 and 10- 3 dilutions to five small
test tubes, separately. Add O. 5 ml of phosphate buffer
solution containing 2 % newborn calf serum to each tube and
incubate in 37°C water bath for one hour. Thirty mice each
weighing 10-12 g are divided into five groups and injected
i. c. with O. 03 ml of the incubated ( at 37°C) virus
suspensions of 10- 7 , 10- 6 , 10-s , 10- 4 and 10- 3
respectively. Each dilution shall be given to six mice.
Observe the mice daily for 14 days. The deaths within 4 days
after inoculation shall be considered as nonspecific. LDso
shall be calculated statistically based on the mice dying from
the disease beyond 5 days af ter inoculation.
(3) Preparation of virus suspension used for neutralization
The virus dilution of 100 LDso estimated in preliminary
determination shall be used as the dilution of virus
suspension for neutralization test in mice. After incubation in
37°C water bath for one hour, the suspension shall contain
32-320 LDso of virus.
Preparation of rabies immunoglobulin standard
solution
Immunoglobulin standard shall be provided by the NCL. The
working standard of immunoglobulin used by the
manufacturer shall be accredited by the NCL. Prepare the
standard solution following the instructions.
Preparation of test sample solution
Dilute the test sample 2-fold serially with phosphate buffer
solution containing 2% newborn calf serum. Dilutions from
1 : 800 to 1 : 102 400 may be generally adopted. But on the
basis of the actual titer of test sample, the lowest dilution
factor may be appropriately decreased or increased. If the
above eight dilutions are adopted, first add 2. 7 ml of diluent
 3512
to O. 3 ml of test sample to prepare a dilution of 1 : 10. Then,
add 3. 5 ml of diluent to O. 5 ml of the 1 : 10 diluted sample to
prepare a dilution of 1 : 80 and add 2. 7 ml of diluent to
O. 3 ml of 1 : 80 diluted sample to prepare test sample
solution (1) at a dilution of 1 : 800. After that, test sample
solutions (2) to (8), at dilutions of 1: 1600, 1: 3200,
1 : 6400, 1 : 12 800, 1 : 25 600 and 1 : 102 400
respectively, shall be prepared by a 2-fold serial dilution
with the addition of O. 5 ml of dilution solution to O. 5 ml of
diluted samples starting from solution (1).
Procedure
Add O. 5 ml of test sample solutions ( 1) to ( 8) into eight
small test tubes separately. Add O. 5 ml of standard of the
eight dilutions into another eight small test tubes separately.
Add O. 5 ml of virus suspension for neutralization test into
each the sixteen tubes, incubate in 37°C water bath for one
hour and use for injection in mice. In addition, the mice of
the same body weight are used to determine the actual LDso
of the virus suspension for neutralization test. The method is
as follows: Use virus suspension for neutralization test as the
original stock to prepare dilutions of 100, 10- 1 , 10- 2 and
10- 3 • Transfer O. 5 ml of each of the above four dilutions to
four small test tubes, add O. 5 ml of phosphate buffer
solution containing 2 % newborn calf serum into each tube
and incubate in 37°C water bath for one hour as the
neutralization virus control. lnoculate i. c. O. 03 ml of the
four dilutions of neutralized test sample, the standard of
immunoglobulin and virus suspension control to each mouse
weighing 10-12 g respectively. Each dilution shall be injected
into six mice. The inoculation of test sample and standard
shall be carried out in an order from low to high dilutions, and
the inoculation of virus control shall be carried out from high to
low dilutions.
Observe the mice for 14 days and record the morbidity and
., 1 r .1 • 1 ·1 rr1 1 .1 • .1 • A 1 r,
deatn ot tne m1ce dauy. 1 ne aeatns wnnm ':!: aays aner
inoculation shall be considered as nonspecific.
Potency CIU / mD of test sample = CB i / B 2 ) X D
Where: B1 = Reciprocal EDso of test sample;
B2 = Reciprocal EDso of standard
of immunoglobulin;
D lnternational unit of standard of
immunoglobulin, IU/ml.
Method 2. Rapid Fluorescent Focos Inhibition Test ( RFFIT)
Method
The potency of rabies immunoglobulin is determined by mixing
different dilutions of test sample and standard of rabies
immunoglobulin with rabies virus suspension, respectively, then
infecting sensitive cells with the mixture, staining with
fluorescein isothiocyanate (FITC) labeled rabies virus antibody
and observing the decrease in fluorescent foci of cells at specified
time, based on the principle that rabies virus can be neutralized
by rabies immunoglobulin.
Reagents
(1) DMEM cell medium containing 5% newborn calf serum:
Add antibiotics and glutamine to DMEM containing 5 %
newborn calf serum to final concentrations of 100 U/ml and
O. 03% respectively just befare use, and adjust the pH to
7. 6 with sodium bicarbonate solution according to the
instructions of DMEM
(2) DMEM cell medium containing 10% newborn calf
serum: Add antibiotics and glutamine in to DMEM containing
10% newborn calf serum to final concentrations of 100 U/ml
and O. 03% respectively just befare use, and adjust the pH
to 7. 6 with sodium bicarbonate solution according to the
instructions of DMEM
(3) Phosphate buffer solution ( PBS): Weigh O. 24 g of
Potency Test for Rabies lmmunoglobulin 3111
potassium dihydrogen phosphate, l. 44 g of disodium
hydrogen phosphate (Na2HP04•12H2Ü) and 8 g of sodium
chloride, dissolve with water and dilute to 1000 ml. Adjust
the pH to 7. 2 with sodium hydroxide. Prepare the solution
just before use.
( 4) 80 % Cold acetone: Measure 80 ml of acetone, add 20
ml of O. 1 mol/L PBS (pH7. 6), mix well and seal up the
container, store at 4 ºC.
(5) 80% Glycerol: Measure 80 ml of glycerol, add 20 ml of
water, mix well and capping the container, store at 4 ºC.
(6) O. 25% Trypsin-EDTA
(7) FITC lableled anti-rabies virus nucleoprotein antibody:
Dilute to working concentration according to the instructions.
Preparation of virus for neutrali7.ation
( 1) Preparation of virus suspension
Dilute CVS-11 strain ( generally freeze-dried) properly and
inoculate into BSR cells growing well at a MOI of O. 1,
culture at 37°C in a 5 % carbon dioxide incubator for one <lay
then at 34 ºC for 2 days. Collect the culture supernatant and
centrifuge at 4ºC, 4000 r/min for 10 minutes to remove the
cell debris. Collect the supernatant, add 10 % newborn calf
serum, mix well and dispense into small tubes. Store at
- 7OºC below for use.
Preliminary titration of virus suspension: Thaw one
container of virus suspension in frozen rapidly by running
water and dilute 5-fold serially on a 24-well microtitre plate
starting from 1 : 5 dilution. Add 100 µl of virus suspension
into 400 µl of DMEM containing inactivated 10 % newborn
calf serum and mix well. Transfer 50 µl of each dilution to a
96-well microtitre plate, in duplicate. Add 50 µ1 of BSR cell
suspension at a concentration of 5X 106 cells /ml into each
well, and incubate at 37°C in a 5% carbon dioxide incubator
for 24 hours. Discard the supernatant and wash the plate
once with PBS. Then, add 50 111 of 80 % cold acetone into
each well, allow to fix at 4ºC for 30 minutes, or at -30ºC for
10 minutes. Discard the acetone and dry the plate by
volatilization. Add 50 µl of FITC-labeled anti-rabies virus
nucleoprotein antibody at the working concentration into each
well and incubate at 37°C for 30 minutes. Wash the plate 3
times with PBS and dry. Add 50 µl of 80 % glycerol into each
well and count the fluorescent focus in each well under
fluorescence microscope. Select a well in which the
fluorescent focus count is less than 30, record the
fluorescent focus counts in the well and in four neighboring
wells and calculate the mean value. The titer of virus
suspension is calculated by the following equation:
Titer of virus suspension (FFU/ml) = (Mean fluorescent
focus count in the well containing virus suspension at the
highest dilution X 5 + Mean fluorescent focus count in the
neighboring wells containing virus suspension at lower
dilutions) / 2 X The lowest dilution factor of virus
suspension in the five wells X 20
(2) Preparation of virus suspension used for neutralization
Take one container of virus suspension and proceed the same
procedures as those for preliminary titration of virus
suspension. Observe and caculate the ratio of fluorescent
focus in each well under fluorescent microscope. The dilution
of virus suspension infecting 80 %-95 % of cells is used for
neutralization test.
Thaw one container of virus suspension in frozen by running
water, dilute with DMEM containing 5 % newborn calf
serum to the virus dilution for neutralization test and keep in
ice bath for use.
Preparation of rabies immunoglobulin standard
solution
Immunoglobulin standard shall be provided by the NCL. The
 3513 Potency Test for Diphtheria Antitoxin in Human Immunoglobulin
working standard of irnmunoglobulin used by the manufacturer
shall be accredited by the NCL Prepare the standard solution
following the instructions. Dilute the rabies immunoglobulin
standard 3-fold serially with DMEM containing 10 %
inactivated newbom calf serum, i. e. add DMEM onto a 96-
well microtitre plate, 100 µl per well, then add 50 µl of test
sample into each well to prepare a 1 : 3 dilution. Mix well
and add 50 µl of the mixture into the DEME in next well to
prepare a 1 : 9 dilution, and so on until a proper dilution is
reached.
Preparation of test sample solution
Dilute the test sample (serum sample shall be pre-inactivated
at 56ºC for 30 minutes) 3-fold serially with DMEM
containing 10% inactivated newbom calf serum, i. e. add
DMEM onto a 96-well microtitre plate, 100 µl per well,
then add 50 µl of test sample into each well to prepare a 1 : 3
dilution. Mix well and add 50 µl of the mixture into the
DEME in next well to prepare a 1 : 9 dilution, and so on
until a proper dilution is reached. Discard 50 µl of the
mixture from the last well.
Procedure
Add 50 µl of virus suspension for neutralization to each well
of dilution of test sample solution and standard solution.
Meanwhile, set upa well containing 100 µl DMEM as blank
control and a well containing 50 µl of virus suspension for
neutralization as control for virus neutralization. Mix well
and incubate at 37°C for one hour for neutralization. Add 50
µl of BSR cell suspension containing 5 X 10 6 cells/ml) into
each well and incubate at 37°C in a 5% carbon dioxide
incubator for 24 hours.
Discard the DMEM, add 100 µl of PBS into each well to
wash the plate. Discard the PBS, add 50 µl of 80 % acetone
pre-cooled to 4 ºC in to each well, allow to fix at 4ºC for 30
minutes or at -30ºC for 10 minutes. Discard the acetone and
dry the plate by volatilization. Add 50 µl of FITC-labeled
anti-rabies virus nucleoprotein antibody at the working
concentration into each well and incubate at 37°C for 30
minutes. Discard the supematant, wash the plate 2-3 times
with PBS, dry and add 50 µl of 80 % glycerol into each well
then observe under fluorescent microscope. Calculate by the
following equation:
lg EDso of standard= lg (1/A) - (0. 5-B) /
(C-B) Xlg n1
Where: A = Dilution of standard with less than 50% of
fluorescent foci;
B Percentage of fluorescent foci in the wells
containing standard with less than 50 % of
fluorescent foci;
C Percentage of fluorescent foci in the wells
containing standard with more than 50 % of
fluorescent foci;
n1 = Dilution factor of standard.
lg ED5o of test sample =
lg O/E) - (O. 5-F) / CG-F) Xlg nz
Where: E = Dilution of test sample with less than 50% of
fluorescent foci;
F = Percentage of fluorescent foci in the wells
containing test sample with less than 50 % of
fluorescent foci;
G Percentage of fluorescent foci in the wells
containing test sample with more than 50 %
of fluorescent foci;
nz = Dilution factor of test sample.
Potency (IU/mD of test sample = lo<I-Kl XL
Where: ] = lg ED5o of standard;
K = lg ED5o of test sample;
L = Potency of standard, IU / ml.
[Note]
(1) The titer of virus suspension for neutralization shall be
not less than 106 FFU/ ml.
(2) The virus suspension shall be diluted in ice bath as far as
possible.
( 3) The test is valid if 80 %-9 5 % of cells in virus control well
are positive in fluorescent staining, and no fluorescence
appears in cell control well.
3513 Potency Test for Diphtheria
Antitoxin in Human Immunoglobulin
The method is based on the principle that sheep erythrocytes
after treatment with aldehyde and tannic acid can adsorb
diphtheria toxoid onto their surfaces. The adsorbed toxoid
can bind the diphtheria antitoxin in test sample and cause
specific agglutination. The potency of diphtheria antitoxin of
test sample is determined by comparison of the agglutination
reaction end points.
Reagent
(1) Physiological saline containing 1% rabbit serum: Collect
rabbit blood aseptically, separa te serum and inactivate at
56ºC for 30 minutes. Mix O. 5 ml of the rabbit serum with
49. 5 ml of physiological saline thoroughly.
(2) Diphtheria antitoxin diagnostic erythrocyte suspension:
Reconstitute freeze-dried diphtheria antitoxin diagnostic
erythrocyte with physiological saline containing 1 % rabbit
serum to prepare a 5 % erythrocyte suspension.
Preparation of diphtheria antitoxin standard solution
Dilute the diphtheria antitoxin standard with physiological
saline containing 1 % rabbit serum to a concentration of O. 2
HAU per ml.
Preparation of test sample solution
Dilute the test sample 4-fold with physiological saline
containing 1 % rabbit serum.
Procedure
Dilute the test sample 2-fold serially with physiological saline
containing 1 % rabbit serum on a UV type microtitre plate
and keep 25 µl of solution in each well. Add 25 µl of
diphtheria antitoxin diagnostic erythrocyte suspension into
each well and mix thoroughly on an oscillator for 30-60
seconds. Bind at 37°C in a wet box for one hour.
Dilute diphtheria antitoxin standard solution 2-fold serially
with physiological saline containing 1 % rabbit serum on a
UV type microtitre plate and keep 25 µl of solution in each
well. Carry out the same procedure as that for test sample
starting from adding 25 µl of diphtheria antitoxin diagnostic
erythrocyte suspension into each well.
Add 25 µl of physiological saline containing 1 % rabbit serum
on a UV type microtitre plate and carry out a negative control
test by the same procedure as that for test sample starting
from adding 25 µl of diphtheria antitoxin diagnostic
erythrocyte suspension into each well.
Typical result "-" shall be observed in the negative control
wells, otherwise the test is invalid and shall be repeated.
The appearance of result "++"is served as the reaction end
point. The diphtheria antitoxin potency of test sample is
calculated by the following equation:
Diphtheria antitoxin potency (HAU/g) of test sample =
(E XD) /F
Where: E= Potency of diphtheria antitoxin standard of the
highest dilution showing the result "+ + ",
 3514 Test far Fe Function in Human lmmunoglobulin
HAU/ml;
D The highest dilution factor of test sample
= chromium chloride solution with O. 9 ml of physiological
showing the result "+ +";
F = lgG content in the test sample of human
immunoglobulin far intravenous injection,
g/ml; or the protein content of test sample of
human immunoglobulin, g/ml.
[Note]
(1) The criterion for judgment is as fallows:
" - ": Erythrocytes are centralized at the bottom of well and
appear as a dense small red point with smooth border.
"+": Most of erythrocytes are centralized at the bottom of
well, and only a few of them are scattered around the
bottom.
"++": A part of erythrocytes are agglutinated, anda loose
small red loop appears at the center of well bottom.
"+ + + ": Most of erythrocytes are agglutinated and
distributed evenly, and only a slightly red loop appears at
the center of bottom of well.
"+ + + + ": All the erythrocytes are agglutinated and
distributed evenly.
(2) The wells of microtitre plate shall be kept clean, and
the abrasion on their surfaces shall be avoided, otherwise the
erythrocytes are not easy to be deposited, and false positive
result may appear easily.
3514 Test for Fe Function in Human
lrnmunoglobulin
The method is based on the principle that the Fab fragment
of specific antibody ( immunoglobulin) can bind to the
corresponding coated antigen on human erythrocyte and
expose the binding site of complement Clq on the Fe
fragment, then activate the subsequent various components
of complement. Eventually, the membrane of erythrocyte is
attacked and disrupted, then hemoglobin is released The Fe
function of the test sample is determined by the calculation of
the f unction index ( I Fe ) of human immunoglobulin activating
complement with the dynamic curve of hemolytic reaction.
Reagent
(1) PBS: Dissolve l. 02 g of anhydrous disodium hydrogen
phosphate, O. 34 g of anhydrous sodium dihydrogen
phosphate and 8. 77 g of sodium chloride in a quantity of
water, adjust pH to 7. 2 with 1 mol/L sodium hydroxide or
hydrochloric acid solution and dilute to 1000 ml with water.
(2) Calcium and magnesium stock solution: Dissolve l. 10 g
of calcium chloride and 5. 08 g of magnesium chloride in 25
ml of water.
( 3 ) Barbital-calcium and magnesium stock solution:
Dissolve 51. 85 g of sodium chloride and 6. 37 g of barbital
sodium in 1000 ml of water. Add 3. 125 ml of calcium and
magnesium stock solution, adjust pH to 7. 3 with 1 mol/L
hydrochloric acid solution and dilute to 1250 ml with water.
Sterilize by filtration and store at 4ºC far use.
( 4) Bovine albumin-barbital buffer: Add O. 15 g of bovine
serum albumin into 20 ml of barbital stock solution, dissolve
in water and dilute to 100 ml. Prepare just before use.
(5) l. 3 mg/L Tannic acid-PBS (pH 7. 2) solution
Solution A: Dissolve 1 mg of tannic acid in 10 ml of PBS
(pH 7. 2).
Solution B: Mix O. 1 ml of solution A with 7. 5 ml of PBS
(pH 7. 2) thoroughly. Prepare just befare use.
( 6 ) 10 % Chromium chloride solution: Dissolve 5 g of
chromium chloride in 50 ml of physiological saline. The solution
may be preserved for not more than 6 months at 4ºC.
(7) 1% Chromium chloride solution: Mix O. 1 ml of 10%
saline thoroughly. Prepare just befare use.
Preparation of sensitized erythrocytes
Solution A: Pool the group O blood, containing
anticoagulant, from at least three healthy donors and wash 3
times with PBS. At the last washing, centrifuge the pooled
blood at 2000 r/min far 10 minutes to separate erythrocytes.
Suspenda quantity of the packed erythrocytes in l. 3 mg/L
tannic acid-PBS ata volume ratio of 1 : 40 and gently shake
in 37°C water bath for 30 minutes. Wash 3 times with PBS.
Then, prepare into a 2. 5 % erythrocyte suspension with PBS.
Solution B: Mix the diphtheria toxoid or epidemic mumps
virus, properly diluted with PBS, with O. 25 ml of 1%
chromium chloride solution at a volume ratio of 10 : 1 and
shake gently in 37°C water bath far 15 minutes.
Mix solutions A and B at a volume ratio of 1 : 4 and shake
gently in 37°C water bath for 30 minutes. Centrifuge and
discard the supernatant. Wash the sediment ( sensitized
erythrocytes) 3 times with PBS. Suspend the erythrocytes
with bovine albumin-barbital buffer solution and adjust to a
suitable concentration at which the absorbance of suspension
is l. 0±0. 1 at 541 nm.
Preparation of reference solution
Adjust the pH of reference to 6. 8-7. O with 1 mol/L sodium
hydroxide solution and dilute the reference to contain 40 mg
of IgG per ml with bovine albumin-barbital buffer solution.
Preparation of test sample solution
Adjust the pH of test sample to 6. 8-7. O with 1 mol/L
sodium hydroxide solution and dilute the test sample to
contain 40 mg of IgG per ml with bovine albumin-barbital
buffer solution.
Procedure
To O. 9 mi oí test sampie soiution, add O. 1 ml of sensitized
erythrocytes and mix well. Shake gently in 37°C water bath
far 30 minutes. Centrifuge and discard the supernatant.
Wash the erythrocyte sediment 3 times with 1 ml of bovine
albumin-barbital buffer. Discard 800 111 of supernatant after
the last centrifugation, add 600 111 of bovine albumin-barbital
buffer preheated to 37ºC and mix well. Two minutes later,
add 200 111 of complement prediluted to 150 CHso per ml and
mix welL Determine the starting absorbance CA.) at 541 nm
by ultraviolet-visible spectrophotometry ( 0401) immediately,
then determine the absorbance every other minute to obtain a
hemolytic reaction dynamic curve with the absorbance of test
sample at 541 nm against the time. Stop the determination
when the absorbance is above the turning point of the curve.
Repeat the above-mentioned procedure using O. 9 ml of
reference and negative control ( bovine albumin-barbital
buffer) instead of the test sample respectively. Calculate the
slopes of curves of reference, test sample and negative
control by the fallowing equation ( 1 ) , and the function
index ( I Fe ) of test sample activating complement by
equation ( 2). The slopes of curves should be no less than
60 % of national reference product activity.
S' =Sexp/A. (1)
he= [ (S ~ - S ~) / ( S: - S ~) ] X 100 % ( 2)
Where: S' =Slope of Sexp after calibration by A.;
A.= Starting absorbance of test sample, reference
or negative control at 541 nm;
Sexp = Maximum slope between three adjacent points
on hemolytic reaction dynamic curve of test sample,
reference or negative control;
I Fe = Function index of test sample activating
complement;
 3515 Potency Test for Anti-human T Lymphocyte Immunoglobulin
S~ =Slope of curve for test sample;
S; = Slope of curve for reference;
S; = Slope of curve for negative control.
3515 Potency Test for Anti-human
T Lymphocyte lmmunoglobulin
CE Rosette Formation-inhibition Test)
The method is based on the principie that anti-human T
lymphocyte immunoglobulin may bind the E-receptor of
human lymphocyte and inhibit the specific binding of sheep
erythrocytes with lymphocyte E receptor. The potency of anti-
human T lymphocyte IgG in test sample is determined
according to its inhibiting rate of binding.
Reagent
(1) Lymphocyte separating solution (Ficoll solution)
(2) Hank solution
(3) Hank solution containing 20% fetal calf serum: On the
date of test, add the fetal calf serum, inactivated at 56ºC for
30 minutes and adsorbed with sheep erythrocytes, into a
quantity of sterilized Hank solution to a final concentration of
20%. Adjust pH to 7. 2-7. 4 with O. 5 mol/L sodium
bicarbonate or hydrochoric acid solution.
( 4) 1 % Sheep erythrocyte suspension: Collect blood from
the jugular veins of sheep, add to Alserver solution and store
for not more than 2 weeks. W ash a quantity of sheep
erythrocytes 3 times with physiological saline and prepare a
1 % sheep erythrocyte suspension with Hank solution
. • · º"' n / r .
1 1 r • .1 r
conrammg ¿u 70 1eta1 can serum just betore test.
(5) Lymphocyte suspension: Mix fresh human intravenous
blood containing heparin as an anticoagulant with an equal
volume of physiological saline thoroughly, and add slowly
onto the surface of an equal volume of lymphocyte separating
solution. Centrifuge at 2000 r/min for 20 minutes. Pipette
out the lymphocyte layer and wash with a quantity of
physiological saline. Centrifuge at 1200 r/min for 10
minutes. Discard the supematant, add a quantity of Hank
solution containing 20 % fetal calf serum to the precipitate
and shake well to obtain a lymphocyte stock solution. Dilute
the stock solution 20-fold with 1 % acetic blue solution and
count the lymphocytes by microscopy. The lymphocyte
suspension is prepared by diluting the lymphocyte stock
solution to a concentration of 5 X 106 lymphocytes per ml,
according to the result of counting, with Hank solution
containing 20 % fetal calf serum.
Preparation of test sample solution
Dilute the test sample according to its potency into several
suitable concentrations with Hank solution containing 20 %
fetal calf serum.
Procedure
Add 100 µl of lymphocyte suspension into 100 µI of test
sample solution of each dilution, in duplicate. Mix well and
allow the mixture to stand in 37°C water bath for 30 minutes.
Add 100 µl of 1 % sheep erythrocyte suspension, mix well
and allow the mixture to stand at room temperature for 15
minutes. Centrifuge at 500 r/min for 5 minutes, allow to
stand overnight at 2-8ºC. Add 100 µI of O. 2% trypan-blue
solution diluted on the date of test, shake well gently and
calculate the formation rate of E rosette by microscopy. A
control test is performed by the above procedure using 100 µl
of Hank solution containing 20 % fetal calf serum instead of
test sample solution. Calculate the E-rosette inhibition rate.
The highest dilution factor of test sample, in which the
E-rosette inhibition rate is more than 25%, is served as the
E-rosette inhibiting potency.
3516 Potency Test for Anti-human
T Lymphocyte Immunoglobulin
(Lymphocytotoxicity Test)
The method is based on the principie that anti-human T
lymphocyte immunoglobulin may bind to human lymphocytes
and destroy them in the presence of complement. The
potency of anti-human T lymphocyte immunoglobulin in the
test sample is determined according to the percentage of dead
lymphocytes.
Reagent
(1) Lymphocyte separation solution (Ficoll solution)
(2) Hank solution
(3) Hank solution containing 20% fetal calf serum: Add
fetal calf serum, inactivated at 56ºC for 30 minutes, to a
quantity of sterile Hank solution to prepare a 20% solution
on the date of test. Adjust pH to 7. 2-7. 4 with O. 5 mol/L
sodium bicarbonate solution or diluted hydrochloric acid
solution.
(4) Lymphocyte suspension: Mix fresh human intravenous
blood containing heparin asan anticoagulant thoroughly with
an equal volume of physiological saline. Add slowly onto the
surface of an equal volume of lymphocyte separating solution
and centrifuge at 2000 r/min for 20 minutes. Pipette out the
lymphocyte layer and wash with a quantity of physiological
saline. Centrifuge at 1200 r/min for 10 minutes and discard
the supernatant. Add a quantity of Hank solution containing
20 % fetal calf serum into the precipitate and shake well to
obtain a lymphocyte stock solution. Dilute the lymphocyte
stock solution 20-fold with 1 % acetic blue solution and count
the lymphocytes by microscopy. Dilute the lymphocyte stock
solution with Hank solution containing 20 % fetal calf serum
into a suspension at a concentration of 5 X 106 lymphocytes
per mi according to the result of counting.
(5) Complement: Normal rabbit serum used as complement
shall have no significant toxicity to the target cells used in the
test. The rabbit serum shall be selected prior to the test by
the following method: To O. 05 mi of lymphocyte
suspension, add O. 05 mi of 1 : 5 diluted rabbit serum and
allow the mixture to stand at 37°C for one hour. Add O. 05
ml of physiological saline containing O. 5% trypan-blue,
incubate at 37°C for 5 minutes and then count lymphocytes
by microscopy. Only the rabbit serum, in which the number
of dead cells is less than 10 % , may be used as complement.
(6) Physiological saline containing O. 5% trypan-blue
( 7 ) 2. 5 % Glutaraldehyde solution ( diluted with Hank
solution)
Preparation of test sample solution
Dilute the test sample with Hank solution containing 20 %
fetal calf serum to severa! suitable concentrations according
to its potency.
Preparation of positive control solution
Heat the porcine plasma or rabbit serum, which have been
immunized with human T lymphocytes, at 60ºC far 10
minutes and dilute 10-fold with physiological saline.
Preparation of negative control
Heat normal porcine plasma or rabbit serum at 60ºC for 10
minutes and dilute 10-fold with physiological saline.
 3518 Potency Test for Human Coagulation Factor VU:
Procedure
To O. 05 ml of test sample solution, add O. 05 ml of
lymphocyte suspension and allow the mixture to stand at
37°C for one hour. Add O. 05 ml of 1 : 5 diluted rabbit
serum, allow to stand at 37°C for 30 minutes. Add O. 05 ml
of physiological saline containing O. 5% trypan-blue, allow
to stand at 37°C for 5 minutes. Count the lymphocytes
immediately by microscopy and calculate the percentage of
dead cells. One hundred lymphocytes are counted in general.
A positive control test is carried out by the above procedure
using O. 05 ml of positive control solution instead of test
sample. A negative control test is carried out by the above
procedure using O. 05 ml of negative control solution instead
of test sample.
Result evaluation
The test is valid if the percentage of dead cells is more than
20% in positive control group and less than 10% in negative
control group. The result "+" is served as the end point for
judgment. The highest dilution factor of test sample which
shows the result of " + " is defined as lymphocytotoxic
potency.
[Note]
(1) The result is evaluated by the percentage of dead cells in
test group:
Percentage of dead cells Result
Less than 1O% (-)
(±)
10%-20%
21%-40% C+)
41%-60% e++)
61%-80% e+++)
Not less than 81 % e++++)
(2) To avoid the experimental error when the test sample is
in a large quantity or there is no enough time to observe· the
result, O. 05 ml of physiological saline containing O. 5 %
trypan-blue may be added to the test sample after the
reaction of antigen, antibody and complement. Allow the
mixture to stand at 37°C for 5 minutes and add O. 05 ml of
2. 5 % glutaraldehyde solution immediately. Then, count by
rnicroscopy at an appropriate time. Alternatively, add O. 05
ml of 2. 5 % glutaraldehyde solution to test sample and allow
the mixture to stand at room temperature for 10 minutes at
first. Then, add O. 05 ml of physiological saline containing
O. 5 % trypan-blue and allow to stand at 37°C for 5 minutes.
Count by microscopy at an appropriate time.
3517 Potency Test for Human
Coagulation Factor ll
( One-step Method)
The potency of human coagulation factor II is deterrnined by one-
step method using human coagulation factor 11 deficiency plasma
as substrate plasma.
Reagent
(1) Diluent: Dissolve 11. 75 g of barbital sodium and 14. 67
g of sodium chloride in a quantity of water, adjust pH to 7. 3
with 1 mol/L hydrochloric acid and dilute to 2000 ml with
water. Add 20 % human albumin to a final concentration of
1 % just befare use.
(2) Calcium-containing thromboplastin
(3) Coagulation factor II deficiency plasma: Human plasma
or artificial substrate plasma, in which human coagulation
factor 11 concentration is less than 1 %.
Preparation of standard solution of human coagulation factor II
Dilute the standard with coagulation factor 11 deficiency
plasma or physiological saline to a concentration of 1 IU
per ml, then make 10-fold, 20-fold, 40-fold and 80-fold
dilutions with diluent respectively, allow to stand in an ice
ha th for use.
Preparation of test sample solution
Dilute the test sample with coagulation factor II deficiency
plasma or physiological saline to a concentration of about
1 IU per ml, then make 10-fold, 20-fold or 40-fold dilution
with diluent, allow to stand in an ice bath for use.
Procedure
Mix O. 1 ml of test sample solution and O. 1 ml of coagulation
factor II deficiency human plasma thoroughly and warm in
37°C water bath for a certain time (3 minutes in general).
Add O. 2 ml of calcium-containing thromboplastin preheated
to 37°C and record the coagulation time.
Repeat the above-mentioned procedure using O. 1 ml of
standard solution of human coagulation factor 1I at various
dilutions instead of the test sample solution.
A linear regression equation is obtained by regressing the
logarithm of potency (IU / mD of human coagulation factor
11 standard solution with the logarithm of corresponding
coagulation time (second). Calculate the potency of human
coagulation factor II in test sample solution and multiply
with the dilution factor to obtain the human coagulation
factor II potency CIU/ mD of test sample.
[Note]
( 1) The correlation coefficient of linear regression shall be
not less than O. 98.
( 2 ) Each dilution of test sample shall be determined in
duplicate, and the difference between the two results shall
not exceed 10% of the mean value, otherwise the test shall
be repeated.
(3) The apparatus directly contacting the standard, test
sample and plasma shall be made of plastic or silicified glass.
( 4 ) The test is carried out with an automatic blood
coagulation analyzer following the instructions.
3518 Potency Test for Human
Coagulation Factor Vil
( One-step Method)
The potency of human coagulation factor VlI is determined by
one-step method using human coagulation factor VU:
deficiency plasma as substrate plasma.
Reagent
(1) Diluent: Dissolve 11. 75 g of barbital sodium and 14. 67
g of sodium chloride in a quantity of water, adjust pH to 7. 3
with 1 mol/L hydrochloric acid and dilute to 2000 ml with
water. Add human albumin to a final concentration of 1 %
just befare use.
(2) Calcium-containing thromboplastin
(3) Coagulation factor VJI deficiency plasma: Human plasma
or artificial substrate plasma, in which human coagulation
factor VU: concentration is less than 1%.
Preparation of standard solution of human coagulation factor -W
Dilute the standard with coagulation factor VU: deficiency
plasma or physiological saline to a concentration of 1 IU per
ml, then make 10-fold, 20-fold, 40-fold and 80-fold
dilutions with diluent respectively, allow to stand in an ice
bath for use.
Preparation of test sample solution
Dilute the test sample with coagulation factor VU: deficiency
plasma or physiological saline to a concentration of about 1
 3519 Potency Test far Human Coagulation Factor
IU per ml, then make 10-fald, 20-fald or 40-fald dilution
with diluent, allow to stand in an ice bath far use.
Procedure
Mix O. 1 ml of test sample solution and O. 1 ml of coagulation
factor VH deficiency plasma thoroughly and warm in 37°C
water bath far a certain time ( 3 minutes in general). Add
O. 2 ml of calcium-containing thromboplastin preheated to
37°C and record the coagula tion time.
Repeat the above-mentioned procedure using O. 1 ml of
standard solution of human coagulation factor VH of various
dilutions instead of the test sample solution.
A linear regression equation is obtained by regressing the
logarithm of potency CIU/ml) of human coagulation factor
VH standard solution with the logarithm of corresponding
coagulation time ( second). Calculate the potency of human
coagulation factor VH in test sample solution and multiply
with the dilution factor to obtain the human coagulation
factor Vil potency CIU/ mD of test sample.
[Note]
(1) The correlation coefficient of linear regression shall be
not less than O. 98.
(2) Each dilution of test sample shall be determined in
duplicate, and the difference between the two results shall
not exceed 10 % of the mean value, otherwise the test shall
be repeated.
(3) The apparatus directly contacting the standard, test
sample and plasma shall be made of plastic or silicified glass.
( 4 ) The test is carried out with an automatic blood
coagulation analyzer fallowing the instructions.
3519 Potency Test for Human
Coagulation Factor 1X
( One-step Method)
The potency of human coagulation factor IX is determined by
one-step method using human coagulation factor IX
deficiency plasma as substrate plasma
Reagent
(1) 3. 8% Sodium citrate solution: Dissolve 9. 5 g of
anhydrous sodium citrate in water and dilute to 250 ml.
(2) Imidazole buffer solution (pH 7. 3): Dissolve O. 68 g of
imidazole and l. 17 g of sodium chloride in 100 ml of water.
Add 42. 2 ml of O. 1 mol/L hydrochloric acid solution and
dilute to 200 ml with water.
(3) Diluent: Mix one volume of 3. 8% sodium citrate with
five volumes of imidazole buffer solution. Add 20 % human
albumin to a final concentration of 1%.
(4) Activated partial thromboplastin time (APTT) reagent
(5) Human coagulation factor IX deficiency plasma: Human
plasma or artificial substrate plasma, in which human
coagulation factor IX concentration is less than 1 %.
( 6) Physiological saline.
(7) O. 05 mol/L Calcium chloride solution: Dissolve 147 g of
calcium chloride (CaClz• 2H2 0) in water and dilute to 1000
ml to prepare a 1 mol/L calcium chloride stock solution.
Dilute the stock solution 20-fold with water to prepare a O. 05
mol/L calcium chloride solution just befare use.
Preparation of standard solution of hmnan coagulation factor 1X
Dilute the standard human coagulation factor IX with
coagulation factor IX deficiency plasma or physiological saline
to a concentration of 1 IU per ml, then make 10-fald, 20-
fald, 40-fold and 80-fald dilutions with diluent respectively,
allow to stand in an ice bath far use.
IX
Preparation of test sample solution
If the test sample contains heparin, the heparin shall be
neutralized with protamine sulfate at first. Dilute the test
sample with coagulation factor IX deficiency plasma or
physiological saline to a concentration of about 1 IU per ml,
then make 10-fold, 20-fald or 40-fald dilution with diluent,
allow to stand in an ice bath far use.
Procedure
Keep O. 1 ml of APTT reagent in 37°C water bath far a
certain time ( 4 minutes in general ) , add O. 1 ml of
coagulation factor IX deficiency plasma and O. 1 ml of test
sample solution, mix well and warm in 37°C water bath far
a certain time ( 5 minutes in general). Add O. 1 ml of O. 05
mol/L calcium chloride solution preheated to 37°C and record
the coagulation time.
Repeat the above mentioned procedure using O. 1 ml of
standard solution of human coagulation factor IX of various
dilutions instead of the test sample solution.
A linear regression equation is obtained by regressing the
logarithm of potency (IU/ml) of human coagulation factor
IX standard solution with the logarithm of corresponding
coagulation time ( second). Calculate the potency of human
coagulation factor IX in test sample solution and multiply
with the dilution factor to obtain the human coagulation
factor IX potency CIU/mD of test sample.
[Note]
( 1) The correlation coefficient of linear regression shall be
not less than O. 98.
( 2 ) Each dilution of test sample shall be determined in
duplicate, and the difference between the two results shall
not exceed 10 % of the mean value, otherwise the test shall
be repeated.
(3) The apparatus directly contacting the standard, test
sample and plasma shall be made of plastic or silicified glass.
The test is carried out with an automatic blood coagulation
analyzer fallowing the instructions.
3520 Potency Test for Human
Coagulation Factor X
( One-step Method)
The potency of human coagulation factor X is determined by
one-step method using human coagulation factor X
deficiency plasma as a substrate plasma.
Reagent
(1) Diluent: Dissolve 11. 75 g of barbital sodium and 14. 67
g of sodium chloride in a quantity of water, adjust pH to 7. 3
with 1 mol/L hydrochloric acid and dilute to 2000 ml with
water. Add human albumin to a final concentration of 1 %
just befare use.
(2) Calcium-containing thromboplastin
(3) Coagulation factor X deficiency plasma: Human plasma
or artificial substrate plasma, in which human coagulation
factor X concentration is less than 1%.
Preparation of standard solution of hmnan coagulation factor X
Dilute the standard with coagulation factor X-deficiency
plasma or physiological saline to a concentration of 1 IU
per ml, then make 10-fold, 20-fald, 40-fold and 80-fald
dilutions with diluent respectively, allow to stand in an ice
bath for use.
Preparation of test sample solution
Dilute the test sample with coagulation factor X deficiency
plasma or physiological saline to a concentration of about 1
IU per ml, then make 10-fald, 20-fald or 40-fald dilution
 3523
with diluent, allow to stand in an ice bath for use.
Procedure
Mix O. 1 ml of test sample solution and O. 1 ml of coagulation ,
factor X deficiency plasma well and warm in 37°C water bath
for a certain time ( 3 minutes in general). Add O. 2 ml of
calcium-containing thromboplastin preheated to 37°C and
record the coagulation time.
Repeat the above-mentioned procedure using O. 1 ml of
standard solution of human coagulation factor X of various
dilutions instead of the test sample solution.
A linear regression equation is obtained by regressing the
logarithm of potency CIU/ml) of human coagulation factor
X standard solution with the logarithm of corresponding
coagulation time ( second). Calculate the potency of human
coagulation factor X in test sample solution and multiply
with the dilution factor to obtain the human coagulation
factor X potency (IU/mD of test sample.
[Note]
(1) The correlation coefficient of linear regression shall be
not less than O. 98.
( 2 ) Each dilution of test sample shall be determined in
duplicate, and the difference between the two results shall
not exceed 10 % of the mean value, otherwise the test shall
be repeated.
(3) The apparatus directly contacting the standard, test
sample and plasma shall be made of plastic or silicified glass.
( 4 ) The test is carried out with an automatic blood
coagulation analyzer following the instructions.
3521 Potency Test for Human
Coagulation Factor W
COne-step Method)
The potency of human coagulation factor VIH is determined by
one-step method using human coagulation factor VIH
deficiency plasma as a substrate plasma.
Reagent
(1) 3. 8% Sodium citrate solution: Dissolve 9. 5 g of
anhydrous sodium citrate in water and dilute to 250 ml.
(2) Imidazole buffer solution (pH 7. 3): Dissolve O. 68 g of
imidazole and l. 17 g of sodium chloride in water and dilute
to 100 ml. Add 42. 2 ml of O. 1 mol/L hydrochloric acid
solution and dilute to 200 ml with water.
(3) Diluent: Mix one volume of 3. 8% sodium citrate with
five volumes of imidazole buffer solution and add 20 % human
albumin to a final concentration of 1%.
(4) Activated partial thromboplastin time (APTD reagent
(5) Human coagulation factor VIH deficiency plasma: Human
plasma or artificial substrate plasma, in which human
coagulation factor V1H concentration is less than 1 %.
(6) O. 05 mol/L Calcium chloride solution: Dissolve 147 g of
calcium chloride (CaClz• 2H 2 0) in water and dilute to 1000
ml to prepare a 1 mol/L calcium chloride stock solution.
Dilute the stock solution 20-fold with water to prepare a O. 05
mol/L calcium chloride solution just before use.
Preparation of standard solution of human coagulation factor "\I
Dilute the standard human coagulation factor VIH with
coagulation factor VIH deficiency plasma to a concentration of
1 IU per ml. Then, make 10-fold, 20-fold, 40-fold and 80-
fold dilutions with diluent respectively, allow to stand in an
ice bath for use.
Preparation of test sample solution
Dilute the test sample with coagulation factor VIH deficiency
plasma to a concentration of about 1 IU per ml, then make
Biological Activity Test on lnterferon
10-fold, 20-fold or 40-fold dilution with diluent, allow to
stand in an ice bath for use.
3522 In vivo 'Iest for Biological Activity
of Recombinant Hmnan Erythropoietin
(Reticulocyte Method)
This method is based on the stimulating effect of human
erythropoietin CEPO) on the formation of reticulocyte. The
number of reticulocytes in mice injected s. c. with EPO
increases with the increasing dosage. The in vivo biological
activity of EPO is determined by <lose-response parallel line
analysis on the count ratio of reticulocyte to erythrocyte.
Reagent
(1) Dipotassium ethylenediamine tetraacetate ( EDTA-K2 )
anticoagulant: Dissolve 100 mg of EDTA-K2 in 10 ml of
physiological saline and mix well. Prepare just before use.
( 2) Diluent: Dissolve O. 1 g of bovine serum albumin in
physiological saline and dilute to 100 ml.
Preparation of standard solution
Reconstitute EPO standard following the instructions and
dilute with diluent to three ( high, medium and low)
concentra tions.
Preparation of test sample solution
Dilute the test sample with diluent to three concentrations
similar to those of standard solution.
Procedure
Inject s. c. the standard and test sample solutions of low,
medium and high dosages (e. g. 10, 20 and 40 IU per
mouse) into the inbred mi ce ( female BALB/ c) aged 6-8
weeks or B6D2Fl mice respectively. Both the standard and
test sample at each dosage are injected into at least four
mice, not more than O. 5 ml per mouse. Bleed three to four
drops of blood from orbit of each mouse on the 4th day and
transfer to a tube containing 200 µl of EDTA-K2
anticoagulant for determining the cell number ratio (Ret % )
of reticulocyte to erythrocyte of each mouse by an automatic
reticulocyte analyzer. Calculate the in vivo biological activity
of test sample by <lose-response parallel line assay ( see
Statistical Methods for Biological Assays described in Chinese
Pharmacopoeia, Volume JI ) using injecting dose ( IU)
against Ret%.
3523 Biological Activity Test
on Interferon
Method I : Cytopathic Inhibition Assay
The method is based on the principle that interferon can
protect human amnion-derived WISH cell from the damage of
vesicular stomatitis virus ( VSV). The biological activity of
interferon is determined by the protective effect curve of live
WISH cells after being stained with crystal violet.
Reagent
(1) MEM or RPMI 1640 medium: Dissolve one bag ( the
specification is 1 L) of MEM or RPMI 1640 medium powder
in water and dilute to 1000 ml. Add 10 5 IU of penicillin, 10 5
IU of streptomycin and 2. 1 g of sodium bicarbonate. Mix
well after dissolution and sterilize by filtration. Store at 4 ºC.
(2) Complete medium: To 10 ml of newborn calf serum,
add 90 ml of MEM or RPMI 1640 medium. Store at 4 ºC.
(3) Testing medium: Mix 7 ml of newborn calf serum with
93 ml of MEM or RPMI 1640 medium. Store at 4ºC.
 3523 Biological Activity Test on lnterferon
( 4) Challenge medium: Mix 3 ml of newborn calf serum
with 97 ml of MEM or RPMI 1640 medium. Store at 4 ºC.
( 5 ) Digestion solution: Dissolve O. 2 g of
ethylenediaminetetraaceticacid disodium, 8. O g of sodium
chloride, O. 2 g of potassium chloride, l. 152 g of disodium
hydrogen phosphate and O. 2 g of potassium dihydrogen
phosphate in water and dilute to 1000 ml. Sterilize at 121 ºC
for 15 minutes.
(6) Staining solution: Dissolve 50 mg of crystal violet in 20
ml of absolute ethanol and dilute to 100 ml with water.
(7) Destaining solution: Dilute 50 ml of absolute ethanol and
O. 1 ml of acetic acid to 100 ml with water.
(8) PBS: Dissolve 8. O g of sodium chloride, O. 20 g of
potassium chloride, l. 44 g of disodium hydrogen phosphate
and O. 24 g of potassium dihydrogen phosphate in water and
dilute to 1000 ml. Sterilize at 121 ºC for 15 minutes.
Preparation of standard solution
Reconstitute the national standard for testing the biological
activity of human interferon following the instructions and
dilute to a concentration of 1000 IU per mi with testing
medium, then make eight 4-fold dilutions serially on a 96-
well cell culture plate, in duplicate. Prepare the solution
under an aseptic condition.
Preparation of test sample solution
Dissolve the test samples based on the labeled volume and
dilute to contain about 1000 IU per ml with testing medium,
then make eight 4-fold dilutions serially on a 96-well cell
culture plate, in duplicate. Prepare the solution under an
aseptic condition.
Procedure
lncubate WISH cells in a culture bottle to form a monolayer
of anchored cells. Transfer the cells into complete medium
for subculture at a ratio of (1 : 2) / (1 : 4), 2-3 times a
week. Discard the medium, wash the cells twice with PBS
and digest with digestion solution. Collect the cells and dilute
with complete medium to prepare a suspension at a
concentration of 2. 5 X 105 -3. 5 X 10 5 cells per ml. Add the
suspension onto a 96-well cell culture plate, 100 µl per well,
and incubate at 37°C in a 5 % carbon dioxide incubator for 4-6
hours. Transfer the prepared standard and test sample
solutions to the plate inoculated with WISH cells, 100 µl per
well and incubate at 37ºC in a 5 % carbon dioxide incubator
for 18-24 hours. Discard the supernatant. Dilute the
vesicular stomatitis virus, stored at -70ºC, with challenge
medium to about 100 CCIDso, inoculate the plate, 100 µl
per well and incubate at 37°C in a 5% carbon dioxide
incubator for 24 hours, until the 50% CPE point of standard
solution appears at a concentration of 1 IU per ml under
microscope. Discard the supernatant, add 50 µl of staining
solution into each well, allow to stand at room temperature
for 30 minutes. Remove the staining solution by washing
with flowing water carefully and adsorb the residual
moisture. Add 100 µl of destaining solution into each well,
allow to stand at room temperature for 3-5 minutes. Mix
well and read absorbance with microtitre plate reader at 570
nm using 630 nm as a reference wavelength. Record the
determination results.
Analyze the data by using four-parameter regression method
or other relevant computer software. Calculate the test result
by the following equation:
Biological activity (IU/mD of test sample=
PrX [ WsXEs> / <DrXEr) J
Where: P r = Biological activity of standard, IU/ml;
Ds = Pre-dilution factor of test sample;
Dr=Pre-dilution factor of standard;
Es= Dilution factor of test sample with response
equivalent to that of standard at 50 % effective
concentration;
Er = Dilution factor of standard at 50% effective
concentration.
Atternative chromogenic tests can be used after equivalent
validation.
Method ll: Reporting gene method ( applicable for Interferon D
In this method, plasmids containing interferon-stimulated
response elements and luciferase genes are transfected in
HEK293 cells to establish the cell line HEK293purolSRE-
Luc, which is used for determination of biological activity.
After the type l interferon binds to the receptors on the cell
membrane, the interferon-stimulated response elements will
be activated by the signal transduction and the luciferase
expression will be turned on. The expression quantity is
positively correlated to the biological activity. Determine the
luminous intensities after the cell lysis solutions and the
luciferase substrates are added, and obtain the <lose-response
curve of the biological activity of the standard solution versus
the chemiluminescence intensity so as to determine the
biological activity of type l interferon.
Reagents
( 1) Complete culture solution: MEM culture solution,
containing 2 mM of L-glutamine, 1 mM of sodium pyruvate,
O. 01 mg/L of nonessential amino acid, 2 µg/mL of
puromycin, 100 U/ ml of penicillin, 100 µg/ mL of
streptomycin and 10% of fetal bovine serum. Store at 4 ºC.
(2) Culture solution for test: Except for the absence of
puromycin, other components are identical with these of
complete culture medium. Store at 4 ºC.
(3) PBS: Dissolve 8. O g of sodium chloride, O. 20 g of
potassium chloride, l. 44 g of disodium hydrogen phosphate,
and O. 24 g of potassium dihydrogen phosphate with water
and dilute to 1000 mL, sterilize at 121 ºC for 15 minutes.
( 4) Digestive fluid: Dissolve O. 2 g of disodium edetate and 2. 5 g
of trypsin with Pffi and dilute to 1000 ml. Conduct the sterile
filtration. Store at 4ºC.
( 5) Luciferase reporter gene assay kit: lt contains the cell
lysis solutions, the luciferase substrates, etc.
Preparation of reference solution
Reconstitute the national standard of the recombinant
interferon for biological activity assay according to the
instruction, and dilute to approximately 10 000 IU per ml
with the culture solution for test. Conduct the 4-fold serial
dilution in the 96-well cell culture plate, with 8 levels of
dilution, 2 wells for each level of dilution. Perform under
aseptic conditions.
Preparation of test solution
Dissolve the substance being examined according to the
labeled amount and dilute to approximately 10 000 IU per ml
with the culture solution for test. Conduct the 4-fold serial
dilution in the 96-well cell culture plate, with 8 levels of
dilution, 2 wells for each level of dilution. Perform under
aseptic conditions.
Procedure
Let the HEK293purolSRE-Luc cell grow in the complete
culture solution with adherence to the wall of the plate.
Passage the cells 2-3 times per week at the ratio of 1 : 4 so
that the cells can grow in the complete culture solution. Take
the cultured cells and discard the culture solution, wash once
 3525 Biological Activity Test for Recombinant Human Granulocyte Colony-stimulating Factor
with PBS and then digest and harvest the cells. Prepare the
cell suspension containing 3. 5 X 105 ........ 4.5X10 5 cells per ml
with the culture solution for test. Transfer the prepared
standard solution and test solution into the 96-well cell
culture plate which can be used for cell culture and
chemiluminescence microplate reader, 100 µL of each
solution in each well. Then inoculate the above cell
suspension into the same 96-well cell culture plate, 100 µL in
each well. Incubate the plate at the condition of 37°C and 5 %
C02 for 18 to 24 hours. Draw the supernatants in the 96-well
cell culture plate completely and carefully, add the cell lysis
solutions and the luciferase substrates according to the
instruction of luciferase reporter gene assay kit, determine
the luminous intensity and record the determination results.
Process the test data using a computer program or four-
parameter regression method and calculate test results according
to the following formula:
Biological activity of the test sample (U/mD =
p XD,XE,
r Dr XEr
Where P r is the biological activity of the standard substance,
in IU/ml;
D, is the pre-dilution factor of the test sample;
Dr is the pre-dilution factor of the standard;
E, is the dilution factor of the substance being
examined which is equivalent to the half-effect
dilution factor of the standard.
Er is the half-effect dilution factor of the standard.
3524 Biological Activity Test for
Recombinant Human Interleukin-2
CCTLL-2 Cell /MTT Colourimetric
1' 8 .1 1"
iv1etnoaJ
The method is based on the principie that the viability of
CTLL-2 cell is dependent on stimulation of interleukin-2
CIL-2) at different potency. The biological activity of IL-2
may be determined according to the growth of CTLL-2 cells.
Reagent
( 1 ) RPMI 1640 medium: Dissolve one bag ( the
specification is 1 L) of RPMI 1640 medium powder in water
and dilute to 1000 ml. Add 10 5 IU of penicillin, 105 IU of
streptomycin and 2. 1 g of sodium bicarbonate. Mix well
af ter dissolution and sterilize by filtration. Sto re at 4 ºC.
(2) Basic medium: Mix 10 ml of newborn calf serum with
90 ml of RPMI 1640 medium. Store at 4ºC.
(3) Complete medium: Add recombinant human IL-2 into
100 ml of basic medium to a final concentration of 400-800
IU. Store at 4 ºC.
(4) PBS: Dissolve 8. O g of sodium chloride, O. 20 g of
potassium chloride, l. 44 g of disodium hydrogen phosphate
and O. 24 g of potassium dihydrogen phosphate in water and
dilute to 1000 ml. Sterilize at 121 ºC for 15 minutes.
(5) Thiazole blue (MTT) solution: Dissolve O. 1 g of MTT
in PBS and dilute to 20 ml. Sterilize by filtration with a O. 22
µm membrane. Store at 4ºC , protected from light.
(6) Lysis solution: 15% sodium dodecyl sulfate (SDS).
Use the solution within 12 months after preparation.
CfLL-2 cell
The CTLL-2 cell culture shall be a weak acidic and slightly
turbid liquid It is used for the biological activity test of
interleukin-2 48-60 hours after processing.
Preparation of veference solution
Reconstitute the national standard for the determination of
biological activity of human interleukin-2 following the
instructions and dilute with basic medium to a concentration
of 200 IU per ml, then make eight 2-fold dilutions serially
on a 96-well cell culture plate, in duplicate. Keep 50 µl of
standard solution in each well and discard the rest. Prepare
the solution under an aseptic conditions.
Preparation of test sample solution
Reconstitute the test samples based on the labeled volume,
and dilute with basic medium to a concentration of about 200
IU per ml, then make eight 2-fold dilutions serially on a
96-well cell culture plate, in duplicate. Keep 50 µl of test
sample solution in each well and discard the rest. Prepare the
solution under an aseptic conditions.
Procedure
Incubate CTLL-2 cells in complete medium at 37°C in a 5 %
carbon dioxide incubator to prepare enough cells for test.
Collect the cells by centrifugation, wash 3 times with RPMI
1640 medium and resuspend at a concentration of 6. OX 105
cells perml in basic medium. Keep at 37°C in 5 % carbon
dioxide incubator for use. Add the cell suspension to a
96-well cell culture plate containing standard and test sample
solutions, 50 µl per well, and incubate at 37°C in a 5%
carbon dioxide incubator for 18-24 hours. Add 20 µl of MTT
solution into each well and incubate the plate at 37°C in a 5 %
carbon dioxide incubator for 4-6 hours. Add 150 µl of lysis
solution into each well and incubate at 37°C in a 5% carbon
dioxide incubator for 18-24 hours. All the above steps shall
be carried out under an aseptic condition. Mix the cell culture
thoroughly and read absorbance with microtiter plate reader
at 570 nm using 630 nm as a reference wavelength. Record
the determination results.
Analyze the data using four-parameter regression method or
other relevan! computer software. Calculate the test result
by the following equation:
Biological activity CIU/mD of test sample
=PrX [ CD.XE.) / CDrXEr) J
Where: Pr=Biological activity of standard, IU/ml;
D.= Pre-dilution factor of test sample;
Dr = Pre-dilution factor of standard;
E.= Dilution factor of test sample with response
equivalent to that of standard at 50 % effective
concentration;
Er = Dilution factor of standard at 50% effective
concentration.
3525 Biological Activity Test for
Recombinant Human Granulocyte
Colony-stimulating Factor
CNFS-60 Cell Strain/MTT
Colourimetric Method)
The method is based on the principie that the growth rate of
murine myeloid leukemia cells ( NFS-60) is dependent on
stimulation of recombinant human granulocyte colony-
stimulating factor ( rhG-CSF) at different potencies. The
biological activity of rhG-CSF is determined according to the
growth of NFS-60 cells.
Reagent
( 1 ) RPMI 1640 medium: Dissolve one bag ( the
specification is 1 L) of RPMI 1640 medium powder in water
and dilute to 1000 ml. Add 10 5 IU of penicillin, 10 5 IU of
streptomycin and 2. 1 g of sodium bicarbonate. Mix well
after dissolution and sterilize by filtration. Store at 4 ºC.
 3526 Biological Activity Test on Recombinant Human Granulocyte/Macrophage Colony-stimulating Factor
(2) Basic medium: Mix 100 ml of newborn calf serum with
900 mi of RPMI 1640 medium. Store at 4ºC.
(3) Complete medium: Add rhG-CSF into basic medium to
a final concentration of 10-20 ng per mi.
( 4) PBS: Dissolve 8 g of sodium chloride, O. 2 g of
potassium chloride, l. 44 g of disodium hydrogen phosphate
and O. 24 g of potassium dihydrogen phosphate in water and
dilute to 1000 mi. Sterilize at 121 ºC for 15 minutes.
(5) Thiazole blue ( MTT) solution: Dissolve O. 10 g of
MTT powder in 20 ml of PBS to prepare a 5. O mg/ mi
solution. Sterilizeby filtration with a O. 22 µm membrane.
Store at 4 ºC , protected from light.
(6) Lysis solution: Dilute 14 mi of hydrochloric acid and 50
ml of Triton X-100 solution to 500 ml with isopropanol.
Store at room temperature, protected from light.
Preparation of standard solution
Reconstitute the standard for the determination of biological
activity of rhG-CSF following the instructions and dilute with
basic medium to a concentration of 50-300 IU per ml, then
make eight 2-fold dilutions serially on a 96-well cell culture
plate, in duplicate. Keep 50 µl of standard solution in each
well and discard the rest. Prepare the solution under an
aseptic condition.
Preparation of test sample solution
Dissolve the test samples based on the labeled volume, and
dilute with basic medium to a concentration of about 50-300
IU per ml, then make eight 2-fold dilutions serially on a 96-
well cell culture plate, in duplicate. Keep 50 µl of test
sample solution in each well and discard the rest. Prepare the
solution under an aseptic conditions.
Procedure
lncubate NFS-60 cell strain in complete medium at 37°C in a
5 % carbon dioxide incubator. Control the concentration of
culture a t a range of l. OX 105 -4. OX 10 5 cells per mi. The
culture which has been incubated for 24-36 hours after
processing shall be used for the biological activity test. Pr~
warm ali the solutions for test to 37°C. Centrifuge enough
culture to collect NFS-60 cells. Wash the cells 3 times with
RPMI 1640 medium, resuspend at a concentration of 2. OX
105 cells per mi in basic medium, and keep at 37°C for use.
Add the cell suspension to a 96-well cell culture plate
containing standard and test sample solutions, 50 µl per
well, and incubate at 37°C in a 5% carbon dioxide incubator
for 40-48 hours. Add 20 µl of MTT solution into each well,
and incubate the plate at 37°C in a 5 % carbon dioxide
incubator for 5 hours. Ali the above steps shall be carried out
under an aseptic condition. Add 100 µl of lysis solution to
each well and mix thoroughly. Read absorbance with
microtitre plate reader at 570 nm using 630 nm as a reference
wavelength. Record the determination results.
Analyze the data by using four-parameter regression method
or other relevant computer software. Calculate the test result
by the following equation:
Biological activity ( IU / ml) of test sample=
PrX [ CDsXEs) / CDrXEr) J
Where: P r = Biological activity of standard, IU/ ml;
Ds=Pr~dilution factor of test sample;
Dr = Pr~dilution factor of standard;
Es = Dilution factor of test sample with response
equivalent to that of standard at 50 % effective
caneen tra tion;
Er = Dilution factor of standard at 50% effective
concentration.
3526 Biological Activity Test on
Recombinant Human Granulocyte/
Macrophage Colony-stimulating Factor
CTF-1 Cell/MTT Colourimetric
Method)
The method is based on the principie that the growth rate of
human erythrocyte leukemia cell ( TF-1) is dependent on
stimulation of recombinant human granulocyte/macrophage
colony-stimulating factor ( rhGM-CSF ) at different
potencies. The biological activity of rhGM-CSF is determined
according to the growth of TF-1 cells.
Reagent
(1) RPMI 1640 medium: Dissolve one bag ( the specification is
1 L) of RPMI 1640 medium powder in water and dilute to
1000 mi. Add 105 IU of penicillin, 105 IU of streptomycin and
2. 1 g of sodium bicarbonate, mix well after dissolution and
sterilize by filtration. Store at 4ºC.
(2) Basic medium: Mix 100 ml of newborn calf serum and
900 ml of RPMI 1640 medium. Store at 4ºC.
(3) Complete medium: Add rhGM-CSF into basic medium
to a final concentration of 5. O ng or 80 IU per mi.
( 4) PBS: Dissolve 8 g of sodium chloride, O. 2 g of
potassium chloride, l. 44 g of disodium hydrogen phosphate
and O. 24 g of potassium dihydrogen phosphate in water and
dilute to 1000 mi. Sterilize at 121 ºC for 15 minutes.
(5) Thiazole blue ( MTT) solution: Dissolve O. 10 g of
MTI powder in 20 ml of PBS to prepare a 5 mg/ml solution.
Sterilize by filtration with a O. 22 µm membrane. Store at
4 ºC , protected from light.
(6) Lysis solution: Dilute 14 ml of hydrochloric acid and 50
ml of Triton X-100 solution to 500 mi with isopropanol.
Preparation of standard solution
Reconstitute the rhGM-CSF standard following the
instructions and dilute with basic medium to a concentration
of 10-20 IU per mi. Make eight 2-fold dilutions serially on a
96-well cell culture plate, in duplicate. Keep 50 µl in each
well and discard the rest. Prepare the solution under an
aseptic condition.
Preparation of test sample solution
Reconstitute the test samples based on the labeled volume,
and dilute with basic medium to a concentration of about 10-
20 IU per ml, then make eight 2-fold dilutions serially on a
96-well cell culture plate, in duplicate. Keep 50 µl in each
well and discard the rest. Prepare the solution under an
aseptic condition.
Procedure
lncubate TF-1 cell strain in complete medium at 37°C in a
5 % carbon dioxide incubator. Control the concentration of
culture at a range of 2. O X 105 -7. OX 10 5 cells per mi. The
culture which has been incubated for 24-36 hours after
passage shall be used for biological activity test. Prewarm ali
the solutions for test to 37°C. Centrifuge enough culture to
collect TF-1 cells. Wash the cells 3 times with basic
medium, resuspend ata concentration of 4. OX 10 5 cells per
mi in basic medium, and keep at 37°C for use. Add the cell
suspension onto a 96-well cell culture plate containing
standard and test sample solutions, 50 µl per well, and
incubate at 37ºC in a 5% carbon dioxide incubator for 48-52
hours. Add 20 µl of MTT solution into each well and
incubate the plate at 37°C in a 5 % carbon dioxide incubator
for 5 hours. Ali the above steps shall be carried out
aseptically. Add 100 µl of lysis solution into each well and
 3528 Biological Activity Test for Recombinant Epidermal Growth Factor
mix well. Read absorbance with microtitre plate reader at
570 nm using 630 nm as a reference wavelength. Record the
determination results.
Analyze the data by using four-parameter regression method
or other relevant computer software. Calculate the test result
by the following equation:
Biological activity CIU/ mD of test sample
=PrX [ CDsXEs) / CDrXEr) J
Where: P r = Biological activity of standard, IU/ml;
Ds = Pre-dilution factor of test sample;
Dr = Pre-dilution factor of standard;
Es= Dilution factor of test sample with response
equivalent to that of standard at 50 % effective
concentration;
Er = Dilution factor of standard at 50% effective
concentration.
3527 Biological Activity Test f or
Recombinant Bovine Basic
Fibroblast Growth Factor
( Cell Proliferation/MTT Colourimetric
Method)
The method is based on the stimulating effect of recombinant
bovine basic fibroblast growth factor ( rbBFGF) on the
growth of murine embryonic fibroblasts (BALB/c 3T3 celD.
The biological activity of rbBFGF is thus determined by the
effect on the growth of BALE/ c 3T3 cells.
Reagent
( 1 ) RPMI 1640 medium: Dissolve one bag ( the
specification is 1 L) of RPMI 1640 medium powder in water
and dilute to 1000 ml. Add 10 5 IU of penicillin, 10 5 IU of
streptomycin and 2. l g of sodium bicarbonate, mix weH
after dissolution and sterilize by filtration. Store at 4 ºC.
( 2) Maintenance medium: Dilute 4 ml of newborn calf
serum to 1000 ml with RPMI 1640 medium.
(3) Complete medium: Dilute 100 ml of newborn calf serum
to 1000 ml with RPMI 1640 medium.
( 4) PBS: Dissolve 8 g of sodium chloride, O. 2 g of
potassium chloride, l. 44 g of disodium hydrogen phosphate
and O. 24 g of potassium dihydrogen phosphate in water and
dilute to 1000 ml. Sterilize at 121 ºC for 15 minutes.
(5) Thiazole blue ( MTT) solution: Dissolve O. 10 g of
MTT powder in 20 ml of PBS. Sterilize by filtration with a
O. 22 µm membrane. Store at 4 ºC , protected from light.
Preparation of standard solution
Reconstitute the standard of rbBFGF following the instructions
and dilute with maintenance medium to a concentration of 40 IU
per ml, then make eight 4-fold dilutions serially on a 96-well cell
culture plate, in duplicate. Prepare the solution under an aseptic
condition.
Preparation of test sample solution
Reconstitute the test sample based on the labeled volume and
dilute with maintenance medium to contain about 40 IU per
ml. Make eight 4-fold dilutions serially on a 96-well cell
culture plate, in duplicate. Prepare the solution under an
aseptic condition.
Procedure
Incuba te BALB/ c 3T3 cell strain in complete medium at 37°C
in a 5 % carbon dioxide incubator. Control the concentration
of culture ata range of l. OX 105 -5. OX 10 5 cells per ml. The
culture which has been incubated for 24-36 hours after
passage shall be used for the determination of biological
activity of rbBFGF. Discard the medium, digest and collect
cells to prepare a suspension of 5. OX 104- l. OX 105 cells per
ml with complete medium. lnoculate the suspension onto a
96-well cell culture plate, 100 µl per well, and incubate at
37ºC in a 5 % carbon dioxide incubator for 24 hours. Replace
the complete medium with maintenance medium and incubate
at 37°C in a 5 % carbon dioxide incubator for another 24
hours. Discard the maintenance medium, add standard and
test sample solutions, 100 µl per well, and incubate at 37°C
in a 5 % carbon dioxide incubator for 64-72 hours. Add 20 µl
of MTT solution into each well and incubate at 37ºC in a 5 %
carbon dioxide incubator for 5 hours. All the above steps shall be
carried out under an aseptic candi tion. Discard the liquid on the
plate, add 100 µl of dimethyl sulfoxide (DMSQ) into each well
and mix thoroughly. Read the absorbance with microtitre plate
reader at 570 nm using 630 nm as a reference wavelength
Record the determination results.
Analyze the data by using four-parameter regression method
or other relevant computer software. Calculate the test result
by the following equation:
Biological activity CIU/mD of test sample=
PrX [ CDsXEs) / CDrXEr) J
Where: Pr=Biological activity of standard, IU/ml;
D s = Pre-dilution factor of test sample;
D r = Pre-dilution factor of standard;
Es= Dilution factor of test sample with response
equivalent to that of standard at 50 % effective
concentration;
Er = Dilution factor of standard at 50% effective
concentration.
3528 Biological Activity Test f or
Recombinant Epidennal Growth Factor
( Cell Proliferation/MTI Colourimetric
Method)
The method is based on the stimulating effect of recombinant
human epidermal growth factor ( rhEGF) on the growth of
murine embryonic fibroblast ( BALB/c 3T3 cell ). The
biological activity of rhEGF is thus determined by the effect
on the growth of BALB/ c 3T3 cells.
Reagent
( 1 ) RPMI 1640 medium: Dissolve one bag ( the
specification is 1 L) of RPMI 1640 medium powder in water
and dilute to 1000 ml. Add 10 5 IU of penicillin, 10 5 IU of
streptomycin and 2. 1 g of sodium bicarbonate, mix well
after dissolution and sterilize by filtration. Store at 4ºC.
( 2) Maintenance medium: Dilute 4 ml of newborn calf
serum to 1000 ml with RPMI 1640 medium.
(3) Complete medium: Dilute 100 ml of newborn calf serum
to 1000 ml with RPMI 1640 medium.
( 4) PBS: Dissolve 8 g of sodium chloride, O. 2 g of
potassium chloride, l. 44 g of disodium hydrogen phosphate
and O. 24 g of potassium dihydrogen phosphate in water and
dilute to 1000 ml. Sterilize at 121 ºC for 15 minutes.
(5) Thiazole blue ( MTT) solution: Dissolve O. 10 g of
MTT powder in 20 ml of PBS. Sterilize by filtration with a
O. 22 µm membrane. Store at 4 ºC , protected from light.
Preparation of standard solution
Reconstitute the standard of rhEGF following the instructions
and dilute with maintenance medium to a concentration of 50 IU
per ml, then make eight 4-fold dilutions serially on a 96-well cell
culture plate, in duplicate. Prepare the solution under an aseptic
condition.
 3529 Biological Activity Test for Recombinant Streptokinase
Preparation of test sample solution
Reconstitute the test samples based on the labeled volume,
and dilute with maintenance medium to a concentration of
about 50 IU per ml, then make eight 4-fold dilutions serially
on a 96-well cell culture plate, in duplicate. Prepare the
solution under an aseptic conditions.
Procedure
lncubate BALB/ c 3T3 cell strain in complete medium at 37ºC
in a 5 % carbon dioxide incubator. Control the concentration
of culture ata range of l. OX105 -5. OX10 5 cells per ml. The
culture which has been incubated for 24-36 hours after
developing shall be used for the determination of biological
activity of rhEGF. Discard the medium, digest and collect
cells to prepare a suspension of 5. OX 104 -8. OX 104 cells per
ml with complete medium. lnoculate the suspension onto a
96-well cell culture plate, 100 µl per well, and incubate at
37°C in a 5% carbon dioxide incubator for 24 hours. Replace
the complete medium with maintenance medium and incubate
at 37°C in a 5 % carbon dioxide incubator for another 24
hours. Discard the maintenance medium, add standard and
test sample solutions, 100 µl per well, and incubate at 37ºC
in a 5% carbon dioxide incubator for 64-72 hours. Add 20 µl
of MTT solution to each well and incubate at 37°C in a 5 %
carbon dioxide incubator for 5 hours. Ali the above steps
shall be carried out under an aseptic condition. Discard the
liquid on the plate, add 100 µl of dimethyl sulfoxide
( DMSO ) in to each well and mix thoroughly. Read the
absorbance with microtitre piate reader at 570 nm using 630 nm
as a reference wavelength. Record the determination results.
Analyze the data by using four-parameter regression method
or other relevant computer software. Calculate the test result
by the following equation:
Biological activity CIU/ml) of test sample=
PrX [ CDsXEs) / CDrXEr) J
Where: Pr=Biological activity of standard, IU/ml;
Ds = Pre-dilution factor of test sample;
Dr = Pre-dilution factor of standard;
Es= Dilution factor of test sample with response
equivalent to that of standard at 50 % effective
concentration;
E r = Dilution factor of standard at 50 % effective
concentra tion.
3529 Biological Activity Test for
Recombinant Streptokinase
The method is based on the principie that a complex formed by
streptokinase with plasminogen can actívate free plasminogen to
become a biologically active plasmin. The biological activity of
recombinant streptokinase is determined quantitatively with the
rings of lysis formed on agarose plate due to the degradation of
human fibrin into soluble fibrin fragments by biologically active
plasmin.
Reagent
( 1) Human thrombin solution: Dissolve human thrombin and
dilute to a concentration of 100 IU per ml with physiological
saline solution. Store at -20ºC.
(2) Human plasminogen solution: Dissolve human plasminogen
with physiological saline and dilute to a concentration of O. 5 mg
per ml. Store at -20ºC.
(3) Human fibrinogen solution: Prepare just befare use.
Preheat human fibrinogen and physiological saline in 37ºC
water bath for 15 minutes befare preparation. Dissolve
fibrinogen in a quantity of physiological saline. Keep the
solution in 37°C water bath for 30 minutes to dissolve the
fibrinogen completely and dilute to a concentration of 6 mg
per mi with physiological saline.
Preparation of standard solution
Reconstitute the national standard for determination of
biological activity of recombinant streptokinase following the
instructions and dilute with physiological saline to the
concentrations of 1000, 250, 62. 5, 15. 6 and 3. 9 IU per mi
respectively.
Preparation of test sample solution
Reconstitute the test sample based on the labeled volume,
and dilute with physiological saline to a concentration of 100
IU or 1 µg per ml.
Procedure
Swell 125 mg of agarose in 23 mi of physiological saline by
boiling, and equilibrate in 55-60ºC water bath. Add 14 µl of
100 IU per mi human thrombin solution and 280 µl of O. 5
mg/ml human plasminogen solution with shaking, then add
2. 2 mi of 6 mg/ mi human fibrinogen solution and shake
continuously until the mixture is turbid. Pour the mixture
into a culture dish, 8 cm in diameter, immediately and
allow the culture dish to stand horizontally. After agarose is
coagulated completely, allow the culture dish to stand at 4ºC
for at least 30 minutes and use within 2 days. Dig wells,
2 mm in diameter, on the coagulated agarose dish containing
fihrin. Add test samples and standard solutions at each
dilution into the wells separately in duplicate, 10 µl per
well, allow the culture dish to stand horizontally at 37°C in a
wet box for 24 hours. Measure the longitudinal and
transverse diameters of each lysis ring twice separately and
calculate the mean diameters. A linear regression equation is
obtained by regressing the logarithm of the standard activity
of each dilution with the logarithm of mean diameter of the
corresponding lysis ring. Calculate the biological activity of
test sample by inserting the logarithm of diameter of its lysis
ring into the linear regression equation.
3530 Biological Activity Test for
Mouse Nerve Growth Factor
Method 1 Incubation of chicken embryo dorsal root ganglia
Reagent
( 1) Rat tail collagen After rat tails are disinfected with
75% alcohol, tail tendon is separated, and cut into pieces,
then immersed and dissolved in O. 1 % glacial acetic acid
solution for 48 hours. Centrifuge at 4000 rpm and 4ºC for 30
minutes, and take the supernatant, sterilize at 121 ºC for 15
minutes, and preserve at -20ºC.
( 2) DMEM culture solution Take commercially available
DMEM medium, add penicillin, streptomycin and L-
glutamine at the final concentrations of 100 IU/ml, 100
IU/ml and 2 mmol/L, respectively, and mix well.
(3) Basic culture solution Take 10 ml of fetal bovine serum
CFBS), add DMEM culture solution 90 ml, and preserve at 4ºC.
Preparation of reference solution and test solution
Take national reference standard of murine nerve growth
factor for examining biological activity, and perform three-
fold serial dilutions with DMEM culture solution for 5-6
dilutions. Perform the same dilution for the filtered test
sample.
 3531 Biological Activity Test for Nimotuzumab lnjection
Test
Take dorsal root ganglia from 7-9 days-old chicken embryo
under aseptic conditions, put into culture flasks coated with
rat tail collagen separately. After adhering to the wall of flask
for 1-2 hours, add standard solutions and test solutions with
different dilutions. Perform the negative control in parallel.
lncubate at 37°C in an incubator with saturated humidity
(5% C0 2 atmosphere) for 24 hours, and observe the growth
of ganglion axons using an inverted microscope. The endpoint
is the highest dilution leading to + + + + growth, the
biological activity unit of the test sample is calculated by the
following formula
Activity unit of test sample (AU/mD =
Activity of working reference substanceX
Test sample dilution at endpoint
Working reference substance dilution at endpoint
[Remark]
Assessment criteria for growth of ganglion axons
"# ": lnhibition of overgrown ganglion;
"++++": Ganglion axons are grown at all surrounding,
long and dense, like tree branches;
"+ + +": Ganglion axons are grown at 2/3 surrounding,
like tree branches;
"++": Ganglion axons are grown at 1/2 surrounding;
"+": A few of ganglion axons are grown;
" - ": No growth of ganglion axons.
Method ll TF-1 cells/MTS colorimetry
The method is based on that the growth of human erythrocyte
leukemia cells ( abbreviated as TF-1 cell ) varies with the
biological activity of murine nerve growth factor (NGF), so that
the biological activity of NGF can be measured.
Reagent
( 1) RPMI 1640 culture solution Take commercially
available RPMI 1640 culture solution, add penicillin and
streptomycin at the final concentrations of 100 IU/ml and
100 IU/ml.
(2) Basic culture solution Add 100 ml of fetal bovine serum
(FBS) into 900 ml of RPMI 1640 culture solution, and
preserve at 4ºC.
(3) Complete culture solution Add murine nerve growth
factor into the basic culture solution to make the final
concentration of 12 U/ml.
( 4) MTS solution Take commercially available MTS, thaw
at 4ºC, dispense into EP tubes at l. 2 ml of aliquot per tube,
and preserve at -20ºC and protect from light.
(5) TF-1 cells lncubate the TF-1 cell strains with complete
culture solution in an incubator ( 5 % C0 2 atmosphere) at
37°C, the cell concentration is controlled at l. OX 10 5 -5. OX
10 5 cells/ml, subculture for 24 hours before use for NGF
biological activity determination.
Preparation of standard solution
Take national standard of murine nerve growth factor for
examining biological activity, perform the reconstitution
according to package insert, and dilute with basic culture
solution to make a solution containing 100 U per 1 ml
(dilution in each step <loes not exceed 10 times). Perform
three-fold serial dilutions in 96-well cell culture plate with 8
dilutions, each dilution in two wells, with 100 µl standard
solution is put in each well respectively, and the excess
solution is discarded. The above procedures are carried out
under aseptic conditions.
Preparation of test solution
The test sample is reconstituted according to label claim, and
diluted with basic culture solution to make a solution
containing 100 U per 1 ml ( dilution in each step <loes not
exceed 10 times). Perform three-fold serial dilutions in 96-
well cell culture plate with 8 dilutions, each dilution in two
wells, with 100 µl standard solution added to each well
respectively, and the excess solution is discarded. The above
procedures are carried out under aseptic conditions.
Test
lncubate the TF-1 cell strains with complete culture solution
in an incubator ( 5 % C02 atmosphere) at 37°C , cell
concentration is controlled at l. O X 105 -5. O X 10 5 cells/ml,
subculture for 24 hours before use for NGF biological activity
determination. The solutions used in the test are preheated to
37°C. Take sufficient TF-1 cell cultures, collect TF-1 cells by
centrifugation, wash three times with basic culture solution,
then re-suspend in the basic culture solution to make a cell
suspension containing 6. OX104 cells, and preserve at 37°C in
5 % C02 atmosphere. In 96-well cell culture plate containing
the standard solution and the test solution, add 100 µl of cell
suspension to each well, and incubate at 37°C in 5% C0 2
atmosphere for 66-72 hours. Add 20 µl of MTS solution into
each well, and incubate at 37°C in 5% C02 atmosphere for 3
hours. The above procedures are carried out under aseptic
conditions. Use plate reader to measure the absorbance at the
wavelength of 490 nm with 550 nm as reference wavelength,
and record the result.
Process the test data using a computer program or four-
parameter regression calculation method, and then calculate
the result using the following equation:
Biological activity of the test sample (U/mD =
DsXEs
PrXDrXEr
Where: Pr is biological activity of the standard substance
(U/mD;
Ds is pre-dilution factor of test sample;
Dris pre-dilution factor of standard substance;
Es is dilution factor of test sample equivalent to
median effective <lose of standard substance.
E, is dilution factor of median effective <lose of
standard substance.
3531 Biological Activity Test for
Nimotuzumab Injection
1 . H292 cell proliferation inhibition
This method is based on the principie that the growth of
human lung cancer lymph node metastasis ( H292) cells is
inhibited by addition of Nimotuzurnab Injection in a concentration-
dependent manner. The bioactivity of Nimotuzumab Injection
then is measured.
Reagent
(1) RPMI 1640 medium: Dissolve 1 bag ( the specification is
1 L) of RPMI 1640 medium powder in water and dilute to
1000 ml; add 10 5 IU of penicillin, 10 5 IU of streptomycin,
and 2. 1 g of sodium bicarbonate. Mix well to dissolve and
then sterilize the medium by filtration. Store at 4ºC. Or use
commercial RPMI 1640 solution.
(2) Maintenance medium: Mix 3 ml of fetal bovine serum
(FBS) with 97 ml of RPMI 1640 medium. Store at 4ºC.
(3) Complete medium: Mix 5 ml of fetal bovine serum
(FBS) with 95 ml of RPMI 1640 medium. Store at 4 ºC.
( 4) Phosphate buffer solution ( PBS): Dissolve 8. O g of
sodium chloride, O. 20 g of potassium chloride, l. 44 g of
dipotassium phosphate and O. 24 g of Monopotassium
 3531 Biological Activity Test for Nimotuzumab Injection
phosphate in water and dilute to 1000 ml. Sterilize at 121 ºC
for 15 minutes. Or use commercial PBS solution.
( 5) O. 25 % disodium ethylenediamine tetraacetate ( ElITA) -
trypsin: Use commercial O. 25% EDTA-trypsin.
( 6) Colour reagent: Mix 540 µl of the reagent from
commercial cell counting kit CCCK-8) solution with 810 µl of
maintenance medium.
Preparation of reference solution
Dilute Nimotuzumab reference with maintenance medium to a
concentration of 300 µg/ ml, then make eight different
concentrations using a 4-fold serial dilution in duplicate.
Prepare the solution under an aseptic condition.
Preparation of test sample solution
Dilute test sample with maintenance medium to a
concentration of 300 µg/ ml, then make eight different
concentrations using a 4-fold serial dilution in duplicate.
Prepare the solution under an aseptic condition.
Testing Procedure
lncubate H292 cells in complete medium at 37°C in a 5 %
C02 incubator. Control the concentration of culture ata range
of l. OX 105-5. O X 10 5 cells per ml. The culture which has
been incubated for 24-36 hours after processing shall be used
for the biological activity test. Discard the medium in the
incubation flask, digest with O. 25 % EDTA-trypsin, collect
cells, and dilute with the complete medium to prepare a
suspension ata concentration of 6X104-8X 104 cells per ml.
Add 100 µl suspension per well onto a 96-well cell culture
plate, and incuba te at 37°C in a 5 % C0 2 incubator for 18-20
hours. Discard the complete medium of the cell culture plate;
then add 200 µl of different concentration standard solution
or sample solution in each well, and incubate at 37°C in a 5 %
C02 incubator for 68-72 hours. Add 30 µl of colour reagent
into each well, mix well, and incubate at 37°C in a 5% C0 2
incubator for 4 hours. Read the absorbance with microtitre
plate reader at 450 nm using 630 nm as a reference
wavelength. Record the result. Add 200 µl maintenance
medium to the well with cells as cell control, add 200 µl
maintenance medium to the well without cells as blank
control. Measure with the same method and record the
results.
Analyze the data using a computer software or four-
parameter regression method, the concentrations of the
reference standard or test sample being examined as the
horizontal coordinate, average absorbance values as the
vertical coordina te. Calculate 50 % effective concentration
CED5o) of test sample and reference standard, then calculate
the test result by the following equation:
Biological activity of test sample=
Standard EDso 7 Test sample EDso X 100 %
Validation Criteria: The S-shaped curve parallel assumption
is not rejected ( P > O. 05) and the fit of the curve, R 2 ,
should be more than O. 92.
Acceptable Criteria: Biological Activity of test sample
compared to reference standard should be not less than 50 % .
][ . Detennination of Relative Binding Activity of
Nimotuzumab lnjection
This method is based on the principie that the binding
activity of Nimotuzumab lnjection with human lung cancer
H125 cells is in a concentration-dependent manner. The
relative binding activity of Nimotuzumab Injection is
performed by flow cytometry.
Reagent
( 1) RPMI 1640 medium: Dissolve 1 bag ( the specification is
1 L) of RPMI 1640 medium powder in water and diluted to
1000 ml, add 10 5 IU of penicillin, 10 5 IU of streptomycin,
and 2. 1 g of sodium bicarbonate. Mix well to dissolve and
then sterilize the medium by filtration. Store at 4 ºC. Or use
commercial RPMI 1640 solution.
(2) Cell medium: Mix 10 ml of fetal bovine serum (FBS)
with 90 ml of RPMI 1640 medium. Store at 4ºC.
(3) lOX Phosphate buffer solution (PBS): Dissolve 19. 7068 g
of dipotassium hydrogen phosphate Trihydrate ( Kz HP0 4 •
3H2 0), 3. 4328 g of Sodium dihydrogen phosphate dihydrate
(NaH2PÜ4• 2H2Ü), and 14. 4 g of sodium chloride (NaCD
in 200 ml of ultrapure water, then sterilize at 121 ºC for 15
minutes.
( 4) PBS: Dilute 100 ml of 10 X PBS with ultrapure water to
1000 ml.
(5) O. 25% disodium ethylenediamine tetraacetate CEDTA) -
trypsin: Use commercial O. 25 % EDTA-trypsin.
(6) 10% sodium azide: Dissolve O. 10 g of sodium azide in 1
ml of ultrapure water.
(7) Diluent: Mix well O. 10 g of bovine serum albumin, 100
µl of 10% sodium azide, and 10 ml of PBS.
( 8 ) 1 % paraformaldehyde solution: Place 5 g of
paraformaldehyde, 250 µl of 1 mol/L sodium hydroxide
solution, and 50 ml of lOXPBS in a 500 ml volumetric flask,
mix well, and dilute to volume with water.
(9) Anti-human fluorescein isothiocyanate (FITC) diluted
solution: Dilute an appropriate amount of anti-human FITC
antibody solution with PBS at ratio of 1 : 20-1 : 30.
Preparation of Reference Solution
Dilute Nimotuzumab reference with the diluent under sterile
conditions to 50, 15, 5. O, 3. O, 2. O, l. O, O. 50, O. 20, and
O. 05 µg/ ml, two wells for each concentration.
Preparation of Test Solution
Dilute the substance being examined with the diluent under
sterile conditions to 50, 15, 5.0, 3.0, 2.0, 1.0, 0.50,
O. 20, and O. 05 µg/ml, two wells for each concentration.
Testing Procedure
lncubate H125 cells in complete medium at 37°C in a 5%
C02 incubator. Control the concentration of culture ata range
of l. OX 105-5. O X 105 cells per ml. The culture which has
been incubated for 24-36 hours after processing shall be used
for the relative binding activity test. Discard the medium in
the incubation flask, digest with O. 25% EDTA-trypsin and
collect cells, then centrifuge at 1100 rpm for 5 minutes at
4 ºC, discard the supernatant, collect and count the cells. The
cell activity ( percentage of live cell in total cells) should be
not less than 80%. Wash cells with 10 ml PBS twice, and
then prepare a cell suspension of 1X10 7 cells per ml. Add 20
µl of different concentration standard or test solutions into a
number of appropriate centrifuge tubes, two tubes for each
concentration. Use two tubes containing 20 µl of diluents as
the blank control. Add 25 µl of the cell suspension separately
into the centrifuge tubes containing different concentrations
of the test solution, standard solution, or blank control
solution, mix well, and incubate at 4ºC for 30 minutes. Add
700 µl of PBS to each tube, centrifuge at 1100 rpm for 5
minutes at 4ºC. Carefully discard the supernatant, and shake
gently on a vortex oscillator. Add 20 µl of anti-human FITC
diluted solution to each tube, mix well, and incubate at 4ºC
for 30 minutes. Add 700 µl of PBS to each tube, centrifuge at
1100 rpm for 5 minutes at 4 ºC. Carefully discard the
supernatant, and shake gently on a vortex oscillator. Add
500 µl of 1 % paraformaldehyde solution to each tu be. Read
the average fluorescence intensity of the cells by flow
cytometry, and record the results.
Analyze the data by using a computer software or four-
parameter regression method, the concentrations of the
 3533
reference standard or test sample being examined as the
horizontal coordinate, average fluorescence intensity as the
vertical coordina tes. Calculate 50 % effective concentration
(EDso) of test sample and reference standard, then calculate
the test result by the following equation:
Relative biological activity of test sample=
Standard ED5o -7-Test sample EDso X 100%
Validation Criteria: The S-shaped curve parallel assumption
is not rejected ( P >O. 05) and the fit of the curve, R 2 ,
should be more than O. 97.
Acceptable Criteria: Relative biological activity of test sample
compared to reference material should be not less than 60 % .
3532 Biological Activity Test for
Recombinant Human Interleukin-11
Clnterleukin B9-11 Cell/MTT
Colourimetric Method)
The method is based on the principle that the proliferation of
murine B9 hybridoma subclone cell ( B9- l l cell line) is
dependent on the concentration of interleukin-11. The
biological activity of recombinant Human lnterleukin-11 may
be determined according to the growth rate of B9-11 cells.
Reagent
(1) RPMI 1640 medium: Dissolve one bag (the specification
is 1 L) of RPMI 1640 medium powder in ultrapure water and
dilute to 1000 ml. Add 2. 1 g of sodium bicarbonate. Mix well
to dissolve and then sterilize the medium by filtration. Store
at 4ºC.
(2) Complete medium: Add 100 ml newborn calf serum into
900 ml of RPMI 1640 medium. Add rhIL-11 to a final
concentration of 50 IU/ ml. Store at 4ºC.
(3) Biological Activity testing medium: Add 50 ml fetal
bovine serum into 950 ml of RPMI 1640 medium.
( 4) PBS: Dissolve 8 g of sodium chloride, O. 2 g of
potassium chloride, l. 44 g of disodium phosphate and O. 24
g of Monopotassium phosphate in ultrapure water and dilute
to 1000 ml. Sterilize it at 121 ºC for 15 minutes.
(5) Thiazole blue (MTT) solution: Dissolve O. 10 g of MTT
in 20 ml of PBS to prepare a 5. O mg/ml solution. Sterilize by
filtration through a O. 22 µm membrane. Store at 4 ºC and
protect it from light.
(6) Lysis solution: dimethyl sulfoxide (DMSO, AR) or 10%
sodium dodecyl sulfate (SDS) containing O. 01 mol/L HCL
Preparation of Reference Solution
Reconstitute the reference for the determination of biological
activity of recombinant human interleukin-11 according to the
instructions. Then dilute the solution with basic medium to a
concentration of 1000 IU/ml or other appropriate concentration.
In a 96-well cell culture plate, make eight different
concentrations using a 4-fold serial dilution in duplicate. Keep
50 µl of standard solution in each well and discard the rest.
Prepare the solutions under an aseptic conditions.
Preparation of Test Sample Solution
Reconstitute test sample and dilute it with basic medium to a
concentration of 1000 IU/ml or other appropriate concentration.
In a 96-well cell culture plate, make eight different
concentrations using a 4-fold serial dilution in duplicate. Keep 50
µl of test sample solution in each well and discard the rest
Prepare the solutions under an aseptic condition.
Testing Procedure
Incuba te B9- ll cells in complete medium at 37°C in a 5 %
C02 incubator. The culture which has been incubated for 24-
72 hours after processing shall be used for the biological
Potency Test for Botulinum Toxin Type A
activity test. Pre-warm ali the solutions for test to 37°C.
Centrifuge enough culture to collect B9-11 cells. Wash the
cells 3 times with RPMI 1640 medium, re-suspend at a
concentration of 2. 0-3. OX 10 5 cells per ml in basic medium
( properly adjust inoculum density according to cell
situation), and keep at 37ºC for use. Add the cell suspension
onto a 96-well cell culture plate containing standard and test
sample solutions, 50 µl per well, and incubate at 37°C in a
5 % C0 2 incubator for 40-48 hours. Add 20 µl of MTT
solution into each well, and incubate the plate at 37°C in a
5 % C02 incubator for 4-5 hours. All the above operations
must be carried out under an aseptic condition. Add 100 µl of
lysis solution into each well and mix thoroughly ( keep
overnight for SDS lysis solution). Read absorbance with
microtitre plate reader at 570 nm, using 630 nm as a
reference wavelength. Record the results.
Analyze the data by using computer software or four-
parameter regression method. Calculate the test result by the
following equation:
Biological activity (IU/mD of test sample=
Pr XD, XE, -7-Dr-7-Er
Where: P r = Biological activity of standard, IU/ml;
D, =Pre-dilution factor of test sample;
Dr = Pre-dilution factor of standard;
E, = Dilution factor of test sample with response
equivalent to that of standard at 50 % effective
concentration;
Er = Dilution factor of standard at 50 % effective
concentration
3533 Potency Test for Botulinum
Toxin Type A
(Method of Parallel Lines)
The potency test is based on the principie that the muscle
paralysis effect of Botulinum Toxin Type A causes mouse
death. The potency of Botulinum Toxin Type A is
determined by LÜ;o assay in mice. For determination of the
LDso, after serial dilutions of test sample and reference,
inject each dilution of test sample and reference into groups
of mice, separately, calculate the LDso, and the, LDso of test
sample is corrected by parallel line analysis . Thus calculate
the total LO;o per container of test sample ( 1 LDso is defined
as 1 potency unit).
Reagent
Diluent: Normal saline
Preparation of test sample and reference solutions
Reconstitute ten to twenty containers of the test sample and
the reference with 2. 5 ml of normal saline separately, mix
well. Prepare not less than five dilutions of the test sample
and the reference at an equal? interval ratio separately, about
half of animals shall die after injected with the median
dilution of the sample.
Procedure
Inject each dilution of test sample and reference solutions into
each of 10 female SPF Kunming mice of 26-30 days old
respectively, each mouse shall be injected i. p. with O. 5ml of
solution. Record the death of animals daily for 4 days
following injection. The result shall be calculated by parallel
line analysis according to the <lose-response curve of survival
rate on the 4th day. The 95% confidence interval shall be
within 50 %-200 % of the estimated potency.
The test shall be repeated if the following case occurs:
Nonspecific death occurs in at least two animals injected with
 3601 Test Requirements far Monitoring SPF Chick Embryos

the same dilution.
The test is valid provided that:
(1) most than 70% of amimals injected with the lowest dilution
of reference on test sample clied.
(2) More than 70% of animals injected with the highest
dilution of reference or test sample survived.
(3) There are no less than 4 valid dilutions far the test
sample and the reference, including the dilution which make
more than half of animals die and the dilution which make
less than half of animals die except the lowest dilution and
the highest dilution.
( 4) There is no significant difference in parallelity and
linearity of the <lose-response curves between the test sample
and the reference.

3600 Specific Biologica Raw Materials/ Animals

Procedure
(1) Sampling
3601 Test Requirements for Fresh egg samples are required far detecting avian lymphoid
Monitoring SPF Chick Embryos leukosis virus and avian encephalomyelitis virus. Serum
samples are required far detecting other pathogenic
Specific Pathogen Free ( SPF) chicks represent that the microorganism. The amount of each serum sample shall be
chicks are bred under a strictly controlled surveillance to at least 1 ml. To avoid contamination, sampling shall fallow
meet the requirements far specified microbiological the aseptic procedures.
examination. SPF chick embryo refers to the embryo which (2) Amount of egg samples
is hatched from a fertilized egg laid by SPF hen and incubated Far detecting lymphoid leukosis virus, 200 eggs shall be
under an appropriate condition far the production of sampled from each flock of chickens, one egg from each hen
biologics. The quality of the SPF chick embryos shall be (If the chicken number in one flock is less than 200, sample
controlled through detecting specific pathogenic shall be taken from each individual).
microorganisms in SPF chick flocks and their eggs. For detecting avian encephalomyelitis virus, at least 50 eggs
Detection oi pathogens and method shall be sampled from each flock, one egg from each hen CH
Detection of pathogens far surveillance on SPF chick embryos the chicken number in one flock is less than 50, samples
and the detection methods used are listed as fallows: shall be taken from each individual).
Far detecting other pathogenic microorganism infections,
No. Pathogenic microorganism Demand Method samples shall be taken at random at a rate of 5 % from each
chicken flock. For the flock consisting of less than 200 chickens,
1 Salmonella Pullorum • SPA, IA, TA
sample shall be taken at a proportion of 10 %-15 %.
2 A vian Influenza Virus (type A) • AGP, HI (3) Storage and shipping of the samples
3 lnfectious Bronchitis Virus • AGP, SN, HI After collection, samples shall be sent to the laboratory far
4 lnfectious Bursal Disease Virus • AGP, SN tests as soon as possible.
5 lnfectious Laryngotracheitis Virus • AGP, SN If samples can not be sent to the laboratory in time, sera
6 Newcastle Disease Virus • HI shall be stored frozen at or below -15ºC. Eggs shall be
7 Fowl Pox Virus • AGP stored at 4-lOºC. The storage period shall not exceed one
week.
8 Marek' s Disease Virus • AGP
Samples shall be marked with distinct labels. Delivery sheets
9 Haemophilus Paragallinarum
10 Pasteurella Multocida
•
o
SPA
AGP
shall be attached indicating the names and numbers of
chicken groups as well as the names and quantity of the
11 Avian Adenovirus Group III • HI samples.
12 Mycoplasma Gallisepticum • SPA, HI During shipping of samples, care shall be taken to avoid
13 Mycoplasma Synoviae • SPA, HI temperature rising and the breakage of eggs .
14 Avian Encephalomyelitis Virus • AGP, EST, SN Result evaluation
15 Lymphoid Leukosis virus • ELISA lf any test result of a sample fails to meet the requirements in
16 Reticuloendotheliosis Virus • AGP this Annex, the sampling batch of chick embryo shall be
17 Avian Reovirus • AGP judged as unqualified.
18 Avian Adenovirus Group I
19 Chicken lnfectious Anaemia Virus •
• AGP
IFA 3602 Requirements for Microbiological
Note: e=Obligatory, result shall be negative Test on Laboratory Animals
O=In case of need, result shall be negative
SPA=Serum Plate Agglutination Test
The Requirements ( quoted from GB14922. 1-2001 ) are
IA = Isolation of pathogen
suitable far guinea pig, hamster, rabbit, dog and monkey,
AGP=Agar Diffusion Test
as well as the mouse and rat above clean level.
HI=Haemagglutination lnhibition Test
ELISA = Enzyme-Linked lmmunosorbent. Assay l. Microbiological grade of laboratory animals
EST=Embryo Sensitive Test ( 1) Conventional ( CV) animals: The CV animals shall be free
SN=Serum Neutralization Test from pathogens of specified anthropozoonosis or fulminating
TA=Test-tube Agglutination Test zoonoses.
IFA= lndirect lmmunofluorescence Assay (2) Clean (CL) animals: Except the pathogens that shall
 3602 Requirements for Microbiological-Test on Laboratory Animals

be excluded for the CV animals, the CL animals shall be free Table 3 Test requirements for pathogens in
from the pathogens seriously harmful to animals or dog and monkey
significantly interfering scientific research.
Grade of animal Pathogen DogMonkey
( 3 ) Specific pathogen free ( SPF) animals: Except the
pathogens that shall be excluded for the CL animals, the SPF cv Salmonella spp.
•• •
•6. •
Pathogenic dermal fungí
SPF animals shall be free from the majar potentially
Bruce/la spp.
infectious pathogens, conditional pathogens or the pathogens
Leptospira spp.
significantly interfering scientific experiments.

••
Shigella spp.
(4) Germ free CGF) animal: They shall be free from any
Mycobacterium tuberculosis
detectable organisms.
2. Test requirements
Leptospira spp. •
Yesinia enterocolitica
•o o
(1) Appearance: The animals shall be healthy and show no Campylobaceter jejuni o o
abnormal signs in appearance. Note: e Obligatory, result shall be negative; O In case of need, result
(2) Pathogens: See Tables 1, 2 and 3. shall be negative; 6. In case of need, immunization is permitted;
(3) Viruses: See Tables 4, 5 and 6. • lmmunization is not permitted, and the result shall be negative.

Table 1 Test requirements for pathogens in mouse and rat Table 4 Test requirements for viruses in
mouse and rat
Grade of animal Pathogen Mouse Rat
GF SPF CL Salmonella spp.
Listeria monocytogenes
•o •o Grade of animal Virus
GF SPF CL Lymphocytic choriomeningitis virus
Mouse Rat
o
Yersinia pseudotuberculosis o o (LCMV)
Yesinia enterocolitica
Pathogenic dermal fungí
o
o
o
o
Hantavirus(HV)
Ectromelia virus(Ect.)
o
•• •
Streptobacillus monili formis o o Mouse hepatitis virus(MHV)
•• ••• ••
Bordetella bronchiseptica Sendai virus(SV)
Mycoplasma spp.
•• Pneumonia virus of mice(PVM)

•• •
Corynebacterium kutscheri Reovirus type III (Reo-3)
Tyzzer's organism
Escherichia coli O 115 a, C, K
•o Minute virus of mice (MVM)
Theiler's mouse encephalomyelitis
•o
(B) virus(TMEV)
o
Pasteurella pneumotropica
Klebsiella pneumoniae
•• •••
Mouse adenovirus (Mad)
Polyoma virus (POLY) o
Staphylococcus aureus
Streptococcus pnemoniae
•o o
Rat parvovirus (KRV)
Rat parvovirus (H-1)
••
~-Hemoiytic streptococcus o o Rat coronavirus (RCV)/Sialodacry-
•
Pseudomonas aeruginosa • • oadenitis virus (SDAV)
No any detectable virus .
• •
e
No any detectable microorganisms
• •
Obligatory, result shall be negative; O In case of need, result shall
Note: e Obligatory, result shall be negative; O In case of need, result
shall be negative.
be negative.

Table 2 Test requirements for pathogens in guinea pig, Table 5 Test requirements for viruses in guinea
harmter and rabbit pig, harmter and rabbit
GuineaHam-
GuineaHam- Grade of animal Vrrus Rabbit
Grade of animal Pathogen Rabbit pig ster
pig ster
GF SPF CL CV Salmonella spp.
Listeria monocytogens
•o •o •o GF SPF CL CV Lymphocytic choriomeningitis e
virus(LCMV)
...
•
Rabbit hemorrhagic disease
Yersinia pseudotuberculosis o o o virus (RHDV)
Yesinia enterocolitica o o o
Pathogenic dermal fungí o o o Sendai virus (SV)
• •
Streptobacillus monili formis o o • Rabbit hemorrhagic
disease virus (RHDV) •
•••
••• • • • •
Pasteurella multocida Sendai virus (SV)
Bordetella bronchiseptica Pneumonia virus of mice
Tyzzer's organism
• (PVM)
Reovirus type III (Reo-3)
• • •
Pasteurella pneumotropica
•• •• •••
Rotavirus (RRV)
Klebsiella pneumoniae
Staphylococcus aureus
•o•o
No any detectable virus
• •
Note: e Obligatory, result shall be negative; A Obligatory,
Streptococcus pnemoniae o immunization is permitted; • Immunization is not permitted, and the

••
P-hemolytic streptococcus o o result shall be negative.
Pseudomonas aeruginosa
• •
No any detectable
microorganisms • •
Note: e Obligatory, result shall be negative; O In case ofneed, result
•
shall be negative.
 3603 Requirements for Parasitological Test on Laboratory Animals

Table 6 Test requirements for viruses in dog Table 2 Test requirements for parasites in guinea
and monkey pig, hamster and rabbit

Grade of animal Virus DogMonkey GuineaHam-

Grade of animal Parasites Rabbit

......
pig ster
SPF cv Rabies virus (RV)
Canine parvovirus (CPV)
...
GF SPF cL¡cv Ectoparasites
• • •
Canine distemper virus (CDV)
Infectious canine hepatitis virus ...
Toxoplasma gondii • • •o
o
Encephalitozoon cuniculi
(ICHV) Eimaria spp . o o
Cercopithecine herpesvirus Type
• Pneumocystis carinii
•
l(BV)
Simian retrovirus D (SRV)
• All helminths • • •
Simian immunodeficiency virus • Flagellates
• • •
(SIV) Ciliates
•
Sirnian T lymphotropic virus
Type l(STLV-1)
• No any detectable parasies

Note: e Obligatory, result shall be negative; O In case of need, result

Simian pox virus (SPV)
• shall be negative .
The above 4 kinds of virases
are not permitted to be used for
•
immunization.
Table 3 Test requirements for parasites in
dog and monkey
Note: e Obligatory, result shall be negative; A Obligatory, the animals
shall be immunized.
Grade of animal Parasites Dog Monkey

SPF cv Ectoparasites
• •
•
1

3603 Requirements for Parasitological

Toxoplasma gondii •
All helminths
• •
Test on Laboratory Animals Entamoeba spp.
Plasmodium spp.
o ••
The Requirements ( quoted from GB14922. 2-2001 ) are Flagellates
• •
suitable for hamster, guinea pig, rabbit, dog and monkey, Note: e Obligatory, result shall be negative; O In case of need, result
11 .1 1 ' 1 1 1 1
as weu as tne mouse ana rat aoove c1ean ieve1. 1 shall be negative.

l. Parasitological grade of laboratory animals

(1) Conventional ( CV) animals: They shall be free from
the specified parasites causing Anthropozoonosis. 3604 Test Requirements for
(2) Clean CCL) animals: Except the parasites that shall be Newborn Calf Serum
excluded for the CV animals, the CL animals shall be free
from the parasites seriously harmful to animals or
The serum refers to that separated from blood collected from
significantly interfering scientific research.
the calf without feeding within 14 hours after birth and made
( 3 ) Specific pathogen free ( SPF) animals: Except the
by aseptic filtering. No additional substances are allowed to
parasites that shall be excluded for the CL animals, the SPF
be added in the manufacturing procedure. Each batch of the
animals shall be free from the majar potentially infectious
serum can not be used for biological production until it is
parasites, conditional pathogenic parasites or the parasites
tested according t_o the following items and the results should
significantly interfering scientific experiments.
meet the requirements.
(4) Germ free CGF) animals: They shall be free from any
For bovine serum treated by validated viral inactivation
detectable organisms.
process, the samples shall be taken for testing of coliphage
2. Test requirements and virus before any inactivation step.
(1) Appearance: The animals shall be healthy and show no
pH It shall be 7. 2-8. O <0631 ).
abnormal signs in appearance.
(2) Parasites: See Tables 1, 2 and 3. Protein content It shall be 35 g/L-50 g/L determined by
Biuret method <0731 , method 3) or other suitable methods.
Table 1 Test requirements for parasites in
mouse and rat Hemoglobin content The hemoglobin content in bovine
serum shall be not more than 200 mg/L determined by
Grade of animal Parasites Mouse Rat spectrophotometry or other suitable methods. The following
•• ••o
GF SPF CL Ectoparasites is the procedure of Spectrophotometry for hemoglobin
Toxoplasma gondii content. Determine the absorbance of the serum sample
Encephalitozoon cuniculi o directly in duplicate using a spectrophotometric of 1 cm path
Pneumoc ystis carinii o o length at the wavelength of absorbance at 576 623 and 700
All helminths • • nm and using distilled water as blank control Calculate the
Flagellates
Ciliates
•• •• hemoglobin content by the following formula using the mean
absorbance value:

No any detectable parasites

Note: e Obligatory, result shall be negative; O In case ofneed, result

• • Hemoglobin content (mg/L) = [ CAs16X115) -
(~23X102) - CA100 X 39. 1) ] X 10;

shall be negative. Where: As16, ~23 and A100 are the mean value of test sample's
absorbance at 576 nm, 623 nm and 700 nm wavelengths.
Osmolarity It shall be 250-330 mOsmol/kg <0632).
 3604
Bacterial endotoxin Not more than 1O EU/ ml ( 1143 , the
limit test of gel-clot method).
Test for proliferation of supporting cells The test is performed
using Sp2/0-Ag14 cells or other suitable cell lines. After
recovery, the cells are subcultured for at least three times in
culture medium prepared with the test sample and shall be
used for the test at the logarithmic phase.
(1) Determination of cell growth curve: Prepare cell culture
medium with the test sample to a concentration of 10%.
Inoculate cells into the medium to a final concentration of
1 X lü4 cells per mL Count live cells every day for 7 days and plot
a cell growth curve. The maximum proliferation concentration of
cells shall be not less than 1 X 106 cells per ml.
(2) Determination of cell doubling time: Calculate the cell
doubling time according to the cell growth curve by the
following equation:
The cell doubling time = T /A A = log2 Y/ X
Where: T = Time for cell growth, hour;
Y = Cell count one day before the growth reaches
the peak value;
X = Number of cells inoculated.
The doubling time of Sp2/0-Agl4 cells shall be not more
than 20 hours.
(3) Determination of cloning rate: Dilute the cells to the
density of 10 viable cells/ml by limit dilution method. Seed
the cells onto a 96-well culture plate, one cell per well and at
least 60 wells per plate should be seeded. Incubate the plate
at 37°C in humidified incubator satured with 5 % carbon
dioxide for one week. Observe the growth of cell clone
regularly and calculate the cloning rate by the following
equation. The cloning rate should be not less than 70 % .
Cloning rate= CA/B) X 100%
Where: A=Number of cell clones;
B= Total number of wells inoculated with cells.
Sterility test Complies with the test for sterility ( 1101 ).
Mycoplasma Complies with the test in 3301. The results
should meet the requirement.
Coliphage Bacteriophage plaque and proliferation method
shall apply. No coliphage shall be detected.
Cell Culture Method and lmmunofluorescence Assay
(1) Preparation of sample: A total of approximately 250 ml
new-born bovine serum is used for test. Prepare the culture
medium with 15 % of test sample for maintaining and cell
passaging throughout the whole test. Use the qualified serum
as negative control.
(2) Preparation of indicator cells: Use monkey-origin cell
(e. g. Vero cells), two kinds of bovine-origin cells (BT and
MDBK cells or primary bovine kidney cells free of virus
contamination), and human diploid cells as indicator cells at
least. After recovery, the indicator cells should be
subcultured at least once prior to use and sufficient quantities
of cell cultures should be prepared for testing.
( 3) Inoculate appropriate quantity of each indicator cell
culture respectively into 75 cm2 culture flasks with medium
containing test sample to make sure the inoculated cells could
reach at least 80 %-90 % confluence after 7 days. Negative
control flasks are conducted simultaneously, lncubate the cell
cultures at 37°C in humidified incubator satured with 5 %
carbon dioxide. Replaced the culture medium once on day 5 if
needed.
( 4) on day 7, subculture each of inoculated indicator cells
into at least two of 75 cm2 flasks and maintain them until day
14. Replaced the culture medium once on day 12 if needed.
(5) On day 13 (or 1 day before the second subculture) or
Test Requirements for Newborn Calf Serum
when the cells in negative control flasks have reached at least
70 % confluence, prepare the positive control cell cultures.
Subculture the cells from one of negative control flasks and
seed into 6-well plates or other suitable cell plates for
cytopathic effect ( CPE ) examination, hemadsorption
( HAd) test and immunofluorescence ( IF) assay. Inoculate
the positive control viruses in the following day.
( 6) Carry out the second subculture on day 14. Cells from
the remain negative control flasks and the test sample flasks
are seeded into 6-well plates or other suitable cell plates. For
CPE examination and HAd test, inoculate the test sample
cells in at least triplicate for each of indicator cells; For IF
assay, inoculate the test sample cells in at least duplicate.
lncubate the cells at least to day 21. The remaining cell
samples are preserved at ::s;;;-60ºC.
(7) lnoculate positive control viruses on day 14. lnoculate
appropriate amount ot positive control viruses onto the cells
prepared in step ( 5) and place at 36ºC ± 1ºC for 2 h in
humidified incubator saturaed with 5 % carbon dioxide. After
adsorption, discard the inoculum and feed the cells with
maintenance media. lncubate the cells at 36ºC ± 1 ºC for 7
days in humidified incubator saturated with 5 % carbon
dioxide for BT cells, bovine diarrhea virus CBVDV) could be
used as a positive control for CPE and bovine Para influenza
virus type 3 (BPl3) as a positive control for HAd: BPl3
bovine adenovirus ( BAV-3), bovine parvovirus ( BPV) and
BVDV are the positive controls for IF assay. For MDBK
cells, reovirus 3 ( RE03 ) and PI3 could be used as the
positive controls for CPE and HAd test. respectively; BPI3,
BAV-3, BVDV and RE0-3 as the positive controls for IF
assay. For Vero cells. BPI3 could be used as the positive
controls for CPE and HAd test; BPl3 and RE0-3 are used
as the positive controls for IF assay. Rabies virus ( RV)
positive control is not required. The positive control viruses
for all IF assay shall be inoculated at 100-300 CCIDso.
(8) CPE should be observed daily for cell cultures inoculated
with negative control and test sample. Carry out CPE
examination. HAd test and IF assay after at least 21 days
post-inoculation or at least seven days arier the last
subculture. The cell cultures inoculated with the positive
control viruses can be detected on the day 7 after inoculation
or when 10%of the cells showed CPE by IF assay. For HAd
test, mixture suspension of red cells from chicken and guinea
pig is used at 2-8ºC and 20-25ºC.
For IF assay, the fixed cells are examined by direct or
indirect IF assay at least for BVDV, BPI3, BAV-3, BPV,
RE03 and RV. All of the results shall be negative.
(9) Evaluation of results: The test is valid if the negative
control cultures shall be absence of CPE and negative for
HAd and IF assay meanwhile. The positive control Cultures
show CPE obviously and positive for HAd test and IF assay.
The test sample is satisfied if the inoculated cultures show no
CPE and negative for HAd test and IF assay. The test
sample is unsatisfactory if the inoculated cultures show any
sign of CPE or positive for HAd test is or positive for any
virus by IF.
For bovine serum to be subjected to virus inactivation, take
the test samples for test prior to inactivation. The serum is
not recommended for biological product use if any of the tests
shows positive result unless the contaminating virus is
identified and shown to be present in an amount that has been
shown to be effectively inactivated in a validation study. If
the BVDV was detected before inactivation, take the sample
again after the inactivation to detect BVDV by sensitive tests.
It shall be not find any evidence of the presence of BVDV.
The bovine serum that is not subjected to virus inactivation
 3605 Culture Media for Biochemical Reactions of Bacteria and Test Method
procedure, the serum is not accepted for use if any of the
tests shows a positive result.
For those bovine viruses where the infectivity assay is not
available, nucleic acid technologies could be used. Nucleic
acid should be extracted from a significant volume of samples
(e. g. 25 ml-50 ml of serum sample) and the statistical limit
for detection in the serum pool should be calculated.
3605 Culture Media for
Biochemical Reactions of
Bacteria and Test Method
The following culture media are commonly used to test the
biochemical reactions arisen from bacteria in the metabolism
of carbohydrates, amino acids and proteins as well as the
utilization of carbon and nitrogen sources.
l. Sugar or alcohol fennentation medium
(1) Formula
Basal medium:
Peptone 10 g
Sodium chloride 5g
O. 5% Acid fuchsin indicator solution 10 ml
(or O. 4% Bromothymol blue solution 6 mD
Water 1000 ml
Sugars or alcohols O. 5 g per 100 ml of basal medium.
(2) Method for preparation
Dissolve peptone and sodium chloride in water by slightly
vvarming. .LL\~djust pH to 7. 3 ± O. 1 ( thc valuc
sterilization). Add acid fuchsin solution ( or bromothymol
blue solution) and mix well. To 100 ml of the solution, add
one kind of sugar, alcohol or glycoside. Mix well. Dispense
the medium into small tubes ( if observation of gas
production is needed, Durham inverted tubes are placed in
small tubes) and sterilize at 116ºC for 15 minutes.
Sugars, alcohols or glycosides commonly used: arabinose,
xylose, rhamnose, glucose, fructose, mannose, galactose,
maltose, lactase, sucrose, trehalose, cellubiose, melibiose,
raffinose, melezitose, inulin, dextrin, starch, mannitol,
dulcitol, sorbitol, inositol, glycerol, salicin, esculin, etc.
(3) Use
The medium is used to differentiate various kinds of bacteria
through the biochemical reactions of sugar fermentation.
Fermentation may produce acid and change the colour of medium
( The colour of medium containing acid fuchsin indicator may
change from colourless to red or even to yellow; the colour of
medium containing bromothymol blue indicator may change from
blue colour to yellow). When gas is produced, small air bubbles
can be seen in Durham inverted tube.
2. Escolio medium
(1) Formula
Peptone 5g
Dipotassium hydrogen phosphate 1g
Ferric citrate o. 5 g
Esculin 3g
Water 1000 ml
(2) Method for preparation
Mix the above ingredients except esculin and dissolve the
solids by slightly warming. Add esculin and mix well.
Adjust pH to 7. 3 ± O. 1 ( the value after sterilization).
Dispense the medium into test tubes and sterilize at 121 ºCfor
15 minutes.
(3) Use
The medium is used to differentiate bacteria based on the test
of esculin hydrolysis. The production of a brownish black
precipitate indicates a positive reaction.
3. Phosphate glucose peptone water medium
(1) Formula
Peptone 7g
Dipotassium hydrogen phosphate 3. 8 g
Glucose 5g
Water 1000 ml
(2) Method of preparation
Mix the above ingredients and dissolve the solids by slightly
warming. Adjust pH to 7. 3 ± O. 1 ( the value after
sterilization). Dispense the medium into small test tubes and
sterilize at 121 ºC for 15 minutes.
(3) Use
The medium is used to differentiate bacteria through methyl
red test (M-R reaction) and acetylmethylcarbinol test (V-P
reaction).
CD Methyl red test ( M-R reaction): lnoculate suspected
colony or slant culture into the phosphate glucose peptone
water medium and incubate atan appropriate temperature for
2-5 days. Add severa! drops of methyl red solution (Dissolve
O. 1 g of methyl red in 300 ml of 95 % ethanol and dilute to
500 ml with water) into the culture tube and observe
immediately. The appearance of bright red or orange red
colour indicates a positive reaction and yellow colour indicates
a negative reaction.
® Acetylmethyl-carbinol test ( V-P reaction) : lnoculate
suspected colony or slant culture into the phosphate glucose
peptone water medium and incubate at an appropriate
temperature for 48 hours. Add 1 ml of crnaphthol ethanol
after solution (Dissolvc 5 g of u.-naphthol in absolute ethanol and
dilute to 100 mD into 2 ml of the culture and mix well.
Then, add O. 4 ml of 40% potassiumhydroxide solution and
mix thoroughly. If a red colour appears instantly or within
severa! minutes, it indicates a positive reaction. If no red
colour appears, it indicates a negative reaction. For a
negative reaction, observe again after incubation in a 35ºC
water bath for 4 hours.
4. Peptone water medium
(1) Formula
Peptone 10 g
Sodium chloride 5g
Water 1000 ml
(2) Method of preparation
Mix the above ingredients and dissolve the solids by slightly
warming. Adjust pH to 7. 3 ± O. 1 ( the value after
sterilization). Dispense the medium in to small test tu bes and
sterilize at 121 ºC for 15 minutes.
(3) Use
The medium is used to differentiate bacteria based on their
ability to decompose tryptophan and produce índole.
lndole test: Inoculate the suspected colony or slant culture
into the peptone water medium and incubate at 35ºC for 24-48
hours, or 4-5 days if necessary. Add several drops of índole
test solution along the wall of test tube. A positive test
shows a rose red colour on the surface of the liquid and a
negative test shows no colour change.
Indole test solution: Dissolve 5 g of p-dimethylaminobenzaldehyde
in 75 ml of pentanol ( or iso-pentanol) and shake
sufficiently. After the dissolution is completed, add slowly
25 ml of concentrated hydrochloric acid dropwise with
constant shaking to prevent the colour from becoming dark
due to the sudden increase of temperature. Alternatively,
add 1 g of p-dimethylaminobenzaldehyde into 95 ml of 95 %
ethanol and shake sufficiently to dissolve completely. Add
slowly 20 ml of concentrated hydrochloric acid dropwise.
 3605 Culture Media for Biochemical Reactions of Bacteria and Test Method

5. Triple sugar iron agar medium colour at the bottom of the medium becomes yellow, it
(1) Formula indicates a positive reaction of glucose fermentation. When
Peptone 20 g the colour of the slant becomes yellow, it indicates a positive
Beef extract 5g reaction of lactase fermentation. When the colour of slant
Lactase 10 g becomes red, it indicates a negative reaction of lactase
Sucrose 10 g fermentation. When a black colour appears at the bottom or
Glucose 1g in the whole medium, it indicates the production of hydrogen
Sodium chloride 5g sulfide.
Ferrous sulfate o. 2 g 7. Urea medium
Sodium thiosulfate o. 2 g (1) Formula
O. 2 % Phenolsulfonphthalein
Peptone 1g
indicator solution 12. 5 ml
Glucose 1g
Agar 12-15 g
Sodium chloride 5g
Water 1000 ml
Dipotassium hydrogen phosphate 2g
(2) Method of preparation
O. 2 % Phenol red solution 6 ml
Mix the above ingredients except lactase, sucrose, glucose,
indicator solution and agar, and dissolve by heating. Adjust 20 % Sterile urea solution 100 ml
pH to 7. 3 ±O. 1 ( the value after sterilization). Add agar, Water 1000 mi
(2) Method for preparation
swell by heating. Then, add the rest ingredients and mix
well. Dispense the medium and sterilize at 121 ºC for 15 Mix the above ingredients well except urea solution. Adjust
minutes, allow to stand to form a short slant with high pH to 6. 9 ±O. 1 ( the value after sterilization). Sterilize the
bottom (2-3 cm). medium at 121 ºC for 15 minutes. Cool clown to 50-55ºC.
(3) Use Add in sterile urea solution ( sterilized by membrane
The medium is used for the preliminary identification of filtration) and mix well. Dispense the medium into sterile
bacteria in Enterobacteriaceae based on the fermentation of test tubes.
sugars and hydrogen sulfide production. (3) Use
Test method and result observation: Streak the suspected The medium is used to differentiate the bacteria by the test of
colony or slant culture onto the medium slant and also urease production.
puncture into the deep bottom of the medium. Incubate at Test method and result observation: Inoculate the suspected
35ºC for 24-48 hours and observe the result. When the colony or a small amount of slant culture onto the urea
colour at the bottom of the medium becomes yellow, it medium. Incubate at an appropriate temperature for 24 hours
indicates a positive reaction of glucose fermentation. When and observe the result. When the colour of the medium
the colour of the slant becomes yellow, it indicates a positive becomes red, it indicates a positive reaction of urease
reaction of lactase and sucrose fermentation. When a black production. If no colour changes, it indicates a negative
colour appears at the bottom or in the entire medium. it reaction. The culture with negative result shall be further
indicates the production of hydrogen sulfide. observed for one week.
6. Kligler double sugar iron medium 8. Phenylalanine agar medium
(1) Formula (1) Formula
Peptone 20 g Disodium hydrogen phosphate 1g
Beef extract 3g Y east extracts 3g
Y eas t extract 3g DL-Phenylalanine 2g
Lactase 10 g (or L-phenylalanine) (1 g)
Glucose 1g Sodium chloride 5g
Sodium chloride 5g Agar 12-15 g
Ferric citrate 0.3g Water 1000 mi
Sodium thiosulfate o. 3 g (2) Method for preparation
O. 2 % Phenolsulfonphthalein Dissolve the above ingredients in water except agar. Adjust
indicator solution 12. 5 mi pH to 7. 3 ±O. 1 ( the value after sterilization). Add agar,
Agar 12-15 g swell by heating. Dispense the medium into test tubes and
Water 1000 ml sterilize at 121 ºC for 15 minutes, allow to stand to form long
(2) Method for preparation slants.
Mix the above ingredients except lactase, indicator solution (3) Use
and agar, and dissolve the solids by heating. Adjust pH to The medium is used to identify bacteria by the test of
7. 3 ±O. 1 ( the value after sterilization). Add agar, swell
phenylalanine deaminase ( or phenyl pyruvic acid test).
by heating. Add the remaining ingredients and mix well.
Test method and result observation: Inoculate a large
Dispense the medium and sterilize at 121 ºC for 15 minutes,
amount of slant culture onto the phenylalanine agar slant.
allow to stand to form a short slant with high bottom (2-3
Incubate atan appropriate temperature for 4 or 18-24 hours.
cm).
Add 4-5 drops of 10 % ferric chloride solution on top of the
(3) Use
slant and let them flow clown along the slant. The
The medium is used for the preliminary identification of
appearance of dark green colour indicates a positive reaction
bacteria in Enterobacteriaceae by test of sugar fermentation
of phenylalaninedeaminase. If no colour changes, it indicates
and hydrogen sulfide production.
a negative reaction.
Test method and result observation: Streak the suspected
colony or slant culture onto the medium slant and also 9. Amino acid decarboxylase test medium
puncture into the deep bottom of the medium. Incubate at (1) Formula
35ºC for 24-48 hours and observe the result. When the CD Basal medium
 3605 Culture Media far Biochemical Reactions of Bacteria and Test Method

Peptone 5g Ammonium sulfate 2g

Yeast extract 3g Dipotassium hydrogen phosphate o. 6 g
Glucose 1g Potassium dihydrogen phosphate o. 4 g
l. 6 % Bromocresol purple indicator Sodium malonate 3g
solution 1 ml O. 4 % Bromothymol blue indicator
Water 1000 ml solution 6 ml
(2) Amino acids Water 1000 ml
L-lysine O. 5 g (Dissolve in alkaline solution) (2) Method far preparation
L-Ornithine O. 5 g (Dissolve in alkaline solution) Dissolve the above ingredients in water except bromothymol blue
L-Arginine O. 5 g (Dissolve in water without adding alkaline solution Adjust pH to 6. 8 ( the value after sterilization). Add
solution) the bromothymol blue solution. Dispense the medium into test
(2) Method far preparation tubes and sterilize at 121 ºC far 15 minutes.
Prepare the basal medium far use. Add the three kinds of (3) Use
amino acids after dissolution to each 100 ml of the basal The medium is used to differentiate the bacteria by judging
medium separately (The final concentration of amino acid is whether the bacteria can utilize sodium malonate as a source
O. 5%). Adjust pH to 6. 8 ( the value after sterilization). of carbon far growth and propagation.
Dispense the medium into small test tubes, 2. 5 ml far each, Test method and result observation: Inoculatethe slant
and add liquid paraffin dropwise onto the medium to farm a culture or broth culture medium into the sodium malonate
layer. At the same time, dispense a portian of the basal medium and incubate at an appropriate temperature far 48
medium into small test tubes as control medium. Sterilize all hours. Observe the result 24 and 48 hours later respectively.
of media at 116ºC far 10 minutes. The colour change of the medium from green to blue
(3) Use indicates a positive reaction. If no colour changes or the
The medium is used to differentiate the bacteria by the test of colour changes from green to yellow, it indicates a negative
decarboxylase and dihydrolase. reaction.
Test method and result observation: Inoculate the suspected 12. Citrate medium
slant culture into the three kinds of media and basal media (1) Formula
separately. Incubate at an appropriate temperature far 24-48 Sodium chloride 5g
hours. Magnesium sulfate o. 2 g
Both the media to be tested and control media shall show Dipotassium hydrogen phosphate 1g
yellow colour at the beginning due to glucose fermentation Ammonium dihydrogen phosphate 1g
and acid production of bacteria to be tested. If the media to Anhydrous sodium citrate 2g
be tested show a purple or purple-red colour during l. O% Bromothymol blue indicator
continuous incubation, it indicates a positive reaction. If solution 10 ml
both the media to be tested and control media still show Agar 14 g
yellow colour at the end of incubation, the reaction shall be Water 1000 ml
judged as negative. ( 2) Methodfarprepara tion
10. Gelatin medium Mix the above ingredients except the indicator solution and
(1) Formula agar and dissolve by slightly warming. Adjust the pH to 6. 9
Peptone 5g ±0. 1 ( the value after sterilization). Add agar, swell by
Beef extract 3g heating. Add the indicator solution and mix well. Dispense
Gelatine 120 g the medium into small test tubes and sterilize at 121 ºC far 15
Water 1000 ml minutes, allow to stand to farm slants.
( 2) Method far prepara tion (3) Use
Mix the above ingredients in water and soak far about 20 The medium is used to differentiate bacteria by judging
minutes. Dissolve the solids by heating and adjust pH to 7. 3± whether the bacteria can utilize citrate as a source of carbon
O. 1 ( the value after sterilization). Dispense the medium into far growth and propaga tion.
small test tubes and sterilize at 121 ºC far 15 minutes. Test method and result observation: Inoculate the suspected
(3) Use colony or slant culture onto the citrate slant and incubate far
The medium is used to differentiate the bacteria by the test of 48-72 hours. The growth of colony on the slant and the
gelatine liquefaction. colour change of the medium from green to blue indicate a
Test method and result observation: Inoculate by puncturing positive reaction. If no growth of colony is faund and the
a small amount of the test slant culture into the gelatine colour of medium is still green, it indicates a negative
medium and incubate at an appropriate temperature far 24 reaction. The culture with negative result shall be further
hours. Take the medium out of the incubator and put into a incubated and observed until the 7th day.
refrigerator far 10-20 minutes. If the medium is still in 13. Nitrate peptone water medium
liquidfarm, the test is positive. If the medium solidifies (1) Formula
again, the test is negative. Sometimes, the reaction of Peptone 10 g
gelatin liquefaction by bacteria is very slowly. If the Y eas t extract 3g
liquefaction of gelatinis not seen, the final judgment of a Potassium nitrate 2g
negative reaction shall be made after 1-2 weeks of continuous Water 1000 ml
incubation. (2) Method far preparation
11. Sodium malonate medium Mix the above ingredients and dissolve in water by heating.
(1) Formula Adjust pH to 7. 3 ± O. 1 ( the value after sterilization).
Yeast extract 1g Dispense the medium into small test tubes and sterilize at
Sodium chloride 2g 121 ºC far 15 minutes.
Glucose o. 25 g (3) Use
 3701 List of National Standards for Biologics

The medium is used to differentiate bacteria by judging 15. Serni-solid agar rnedium
whether the bacteria can reduce nitrate to nitrite. (1) Formula
Test method and result observation: lnoculate the culture to Peptone 10 g
be tested into the nitrate peptone water medium and incubate Beef extract 3g
at an appropriate temperature for 24 hours. Mix the Sodium chloride 5g
following two solutions CA and B) at equal volumes. To Agar 4g
each culture, add O. 1 ml of the mixture. If a red colour Water 1000 ml
appears, it indicates a positive reaction. If no red colour (2) Method for preparation
appears, it indicates a negative reaction. Mix the above ingredients except agar and dissolve the solids
Solution A: Dissolve 5 g of a-naphthylamine in 1000 ml of 5 by slightly warming. Adjust the pH to 7. 2 ±O. 2 Cthe value
mol/L acetic acid. after sterilization). Add agar, swell by heating. Dispense
Solution B: Dissolve 8 g of sulfanilic acid (p-aminobenzene the medium into small test tubes and sterilize at 121 ºC for 15
sulfonic acid) in 1000 ml of 5 mol/L acetic acid. minutes and solidify by standing vertically.
(3) Use
14. Litrnus rnilk rnedium
(1) Formula
The medium is used to observe the motility of bacteria as
Skimmed milk 10 g well as to preserve bacterial strains.
10% Litmus solution O. 65 ml Test for the mobility of bacteria: Inoculate by puncturing
Water 100 ml the suspected slant culture into the semi-solid nutrient agar
(2) Method for preparation medium and incubate at an appropriate temperature for 24
Dissolve the skimmed milk in water and add in 10% litmus hours. If the growth of bacteria diffuses around the
solution. Dispense the medium into small test tubes and puncture line, the result is positive. Otherwise, it is
sterilize at 116ºC for 10 minutes. negative and the culture shall be further incubated and
(3) Use observed for 2-3 days.
The medium is used to examine the bacteria{ or their
solidification and fermentation effects on milk.

3700

3701 Lisi of Naiionai Standards for Blologics

(far Potency, Titer, Activity, Concentration and Content Tests)

Name Usage
Purified Protein Derivative of BCG Standard Potency test
Purified Protein Derivative of Tuberculin Standard Potency test
Bacteria Turbidity Standards Bacterial content
Ref erence of BCG Content Bacterial content
References of Bacteria Content of Plague Vaccine, Brucellosis Vaccine and Anthrax Bacteria content
Vaccine (Live) for Percutaneous Scarification
Diphtheria Toxoids Standard Potency test
Tetanus Toxoids Standard Potency test
Pertussis Vaccine Standard Potency test
Reference for Content Test of Typhoid Vi Polysaccharide Content test
Reference of Recombinant Hepatitis B Vaccine CYeast) Relative potency test (in vitro)
Rabies Vaccine Standard Potency test
Reference of J apanese Encephalitis Vaccine Clnactivated) Potency test
Reference of Japanese Encephalitis Vaccine (Live) Virus titration
Reference of Poliomyelitis Vaccine CType I , Live) Virus titration
Reference of Poliomyelitis Vaccine CType II , Live) Virus titration
Reference of Poliomyelitis Vaccine CType fil, Live) Virus titration
Reference of Measles Vaccine (Live) Virus titration
Reference of Rubella Vaccine (Live) Virus titration
Reference of Mumps Vaccine (Live) Virus titration
Anti-HBs Standard (Plasma) Antibody potency test
 3701 List of National Standards for Biologics

continued
Name Usage
Anti-HBs Standard (Human Immunoglobulin) Antibody potency test
Human Rabies Immunoglobulin Standard Antibody potency test
Human Diphtheria lmmunoglobulin Standard Antibody potency test
Human Tetanus lmmunoglobulin Standard Antibody potency test
Tetanus Antitoxin Standard Antibody potency test
Reference of Botulinum (Type A) Antitoxins Antibody potency test
Reference of Botulinum (Type B) Antitoxins Antibody potency test
Reference of Botulinum (Type E) Antitoxins Antibody potency test
Reference of Botulinum (Type F) Antitoxins Antibody potency test
Reference of Gas-gangrene (Cl. welchii) Antitoxin Antibody potency test
Reference of Gas-gangrene (Cl. oedematiens) Antitoxin Antibody potency test
Reference of Gas-gangrene (Cl. histolyticum) Antitoxin Antibody potency test
Reference of Gas-gangrene (Cl. septicum) Antitoxin Antibody potency test
Naja naja (atra) Antivenin Standard Antibody potency test
Agkistrodon acutus Antivenin Standard Antibody potency test
Agkistrodon halys Antivenin Standard Antibody potency test
Bungarus multicinctus Antivenin Standard Antibody potency test
Human Thrombin (Freeze-dried) Standard Activity test
Human Coaguiation Factor Vill Standard Activity test
Human Coagulation Factor II , W, IX, X Standard Activity test
Recombinant Human lnterleukin-2 Standard Activity test
Recombinant Human lnterferon alb Standard Activity test
Recombinant Human Epidermal Growth Factor Standard Activity test
Recombinant Streptokinase Standard Activity test
Recombinant Human Granulocyte Colony Stimulating Factor Standard Activity test
Recombinant Human Granulocyte/Macrophage Colony Stimulating Factor Standard Activity test
Recombinant Bovine Basic Fibroblast Growth Factor Standard Activity test
Recombinant Human lnterferon a2a Standard Activity test
Recombinant Human Erythropoietin Standard Activity test
Recombinant Human lnterferon a2b Standard Activity test
Protein Content Standard (Human Albumin) Protein content test
Prekallikrein Activator Standard PKA content test
Reference for Host Bacteria! Protein Test CE. coli) Residual host bacteria! protein test
Residual DNA (Vero CelD (for Hybridization) Standard Residual DNA test (Vaccine)
Residual DNA (CHO CelD Standard Residual DNA test
Residual DNA Standard CE. coli) Residual DNA test
Reference of Anti-A and Anti-B Blood Grouping Reagents (Monoclonal Antibody) Quality control for diagnostic kit
Reference of Hepatitis B Surface Antigen Quality control for diagnostic kit
Referen~e of Diagnostic Kit for Antibody to Hepatitis C Virus Quality control for diagnostic kit
Reference of Diagnostic Kit for Antibody to Human Immunodeficiency Virus Quality control for diagnostic kit
Reference of Diagnostic Kit for Syphilis (Non-specific) Quality control far diagnostic kit
Reference of Diagnostic Kit for Syphilis (Specific) Quality control for diagnostic kit
 8001 Reagents

8000 Reagents and Reference Substance

Acetonitrile [CH3CN 41. 05]

8001 Reagents
Reagents are substances used for various assays and tests
described in the pharmacopoeia. However, adsorbents,
supports and packing materials used in chromatography are
not included. With the exception of biochemical reagents and
indicators, chemical reagents are classified into 4 grades:
primary standard, guaranteed reagent, analytical pure and
chemical pure. The following rules may be followed in
choosing the grade of reagent for different purpose.
(1) Primary standard should be used for standardization of
volumetric solutions.
(2) Reagents of "analytical pure" or "chemical pure" grade
may be used for the preparation of volumetric solutions, but
in case of the concentration of the solution is determined
according to the weight of the solute without standardization,
"primary standard" should be used.
(3) "Guaranteed reagents" or "analytical pure" grade should
be used for the preparation of standard solutions used for
limit tests of impurities.
(4) Reagents of "analytical pure" or "chemical pure" grade
may be used for the preparation of test solutions and buffer
solutions, etc..
Acacia White or slightly yellow granules or powder. Freely
soluble in water and forms viscous liquid; insoluble in
ethanol.
Acetaldehyde [CH3CHO 44. 05]
A clear, colourless liquid; suffocating odour, freely volatile;
inflammable; easily oxidized into acetic acid; polymerized to
form turbidity or sediment after long storage.
Miscible with water, ethanol, chloroform or ether.
Acetanilide [C8 Hg NO 135. 16]
Lustrous scale crystals or a white power; taste, slightly
burning; volatile at about 95ºC.
Freely soluble in ethanol, chloroform, ether, acetone or hot
water; slightly soluble in water; practically insoluble in petroleum
ether.
Acetic Acid [Cz H4 0 2 60. 05]
A clear, colourless liquid; it contains 36%-37% of C2H4Ü2
(g/g).
Miscible with water, ethanol or ether; insoluble in carbon
disulfide.
Acetic Acid, Glacial [CH3COOH 60. 05]
A clear, colourless liquid; odour, irritant; corrosive;
solidifies to icy crystals below its congealing point (16. 7°C ).
Miscible with water or ethanol.
Acetic Anhydride [ CCH3C0)20 102. 09]
A clear, colourless liquid.
Miscible with chloroform, ether or glacial acetic acid; soluble
in water to form acetic acid; soluble in ethanol to formethyl
aceta te.
Acetone [CH3COCH3 58. 08]
A clear, colourless liquid; odour, characteristic; highly
volatile; inflammable.
Soluble in water or ethanol.
A clear, colourless liquid; slightly ether-like, odour; inflamrnable.
Miscible with water or ethanol.
Acetylacetone [CH3COCH2COCH3 100. 12]
A colourless or pale yellow liquid with slight acetone and
acetic acid odour; inflammable.
Miscible with water, ethanol, ether or chloroform.
Acetyl Chloride [CH3COC1 78. 50]
A colourless fumy liquid; pungent, odour; inflammable;
highly irritant to skin and mucous membrane; decomposes
vigorously in water or ethanol.
Soluble in chloroform, ether, benzene, petroleum ether or
glacial acetic acid.
N-Acetyl-L-Tyrosine Ethyl Ester [C13 H11 N0 4 251. 28]
A white powder.
Biochemical reagent for determination of the potency of
chymotrypsin.
Acrylamide [C3HsNO 71. 08]
White, flake crystals.
&:>luble in water, ethanol, ether, acetone or chloroform; slightly
soluble in toluene; insoluble in benzene or n-heptane.
Agar Complies with the requirements prescribed in the
monograph of the Chinese Pharmacopoeia.
Agarose
White or pale yellow granules or powder; hygroscopic. &:>luble in
hot water.
Alcohol
See Ethand.
Alizarin Fluoro-blue [ C19 H1s NOs 385. 33 J
An orange powder.
Slightly soluble in water, ethanol or ether.
Alizarin Red [C14H1Na01S•H20 360. 28]
A yellowish-brown or orange powder.
Freely soluble in water; slightly soluble in ethanol; insoluble
in benzene or chloroform.
Aluminium Hydroxide [Al (0H) 3 78. 00]
A white powder; odourless.
Soluble in hydrochloric acid, sulfuric acid or sodium
hydroxide; insoluble in water or ethanol.
Aluminium Nickel Alloy ( Raney' s Alloy)
Grey metallic substance.
Its aluminium dissolves in solution of sodium hydroxide to
liberate hydrogen, the remaining nickel is active.
Aluminium Nitrate [Al CN03 )3•9H2 O 375. 13]
White crystals; hygroscopic; induces combusion and
explosion when heated with organic substances.
Freely soluble in water and ethanol; slightly soluble in
acetone; insoluble in ethyl acetate and pyridine.
Aluminium Oxide [Alz 0 3 101. 96]
A white powder; tasteless; hygroscopic.
Soluble in sulfuric acid; slowly soluble in sodium hydroxide
solution with the formation of hydroxides; insoluble in water,
ethanol or ether.
AluminiumSulfate [Alz (SQ4)3•l8H20 666.43]
White lustrous crystals or a crystalline powder.
 326 8001 Reagents
Soluble in water; insoluble in ethanol.
Aluminium Trichloride [AlCb 133. 34]
White or pale yellow crystals ora crystalline powder; odour,
characteristic of hydrochloric acid; fuming on exposure to
air; liberation of much heat or even explosion occurs m
contact with water; hygroscopic; corrosive.
Soluble in water or ether.
Amido Black 10 B [C22Hl4N6Naz09Sz 616. 50]
A brownish-black powder.
Soluble in water, ethanol or ether to form a bluish-black
solution, soluble in sulfuric acid to form a green solution;
slightly soluble in acetone.
4-Aminoantipyrine [Cu 813 N3 O 203. 24]
Pale yellow crystals.
Soluble in water, ethanol or benzene; slightly soluble m
ether.
p-Arninobenzoic Acid [C1 H1 NOz 137. 14]
White crystals; turns pale yellow on exposure to air or to
light.
Freely soluble in boiling water, ethanol, ether or acetic acid;
very slightly soluble in water.
7-Aminodesacetoxycephalosporanic Acid [ Cs H10 Nz 03 S
214.25]
A white or faintly yellow crystalline powder.
Insoluble in water, ethanol or acetone; soluble in strong acid
and alkali.
1-Amino-2-naphthol-4-sulfonic Acid [C10 H9N04S 239. 25]
White or grey crystals; discoloured on exposure to light;
hygroscopic.
Soluble in hot sodium bisulfite solution or in alkaline
solution, easily oxidized; insoluble in water, ethanol or
ether.
p-Aminophenol [C6H1NO 109. 13]
A white or yellow crystalline powder; discoloured gradually
on exposure to air or light.
Soluble in hot water or ethanol.
Ammonia [NH 3 17]
Obtained by heating ammonium salt (ammonium chloride) with
strong base ( calcium hydroxide ) , or heating concentrated
ammonia solution then the gas is dried with calcium oxide.
Colourless; odour, characteristic of ammonia; liquif ying
at -33ºC, congealing to colourless crystals at -78ºC.
Very soluble in water with the liberation of much heat.
Ammonium Acetate [NH4 C2 H3 02 77. 08]
White granules or crystals; hygroscopic.
Soluble in water or ethanol; slightly soluble in acetone.
Ammonium Aurintricarboxylate [C22 H 23 N 30 9 473. 44]
A brownish-yellow or dark red powder or granules.
Soluble in water or ethanol.
Ammonium Carbonate
A mixture of ammonium bicarbonate and ammonium carbamate;
white transparent mass or powder; odour, ammonia-like.
Soluble in water; decomposes in hot water; insoluble in
ethanol or concentrated ammonia solution.
Anunonimnc.ericSulfate [Ce (SQ4)2•2 <Na)2S04•4H20
668.58]
A yellow or orange-yellow crystalline powder.
Soluble in solutions of acid; slightly soluble m water;
insoluble in acetic acid.
Ammonium Chloride [NH4 Cl 53. 49 J
White crystals or a crystalline powder.
Soluble in water or glycerin; slightly soluble in ethanol.
Ammonium Citrate Dibasic [ (NH4 )z HC6 Hs 01 226. 19]
Colourless fine crystals or white granules.
Soluble in water, slightly soluble in ethanol.
Ammonium Citrate, Tribasic [C6 H11N301 243. 22]
A white powder; deliquescent.
Freely soluble in water; insoluble in ethanol, acetone or
ether.
Ammonium Ferric Citrate [C12 H22FeN3 014 488. 16]
A brownish red or green flake or powder; deliquescent. It
reduces to ferrous on exposure to light.
Soluble in water; insoluble in ethanol or ether.
Ammonium Fonnate [CHsN02 63. 06]
Colourless crystals or granules; deliquescent; soluble in
water or ethanol.
AmmoniumMolybdate [ (NH4)6Mo1024•4H20 1235. 86]
Colourless or pale yellowish-green crystals.
Soluble in water, insoluble in ethanol.
Ammonium Nitrate [NH4 N03 80. 04]
White, transparent crystals ora white powder.
Freely soluble in water; slightly soluble in ethanol.
Ammonium Oxalate [ (NH4)2C204•H20 142. 11]
White crystals; decompose on heating.
Soluble in water; slightly soluble in ethanol.
Ammonium Persulfate [ (NH4)2SzOs 228. 20]
White, transparent crystals or powder; odourless; strong
oxidizing agent.
Freely soluble in water.
Ammonium Phosphate Monobasic [NH4H2P04 115. 03]
Colourless crystals or a white crystalline powder; tasteless.
It loses 8 % of ammonia on exposure to air.
Slightly soluble in ethanol; insoluble in acetone.
Ammonium Reineckate [NH4 Cr ( NH3) 2 ( SCN) 4• H2 O
354.45]
Red or deep red crystals; decompose in aqueous solution with
formation of a blue colour and liberate hydrogen cyanide.
Soluble in hot water and in ethanol; slightly soluble in water.
Ammonium Sulfamate [NH2 S03 NH4 114. 13]
White crystals; hygroscopic.
Freely soluble in water; slightly soluble in ethanol.
Ammonium Sulfate [ CNH4)2S04 132. 14]
White crystals or granules.
Soluble in water; insoluble in ethanol or in acetone.
Ammonium Thiocyanate [NH4SCN 76. 12]
White crystals.
Freely soluble in water and ethanol; soluble in methanol and
acetone.
Practically insoluble in chloroform or ethyl acetate.
Ammonium Vanadate [NH4 V03 116. 98]
A white or slightly yellow crystalline powder.
Freely soluble in hot water or dilute ammonia solution;
slightly soluble in cold water; insoluble in ethanol.
Arnyl Acetate [CH 3C00Cs Hn 130. 19]
A clear, colourless liquid; fruit-like, odour; inflammable.
Miscible with ethanol or ether; slightly soluble in water.
Aniline [C6HsNH2 93.13]
A colourless or pale yellow, clear oily liquid; odour,
characteristic; it turns brown gradually on exposure to light
and air; inflammable.
Miscible with ethanol, ether or benzene; slightly soluble in water.
Anilinoacetic Acid ( Phenylglycine) [ Cs Hg N02 151. 16]
White or pale yellow crystals.

Soluble in water; slightly soluble in ethanol or ether.
P-Methoxybemaldehyde (Anisaldehyde) [CH3 0Cs f4 CHO
136. 15]
A colourless oily liquid.
Miscible with alcohols or ether; slightly soluble in water.
Anthrone [C14H100 194.23]
White crystals.
Soluble in ethanol, benzene or hot sodium hydroxide
solution; insoluble in water.
Antimony Trichloride [SbCb 228. 11]
White crystals; fuming on exposure to air; hygroscopic;
corrosive.
Soluble in ethanol, acetone, ether or benzene; soluble m
water and produces sediment of antimony hydroxide.
Antimony Po~ium Tartrate [ C H4 K01 Sb. 1/2 H2 O
333.93]
White transparent crystals or a white powder; odourless;
taste, slightly sweet; efflorescent.
Soluble in water; insoluble in ethanol.
Antithrombin :m:
White freeze drying mass. Extracted from human plasma and
purified by affinity chromatography.
Arsenic Trioxide [As2 Ü3 197. 84]
A white crystalline powder; odourless; tasteless; when
slowly heated, sublimes but does not decompose.
Soluble in boiling water, sodium hydroxide solution or
sodium carbonate solution; slightly soluble in water;
practically insoluble in ethanol, chloroform or ether.
Ascorbic Acid [CG Hs 06 176. 13]
Complies with the requirements prescribed in the monograph
of the Chinese Pharmacopoeia, volume 11.
Auric Chloride [HAuCl4•3H2 O 393. 83]
Bright yellow or orange crystals.
Soluble in water, ethanol or ether; slightly soluble m
chloroform.
Azo Violet [C12H9N3Ü4 259. 22]
A reddish-brown powder.
Soluble in acetic acid, sodium hydroxide solution or toluene.
Barbital [CsH12N2Ü3 184. 19]
White crystals or powder; taste, slightly bitter.
Soluble in hot water, ethanol, ether or alkaline solution.
Barbital Sodium [CsHuN2NaÜ3 206.18]
White crystals or powder; taste, bitter.
Soluble in water; slightly soluble in ethanol; insoluble m
ether.
Barium Chloride [BaClz• 2H2 O 244. 26]
White crystals or granules.
Freely soluble in water or methanol; practically insoluble in
ethanol, acetone or ethyl acetate.
Barium Hydroxide [Ba <OH>2•8H20 315. 46]
White crystals; absorbs carbon dioxide easily to form barium
carbonate.
Freely soluble in water; slightly soluble in ethanol.
Barium Nitrate [Ba CNQ3)2 261. 34]
White crystals or crystalline powder; burns and detonates
when being in contact, rubbing or bumping with organic
substances.
Soluble in water; insoluble in ethanol.
Barium Perchlorate [Ba CC104 )2•3H2 O 390. 32]
Colourless crystals. Poisonous.
Soluble in water or methanol; slightly soluble in ethanol,
8001 Reagents
ethyl acetate or acetone; practically insoluble in ether.
Beef Extract
A yellowish-brown to dark brown pasty substance; odour,
characteristic of beef; taste sour.
Soluble in water.
Chloride Not more than 6 % of the total solids, calculated
as NaCl.
Nitrate Decolourize a solution of beef extract Cl-10) by
boiling with active charcoal, filter, add 1 drop of the filtrate
to 3 drops of a solution of diphenylamine in sulfuric acid
0-100), no blue colour is developed.
Ethanol insoluble substance To 25 ml of a solution of beef
extract 0 -10) add 50 ml of ethanol, shake thoroughly and
filter, wash the residue with dilute ethanol (2-3), dry it at
105ºC for 2 hours, the residue is not more than 10 % of the
total solids.
Ethanol soluble nitrogen The nitrogen content of the
filtrate obtained in the test for ethanol insoluble substance is
not less than 6 % of the ethanol soluble substance.
Total solids Mix 10 ml of a solution of beef extract 0 -
10) with clean sand or asbestos, dry at 105ºC for 16 hours,
the residue is not less than O. 75 g.
Residue on ignition Not more than 30 % of the total solids
<0841>.
Beef Extract Powder
A beige powder; hygroscopic; soluble in water.
Beef Hemoglobin
Dark brown crystals or crystalline powder.
Soluble in water or dilute acid.
Purity One band should be obtained af ter developing by
electrophoresis on cellulose acetate membrane.
Total Nitro gen Not less than 16. O% ( 0704, Method 1).
Loss on drying When dried to constant weight at l05ºC,
loses not more than 10. 5 % of its weight ( 0831 ) .
Residue on ignition Not more than l. O% ( 0841 ) .
Benzalkonium Chloride
A white or pale yellow powder or gelatinous flakes.
Very soluble in water, ethanol or acetone; slightly soluble in
benzene; practically insoluble in ether.
Benzene [C6 H6 78. 11]
A colourless clear liquid; odour, characteristic;
inflammable.
Miscible with ethanol, ether, acetone, carbon tetrachloride,
carbon disulfide or acetic acid; slightly soluble in water.
Boiling point: 80. 1ºC.
Benzidine [H2 NC6 H4 C6 H4 NH2 184. 24]
A white or faintly red crystalline powder; darkens on
exposure to air and light.
Freely soluble in boiling ethanol; sparingly soluble in ether;
slightly soluble in boiling water; very slightly soluble in cold
water.
Benzidine Acetate [ C14 H 16 Nz 02 244. 29]
White or pale yellow crystals or powder.
Soluble in water, acetic acid or hydrochloric acid; very
slightly soluble in ethanol.
Benzoic Acid [C6HsCOOH 122. 12]
Complies with the requirements prescribed in the monograph
of the Chinese Pharmacopoeia.
Benzoquinone [ Cs H4 0 2 108. 1OJ
Yellow crystals; odour, characteristic; sublima ble.
Soluble in ethanol or ether; slightly soluble in water.
N-Benzoyl-L-Arginine Ethyl Ester Hydrochloride
[ C1s H23 ClN4 03 342. 82]
 8001 Reagents
White or off-white crystalline powder.
Very soluble in water or dehydrated ethanol.
Benzoyl-DL-argrinyl-naphthylamine Hydrochloride
[C22 Hzs Ns 02• HCl 439. 94]
White crystals.
Soluble in water or ethanol.
Benzoyl Chloride [C6HsCOCl 140. 57]
A colourless, clear liquid; irritant; corrosive; fuming on
exposure to moist air; its vapour is corrosive and lachrymatory.
Miscible with ether or carbon disulfide; decomposes in water
or ethanol.
Bismuth Subgallate [GHsBi06•H20 430.12]
Yellow powder; odourless; tasteless.
Soluble and decomposes in dilute mineral acid or dilute alkali
hydroxide solution; practically insoluble in water, ethanol,
ether or chroloform.
Bismuth Subnitrate [ 4BiN03 (OH ) 2 • BiO (OH)
1461. 99]
A white powder; heavy; odourless; tasteless; slightly
hygroscopic.
Soluble in hydrochloric acid, nitric acid, dilute sulfuric acid
or acetic acid; practically insoluble in water or ethanol.
Bis ( cyclohexanone) oxalyldihydrazone [ C14 Hzz N4 02
278.36]
White crystals.
Soluble in hot methanol or ethanol; insoluble in water.
Blue Dextran 2000
Prepared from freeze drying of dextran T 2000 ( the average
molecular weight of about 2 000 000 ) by introduction of
polycyclic chromophore that colours the substance blue.
Freely soluble in water and electrolyte solutions.
Borax [Na2B401•lOH20 381. 37]
White crystals or granules; hard.
Soluble in water and glycerin; insoluble in ethanol or acid.
Boric Acid [H3 BQ3 61. 83]
White transparent crystals or a crystalline powder; with
pear-like luster.
Freely soluble in hot water, hot ethanol or hot glycerin;
soluble in water or ethanol; slightly soluble in acetone or
ether.
Brilliant Green [Cz1 H33Nz• HS04 482. 64]
Lustrous golden crystals.
Soluble in water or ethanol, the colour of solution is green.
Bromine [Brz 159. 81]
A deep red liquid, odour, asphyx.iating irritant; fuming;
freely volatile.
Miscible with ethanol, chloroform, ether benzene or carbon
disulfide; slightly soluble in water.
Bromocresol Green [ Cz1 Hl4 Br4 Os S 698. 02]
A pale yellow or brown powder.
Soluble in ethanol or dilute alkaline solution; insoluble m
water.
Bromocresol Purple [C21 Hl6Br20sS 540. 23]
A pale yellow or pale red crystalline powder.
Soluble in ethanol or dilute alkaline solution; insoluble in
water.
Bromophenol Blue [ Cl9 H10 Br4 Os S 669. 97]
A yellow powder.
Soluble in ethanol, ether, benzene or dilute alkaline solution;
slightly soluble in water.
Bromothyrnol Blue [ Cz1 Hzs Brz Os S 624. 39]
A white or pale red crystalline powder.
Freely soluble in ethanol, dilute alkaline solution or ammonia
solution; slightly soluble in water.
2, 3-Butanedione [C4 HG 02 86. 09]
A yellowish-green liquid; odour, characteristic.
Miscible with ethanol or ether; soluble in water.
Butanol (n-Butanol) [CH3 (CH2 )3 OH 74. 12]
A clear, colourless liquid; odour, characteristic; inflammable;
highly refractive.
Miscible with ethanol, ether or benzene; soluble in water.
Boiling point: 117-118ºC.
t-Butanol [ (CH3)3COH 74. 12]
White crystals, or liquid when contain small amount of
water; odour, camphor like; hygroscopic; inflammable.
Miscible with ethanol or ether; soluble in water.
Boiling point: 82. 4ºC.
Butanone [CH3COCz Hs 72. 11]
A colourless liquid; freely volatile; inflammable; azeotropic
with water; acutely irritant to mucous membrane of nose and
eyes.
Miscible with ethanol or ether.
Butyl Acetate [CH3COO(CH2)3CH3 116.16]
A clear, colourless liquid.
Miscible with ethanol or ether; insoluble in water.
Butylated Hydroxytoluene [C1s H24 O 220. 4]
Colourless crystals or a white crystalline powder.
Melting point: about 70ºC.
Cadmium Aeetate [Cd(CzH302)2•2H20 266. 53]
White crystals.
Freely soluble in water; soluble in ethanol; very slightly
soluble in ether.
Cadmium lodide [Cdlz 366. 22]
White or pale yellow crystals ora crystalline powder.
Soluble in water, ethanol, ether, ammonia solution or acids.
Cadmium Nitrate [Cd(N03 )z•4Hz O 308. 49]
White needle or prismatic crystals; deliquescent. Hypergolic
and detonable when mixed with organic substances.
Freely soluble in water, soluble in ethanol, acetone or ethyl
acetate; practically insoluble in concentrated nitric acid.
Caffeine [CsH10N402•H20 212. 21]
White or yellowish-green needle crystals with silky luster;
odourless; taste bitter; efflorescent.
Freely soluble in hot water or chloroform; sparingly soluble
in water, ethanol or acetone; slightly soluble in ether.
Calcein [~o H24 Nz Naz 013 666. 51]
A bright yellow powder.
Soluble in water; insoluble in dehydrated ethanol or ether.
Calcium Carbonate [CaC03 100. 09]
A white crystalline powder.
Soluble in acid; insoluble in water and ethanol.
Calcon Carboxylic Acid
Brown to black colour crystals or a brown powder.
Freely soluble in alkaline solution and ammonia solution;
slightly soluble in water.
Calcium Chloride [CaClz•2H20 147. 01]
White granules or lumps; hygroscopic.
Freely soluble in water or ethanol.
Calcium Chloride, Anhydrous [CaClz 110. 99]
White granules or fused masses; highly hygroscopic.
Freely soluble in water or ethanol; liberates much heat when
dissolved in water.

Calcium Fluoride [CaF2 78. 08]
A white powder or cubic crystals; luminous when heated.
Soluble in concentrated mineral acids to liberate hydrogen-
fluoride; insoluble in water.
Calcium Hydroxide [Ca(OH)2 74. 09]
A white crystalline powder; absorbs carbon dioxide easily to
form calcium carbonate.
Slightly soluble in water.
Calcium orthophosphate [Ca3 (P04 )z 310. 20]
A white amorphous powder; tasteless. Stable in air,
decomposes in hot water. Soluble in dilute hydrochloride acid
or nitric acid; practically insoluble in water, ethanol or acetic
a cid.
Calcium Sulfate [CaS04•2H20 172.17]
A white crystalline powder.
Soluble in solutions of ammonium salts, sodium thiosulfate,
sodium chloride or acids; insoluble in water or ethanol.
Calcon [C20H13N2Na05S 416. 39]
A brown or brownish-black powder.
Soluble in water or ethanol.
Carbon Active [C 12. 01]
A black fine powder; odourless, tasteless; high adsorption
capacity of organic pigment and nitrogen base.
Insoluble in any solvents.
Calcon Carboxylic Acid
Brown to black crystals or a brown powder.
Easily soluble in alkaline solution or concentrated ammonia
solution.
Camphor [C10H1sO 152. 25]
A white crystalline powder or colourless translucent hard
solid, easily reduced to fine powder after adding a little
ethanol, chloroform or ether; odour, pungent; taste,
pungent then cool and refreshing; easily volatilizes at normal
temperature, burns with black fume and luminous flame.
Very soluble in chloroform; freely soluble in ethanol, ether,
fatty oil or volatile oil; very slightly soluble in water.
Camphor oil
Natural oil with strong odour of camphor.
Soluble in ether or chloroform; insoluble in ethanol.
Camphor Sulfonic Acid [ C10 H1s 04 S 232. 30]
White columnar crystals.
Slightly soluble in glycerin, glacial acetic acid or ethyl
acetate; very slightly soluble in ethanol; practically insoluble
in ether.
Carbon Disulfide [ CSz 76. 14]
A clear, colourless liquid; the pure substance has an ether-
like odour; the usual commercial grade has a foul smell;
inflammable; decomposes on standing for a long time.
Freely soluble in ethanol or ether; insoluble in water. It can
dissolve iodine, bromine, sulphur, fat, rubber etc.
Boiling point: 46. 5 ºC.
Carbon Tetrachloride [CC14 153. 82]
A clear, colourless liquid; odour, characteristic; heavy.
Miscible with ethanol, chloroform, ether or benzene; very
slightly soluble in water.
Casein
A white or pale yellow granular powder; odourless.
Insoluble in water or other neutral solvent; freely soluble in
commonia solution or sodium hydroxide solution.
Alkalinity Shake 1 g of the casein with 20 ml of water for
10 minutes and filter, the filtrate exhibits no alkaline reaction
with litmus paper.
8001 Reagents
Nitrogen 15. 2%-16. o%, calculated on the dried basis
(0704).
Fats Not more than O. 5 % ( 0713).
Water soluble matter Not more than O. 1%.
Loss on drying Not more than 10. O% ( 0831).
Residue on ignition Not more than 1 % ( 0841 ) .
Casein tryptone
A slightly yellow powder.
Derived by digestion of casem with trypsin; easily
hygroscopic.
Soluble in boiled water.
Catechol [Cs Hs 02 110. 11]
Colourless or pale grey crystals or a crystalline powder;
volatilizes with steam.
Freely soluble in water, ethanol or benzene.
Catechol Violet [ C19 Hl4 01 S 386. 38]
A reddish-brown crystalline powder with metallic lustre.
Freely soluble in water or ethanol.
Ceric Ammonium Nitrate ( Ammonium Ceric Nitrate
[ (NH4 )zCe(N03 )s 548. 22]
Reddish-orange crystals; strong oxidizing agent.
Soluble in water or ethanol; insoluble in concentrated nitric
a cid.
Ceric Sulfate [Ce(S04 )z 332. 24]
Deep yellow crystals.
Soluble in hot acid solution; slightly soluble m water,
decomposes with the formation of basic salt.
Cerous Nitrate [Ce(N03)3•6H20 434. 22]
White, transparent crystals.
Soluble in water, ethanol or acetone.
Cesium Chloride [CsCl 168. 36]
Colourless cubic crystals or a white crystalline powder;
deliquescent.
Freely soluble in water; slightly soluble in ethanol.
Cetrimonium Bromide [Cl6H33N(CH3)3Br 364. 45]
A white crystalline powder.
Soluble in water; slightly soluble in ethanol; insoluble m
ether.
Chloral Hydrate [CzH3Ch02 165. 40]
White crystals; odour, pungent; irritant to skin; volatilizes
gradually on exposure to air; turns to yellow after long time
storage.
Soluble in ethanol, chloroform or ether; soluble in water
with hydrolysis.
Chloramine T [Cr H1ClNNa02S•3H20 281. 69]
A white crystalline powder; odour, faintly chlorine-like.
Soluble in water; insoluble in chloroform or benzene.
Chlorinated Lime ( Bleaching powder)
A greywhite granular powder; odour, chlorine-like; It
absorbs water and carbon dioxide and decomposes slowly on
exposure to air.
Chlorine [Clz 70. 90]
Obtained by the action of hydrochloric acid and manganese
dioxide.
Yellowish-green gas; odour, strong suffocating.
Freely soluble in carbon disulfide or carbon tetrachloride;
soluble in water or alkaline solution.
p-Chloroaniline [CsH 6 ClN 127.57]
White or deep yellow crystals.
Soluble in hot water, ethanol, acetaldehyde or acetone.
Chloroform [CHCh 119. 38]
 8001 Reagents
A colourless, dear liquid; heavy; refractive; volatilizes easily.
Miscible with ethanol, ether, benzene or petroleum benzin;
slightly soluble in water.
Chloroform, Ethanol Free [CHCb 119. 38]
Wash 500 ml of chloroform with three portions of water,
each of 50 ml. Dry the chloroform layer over anhydrous
sodium sulfate for more than 12 hours, filter with absorbent
cotton and distill.
This solution should be freshly prepared.
p-Chlorophenol [ C6 Hs ClO 128. 56]
White crystals; odour, phenolic.
Freely soluble in ethanol or ether; slightly soluble in water.
Chloroplatinic Acid [Hz PtC16 • 6 H 2O 517. 90]
Orange red crystals; highly deliquescent.
Freely soluble in water; soluble in chloroform, acetone or
ether.
Chlorosulfonic Acid [S02ClOH 116. 52]
A colourless or slightly yellow liquid; corrosive and strongly
irritant; fuming in air; explosive when dripped into water;
decomposes to sulfuric acid and hydrochloric acid in water,
also decomposes in alcohols or acids.
Cholesterol [C21 H46 O 386. 66]
Monohydrate of cholesterol is white or pale yellow laminar
crystals; anhydrous cholesterol is obtained at 70-80ºC ; it
turns to yellow on exposure to air.
Soluble in benzene, petroleum benzin or vegetable oil;
slightly soluble in ethanol; practically insoluble in water.
Choodroitinase ABC
Extracted mainly from ordinary proteus. It can degrade
chondroitin sulfate.
A white to brown or pale orange powder.
Soluble in water.
Chrome Azurol S [C23H13ClzNa309S 605. 31]
A brown powder.
Soluble in water to form a brownish-yellow solution; shows
lower solubility in ethanol than in water with reddish-brown
colour.
Chromic Acid [H2 Cr04 118. 01]
An aqueous solution of chromium trioxide.
Chromium Trioxide [Cr03 99. 99]
Dark red crystals; powerful oxidizing agent and corrosive;
hygroscopic; may cause inflame in contact with organic
substances.
Freely soluble in water; soluble in sulfuric acid.
Chromogenic Substrate S-2238 [ H-D-Phe-Pip-Arg-pNA •
2HC1 625. 6]
White freeze-drying mass. It's the specific chromogenic
substrate of factor II •.
Chromogenic Substrate S-2765 [ N-a-Z-D-Arg-Gly-Arg-
pNA • 2HCl 714. 6]
White freeze-drying mass. It's the specific chromogenic
substrate of factor X..
Chromotropic Acid [ C10 Hs Os Sz• 2 Hz O 356. 33]
White crystals.
Soluble in water.
Cinchonine [ Cl9 Hz2 Nz O 294. 40]
White crystals ora powder; taste, slightly bitter; darkens on
exposure to light.
Soluble in ethanol or chloroform; slightly soluble in ether;
practically insoluble in water.
Citric Acid [C6 H 8 01• Hz O 210. 14]
White crystals or granules; efflorescent; hygroscopic.
Freely soluble in water or ethanol.
CobaltousAcetate [CO CC2H302)2•4H20 249.08]
Violet-red crystals.
Soluble in water, ethanol, dilute acid or amyl acetate.
Cobaltous Chloride [ CoCb• 6 Hz O 237. 93 J
Red or purplish-red crystals.
Freely soluble in water or ethanol; soluble m acetone;
slightly soluble in ether.
Cobaltous Nitrate [Co CN03 )z• 6H2 O 291. 03]
White crystals or crystalline granules.
Freely soluble in water or ethanol; slightly soluble in acetone
or ammonia solution.
Concentrated Ammonia Solution ( Strong ammonia solution )
[NH4 OH 35. 05]
A colourless, dear liquid; corrosive; contains 25%-28%
(g/g) of NH3.
Miscible with ethanol or ether.
Concentrated hydrogen Peroxide Solution ( 30 %)
34.01]
A colourless, clear liquid; strong oxidizing agent and
corrosive.
Miscible with water or ethanol.
Congo Red [C32H22N6Na206Sz 696. 68]
A reddish-brown powder.
Soluble in water or ethanol.
~ie Brilliant Blue G 250 [ C41 H4s N3 Na01 Sz
854.04]
A violet crystalline powder.
Soluble in hot water or ethanol; slightly soluble in water.
~ie Brilliant Blue R 250 [ C4s H44 N3 Na01 Sz
825.99]
A violet powder.
Slightly soluble in hot water or ethanol; insoluble in water.
Corn Flour
A yellow powder made of yellow maize.
Insoluble in water.
m-Cresol Purple [C21 H1sOsS 382. 44]
A reddish-yellow or brownish-green powder.
Freely soluble in methanol, ethanol or sodium hydroxide
solution; slightly soluble in water.
Cresol Red [C21 H1sOsS 382. 44]
A deep red, reddish-brown or dark green powder.
Freely soluble in ethanol or dilute sodium hydroxide solution;
slightly soluble in water.
0-Cresol [CH3C6 H4 OH 108. 14]
A colourless liquid or crystal; odour, phenolic; corrosive and
poisonous; changes into brown gradually on exposure to air
and light.
Soluble in ethanol, ether or chloroform; slightly soluble in
water.
Melting point: 30ºC.
Crystal Violet [C2s H30ClN3 407. 99]
A dull green powder with metallic luster.
Soluble in water, ethanol or chloroform; insoluble in ether.
Copper [Cu 63. 55]
Red brown slices, granules, crumbs or powder; lustrous;
stable under dry air and room temperature, produces basic
salts on exposure to moist air for a long time.
Freely soluble in hot sulfuric or nitric acid; soluble in
ammonia solution and produces complex salt.
 8001 Reagents I' SJ'{
Cupric Acetate [Cu (~H3Ü2)2•H20 199. 65] discoloured.
Dark green crystals. 2, 3-Diaminonaphthalene ( 2, 3-Naphthalene diamine )
Soluble in water or ethanol; slightly soluble in ether or [C10 H10N2 158. 20]
glycerin. Foliaceous crystals.
Cupric Carbonate ( Basic) Soluble in ethanol or ether.
COH) 2 221. 12] Diammoniwn Hydrogen Phosphate
Green or blue amorphous powder or greyish-green crystals;
132.06]
poisonous.
White crystals ora crystalline powder.
Soluble in dilute acid or ammonia solution; insoluble in water
lt loses ammonia on exposure to air and turns into
or ethanol.
ammonium dihydrogen phosphate.
Cupric Chloride [CuClz•2H20 170. 48] Soluble in water; insoluble in ethanol.
Pale Bluish-green crystals.
Dibutyl Phthalate [C16 H22 0 4 278. 35]
Soluble in water, ethanol or methanol; slightly soluble m
A colourless or pale yellow oily liquid.
acetone or ethyl acetate.
Freely soluble in ethanol, acetone, ether or benzene;
Cupric Nitrate [Cu (NÜ3)2•3H20 241. 60] practically insoluble in water.
Blue, columnar crystals; burns and detonates when heated,
0-Dianisidine [ CCH3 OC6 H3 NH2 ) 2 244. 29]
rubbed or bumped with pulverized carbon, sulphur or other
White crystals.
combustible substances.
Soluble in ethanol, ether or benzene; insoluble in water.
Soluble in water or ethanol.
Dichloromethane [CH2Clz 84. 93]
Cupric Sulfate [CuSÜ4•5H20 249. 69]
A colourless liquid; odour, ether-like.
Blue crystals, granules or crystalline powder.
Miscible with ethanol, ether or dimethylformamide;
Soluble in water; slightly soluble in eJ:hanol.
sparingly soluble in water.
Cupric Sulfate, Anhydrous [CuS04 159. 61] Boiling point: 40-41 ºC.
Greyish-white or greenish-white crystals or an amorphous
powder; hygroscopic. 2, 6-Dichloroindophenol Sodiwn [C12H6ClzNNa02•2H20
Soluble in water; practically insoluble in ethanol. 326. 11]
Grass green, fluorescent crystals ora dark green powder.
Curcwna Powder ( Tunneric Powder) Freely soluble in water or ethanol; insoluble in chloroform or
A powder of the rhizome of Curcuma longa L. (Fam. Gingi- ether.
beraceae), containing 5% volatile oil, yellow curcumin,
starch and resin. 2, 6-Dichloroquinone Chlorimide [C6 H2CbNO 210. 45]
A greyish-yellow crystalline powder.
Cyclohexane [CG H12 84. 16] Freely soluble in chloroform or ether; soluble in hot ethanol
A colourless, clear liquid; inflammable. or dilute sodium hydroxide solution; insoluble in water.
Miscible with methanol, ethanol, acetone, ether, benzene or
carbon tetrachloride; practically insoluble in water. Diethylamine [ CC2Hs)2NH 73.14]
Boiling point: 80. 7°C. A colourless liquid; odour, ammonia-like; strongly alkaline;
corrosive; freely volatile; inflammable.
Cysteine Hydrochloride [CH2 CSH)CHCNH2) COOH • HCl Miscible with water or ethanol.
157. 62]
White crystals. Diethyl Ether, Anhydrous [ ( ~ Hs ) 2 O 74. 12 J
Soluble in water or ethanol. See Ether, but with less content of water.

Cyclohexanone [CG H10 O 98. 14] Digitonin [CsG H92 Ü29 1229. 33]
A colourless oily liquid; odour resembling peppermint or White crystals.
acetone; its vapour mixed with air is explosive. Sparingly soluble in dehydrated ethanol; slightly soluble in
Miscible with ethanol or ether; slightly soluble in water. ethanol; practically insoluble in water, chloroform or ether.

L-Cystine [ C6 H12 N2 04 Sz 240. 30] p-Dihydrocybezene see Hydroquinone.

White crystals. 1, 3-Dihydroxynaphthalene [C10 Hs 0 2 160. 17]
Soluble in acid or alkali solution; practically insoluble m Pink-red flake crystals; soluble in water, ethanol or ether.
water or ethanol.
2, 7-Dihydroxynaphthalene (2, 7-Naphthalenediol)
Dehydrated Ethanol See Ethanol, Absolute. 160. 17]
Dextrin White needle or scale crystals; the colour of solution darkens
Complies with the requirements prescribed in monograph of quickly on exposure to air.
the Chinese Pharmacopoeia. Soluble in hot water, ethanol or ether; slightly soluble m
chloroform or benzene.
p-Diaminobemene (p- Phenylenediamine)
108. 14] 3, 5-Dihydroxy Toluene [GHs02•H20 142.14]
White or pale red crystals; darkens on exposure to air; White crystals; easily oxidized to red in air; odour,
sublimes easily on heating. unpleasant; tas te, sweet.
Soluble in ethanol, chloroform or ether; slightly soluble in Soluble in water or ethanol; slightly soluble in benzene,
water. chloroform or carbon disulfide.
Diaminobenzidine Hydrochloride [C12 H14 N4 • 4HC1•2H2 O 3, 3' -Dimethoxybenzidine [ C14 Hl6 Nz 02 244. 28 J
396. 14] White crystals; with a purple gloss in the air; soluble m
A white or grey powder. ethanol or ether; insoluble in water.
Soluble in water, its aqueous solution is readily oxidized and Melting point: 137-138ºC.
 8001 Reagents
Dimethylacetamide [C4 H 9 NO 87. 12]
A clear, colourless liquid; miscible with water and with
many organic solvents.
p-Dimethylaminobemaldehyde [~ Hn NO 149. 19]
White or pale yellow crystals; odour, characteristic;
gradually turns to red on exposure to light.
Soluble in ethanol, acetone, chloroform, ether or acetic acid;
slightly soluble in water.
Dimethylformamide [HCON CCH3 )2 73. 09]
A colourless liquid with slight ammonia odour.
Miscible with water, ethanol, chloroform or ether.
Dimethylglyoxime [CH3C(NOH)C(NOH)CH3 116. 12]
A white powder.
Soluble in ethanol or ether; insoluble in water.
N, N-Dimethyl-p-phenylenediamine Dihydrochloride
[CsH12N2•2HCl 209. 12]
A white or greyish-white crystalline powder; darkens
gradually on exposure to air; hygroscopic.
Soluble in water or ethanol.
Dimethylsulfoxide [CCH3)2SO 78. 14]
A colourless, viscous liquid; very hygroscopic; taste,
slightly bitter; violent reaction occurs in contact with
chlorine at room temperature.
Soluble in water, ethanol, acetone, chloroform, ether or
benzene.
Dimethyl Yellow [C14 H1sN3 225. 29]
A golden yellow crystalline powder.
Soluble in ethanol, chloroform, ether, benzene, petroleum
ether or sulfuric acid; insoluble in water.
2, 4-Dinitroaniline [ C6 Hs N3 04 183. 12]
Yellow or yellowish-green crystals.
Soluble in chloroform or ether; slightly soluble in ethanol;
insoluble in water.
m-Dinitrobenzene [C6 H4 (N02 )z 168. 11]
A pale yellow crystal; inflammable.
Freely soluble in chloroform, ethyl acetate or benzene;
soluble in ethanol; slightly soluble in water.
3, 5-Dinitrobenzoic Acid [C, H4N206 212. 12]
White or pale yellow crystals; volatilizes with steam.
Freely soluble in ethanol or glacial acetic acid; slightly
soluble in water, ether, benzene or carbon disulfide.
2, 4-Dinitrochlorobenzene [C6 H3ClN204 202. 55]
Yellow crystals. It may cause explosion when heated to high
temperature.
Freely soluble in hot ethanol; soluble in ether, benzene or
carbon disulfide; insoluble in water.
2, 4-Dinitrofluorobenzene [C6 H3FN204 186. 11]
Light yellow crystals or an oily liquid; darkens on exposure
to light and on long storage; soluble in ether; insoluble in
water.
Melting point: 26ºC.
2, 4-Dinitrophenol [C6H4N20s 184.11]
Yellow, rhombic crystals; sublimes on heating.
Soluble in ethanol, ether, chloroform or benzene; very
slightly soluble in cold water.
2, 4-Dinitrophenylhydrazine [Cs H6 H4 04 198. 14]
A red crystalline powder; stable in acid solution, unstable in
alkaline solution.
Soluble in hot ethanol, ethyl acetate, aniline or dilute mineral
acids; slightly soluble in water and ethanol.
Dioctyl Phthalate [Cz4H3s04 390. 56]
A colourless or pale yellow oily liquid; odour, slight
characteristic.
Miscible with organic solvent; insoluble in water.
Dioctyl Sodium Sulfosuccinate [ C20 H31 Na01 S 444. 57]
White, wax-like solids.
Soluble in water, methanol, acetone, benzene, or carbon
tetrachloride. Easily hydrolyzes in alkaline solution.
Dioxane [C4 Hs 02 88. 11]
A colourless liquid; odour, ether-like; inflammable; freely
absorbs oxygen to form peroxide.
Miscible with water or most of organic solvents.
Boiling range: 100-103ºC.
Diphenylamine [ (C6Hs )z NH 169. 23]
White crystals; odour, aroma tic; gradually discolours on
exposure to light.
Soluble in ether, benzene, glacial acetic acid or carbon
disulfide; insoluble in water.
Diphenylcarbazide [C, H 5 NHNHCONHNHC, Hs 242. 28]
A white, crystalline powder; gradually tums to red in air.
Soluble in hot ethanol, acetone or glacial acetic acid; very
slightly soluble in water.
Dipotassium Hydrogen Phosphate [K2 HP04 17 4. 18]
White granules or a crystalline powder.
Freely soluble in water; slightly soluble in ethanol.
2, 2'-Dipyridyl [Cs H4N •Cs H4N 156. 19]
A white or pale red crystalline powder.
Freely soluble in ethanol, chloroform, ether benzene or
petroleum benzin; slightly soluble in water.
Disodium Ethylenediaminetetraacetate [ C10 H14 Nz Na2 Os •
2H2 O 372. 24]
A white crystalline powder.
Soluble in water; very slightly soluble in ethanol.
Disodium Hydrogen Phosphate [ Na2 HP04 • 12H20
358. 14]
White crystals ora granular powder; efflorescent.
Soluble in water; insoluble in ethanol.
Disodium Hydrogen Phosphate, Anhydrous [Na2 HP04
141. 96]
A white crystalline powder; hygroscopic; it can absorb 2-7
mol of water on exposure to air for a long time.
Freely soluble in water; insoluble in ethanol.
Distilled Water, Ammonia Free
To 1000 mi of distilled water add 1 mi of dilute sulfuric acid
and 1 mi of potassium permanganate TS, distil.
To 50 ml of the distillate add 1 ml of alkaline mercuric
potassium iodide TS, no colour is produced.
Ditertbutyl-p-cresol [ [CCH 3 ) 3 C]2 C6 H2 CCH3) OH
220.35]
White or pale yellow crystals; soluble in ethanol or
petroleum ether; insoluble in water or alkaline solution.
Dithizone [ C6 Hs NHNHCSNNC6 Hs 256. 33]
A bluish-black crystalline powder.
Soluble in chloroform or carbon tetrachloride; insoluble m
water.
Eosin Sodium [C20H6Br4Na20s 691. 86]
A red powder.
Freely soluble in water; an aqueous solution exhibits a red
fluorescence; slightly soluble in ethanol; insoluble in ether.
Eriochrome Black T [CzoH1 2N3Na01S 461. 39]
 8001 Reagents

A brownish-black powder. Deep green or purplish-blue crystalline powder.

Soluble in water or ethanol. Soluble in water, ethanol or ether.
Ethanol [Cz Hs OH 46. 07] Factor X.
A clear, colourless liquid; freely volatile; inflammable. White freeze-drying mass. Extracted and purified from
Miscible with water, ether or benzene. bovine plasma.
Ethanol, A)N)lute (dehydrated) [CzHsOH 46. 07] Fast Bine BB Salt [C17H1sClN3Ü3•l/2ZnClz 415. 96]
A clear, colourless liquid; odour, alcoholic; inflammable; A pale cream-red powder.
hygroscopic, it contains not more than O. 3% of water. Ferric Ammonium Sulfate [ FeNH4 ( S04 )z • 12H2 O
Miscible with water, acetone or ether. 482.20]
Boiling point: 78. 5ºC. White to pale violet crystals.
Ethanol, Aldehyde Free Soluble in water; insoluble in ethanol.
Place 2. 5 g of lead acetate into a conical flask with a stopper, Ferric Chloride [FeCb•6H20 270. 30]
dissolve with 5 ml of water, add 1000 ml of ethanol, mix Brownish-yellow or orange-yellow crystal-like mass; highly
well, add slowly 25 ml of ethanolic potassium hydroxide hygroscopic.
solution 0-5), stand for 1 hour, shake thoroughly, stand Freely soluble in water, ethanol, acetone, ether or glycerin.
for 12 hours, pour out the supernatant and distill.
Measure 25 ml of the Ethanol, Aldehyde Free into a conical Ferric Nitrate [Fe (NÜ3h•9H20 404. 02]
flask, add 75 ml of dinitrophenylhydrazine TS, heat and Light purple to greyish-white crystals; slight deliquescent;
reflux on water bath for 24 hours, evaporate ethanol, add decomposes below lOOºC.
200 ml of 2% ( ml/ml) sulfuric acid solution, stand for Freely soluble in water, ethanol or acetone; slightly soluble
24 hours, no crystal is produced. in nitric acid.

Ethanol, Neutral Ferrous Sulfate [FeSÜ4•7H20 278. 02]

Add 2 ,..._, 3 drops of phenolphthanlein IS to ethanol, titrat Pale blue-green crystals or granules.
with sodium hydroxide (O. 1 mol/L) VS until the colour Soluble in water; insoluble in ethanol.
changes to pink. Fluorane [CzoH12Ü3 300.31]
Ether [C2HsOCzHs 74. 12] Fluorescein [CzoH12Üs 332. 11]
A clear, colourless liquid; taste, paralysing sweet and An orange or red powder.
astringent; freely volatile; inflammable; anaesthetic; Soluble in hot ethanol, glacial acetic acid, sodium carbonate
oxidized to peroxide on exposure to light or on long solution or sodium hydroxide solution; insoluble in water,
storage. Boiling point: 34. 6°C. chloroform or benzene.
Ether Dehydrated [ ( C2 Hs) 2O 74. 12] Fonnaldehyde Solution [HCHO 30. 03]
As described under Ether, but its water content is less than A colourless liquid; odour, pungent; it becomes muddy with
that of Ether. polymerization when cooled.
Ethoxychrysoidine Hydrochloride [ Cl4 H16 N4 O • HCl Slowly oxidized to formic acid on exposure to air. It contains
292. 77] about 37 % of HCHO.
A deep reddish-brown or blackish-brown powder. Miscible with water or ethanol.
Soluble in water or ethanol. Formamide [HCONH2 45. 04]
Ethyl Acetate [CH3CQOC2Hs 88.11] A colourless, slightly viscous liquid with faint odour of
A clear, colourless liquid. ammonia; hygroscopic; irritant.
Miscible with acetone, chloroform or ether; soluble in Miscible with water or ethanol.
water. Fonnic Acid [HCOOH 46. 03]
Ethyl Cyanoacetate [CH2 (CN) CQCz Hs 113. 12] A clear, colourless liquid; odour, pungent; corros1ve to
A colourless liquid; odour, characteristic of ester; taste, skin.
slightly sweet. lt contairis not less than 85 % of HCOOH.
Miscible with ethanol or ether; soluble in ammonia solution Miscible with water, ethanol, ether or glycerin.
or alkaline solution; insoluble in water. Fonnic Acid, Anhydrous [HCOOH 46. 03]
Ethyl Fonnate [HCOOC2 Hs 74. 08] A clear, colourless liquid; odour, pungent; highly corrosive;
A mobile, flammable liquid; irritant to skin and mucas strongly acidic; it contains not less than 98% of HCOOH.
membranes; anaesthetic in high dosage. Miscible with water, ethanol or ether.
Miscible with ethanol or ether; soluble in about 10 parts of Fuchsin Basic (Magenta)
water and gradually decomposes into formic acid and Dark green crystals with metallic lustre.
ethanol. Soluble in water or ethanol; insoluble in ether.
Glycol Monoethyl Ether [C3 HsÜz 76. 10] Furfural [Cs H4Üz 96. 09]
Colourless liquid; odour, pleasant; toxic. A colourless or pale yellow oily liquid; easily turns to brown
Miscible with water, ethanol, ether, glycerin, acetone and on exposure to air or light.
dimethylformamide. Miscible with water, ethanol or ether.
Boiling point: 124. 3ºC. GallicAcid [~H60s•H20 188.14]
N-Ethylmaleimide [C6 H1 N02 125. 12] White or pale brown crystals ora powder.
White crystals. Soluble in hot water, ethanol or ether; insoluble m
Freely soluble in ethanol or ether; slightly soluble in water. chloroform or benzene.
Ethyl 1Mrabrumphenolphthalein Potammn [ C!z 813 Br4 K04 Gelatin
700.06] Pale yellow to yellow, translucent and faintly lustrous granules or
 8001 Reagents
thin sheets; odourless. Easily decomposed by microorganisms
when moistened. Swell and softens when immersed in water
far a long duration, its weight increases gradually five to ten
times dueto the absorption of water. Soluble in hot water,
acetic acid or a hot mixture of glycerol and water; insoluble
in ethanol, chloroform or ether.
Glucose [C6 H12 06· H2 O 198. 17]
Complies with the requirements prescribed in the monograph
of the Chinese Pharmacopoeia.
Glutaradehyde [C5 Hs 02 100. 12]
A clear, colourless oily liquid; soluble in water, ethanol or
ether.
Glutamate Dehydrogenase
White powder. Formular weight is 260 Kda (gel) , activity
is more than 500 units/mg protein.
Glycerin (Glycerol) [C3Hs03 92. 09]
A clear, colourless, viscous liquid; odourless; taste, sweet;
hygroscopic.
Miscible with water or ethanol.
Glycerol Monostearate [C21 H4204 358. 57]
A white or slightly yellow waxy salid; odour, pleasant;
soluble in hot organic solvents, as ethanol, ether or acetone;
insoluble in water.
Melting point: 56-58ºC.
Glycine [C2H5N02 75. 07]
A white crystalline powder.
Soluble in water or pyridine; slightly soluble m ethanol;
practically insoluble in ether.
Heptane (n-Heptane) [G H 16 100. 20]
A colourless, clear liquid, inflammable.
Miscible with ethanol, chloroform or ether; insoluble m
water.
Boiling point: 98. 4ºC.
n-Hexane [CG H14 86. 18]
A clear, colourless liquid; odour, characteristic; highly
volatile; irritant to respiratory tract.
Miscible with ethanol or ether, insoluble in water.
Boiling point: 69ºC.
2, 4, 6, 2', 4', 6'-Hexanitrodiphenylamine [C12H5N1012
439.22]
Yellow crystals; It may cause explosion when heated or
strong impact; soluble in nitric acid; slightly soluble in
acetone; insoluble in water, ethanol, ether or chloroform.
Holmium Oxide [Ho2 03 377. 86]
Yellow salid; slightly hygroscopic; dissolves in acid with
formation of a yellow salt.
Freely soluble in water.
Hydrazine Sulfate [ (NH2 )2• H 2S0 4 130. 12]
White crystals or powder.
Freely soluble in hot water; slightly soluble in water or ethanol.
Hydrochloric Acid [HCl 36. 46]
A colourless, clear liquid; odour, pungent and
characteristic; corrosive; fuming on exposure to air. It
contains 36 %-38 % (g/ g) of HCI.
Miscible with water or ethanol.
Hydrofluoric Acid [HF 20. 01]
A colourless, fuming liquid; odour, pungent;
corrosive to metal or glass.
Miscible with water or ethanol.
Hydroiodic Acid [ HI 12 7. 91]
An aqueous solution of hydrogen iodide, colourless; Due to
the precipitation of iodine on exposure to light and on long
storage, the colour changes from slightly yellow to brown;
corrosive; odour, irritant.
Miscible with water or ethanol.
Hydroquinone [CG H4 (OH)2 110. 11]
White or almost white crystals; discoloured on exposure to
light.
Freely soluble in hot water; soluble in water, ethanol or ether.
p-Hydroxydiphenyl [C6 H5 C6H40H 170. 21]
Almost white crystals.
Freely soluble in ethanol or ether; soluble in alkali solution;
insoluble in water.
Hydroxylamine Hydrochloride [NH2 OH• HCl 69. 49]
White crystals; decampases easily when moistened,
corros1ve.
Soluble in water, ethanol or glycerin.
5-Hydroxymethyl Furfural [C6 H6 03 126. 11]
A needle crystal.
Freely soluble in methanol, ethanol, acetone, ethyl acetate
or water; soluble in benzene, chloroform or ether; slightly
soluble in petroleum ether.
p-Hydroxyphenylglycine [Cs Hg N03 167. 16]
White lustrous flakes.
Freely soluble in hydrochloric acid solution 0-5); soluble
in acid or alkaline solution; practically insoluble in water,
ethanol, ether, acetone, chloroform, benzene, glacial acetic
acid or acetate easter.
8-~ [Ci Hd'~O 145. 16]
A white or pale yellow crystalline powder. Odour, phenolic;
darkens easily on exposure to light.
Freely soluble in ethanol, acetone, chloroform, benzene or
mineral acid; practically insoluble in water.
Hypophosphorous Acid [ H3 P02 66. 00]
White, transparent crystals; colourless oily liquid is formed
when over cooled; odourless; hygroscopic. It is a strong
reducing agent.
Soluble in water, ethanol or ether.
lmidazole [ C3 H4 N2 68. 08 ]
White, nearly transparent crystals.
Freely soluble in water, ethanol or pyridine; slightly soluble
in benzene; very slightly soluble in petroleum ether.
lndigoCarmine [C16HsN2Na20sSz 466.36]
Blue crystals or powder with metallic lustre.
Slightly soluble in water; insoluble in ethanol.
lodine [12 253. 81]
Purplish-black scale crystals or masses with metallic lustre.
Soluble in ethanol, ether or solution of potassium iodide;
very slightly soluble in water.
lodine Monochloride [ICl 162. 36]
A brownish-red, oily liquid or dark-red crystals; strongly
irritant; with odour of chlorine and iodine; corrosive and
oxidati ve.
lodine Pentoxide D2 05 333. 81]
A white crystalline powder; readily decampases on exposure
to light; hygroscopic.
Freely soluble in water to formiodic acid; insoluble in
dehydrated ethanol, chloroform, ether or carbon disulfide.
highly lodine Trichloride [ICb 233. 26]
Yellow or pale brown crystals; odour, strongly pungent;
volatile at room temperature; readily decomposes in contact
with water; hygroscopic; corrosive.
Soluble in water, ethanol, ether or benzene.

Iron [Fe 55. 85]
A silky grey, or greyish black amorphous powder; easily
oxidized on exposure to moist air.
Soluble in dilute acid; insoluble in concentrated acid and
dilute alkaline solution.
lsatin [Cs Hs N02 147. 13]
Dark red crystals or a crystalling powder; taste, bitter;
sublima ble.
Soluble in ether or boiling water; freely soluble in boiling
ethanol; practically insoluble in cold water.
Isoamyl Acetate [CH3COOCH2CH2CH CCH3) 2 130.19]
A clear, colourless liquid; odour, characteristic resembling
that of banana.
Miscible with ethyl acetate, ethanol, amyl alcohol, ether,
benzene or carbon disulfide; very slightly soluble in water.
Isoamylol [ CCH3)2CHCH2CH20H 88. 15]
Colourless liquid; odour, characteristic; inflammable.
Miscible with organic solvent; slightly soluble in water.
Boiling point: 132ºC.
Isobutanol [ CCH3)2CHCH20H 74. 12]
A colourless, clear liquid; strongly refractive; inflammable.
Miscible with water, ethanol or ether.
Boiling range: 107. 3-108. 3ºC.
Isobutyl Acetate [CH3COOCH2CH CCH3)2 116. 16]
A colourless liquid; inflammable.
Miscible with ethanolor ether; insoluble in water.
Isoniazid [Cs H1 N3 O 137. 14]
Complies with the requirements prescribed in the monograph
of the Chinese Pharmacopoeia, volume 11.
Isooctane
See Trimethylpentane.
lsopropanol [ CCH3 )zCHOH 60. 10]
A colourless, clear liquid; odour, characteristic; taste,
slightly bitter.
Miscible with water, ethanol or ether.
Boiling range: 82. 0-83. OºC.
Isopropyl Ether [Cs Hl4 O 102. 18]
A colourless, clear liquid; inflammable.
Miscible with ethanol, chloroform, ether or benzene;
slightly soluble in water.
Isopropyl Myristate [C11H3402 270. 46]
A colourless liquid; soluble in ethanol, ether, acetone,
chloroform or toluene; insoluble in water, glycerol or 1, 2-
propanediol.
It decomposes at about 208ºC.
Kerosene, Refined
A colourless or pale yellow oily liquid; odour, characteristic.
Miscible with chloroform, benzene or carbon disulfate;
insoluble in water or ethanol.
Wash 300 mi of commercial kerosene with 20 mi of crude
sulfuric acid in a 500 mi separator far 4-5 times, until the
acid layer is pale black, separate the kerosene layer and wash
with water. Then wash it with 20 mi of sodium hydroxide
solution O- 5), wash it with water and dehydrate with
anhydrous calcium chloride, transfer it into a distilling flask,
distill under an air condenser. on a sand bath. Collect the
distillate in the range of 160-250ºC.
Kieselguhr
A white or almost white powder; strongly adsorptive powder
and good filter medium.
Insoluble in water, acid or alkali solution.
Lactic Acid [CH3CH (OH) COOH 90. 08]
8001 Reagents
Complies with the requirements prescribed in monograph of
the Chinese Pharmacopoeia.
Lanthanum Nitrate [La (NÜ3) 3• 6H 2O 433. 01]
White crystals.
Soluble in water, ethanol or acetone.
Lanthanum Oxide [La2 0 3 325. 84]
A white or almost white amorphous powder; absorbs carbon
dioxide on exposure to air. It is dissolved in dilute mineral
acid to generate salts; insoluble in water.
Lactose [C12 H22 Ou• H2 O 360. 31]
White crystalline granules or a crystalline powder;
odourless; taste, slightly sweet. Freely soluble in water;
insoluble in ethanol, chloroform or ether.
LeadAcetate [Pb CC2H3Ü2)2•3H 20 379.34]
White crystals or a powder.
Freely soluble in water or glycerin; soluble in ethanol.
Lead Dioxide [Pb02 239. 21]
A dark brown powder.
Lead Monoxide [PbO 223. 20]
A yellow to brownish-yellow powder or crystals.
At 300-500ºC , it converts into Pb3 0 4 , but at higher
temperature reverts to PbO.
Soluble in hot solution of sodium hydroxide, acetic acid or
dilute nitric acid.
Lead Nitrate [Pb CN0 3) 2 331. 21]
White crystals; burns and detonates when in contact, rubbed
or bumped with organic substances.
Soluble in water; slightly soluble in ethanol.
Liquefied Phenol
To 90 g of phenol add a little water, heat slowly on a water
bath until liquefied, cool, add sufficient water to produce
100 mi.
Lithium Carbonate [Li2CÜ3 73. 89]
A white powder or crystals; light.
Soluble in dilute acid; slightly soluble in water; insoluble in
ethanol or acetone.
Lithium Chloride [LiCI 42. 39]
A white crystalline powder.
Soluble in water, ethanol, acetone, ether, isoamyl alcohol or
sodium hydroxide solution.
Lithium Hydroxide [LiOH • H2 O 41. 95]
White small monoclinic crystals; odour, pungent; absorbs
water and carbon dioxide on exposure to air; soluble in
water; slightly soluble in ethanol.
Lithium Lactate [LiC3H 5 03 96. 01]
A white powder; odourless.
Soluble in water.
Lithium Sulfate [Li2S0 4•H20 127. 96]
White crystals.
Soluble in water; practically insoluble in ethanol.
Litmus
A blue powder or mass.
Partly soluble in water or ethanol.
Magnesium [Mg 24. 31]
A silky white powder with metallic lustre.
Soluble in acid; insoluble in water.
Magnesium Acetate [ CCH3COQ)2Mg 142. 39]
White crystals; Hygroscopic.
Freely soluble in water or ethanol.
 8001 Reagents
Macrogol 6000
A white waxy solid slice or granular powder; odour,
characteristic.
Soluble in water or ethanol; insoluble in ether.
Magnesium Chloride [MgClz• 6 Hz O 203. 30J
White crystals ora powder; hygroscopic.
Soluble in water or ethanol.
Magnesium Nitrate [Mg CN03)z•6HzO 256. 42]
White crystals; hygroscopic. Its aqueous solution is neutral,
decomposes at 330ºC, bums when mixed with inflammable
organic substances, imflammable and detonable. Soluble in
ethanol, ammonia or water.
Magnesium Oxide [MgO 40. 30]
A white very fine powder; odourless; easily absorbs water
and carbon dioxide on exposure to air, produces magnesium
hydroxide with water.
Soluble in dilute acid; very slightly soluble in pure water;
insoluble in ethanol.
Magnesium Sulfate [MgS04•7HzO 246. 48]
White crystals ora powder; efflorescent.
Freely soluble in water; slowly soluble in glycerin; slightly
soluble in ethanol.
Maize Starch
It is a white with slightly yellow powder made of maize by
wet grinding; lustrous.
White maize starch is white and lustrous; yellow maize
starch white with slightly yellowish shadow.
Insoluble in cold water and ethanol.
Malachite Green [2G3 Hzs Nz• 3G Hz 04 929. 04]
Green flake crystals with metallic lustre.
Freely soluble in hot water or ethanol; very slightly soluble
in water.
Malonic Acid [C3 H404 104. 06]
White transparent crystals; strongly irritant.
Soluble in water, methanol, ethanol, ether or pyridine.
Maltose [ C1z Hzz 011 342. 30]
White crystals (f3-type); odour, sweet.
Freely soluble in water; slightly soluble in ethanol; insoluble
in ether.
Specific rotation [a]o: + 125º-+ 137º.
Manganese Dioxide [Mn02 86. 94]
Black crystals or a powder. It can cause buming or explosion
when heated or rubbed with organic substances or other
reductive substances.
Insoluble in water, nitric acid or cold sulfuric acid; soluble in
nitric acid or dilute sulfuric acid in presence of hydrogen
peroxide or oxalic acid.
Manganese Sulfate [MnS04• Hz O 169. 02]
Pink crys tals.
Soluble in water; insoluble in ethanol.
Mannitol [C6 Hi4 06 182. 17]
White crystals; odourless; taste, sweet.
Freely soluble in water; slightly soluble m ethanol;
practically insoluble in ether.
Mercaptoacetic Acid [ C2 H4 02 S 92. 12 J
A sulfur-containing organic compound; colourless clear liquid
with a strong irritating smell.
Miscible with water, ethanol and ether; soluble in common
solven t.
Mercuric Acetate [Hg (Cz H3 02 )z 318. 68]
White crystals ora powder; odour, resembling acetic acid.
Soluble in water or ethanol.
Mercuric Bromide [HgBrz 360. 40]
White crystals ora crystalline powder.
Freely soluble in hot ethanol, hydrochloric acid,
hydrobromic acid or solution of potassium bromide; slightly
soluble in chloroform or ether.
Mercuric Dichloride [HgCb 271. 50]
White crystals or a crystalline powder, slightly volatile at
room temperature. It decomposes to mercurous chloride on
exposure to light.
Soluble in water, ethanol, acetone or ether.
Mercuric Iodide, Red [Hglz 454. 40]
A emerald red powder; odourless; heavy.
Soluble in ether, sodium thiosulfate solution and potassium
iodide solution; slightly soluble in dehydrated ethanol;
insoluble in water.
Mercuric Nitrate [Hg CN03 )z• H2 O 342. 62]
A white or slightly yellow crystalline powder, with an odour
of nitric acid; hygroscopic.
Freely soluble in water or dílute nitric acid; a precipitate of
basic salt is formed in a large amount of water or boiling
water.
Mercurous Nitrate [HgN03• Hz O 280. 61]
White crystals; usually with an odour of nitric acid.
Freely soluble in water or dilute nitric acid.
A precipitate of basic salt is formed in a large amount of
water.
Mercuric Oxide, .Yellow ( Yellow Mercuric Oxide) [HgO
216.59]
A yellow or orange powder; heavy; darkens gradually on
exposure to light.
Freely soluble in dilute sulfuric acid, dilute hydrochloric acid
or dilute nitric acid; insoluble in water, ethanol, acetone or
ether.
Mercuric Sulfate [HgS04 296. 68]
White granules ora crystalline powder; odorless; toxic.
Soluble in hydrochloric acid, hot diluted sulfuric acid, and
concentrated sodium chloride solution.
Mercury [Hg 200. 59]
A silver-white lustrous, liquid metal; heavy; slightly volatile
at room temperature; produces amalgam with metals other
than iron.
Soluble in dilute nitric acid; insoluble in water.
Metalphthalein (Phthalein Purple) [ ~z H3z Nz 01z 636. 58]
A pale yellow or pale brown powder.
Sensitivity To 10 mg add 1 ml of concentrated ammonia
solution, dilute with water to 100 ml and mix well. Mix 5 ml
of this solution with 95 ml of water, 4 ml of concentrated
ammonia solution, 50 ml of ethanol and O. 1 ml of O. 1 mol/L
barium chloride solution. The mixture turns to bluish-purple
colour and should be colourless with adding O. 15 ml of
O. 1 mol/L EDTA solution.
Methanol [CH30H 32. 04]
A clear, colourless liquid; volatile; inflammable. lt contains
O. 1 % of water.
Miscible with water, ethanol or ether.
Boiling range: 64-65ºC.
Methanol, Anhydrous [CH30H 32. 04]
A clear, colourless liquid; volatile; burns with a bluish flame
without fume. It contains not more than O. 05 % of water.
Miscible with water, ethanol or ether.
Boiling point: 64. 7°C.
p-Methoxybenzaldehyde see Anisaldehyde.

Methyl Acetate [CH3COOCH3 74. 08]
A clear, colourless liquid.
Miscible with water, ethanol or ether.
Methylamine Hydrochloride [CH3NH 2•HCl 67. 52]
White or almost white crystals; hygroscopic.
Soluble in water or dehydrated ethanol.
N, N' -Methylene Bisacrylamide [ C; H10 Nz 02 154. 17]
A white crystalline powder; hydrolyses in aqueous solution
with formation of ammonia and acrylic acid.
Sparingly soluble in water.
Methylene Bine [Cl6H1sClN3S•3H20 373. 90]
Deep emerald green crystals or a dark brown powder; with
bronze lustre.
Freely soluble in hot water.
Methyl Isobutyl Ketone [CH3COCH2CH(CH3)2 100.16]
A colourless liquid; inflammable.
Miscible with ethanol, ether or benzene; slightly soluble in
water.
p-Methylaminophenol Sulfate [ C14 H1s Nz 02 • Hz S04
344.39]
White crystals; turns grey on exposure to light.
Soluble in water; insoluble in ethanol or ether.
Methyl p-Hydroxybenzoate [Cs Hs03 152. 14]
Colourless crystals or white crystalline powder; odourless or
slightly irritant.
Soluble in ethanol, ether or acetone; slightly soluble in
benzene or carbon tetrachloride; practically insoluble m
water.
Methyl Orange [C14 H14N3Na03S 327. 34]
Orange crystals or a powder.
Freely soluble in hot water; practically insoluble in ethanol.
Methyl Red [C1sH1sN302 269. 30]
Purplish-red crystals.
Soluble in ethanol or acetic acid; insoluble in water.
4-Methylumbelliferyl-JH>-glucuronide, MUG [C1s H16 09
376.3]
White needle crystals.
Soluble in water, ethanol or ether; decomposes m dilute
sodium hydroxide solution.
Microcrystalline Cellulose [ ~n H1on+2 Osn+ i]
A white or off-white powder; odourless; tasteless.
Insoluble in water, ethanol, acetone or toluene.
Morphine, Anhydrous [C11 H19 N03 285. 34]
Rhombic, short columnar prism crystals ( crystallized from
anisole); decomposes at 254ºC.
Morin [ C1s H10 01 302. 23]
Light yellow needle crystals; turns brown on exposure to air.
Freely soluble in ethanol; soluble in alkalic solution; slightly
soluble in acetic acid and ether.
a-Naphthol ( 1-Naphthol) [C10 H1 OH 144. 17]
White or pale pink crystals or a powder; odour, phenolic;
darkens gradually on exposure to light.
Freely soluble in ethanol, chloroform, ether, benzene or
alkali solution; slight soluble in water.
p-Naphthol (2-Naphthol) [C10 H1 OH 144. 17]
White or pale yellow crystals or a powder; odour,
characteristic; discoloured easily on exposure to light.
Freely soluble in ethanol, ether, glycerin or sodium
hydroxide solution; soluble in hot water; slightly soluble in
water.
8001 Reagents
a-Naphtholbenzein [Cz1 Hzo03 392. 45]
A reddish-brown powder.
Soluble in ethanol, ether, benzene or glacial acetic acid;
insoluble in water.
a-Naphthylamine [C10H1NH2 143.19]
White needle crystals or a powder; odour, unpleasant;
changes gradually to faint red on exposure to air; easy
sublimation; volatile with the water vapor.
Freely soluble in ethanol or ether, slightly soluble in water.
a-Naphthylamine Hydrochloride [C10HgN•HCl 179. 65]
A white crystalline powder; discoloured on exposure to air.
Soluble in water, ethanol or ether.
N-Naphthylethylenediamine Dihydrochloride [ C12 H14 Nz •
2HC1 259. 18]
White or faintly red crystals.
Freely soluble in hot water, ethanol or dilute hydrochloric
acid; slightly soluble in water, dehydrated ethanol or
acetone.
Neutral Red [C1s H 17 N4 Cl 288. 78]
A dark green or brownish-black powder.
Soluble in water or ethanol.
Nickel Anunonium Sulfate [NiS04 • (NH4 )2 S04 • 6H2 O
394.99]
Bluish-green crystals.
Soluble in water; insoluble in ethanol.
Nickel Nitrate [Ni (NQ3)2•6H20 290. 79]
Green crystals. Its aqueous solution is acidic.
Freely soluble in water; soluble in ethanol or ethylene glycol;
slightly soluble in acetone.
Nickel Sulfate [NiS04•7H20 280. 86]
Green, transparent crystals.
Soluble in water or ethanol.
p-Nicotinamide Adenine Dinucleotide, Reduced, Disodium Salt
[Cz1 H21 N1Na2014 P2]
White to slight yellow powder. Soluble in water.
Nicotinyl L-tyrosyl-hydrazide [ C1s Hl6 N4 03 300. 32]
White crystals.
Soluble in hot ethanol.
Ninhydrine [~H604 178.14]
A white or pale yellow crystalline powder; hygroscopic;
discoloured gradually on exposure to light and air.
Soluble in water or ethanol; slightly soluble in chloroform or
ether.
Nitric Acid [HN03 63. 01]
A colourless, clear liquid; fumes on exposure to air; odour,
suffocating irritant; tums to brown with formation of
nitrogen tetroxide on exposure to light.
It contains 69%-71% (g/g) of HN03.
Miscible with water.
Nitric Acid, Fuming [HN0 3 63. 01]
A clear, colourless or slightly yellowish-brown liquid; highly
oxidative and corrosive; A reddish-yellow fog of nitrogen
dioxide and nitrogen tetroxide is produced on contact with
atr.
Miscible with water.
p-Nitroaniline [~H6N202 138.13]
Yellow crystals or powder.
Freely soluble in methanol; soluble m ethanol or ether;
insoluble in water.
Nitrobenzene [C6HsN02 123.11]
A colourless or pale yellow oily liquid; odour, bitter almond-
 8001 Reagents
like.
Freely soluble in ethanol, ether, benzene or oil; very slightly
soluble in water.
Nitromethane [CH3N02 61. 04]
A colourless, oily liquid; inflammable; its vapour forros
explosive mixture with air.
Miscible with water, ethanol or alkali solution.
p-Nitrophenol [CG Hs N03 139. 11]
White or pale yellow crystals; sublimable; inflammable.
Freely soluble in ethanol, chloroform, ether or sodium hydroxide
solution; slightly soluble in water.
p-Nitrophenyl-azo-resorcinol [C12 Hg N3 04 259. 22]
A reddish-brown powder.
Slightly soluble in boiling ethanol, acetone, ethyl acetate and
toluene; insoluble in water. Soluble in dilute alkali solution.
n-Octanol [Cs H11 OH 130. 23]
A colourless liquid; odour, characteristically aromatic.
Miscible with ethanol, ether or chloroform; insoluble m
water.
Boiling range : 194-195ºC.
n-Octylamine [CH3 (CH2 )1 NH2 129. 24]
A colourless liquid; odour, ammonia-like; soluble in ethanol
or ether; slightly soluble in water.
Olive Oil
A pale yellow or slightly green liquid.
Miscible with chloroform, ether or carbon disulfide; slightly
soluble in ethanol; insoluble in water.
Orange N [C1sH14N3Na03S 375. 38]
A yellow powder.
Soluble in water or ethanol.
Oxalic Acid [H2Cz04•2H20 126. 07]
White, transparent crystals or crystalline granules;
efflorescent.
Freely soluble in water or ethanol; insoluble in chloroform or
benzene.
Ox Bile Salt
A white or pale yellow power; taste, bitter and sweet;
hygroscopic.
Freely soluble in water or ethanol.
Palladium Chloride [PdClz 177. 33]
Red, needle crystals; hygroscopic.
Soluble in water, ethanol, acetone or hydrobromic acid.
Pancreatic Digest of Casein ( Pancreatin Hydrolysate)
Yellow granules. Casein hydrolyzed with pancreatin
decolourized and purified with active carbon. lt is used as
culture medium of bacteria, especially in test for sterility.
Pancreatin
Complies with the requirements prescribed in the monograph
of the Chinese Pharmacopoeia.
Papaic Digest of Soybean Meal
lt is the enzyme, which can digest protein, obtained from
immature papaya.
A yellow or slightly yellow powder, soluble in water.
Paraffin Liquid
A colourless oily liquid; almost odorless; tasteless. Miscible
with most of fats, soluble in ether or chloroform; insoluble
in water or ethanol.
Pararosaniline Hydrochloride [ C19 H1 s ClN3 323. 8]
Crystals with green luster ora brownpowder.
Easily soluble in ethanol to forro a scarlet colour and in hot
water to forro a red colour; slightly soluble in cold water,
insoluble in ether.
1-Pentanol (n-Pentanol) [CsH120 88.15]
A clear, colourless liquid; odour; characteristic and
pungent. Its vapour can forro explosive mixture with air.
Miscible with ethanol or ether; slightly soluble in water.
Boiling point: 138. 1ºC.
Pepsin
White to yellowish flakes or granules; tas te, slightly sour
and salty; hygroscopic.
Freely soluble in water; practically insoluble in ethanol,
chloroform or ether.
Peptone
A yellow or pale yellow powder; odourless; taste, slightly
bitter.
Soluble in water; insoluble in ethanol or ether.
Peptone from poultry
A yellow or slightly yellow powder; soluble in water.
Perchloric Acid [HC104 100. 46]
A colourless, clear liquid; strong oxidant; very hygroscopic;
volatile and corrosive.
Miscible with water.
Periodic Acid [HI04•2H20 227. 94]
Colourless monoclinic crystals; hygroscopic; changes to
slightly yellow on exposure to air; oxidative.
Freely soluble in water; soluble in ethanol; slightly soluble in
ether.
Petroleum Ether
A ciear coiouriess iiquid; odour, characteristic;
inflammable; low boiling fractions of petroleum ether is
highly volatile.
Miscible with anhydrous ethanol, ether or benzene; insoluble
in water.
Boiling range: 30-60ºC; 60-90ºC; 90-120ºC.
o-Phenanthroline [C12 Ha Nz• Hz O 198. 22]
White or pale yellow crystals or a crystalline powder;
darkens after long standing.
Soluble in ethanol or acetone; slightly soluble in water;
insoluble in ether.
Phenol [C6 Hs OH 94. 11]
Colourless or faintly pink needle crystals or a crystalline
mass; odour, characteristic; hygroscopic; gradually darkens
on exposure to light and air. Freely soluble in ethanol,
chloroform, ether, glycerin and volatile oils; soluble in
water; sparingly soluble in liquid paraffin.
Phenol Red [Cl9 Hl4 Os S 354. 38]
A deep red crystalline powder.
Soluble in ethanol; insoluble in water, chloroform or ether;
soluble in sodium hydroxide solution or sodium carbonate
solution.
Phenoxyethanol [~ H 5 OCH2CH2 OH 138. 17]
A colourless, clear liquid; odour, aromatic.
Freely soluble in ethanol, ether or sodium hydroxide
solution; slightly soluble in water.
Phenylacetamid [Cs Hg NO 135. 16]
White crystals; soluble in hot water or ethanol, slightly
soluble in cold water or ether.
Melting point: 156-160ºC.
Phenylhydrazine [CG HsN2 108. 14]
A yellow oily liquid, turns to scale crystals at a temperature
below 23ºC; turns to brown on exposure to air and light;
corrosive; inflammable.
Miscible with ethanol, ether, chloroform or benzene; soluble

in dilute acid; slightly soluble in water or petroleum benzin.
Phenylhydrazine Hydrochloride [ C6 Hs Nz• HCl 144. 60]
White or white transparent crystals; sublimable.
Freely soluble in water; soluble in ethanol, practically
insoluble in ether.
Phloroglucinol [C6 H3 (OH)3•2H2 O 162. 14]
A white or pale yellow crystalline powder; taste, sweet. It
turns to pale red on exposure to light.
Freely soluble in ethanol or ether; slightly soluble in water.
Phenolphthalein [Czo H14 04 318. 33]
A white powder.
Soluble in ethanol; insoluble in water.
Phenolsulfonphthalein [C19 H14 Os S 354. 38]
A deep red crystalline powder.
Soluble in ethanol, solution of sodium hydroxide or sodium
carbonate; insoluble in water, chloroform or ether.
L-a-Phosphatidyl Choline, from soyabean [ C44 Hss Nsg
790. 16]
A yellow to brown waxy solid; soluble in ethanol, ether,
chloroform or petroleum ether; slightly soluble in benzene;
insoluble in acetone, water or cold vegetable oíl. It swells in
water to colloidal liquid.
Phosphorus Pentoxide [P2 Os 141. 94]
A white powder, odour alliaceous; corrosive; highly
hygroscopic.
Phosphomolybdic Acid [P20s•20Mo03•51H20 3939. 49]
Bright yellow crystals.
Soluble in water, ethanol or ether.
Phosphoric Acid [H3P04 98. 00]
A clear, colourless, viscous liquid; corrosive.
Soluble in water.
Phosp~tic Acid [P20s•20W03•28H20 5283. 34]
White or pale yellow crystals.
Soluble in water, ethanol or ether.
o-Phthalaldehyde [Cs H6 02 134. 13]
Pale yellow needle crystals.
Soluble in water, ethanol or ether; slightly soluble m
petroleum benzin.
Picrolonic Acid [C10HsN40s 264. 21]
Yellow flake crystals.
Soluble in ethanol; slightly soluble in water.
Polyethylene Glycol 1500
A white or cream waxy solid; odour, characteristic; melts on
heating.
Soluble in water or ethanol.
Polyethylene Glycol Adipate HO [ CH2 CH2 OCO ( CH2 )4
COO]nH
White powder or crystals.
Soluble in chloroform; insoluble in water, ethanol or ether.
Polyethylene Glycol Glutarate
[HO(CH2CH20CO (CH2)3COOJnH 600-800]
A brownish black viscous liquid.
Soluble in acetone or chloroform.
Polysorbate 80
A pale red, oily liquid; odour, fat-like.
Soluble in water or most of organic solvents; insoluble in
paraffin oil or vegetable oil.
Potassium Acetate [KC2 H3 02 98. 14]
White crystals or powder; hygroscopic.
Freely soluble in water or ethanol.
8001 Reagents
Potassium Aluminium Sulfate [ AlK ( S04 )z • 12H2 O
474.39]
White, transparent crystals or powder; odourless; taste,
slightly sweet and astringent.
Freely soluble in water or glycerin; insoluble in ethanol or
acetone.
Potassium Biphthalate [KHC6 H4 (C00)2 204. 22]
A white crystalline powder.
Soluble in water; slightly soluble in ethanol.
Potassium Bisulfate [KHS04 136. 17]
White crystals; its aqueous solution is strongly acidic.
Soluble in water.
Potassium Bitartrate [KHC H4 06 188. 18]
White transparent crystals ora crystalline powder.
Soluble in water; insoluble in ethanol.
Potassium Bromate [KBr03 167. 00]
White crystals or powder.
Soluble in water; insoluble in ethanol.
Potassium Bromide [KBr 119. 00]
A white crystalline powder.
Soluble in water, boiling ethanol or glycerin; slightly soluble
in ethanol.
PotassiumCarbonate [K2C03•lH20 165.23]
White crystals or granules; hygroscopic.
Soluble in water; insoluble in ethanol.
Potassium Carbonate, Anhydrous [K2C03 138. 21]
White crystals or powder; hygroscopic.
Soluble in water; its aqueous solution is strongly alkaline;
insoluble in ethanol.
Potassium Chlorate [KC103 122. 55]
White, transparent crystals ora powder.
Freely soluble in boiling water; soluble in water or glycerin;
practically insoluble in ethanol.
Potassium Chloride [KCl 74. 55]
White crystals or a crystalline powder.
Freely soluble in water or glycerin; slightly soluble m
ethanol; insoluble in acetone or ether.
Potassium Chromate [K2Cr0 4 194. 19]
Pale yellow crystals.
Soluble in water; insoluble in ethanol.
Potassium Cyanide [KCN 65. 12]
White granules or clinkers.
Soluble in water; slightly soluble in ethanol.
Potassium Dichromate [K2Cr20 294. 18]
Lustrous orange-red crystals; taste, bitter; strongly
oxidative.
Soluble in water; insoluble in ethanol.
Potassium dihydrogen Phosphate [KH2P0 4 136. 09]
White crystals or a crystalline powder.
Soluble in water; insoluble in ethanol.
Potassium Ferricyanide [K3 Fe(CN)2 329. 25]
Red crystals; decompose easily on exposure to light, air and
acid.
Soluble in water; slightly soluble in ethanol.
Potassium Ferrocyanide [K4Fe(CN)6•3H20 422. 39]
Yellow crystals or granules. Its aqueous solution readily
deteriora tes.
Soluble in water; insoluble in ethanol.
Potassium Hyaluronate
A white, loose, flocky mass or flake.
Freely soluble in water.
 8001 Reagents

Loss on drying When dried to constant weight in vacuum Potato Starch [ ( C6 H10 Os ) n ]
over phosphorous pentoxide, loses not more than 10 % of its A white, amorphous powder; odourless; tasteless; highly
weight ( 0831 ) . hygroscopic.
Total nitrogen 3%-4% calculated on the dried basis ( 0704 Insoluble in water and ethanol; forms slightly blue collosol in
method 1 >. hot water.
Residue on ignition 14%-18% calculated on the dried basis Procaine Hydrochloride [ C13 H20 Nz 02• HCl 272. 78]
<0841 >. Complies with the requirements prescribed in the monograph
Viscosity Kinematic viscosity of O. 15% aqueous solution, of the Chinese Pharmacopoeia.
5-6 mm 2 / s ( 0633 method 1 ) .
pH value 6. 0-7. O, using O. 15% aqueous solution ( 0631 ). Pronase E
Molecular weight : 15 000-27 000
Po~ium Fluoride [KF 58. 10] A white or slightly brown powder. A nonspecific protease
White crystals; hygroscopic. isolated from Streptomyces griseus.
Freely soluble in water; soluble in hydrofluoric acid or The molecular weight is commonly 20 000.
ammonia solution; insoluble in ethanol. Freely soluble in brine and dilute salt solution.
Po~ium hydroxide [KOH 56. 11] The optimal ph value: 7. 8-8. O.
White granules or sticks; absorbs carbon dioxide easily to Propanol (n-propanol) [CH3CH2CH20H 60.10]
form potassium carbonate; hygroscopic. A clear, colourless liquid; inflammable.
Soluble in water or ethanol. Miscible with water, ethanol or ether.
Po~ium lodate [KI0 3 214. 00] Boiling point: 97. 2ºC.
White crystals or a crystalline powder. Propylene Glycol [ C3 Hs 02 76. 1O]
Soluble in water or dilute sulfuric acid; insoluble in ethanol. A colourless, viscous liquid; taste, slightly acrid.
Miscible with water, acetone or chloroform.
Po~ium lodide [KI 166. 00]
White crystals or powder. Propyl p-Hydroxybenzoate [C10 H12 03 180. 20]
Soluble in water, ethanol, acetone or glycerin; insoluble in White crystals.
ether. Freely soluble in ethanol or ether; Slightly soluble in boiling
water; practically insoluble in water.
Potmimn Naphthoquinooe sulfonate [C10HsKOsS 276. 31]
Golden yellow crystals. Protocatechuic Acid [G H6 04 154. 12]
Soluble in 50 % ethanol; slightly soluble in water. White or faintly brown crystals; discoloured on exposure to
a1r.
Po~iurn Nitrate [KN0 3 101. 10] Soluble in ethanol or ether, slightly soluble in water.
White crystals or powder; burns and detonates when m
Purified Water, Ammonia Free
contact, rubbed or bumped with organic substances.
Distill 1000 ml of purified water with 1 ml of diluted
Soluble in water; slightly soluble in ethanol.
sulphuric acid and 1 ml of potassium permanganate TS.
Po~iurn Periodate [KI04 230. 00] Add 1 ml of alkaline mercuric potassium iodide TS to 50 ml
A white, crystalline powder. of the Purified Water, Ammonia Free, no colour is
Soluble in hot water; slightly soluble in water. developed.
Po~ium Permanganate [KMn04 158. 03] Pyridine [Cs Hs N 79. 10]
Dark purple crystals with metallic luster; strong oxidant; A colourless, clear liquid; odour, disagreeable; taste,
decomposes in ethanol, concentrated acids or other organic peppery; hygroscopic; inflammable.
solvents with liberation of oxygen. Miscible with water, ethanol, ether or petroleum ether.
Soluble in water. Pyridine, Anhydrous [Cs HsN 79. 10]
Po~ium Pyroantimonate [K2 H2 Sb2 01 435. 73] To 200 ml of pyridine reagent add 40 ml of benzene, m1x
White granules ora crystalline powder. well and distil on a sand bath.
Freely soluble in hot water; slightly soluble in cold water; Collect the distillate at 115-116ºC, tightly closed, spare.
insoluble in ethanol. Pyrogallol [C6 H3 (OH)3 126. 11]
Po~ium Sodiurn Tartrate [KNaC4 ~ 06• 4H2 O 282. 22] White lustrous crystals.
White, transparent crystals ora crystalline powder. Soluble in water, ethanol or ether; slightly soluble m
Soluble in water; insoluble in ethanol. chloroform, benzene or carbon disulfide.

Po~iurn Sulfate [K2 S04 17 4. 26] Quinaldine Red [C21 H23 IN2 430. 33]
White crystals ora crystalline powder. A deep red powder.
Soluble in water or glycerin; insoluble in ethanol. Soluble in ethanol; slightly soluble in water.
Quinalizarin [C14 Hs 06 272. 21]
Po~iurn Tetrahydroborate [KBH4 53. 94]
Red or dark red crystals or a powder with green metallic
A white crystal. Stable in air.
lustre.
Easily soluble in water.
Soluble in acetic acid with yellow colour; in sulfuric acid with
Po~ium Trihydrogen Oxalate ( Po~iurn Tetroxalate ) bluish-violet colour; in aqueous alkaline solution with
[KH3(C204)2•2H20 254.19] reddish-violet colour; insoluble in water.
White crystals or a crystalline powder.
Quinine Sulfate [ (C20 H24N202 )z• H2S04•2H20 782. 96]
Soluble in water; slightly soluble in ethanol.
White, fine needle crystals; odourless; taste, very bitter;
Po~ium Thiocyanate [KSCN 97. 18] gradually discoloured on exposure to light; its aqueous
White crystals. solution yields neutral reactions.
Soluble in water or ethanol. Freely soluble in a mixture of chloroform-dehydrated ethanol

(2 : 1); slightly soluble in water, ethanol, chloroforrn or
ether.
Radix Arnebiae, Redix Lithospenni
See the monograph of Radix in Volume I
Red Mercuric lodide [Hgl2 454. 40]
A bright red powder; heavy; odourless.
Soluble in ether, sodium thiosulfate solution or potassium
iodide solution; Slightly soluble in dehydrated ethanol;
insoluble in water.
Resazurin [C12 H1 N04 229. 19]
Deep red crystals with green lustre.
Soluble in dilute sodium hydroxide solution; slightly soluble
in ethanol or glacial acetic acid; insoluble in water or ether.
(p-Nitrophenyl-azo) -resorcinol [ C12 Hg N3 04 259. 22]
A reddish-brown powder.
Slightly soluble in boiling ethanol, acetone, acetate easter or
toluene; insoluble in water; soluble in dilute alkaline
solution.
Resorcinol [C6 H4 COH)2 110. 11]
White transparent crystals; turns to pale red on exposure to
light, air or in contact with iron.
Soluble in water, ethanol or ether.
Rhodamine B [ C2a H31 ClN2 03 479. 02]
Green lustrous crystals or a reddish-violet powder.
Freely soluble in water with a bluish-red colour; its diluted
solution is strongly fluorescent; freely soluble in ethanol;
slightly soluble in hydrochloric acid or sodium hydroxide
solution.
Rose Bengal Sodium Salt [C20H2Cl4LNa20s 1017. 6]
A brownish red powder; soluble in water with a purple
colour; non-fluorescence; soluble in sulfuric acid with a
brown colour.
Ruthenium Red [Ru2 (OH)2Cl4•'7NH3•3H2 O 551. 23] or
[ CNH3)sRuO-RuCNH3)4-0-Ru CNH3)sCl6 786. 35]
A brownish-red powder.
Soluble in water, insoluble in ethanol and glycein.
Salicylic Acid [C1H603 138. 12]
White crystals or powder; taste, sweet followed by acrid;
gradually discolours on exposure to light; sublimes at 76ºC.
Soluble in ethanol and ether; slightly soluble in water.
Salicylaldehyde [C6H4 COH) CHO 122. 12]
A colourless or pale brown oily liquid; bitter almond-like
odour.
Soluble in ethanol, ether and benzene; slightly soluble in
water.
Sarcolysin [ C13 H1a Ch N2 02 305. 20 J
Needle crystals.
Soluble in ethanol or ethylene glycol, practically insoluble in
water.
Selenious Acid [H2 Se03 128. 97]
White crystals; hygroscopic; reduced to selenium by most of
the reducing agents.
Freely soluble in water or ethanol; insoluble in ammonia
solution.
Semicarbazide Hydrochloride [ NH2 CONHNH2 HCl
111. 53]
White crystals.
Freely soluble in water; insoluble in ethanol and ether.
Silico Gel (Silicic Acid) [mSi02•nH2O]
A white, translucent, or milky white granules or pellets;
hygroscopic; usually contains about 3%-7 % of water;
moisture absorbing capacity is about 40%.
8001 Reagents
Silicon Dioxide [Si0 2 60. 08]
A colourless transparent crystal or powder.
Soluble in excessive hydrofluoric acid, practically insoluble in
water or acids.
Silicowolframic Acid (Silicotungstic Acid) [Si02• 12W03•
26H20 3310. 66]
White or pale yellow crystals; hygroscopic.
Freely soluble in water or ethanol.
Silver Diethyldithiocarbamate [ (Cz Hs )2 NCSz Ag 256. 14]
Pale yellow crystals.
Freely soluble in pyridine; soluble in chloroform; insoluble in
water, ethanol, acetone or benzene.
Silver Nitrate [AgN0 3 169. 87]
White, transparent, flaky crystals.
Freely soluble in ammonia solution; soluble m water or
ethanol; slightly soluble in ether or glycerin.
Silver Oxide [Ag2 O 231. 74]
A brownish-black powder; heavy; slowly decomposes on
exposure to light; inflammable.
Freely soluble in dilute nitric acid or ammonia solution;
practically insoluble in water or ethanol.
Soda Lime
A mixture of sodium hydroxide and calcium oxide, pink
small granules with the addition of special indicator; the
colour fades gradually on absorption of carbon dioxide.
Sodium Acetate [NaCzH 302•3H20 136. 08]
White transparent crystals or white granules; efflorescent.
Soluble in water.
Sodium Acetate, Anhydrous [NaC2H 302 82. 03]
A white powder; hygroscopic.
Freely soluble in water; soluble in ethanol.
Sodium Alizarinsulfonate [Cl4 H1 Na01 S• H 2O 360. 28]
An orange or yellowish-brown powder.
Freely soluble in water; slightly soluble in ethanol; insoluble
in chloroform or benzene.
Sodium Ammonium Phosphate [Na CNH4 )2 P04 • 4H 2 O
226. 10]
White crystals or granules; efflorescent and loss part of
ammoma.
Soluble in water; insoluble in ethanol.
Sodium Bicarbonate [NaHC03 84. 01]
A white crystalline powder.
Soluble in water; insoluble in ethanol.
Sodium Bisulfite [NaHS03 104. 06]
A white crystalline powder; odour, resembling sulfur
dioxide; oxidized to form sulfate on exposure to air.
Soluble in water; slightly soluble in ethanol.
Sodium Bitartrate [NaHC4H406•H 20 190. 09]
A white crystalline powder; taste, sour.
Freely soluble in hot water; insoluble in water or ethanol.
Sodium Borohydride [NaBH 4 37. 83]
A white crystalline powder; hygroscopic.
Soluble in water, ammonia solution, ethylenediamine or
pyridine; insoluble in ether.
Sodium Bromide [NaBr 102. 89]
White crystals or a powder.
Soluble in water; slightly soluble in ethanol.
Sodium Carbonate [Na2C03•lOH20 286.14]
White transparent crystals.
Soluble in water or glycerin; insoluble in ethanol.
Sodium Carbonate, Anhydrous [Na2C03 105. 99]
 8001 Reagents
White powder or granules; absorbs one mol of water on
exposure to air.
Soluble in water; to produce strongly alkaline solution;
insoluble in ethanol.
Sodiurn Carbonate, Monohydrate [Na2CÜ3•H20 124. 00]
White prismatic crystals; hygroscopic; loses water of
crystallization at lOOºC.
Freely soluble in water; insoluble in ethanol.
Sodiurn Carboxymethylcellulose
A white powder or small granules; hygroscopic; easily
disperses and swells in hot water; the viscosity of a 1 %
solution is O. 005-2. O Pa •s.
Sodiurn Chloride [NaCl 58. 44]
White crystals ora crystalline powder; hygroscopic.
Soluble in water and glycerin; very slightly soluble in ethanol
or hydrochloric acid.
Sodiurn Chromotropate [C10H 6Na20 8 5¿•2H20 400. 29]
A white or grey powder.
Soluble in water with a pale brown colour.
SodiurnCitrate [C6HsNa3Ü7•2H20 294.10]
White crystals ora powder.
Freely soluble in water; insoluble in ethanol.
Sodiurn Cobaltinitrite [Na3Co(NÜ2)5 403. 94]
A yellow or yellowish-brown crystalline powder; readily
decampases.
Very soluble in water; slightly soluble in ethanol.
Sodiurn Deoxycholate [C24H39NaÜ4 414. 56]
A white crystalline powder; taste bitter..
Freely soluble in water; slight soluble in alcohols; insoluble
in ether.
Sodiurn Diethyldithiocarbamate [ ( C2 Hs) z NCS¿ Na• 3 H2 O
225.31]
White crystals; its aqueous solution is alkaline and gradually
decomposes to produce a turbidity due to the liberation of
carbon disulfide in contact with acid.
Freely soluble in water; soluble in ethanol.
Sodiurn dihydrogen Phosphate [NaH 2PÜ4•H20 137. 99]
White crystals or granules.
Freely soluble in water; practically insoluble in ethanol.
Sodiurn Diphenylamine-4-Sulfonate ( Sodiurn Diphenylamine
Sulfonate) [ C12 H10 NNa03 S 271. 27]
A white, crystalline powder; gradually discolours on
exposure to air, tums blue in acid; soluble in water or hot
ethanol; insoluble in ether, benzene, toluene or carbon
disulphide.
Sodiurn Ferricyanide, Ammoniated [Na3 [Fe(CN)s NH3}
3H2 O 325. 98]
Y ellow crystals.
Soluble in water.
Sodiurn Fluoride [NaF 41. 99 J
A white powder or cubic crystals.
Soluble in water; its aqueous solution is corrosive to glass;
insoluble in ethanol.
Sodiurn Formate [HCOONa • 2H 2O 104. 04]
White crystals; odour, faint formic acid; hygroscopic.
Soluble in water or glycerin; slightly soluble in ethanol.
Sodiurn Heptanesulfonate [ Cr H1s NaÜ3 ~·Hz O 220. 27]
Sodiurn Hexanesulfonate [C6H 13 Na03S 188. 18]
A white powder.
Soluble in water.
Sodiurn Hydrosulfite [Na2 S¿ 0 4 17 4. 11]
A white or almost white powder; odour, characteristic;
hygroscopic; decampases and burns on exposure to air or heat.
Freely soluble in water; insoluble in ethanol.
Sodiurn Hydroxide [NaOH 40. 00]
White granules or flakes; easily absorbs carbon dioxide and
water; hygroscopic.
Freely soluble in water, ethanol or glycerin.
Sodiurn Hypochlorite Solution [NaOCl 74. 44]
A clear, pale yellowish-green liquid; corrosive; strongly
oxidative and alkaline.
Miscible with water.
Sodiurn lodide [Nal 149. 89]
White crystals ora powder.
Soluble in water, ethanol or glycerin.
Sodiurn Laurylsulfate [ CH3 ( CH2 ) io CH2 OS03 Na
288.38]
White or pale yellow crystals or a powder; odour,
characteristic; decampases on exposure to moist and warm
air. lt is a mixture of 85 % sodium laurylsulfate and other
sodium alkyl sulfates.
Freely soluble in water, its 10% aqueous solution is not clear
at low temperature; soluble in hot ethanol.
Sodiurn Mercaptoacetate [ Cz H3 NaOz S 114. 1O]
A white powder; freeiy soiubie in water; siightly soiubie in
ethanol.
Sodium Metal [Na 22. 99 J
A silver-white metal, cube structure; the new section
luminescence, gradually tums dark gray on exposure to air;
soft, lightweight, decampase by reacting with water to
produce sodium hydroxide, hydrogen and generates heat. It
can cause buming with a bright yellow flame.
Sodium Molybdate [Na2MoÜ4•2H20 241. 95]
A white crystalline powder; loses water of crystallization at
lOOºC.
Soluble in water.
Sodium ~Naphthalenesulfonate [C10H7Naü3S 230. 22]
White crystals or powder.
Soluble in water; insoluble in ethanol.
Sodium 1, 2-Naphthoquinone-4-sulfonate [ Cio Hs Na Os S
260. 20]
White crystals.
Freely soluble in water; slightly soluble in ethanol.
Sodiurn Nitrate [NaNÜ3 84. 99 J
Colourless, transparent crystals or white granules; bums and
detonates in contact, rubbed or bumped with organic
substances.
Soluble in water; slightly soluble in ethanol.
Sodiurn Nitrite [NaN02 69. 00]
White or pale yellow crystals or granules; hygroscopic;
bums and detonates in contact with organic substances,
evolving noxious and irritant gas of nitrogen oxide and
nitrogen peroxide.
Soluble in water; slightly soluble in ethanol or ether.
Sodiurn Nitroprusside [Naz Fe(NQ) CCN)s• 2Hz O 297. 95]
Transparent, dark red crystals; its aqueous solution
decampases gradually and tums to green.

Soluble in water; slightly soluble in ethanol.
SodilUll 1-Nitmi0-2-naphthol-3 1 6-d&dfonate [C10HsNNaiOsSz
377.26]
Golden yellow crystals or a crystalline powder.
Soluble in water; slightly soluble in ethanol.
Sodium Octanesulfonate [CsH11Na03S 216. 28]
Sodium Oxalate [Na2G04 134. 00]
A white crystalline powder.
Soluble in water; insoluble in ethanol.
Sodium Pentanesulfonate [Cs Hu Na03S• H2 O 192. 21]
Whi te crys tals.
Soluble in water.
Sodium Periodate [Nal04 213. 89]
A white, crystalline powder.
Soluble in water, hydrochloric acid, nitric acid, sulfuric acid
or acetic acid; insoluble in ethanol.
Sodium Phosphate [Na3P04•12H20 380. 12]
Colourless or white granules.
Freely soluble in water; slightly soluble in ethanol.
Sodium Pyrosulfite [Na2 Sz Os 190. 11]
White crystals or a powder with slight odour of sulfur
di oxide; hygroscopic.
Soluble in water or glycerin; slightly soluble in ethanol.
Sodium Salicylate [C1HsNa03 160.10]
White scales or powder; odourless, turns to pink on
exposure to light for a long time.
Freely soluble in water and glycerin; soluble in ethanol;
practically insoluble in chloroform, ether or benzene.
Sodium Selenite [Na2 Se0 3 172. 94]
White crystals or a crystalline powder; efflorescent; readily
reduced by reducing agent.
Freely soluble in water; insoluble in ethanol.
Sodium Sulfate [Na2SÜ4 142. 04]
A white granular powder; absorbs a molecule of water in the
moist air.
Soluble in water or glycerin; insoluble in ethanol.
Sodium Sulfate, Anhydrous [Na2 S04 142. 04]
A white crystalline powder; hygroscopic.
Soluble in water; insoluble in ethanol.
Sodium Sulfide [Na2S•9H20 240. 18]
White crystals; its aqueous solution is alkaline.
Soluble in water; slightly soluble in ethanol; insoluble m
ether.
Sodium Sulfite [Na2 SQ3• 7H2 O 252. 15]
White, transparent crystals; odour, resembling sulfurous
acid; efflorescent; oxidized to form sodium sulfate on
exposure to air.
Soluble in water; very slightly soluble in ethanol.
Sodium Sulfite 1 Anhydrous [Na2 S03 126. 04]
White small crystals or powder.
Soluble in water and glycerin; very slightly soluble m
ethanol.
Sodium Taurocholate [C26H44NNa01S 537. 69]
White crystals; taste, sweet followed by bitter.
Freely soluble in water; soluble in ethanol.
Sodium Tellurite [Na2 Te03 221. 58]
A whi te powder.
Freely soluble in hot water; slightly soluble in water.
8001 Reagents
Sodium Tetraphenylborate [ (C6 Hs )4BNa 342. 22]
White crystals; odourless.
Freely soluble in water, methanol, anhydrous ethanol or
acetone.
Sodium Thioglycollate [CH2 (SH) COONa 114. 10]
White crystals; odour, slight; hygroscopic.
Freely soluble in water; slightly soluble in ethanol.
Sodium Thiosulfate [Na2 Sz Ü3• 5H2 O 248. 19]
Colourless, transparent crystals or white granules.
Soluble in water with endothermic reaction; slightly soluble
in ethanol.
SodilUll Wolframate (SodilBll Tungstate) [Na2 WQ4• 2H2 O
329.86]
A white crystalline powder; efflorescent.
Soluble in water; insoluble in ethanol.
Soluble Starch
A white powder; odourless; tasteless.
Soluble in boiling water; insoluble in water, ethanol or
ether.
Solvent Blue 19
It is a mixture of 1-amino-4-anilinoanthraquinone and
1-methylamino-4-anilinoanthraquinone.
Sorbitan Monooleate ( Span80)
A light pink or red brown oily liquid; odour, charactristic;
insoluble in water. It becomes emulsion solution after
dispersed in hot water.
Starch [ (C6 H10 Os),, (162. 14),, See Patato Starch and
Soluble Starch. ]
Stannous Chloride [SnClz• 2H2 O 225. 65]
White crystals.
Soluble in water, ethanol or sodium hydroxide solution.
Strontinum Chloride [SrClz•6H20 266. 64]
Colourless crystals or granules; odourless; volatilizes on
exposure to air; hygroscopic on exposure to moist air.
Freely soluble in water; soluble in ethanol.
Strontium Hydroxide [Sr (OH)2•8H20 265. 76]
Colourless or white crystals; deliquescent; absorbs carbon
dioxide from air to form carbonate. It loses seven molecules
of water in dry air.
Soluble in hot water or acid; slightly soluble in water.
Succinic Acid [H2C4H4Ü4 118. 09]
White crystals.
Soluble in hot water; slightly soluble in ethanol, acetone or
ether; insoluble in benzene, carbon disulfide, carbon
tetrachloride or petroleum benzin.
Sucrose [ C12 H22 Üu 342. 30]
Colourless crystals or white crystalline, loose powder;
odourless; tas te, sweet.
Very soluble in water; slightly soluble in ethanol; insoluble
in chloroform or ~ther.
Sudan][ [C6HsNNC6H4N: NC10H60H 352. 40]
A reddish brown powder.
Soluble in chloroform or glacial acetic acid; slightly soluble in
ethanol; insoluble in water.
Sudan N [C24H20N4Q 380. 45]
A dark brown powder.
Soluble in ethanol, chloroform, ether, benzene or phenol;
slightly soluble in acetone; insoluble in water.
Sulfamic Acid [NH2•S0 3H 97. 09]
 8001 Reagents
White crystals.
Soluble in water, hydrolyses easily with formation of
ammonium bisulfate; slightly soluble in methanol or ethanol;
insoluble in ether or acetone.
Sulfanilamide [C6HaN202S 172. 21]
White foliar or needle crystals or a powder.
Soluble in boiling water, ethanol, acetone, glycerin,
hydrochloric acid or solution of caustin alkali; slightly soluble
in water; insoluble in chloroform, ether or benzene.
Sulfanilic Acid [C6H1N03S 173.19]
A white or almost white powder, discoloured on exposure to
light.
Freely soluble in solution of ammonia, sodium hydroxide or
sodium carbonate; soluble in hot water; slightly soluble in
water.
Sulf~icylic Acid [GH606S•2H20 254. 22]
White crystals ora crystalline powder; instantly tums to red
with trace of iron; decompose to phenol or salicylic acid at
high temperature.
Freely soluble in water or ethanol; soluble in ether.
Sulfur [S 32. 06]
Sorts of allotrope of sulfur, yellow fine powder; combustible.
Soluble in benzene, toluene, carbon tetrachloride or carbon
disulfide; slightly soluble in ethanol or ether; insoluble in
water.
Sulfuric Acid [H2 so4 98. 08]
A clear; colourless; viscous liquid; generates much heat
when mixed with water or alcohol. It contains 95%-98% (g/g)
of H2S04.
Miscible with water or ethanol.
Relative density: about l. 84.
Sulfuric Acid, Nitrogen Free
Introduce sulfuric acid to a porcelain evaporating dish heated
on a sand bath until the vapor of sulfur trioxide is evolved
(about 2 hours), and continue to heat for more 15 minutes;
then allow to cool in an empty desiccator.
Sulfurous Acid [H2 SQ3 82. 07]
A colourless transparent liquid; asphyxiating odour of sulfur
dioxide; unstable, easily decomposed.
Miscible with water.
Talcum Powder
Complies with the requirements prescribed in monograph of
the Chinese Pharmacopoeia.
Tannic Acid [G6 Hs2 046 1701. 22]
A pal e yellow or pal e brown loose powder; odour,
characteristic; gradually darkens on exposure to air or light.
Soluble in water or ethanol.
Tartaric Acid [H2C4H406 150. 29]
White, transparent crystals ora white crystalline powder.
Soluble in water, methanol, ethanol, propanol or glycerin;
slightly soluble in ether; insoluble in chloroform.
Tertiary Butanol [ (CH3)3COH 74.12]
White crystals; it is a liquid when containing a little of
water; odour, camphor-like; hygroscopic; inflammable.
Miscible with ethanol or ether; soluble in water.
Boiling point: 82. 4 ºC.
Tetrabromophenolphthalein Potassium Ethyl Ester
[ C22 Hl3 Br4 K04 700. 06]
A dark green or bluish-violet crystalline powder.
Soluble in water, ethanol or ether.
Tetrabutylammonium Bromide [ (C4 Hg )4 NBr 322. 37]
White crystals; deliquescent; soluble in water, ethanol,
ether or acetone.
Tetrabutylammonium Hydroxide Solution [ Cl6 H31 NO
259.48]
A pure, colourless liquid; odour, ammonia-like; strongly
alkaline; easy to absorb carbon dioxide; usually prepared into
10 % or 20 % of solution.
Tetrabutylammonium Iodide [ <CH9)4NI 369. 37]
White or pale yellow crystals.
Freely soluble in ethanol; soluble in water; slightly soluble in
chloroform.
n-Tetradecane [CH3 (CH2 )12CH3 198. 39]
A clear, colourless liquid.
Miscible with ethanol or ether; insoluble in water.
Tetraethylammonium Hydroxide [ ( ~ Hs ) 4NOH 14 7. 26]
The free base only exists in solution or as a hydrate. Usually
it is used as a solution of 1O% , 25 % or 60 % . The aqueous
solution is colourless, strongly corrosive; very strongly
alkaline; freely absorbs carbon dioxide from air.
Tetraheptylammonium Bromide [ (G H1s )4 NBr 490. 71]
Chromatographic pure.
melting point: 89-91 ºC.
Tetrahydrofuran [C4 Ha O 72. 11]
A colourless liquid; odour, ether-like; inflammable; easily
forms peroxide during storage.
Miscible with water, ethanol, acetone or ether.
Boiling point: 66°C.
Tetramethylammonium Hydroxide [ (CH3)4NQH 91.15]
A colourless, clear liquid; absorbs carbon dioxide easily;
corrosive.
Soluble in water or ethanol.
Tetramethylethylenediamine [C6 H16 N2 116. 21]
A clear, colourless liquid.
Miscible with water and ethanol.
Tetrazolium Blue [C40H32CbNa02 727. 65]
Colourless or yellow crystals.
Freely soluble in methanol, ethanol or chloroform; slightly
soluble in water.
Thallous Chloride [TlCl 239. 85]
A white crystalline powder; poisonous.
Tums to purple on exposure to air or light.
Soluble in boiled water or 260 times cold water; insoluble in
ethanol. lts solubility in water is decreased when mixed with
hydrochloric acid.
Thallons Nitrate [TlN03 266. 40]
White or colourless crystals; poisonous; decompose at
450ºC.
Very soluble in hot water; soluble in cold water; insoluble in
alcohol.
Thioacetamide [CH3CSNH2 75. 13]
Colourless or white flaky crystals.
Soluble in water, ethanol or benzene; slightly soluble m
ether.
Thioglycollic Acid [CH2 (SH)COOH 92. 12]
A clear, colourless liquid; odour, irritant.
Miscible with water, ethanol, ether or benzene.
Thiourea [NH2CSNH2 76. 12]

White rhombic crystals or needle crystals; taste, bitter.
Soluble in water or ethanol; slightly soluble in ether.
Thrombin (Fila )
White freeze-drying mass. Extracted and purified from
bovine plasma or human plasma.
Thorin [ Cl6 H11 AsN2 Na2 010 Sz 5 76. 30]
Red crystals.
Freely soluble in water; insoluble in organic solvent.
Thorium Nitrate [Th (N03)4•4H20 552. 12]
White crystals or a crystalline powder; strong oxident;
radioactive; The aqueous solution has acidity.
Soluble in water and ethanol.
Thymol [C10 Hl4 O 150. 22]
White crystals.
Very slightly soluble in water.
Thymol Bine [Cz1H300sS 466. 60]
A brownish-green crystalline powder.
Soluble in ethanol; insoluble in water.
Thymolphthalein [ Czs H30 04 430. 54]
A white powder.
Soluble in ethanol; insoluble in water.
Titanium Dioxide [Ti02 79. 88]
A white powder.
Soluble in hydrofluoric acid or hot concentrated sulfuric acid;
insoluble in water, hydrochloric acid, nitric acid or dilute
sulfuric aicd.
Titanium Trichloride [TiCh 154. 24]
Dark violet-blue crystals; hygroscopic; unstable, dry powder
in the air easy firing, react easily in the moist air and
dissociate quickly.
Soluble in alcohols; practically insoluble in ethers.
Titan Yellow [CisH19NsNa206S4 695. 73]
A pale yellow or brown powder.
Soluble in water, ethanol, sulfuric acid or sodium hydroxide
solution.
Toluene [C6HsCH3 92.14]
A clear, colourless liquid; odour, benzene-like; inflammable.
Miscible with ethanol or ether.
Boiling point: 110. 6ºC.
p-Toluenesulfonic Acid [CH3~ H4S03 H • H20 190. 22]
White crystals.
Freely soluble in water; soluble in ethanol or ether.
o-Toluidine [ G Hg N 107. 16 J
A pale yellow liquid; tums to brownish-red gradually on
exposure to light and air.
Soluble in ethanol, ether or dilute acid; slightly soluble in
water.
Toluidine Bine [ C1s H 16 ClN3 S 305. 83 J
A dark green powder, with bronze lustre.
Freely soluble in water; slightly soluble in ethanol; very
slightly soluble in chloroform; practically insoluble in ether.
p-byl-lrArginiIE ~ Ester Hydrochbide [Ci4 1-6 Ni 04 s.
HCl 378. 88]
White crystals.
Soluble in water or methanol.
Tragacanth
A white or faint yellow powder; odourless.
Soluble in alkali solution or hydrogen peroxide solution;
insoluble in ethanol.
Trichloroacetic Acid [CChCOOH 163. 39]
Colourless crystals; odour, characteristic; hygroscopic;
8001 Reagents
corrosive; its aqueous solution is strongly acidic.
Freely soluble in ethanol or ether; soluble in water.
Triethanolamine [N CCH2CH20H)3 149. 19]
A colourless or pale yellow, viscous liquid. Tums to brown
on standing for a long time; absorbs water and carbon
dioxide on exposure to air; it is strongly alkaline.
Miscible with water or ethanol.
Triethylamine [ ( Cz Hs ) 3 N 10 l. 19]
A colourless liquid; odour, strong ammonia.
Miscible with ethanol or ether; slightly soluble in water.
Boiling point: 89. 5ºC.
Triethylenediamine [ ~ H12 N2• 6H2 O 220. 27]
White or slightly yellow crystals; odour, characteristic;
hygroscopic.
Freely soluble in water, methanol or ethanol.
Trifluoroacetic Acid [CF3 COOH 114. 02]
A colourless, fuming liquid; hygroscopic; strongly corrosive.
Freely soluble in water, ethanol, acetone or ether.
Trimethylpentane [ CCH3 )3CCH2CH CCH3 )z 114. 23]
A clear, colourless liquid; inflammable. It can form explosive
mixture with air.
Soluble in acetone, chloroform, ether or benzene; insoluble
in water.
Boiling point: 99. 2ºC.
Trinitrophenol [C6 H3 N3 01 229. 11]
Pale yellow crystals; odourless; taste, bitter; violet
explosion occurs at high temperature or when bumped or
rubbed in dryness.
Soluble in hot water, ethanol or benzene.
Trioctyl Phosphate [ CCs Hl7 )a• P04 434. 64]
A colourless or pale yellow oily liquid.
Soluble in ethanol, acetone or ether.
Triphenyltetrazolium Chloride [Cl9 H1sClN4 334. 81]
White crystals; darkens gradually on exposure to light.
Soluble in water, ethanol or acetone; insoluble in ether.
Trometamol [C4 Hn N03 121. 14]
White crystals with strong alkalinity.
Soluble in water; insoluble in ether.
T~in
A white, almost white or pale yellow powder.
Soluble in water; insoluble in ethanol.
Tyrosine [~ Hn N03 181. 19]
White crystals.
Soluble in water; insoluble in ethanol or ether.
Tryptone
A beige powder; easily deliquescent.
Soluble in water; insoluble in ethanol or ether.
Uranyl Acetate [U02 CC2H302)2•2H20 424. 15]
A yellow crystalline powder.
Soluble in water; slightly soluble in ethanol.
Urea [NH2CONH2 60. 06]
White crystals or powder; odour, ammoniacal.
Soluble in water, ethanol or benzene; practically insoluble in
chloroform or ether.
Urotropine [C6H12N4 140.19]
White crystals; odourless.
Soluble in water, ethanol or chloroform; slightly soluble in
ether.
Valine [CsH11N02 117.15]
White crystals; sublimable.
Soluble in water; insoluble in ethanol or ether.
 8002 Test Solutions
Vanadium Pentoxide [V2 Os 181. 88]
An orange yellow crystalline powder or reddish-brown needle
crystals.
Solube in acid solution or alkali solution; slightly soluble in
water; insoluble in ethanol.
Vanillin [Cs Hs Ü3 152. 15]
White crystals; odour, fragrant.
Freely soluble in ethanol, chlorofarm, ether, glacial acetic
acid or pyridine, soluble in oil or sodium hydroxide solution.
Water, Nitrate-free and Nitrite-free
Purified water, ammonia free, or deionized water.
Nitrate, Nitrite Carry out the test under Purified Water,
no colour is developed.
Xanthydrol [Ci3H1002 198. 22]
A pale yellow crystalline powder.
Soluble in ethanol, chlorofarm or ether; insoluble in water.
Xylene [ C; H4 ( CH3 ) 2 106. 17]
A clear, colourless liquid; odour, characteristic;
inflammable. It is a mixture of the three isomers of o-, m-
and p-xylene.
Miscible with ethanol, chloroform or ether; insoluble in
water.
Boiling point: 137-140ºC.
Xylene Cyanol Blue FF [CzsH21N2Na06Sz 538. 62]
A brown or bluish-black powder.
Freely soluble in ethanol; soluble in water.
Xylenol Orange [C31H2sN2N~Ü13S 760. 59]
A reddish-brown crystalline powder; hygroscopic.
Freely soluble in water; insoluble in ethanol.
Yeast Extract
A reddish-yellow to brown powder; odour, characteristic,
but no rancid smell.
Soluble in water to yield a weak acidic reaction.
Chloride Not more than 5 % , calculated as NaCl ( 0801 ) .
Nitrogen 7. 2%-9. 5%, calculated on the dried basis ( 0704 ).
Coagulable Protein No sediment is produced when its
aqueous solution 0-20) is filtered and boiled.
Loss on drying Not more than 5. 0% (0831 ).
Residue on ignition Not more than 15 % ( 0841 ) .
Zinc [Zn 65. 39]
Greyish-white granules with metallic lustre.
Soluble in dilute acid with liberation of hydrogen; soluble
slowly in ammonia solution or sodium hydroxide solution.
Zinc Acetate [Zn(CzH3Ü2)2•2H20 219. 51]
White crystals.
Freely soluble in water or boiling ethanol; slightly soluble in
ethanol.
Zinc Chloride [ZnCl 136. 30]
A white crystalline powder or clinkers.
Freely soluble in water; soluble in ethanol, acetone or ether.
Zincon [C20 H1s N4 Na06 S 462. 42]
A brown, crystalline powder.
Soluble in ethanol or sodium hydroxide solution; insoluble in
water.
Zinc Oxide [ZnO 81. 39]
A white or pale yellow powder.
Soluble in dilute acid, concentrated alkali solution or
ammonia solution; insoluble in water or ethanol.
Zinc Sulfate [ZnS04• 7H2O 287. 56]
White crystals, granules or powder.
Freely soluble in water; soluble in glycerin; slightly soluble
in ethanol.
ZirconiumNitrate [Zr (NQ3)4•5H20 429.32]
White crystals; hygroscopic; decampases at lOOºC.
Freely soluble in water; soluble in ethanol.
Zirconyl Chloride [ZrOClz• 8H2 O 322. 25]
White silky or needle crystals; Its aqueous solution is acidic.
Freely soluble in water or ethanol; slightly soluble in
hydrochloric acid
Polyethylene Glycol Glutarate [ HO ( CH2 CH2 oca
( CH2 ) 3COO) n H 600-800 ]
Dark brown viscous liquid.
Soluble in acetone or chlorofarm.
8002 Test Solutions
Acetic Acid, Dilute
Dilute 60 ml of glacial acetic acid with water to 1000 ml.
N-Acetyl-L-tyrosine Ethyl Ester TS
Dissolve 24. O mg of N-Acetyl-L-tyrosine ethyl ester in
O. 2 ml of ethanol, add 2 ml of phosphate buffer solution
( mix 38. 9 ml of O. 067 mol/L potassium dihydrogen
phosphate with 61. 6 ml of O. 067 mol/L disodium hydrogen
phosphate, pH7. O) and 1 ml of the indicator solution (mix
equal quantities of O. 1 % alcoholic methyl red solution and
O. 05 % alcoholic methylene blue solution) , dilute with water to
10 ml.
Acid Ferric Ammonium Sulfate TS
Dissolve 20 g of ferric ammonium sulfate m 9. 4 ml of
sulfuric acid, dilute with water to 100 ml.
Acid Stannous Chloride TS
Dissolve 20 g of stannous chloride in hydrochloric acid to
make 50 ml and filter.
The solution should be used within three months.
Acid Zirconium Afüarin 1S
Dissolve 70 mg of sodium alizarinsulfonate in 50 ml of water,
and then add the solution slowly to 50 ml of O. 6 % zirconyl
chloride (ZrOClz• 8H2 0) solution. Dilute with a mixed acid
solution which contains 123 ml of hydrochloric acid and 40 ml
of sulfuric acid per 1000 ml to 1000 ml. Allow to stand far 1
hour.
Alizarin Fluoro Blue 1S
Add 12. 5 ml of sodium hydroxide solution (l. 2-100) to
O. 19 g of alizarin fluoro blue, add 800 ml of water and
O. 25 g of sodium acetate crystals, adjust to about pH5. 4
with dilute hydrochloric acid and dilute with water to make
1000 ml, mix well.
Alkaline Cupric Citrate 1S
( 1) Dissolve 17. 3 g of cupric sulfate and 115. O g of citric
acid in 200 ml of warm or slightly warm water.
(2) Dissolve 185. 3 g of anhydrous sodium carbonate (dried
at 180ºC far 2 hours) in water to make 500 ml.
Add 50 ml of solution ( 2) to 20 ml of solution ( 1) with
constant shaking befare use, cool and then dilute with water
to 100 ml.
Alkaline Cupric Tartrate 1S
(1) Dissolve 6. 93 g of crystalline cupric sulfate in water to
make 100 mi.

(2) Dissolve 34. 6 g of crystalline potassium sodium tartrate
and 10 g of sodium hydroxide in water to make 100 ml.
Mix equal volume of solution (1) and solution (2) before use
Alkaline Mercuric Po~ium lodide TS
Dissolve 10 g of potassium iodide in 10 ml of water, add
slowly with stirring a saturated solution of mercuric chloride
until a red precipitate remains undissolved. Add 30 g of
potassium hydroxide and allow to dissolve, then add 1 ml or
more of the saturated solution of mercuric chloride. Dilute
with water to 200 ml. Allow the precipitate to settle and use
the supernatant liquid.
Add 2 ml of this solution to 50 ml of water which contains
O. 05 mg of ammonia, a yellowish-brown colour is formed
immediately.
Alkaline Hydroxylamine TS
(1) Dissolve 12. 5 g of sodium hydroxide in dehydrated
methanol to make 100 ml.
( 2 ) Add 100 m1 of dehydrated methanol to 12. 5 g of
hydroxylamine hydrochloride and heat under reflux to dissolve.
Mix equal parts of the two solutions and filter before use.
This solution should be freshly prepared and used within
4 hours.
Alkaline JJ-Naphthol TS
Dissolve O. 25 g of ~-naphthol in 10 ml of sodium hydroxide
solution 0-10). This solution should be freshly prepared.
Alkaline Pyrogallol TS
Dissolve O. 5 g of pyrogallol in 2 ml of water. Add 8 ml of
water which contains 12 g of potassium hydroxide, mix well.
This solution should be freshly prepared.
Alkaline Sodium Hydrosulfite TS
Dissolve 50 g of sodium hydrosulfite in 250 ml of water
. Dissolve 28. 57 g of potassium hydroxide in water to make
40 ml. Mix these two solutions. This solution should be
freshly prepared.
Alkaline Sodium Nitroprusside TS
Dissolve 1 g of sodium nitroprusside and 1 g of sodium
carbonate in water to make 100 ml.
Alkaline Tetrazolium Blue TS
Mix 10 ml of O. 2 % methanol solution of tetrazolium blue
with 30 ml of 12 % methanol solution of sodium hydroxide
before use.
Alkaline Trinitrophenol TS
To 20 ml of 1% trinitrophenol solution, add 10 ml of 5% sodium
hydroxide solution, then dilute with water to 100 ml.
This solution must be freshly prepared.
1-Arnino-2-naphthol-4-sulfonic acid TS
Mix thoroughly 5 g of sodium sulfite anhydrous with 94. 3 g
of sodium bisulfite and O. 7 g of l-amino-2-naphthol-4-
sulfonic acid. Dissolve l. 5 g of the mixture in 10 ml of water
before use, filter if necessary.
Aluminium trichloride TS
Dissolve 1 g of aluminium chloride in ethanol to make 100 ml.
Amrnonia TS
Dilute 400 ml of concentrated ammonia solution with water
to make 1000 ml.
Ammonia Concentrated TS
Use concentrated ammonia solution directly.
Ammoniated Ammonium Chloride TS
Dilute concentrated ammonia TS with equal volume of water,
add ammonium chloride to make a saturated solution.
Ammoniated Cupric Chloride TS
Dissolve 22. 5 g of cupric chloride in 200 ml of water, add
8002 Test Solutions
100 ml of concentrated ammonia TS and mix well.
Ammoniated Cupric Sulfate TS
Add ammonia TS dropwise to cupric sulfate TS until the
freshly formed precipitate is almost dissolved, allow to settle
and decant the clear solution. The solution should be freshly
pre pared.
Ammoniated Nickel Nitrate TS
Dissolve 2. 9 g of nickel nitrate in 100 ml of water, add 40 ml
of ammonia TS, shake and then filter.
Ammoniated Silver Nitrate TS
Dissolve 1 g of silver nitrate in 20 ml of water, add ammonia
TS dropwise with stirring until the precipitate is almost
completely dissolved, filter.
Preserve the solution in amber coloured glass bottle,
protected from light.
Ammoniated Sodium Ferricyanide TS
Dissolve 1 g of ammoniated sodium ferricyanide
[Na2Fe (CN)sNH3•H20] in water to make 100 ml.
Ammonium Acetate TS
Dissolve 10 g of ammonium acetate in water to make 100 ml.
Ammonium Carbonate TS
Dissolve 20 g of ammonium carbonate with 20 ml of
ammonia TS in water to make 100 ml.
Ammonium Ceric Ni trate TS (Ceric Ammonium Nitrate TS)
Dissolve 25 g of ammonium ceric nitrate in dilute nitric acid
to make 100 ml.
Ammonium Chloride TS
Dissolve 10. 5 g of ammonium chloride m water to make
100 ml.
Ammonium Magnesium Chloride TS
Dissolve 5. 5 g of magnesium chloride and 7 g of
ammonium chioride in 6 5 mi oí water and then add 3 5 mi
of ammonia TS. Allow to stand in glass bottle for several
days, filter.
The solution should be filtered, if turbidity occurs.
Ammonium Mercuric Thiocyanate TS
Dissolve 5 g of ammonium thiocyanate and 4. 5 g of mercuric
chloride in water to make 100 ml.
Ammonium Molybdate TS
Dissolve 10 g of ammonium molybdate m water to make
100 ml.
Ammonium Molybdate in Sulfuric Acid TS
Dissolve O. 1 g of ammonium molybdate in 10 ml of sulfuric
acid.
Ammonium Molybdate-Sulfuric Acid TS
Dissolve 2. 5 g of ammonium molybdate in a mixtre of 15 ml
of sulfuric acid and water to make 100 ml.
This solution should be used within two weeks.
Ammonium Oxalate TS
Dissolve 3. 5 g of ammonium oxalate in water to make
100 ml.
Ammonium Polysulfide TS
Add sulfur to ammonium sulfide TS and make a saturated
solution.
Ammonium Reineckate TS
Dissolve O. 5 g of ammonium reineckate in 20 ml of water,
shake for 1 hour, filter.
This solution should be freshly prepared and used within 48
hours.
 8002 Test Solutions

Ammonium Sulfide 1S Calcium Hydroxide 1S

To a solution of 60 ml of ammonia TS saturated with Put 3 g of calcium hydroxide in glass bottle, add 1000 ml of
hydrogen sulfide, add 40 ml of ammonia TS. water, well-stoppered, allow to stand for 1 hour with
Preserve in amber coloured glass bottle, protected from frequent shaking vigorously, use the supernatant liquid as the
light. test solution.
This solution is unsuitable far use if a copious precipitate of Calcium Sulfate 1S
sulfur is present. Use the saturated solution of calcium sulfate directly.
Ammonium Thiocyanate 1S Cerous Nitrate 1S
Dissolve 8 g of ammonium thiocyanate m water to make Dissolve O. 22 g of cerous nitrate in 50 ml of water, add
100 ml. O. 1 ml of nitric acid and 50 mg of hydroxylamine
Ammonium vanadate 1S hydrochloride, dilute with water to make 1000 ml and
Dissolve O. 25 g of ammonium vanadate m water to make mix well.
100 ml.
Chloral Hydrate 1S
Anisaldehyde 1S Dissolve 50 g of chloral hydrate in a mixture of 15 ml of
Dissolve O. 5 ml of anisaldehyde in 50 ml of acetic acid, add water and 10 ml of glycerin.
1 ml of sulfuric acid and mix well.
Chlorine 1S
This solution should be freshly prepared.
Use the saturated solution of chlorine in water.
Anthrone 1S This solution should be freshly prepared.
Dissolve O. 7 g of anthrone in 50 ml of sulfuric acid and dilute
Chloroplatinic Acid ( Platinic Chloride) 1S
with sulfuric acid solution (70-100) to 500 ml.
Dissolve 2. 6 g of chloroplatinic acid in water to make 20 ml.
Antimony Trichloride 1S
Chromic Nitrate 1S
Use the saturated chlorofarm solution of antimony
(1) Add 10 ml of nitric acid to 100 ml of water and mix
trichloride.
well.
Arnebia 1S (2) Di.ssolve 10 g of chromium trioxide in 100 ml of water.
Extract 1 O g of coarse powder of arnebia with 100 ml of Mix equal volume of the two solutions befare use.
90 % ethanol far 24 hours and filter. Add equal volume of
Chromotropic Acid 1S
glycerin to the filtrate, mix, allow to stand for 2 hours and
r·L Dissolve 50 mg of sodium chromotropate in 100 ml of a cold
1uter.
mixture of sulfuric acid and water (9 : 4).
Preserve in amber coloured glass bottle Use within 2 months.
This solution should be freshly prepared befare use.
Auric Chloride 1S
Citric-Acetic Anhydride 1S
Dissolve 1 g of auric chloride in 35 ml of water.
Dissolve 2 g of citric acid in 100 ml of acetic anhydride.
Barium Chloride 1S
Cobaltous Acetate 1S
Dissolve 5 g of fine powder of barium chloride in water to
Dissolve O. 1 g of cobaltous acetate in methanol to make
make 100 ml.
100 ml.
Barium Hydroxide 1S
Dissolve barium hydroxide in freshly boiled and cooled water Cobaltous Chloride 1S
Dissolve 2 g of cobaltous chloride m 1 ml of hydrochloric
to make a saturated solution.
This solution should be freshly prepared. acid, add water to make 100 ml.

Barium Nitrate 1S Copper-Pyridine 1S

Dissolve 6. 5 g of barium nitrate in water to make 100 ml.
Boric Acid 1S
Use the saturated acetone solution of boric acid.
Brilliant Green 1S
Dissolve O. 1 g of brilliant green in 100 ml of water.
Bromine 1S
To 2-3 ml of bromine in a glass bottle which is stoppered and
lubricated with vaseline add 100 ml of water to make a
saturated solution by shaking.
This solution should be protected from light.
Bromine-Potassium Bromide 1S
Dissolve 30 g of bromine and 30 g of potassium bromide in
water to make 100 ml.
Bromothymol Blue 1S
Dissolve O. 3 g of Bromothymol Blue in 5 ml of 1 mol/L
sodium hydroxide solution, dilute with water to 1000 ml.
Cadmium lodide 1S
Dissolve 5 g of cadmium iodide in water to make 100 ml.
Calcium Chloride 1S
Dissolve 7. 5 g of calcium chloride in water to make
100 ml.
Dissolve 4 g of cupric sulfate in 90 ml of water and add 30 ml
of pyridine. This solution should be freshly prepared.
Cupric Acetate 1S
Dissolve O. 1 g of cupric acetate in 5 ml of water which
contains a few drops of acetic acid. Dilute with water to
100 ml and filter.
Cupric Acetate Concentrated 1S
Dissolve 13. 3 g of cupric acetate in a mixture of 195 ml of
water and 5 ml of acetic acid.
Cupric Sulfate 1S
Dissolve 12. 5 g of cupric sulfate in water to make 100 ml.
Cupric Iodotartrate 1S
Dissolve successively 7. 5 g of cupric sulfate, 25 g of
potassium sodium tartrate, 25 g of anhydrous sodium
carbonate, 20 g of sodium bicarbonate and 5 g of
potassium iodide in 800 ml of water. Dissolve O. 535 g of
potassium iodate in water and add this solution to the
mixture mentioned above. Dilute with water to 1000 ml.
Cyanogen Bromide 1S
Add O. 1 mol/L ammonium thiocyanate solution dropwise to
bromine TS until it is colourless.
This solution should be freshly prepared. It is poisonous.

Diaminonaphthylene TS
Dissolve O. 1 g of 2, 3-diaminonaphthylene and O. 5 g of
hydroxylamine hydrochloride in 100 ml of O. 1 mol/L
hydrochloric acid solution, heat if necessary, cool and filter.
This solution should be freshly prepared and protected from light.
Diazotized Sulfanilic Acid TS
Dissolve l. 57 g of sulfanilic acid in 80 ml of water and 1O ml
of dilute hydrochloric acid by heating on a water bath. Cool to
15ºC and add 6. 5 ml of sodium nitrite solution 0-10) with
stirring and dilute with water to 100 ml.
This solution should be freshly prepared.
Diazotized Dinitroaniline TS
Dissolve 50 mg of 2, 4-dinitroaniline in l. 5 ml of
hydrochloric acid, add l. 5 ml of water, cool in ice bath,
then add 5 ml of 10% sodium nitrite solution dropwise with
shaking.
Diazotized p-Nitroaniline TS
Dissolve O. 4 g of p-nitroaniline in a mixture of 20 ml of
dilute hydrochloric acid and 40 ml of water, cool to 15°C and
add 10 % sodium nitrite solution until 1 drop of the solution
turns blue to starch potassium iodide TP.
This solution should be freshly prepared.
2, 6-Dichloroindophenol Sodiurn TS
Dissolve O. 1 g of 2, 6-dichlorophenol indophenol sodium in
100 ml of water and filter.
2, 6-Dichloroquinone Chlorimide TS
Dissolve 1 g of 2, 6-dichloroquinone chlorimide in 200 ml of
ethanol.
Dimethyl-p-Phenylenediamine Hydrochloride TS
Dissolve O. 1 g of dimethyl-p-phenylenediamine hydrochloride
in 10 ml of water. This solution should be freshly prepared
with a small amount. Preserve this solution in cold and dark
piace. Do not use when turns to reddish-brown coiour.
p-Dimethylaminobenzaldehyde TS
Dissolve O. 125 g of p-dimethylaminobenzaldehyde in a cooled
mixture of 65 ml of nitrogen-free sulfuric acid and 35 ml of
water, add O. 05 ml of ferric chloride TS and mix well.
This solution should be used within 7 days.
Dimethylglyoxime TS
Dissolve 1 g of 2, 3-dimethylglyoxime in 100 ml of ethanol.
Dinitrobenzene TS
Dissolve 2 g of m-dinitrobenzene in ethanol to make 100 ml.
Dinitrobenzoic Acid TS
Dissolve 1 g of 3, 5-dinitrobenzoic acid in ethanol to make
100 ml.
Dinitrophenylhydrazine TS
Dissolve l. 5 g of 2, 4-dinitrophenylhydrazine in 20 ml of
sulfuric acid solution O - 2) , dilute with water to 100 ml
and filter.
Dinitrophenylhydrazine Dilute TS
Dissolve O. 15 g of 2, 4-dinitrophenylhydrazine in 100 ml of
aldehyde free ethanol which contains O. 15 ml of sulfuric
acid.
Dioctyl Sodiurn Sulfosuccinate TS
Dissolve O. 9 g of dioctyl sodium sulfosuccinate in 50 ml of
water with gentle heating. Cool to room temperature, dilute
with water to 200 ml.
Diphenylamine TS
Dissolve 1 g of diphenylamine in 100 ml of sulfuric acid.
Dipyridyl TS
1
Dissolve O. 2 g of 2, 2 -dipyridyl, 1 g of sodium acetate
8002 Test Solutions
crystal and 5. 5 ml of glacial acetic acid in water to make
100 ml.
Disodiurn Hydrogen Phosphate TS
Dissolve 12 g of crystalline disodium hydrogen phosphate in
water to make 100 ml.
Ethanol, Dilute TS
Dilute 529 ml of ethanol with water to 1000 ml.
It contains 49. 5%-50. 5% (ml/mD of C2HsOH at 20ºC.
Ethanolic Ammonia TS
Add concentrated ammonia solution to dehydrated ethanol. It
contains 9-11 g of NH3 per 100 ml.
Preserve in rubber stoppered bottle.
Ethanolic p-Dimethylaminobenzaldehyde TS
Dissolve 1 g of p-dimethylaminobenzaldehyde in 9. O ml of
ethanol and 2. 3 ml of hydrochloric acid, dilute with ethanol
to 100 ml.
Ethanolic Dinitrophenylhydrazine TS
Dissolve 1 g of dintrophenylhydrazine in 1000 ml of ethanol,
add 10 ml of hydrochloric acid slowly and mix well.
Ethanolic Hydroxylamine Hydrochloride TS
Mix one portian of hydroxylamine hydrochloride solution
( 34. 8 - 100 ) , one portian of sodium acetate-sodium
hydroxide TS with four portions of ethanol.
Ethanolic Mercuric Bromide TS
Dissolve 2. 5 g of mercuric bromide in 50 ml of ethanol by
gentle heat.
Preserve in glass-stoppered bottle, protected from light.
Ethanolic Po~iurn Hydroxide 'IS
Use ethanolic potassium hydroxide (0. 5 mol/L) VS.
Ethanolic Silver Nitrate TS
Dissolve 4 g of silver nitrate in 10 ml of water, dilute with
ethanol to 100 ml.
Ethanolic Sodiurn Nitrite TS
Dissolve 5 g of sodium nitrite in 60 % solution of ethanol to
make 1000 ml.
Ethanolic Sulfuric Acid TS
Dilute 57 ml of sulfuric acid to 1000 ml with ethanol. It
contains 9. 5%-10. 5% (g/mD of H2SÜ4.
Ferric Chloride TS
Dissolve 9 g of ferric chloride in water to make 100 ml.
Ferric Perchlorate TS
Add O. 8 g of ferrous powder gradually in several portions in
10 ml of 70% perchloride acid, dissolve by briefly heating,
cool, dilute with dehydrated ethanol to 100 ml. Befare use,
measure 20 ml of the above solution, add 6 ml of 70 %
perchloride acid, dilute with dehydrated ethanol to 500 ml.
Ferric Salicylate TS
( 1) Dissolve O. 1 g of ferric ammonium sulfate in 2 ml of
dilute sulfuric acid and add water to make 100 ml;
(2) Dissolve l. 15 g of sodium salicylate in water to make
100 ml;
(3) Dissolve 13. 6 g of sodium acetate in water to make
100 ml.
( 4) Mix 1 ml of ferric ammonium sulfate solution, O. 5 ml of
sodium salicylate solution, O. 8 ml of sodium acetate solution
with O. 2 ml of dilute acetic acid befare use, add water to
make 5 ml and mix well.
Ferric sulfate TS
Mix 5 g of ferric sulfate with sorne water, add 20 ml of
sulfuric acid and shake thoroughly, dilute with water to
100 ml.
 8002 Test Solutions
Ferrous sulfate TS
Dissolve 8 g of ferrous sulfate crystal with 100 ml of the
boiled and cooled water, freshly prepared.
Folio pheool TS
Folin phenol TS A Mix 4% solution of sodium carbonate
and O. 2 mol/L sodium hydroxide solution with equal volume
as solution I . Mix O. 04 mol/L copper sulfate solution and
2 % solution of sodium tartrate as solution 11 . Mix the
solution I and solution 11 with the proportion of 50 : l.
Folin phend TS B Dissolve 100 g of sodium tungstate and
25 g of sodium molybdate in 700 ml of water, add 100 ml of
hydrochloric acid and 50 ml of 85 % solution of
orthophosphoric acid and heat the mixture under a reflux
condenser for 10 hours, stand to cool.
Add 150 g of lithium sulphate, 50 ml of water and a few
drops of bromine and boil to remove excess bromine ( about
15 minutes), cool, dilute to 1000 ml with water and
filter. The filtrate should be store in brown bottle and diluted
with one volume of water and mix well before use.
Folio TS
Heat 10 g of sodium tungstate under reflux, 2. 5 g of sodium
molybdate, 70 ml of water, 5 ml of 85% phosphoric acid solution
and 10 ml of hydrochloric acid in a 200 ml flask gently for about 10
hours, cool, add 15 g of lithium sulfate, 5 ml of water and 1 drop
of bromine VS. fuil the mixture for about 15 minutes, or until the
excess bromine is expelled. Cool, dilute with water to 100 ml and
filter. The filtrate is kept in amber coloured glass bottle in a
refrigerator.
Dilute 2. 5 rnl of the stock solution with water to 10 ml
before use, shake thoroughly.
Formaldehyde TS
Use "Formaldehyde Solution" directly.
Formaldehyde-Sulfuric Acid TS
Add 1 drop of formaldehyde TS to 1 ml of sulfuric acid and
mix well.
This solution should be freshly prepared before use.
Fuchsin-Pyrogallol TS
Dissolve O. 1 g of fuchsin basic in 50 ml of boiling water, cool, add
2 ml of a saturated solution of sodium bisulfite, and allow to stand
for 3 hours. Add O. 9 ml of hydrochloric acid and allow to stand
ovemight. Add O. 1 g of pyrogallol, shake until dissolution is
effected, dilute with water to 100 ml.
Fuchsin-Sulfurous Acid TS
Dissolve O. 2 g of fuchsin basic in 100 ml of hot water, add
20 ml of sodium sulfite solution ( 1 - 10) and 2 ml of
hydrochloric acid, then dilute with water to 200 ml, add O. 1
g of activated carbon, stir and filter promptly. Allow to
stand for at least 1 hour.
This solution should be freshly prepared.
Furfural TS
Dilute 1 ml of furfural with water to 100 ml.
This solution should be freshly prepared.
Glycerio Dilute TS
Dilute 33 ml of glycerin with water to 100 ml. Add a small
piece of camphor or a drop of liquefied phenol.
Glycerin-acetic Acid TS
Mix 1 volume of glycerin, 1 volume of 50% acetic acid
solution and 1 volume of water.
Glycerin-ethanol TS
Mix 1 volume of glycerin and 1 volume of dilute ethonal.
Glycerol Starch Lubricant
Dissolve 9 g of starch soluble starch in 22 g of glycerol, heat
to 140ºC, keep stirring for 30 minutes. This solution should
be cold before use.
Hemoglobio TS
Dissolve 1 g of bovine hemoglobin in hydrochloric acid
solution (dilute 65 ml of 1 mol/L hydrochloride acid solution
with water to 1000 mD to make 100 ml and mix.
Preserve in refrigerator and use within 2 days.
Hydrochloric Acid TS
Dilute 8. 4 ml of hydrochloric acid with water to make
100 ml.
Hydrochloric Acid Dilute TS
Dilute 234 ml of hydrochloric acid with water to 1000 ml.
lt contains 9. 5%-10. 5% of HCl.
Hydrogeo Peroxide TS
Dilute 30 % concentrated hydrogen peroxide solution with
water to a 3% solution.
Hydrogeo Sulfide TS
Use the saturated solution of hydrogen sulfide.
Preserve in amber coloured glass bottle and protected from
the light. lt is unsuitable to be used if it does not possess an
odour of H2S, or if it does not produce a copious precipitate
of sulfur when added to an equal volume of ferric chloride
TS.
p-Hydroxydipheoyl TS
Dissolve l. 5 g of p-hydroxydiphenyl in 10 ml of 5 % sodium
hydroxide solution and dilute with water to 100 ml.
This solution may be kept several months in amber coloured
glass bottle.
Hydroxylamioe Hydrochloride TS
Dissolve 3. 5 g of hydroxylamine hydrochloride m 60 %
ethanol to make 100 ml.
Hydroxylamioe Sodium Acetate TS
Dissolve O. 2 g of hydroxylamine hydrochloride and O. 2 g of
anhydrous sodium acetate in 100 ml of methanol.
This solution should be freshly prepared.
Iodigo Carmioe TS
Dissolve indigo carmine in a mixture of 12 ml of sulfuric acid
and 80 ml of water to make a solution which contains O. 09-
0. 11 g of C16HsN2Ü2 (SÜ3Na)2 per 100 ml.
Iodole Medium TS
Dissolve 5. O g of 4-dimethylaminobenz.aldehyde in 75 ml of
pentanol (or butanoD, shake thoroughly, mix well, and then
add 25 ml of concentrated hydrochloric acid slowly with shaking
to avoid the colour change by sudden heat. Or Dissolve l. O g of
4-dimethylaminobenz.aldehyde in 95 ml of 95 % ethanol, nux
well, then slowly added O. 25 of hydrochloric acid.
lodioe Potassium Iodide TS
Dissolve O. 5 g of iodine and l. 5 g of potassium iodide in
25 ml of water.
lodioe TS
Use iodine (O. 05 mol/L) VS directly.
lodioe TS ( for Microbial Limit Tests)
Dissolve 6 g of iodine and 5 g of potassium iodide in 20 ml of
water.
lodioe Mooochloride TS
Dissolve O. 14 g of potassium iodide and 90 mg of potassium
iodate in 125 ml of water, add 125 ml of hydrochloric acid.
Preserve in well-closed glass bottle and store in a cool place.
Isatio TS
Dissolve O. 1 g of isatin in 10 ml of acetone. Add 1 ml of
glacial acetic acid and mix well.

Isoniazid TS
Dissolve O. 25 g of isoniazid in O. 31 ml of hydrochloric acid
and sufficient methanol eor dehydrated ethanol) to make
500 ml.
Lanthanum TS
Dissolve 5 g of lanthanum oxide in 25 ml of hydrochloric
acid, dilute it with water to make 100 ml. Allow the solution
to stand far 24 hours.
Lead Acetate TS
Dissolve 10 g of lead acetate in freshly boiled and cooled water,
add acetic acid dropwise to make the solution clear, and then dilute
with freshly boiled and cooled water to 100 ml.
Lead Subacetate TS
Add 10 ml of water to 14 g of lead monoxide, triturate until
a paste is farmed. Transfer it to a glass bottle with 10 ml of
water, add 70 ml of a solution which contains 22 g of lead
acetate, shake vigorously far 5 minutes. Allow the mixture to
stand far 7 days with frequent shaking, filter, add freshly
boiled and cooled water to make 100 ml.
Lead Subacetate, Dilute TS
Dilute 4 ml of lead subacetate TS with freshly boiled and
cooled water to 100 ml.
Magnesium Sulfate TS
Dissolve 12 g of magnesium sulfate crystals (MgS0 4 • 7H2 0)
in water to make 100 ml.
Magnesium Sulfate, Dilute TS
Dissolve 2. 3 g of magnesium sulfate m water to make
100 ml.
Mercuric Acetate TS
Dissolve 5 g of mercuric acetate, finely powdered, in warm
glacial acetic acid to make 100 ml.
Preserve this solution in a well-closed amber coloured glass
bottle.
Mercuric Dichloride TS
Dissolve 6. 5 g of mercuric chloride in water to make 100 ml.
Mercuric Nitrate TS
Dissolve 40 g of yellow mercuric oxide in a mixture of 32 ml
of nitric acid and 15 ml of water.
Preserve in stoppered glass bottle and protected from light.
Mercuric Potassium Iodide TS
Dissolve l. 36 g of mercuric chloride in 60 ml of water and
dissolve 5 g of potassium iodide in 10 ml of water, mix these
two solutions and dilute with water to 100 ml.
Mercuric Sulfate TS
Mix 5 g of yellow mercuric oxide with 40 ml of water, add
20 ml of sulfuric acid slowly with stirring, then add another
40 ml of water and mix well.
Mercurous Nitrate TS
Dissolve 15 g of mercurous nitrate in a mixture of 90 ml of
water and 10 ml of dilute nitric acid.
This solution should be kept in amber coloured glass bottle
with the addition of one drop of mercury.
p-Methylarninophenol TS
Dissolve O. 2 g of p-methylaminophenol sulfate in 100 ml of
water. Add 20 g of sodium pyrosulfite and allow it to
dissolve.
Preserve in a stoppered amber coloured glass bottle and use
within two weeks.
Modified Potassium Iodobismuthate TS
Dissolve 1 ml of potassium Iodobismuthate in 2 ml of O. 6 mol/L
hydrochloric acid solution, dilute with water to 10 ml.
8002 Test Solutions
Molybdenum Phosphotungstate TS
Boil 70 ml of water under reflux, 5 ml of phosphoric acid,
10 g of sodium tungstate and 2. 4 g of phosphomolybdic acid
far 2 hours. Cool, dilute with water to 100 ml and shake
thoroughly.
Preserve in a glass bottle, protected from light.
a-Naphthol TS
Add 6. 5 ml of sulfuric acid slowly to 10. 5 ml of 15%
ethanolic solution of crnaphthol, mix well and then add
40. 5 ml of ethanol and 4 ml of water, mix well.
Ninhydrin TS
Dissolve 2 g of Ninhydrin in ethanol to make 100 ml.
Nitric Acid Dilute TS
Dilute 105 ml of nitric acid with water to 1000 ml.
It contains 9. 5 %-10. 5 % of HN0 3 •
Oxalic Acid TS
Dissolve 6. 3 g of oxalic acid in water to make 100 ml.
Perchloric Acid TS
Add 500 ml of water to 13 ml of 70 % perchloric acid. Adjust
pH to O. 5 with 70 % perchloric acid.
Phenoldisulfonic Acid TS
To 3 g of freshly distilled phenol add 20 ml of sulfuric acid
and heat in water bath far 6 hours. Transfer the product to a
glass-stoppered bottle while it is not solidified. Befare use
heat gently in a water bath until it is molten.
Phenylhydrazine Sulfate TS
Dissolve 60 mg of phenylhydrazine hydrochloride in 100 ml
of sulfuric acid solution Cl-2).
Phloroglucinol TS
Dissolve O. 5 g of phloroglucinol in ethanol to make 25 ml.
Preserved in glass-stoppered bottle, protected from light.
Phioroglucinoi hydrochloric acid 1S
Dissolve O. 1 g of phloroglucinol hydrochloric acid in 1 ml of
ethanol, add 9 ml of hydrochloric acid and mix well.
This solution should be freshly prepared.
Phosphomolybdic Acid TS
Dissolve 5 g of phosphomolybdic acid in dehydrated ethanol
to make 100 ml.
Phosphomolybdotungstic acid solution
Dissolve 100 g of sodium tungsten and 25 g of sodium molybdate
in 700 ml of water, add 100 ml of hydrochloric acid and 50 m1 of
phosphate acid, heat reflux far 10 hours, stand to cool. Add
150 g of lithium sulphate, 50 ml of water and O. 2 m1 of
bromine, boil to remove excess bromine (about 15 minutes),
cool, dilute to 1000 ml with water and filter. The filtrate should
not be green (If it turns green after preservation, add O. 2 ml of
bromine and boil to remove excess bromine).
Phosphotun~tic Acid TS
Dissolve 1 g of phosphotungstic acid in water to make
100 ml.
Phthalaldehyde TS
Dissolve l. O g of phthalaldehyde in 5 ml of methanol and 95 m1
of O. 4 mol/L boric acid solution (adjust to pH 10. 4 with 45%
sodium hydroxide solution ) with shaking. Add 2 ml of
thioglycollic acid and adjust to pH 10. 4 with 45 % sodium
hydroxide solution.
Potassium Acetate TS
Dissolve 10 g of potassium acetate in water to make 100 ml.
Potassium Carbonate 1S
Dissolve 7 g of anhydrous potassium carbonate in water to
make 100 ml.
 8002 Test Solutions
Po~ium Chlorate 1S
Use the saturated solution of potassium chlorate in nitric acid
Po~ium chromate 1S
Dissolve 5 g of potassium chromate in water to make 100 mi.
Po~ium Cyanide 1S
Dissolve 10 g of potassium cyanide in water to make 100 mi.
Po~ium Dichromate 1S
Dissolve 7. 5 g of potassium dichroma.te in water to make 100 mi.
Po~ium Ferricyanide 1S
Dissolve 1 g of potassium ferricyanide in 10 mi of water.
This solution should be freshly prepared.
Po~ium Ferricyanide Dilute 1S
To 10 ml of 1 % potassium ferricyanide solution add O. 5 ml
of 5 % ferric chloride solution and 40 ml of water, shake
thoroughly.
Po~ium Hexacynoferrate 1S
Wash 5 g of potassium hexacynoferrate with a small quantity
of water previously, dissolve it in water to make 100 ml.
This solution should be freshly prepared.
Po~ium Hydroxide 1S
Dissolve 6. 5 g of potassium hydroxide in water to make
100 ml.
Po~ium Iodide 1S
Dissolve 16. 5 g of potassium iodide in water to make
100 ml.
This solution should be freshly prepared.
Pobmium lodobismuthate, Dilute TS
Dissolve O. 85 g of bismuth subnitrate in a mixture of 10 ml
of glacial acetic acid and 40 ml of water. Befare use, add
5 ml of potassium iodide solution ( 4 - 10) and 20 ml of
glacial acetic acid to 5 ml of the solution mentioned above,
dilute with water to 100 ml.
Po~ium lodobismuthate 1S
Dissolve O. 85 g of bismuth subnitrate in a mixture of 10 mi
of glacial acetic acid and 40 mi of water, and then add 20 mi
of potassium iodide solution (4-10) and mix well.
Po~ium lodoplatinate 1S
Dissolve 20 mg chloroplatinic in 2 ml of water, add 25 ml of
4% potassium iodide solution, shake to dissolve if a
precipitate is produced, then add water to make 50 ml and
shake thoroughly.
Po~ium lodoplatinate, Concentrated 1S
Dissolve O. 15 g of chloroplatinic acid and 3 g of potassium
iodide in water to make 60 mi.
Po~ium permanganate 1S
Use potassium permanganate (0. 02 mol/L) VS directly.
Po~ium Pyroantimonate 1S
Dissolve 2 g of potassium pyroantimonate in 85 ml of hot
water, cool rapidly, add 10 mi of potassium hydroxide
solution (3-20). After 24 hours, filter. Dilute it in water
to make 100 mi solution.
Po~ium Sulfate 1S
Dissolve 1 g of potassium sulfate in water to make 100 ml.
Resorcinol 1S
Dissolve 1 g of resorcinol in hydrochloric acid to make
100 ml.
Rose Bengal Sodium 1S
Dissolve O. 1 g of rose bengal sodium in 75 ml of water.
Ruthenium Red 1S
Add ruthenium red to 1-2 ml of 10% sodium acetate solution
until a wine-red colour is obtained.
This solution should be freshly prepared.
Semicarbazide Hydrochloride 1S
Triturate 2. 5 g of semicarbazide hydrochloride with 3. 3 g of
sodium acetate, transfer the mixture to a conical flask with
30 ml of methanol, allow to stand for 30 minutes at a
temperature below 4ºC , filter and add methanol to make
100 ml.
Silicowolframic Acid 1S ( Silicot~tic Acid 1S)
Dissolve 10 g of silicotungstic acid in water to make 100 ml.
Silver Diethyldithiocarbamate 1S
To O. 25 g of silver diethyldithiocarbamate add a suitable
amount of chloroform and l. 8 mi of triethylamine, dilute
with chloroform to 100 mi and dissolve it by stirring. Allow
the solution to stand ovemight, and then filter with
absorbent cotton wool.
Preserve in amber coloured glass bottle and store in a cool
place.
Silver Nitrate 1S
Use silver nitrate (O. 1 mol/L) VS directly.
Sodium Acetate 1S
Dissolve 13. 6 g of crystalline sodium acetate m water to
make 100 ml.
Sodium Acetate-Sodium Hydroxide 1S
Dissolve 10. 3 g of sodium acetate and 86. 5 g of sodium
hydroxide in water to make 1000 mi.
Sodium Bicarbonate 1S
Dissolve 5 g of sodium bicarbonate in water to make 100 ml.
Sodium Bisulfite 1S
Dissolve 10 g of sodium bisulfite in water to make 30 mi.
This solution should be freshly prepared.
Sodium Bitartrate 1S
Dissolve 1 g of sodium bitartrate in water to make 10 mi.
This solution should be freshly prepared.
Sodium Carbonate 1S
Dissolve 12. 5 g of sodium carbonate monohydrate or 10. 5 g
of anhydrous sodium carbonate in water to make 100 ml.
Sodium Cobaltinitrite 1S
Dissolve 10 g of sodium cobaltinitrite in water to make 50 mi
and filter.
Sodium Diethyldithiocarbamate 1S
Dissolve O. 1 g of sodium diethyldithiocarbamate in 100 mi of
water and filter.
Sodium Fluoride 1S
Dissolve O. 5 g of sodium fluoride in 100 ml of O. 1 mol/L
hydrochloric acid solution.
This solution should be freshly prepared.
Sodium Hydroxide 1S
Dissolve 4. 3 g of sodium hydroxide in water to make 100 mi.
Sodium Hypobromite 1S
Add 5 mi of bromine to a solution of 20 g of sodium hydroxide in
75 mi of water. Dilute with water to 100 mi and mix well.
This solution should be freshly prepared.
Sodium Hypochlorite 1S
Dissolve appropriate amount of sodium hypochlorite in water. The
final filtrate contains not less than 4% of NaClO.
Preserve in amber coloured glass bottle, protected from
light.
Sodium Nitrite 1S
Dissolve 1 g of sodium nitrite in water to make 100 ml.

Sodium Nitrop~ide TS
Dissolve 1 g of sodium nitroprusside in water to make 20 ml.
This solution must be freshly prepared.
Sodium Nitrop~ide Acetaldehyde TS
Mix 10 ml of 1% sodium nitroprusside solution with 1 ml of
acetaldehyde.
Sodium Periodate TS
Dissolve l. 2 g of sodium periodate in 100 ml of water.
Sodium Sulfite TS
Dissolve 20 g of anhydrous sodium sulfite in 100 ml of
water. This solution should be freshly prepared.
Sodium Sulfide TS
Dissolve 1 g of sodium sulfide in water to make 10 ml.
This solution should be freshly prepared.
Sodium ( Potassium) Tellurite TS
Dissolve O. 1 g of sodium ( potassium) tellurite in 10 ml of
freshly boiled cooling water ( 50ºC).
Sodium Tetraphenylborate TS
Dissolve O. 1 g of sodium tetraphenylborate in water to make
100 ml.
Sodium Thiosulfate TS
Use sodium thiosulfate (O. 1 mol/L) VS directly.
Sodium Tungstate TS
Dissolve 25 g of sodium tungstate in 72 ml of water. Add
2 ml of phosphoric acid and mix well.
Stannous Chloride TS
Dissolve l. 5 g of stannous chloride in 10 ml of water and a
small amount of hydrochloric acid.
This solution should be freshly prepared.
Sudan][ TS
Dissolve O. 01 g of sudan fil in 5 ml of 90% ethanol. Add
5 ml of glycerin and mix well.
Preserve in amber coloured glass bottle, use within
2 months.
Sulfanilamide TS
Dissolve 50 mg of sulfanilamide in 10 ml of 2 mol/L hydrochloric
acid solution.
Sulfanilic a-Naphthylamine TS
Dissolve O. 5 g of anhydrous sulfanilic acid in 150 ml of acetic
acid. Dissolve O. 1 g of a-naphthylamine hydrochloride in
150 ml of acetic acid. Mix these two solutions.
This solution will show pink colour after standing far a long
time, add zinc powder to decolourize it befare use.
Sulfuric Acid, dilute, TS
Dilute 57 ml of sulfuric acid with water to 1000 ml.
It contains 9. 5 %-10. 5 % of H2 504.
Tannic Acid TS
Dissolve 1 g of tannic acid in 1 ml of ethanol and dilute with
water to 100 ml.
This solution should be freshly prepared.
Tatrarnethylammonium hydroxide TS
Dilute 1 ml of 10% tetramethylammonium hydroxide
solution with dehydrated ethanol to make 10 ml.
Thioacetamide TS
Dissolve 4 g of thioacetamide in water to make 100 ml. Store
in refrigerator. Add l. O mi of thioacetamide solution to
5. O ml of a mixture consisting of 15 ml of 1 mol/L sodium
hydroxide solution, 5. O ml of water and 20 ml of glycerin
befare use, heat far 20 seconds in a water bath, cool and use
immediately.
8003 Test Papers
Thiourea TS
Dissolve 10 g of thiourea in water to make 100 ml.
Titanium Sulfate TS
Dissolve O. 1 g of titanium dioxide in 100 mi of sulfuric acid
by heating, allow to cool.
p-Tosyl-L-Arginine Methyl Ester Hydrochloride TS
Dissolve 98. 5 mg of p-tosyl-L-arginine methyl ester hydrochloride
in 5 ml of trometamol buffer solution (pH 8. 1) , add O. 25 ml of
indicator solution (mix O. 1% alcoholic methyl red solution with
an equal volume of O. 05 % alcoholic methylene blue solution) and
dilute with water to 25 ml.
Trichloroacetic Acid TS
Dissolve 6 g of trichloroacetic acid in 25 mi of chloroform,
add O. 5 ml of concentrated hydrogen peroxide solution and
mix well.
Trinitrophenol TS
A saturated solution of trinitrophenol in water.
Trinitrophenol Lithium TS
Dissolve O. 25 g of lithium carbonate and O. 5 g of
trinitrophenol in 80 ml of boiling water, cool and dilute with
water to 100 ml.
Triphenyltetrazolium Chloride TS
Dissolve 1 g of triphenyltetrazolium chloride in dehydrated
ethanol to make 200 ml.
Vanadium Pentoxide TS
Dissolve vanadium pentoxide in phosphoric acid with shaking
vigorously far 2 hours to produce a saturated solution. Filter
with a sinter-glass funnel, dilute 1 volume of filtrate with 3
volume of water and mix well.
Vanillin Sulfuric Acid TS
Dissolve O. 2 g of vanillin in 10 ml of sulfuric acid.
Vanillin TS
Dissolve O. 1 g of vanillin in 10 ml of hydrochloric acid.
Xanthydrol-Methanol TS
Dissolve 85 % xanthydrol solution in 100 ml of methanol.
Zinc Chloride-lodine TS
Dissolve 20 g of zinc chloride and 2 g of potassium iodide in
10 ml of water, then saturated with iodine.
Preserve in amber coloured glass bottle.
Zinc Uranyl Acetate TS
Dissolve 10 g of uranyl acetate in 5 mi of glacial acetic acid
and 50 mi of water by heating. Dissolve 30 g of zinc acetate
in 3 ml of glacial acetic acid and 30 ml of water with gentle
heating. Mix the two solutions, cool and filter.
Zirconium Afüarin TS
Dissolve 5 mg of zirconium nitrate in a mixture of 5 ml of
water and 1 mi of hydrochloric acid. Dissolve 1 mg of sodium
alizarinsulfonate in 5 ml of water. Mix these two solutions.
8003 Test Papers
Ammoniated-Silver Nitrate TP
Immerse a strip of filter paper in ammoniated-silver nitrate
TS until thoroughly wetted.
Benzidine-Cupric Acetate TP
To 9 ml of a saturated solution of benzidine acetate add 7 ml
of water and 16 mi of O. 3% cupric acetate solution, immerse
a strip of filter paper in the mixture until thoroughly
wetted. Remove the paper and allow to dry.
 8004 Buffer Solutions
Blue Litmus TP
Immerse a strip of filter paper in litmus IS until thoroughly
wetted. Remove the paper and allow to dry.
Sensitivity Introduce O. 5 mi of O. 1 mol/L hydrochloric
acid in a beaker. Add 100 mi of freshly boiled and cooled
water and mix. Drop a strip of blue litmus TP 10-12 mm in
width in the solution and stir continuously. The colour of the
paper changes within 45 seconds.
Cadmiurn Acetate TP
Dissolve 3 g of cadmium acetate in 100 mi of ethanol, add
ammonia TS until the majority of the precipitate is dissolved.
Filter and immerse a strip of filter paper in the filtrate.
Remove the paper before use and allow to dry.
Congo Red TP
Immerse a strip of filter paper in congo red IS until
thoroughly wetted, remove the paper and allow to dry.
Curcuma TP ( Tunneric TP)
lmmerse a strip of filter paper in curcuma IS until thoroughly
wetted. Lay the paper on a glass plate and dry at lOOºC.
Lead Acetate TP
Immerse a strip of filter paper in lead acetate TS until
thoroughly wetted. Remove the paper and allow to dry at
lOOºC.
Mercuric Bromide TP
Immerse a strip of filter paper in alcoholic mercuric bromide
TS for 1 hour. Remove the paper and allow to dry in a dark
place.
Mercuric Dichloride TP
Immerse a strip of filter paper in a saturated solution of
mercuric dichloride for 1 hour, then remove the strip and
allow to dry at 60ºC in a dark place.
Mercuric Nitrate TP
To 45 ml of a saturated solution of mercuric nitrate add 1 mi
of nitric acid. Immerse a strip of filter paper in the solution
until thoroughly wetted. Remove the paper and allow to dry.
Nickel hydroxide TP
Dip the filter paper into 30 % nickel sulfate in concentrated
ammonia solution, take out and dry. Then immerse in
1 mol/L sodium hydroxide solution for a few minutes to
make the filter paper covered with homogeneous
precipitation of nickel hydroxide. Take out the filter paper
and wash it with water (Can not dry). Store it in the wet
cotton reserve.
Red Litmus TP
Immerse a strip of filter paper in litmus IS, add a small
amount of hydrochloric acid until a red colour is obtained.
Remove the paper and allow to dry.
Sensitivity Introduce O. 5 mi of O. 1 mol/L sodium
hydroxide solution to a beaker, add 100 mi of freshly boiled
and cooled water and mix. Drop a strip of red litmus TP 10-
12 mm in width in the solution and stir continuously. The
colour of the paper changes within 30 seconds.
Starch-Iodide TP
lmmerse a strip in 100 mi of freshly prepared starch IS,
which contains O. 5 g of potassium iodide, until thoroughly
wetted. Remove the paper and allow to dry.
Trinitrophenol TP
lmrnerse a strip of filter paper in a saturated solution of
trinitrophenol until thoroughly wetted. Remove the paper and
allow to dry.
Immerse the paper in sodium carbonate solution ( 1-10)
until wetted uniformly before use.
8004 Buffer Solutions
O. 5 % sodiurn laurylsulfate in Phosphate BS
Take 6. 9 g of sodium dihydrogen phosphate, O. 9 g of sodium
hydroxide and 5 g of sodium laurylsulfate, add 800 ml of water
and sonicate for 30 minutes, adjust to pH 6. 8 with 2 mol/L
sodium hydroxide solution, then dilute with water to 1000 ml.
O. 1 mol/L trometamol BS
Dissolve 121 g of trometamol in 900 ml of water, adjust to
pH 7. 2 with 25% citric acid solution and dilute with water to
1000 mi.
2-0xopentanedioic acid BS
Dissolve 220 mg of 2-0x.opentanedioic acid in 60 ml of
hydrochloric acid triethanolamine buffer solution (pH 8. O) (Take
1 ml of triethanolaminde, add 60 ml of ammonia free distilled
water and adjust to pH 8. O with diluted hydrochloric acid) .
Acetate BS (pH 3. 5)
Dissolve 25 g of ammonium acetate in 25 ml of water, add
38 ml of 7 mol/L hydrochloric acid solution. Adopt the
potentiometric method to adjust accurately to pH 3. 5 with
2 mol/L hydrochloric acid solution or 5 mol/L ammonia
solution and dilute with water to 100 mi.
Ammonium Acetate BS ( pH 4. 5)
Dissolve 7. 7 g of ammonium acetate in 50 ml of water. Add
6 mi of glacial acetic acid and dilute with water to 100 mi.
Ammonium Acetate BS (pff 4. 8)
Dissolve 77 g of ammonium acetate in 200 mi of water. Add
57 ml of glacial acetic acid and dilute with water to 1000 mi.
Ammonium Acetate BS ( pH 6. O)
Dissolve 100 g of ammonium acetate in 300 ml of water, add
7 mi of glacial acetic acid and mix well.
Ammonium Acetate-Ethanol BS (pH 3. 7)
Mix 15. O mi of 5 mol/L acetic acid solution with 60 mi of
ethanol and 20 ml of water, and adjust to pH 3. 7 with
10 mol/L ammonium hydroxide solution, dilute with water
to 1000 mi.
Ammonia-Ammonium Chloride BS (pH 8. O)
Dissolve l. 07 g of ammonium chloride in water to make
100 ml, adjust to pH 8. O with dilute ammonia solution 0-
30).
Ammonia-Ammonium Chloride BS (pH 10. 0)
Dissolve 5. 4 g of ammonium chloride in 20 mi of water, add
35 ml of concentrated ammonia solution. Dilute with water
to 100 mi.
Barbital BS ( pH 7. 4)
Dissolve 4. 42 g of sodium barbital in 400 ml of water, adjust
to pH 7. 4 with 2 mol/L hydrochloric acid.
Barbital BS (pH 8. 6)
Dissolve 5. 52 g of barbital and 30. 9 g of sodium barbital in
water to make 2000 ml.
Barbital-Sodium Chloride BS (pH 7. 8)
Dissolve 5. 05 g of sodium barbital and 3. 7 g of sodium
chloride in water, heat to dissolve O. 5 g of gelatin in water,
combine the two solution, adjust to pH 7. 8 with O. 2 mol/L
hydrochloric acid solution. Dilute with water to 500 mi.
Borate-Potassium Chloride BS ( pH 9. O)
Dissolve 3. 09 g of boric acid in 500 mi of O. 1 mol/L
potassium chloride solution, and add 210 mi of O. 1 mol/L
sodium hydroxide solution.

Borax-Calciwn Chloride BS ( pH 8. O)
Dissolve O. 572 g of borax and 2. 94 g of calcium chloride in
about 800 ml of water, and adjust to pH 8. O with about
2. 5 ml of 1 mol/L hydrochloric acid solution. Dilute with
water to 1000 ml.
Borax-Sodiwn Carbonate BS (pH 10. 8-11. 2)
Dissolve 5. 30 g of anhydrous sodium carbonate in water to
make 1000 ml. Dissolve l. 91 g of borax in water to make
100 ml. Mix 973 ml of sodium carbonate solution with 27 ml
of borax solution before use, shake thoroughly.
Citrate BS
Dissolve 4. 2 g of citric acid in 40 ml of 1 mol/L 20 %
ethanolic sodium hydroxide solution. Dilute with 20 %
ethanol solution to 100 ml.
Citrate BS CpH 6. 2)
Adjust the pH value of 2. 1 % citric acid solution to 6. 2 with
50 % sodium hydroxide solution.
Citrate-Disodiwn Hydrogen Phosphate BS (pH 4. O)
(a) Dissolve 21 g of citric acid or 19. 2 g of anhydrous citric
acid in water to make 1000 ml, preserve in a refrigerator.
( b) Dissolve 71. 63 g of disodium hydrogen phosphate in
water to make 1000 ml.
To 61. 45 ml of solution (a) add 38. 55 ml of solution (b)
and mix well.
Citrate-Disodiwn Hydrogen Phosphate BS (pH 7. 0)
(a) Dissolve 21 g of citric acid or 19. 2 g of anhydrous citric
acid in water to make 1000 ml, preserve in a refrigerator.
(b) Dissolve 71. 63 g of disodium hydrogen phosphate in
water to make 1000 ml.
To 17. 65 ml of solution (a) add 82. 35 ml of solution (b)
and mix well.
Lithium-Acetate BS (pH 3. O)
Add 800 ml of water to 50 ml of glacial acetic acid and mix.
Adjust to pH 3. O with lithium hydroxide, dilute with water
to 1000 ml.
Phosphate BS
Dissolve 38. O g of sodium dihydrogen phosphate and 5. 04 g
of disodium hydrogen phosphate in water to make 1000 ml.
Phosphate BS ( pH 2. O)
(a) Dilute 16. 6 ml of phosphoric acid with water to 1000 ml
and mix well.
( b) Dissolve 71. 63 g of disodium hydrogen phosphate in
water to make 1000 ml.
To 72. 5 ml of solution (a) add 27. 5 ml of solution (b) and
mix well.
Phosphate BS (pH 2. 5)
Dissolve 100 g of potassium dihydrogen phosphate in 800 ml
of water, adjust to pH 2. 5 with hydrochloric acid. Dilute
with water to 1000 ml.
Phosphate BS (pH 5. O)
Adjust the pH of O. 2 mol/L sodium dihydrogen phosphate
solution to 5. O with sodium hydroxide TS.
Phosphate BS ( pH 5. 8)
Dissolve 8. 34 g of potassium dihydrogen phosphate and
O. 87 g of dipotassium hydrogen phosphate in water to make
1000 ml.
Phosphate BS (pH 6. 5)
Dissolve O. 68 g of potassium dihydrogen phosphate in
15. 2 ml of O. 1 mol/L sodium hydroxide solution, dilute
with water to 100 ml.
Phosphate BS (pH 6. 6)
Dissolve l. 74 g of sodium dihydrogen phosphate, 2. 7 g of
8004 Buffer Solutions
disodium hydrogen phosphate and l. 7 g of sodium chloride in
water to make 400 ml.
Phosphate-Pancreatin BS (pH 6. 8)
(a) Dissolve 6. 8 g of potassium dihydrogen phosphate in
500 ml of water and adjust to pH 6. 8 with O. 1 mol/L
sodium hydroxide solution.
(b) Dissolve 10 g of pancreatin in water.
Mix the two solutions and dilute with water to 1000 ml.
Phosphate BS ( pH 6. 8)
Add 118 ml of O. 2 mol/L sodium hydroxide solution to
250 ml of O. 2 mol/L potassium dihydrogen phosphate
solution, dilute with water to 1000 ml and shake thoroughly.
Phosphate BS ( pH 7. O)
Dissolve O. 68 g of potassium dihydrogen phosphate in
29. 1 ml of O. 1 mol/L sodium hydroxide solution and dilute
with water to 100 ml.
Phosphate BS ( pH 7. 2)
Mix 50 ml of O. 2 mol/L potassium dihydrogen phosphate
solution with 35 ml of O. 2 mol/L sodium hydroxide solution,
dilute with freshly boiled and cooled water to 200 ml and mix
well.
Phosphate BS ( pH 7. 3)
Dissolve l. 9734 g of disodium hydrogen phosphate and
O. 2245 g of potassium dihydrogen phosphate in water to
make 1000 ml. Adj ust to pH 7. 3.
Phosphate BS ( pH 7. 4)
Dissolve l. 36 g of potassium dihydrogen phosphate in 79 ml
of O. 1 mol/L sodium hydroxide solution, dilute with water
to 200 ml.
Phosphate BS ( pH 7. 6)
Dissolve 27. 22 g of potassium dihydrogen phosphate in water
to make 1000 m!.
Mix 50 ml of this solution with 42. 4 ml of O. 2 mol/L
sodium hydroxide solution, dilute with water to 200 ml.
Phosphate BS (pH 7. 8)
(a) Dissolve 35. 9 g of disodium hydrogen phosphate m
water to make 500 ml.
(b) Dissolve 2. 76 g of sodium dihydrogen phosphate m
water to make 100 ml.
To 91. 5 ml of solution (a) add 8. 5 ml of solution (b), mix
well.
Phosphate BS (pH 7. 8-8. O)
Dissolve 5. 59 g of dipotassium hydrogen phosphate and
O. 41 g of potassium dihydrogen phosphate in water to make
1000 ml.
Phosphate-Triethylamine BS ( pH 3. 2)
Mix about 4 ml of phosphoric acid with about 7 ml of
triethylamine, dilute with 50% methanol to 1000 ml, adjust
to pH 3. 2 with phosphoric acid.
Phthalate BS (pH 5. 6)
Dissolve 10 g of potassium biphthalate in 900 ml of water
with stirring. Adjust to pH 5. 6 with sodium hydroxide TS
Cor dilute hydrochloric acid). Dilute with water to 1000 ml
and mix well.
Potassiwn Acetate BS ( pH 4. 3)
Dissolve 14 g of potassium acetate in 20. 5 ml of glacial acetic
acid, and dilute with water to 1000 ml.
Potassiwn biphthalate-Sodiwn hydroxide BS ( pH 5. O)
Adjust the pH of 100 ml of O. 2 mol/L potassium biphthalate
solution to 5. O with about 50 ml of O. 2 mol/L sodium
hydroxide solution.
 8005 lndicator Solutions
Sodium Acetate BS
Dilute 4 ml of Sodium Aceta te BS ( pH 3. 6) with water to
100 ml.
Sodium Acetate BS (pH 3. 6)
Dissolve 5. 1 g of sodium acetate in 20 ml of glacial acetic
acid, dilute with water to 250 ml.
Sodium Acetate BS (pH 3. 7)
Dissolve 20 g of anhydrous sodium acetate in 300 ml of
water, add 1 ml of bromophenol blue IS and 60-80 ml of
glacial acetic acid until the colour changes from blue to pure
green, and then dilute with water to 1000 ml.
Sodium Acetate BS ( pH 3. 8)
Mix 13 ml of 2 mol/L sodium acetate solution with 87 ml of
2 mol/L acetic acid solution. Add O. 5 ml of cupric sulfate
solution which contains 1 mg of Cu per ml. Dilute with water
to 1000 ml.
Sodium Acetate BS ( pH 4. 5)
Dissolve 18 g of sodium acetate in 9. 8 ml of glacial acetic
acid, and dilute with water to 1000 ml.
Sodium Acetate BS ( pH 4. 6)
Dissolve 5. 4 g of sodium acetate in 50 ml of water, adjust to
pH 4. 6 with glacial acetic acid, dilute with water to 100 ml.
Sodium Acetate BS ( pH 6. O)
Dissolve 54. 6 g of sodium acetate in 20 ml of 1 mol/L acetic
acid solution. Dilute with water to 500 ml.
Sodium Formate BS ( pH 3. 3)
To 25 ml of 2 mol/L formic acid solution add 1 drop of
phenolphthalein IS, neutralize with 2 mol/L sodium
hydroxide solution, and then add 75 ml of 2 mol/L formic
acid solution. Dilute with water to 200 ml. The pH of the
solution is 3. 25-3. 30.
Triethylamine BS ( pH 3. 2)
Mix 8 ml of phosphoric acid with 14 ml of triethylamine.
Dilute with water to 1000 ml, adjust to pH 3. 2 with
triethylamine, add 500 ml of water, mix well.
Trometamol BS ( pH 8. O)
Dissolve 12. 14 g of trometamol in 800 ml of water with
stirring, dilute with water to 1000 ml. Adjust to pH 8. O
with 6 mol/L hydrochloric acid solution.
Trometamol BS ( pH 8. 1)
Dissolve O. 294 g of calcium chloride in 40 ml of O. 2 mol/L
trometamol solution. Adjust to pH 8. 1 with 1 mol/L
hydrochloric acid solution, and then dilute with water to
100 ml.
Trometamol BS (pH 9. O)
Dissolve 6. 06 g of trometamol, 3. 65 g of lysine
hydrochloride, 5. 8 g of sodium chloride and O. 37 g of
disodium edetate in 1000 ml of water, adjust to pH 9. O.
Tromethane hydrochloride BS ( pH 7. 2)
Solution A: dissolve 15. 8 g of tromethane hydrochloride in
100 ml of water for bacteria! endotoxins test. Solution B:
dissolve l. 2 g of trometamol in 10 ml of water for bacteria!
endotoxins test.
Mix 100 ml of Solution A and 10 ml of Solution B and dilute
with water for bacteria! endotoxins test to 550 ml, adjust to
pH 7. 2 with O. 1 mol/L hydrochloric acid solution or
O. 1 mol/L sodium hydroxide solution. Then pack with
pyrogen free infusion bottle, gland with the plug and sterilize
for 15 minutes at 121 ºC.
Urotropine BS
Dissolve 75 g of urotropine in water, add 4. 2 ml of concentrated
ammonia solution, and then dilute with water to 250 ml.
8005 Indicator Solutions
Azo Violet IS
Dissolve O. 1 g of azo violet in 100 ml of dimethylformamide.
Brilliant Green IS
Dissolve O. 5 g of brilliant green in 100 ml of glacial acetic
acid.
Colour changes from yellow to green (pH 0-2. 6).
Bromocresol Green IS
Dissolve O. 1 g of bromocresol green in 2. 8 ml of O. 05 mol/L
sodium hydroxide solution, and dilute with water to 200 ml.
Colour changes from yellow to blue (pH 3. 6-5. 2).
Bromocresol Purple IS
Dissolve O. 1 g of bromocresol purple in 20 ml of O. 02 mol/L
sodium hydroxide solution, and dilute with water to 100 ml.
Colour changes from yellow to purple (pH 5. 2-6. 8).
Bromocresol Purple IS (for Microbial Limit Tests)
Dissolve l. 6 g bromocresol purple in 100 ml of 95% ethanol.
Colour changes from yellow to purple (pH 5. 2-6. 8).
Bromophenol Blue IS
Dissolve O. 1 g of bromophenol blue in 3. O ml of O. 05 mol/L
sodium hydroxide solution, and dilute with water to 200 ml.
Colour changes from yellow to bluish green (pH 2. 8-4. 6).
Bromothymol Blue IS
Dissolve O. 1 g of bromothymol blue in 3. 2 ml of O. 05 mol/L
sodium hydroxide solution, and dilute with water to 200 mi.
Colour changes from yellow to blue (pH 6. 0-7. 6).
Calcein Indicator Mixture
Mix uniformly O. 1 g of calcein with 10 g of potassium
chloride by grinding.
Calcon Indicator Mixture
Mix uniformly O. 1 g of calcan with 10 g of anhydrous sodium
sulfate by grinding.
Catechol Violet IS
Dissolve O. 1 g of catechol violet in 100 ml of water.
Colour changes from yellow to violet and then to purplish-red
CpH 6. 0-7. 0-9. O).
Congo Red IS
Dissolve O. 5 g of congo red in 100 ml of 10 % ethanol.
Colour changes from blue to red (pH 3. 0-5. 0).
Cresol Red IS
Dissolve O. 1 g of cresol red in 5. 3 ml of O. 05 mol/L sodium
hydroxide solution, and dilute with water to 100 ml.
Colour changes from yellow to red (pH 7. 2-8. 8).
m-Cresol Purple IS
Dissolve O. 1 g of m-cresol purple in 10 ml of O. 01 mol/L
sodium hydroxide solution, and dilute with water to 100 ml.
Colour changes from yellow to purple (pH 7. 5-9. 2).
Cresol Red-Thymol blue IS
Mix 1 volume of cresol red IS with 3 volume of a O. 1 %
thymol blue solution.
Crystal Violet IS
Dissolve O. 5 g of crystal violet m 100 ml of glacial acetic
acid.
Curcuma IS (Tunneric IS)
Extract 20 g of curcuma powder with 4 quantities of each of
100 ml of water to remove water soluble matter and dry the
residue at lOOºC. Macerate with 100 ml of ethanol for a few
days and filter.

Dimethyl Yellow IS
Dissolve O. 1 g of dimethyl yellow in 100 ml of ethanol.
Colour changes from red to yellow ( pH 2. 9-4. O).
Dimethyl Yellow-methylene Blue IS
Dissolve 15 rng of dimethyl yellow and 15 mg of methylene
blue in 100 rnl of chloroform with shake ( heat gently if
necessary), and filter.
Dimethyl Yellow-solvent blue 19 IS
Dissolve 15 rng of dimethyl yellow and 15 mg of solvent blue
19 in 100 ml of chloroform.
Diphenylcarbazide IS
Dissolve 1 g of diphenylcarbazide in 100 ml of ethanol.
Dithizone {Diphenylthiocarbazone) IS
Dissolve 50 mg of Diphenylthiocarbazone in 100 ml of
ethanol.
Eriochrome Black T lndicator Mixture
Mix uniformly O. 1 g of eriochrome black T with 10 g of
sodium chloride by grinding.
Ethoxychrysoidine IS
Dissolve O. 1 g of ethoxychrysoidine in 100 ml of ethanol.
Colour changes frorn red to yellow (pH 3. 5-5. 5).
Tetrabromo-phenolphthalein Pobmium Ethyl Ester IS
Dissolve O. 1 g of tetrabromophenolphthalein potassium ethyl
ester in 100 rnl of glacial acetic acid.
Eosin Sodium IS
Dissolve O. 5 g of eosin sodium in 100 ml of water.
Eosin Sodium IS (for Microbial Limit Tests)
Dissolve 2. O g of eosin sodium in 100 ml of water.
Ferric Ammonium Sulfate IS
Dissolve 8 g of ferric ammonium sulfate in 100 ml of water.
Fluorescein IS
Dissolve O. 1 g of fluorescein in 100 ml of ethanol.
Hydroxynaphthol blue IS
Dissolve O. 5 g of Hydroxynaphthol blue in 50 ml of water,
add 2 drops of O. 1 mol/L sodium hydroxide solution.
Litmus IS
Add 40 ml of ethanol to 10 g of litmus powder, heat under
reflux for 1 hour. Allow to settle and decant off the
supernatant liquid. Repeat the operation twice using 30 mi of
ethanol each time. Wash the residue with 10 ml of water and
decant off the washing liquid. Boil the residue with 50 ml of
water, allow to cool and filter.
Colour changes from red to blue ( pH 4. 5-8. O).
Malachite Green IS
Dissolve O. 3 g of malachite green in 100 ml of glacial acetic
acid.
Colour changes from yellow to green ( pH 0-2. O) ; from
green to colourless (pH 11. 0-13. 5).
Metalphthalein IS
Dissolve 1 g of metalphthalein and small amount of ammonia
TS in 100 ml of water.
Methyl Orange IS
Dissolve O. 1 g of methyl orange in 100 ml of water.
Colour changes from red to yellow (pH 3. 2-4. 4).
Methyl Orange-Methylene Blue IS
To 20 ml of rnethyl orange IS add 8 ml of O. 2% solution of
methylene blue, and mix well.
Methyl orange-Xylene Cyanol Blue FF IS
Dissolve O. 1 g of methyl orange and O. 1 g of xylene cyanol
blue FF in 100 ml of ethanol.
8005 lndicator Solutions
Methyl Red IS
Dissolve O. 1 g of methyl red in 7. 4 ml of O. 05 mol/L sodium
hydroxide solution and dilute with water to 200 ml.
Colour changes from red to yellow (pH 4. 2-6. 3).
Methyl Red-Bromocresol Green IS
To 20 ml of O. 1 % ethanolic solution of methyl red add 30 ml
of O. 2 % ethanolic solution of bromocresol green, and mix
well.
Methyl Red-Methylene blue IS
To 20 ml of O. 1% ethanolic solution of methyl red add 8 ml
of O. 2% solution of methylene blue, and mix well.
Methylene Blue IS
Dissolve methylene blue O. 5 g with water to 100 ml.
Naphtholbell7.ein IS
Dissolve O. 5 g of a-naphtholbenzein in 100 ml of glacial
acetic acid.
Colour changes from yellow to green (pH 8. 5-9. 8).
Neutral Red IS
Dissolve O. 5 g of neutral red in 100 ml of water and filter.
Colour changes from red to yellow (pH 6. 8-8. O).
Neutral Red IS {for Microbial Limit Tests)
Dissolve l. O g of neutral red, grinding well, in 60 ml of 95%
ethanol, add water to 100 ml.
Colour changes from red to yellow ( pH 6. 8-8. O).
Nile Blue IS
Dissolve 1 g of nile blue in 100 ml of glacial acetic acid.
Colour changes from blue to red (pH 10. 1-11. 1).
p-Nitrophenol IS
Dissolve O. 25 g of p-nitrophenol in 100 ml of water.
Orange NIS
Dissolve O. 5 g of orange N in 100 ml of glacial acetic acid.
Colour changes from red to yellow (pH l. 4-3. 2).
o-Phenanthroline IS
Dissolve O. 5 g of ferrous sulfate in 100 ml of water, add
2 drops of sulfuric acid and O. 5 g of o-phenanthroline. Shake
thoroughly.
This solution should be freshly prepared.
Phenol Red IS
Dissolve 100 mg phenol red in 100 ml of ethanol (Filter if
necessary).
Phenol sulfonphthalein IS
Dissolve O. 1 g of phenol sulfonphthalein in 5. 7 ml of
O. 05 mol/L sodium hydroxide solution, and dilute with
water to 200 ml.
Colour changes from yellow to red (pH 6. 8-8. 4).
Phenol Sulfonphthalein IS (for Microbial Limit Tests)
Dissolve l. O g of phenol sulfonphthalein in 2. 82 ml of 1 mol/L
sodium hydroxide solution, and dilute with water to 100 ml.
Colour changes from yellow to red (pH 6. 8-8. 4).
Phenolphthalein IS
Dissolve 1 g of phenolphthalein in 100 ml of ethanol.
Colour changes from colourless to red (pH 8. 3-10. 0).
Phthalein Purple IS
Adjust to pH of 10 ml of water to 11 with ammonia solution,
and then add 10 mg of phthalein purple and dissolve.
Pobmium Chromate IS
Dissolve 10 g of potassium chromate in 100 ml of water.
Quinaldine Red IS
Dissolve O. 1 g of quinaldine red in 100 ml of methanol.
Colour changes from colourless to red (pH l. 4-3. 2).
 8006 Volumetric Solutions
Quinaldine Red-Methylene Blue IS
Dissolve O. 3 g of quinaldine red and O. 1 g of methylene blue
in 100 ml of anhydrous methanol.
Sodium Alizarinsulfonate IS
Dissolve O. 1 g of sodium afüarinsulfonate in 100 ml of water.
Colour changes from yellow to purple (pH 3. 7-5. 2).
Sodium Diphenylamine Sulfonate IS
Dissolve O. 2 g of sodium diphenylamine sulfonate in 100 ml
of water.
Solvent Blue 19 IS
Dissolve O. 5 g of solvent blue 19 in 100 ml of glacial acetic acid.
Starch IS
Add 5 ml of water to O. 5 g of soluble starch and mix well,
then pour the suspension slowly into 100 ml of boiling water
with stirring. Boil far 2 minutes, cool and decant the
supernatant liquid.
This solution should be freshly prepared.
Starch lodide IS
Dissolve O. 2 g of potassium iodide in 100 ml of freshly
prepared starch IS.
Sudan NIS
Dissolve O. 5 g of sudan N in 100 ml of chloroform.
Thymol Blue IS
Dissolve O. 1 g of thymol blue in 4. 3 ml of O. 05 mol/L
sodium hydroxide solution, and dilute with water to 200 ml.
Colour changes from red to yellow ( pH l. 2-2. 8); from
yellow to bluish-violet ( pH 8. 0-9. 6).
Thymolphthalein IS
Dissolve O. 1 g of thymolphthalein in 100 ml of ethanol.
Colour changes from colourless to blue (pH 9. 3-10. 5).
Xylenol Orange IS
Dissolve O. 2 g of xylenol orange in 100 ml of water.
This solution should be freshly prepared.
Zinc-Starch Iodide IS
Mix 100 ml of water, 5 ml of potassium iodide solution (3-
20) with 1O ml of zinc chloride solution ( 1-5) , boil and
add starch suspension (to 5 g of soluble starch add 30 ml of
water and mix welD with stirring, and continue to boil far
2 minutes, allow to cool.
Preserve in a well-closed bottle, stored in a cool place.
8006 Volumetric Solutions
Ammonium Ferrous Sulfate (O. 1 mol/L) VS
[Fe(NH4)2(SQ4)2•6H 2 0 392.13] 39. 21 g-1000 ml
Preparation Dissolve 40 g of ammonium ferrous sulfate in
a previously cooled mixture of 40 ml of sulfuric acid and
200 ml of water, dilute with water to 1000 ml and mix well.
Standardization To 25 ml of ammonium ferrous sulfate
solution, accurately measured, add two drops of o-phenanthroline
IS and titrate with ceric sulfate (O. 1 mol/L) VS until the
colour tums from pale red to pale green. Calculate the
concentration of ammonium ferrous sulfate VS according to
the volume consumed.
This solution should be standardized befare use.
Ammonium Thiocyanate (O. 1 mol/L) VS
[NH4SCN 76.12] 7. 612 g-1000 ml
Preparation Dissolve 8. O g of ammonium thiocyanate in
water to make 1000 ml, and mix well.
Standardization To 25 ml of sil ver nitra te (O. 1 mol/L)
VS, accurately measured, add 50 ml of water, 2 ml of nitric
acid and 2 ml of ferric ammonium sulfate IS; titrate with
ammonium thiocyanate VS to the appearance of pale reddish-
brown colour which persists on shake vigorously. Calculate
the concentration of the ammonium thiocyanate VS according
to the volume consumed. Sodium thiocyanate (0. 1 mol/L)
VS or potassium thiocyanate (O. 1 mol/L) VS can be used
in place of the ammonium thiocyanate (O. 1 mol/L) VS.
Barium Chloride (O. lmol/L) VS
[BaClz•2H20 244. 26] 24. 43 g-1000 ml
Preparation Dissolve 24. 4 g of barium chloride in water to
make 1000 ml, and mix well.
Standardization To 25 ml of barium chloride (0. lmol/L)
VS, accurately measured, add 60 ml of water, 3 ml of
ammonia concentrated TS and O. 5-1 mg of phthalein purple;
ti trate with disodium edetate ( O. 05 mol/L) VS to the
appearance of purple colour begins to fade. Add 50 ml of
ethanol, continue to titrate until the purple blue colour
disappears. Perform a blank determination and make any
necessary correction. Each ml of disodium edetate
(0. 05 mol/L) VS is equivalent to 12. 22 mg of barium
chloride. Calculate the concentration of the barium chloride
(0. lmol/L) VS according to the volume of the disodium
edetate (O. 05 mol/L) VS consumed.
Barium Perchlorate (O. 05 mol/L) VS
[Ba(Clü4)2•3H20 390. 32] 19. 52 g-1000 ml
Preparation To 15. 8 g of barium hydroxide add 75 ml of
water and 7. 5 ml of perchloric acid, adjust to pH 3. O with
perchloric acid and filter if necessary. Add 150 ml of ethanol
and dilute to 250 ml with water. Dilute to 1000 ml with
acetate buffer solution ( dissolve 10 g of anhydrous sodium
acetate in 300 ml of water, adjust to pH 3. 7 with glacial
acetic acid and dilute with water to 1000 mD.
Standardization To 5 ml of sulfuric acid (O. 05 mol/L)
VS, accurately measured, add 5 ml of water, 60 ml of
ethanol, O. 5 ml of O. 1 % alizarin red solution as the indicator
solution and 50 ml of acetate buffer solution described above.
Titrate with barium perchloride (O. 05 mol/L) VS until the
colour changes to orange red and calculate the concentration
of barium perchlorate VS according to the volume consumed.
Bemalkonium Chloride (O. 01 mol/L) VS
Preparation Dissolve 3. 8 g of benzalkonium chloride in
water, add 10 ml of sodium acetate BS (pH 3. 7) and dilute
with water to 1000 ml. Mix well.
Standardization Dissolve about O. 18 g of analytical pure
potassium chloride, accurately weighed, previously dried far
1 hour at l 50ºC , in sodium acetate BS ( pH 3. 7) to make
250 ml. Mix well. Transfer accurately 20 ml to a 50 ml
volumetric flask, add accurately 25 ml of sodium
tetraphenylborate (0. 02 mol/L) VS, dilute with water to
50 ml and mix well. Filter with dry filter paper, discard the
initial filtrate. Transfer accurately 25 ml of the successive
filtrate to a 150 ml conical flask, add O. 5 ml of bromophenol
blue IS. Ti trate with benzalkonium chloride (O. 01 mol/L)
VS to a blue colour. Each ml of benzalkonium chloride
(O. 01 mol/L) VS is equivalent to O. 7445 mg of potassium
chloride.
Bismuth Nitrate (O. Olmol/L) VS
[Bi(NÜ3)3•5H20 485.10] 4. 851 g-1000 ml
Preparation Dissolve 4. 86 g of bismuth nitrate with
100 ml of nitric acid dilute TS, dilute with water to 1000 ml,
and mix well.
Standardization To 25 mi of bismuth nitrate VS,
accurately measured, add 50 mi of water and three drops of
xylenolorange IS and titrate with disodium edetate
(0. Olmol/L) VS until the colour turns from red to yellow.
Calculate the concentration of bismuth nitrate VS according
to the volume consumed.
Bromine (O. OS mol/L) VS
[Br2 159. 81] 7. 990 g-1000 mi
Preparation Dissolve 3. O g of potassium bromate and 15 g of
potassium bromide in water to make 1000 mi, and mix well.
Standardization To 25 mi of bromine VS, accurately
measured, in an iodine flask add 100 mi of water and 2. O g
of potassium iodide, shake thoroughly. Add 5 ml of
hydrochloric acid, stopper the flask and allow to stand in the
dark far 5 minutes. Titrate with sodium thiosulfate
(0. 1 mol/L) VS, add 2 mi of starch IS towards the end
point of titration, continue to titrate until the blue colour
disappears. Calculate the concentration of the bromine VS
according to the volume consumed.
When the room temperature is ahove 25 ºC , the reacting
liquid must be cooled to about 20ºC. Bromine VS should be
standardized immediately befare use.
If bromine (O. 005 mol/L) VS is needed, dilute bromine
(O. 05 mol/L) VS with water and standardize accordingly.
Storage Preserve in well closed amber coloured glass bottle
with glass stopper, stored ata cool place.
Ceric Sulfate (O. 1 mol/L) VS
[Ce(S04 )z•4H2 O 404. 30] 40. 43 g-1000 mi
Preparation Dissolve 42 g of ceric sulfate (or 70 g of ceric
ammonium sulfate) in 500 ml of water containing 28 mi of
sulfuric acid by heat, cool, dilute with water to 1000 mi.
Standardization Weigh accurately about O. 2 g of sodium
oxalate primary standard, previously dried to constant
weight at 105ºC, and dissolve it in 75 mi of water. Add 6 mi
of sulfuric acid solution ( Mix 20 mi of sulfuric acid with
50 mi water, cool), while still shaking; add 10 mi of
hydrochloric acid, heat up to 70-75ºC, titrate with sodium
thiosulfate (0. lmol/L) VS until the colour turns to light
yellow. Each mi of ceric sulfate (O. 1 mol/L) VS is
equivalent to 6. 700 mg of sodium oxalate. Calculate the
concentration of the ceric sulfate (O. 1 mol/L) VS according
to the volume consumed and weight of the sodium oxalate.
If ceric sulfate (O. 01 mol/L) VS is needed, dilute ceric
sulfate (O. 1 mol/L) VS with water containing 2. 8%
(mi/ mi) of sulfuric acid.
Disodium Edetate (O. OS mol/L) VS
[C10H14N2Na20s•2H20 372. 24] 18. 61 g-1000 mi
Preparation Dissolve 19 g of disodium edetate in water to
make 1000 mi and mix well.
Standardization Di.ssolve O. 12 g of zinc oxide primary
standard, previously ignited to constant weight at about 800°C and
accurately weighed, in 3 mi of dilute hydrochloric acid. Add 25 mi
of water and 1 drop of O. 025 % ethanolic solution of methyl red,
and add ammonia TS until a slight yellow colour is obtained. Add
25 mi of water, 10 mi of ammonium chloride BS (pH 10. O) and
a small amount of eriochrome black T indicator. Titrate with
disodium edetate VS until the colour changes from violet to pure
blue. Perform a blank determination and make any necessary
correction. Each mi of disodium edetate (O. 05 mol/L) VS is
equivalent to 4. 069 mg of ZnO. Calculate the concentration of the
disodium edetate (O. 05 mol/L) VS according to the volume
consumed and weight of the zinc oxide.
Storage Preserve in a glass stoppered bottle, avoid to
contact with rubber.
Ethanolic Potassium Hydroxide (O. S mol/L or O. 1 mol/L)
vs
[KOH 56.11] 28. 06 g-1000 mi; 5. 611 g-1000 mi
Preparation (1) Ethanolic Potassium Hydroxide (O. 5 mol/L)
vs
Di.ssolve 35 g of potassium hydroxide in a conical flask, in
aldehyde-free ethanol to make 1000 mi, well clase with a rubber
stopper and allow to stand far 24 hours. D:x:ant the supernatant
liquid immediately and transfer into a rubber stoppered amber
coloured glass bottle.
(2) Ethanolic Potassium Hydroxide (O. 1 mol/L) VS
Di.ssolve 7 g of potassium hydroxide in a conical flask, in
aldehyde-free ethanol to make 1000 mi, well clase with a rubber
stopper and allow to stand far 24 hours. D:x:ant the supernatant
liquid immediately and transfer into a rubber stoppered amber
coloured glass bottle.
Standardizatinn (1) Etharolic Potassium Hydroxide (0. 5 rml/L)
vs
Dilute 25 mi of hydrochloric acid (O. 5 mol/L) VS with 50
mi of water, add a few drops of phenolphthalein IS. Titrate
with ethanolic potassium hydroxide ( O. 5 mol/L ) VS.
Calculate the concentration of the ethanolic potassium
hydroxide VS according to the volume consumed.
(2) Ethanolic Potassium Hydroxide (0. 1 mol/L) VS
Dilute 25 mi of hydrochloric acid (O. 1 mol/L) VS with
50 mi of water, add a few drops of phenolphthalein IS.
Ti trate with ethanolic potassium hydroxide (O. 1 mol/L)
VS. Calculate the concentration of the ethanolic potassium
hydroxide VS according to the volume consumed.
This solution should be standardized befare use.
Storage Preserve in well closed amber coloured glass bottle
with rubber stopper.
Hydrochloric Acid ( 1, O. S, O. 2 or O. 1 mol/L) VS
[HCI 36. 46] 36. 46 g-1000 mi; 18. 23 g-1000 mi;
7. 292 g-1000 mi; 3. 646 g-1000 mi
Preparation (1) Hydrochloric Acid (1 mol/L) VS Dilute
90 mi of hydrochloric acid with water to 1000 mi and mix well.
( 2) Hydrochloric Acid (O. 5, O. 2 or O. 1 mol/L) VS
Dilute 45 mi, 18 mi or 9. O mi of hydrochloric acid with
water to 1000 mi respectively and mix well.
Standardization ( 1 ) Hydrochloric Acid ( 1 mol/L) VS
Weigh accurately about l. 5 g of anhydrous sodium carbonate
primary standard, previously dried to constant weight at 270-
300ºC, and dissolve it in 50 mi of water. Add 10 drops of
methyl red-bromocresol green IS, titrate with hydrochloric
acid ( 1 mol/L) VS until the colour turns from green to
purplish-red. Boil far 2 minutes, cool to room temperature
and continue the titration until the colour turns from green to
dark purple. F.ach mi of hydrochloric acid (1 mol/L) VS is
equivalent to 53. 00 mg of anhydrous sodium carbonate.
Calculate the concentration of the hydrochloric acid ( 1 mol/L)
VS according to the volume consumed and weight of the
anhydrous sodium carbonate.
(2) Hydrochloric Acid (O. 5 mol/L) VS Carry out the
titration described far Hydrochloric Acid ( lmol/L) VS
using about O. 8 g of anhydrous sodium carbonate primary
standard. Each mi of hydrochloric acid (O. 5 mol/L) VS is
equivalent to 26. 50 mg of anhydrous sodium carbonate.
(3) Hydrochloric Acid (O. 2 mol/L) VS Carry out the
titration described far Hydrochloric Acid ( 1 mol/L) VS
using about O. 3 g of anhydrous sodium carbonate primary
standard. Each mi of hydrochloric acid (O. 2 mol/L) VS is
 8006 Volumetric Solutions
equivalent to 10. 60 mg of anhydrous sodium carbonate.
(4) Hydrochloric Acid (0. 1 mol/L) VS Carry out the
titration described for Hydrochloric Acid ( 1 mol/L) VS
using about O. 15 g of anhydrous sodium carbonate
primary standard. Each ml of hydrochloric acid ( O. 1
mol/L) VS is equivalent to 5. 3 O mg of anhydrous
sodium carbonate.
If hydrochloric acid (O. 05, O. 02 or O. 01 mol/L) VS is
needed, dilute hydrochloric acid (1 or O. 1 mol/L) VS with
water, standardize if necessary.
Lead Nitrate (O. 05 mol/L) VS
[Pb(NQ3)2 331. 21] 16. 56 g-1000 ml
Preparation Weigh accurately 17. 5 g of lead nitrate to a
1000 ml volumetric flask, dissolve and dilute with water to
volume, and mix well.
Standardization To 25 ml of lead nitrate VS, accurately
measured, add 3 ml of glacial acetic acid, 5 g of
hexamethylenetetramine, 70 ml of water, 2 drops of xylenol
orange IS ( 2 g/L ) and titrate with disodium edetate
(O. 05 mol/L) VS until the colour turns to bright yellow.
Calculate the concentration of lead nitrate VS according to the
volume consumed.
If lead nitrate (O. 001 mol/L) VS is needed, dilute lead
nitrate ( O. 05 mol/L) VS with water and standardize
accordingly.
Storage Preserve in a well closed amber coloured glass bottle.
lodine (O. 05 mol/L) VS
D2 253. 81] 12. 69 g-1000 ml
Preparation Dissolve 13. O g of iodine and 36 g of
potassium iodine in 50 ml of water, add 3 drops of
hydrochloric acid and dilute with water to 1000 ml, mix well
and filter with a sintered glass filter.
Standardization To 25 ml of iodine (O. 05 mol/L) VS,
accurately measured, in an iodine flask, add 100 ml of water
and 1 ml of hydrochloric acid solution ( 9- 100) , shaking
gently, titrate with sodium thiosulfate (O. 1 mol/L) VS
towards the end point of titration, add 2 ml of starch IS and
continue to titrate until the blue colour disappears. Calculate
the concentration of the iodine VS according to the volume of
the sodium thiosulfate VS consumed.
If iodine (O. 025 mol/L) VS is needed, dilute the iodine
(O. 05 mol/L) VS with water.
Storage Preserve in well closed with glass stopper amber
coloured glass bottle, store at a cool place.
Lithium Methoxide (O. 1 mol/L) V~
[CH30Li 37. 97] 3. 797 g-1000 ml
Comply with the preparation, the standardization and the
storage described under sodium methoxide (O. 1 mol/L)
VS, use O. 694 g of freshly cut metallic lithium.
Mercuric Ni trate (O. 02 mol/L or O. 05 mol/L) VS
[Hg(NQ3)2•H 20 342. 62] 6. 85 g-1000 ml; 17. 13 g-
1000 ml
Preparation ( 1) Mercuric Nitrate (O. 02 mol/L) VS Dissolve
6. 85 g of mercuric nitrate in 20 ml of 1 mol/L nitric acid solution,
dilute with water to 1000 ml and mix well.
(2) Mercuric Nitrate (0. 05 mol/L) VS Dissolve 17. 2 g
of mercuric nitrate in 400 ml of water and 5 ml of nitric acid,
filter, dilute with water to 1000 ml and mix well.
Standardization ( 1) Mercuric Nitrate (0. 02 mol/L) VS
Weigh accurately about 15 mg of sodium chloride primary
standard previously dried to constant weight at llOºC, and
dissolve it in 50 ml of water, carry out the method for
potentiometric titration ( 0801), titrate with mercuric nitrate
(O. 02 mol/L) VS with stirring, using platinum electrode as
an indicator electrode and mercury-mercurous sulfate as a
reference electrode. Each ml of mercuric nitrate
(O. 02 mol/L) VS is equivalent to 2. 338 mg of sodium
chloride. Calculate the concentration of the mercuric nitrate
(O. 02 mol/L) VS according to the volume consumed and
weight of the sodium chloride.
(2) Mercuric Nitrate (O. 05 mol/L) VS Weigh accurately
about O. 15 g of sodium chloride primary standard previously
dried to constant weight at llOºC, and dissolve it in 100 ml
of water, add 1 ml of diphenylcarbazide IS and titrate with
mercuric nitrate VS, shake vigorously until a pale rose violet
colour appears. Each ml of mercuric ni trate (O. 05 mol/L)
VS is equivalent to 5. 844 mg of sodium chloride. Calculate
the concentration of the mercuric nitrate (O. 05 mol/L) VS
according to the volume consumed and weight of the sodium
chloride.
Methanolic Potassium Hydroxide (O. 1 mol/L) VS
[KOH 56. 11] 5. 611 g-1000 ml
Preparation Dissolve 6. 8 g of potassium hydroxide in 4 ml
of water, add methanol to 1000 ml, well close with a rubber
stopper and allow to stand for 24 hours. Decant the
supematant liquid immediately and transfer into a rubber
stoppered amber coloured glass bottle.
Standardization Comply with standardization described
under ethanolic potassium hydroxide (O. 5 mol/L) VS.
Storage Preserve in well closed amber coloured glass bottle
with rubber stopper.
Oxalic Acid (O. 05 mol/L) VS
[CzH204•2H20 126. 07] 6. 304 g-1000 ml
Preparation Dissolve 6. 4 g of oxalic acid in water to make
1000 ml and shake thoroughly.
Standardization Add 200 ml of water and 10 ml of sulfuric
acid to 25 ml of oxalic acid VS, accurately measured. Titrate
with potassium permanganate (O. 02 mol/L) VS towards the
end point of titration, heat the solution to 65ºC and continue the
titration until the pale red colour persists for 30 seconds. The
temperature of the solution should not be lower than 55ºC at the
end of titration. Calculate the concentration of oxalic acid VS
according to the volume consumed.
If oxalic acid (O. 25 mol/L) VS is needed, weigh about
32 g of oxalic acid, prepare and standardize as directed above
but titrate with potassium permanganate (O. 1 mol/L) VS.
Storage Preserve in well closed amber coloured glass bottle
with glass stopper.
Perchloric Acid (O. 1 mol/L) VS
[HC104 100. 46] 10. 05 g-1000 ml
Preparation To 750 ml of anhydrous glacial acetic acid
( calculate on water content, each g of water need to add
5. 22 ml of acetic anhydride), add 8. 5 ml of perchloric acid
(70%-72%) and mix well. Add 23 ml of acetic anhydride
dropwise with shake, cool and dilute with anhydrous glacial
acetic acid to 1000 ml, mix well and allow to stand for
24 hours. If the reactant is susceptible to acetylation, the
water content of perchloric acid VS should be determined
(0832, method 1 (1) ) , and adjusted to O. 01%-0. 2% by
the addition of water or acetic anhydride.
Standardization Weigh accurately about O. 16 g of
potassium biphthalate primary standard, previously dried to
constant weight at 105ºC, and dissolve it in 20 ml of
anhydrous glacial acetic acid. Add 1 drop of crystal violet IS and

titrate slowly with perchloric acid (O. 1 mol/L) VS to a blue
end point. Perform a blank determination and make any
necessary correction. Each ml of perchloric acid (O. 1 mol/L)
VS is equivalent to 20. 42 mg of potassium biphthalate. Calculate
the concentration of the perchloric acid ( O. 1 mol/L ) VS
according to the volume consumed and weight of the potassium
biphthalate.
If perchloric acid (O. 05 or O. 02 mol/L) VS is needed,
dilute perchloric acid ( O. 1 mol/L) VS with anhydrous
glacial acetic acid and standardize, if necessary.
Perchloric Acid in Dioxane (O. 1 mol/L) VS Dissolve
8. 5 ml of perchloric acid ( 70%-72%) in 100 ml of
isopropanol and dilute with dioxane to 1000 ml.
Weigh accurately about O. 16 g of potassium biphthalate
primary standard, previously dried to constant weight at
105ºC, and dissolve it in 25 ml of propylene glycol and 5 ml
of isopropanol by warming. Cool, add 30 ml of dioxane and a
few drops of methyl orange-xylene cyanol blue FF IS, titrate
with perchloric acid in dioxane VS until the colour tums from
green to bluish-grey. Perform a blank determínatíon and
make any necessary correctíon.
Storage Preserve in well closed amber coloured glass bottle.
Po~ium Bromate (O. 01667 mol/L) VS
[KBr03 167. 00] 2. 784 g-1000 ml
Preparation Díssolve 2. 8 g of potassium bromate in water
to make 1000 ml and mix well.
Standardization To 25 ml of potassium bromate VS,
accurately measured, in an íodine flask add 2. O g of
potassíum iodíde and 5 ml of dilute sulfuric acid, stopper the
flask and mix well. Allow to stand in the dark for 5 minutes,
dílute wíth 100 ml of water, titrate wíth sodíum thiosulfate
(0. 1 mol/L) VS towards the end point of titration, add 2 ml
of starch IS, continue to titrate until the blue colour
disappears. Calculate the concentration of the potassium
bromate VS according to the volume consumed.
If the room temperature is above 25ºC, the reacting liquid
and the water of dilution must be cooled to about 20ºC.
Po~ium Dichromate (0. 01667 mol/L) VS
[K2Cr2Ü1 294. 18] 4. 903 g-1000 ml
Preparation Weigh accurately 4. 903 g of potassium
dichromate primary standard, previously dried to constant
weight at 120°C, and dissolve it in water to make 1000 mi
and mix well.
Po~ium lodate (O. 05 mol/L or O. 01667 mol/L) VS
[KIQ 214. 00] 10. 700 g-1000 ml; 3. 5667 g-1000 rnl
Preparation Potassium Iodate (O. 05 mol/L) VS Weigh
accurately 10. 700 g of potassium iodate primary standard,
previously dried to constant weight at 105ºC, and dissolve it
in water to make 1000 ml, mix well.
Potassium Iodate (O. 01667 mol/L) VS Weigh accurately
3. 5667 g of potassium iodate primary standard, previously
dried to constant weight at 105ºC, and dissolve it in water to
make 1000 ml, mix well.
Po~ium Permanganate (O. 02 mol/L) VS
[KMn04 158. 03] 3. 161 g-1000 ml
Preparation Boíl about 3. 2 g of potassium permanganate
with 1000 ml of water for 15 minutes, allow to stand in a
well closed vessel for at least 2 days, filter with a sintered
glass filter and mix well.
Standardization Weigh accurately about O. 2 g of sodium
oxalate primary standard, previously dried to constant
weight at 105ºC, and dissolve it in 250 ml of freshly boiled
8006 Volumetric Solutions
and cooled water and 10 ml of sulfuric acid with stirring.
Add quickly from a burette about 25 ml of potassium
permanganate (0. 02 mol/L) VS with shaking to avoid the
production of precipitate, after the fading of its colour, heat
the solution to 65ºC and contínue to titrate until the pale red
colour persists for 30 seconds. The temperature of the
solution should not be lower than 55ºC at the end of
titration. Each ml of potassium permanganate (O. 02 mol/L)
VS is equivalent to 6. 70 mg of sodium oxalate. Calculate the
concentration of the potassium permanganate (O. 02 mol/L)
VS according to the volume consumed and weight of the
sodium oxalate.
If potassium permanganate (O. 002 mol/L) VS is needed,
dilute potassium permanganate ( O. 02 mol/L) VS with
water, boíl and cool. Filter and standardize, íf necessary.
Storage Preserve in well closed amber coloured glass bottle
with glass stopper.
Silver Nitrate (O. 1 mol/L) VS
[AgN03 169. 87] 16. 99 g-1000 ml
Preparation Dissolve 17. 5 g of sil ver nítrate in water to
make 1000 ml and mix well.
Standardization Weígh accurately about O. 2 g of sodíum
chloride primary standard, previously dried to constant
weight at llOºC, and dissolve it in 50 ml of water. Add 5 ml
of dextrin solution 0-50), O. 1 g of calcium carbonate and
8 drops of fluorescein IS, titrate with sílver nitrate
(O. 1 mol/L) VS untíl the colour of the opalescent liquid
tums from yellowish-green to pale red. Each ml of sílver
nítrate ( O. 1 mol/L) VS is equivalent to 5. 844 mg of
sodíum chloride.
If silver nitrate (O. 01 mol/L) VS is needed, dilute silver
nitrate (O. 1 mol/L) VS with water immediately befare use.
Storage Preser1e in a v1ell closed amber coloured glass
bottle with glass stopper.
Sodium Acetate (O. 1 mol/L) VS
[Cz H3Na02 82. 04] 8. 204 g-1000 ml
Preparation Dissolve 5. 3 g of anhydrous sodíum carbonate
in 100 ml of anhydrous glacial acetic acid (calculate on water
content, each g of water need to add 5. 22 ml of acetic
anhydride) with stírring, add anhydrous glacial acetíc acíd to
1000 ml and mix well.
Standardization To 15 ml of perchloríc acid (0. 1 mol/L)
VS, accurately measured, add a few drops of crystal víolet
IS, ti trate wíth sodíum aceta te (O. 1 mol/L) VS until the
colour turns to green. Calculate the concentration of sodíum
acetate VS according to the volume consumed.
Sodium Hydroxide ( lmol/L, O. 5 mol/L or O. 1 mol/L) VS
[NaOH 40. 00] 40. 00 g-1000 ml; 20. 00 g-1000 ml;
4. 000 g-1000 ml
Preparation Shake sodium hydroxíde with water to make a
saturated solutíon, cool, transfer to a polyvinyl plastic bottle
and allow to stand for several days.
Dílute 56, 28 or 5. 6 ml respectívely of the saturated and
clarified sodíum hydroxide solutíon wíth freshly boíled and
cooled water to 1000 ml, and mix well.
Standardization <D &xlium Hydroxíde ( 1 mol/L) VS Weígh
accurately about 6 g of potassíum bíphthalate primary
standard, previously dríed to constant weight at 105ºC, add
50 ml of freshly boiled and cooled water and shake
thoroughly. Add 2 drops of phenolphthalein IS and titrate
with sodíum hydroxide ( 1 mol/L) VS to a pink end point.
Potassium biphthalate should have been completely dissolved
 8006 Volumetric Solutions
towards the end point of titration.
Each ml of sodium hydroxide (1 mol/L) VS is equivalent to
204. 2 mg of potassium biphthalate. Calculate the
concentration of the sodium hydroxide ( 1 mol/L) VS
according to the volume consumed and weight of the
potassium biphthalate.
(2) Sodium Hydroxide (0. 5 mol/L) VS Weigh accurately
about 3 g of potassium biphthalate primary standard,
previously dried to constant weight at 105ºC. Standardize as
directed above. Each ml of sodium hydroxide (O. 5 mol/L)
VS is equivalent to 102. 1 mg of potassium biphthalate.
(3) Sodium Hydroxide (0. 1 mol/L) VS Weigh accurately
about O. 6 g of potassium biphthalate primary standard,
previously dried to constant weight at 105ºC. Standardize as
directed above. Each ml of sodium hydroxide (O. 1 mol/L)
VS is equivalent to 20. 42 mg of potassium biphthalate.
If sodium hydroxide (O. 05, O. 02 or O. 01 mol/L) VS is
needed, dilute sodium hydroxide (O. 1 mol/L) VS with
freshly boiled and cooled water. Standardize by hydrochloric
acid (0. 05, O. 02 or O. 01 mol/L) VS, if necessary.
Storage Preserve in a tightly closed polyvinyl plastic
bottle, insert 2 glass tubes through the stopper, connect one
of the tubes to a soda lime tube, use the other tube for the
outlet of the volumetric solution.
Sodium Methoxide (O. 1 mol/L) VS
[CH30Na 54. 02] 5. 402 g-1000 ml
Preparation Place 150 ml of dehydrated methanol (it contains
water less than O. 2%) in a vessel cooled with ice water, add
2. 5 g of freshly cut metallic sodium in portions and dissolve it
completly, add dehydrated benzene ( it contains water less than
O. 02%) to make 1000 ml and mix well.
Standardization Weigh accurately about O. 4 g of benzoic
acid primary standard. previously dried to constant weight in
a vaccum desiccator over phosphorus pentoxide, and dissolve
it in 15 ml of dehydrated methanol. Add 5 ml of dehydrated
benzene and 1 drop of a 1 % solution of thymol blue in
dehydrated methanol, titrate with sodium methoxide VS to a
blue end point. Perform a blank determination and make any
necessary correction.
Each ml of sodium methoxide (O. 1 mol/L) VS is equivalent
to 12. 21 mg of benzoic acid. Calculate the concentration of
the sodium methoxide (O. 1 mol/L) VS according to the
volume consumed and weight of the benzoic acid.
Sodium methoxide VS must be standardized immediately
before use, protected from carbon dioxide and avoid
volatilization of solvent.
Storage Preserve in a well closed container of a titrating
assembly, protected from carbon dioxide and moisture.
Sodium Nitrite (O. 1 mol/L) VS
[NaN02 69. 00] 6. 900 g-1000 ml
Preparation Dissolve 7. 2 g of sodium nitrite and O. 10 g of
anhydrous sodium carbonate in water to make 1000 ml and
mix well.
Standardization Weigh accurately about O. 5 g of sulfanilic
acid primary standard, previously dried to constant weight at
120ºC, and dissolve it in 30 ml of water and 3 ml of
concentrated ammonia TS. Add 20 ml of hydrochloric acid
solution O - 2) with stirring, plunge the burette into the
solution so that about 2/3 of the tip is under the liquid
surface, titrate with sodium nitrite VS quickly with stirring
at a temperature below 30ºC . Withdraw the burette tip
towards the end point of titration, rinse it with water and add
the washings to the solution, continue the titration slowly,
adopt the dead-stop method ( 0701 ) for end point
determination. Each ml of sodium nitrite (O. 1 mol/L) VS
is equivalent to 17. 32 mg of sulfanilic acid. Calculate the
concentration of the sodium nitrite (O. 1 mol/L) VS
according to the volume consumed and weight of the
sulfanilic acid.
If sodium nitrite (O. 05 mol/L) VS is needed, dilute sodium
nitrite (O. 1 mol/L) VS with water and standandize, if
necessary.
Storage Preserve in well closed amber coloured glass bottle
with glass stopper.
Sodium Tetraphenylborate (O. 02 mol/L) VS
[(CsHs)4BNa 342. 22] 6. 845 g-1000 ml
Preparation Dissolve 7. O g of sodium tetraphenylborate in
50 ml of water with shake. Add freshly prepared aluminum
hydroxide gel (dissolve l. O g of aluminum chloride in 25 ml
of water, add sodium hydroxide TS dropwise with stirring
until the pH is 8-9) , 16. 6 g of sodium chloride and stir
thoroughly. Add 250 ml of water and shake for 15 minutes,
allow to stand for 10 minutes and filter. Add sodium
hydroxide TS dropwise to the filtrate until the pH is 8-9, and
then dilute with water to 1000 ml, mix well.
Standardization To 10 ml of sodium tetraphenylborate VS
add 10 ml of sodium aceta te BS ( pH 3. 7) and O. 5 ml of
bromophenol blue IS, and then titrate with benzalkonium
chloride (O. 01 mol/L) VS until the colour turns to blue.
Perform a blank determination and make any necessary
correction. Calculate the concentration of sodium
tetraphenylborate ( O. 02 mol/L ) VS according to the
volume consumed.
This solution should be standardized before use.
If sodium tetraphenylborate (O. 01 mol/L) VS is needed,
dilute sodium tetraphenylborate (O. 02 mol/L) VS with
water before use, standardize if necessary.
Storage Preserve in well closed amber coloured glass
bottle.
Sodium Thiosulfate (O. 1 mol/L or O. 05 mol/L) VS
[Na2Sz03•5H20 248.19] 24. 82 g-1000 ml; 12. 41 g-
1000 ml
Preparation Sodium thiosulfate ( O. 1 mol/L) VS
Dissolve 26 g of sodium thiosulfate and O. 20 g of anhydrous
sodium carbonate in freshly boiled and cooled water to make
1000 ml; shake thoroughly, allow to stand for 1 month,
filter.
Sodium thiosulfate (O. 05 mol/L) VS Dissolve 13 g of
sodium thiosulfate and O. 10 g of anhydrous sodium carbonate
in freshly boiled and cooled water to make 1000 ml; shake
thoroughly, allow to stand for 1 month, filter. Or dilute
Sodium thiosulfate (O. 1 mol/L) VS in freshly boiled and
cooled water.
Standardization ( 1) Sodium thiosulfate (O. 1 mol/L) VS
Weigh accurately about O. 15 g of potassium dichromate
primary standard, previously dried to constant weight at
120ºC, and dissolve it in 50 ml of water in an iodine flask.
Add 2. O g of potassium iodide and dissolve with gentle
shake, add 40 ml of dilute sulfuric acid. Stopper the flask
and shake thoroughly, allow to stand in the dark for
10 minutes, dilute with 250 ml of water and titrate with
sodium thiosulfate (0. 1 mol/L) VS until the end point is nearly
reached, add 3 ml of starch IS and continue to titrate until the
colour turns from blue to brilliant green. Perform a blank
determination and make any necessary correction. Each ml of
sodium thiosulfate (O. 1 mol/L) VS is equivalent to 4. 903 mg
 8061 Ref erence Standards, Reference Drugs and Reference Extractives

of potassium dichromate. Calculate the concentration of the
sodium thiosulfate (O. 1 mol/L) VS according to the volume
consumed and weight of the potassium dichromate.
(2) Sodium thiosulfate (O. 05 mol/L) VS Weigh accurately
about 75mg of potassium dichromate primary standard.
Standardize as directed above. Each ml of sodium thiosulfate
(O. 05 mol/L) VS is equivalent to 2. 452 mg of potassium
dichromate.
When the room temperature is above 25ºC, the reacting
liquid and water of dilution must be cooled to about 20ºC.
If sodium thiosulfate (O. 01 mol/L or O. 005 mol/L) VS is
needed, dilute sodium thiosulfate (O. 1 mol/L or O. 05 mol/L)
VS with freshly boiled and cooled water immediately before use,
standardize if necessary.
Sulfuric Acid (O. 5 , O. 25 , O. 1 or O. 05 mol/L) VS
[H 2 S0 4 98.08] 49.04 g-1000 ml; 24.52 g-1000 ml;
9. 81 g-1000 ml; 4. 904 g-1000 ml
Preparation Sulfuric acid (O. 5 mol/L) VS Add slowly
30 ml of sulfuric acid to water with stirring, cool to room
temperature, dilute with water to 1000 ml, mix well.
Sulfuric acid (O. 25, O. 1 or O. 05 mol/L) VS Add slowly
15 ml, 6. O ml or 3. O ml of sulfuric acid to water with
stirring, dilute with water to 1000 ml, mix well.
Standardization Standardize as described under hydrochloric
acid (1, O. 5, O. 2 or O. 1 mol/U VS.
If sulfuric acid (O. O1 mol/L) VS is needed, dilute sulfuric
acid (0. 5, O. 1 or O. 05 mol/L) VS with water, standardize
if necessary.
Tetrabutylammonium Hydroxide (O. 1 mol/L) VS
[CC4H 9 ) 4NOH 259. 48] 25. 95 g-1000 ml
Preparation Dissolve 9. 115 g of tetramethylammonium
hydroxide in water to make 1000 ml and mix well.
Standardization Weigh accurately about O. 3 g of benzoic
acid, previously dried 24 hours in a silica gel desiccator, and
dissolve in 90 ml of dimethylformamide. Add 3 drops of a O. 1 %
solution of thymol blue in dehydrated methanol, and titrate to a
blue end point with tetramethylammonium hydroxide VS.
Perform a blank determination and make any necessary
correction. Each ml of tetramethylammonium hydroxide
(O. 1 mol/U VS is equivalent to 12. 21 mg of benzoic acid.
Calculate the concentration of the tetramethylammonium
hydroxide (0. 1 mol/L) VS according to the volume consumed
and weight of the benzoic acid.
Storage Preserve in well closed container, keep away from
the carbon dioxide and moisture in air.
Zinc (O. 05mol/L) VS
[Zn 65.39] 3.270g-1000 ml
Preparation Dissolve 15 g of zinc sulfate ( equivalent to
about 3. 3 g of zinc) in 10 ml of hydrochloric acid dilute TS,
dilute with water to 1000 ml and mix well.
Standardization To 25 ml of zinc VS, accurately
measured, add one drop of O. 025 % methyl red ethanol
solution, add ammonia TS until the colour turns to light
yellow. Add 25 ml of water, 10 ml of ammonia-ammonium
chloride BS ( pH 10. O ) and a little amount of
eriochromeblack T indicator mixture. Titrate with disodium
edetate (O. 05mol/L) VS until the colour turns from purple
to pure blue. Perform a blank determination and make any
necessary correction. Calculate the concentration of zinc VS
according to the volume consumed.

Preparation Dissolve 40 g of tetrabutylammonium iodide

in 90 ml of dehydrated methanol in a stoppered glass 8061 Reference Standards,
conical flask. Place in an ice bath, add 20 g of finely Reference Drugs and Reference Extractives
powdered silver oxide, stopper the flask tightly and shake
vigorously for 60 minutes. Centrifuge a few ml and test the
Reference Standards
supernatant liquid for iodide. If the test is positive, add an
1, 3-0-Dicaffeoylquinic acid
additional 2 g of silver oxide to the above mixture and shake
1, 8-Dihydroxyanthraquinone
vigorously for 30 minutes. When all of the iodide has
1-Methyl hydantoin
reacted, filter through a sintered glass funnel, rinse the
2, 3, 5, 4' -Tetrahydroxystil-bene-2-0-,B-D-glucoside
flask and the funnel with three portions of 5 O ml of
2-Undecanone
dehydrated toluene, add the rinsings to the filtrate. Dilute
3, 5-Dicaffeoylquinic acid
with a mixture of dehydrated toluene and dehydrated
3', 6-Disinapoyl sucrose
methanol (3 : 1) to 1000 ml and mix well. Flush the
3, 29-Dibenzoylkarounitriol
solution for 1 O minutes with dry nitrogen which is free
3-Hydroxymorindone
from carbon dioxide. If the solution is not clear, add a few 4, 5-Dicaffeoylquinic acid
ml of dehydrated methanol. 4-Methoxybenzaldehyde (p-Anisaldehyde)
Standardization Weigh accurately about 90 mg of benzoic 4-Methoxysalicylaldehyde
acid primary standard, previously dried to constant weight in 4- Hydroxybenzyl alcohol
a vaccum desiccator over phosphorous pentoxide, and 4- Hydroxyphenylacetic acid
dissolve in 10 ml of dimethylformamide. Add 3 drops of a 4 ' - Hydroxyacetophenone
1
O. 3% solution of thymol blue in dehydrated methanol, and 4 -0-,B-D-glucosyl-5-0-methylvisammioside
titrate to a blue end point with tetrabutylammonium 5-Ethoxychelerythrine
hydroxide VS ( adopt potentiometric method to indicate the 5-Hydroxymethyl furfural
end point). Perform a blank determination and make any 5-Methyl mellein
necessary correction. Each ml of tetrabutylammonium 6-Gingerol
hydroxide (O. 1 mol/L) VS is equivalent to 12. 21 mg of 8-Acetylshanzhiside methyl ester
benzoic acid. Calculate the concentration of the 8-Gingerol
tetrabutylammonium hydroxide (O. 1 mol/L) VS according 10-Gingerol
to the volume consumed and weight of the benzoic acid. 23-Acetate alisol B
a-Cyperone
Storage Preserve in well closed container, keep away from
a-Hederin
the carbon dioxide and moisture in air.
a-Linolenic acid
Tetramethylammonium hydroxide (O. 1 mol/L) VS a-Pinene
[CCH 3 )4NQH 91.15] 9.115 g-1000 ml a-Terpineol
 8061 Reference Standards, Reference Drugs and Reference Extractives

a-Terthiophene Brucine
f3, {3 1-Dimethylacrylalkanin Buddlejasaponin N b
{3-Caryophyllene Buddleoside
{3-Ecdysterone Bufogenin
{3-Pinene Bullatine A
{3-Sitosterol Butanedioic acid
r-Schisandrin Caffeic acid
Acephate Caffeoyl acetate
Acetyl asperulosidic acid methyl ester Calceolarioside B
Acetylharpagide Calcium carbonate
Aconitine Calendoflavoside ( Isorhamnetin-3-0-neohesperidoside)
Adenine Camphene
Adenosine Camphor
Adonifoline Cantharidin
Aesculetin Capsaicine
Aesculin Cardamonin
Agarotetrol Carvacrol
Agrimol B Catalpol
Alantolactone Cephaeline hydrochloride
Aleuritopsin (+)-Ca techin
Allicin Chenodeoxycholic acid
Alnustone Chikusetsusaponin Na
Aloe-emodin Chlorogenic acid
Aloin Chlorophenothane (DDT)
Alpinetin Cholic acid
Amentoflavone Chonglou saponin I ( poly phyllin I )
Ammonium glycyrrhizinate Chonglou saponin 11 ( poly phyllin 11 )
Amygdalin Chonglou saponin VI (poly phyllin VD
Andrographolide Chonglou saponin VJ[ ( poly phyllin VJ[)
Anhydrous glucose Chrysin
Anisodamine Chrysophanol
Anisodamine hydrobromide Cineole
Anwuligan Cinnamaldehyde
Arbutin Cinnamic acid
Arctiin Cinobufagin
Arecoline Citric acid
Arecoline hydrobromide Codeine phosphate
Arginine Columbianadin
Aristolochic acid Columbin
Artemisinin Complanatoside A
Asarinin Corynoline
Asiaticoside Costunolide
Asperosaponin VI ( Akeboside D) Crocin- I
Astilbin Crocin- 11
Astragaloside N Crotonoside
Atractylodin Cryptotanshinone
Atropine sulfate Curculigoside
Aurantio-obtusin Curcumenol
Baicalein Curcumin
Baicalein-7-0-diglusoside Curdione
Baicalin Cyasterone
Belamcandin ( T ectoridin) Cyclovirobuxine D
Benzene hexachloride CBHC) Cypermethrin
Benzoic acid Daidzein
Benzoylaconine Daidzin
Benzoylhypaconitine Daphnetin
Benzoylmesaconine Daphnoretin
Berberine hydrochloride Dauricine
Bergenin D-Borneol
Betaine Daurisoline
Bilirubin Dehydroandrographolide
Bilobalide Dehydrocostuslasctone
Bisdemethoxycurcumin Dehydrodiisoeugenol
Blinin Deltamethrin
Borneol Demethoxycurcumin
Borneo} and Isoborneol Dendrobine
Bornyl acetate Dendrophenol
Bovine serum albumin Deoxycholic acid
 8061 Reference Standards, Reference Drugs and Reference Extractives

Deoxyschizandrin Ginsenoside Rbi

Diethion Ginsenoside Rb3
Dihydrocapsaicin Ginsenoside Rg1
Dimethoate Glucosamine hydrochloride
Dimethyl-dichloro-vinyl phosphate Glucosyl vitexin
Dimpylate Glyceryl trilinoleate
Dioscin Glyceryl trioleate
Diosgenin Glycine
Dipsacoside B Glycyrrhetinic acid
D, L-Alanine Glycyrrhizic acid
Dracohodin perochlorate Griffonilide
Ecdysterone Guan-fu base A
Echinacoside Guanosine
Ecliptasaponin A Gypsogenin-3-0-13-D-glucuronide
Eleutheroside E Harpagide
Ellagic acid Harpagoside
Emetine hydrochloride Hydrochlorothiazide
Emodin Hederagenin
Engeletin Hesperidin
ent-Kaurenoic acid Heterclitin D
Ephedrin hydrochloride Histidine
( - )-Epicatechin Honokiol
(- )-Epicatechin gallate Hosenkoside A
( - )-Epigallocatechin gallate Hosenkoside K
(R, S)-Epigoitrin Hupehenine
Ergosterol Hydroxysafflor yellow A
Erianin Hyodeoxycholic acid
Esculentoside A Hyoscyamine
Ethyl-p-methoxycinnamate Hyoscyamine sulfate
Eugenol Hypaconitine
Eupalinolide A Hyperoside
Euphadienol lcariin
Euphobiasteroid Imperatorin
Evodiamine Indigo
Evodine Indirubin
F angchinoline Irisfloremin
Farrerol Isoalantolactone
Fraxetin Isoborneol
Fenvalerate Isoferulic a cid
Ferrous sulfate Isofraxidin
Ferulic acid Isoimperatorin
Fissistigine A Isopsoralen
Folimat Isoquerci trin
Formononetin Isorhamnetin
Forsythin Isorhynchophylline
Forsythoside A J atrorrhizine hydrochloride
Forsythoside B Jujuboside A
Fraxinellon Jujuboside B
Fructose Kaempferol
Furanodiene Kaempferol-3-0-rutinoside
Galangin Kirenol
Gallic acid Kochioside l e
Gastrodin L-Citrulline
Geniposide Leonurine hydrochloride
Geniposidic acid Levislolide A
Genistin L-Hydroxyproline
Genkwanin Liensinine
Gentiopicrin Liensinine perchlorate
Germacrone Ligustilide
Ginkgolic acid Ligustroflavone
Ginkgolide A Linalool
Ginkgolide B Linderane
Ginkgolide C Liquidambaric acid
Ginkgoneolic acid Liquiritin
Ginsenoside Rd Liriopemuscaribaily baily saponins C
Ginsenoside Re Lithospermoside
Ginsenoside Rf L-Leucine
Ginsenoside Ro Lobetyolin
 8061 Reference Standards, Reference Drugs and Reference Extractives

Loganin Peimisine
Loureirin A Perillaldehyde
Loureirin B Perillene
L-Proline Phellodendrine hydrochloride
Lucidin Physalin L
Luteolin Physcion
Luteolin-7-0-glucoside Picfeltarraenin I A
Lysine Picrinine
Macranthoidin B Picroside I
Madecassoside Picroside JI
Magnolin Pigenin
Magnolol Pingpeimine A
Malathion Pinocembrin
Maltopentose Pinoresinol diglucoside
Mannitol Piperine
Matrine Plantamajoside
Methidathion Platycodin D
Mentha arvensis oil Podophyllotoxin
Menthol Pogostone
(-)-Menthone Poliumoside
Mesaconitine Polydatin
Methamidophos Polygalacic acid
Methoxybenzene Polygalasaponin F
Methyl parathion Polygalaxanthone IIl
Methylsalicylate Praeruptorin A
Mogroside V Praeruptorin B
Mollugin prim-0-Glucosylcimifugin
Monocrotaline Proline
Monocrotophos Protocatechuic acid
Monotropein Protocatechuic aldehyde
Morphine Protopine
Morphine hydrochloride Pseudoephedrine hydrochloride
Morroniside Pseudoginsenoside Fn
Mulberroside A Pseudolaric acid B
Murrayone Pseudoprotodioscin
Muscone Psoralen
Myricitrin Puerarin
Nardosinone Pulegone
Naringin Purpurin
Neohesperidin Quercetin
Nitidine chloride Quercetin-3-0-,B-D-glucose-7-0-,B-D-gentiobioside
Nodakenin Quercitroside
Norisoboldine Quintozene
Notoginsenoside R1 Raddeanin A
Notopterol Rhapontin
Nuciferine Rhein
Nystose Rhoifolin
Obaculactone Ricinic acid methyl este
Obacunone Ricinoleic acid
Oleanolic acid Roburic acid
Oleic acid Rosmarinic acid
Oridonin Ruscogenin
Orientin Rutaecarpine
Osthol Rutin
Oxymatrine Saikosaponin a
Paeoniflorin Saikosaponin d
Paeonol Salicylic acid
Palmatine hydrochloride Salidroside
Panaxadiol Salvianolic acid B
Panaxatriol Sarsasapogenin
Papaverine hydrochloride Sauchinone
Parathion Schaftoside
Patchouli alcohol Schisandrin
Pectolinarigenin Schisantherin A
Pectolinarin Scopolamine
Pedunculoside Scopolamine hydrobromide
Peimine Scopoletin
Peiminine Scutellarin
 8061 Reference Standards, Reference Drugs and Reference Extractives

Senegenin Abutili Semen

Sesamine Acanthopanacis Senticosi / Radix et Rhizoma seu Caulis
Shanzhiside methyl ester Achilleae Herba
Shikonin Achyranthis Asperae Radix et Rhizoma
Shionone Achyranthis Bidentatae Radix
Silybin Aconiti Brachypodi Radix
Sinapine cyanide sulfonate Aconiti Coreani Radix
Sinomenine Acori Calami Rhizoma
Sipeimine Acori Tatarinowii Rhizoma
Sipeimine-3-{3-D-glucoside Adenophorae Radix
Sodium aescinate Ailanthi Cortex
Sodium Danshensu Akebiae Fructus
Sodium sulphate
Alangii Radix Gracilis
Sodium taurocholate
Albiziae Cortex
Sophoricoside
Albiziae Flos
Sophoridin
Alismatis Rhizoma
Specnuezhenide
Spicatoside B Allii Macrostemonis Bulbus
Spinosin Alpiniae Officinarum Rhizoma
Stachydrine hydrochloride Alpiniae Oxyphyllae F ructus
Stevioside Alstoniae Scholaris Folium
Strychnine Amomi Fructus
Swertiamarin Ampelopsis Radix
Synephrine Andrographis Herba
Syringoside Angelicae Dahuricae Radix
(-)-Syringaresinol-4-0-B-D-furanapiosyl-( 1-2 )-{3-D- Angelicae Pubescentis Radix
glucopyranoside Angelicae Sinensis Radix
Tanshinone I Anisi Stellati Fructus
Tanshinone II A Apocyni Veneti Folium
Taraxerone Aquilariae Lignum Resinatum
Taurine Archidendri Folium
Taurohyodeoxycholic acid Arctii Fructus
T auroursodeoxycholic acid Arecae Semen
Taxífolin Arisaematis Rhizoma
Tenacissoside H Aristolochiae Fructus
Tenuífolin Artemisia Scoparia Herba
Terpinen-4-ol Artemisiae Annuae Herba
Tetrahydropalmatine Asari Radix et Rhizoma
Tetrandrine Aspongopus
Threonine
Asteris Radix et Rhizoma
Thymol
Astragali Complanati Semen
Tiliroside
Astragali Radix
Timosaponin B II
Atractylodis Macrocephalae Rhizoma
Toddaloactone
Toosendanin Atractylodis Rhizoma
Tracheloside Aucklandiae Radix
trans-Anethole Aurantii Fructus
T rigonelline Aurantii Fructus Immaturus
Tubemoside I Belamcandae Rhizoma
T ussilagone Benzoinum
Typhaneoside Ber~ridis Cortex
Tyrosine Bistortae Rhizoma
Uridine Bletillae Rhizoma
U rsodeoxycholic acid Bos Taurus Domesticus Gmelin / Bubalus Bubalis L.
U rsolic acid Bovis Calculus Artifactus
Vaccarin &vis Fel
Valepotriate Brassicae Pollen
Valine Bruceae Fructus
Vanillic acid Buddlejae Flos
Verbascoside Bufonis Corium
Vitamin Di Bufonis Venenum
Vitexicarpin Bupleuri Radix
Vitexin Callicarpae Caulis et Folium
Vitexin-2' -0-rhamnoside Callicarpae Macrophyllae Folium
Wedelolactone Callicarpae Nudiflorae Folium
Wogonin Camelliae Sinensis Folium
Reference nn. Campsis Flos
Anemarrhenae Rhizoma Canarii F ructus
 8061 Reference Standards, Reference Drugs and Reference Extractives

Cannabis Semen Dioscoreae Spongiosae Rhizoma

Capra Hircus L. / Ovisaries L. Dipsaci Radix
Caprae Fel / Ovis Fel Dracaenae Combodianae Resina
Carotae Fructus Dracocephali Tangutici Herba
Carpesii Fructus Draconis Sanguis
Carpesii Herba Dryopteridis Crassirhizomatis Rhizoma
Carthami Flos Ecliptae Herba
Caryophylli Flos Eriobotryae Folium
Cassiae Semen Eriocauli Flos
Catechu Erycibes Caulis
Caulis Fissistigmae et Radix Eucommiae Cortex
Celosiae Cristatae Flos Eucommiae Folium
Cervi Cornu Pantotrichum Euodiae Fructus
Chaenomelis F ructus Euonymi Herba
Changii Radix Eupatorii Herba
Chebulae Fructus Euphorbiae Hirtae Herba
Chebulae Fructus lmmaturus Euphorbiae Humifusae Herba
Chrysanthemi Flos Euphorbiae Pekinensis Radix
Chrysanthemi lndici Flos Eupolyphaga / Steleophaga
Chuanxiong Rhizoma Euryales Semen
Cichorii Herba / Cichorii Radix Fagopyri Dibotryis Rhizoma
Cimicifugae Rhizoma Farfarae Flos
Cinnamoni Cortex Fici Microcarpae Folium
Cirsii Herba Fici Simplicissimae Radix
Cirsii Japonici Herba Forsythiae Fructus
Cistanches Herba Fritillariae Cirrhosae Bulbus
Citri Fructus Fritillariae Hupehensis Bulbus
Citri Grandis Exocarpium Fritillariae Pallidiflorae Bulbus
Citri Reticulatae Folium Fritillariae Thunbergii Bulbus
Citri Reticulatae Pericarpium Fritillariae Ussuriensis Bulbus
Citri Reticulatae Pericarpium Viride Galangae Fructus
Citri Sarcodactylis Fructus Galla Chinensis
Clematidis Radix et Rhizoma Ganoderma
Cnidii Fructus Gardeniae Fructus
Codonopsis Radix Gaultheriae Yunnanensis Herba
Coicis Semen Gastrodiae Rhizoma
Commelinae Herba Gecko
Coptidis Rhizoma Gendarussae Herba
Corni Fructus Genkwa Flos
Corydalis Bungeanae Herba Gentianae Macrophyllae Radix
Corydalis Rhizoma Ginkgo Folium
Crataegi Fructus Ginseng Gaulis et Folium
Croci Stigma Ginseng Radix et Rhizoma
Crotonis Fructus Ginseng Radix et Rhizoma Rubra
Cudraniae Radix Glechomae Herba
Cudraniae Radix et Caulis Gleditsiae Fructus Abnormalis
Curculiginis Rhizoma Gleditsiae Sinensis Fructus
Curcumae Longae Rhizoma Gleditsiae Spina
Curcumae Radix Glycyrrhizae Radix et Rhizoma
Curcumae Rhizoma Gossampini Flos
Cuscutae Semen Hedyotidis Chrysotrichae Herba
Cyathulae Radix Hedyotidis Diffusae Herba
Cynanchi Atrati Radix et Rhizoma Hedysari Radix
Cynanchi Paniculati Radix et Rhizoma Hericium
Cynomorii Herba Hirudo
Cyperi Rhizoma Holarrhenae Antidysentericae Semen
Dalbergiae Odoriferae Lignum Hordei Fructus Germinatus
Daphnes Cortex Houttuyniae Herba
Dendrobii Officinalis Caulis Hypecoe Leptocarpe Herba
Desmodii Styracifolii Herba Hyperici Perforati Herba
Dianthi Chinensis Herba Ilicis Cornutae Folium
Dianthi Herba Ilicis Hainanensis Folium
Dichroae Radix lmpatientis Semen
Dictamni Cortex lmperatae Rhizoma
Dioscoreae Hypoglaucae Rhizoma lndigo Naturalis
Dioscoreae Rhizoma lnulae Flos
 8061 Reference Standards, Reference Drugs and Reference Extractives

Inulae Herba Peucedani Radix

Inulae Racemosae Radix Pharbitidis Semen
Inulae Radix Phellodendri Amurensis Cortex
Iridis T ectori Rhizoma Phellodendri Chinensis Cortex
Isatidis Radix Pheretima
]ujubae Fructus Phlomidis Younghusbandii Radix
Kadsurae Caulis Phragmitis Rhizoma
Kaempferiae Rhizoma Phyllanthi Fructus
Kaki Folium Physalis Calyx seu Fructus
Kalimeridis Herba Picrasmae Ramulus et Folium
Kansui Radix Picriae Herba
Knoxiae Radix Picrorhizae Rhizoma
Lamiophlomidis Herba Pinelliae Rhizoma
Lasiosphaera / Calvatia Piní Massonianae Folium
Leonuri Herba Piperis Fructus
Lignum Santalí Albi Piperis Kadsurae Caulis
Ligustici Rhizoma et Radix Piperis Longi Fructurs
Ligustri Lucidi Fructus Platycodonis Radix
Lilii Bulbus Polygalae Radix
Linderae Radix Polygonati Rhizoma
Liquidambaris Resina Polygoni Cuspidati Rhizoma et Radix
Litseae Fructus Polygoni Multiflori Caulis
Lobeliae Chinensis Herba Polygoni Multiflori Radix
Longan Arillus Polygoni Multiflori Radix Praeparata
Lonicerae Flos Po ria
Lonicerae J aponicae Caulis Portulacae Herba
Lo ni cera e ] aponicae Flos Potentillae Chinensis Herba
Lycii Cortex Prunellae Spica
Lycii Fructus Prunus Mume
Lycopodii Herba Psammosilenes Radix
Lygodii Spora Pseudolaricis Cortex
Magnoliae Flos Pseudostellariae Radix
Manis Squama Psoraleae Fructus
Margarita Pteridis Multifidae Herba
Medulla Junci Puerariae Lobatae Radix
Melastomatis Dodecandri Herba Pulsatillae Radix
Melastomatis Normalis Radix Pyrolae Herba
Meliae Cortex Quercidentatae Folium
Melis Adeps Quisqualis F ructus
Menispermi Rhizoma Quisqualis Semen
Mori Cortex Rabdosiae Rubescentis Herba
Mori Folium Ranunculi Ternati Radix
Morindae Officinalis Radix Raphani Semen
Moutan Cortex Rehmanniae Radix
Mume Fructus Rehmanniae Radix Praeparata
Myristícae Semen Rhapontici Radix
Myrrha Rhei Radix et Rhizoma
Nelumbinis Rhizomatis Nodus Rhododendri Daurici Folium
Nelumbinis Semen Rhododendri Mollis Flos
Nigellae Semen Rhodomyrti Radix
Notoginseng Radix et Rhizoma Ricinuscommunis
Notopterygii Rhizoma et Radix Rosae Lavigatae Radix
Omphalia Rubi Lignum Ramuli
Ophiopogonis Radix Rubiae Radix et Rhizoma
Paeoniae Radix Alba Saigae T ataricae Cornu
Paeoniae Radix Rubra Salviae Miltiorrhizae Radix et Rhizoma
Panacis Quinquefolii Radix Saposhnikoviae Radix
Papaveris Pericarpium Sappan Lignum
Paridis Rhizoma Sarcandrae Herba
Pegaeophyti Radix et Rhizoma Sargentodoxae Caulis
Penthori Chinensis Herba Sauropi Folium
Perillae Caulis Saururi Herba
Perillae Folium Saussureae Involucratae Herba
Perillae Fructus Schefflerae Kwangsiensis Caulis et Folium
Periplocae Cortex Schisandrae Chinensis Fructus
Persicae Ramulus Schisandrae Sphenantherae Fructus
 8062 Reference Standards

Schizonepetae Herba Reference Extractives

Schizonepetae Spica Chinese Mahonia Stem Extract
Scrophulariae Radix Coix Seed Oil
Scutellariae Barbatae Herba Costusroot Essential Oil
Scutellariae Radix Curcuma Oil
Securidacae Herba Dioscorea Panthaica Extract
Sedi Herba Dioscorea Nipponica Saponin Extract
Selaginellae Herba Fermented Cordyceps Powder
Senecionis Cannabifolii Herba Ginkgo Leaves Extract
Senecionis Scandentis Herba Notoginseng Total Saponins
Sennae Folium Patchouli Oil
Serpentis Fel Peppermint Oil
Siegesbeckiae Herba Perilla Leaf Oil
Siphonostegiae Herba Sandal Wood Oil
Siraitiae Fructus Spine Date Seed Extract
Smilax China L. Star Anise Oil
Sojae Semen Nigrum Total Amino Acids
Sojae Semen Praeparatum Total Ginkgolic Acids
Sophorae Flos Turpentine Oil
Sophorae Flos Immaturus Vitex Oil
Sophorae Tonkinensis Radix et Rhizoma Xylaria Powder
Sparganii Rhizoma Yunnan Baiyao Extract
Spatholobi Caulis
Spirodelae Herba 8062 Reference Standards
Stevlae Rebaudianae Folium
Streptocaulon Griffithii Radix
Aceglutamide
Styrax
Acetaminophen ( Paracetamol)
Swertiae Bimaculatae Herba
Acetanilide
Swertiae Mileensis Herba
Acetazoiamide
Swertiae Mussotii Herba
Acetone
Syringae Cortex
5- (Acetylamino)-N, N'-bis (2, 3-dihydroxypropyl)-2, 4, 6-
Taccae Esquirolii Rhizoma
triiodobenzene-l, 3-dicarboxamide
Tamaricis Cacumen
5-Acetylamino-2, 4, 6-triiodobenzene-l, 3-dicarboxamide
Taraxaci Herba
N-Acetyl-cys1 -calcitonin (Salmon)
Testudinis Carapax et Plastrum
Acetylcysteine
Thlaspi Herba
Acetylspiramycin
Tinosporae Sinensis Caulis
Aciclovir
Toosendan Fructus
Acitretin
Torreyaae Semen Adamantane
Trachelospermi Caulis et Folium Adenosine Cyclophosphate
Tribuli Fructus Adipiodone
Trichosanthis Fructus Adrenaline
Trichosanthis Pericarpium {3-Alanine
Trichosanthis Radix Alarelin Acetate
Trigonellae Semen Albendazol
Tripterygii Hypoglauci Radix Alcohol
Trollii Chinensis Flos Aldehyde
Turpiniae Folium Alfacalcidol
Typhonii Rhizoma Alfuzosin Hydrochloride
Uncariae Ramulus Cum Uncis Alkene
Vaccariae Semen Almitrine Bismesylate
Verbenae Herba Alprostadil
Violae Herba Altretamine
Visci Herba Amidotrizoic Acid (Diatrizoic Acid)
Viticis Negundo Folium Amikacin
Viticis Negundo Fructus Amikacin lmpurity A
Vladimiriae Radix Amiloride Hydrochloride
Wenyujin Rhizoma Concisum 5-Amino-N, N'-bis ( 2, 3-dihydroxypropyl ) -2, 4, 6-
Wikstroemiae lndicae Radix et Rhizoma triiodobenzene-l, 3-dicarboxamide
Xanthii Fructus p-Aminobenzenesulphonic Acid (sulphanilic acid)
Zanthoxyli Pericarpium Aminobenzoic Acid (Procain lmpurity A)
Zanthoxyli Radix ( +) 2-Aminobutanol (Ethambutol lmpurity A)
Zingiberis Rhizoma Aminocaproic Acid
Zingiberis Rhizoma 2-Amino-2'-chloro-5-nitrobenzophenone (Lorazepam lmpurity D
Ziziphi Spinosae Semen 4-Amino-6-chlorobenzene-l, 3-disulphonamide
 8062 Reference Standards

( 2-Amino-5-chlorophenyl) phenylmethanone ( aminochloroben- Betamethasone

wphenone) Betamethasone Dipropionate
7-Aminodesacetoxycephalos Poranic Acid ( 7-ADCA) Betamethasone Sodium Phosphate
7- [ ( 2-Aminoethyl ) amino J -1-ethyl-6-fluoro-4-oxo-1, Bezafibrate
4-dihydroquinoline-3-carboxylic acid. Bifendate
Aminoglutethimide Bifanazole
CRS )-2-Amino-3-hydroxypropanohydrazide Hydrochloride Bifanol
( 2-Amino-5-nitrophenyl) phenylmethanone. (RS)-(biphenyl-4-yl) phenylmethanol CBifanazole lmpurity
Aminomethylbenzoic Acid A)
m-Aminophenol Bilirubin
Amiodarone Hydrochloride (2RS)-2- (Biphenyl-4-yl) propanoic Acid
Amitriptyline Hydrochloride Bisacodyl
Amoxicillin Bisoprolol Fumarate
Amoxicillin and Clavulanate Potassium far System Suitability Bleomycin AS Hydrochloride
Amoxicillin far System Suitability Bornyl Acetate
Amphotericin B Bromhexine Hydrochloride
Ampicillin Buflomedil Hydrochloride
Ampicillin far System Suitability Bulleyaconitine A
Amrinone Bumetanide
Anhydrous Ethanol (EthanoD Bupivacaine Hydrochloride
Anhydrous Glucose Buprenorphine Hydrochloride
Anisic acid Butenafine Hydrochloride
Anisodamine Hydrobromide p-Butylaminobenzoic Acid
Antazoline Hydrochloride [2-tert-Butylamino-1- ( 4-hydroxy-3-methylphenyl) ethanol]
Aprotinin ~-3-cefaclor
Arginine Hydrochloride Butylparaben
Artemether Caffeine
Artemisinin Calcitonin (salman)
Artes una te Calcium Dobsilate
Asparaginase I Calcium Folinate
Asparaginase II Calcium Gluconate
Asparagine Calcium lactate
Aspartic Acid Calcium Pantothenate
Aspirin/ Acetylsalicylic Acid Camphor
Atenoioi Canrenone
Atracurium Besilate Capreomycin
Atropine Sulfate Captopril
Azithromycin Captopril-Disulphide [ ( 25, 2' S) -1, 1' - [ disulphanediylbis
Azithromycin lmpurity A (Azaerthromya' n A) [ (25) -2-methyl-1-oxopropane-3, 1-diyl] -bis [pyrrolidine-
Azithromycin lmpurity S (Erythromya' n A-E-oxime) 2-carboxylic] acid]
Azithromycin far System Suitability Carbachol
Azlocillin Carbamazepine
Azobenzene Carbamazepine System Suitability Mixture
Aztreonam Carbocisteine
Bacitracin Carboplatin
Beclometasone Di pro pionate Carboprost Methylate
Bendazac Lysine Carboprost Methylate 15-Epimer
Bendrofluazide Carmofur
Benorilate Carteolol Hydrochloride
Benproperine Phosphate Carvedilol
Benserazie Hydrochloride Case in
Benzaldehyde Cefaclor
Benzalkonium Bromide Cefadroxil
Benza thine Benzyl penicillin Cefalexin
Benzbromarone Cefalotin
Benzoic Acid Cefamandole Nafate
1-Benzoyl-3-methyl-5-aminopyrazol Cefathiamidine
Benzoyl Peroxide Cefazoline
Benzyl Alcohol Cefazoline lmpurity A
(IS, 2R )-1-Benzyl-3-dimethylamino-2-methyl-1-phenylpropyl Cefazoline lmpurity E
acetate Cefdinir
Benzylpenicillin Cefepime
Benzylpenicillin far System Suitability Cefetamet Pivoxile
Berberine Hydrochloride Cefixime
Betacyclodextin Cefodizime
Betahistine Hydrochloride Cefanicid Sodium
 8062 Reference Standards

Cefoperazone Clavulanic Acid

Cefoperazone B Clemastine Fumarate
Cefoperazone S-isorner Clenbuterol Hydrochloride
Cefotaxirne Clindamycin
Cefotaxirne for Systern Suitability Cliopquinol
Cefoxitin Cliopquinol for System Suitability
Cefpodoxime proxetil Clobetasol Propionate
Cefprozil Clodronate Disodium
Cefradine Clofazimine
Ceftazidime Clofibrate
Ceftezole Clomifene Citrate
Ceftizo xi me Clomipramine Hydrohloride
Ceftriaxone Clonazepam
Ceftriaxone E-isorner Clonidine Hydrochloride
Cefuroxirne Clorprenaline Hydrochloride
Cefuroxirne Axetil Clotrimazole
Cellulose Acetate Cloxacillin
Cephaeline Clozapine
Cephalomannine Cobamamide
Chenodeoxycholic Acid Codeine Phosphate
Chlorarnbucil Codergocrine Mesilate
Chlorarnphenicol Colchicine
Chlorarnphenicol Palmitate (Crystal forrn A) Colistin
Chlorarnphenicol Palmitate (Crystal forrn B) Cortisone
Chlorhexidine Acetate Cortisone Acetate
Chlormadinone Acetate m-Cresol
p-Chloroacetanilide o-Cresol
p-Chloroaniline p-Cresol
p-Chlorophenol
Crospovidone
Chlorobutanol
Crotamitone
[5-Chloro-2- [ [2- (diethylarnino) ethyl] amino] phenyl]
Curcumenol
(2-fluorophenyD rnethanone ( 2Z) -2-Cyano-3-hydroxy-N- [ 4- ( trifluoromethyl) phenyl J
7-Chloro-1-ethyl-6--fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic
but-2-enamide
Acid
Cyclandelate
7-Chloro-5- (2-fluorophenyl) -1, 3-dihydro-2H-1, 4-bemodiaz-
Cyclohexane
epin-2-one
Cyclohexylamine
Chloroform
Cyclopenthiazide
2-Chloro-N, N-diethylethanamine
1-Cyclopropyl-7-chloro-6- [ (2-aminoethyl) amino] -4-oxo-1, 4-
4-Chloro-N- [ 2- ( 4-hydroxyphenyl ) ethyl ] benzamide
dihydro-3-quinoline-carboxylic acid (Ciprofloxacin lrnpurity D
( chlorobenzoyltyramine)
1-Cyclopropyl-7-chloro-4-oxo-6-- ( piperazin-1-yl) -1, 4-dihydroq-
5-Chloro-l -methyl-4-nitroimidazole
uinoline-3-carboxylic acid ( Ciprofloxacin lmpurityD)
4- [ 2- ( 5-Chloro-2-methoxy-benz.arnido) ethyl] benzenesulf-
1-Cyclopropyl-6-fluoro-7- [ (2-aminoethyl) amino] -4-oxo-
onamide ( Glibenclamide Impurity D
2-Chloro-4-Ni troaniline ( Niclosamide lmpuri ty A) 1, 4-dihydroquinoline-3-carboxylic acid ( Ciprofloxacin
3-Chloropropane-1 , 2-diol lmpurityC)
Chloroquine Phosphate 1-Cyclopropyl-6-fluoro-7-chloro-4-oxo-1, 4-dihydroquinoline-
Chlorothiazide 3-carboxylic acid (Ciprofloxacin lmpurity A)
Chlorphenamine Maleate 1-Cyclopropyl-6--fluoro-7- (piperazin-1-yl) quinolin-4 OH) -one
Chlorprothixene 1-Cyclopropyl-4-oxo-7- (piperazin-1-yl) -1, 4-dihydroquinoline-3-
Chlortalidone carboxylic Acid (Ciprofloxacin lrnpurity B)
Chlortetracycline Hydrochloride Cyclosporin
Cholesterol Cyclosporin for System Suitability
Cholic Acid Cydlophosphamide
Choline Chloride Cysteine Hydrochloride
Chondroitin Sulfate Cystine
Chorionic Gonadotrophin Cytarabine Hydrochloride
Ciclopirox Olamine 5 '-Cytidylic Acid
Cilostazol Danazol
Cimetidine Dapsone
Cinchonidine Daunorubicin
Ciprofibrate 10-Deacetyl-7-epipaclitaxel
Ciprofloxacin Decloxizine
Cisatracurium Besilate Dehydroandrograpolide Succinate
Cisplatin Dermatan Sulfate
Citicoline Sodium Desfluoroparoxetine [ (35, 4R) -3- [ (1, 3-benzodioxol-5-
Clarithromycin yloxy) methyl] -4-phenylpiperidine ]
 8062 Reference Standards

Deslanoside Doxycycline
Deslanoside e Droperidol
[Des-thy-ol8 J -Octreotide Econazole Nitrate
Desmopressin impurity 4-Ehtylcatechol
Desoxycortone Acetate Eicosapentaenoic Acid Ethyl Ester
Dexamethasone Emetine Hydrochloride
Dexamethasone Acetate Enalaprilat
Dexamethasone Phosphate Enalapril Diketopi perazine
Dexamethasone Sodium Phosphate Enalapril Maleate
Dextromethorphane Hydrobromide Enflurane
Dextropropoxyphene Napsylate Enoxacin
Diaveridine Ephedrine Hydrochlorjide
Diazepam Epirubicin Hydrochloride
Diclofenac Sodium 7-Epipacli taxel
Dicyanodiamide (-) 4, 5 a-Epoxy-3, 14-dihydroxymorphinan-6-one (noroxymo~
Dicyclohexyl Phthalate phone)
Diethanolamine Ergocalciferol
Diethylene Glycol Ergometrine Maleate
Diethyl Phthalate Erythromycin
Diethylstilbestrol Erythromycin Ethylsuccinate
Difenidol Hydrocloride Estazolam
( ± ) -9, 10-Difluoro-3-methyl-7-oxo-2, 3-dihydro-7H- Estradiol
pyrido [ 1, 2 , 3-de] -1 , 4-benzoxazine-6-carboxylic acid Estradiol Benzoate
Digitoxin Estradiol Valerate
Digoxin Estrone
Dihydroartemisinin Etacrynic Acid
Dihydroetorphine Hydrochloride Etamsylate
Dihydrophenlglycine Ethacridine Lactate
( ± ) 3, 4-Dihydroxy-2'-methylamino acetophenone-3, Ethambutol Hydrochloride
4-dineopentanoic perchlorate ( Dipivefrin hydrochloride Ethanolamine
impirity I) Ethinylestradiol
ORS) -1- (3, 5-Dihydroxyphenyl) -2- [ (1-methylethyl) Ethyl [ [ 4- [ 2- [ ( 5-chloro-2-methoxybenzoyl) amino J
amino] ethanol ethyl] phenyl ] sulphonyl ] carbamate ( Glibenclamide
Diltiazem Hydrochloride lmpurityB)
Dimegiumine Gadopentetate Ethyi Acryiate
Dimenhydrinate Ethyl Benzoate
N, N-Dimethylaniline Ethylene Glycol
2, 3-Dimercaptosuccinic Acid Ethyl Hydroxybenzoate
1- ( 3, 4-Dimethoxybenzyl ) -2- ( 3, 11-dioxo-4, 10- Ethylparaben
dioxatridec-12-enyl) -6, 7-dimethoxy-2-methyl-1, 2, 3, Etidronate Disodium
4-tetrahydroiso-quinolinium Etimicin
4-Dimethylamino-3-methyl-1, 2-diphenylbutan-2-ol Hydrochloride Etofylline
2, 3-Dimethylaniline Etomidate
2, 6-Dimethylaniline Famciclovir
2, 5-Dimethylphenol Famotidine
2, 6-Dimethylphenol Felodipine
Dimethyl Phthalate Felodipine lmpurity I [Ethyl methyl 4- (2, 3-dichlorophenyl) -
Dimethyl Sulfone 2, 6-dimethylpyridine-3, 5-dicarboxylate]
Dimethyl Sulfoxide Fenfluramine Hydrochlordie
Dioxane Fenofibrate
Dipenoxylate Hydrochloride Fenofibrate lmpurity I [ ( 4-Chlorophenyl) ( 4-hydroxyphenyl)
Diphenhydramine Hydrochloride methanone]
Diphenyl- ( 2-chlorophenyl ) -methanol ( Clotrirmazole Fenofibrate lmpurity II [2- [4- (4-Chlorobenzoyl) phenoxy] -2-
lmpurity I) methylpropanoic Acid]
Dipivefrin Hydrochloride Fentanyl Citrate
Diprophylline F erulic Acid
Dipropylene Glycol Flavoxate Hydrochloride
Dipyridamole Fleroxacin
Dithranol Fluconazole
Dobutamine Hydrochloride Flucytosine
Docosahexaenoic Acid Ethyl Ester Fludrocortisone Acetate
Domiphen Bromide Flumazenil
Dopamine Hydrochloride Flunarizine Hydrochloride
Doxapram Hydrochloride Fluocinonide
Doxepin Hydrochloride Fluorane
Doxorubicin Hydrochloride Fluorescein Sodium
 8062 Reference Standards

( RS) -9, 10-Difmoro-3-methyl-7-oxo-2, 3-dihydro-7H- Hydrochlorothiazide

pyrido [1, 2, 3-de J -1, 4-benzoxazine-6-carboxylic Acid Hydrocortisone
( Ofloxacin lmpurity A) Hydrocortisone Acetate
( RS ) -9-Fluoro-3-methyl-7-oxo-10- ( piperazin-1-yl ) -2, Hydrocortisone Butyrate
3-dihydro-7 H -pyrido [ 1 , 2, 3-de J -1 , 4-benzoxazine-6- Hydrocortisone Sodium Succinate
carboxylic Acid ( Ofloxacin lmpurity E) Hydropuinone
Fluoroquinolonic Acid ( 7-Chloro-1-cyclopropyl-6-fluoro-4- 4-Hydroxybenzoic Acid
oxo-1, 4-dihydroquinoline-3-carboxylic Acid) 4-Hydroxyisophthalic Acid
Fluorouracil 3-Hydroxy-l-benzylindazol ( 1-benzyl-1 H-indazol-3-oD
Fluphenazine Decanoate 2-Hydroxy-l, 2-diphenylethanone
Fluphenazine Hydrochloride 1- ( Hydroxy-3-methylbenzyl ) -2- ( tertiary butylamino )
Flurazepam Hydrochloride ethanol
Flurbiprofen 5-Hydroxymethyl-2-Furaldehyde
Folie Acid 4- (4-Hydroxyphenyl) butan-2-one
Fosfomycin 2- (4-hydroxyphenyD acetamide (Atenolol lmpurity A)
Fructose Hydroxyprogesterone Caproate
Ftibamzone 9- Hydroxypropantheline Bromide
Fumaric Acid 6p-Hydroxy-tropan-3a-ol-atropate
Furazolidone Hydroxyzine Hydrochloride
Furosemide Hymecromone
Galactose Hyodeoxycholic Acid
Galanthamine Hydrobromide lbuprofen
Ganciclovir Ifosfamide
Gemfibrozil Ifosfamide lmpurity I [ 3- ( 2-Cltloroethyl) - [ ( 2-chloropropyl)
Gentamicin amino] tetrahydro-2H-1, 3, 2-oxazaphosphorine 2-oxide]
Gentamicin C1. Hosfamide lmpurity Il [ 3- ( 2-ChloroacetyD - [ ( 2-chloroethyl)
Germacrone amino] tetrahydro-2H-1, 3, 2-oxazaphosphorine 2-oxide]
Glibenclamide Imidazole
Gliclazide Imipramine Hydrochloride
Giipizide indapamide
Gliquidone lndometacin
Gliquidone Sulphonamide lnosine
Glucide lnsulin
Glucose lnsulin Monomer-Dimer
Glutamic Acid lnsulin monomer-dimer
Glutamine lohexol
Glycerol Iotalamic Acid
Glyceryl Behenate lpriflavone
Glycine Isocarboxazid
Glycollic Acid L-lsolecine
Glycopyrrone Bromide Cis-Isomer of Tramadol Hydrochloride
Glycyrrhizinate Ammonium Isoniazid
Glycyrrhizinate Diammonium Isoprenaline Hydrochloride
Glycyrrhizinate Dipotassium Isopropyl 4-aminoboenzoate
Granisetron Hydrochloride Isopropyl Alcohol ( IsopropanoD
Griseofulvin Isosorbide
Guanine Isosorbide 2-Nitrate
Halcinonide Isosorbide Dinitrate
Haloperidol Isosorbide Mononitrate
Heparin Sodium Isotretinoin
a-Hexachlorocyclohexane ltraconazole
1, 1, 1, 3, 3, 3-Hexafluoro-2-propanol J atrorrhizine Hydrochloride
1- ( Hexahydrocyclopenta [ e J pyrrol-2 ( lH ) -yl ) -3- Josamycin
[ ( 2-methylphenyl) sulphonyl] urea Kanamycin
Hexanolactam Kanamycin B
Histamine Diphosphate Ketamine Hydrohloride
Histidine Hydrochloride Ketoconazole
Homatropine Hydrobromide Ketoprofen
Homoharringtonine Ketotifen Fumarate
Homopiperonylonitrile Kitasamycin
Human Growth Hormone Monomer-Dimer Laetose
Human lnsulin Monomer-Dimer Lactulose
Huperzine A Latamoxef
Hyaluronidase Laurocapram
Hydralazine Hydrochloride Leflunomide
Hydrazine Sulfate Leucine
 8062 Reference Standards

Levamisole Hydrochloride Methyl Docosanoate (Methyl Behenate)

Levodopa Methyldopa
Levofloxacin Methyl Eicosenoate
Levonorges trel Methylene Dichloride
Lidocaine Methyl Erueate
Ligustrazine Phosphate Methyl Lignocerate
Linalool (3, 7-Dimethylocta-l, 6-dien-3-oD Methyl Linoleate
Lincomycin Methyl Linolenate
Lindane Methyl Methacrylate
Lisinopril Methyl Myristate (Methyl Tetradecanoate)
Lithium Carbonate 4-Methylamino Phenazone ( 4-Methylamino-l, 5-dimethyl-2-
Lithocholic Acid phenyl-1, 2-dihydro-3H-pyrazol-3-one)
Lofexidine Hydrochloride 3-Methylflavone-8-carboxylic acid
Lomefloxacin 2-Methyl-5-nitroimidazole (Metronidazole lmpurity I)
Lomustine 1-Methylpiperazini (Diethylcarbamazine lmpurity A)
Loperamide Hydrochloride 2-Methylpyridine (Sucralfate lmpurity A)
Lorazepam 3-Methyl-N- [ 4- ( trifluoromethyl) phenyl J isoxazole-4-
Lovastatin carboxamide
Lycoramine Hydrobromide Methyl Oleate
Lysine Acetate Methyl Palmitate
Lysine Hydrochloride Methyl Palmitoleate
Lysolecithin Methylparaben
Malaridine Phosphate (Pyronaridine phosphate) 4- [2- (5-Methylpyrazin-2-carboxamido) ethyl] benzenesul-
Malic Acid phonamide
DL-Malic Acid N-Methylparoxetine
L-Malic Acid Methyl Salicylate
Mal tose Methyl Stearate
Maltotriose Methyltestosterone
Mannitol Metildigoxin
Maprotiline Hydrochloride Metoclopramide
Mebendazole Metoprolol Tartrate
Mebendazole Contain 10% of Polymorph A Metronidazole
Meclofenoxate Hydrochloride Mexiletine Hydrochloride
Meclozine Hydrochloride Mexiletine lmpurity A
Medroxyprogesterone Acetaie Mezlocillin
Mefenamic Acid Mezlocillin Sidium
Megestrol Acetate MiconazoleNitrate
Megnesium Valproate Micronomicin
Meleumycin Midazolam
Meloxicam Midazolam Maleate
Melphalan ( Sarcolysin) Minocycline
Menadiol Acetate p-Minophenol
Menadione Sodium Bisulfate Minoxidil
Menotropin Mitomycin
Menthol Mitoxantrone Hydrochloride
Menthyl Acetate Moclobemide
Mercaptopurine Morphine
Meropenem Morphine Sulfate
Mestanolone Naftifine Hydrochloride
Metacycline Naloxone Hydrochloride
Metamizole Sodium Nandrolone Phenylpropionate
Methacrylic Acid Copolymer I Naphazoline Hydrochloride
Methacrylic Acid Copolymer 11 Naproxen
Methactylic Acid Naproxen Sodium
Methadone Hydrochloride Nefopam Hydrochloride
Methionic Recombinant Human Growth Hormone Neoamine
Methionine Neomycin
Methotrexate Neostigmine Mesylate
6-Methoxy-2-acetonaphthone (Naproxen lmpurity l ) Netilmicin
10-Methoxy-4- (1-methylpiperidin-4-yl) -4H-benzo [ 4, 5] Nicardipine Hydrochloride
cyclohepta [l, 2-b J thiophen-4-ol Nicardipine Hydrochloride lmpurity I [2- (Benzylmethylamino)
Methyl 3, 5-Diamino-6-chloropyrazine-2-carboxylate ethyl methyl-2, 6-dimethyl-4- ( m-nitrophenyl ) -3, 5-
Methyl 12-Hydroxystearate pyridinedicarboxylate]
Methyl 5-Methyl-3-isoxazole-carboxylate Nicotinamide
Methyl 12-0xostearate Nifedipine
Methyl Arachidate ( Methyl Eicosanoate) Nifedipine lmpurity A
 8062 Reference Standards

Nifedipine Impurity B Phenformin Hydrochloride

Nifedipine Impurity I ( Dimethyl 2, 6-dimethyl-4- ( 2- Phenobarbital
nitrophenyD pyridine-3, 5-dicarboxylate) Phenol
Nifedipine Impurity Il ( Dimethyl 2, 6-dimethyl-4- Phenolphthalein
( 2-nitrosophenyl) pyridine-3, 5-dicarboxylate) Phenoxybenzamine Hydrochloride
Nilestriol Phenoxy Methylpenicillin
Nimesulide Phenprobamate
Nimodipine Phentolamine Mesylate
Nimodipine Impurity I ( 2-methoxyethyl 1-methylethyl 2, 6- Phenylalanine
dimethyl-4- ( 3-nitrophenyl) pyridine-3, 5-dicarboxylate) N-Phenyl-1- (2-phenylethyl) piperidin-4-amine
Nisodipine Impurity I [ ( RS ) - Isobutyl methyl 2, 6- Phenylephrine Hydrochloride
dimethyl-4- ( 2-nitrophenyl) pyridine-3, 5-dicarboxylate] a-Phenylglycine
Nisodipine Impurity 11 [ CRS) -lsobutyl methyl 2, 6-dimethyl-4- Phenytoin
(2-nitrosophenyl) pyridine-3, 6-dicarboxylate] Phenytoin Sodium
Nisoldipine Phospha tidylcholine
Nitrazepam Phosphatidylethanolamine
Nitrendipine Phosphatidyl Inositol
Nitrendipine Impurity I [ ethyl methyl 2, 6-dimethyl-4- Phosphoric Acid
( 3-nitrophenyl) pyridine-3, 5-dicarboxylate] Phosphorus acid
Nitrilotriacitic Acid Phthalazine
5-Nitro-N, N'-bis (2, 3-dihydroxypropyl) -1, 3-dicarboxamide Phthalic acid
a-p-hydroxyphenylglycine Pilocarpine Nitrate
( 5-Nitro-2-furyl) methylene diacetate (Nitrofurfural diacetate) Pipemidic Acid
Nitrofural Piperacillin
Nitrofurantoin Piperaxine
Nitroglycerin Piperaxine Feralate
l-p-Nitropheny-2-amino-1, 3-propanediol Piracetam
Nonoxinol Piroxicam
Norepinephrine Bitartrate Pituitrin
Norethisterone Pizotifen
Norethisterone Enanthate Poloxamer
Norfloxacin Polymixin B
Norgestrel Polyxoyl ( 40) Stearate
Norvancomycin Potassium Chloride
Noscapine Potassium Dihydrogen Phosphate
Octreotide Acetate Potassium Iodide
Ofloxacin Praziquantel
Omeprazole Prazosin Hydrochloride
Omeprazole Sulphone Prednisolone
Ondansetron Hydrochloride Prednisolone Acetate
Oversulfated Chondroitin Sulfate Prednisone
Oxacillin Prednisone Acetate
Oxaprozin Primidone
Oxazepam Probenecid
Oxybutynin Hydrochloride Procaine Hydrochloride
Oxytetracycline Procaterol Hydrochloride
Oxytocin Progesterone
Paclitaxel Proglumide
Palmatine Proline
Pamidronate Disodium Promethazine Hydrochloride
Pamoic Acid Propafenone Hydrohloride
Pancreatic Kininogenase Propantheline Bromide
Pantoprazole Sodium Propranolol Hydrochloride
Papaverine Hydrochloride Propylene Oxide
Paracetamol ( Acetaminophen) Propylparaben (Propyl Hydroxybenzoate)
Paromomycin Propylthiouracil
1, 2-Propanediol Prostaglandin Ai (7-[ClR, 25)-2-[ClE, 3S)-3-hydroxyoct-
1, 3-Propanediol 1-enyl]-5-oxocyclopent-3-enyl]heptanoic acid)
Paroxetine Hydrochloride Prostaglandin Bi ( 7-[ 2-[ ( lE, 3S )-3-hydroxyoct-1-enyl]-5-
Pefloxacin oxocyclopent-1-enyl]heptanoic acid)
Penfluridol Protionamide
Penicillamine Pseudoephedrine Hydrochloride
Pentoxifylline Puerarin
Pentoxyverine Citrate Pyrantel Pamoate
Perphenazine Pyrazinamide
Pethidine Hydrochloride N-( Pyridin-4-ylmethyD ethanamine
 8062 Reference Standards

Pyridostigmine Bromide Sulbactam

Pyridoxine Hydrochloride (Vitamin Es) Sul benicillin
Pyruvic Acid (2-0xopropanoic Acid) Sulfadiazine
Quinapril Hydrochloride Sulfadimidine
Quinapril Hydrochloride lmpurity l [ Ethyl C35- [ 2 Sulfamethoxazole
(R * ) , 3a, llaB] J -1, 3, 4, 6, 11, lla-hexahydro-3- Sulfanilamide
methyl-l, 4-dioxo-a- ( 2-phenylethyl) -2H-pyrazino [ 1, Sulfapyridine
2-b] isoquinoline-2-acetate] Sulindac
Quinestrol Sulpirid
Quinidine Sultamicillin
Raceanisodamine Suxamethonium Chloride
Racerepaglinide Tamoxifen Citrate E-lsomer
Ranitidine Hydrochloride Taurine
Raubasine Tazarotene
Recombinant Human Growth Hormone Tazobactam
Recombinant Human lnsulin Tegafur
Repaglinide Teicoplanin
Reserpine Terbutaline Sulfate
Resorcinol T erfenadine
Ribavirin Testostdrone Undecanoate
Riboflavin (Vitamin Bt) Testosterone Propionate
Ribostamycin Tetracaine Hydrochloride
Rifampicin Quinone Tetracycline Hydrochloride
Rifampin Thalidomide
Rotundine Hydrochloride Thebaine
Roxithromycin Theobromine
Rutundine Theophylline
Sacharin lmprurity A Thiamazole
Salbutamol Thiamphenicol
Salbutamol Sulfate 2-Thioletomidate
Salicylic Aicd Thiopental
Salicylic Aicd Tablets Thioridazine Hydrochloride
Scopolamine Butylbromide Threomine
Scopolamine Hydrobromide Thrombin
Serine Thymalfasin
Sevoflurane Thymol
Simvastatin Ticlopidine Hydrochloride
Sisomicin Tinidazole
Sodium Acetate Tioguanine
Sodium Aminosalicylate Tobramycin
Sodium Ascorbate Tolazoline Hydrochloride
Sodium Cromoglicate o-Toluenesulphonamide
Sodium Cyclamate p-Toluenesulphonamide (Saccharin lmpurity B)
Sodium Fluorate Tramadol Hydrochloride
Sodium Glycididazole T ranexamic Acid
Sodium Houttuyfonate Cis-Tranexamic Acid
Sodium lodide Tranilast
Sodium Nitroprusside T rans-paroxetine
Sodium Prasterone Sulfate Tretinoin
Sodium Selenite (Seleniuro Sulfide lmpurity) 4-Trifluoromethyl Aniline
Sodium Stibogluconate T riamcinolone
Sodium Valproate Triamcinolone Acetonide
Somatostatin Triamcinolone Acetonide Acetate
1, 4-Sorbitan Triamterene
Sorbitan Trioleate Triazolam
Sorbitol T richloroethylene
Sotalol Hydrochloride Triethanolamine
Sparfloxacin Triglyceride
Spectinomycin Trihexyphenidyl Hydrochloride
Sphingomyelin Trihydroxybenzyl Benserazide Hydrochloride [ ( RS ) -2-
Spironolactone amino-3-hydroxy-2', 2'-bis ( 2, 3, 4-trihydroxybenzyl)
Stanozolol propanohydrazide]
Steviosin T rimethoprim
Streptomycin Tripropylene Glycol
Strophanthin G T ropicamide
Sucrose Trypsin
 9001 Guidelines for the Stability Testing of Drug Substances and Preparations
Tryptophan
Tyrosine
Ubenimex
Ubidecarenone
Ulinastatin
Undecylenic Acid
Urea
Urokinase
U rsodeoxycholic Acid
Valaciclovir Hydrochloride
Valsartan
Valsartan Enantiomer
Vancomycin
Vancomycin Hydrochloride
Vanillin
Verapamil Hydrochloride
Vinblastine Sulfate
9000
9001 Guidelines for the Stability
Testing of Drug Substances
and Preparations
The purpose of stability testing is to investigate quality
variations of drug substances or drug preparations with time
under the influence of a variety of environmental factors,
including temperature, humidity and light, which substantia-
tes the recommended conditions for manufacture, package,
storage, transportation and shipment and helps to establish
shelf lives of drugs.
The basic requirements for the stability testing include:
( 1) Affecting factor testing, accelerated testing and long-
term testing. One batch of drug substances or preparations is
required for affecting factor testing. Three batches are
required for accelerated and long-term testing. ( 2) Drug
substances for stability testing should be from pilot scale,
and the amount required for testing should be equivalent to
that for stability testing of their preparations. The
manufacturing process of drug substances should be identical
to that for full scale production. Drug preparations used for
stability testing should be from scale-up pilot production with
the same formulation and manufacturing process as those of
full scale production. Generally, the amount for scale-up
pilot production of tablets or capsules should be 10 000 at
least. For large-volume preparations, such as intravenous
injection, at least 10 times of the total amount for each
testing are needed. Far sorne special preparations or dosage
forms, the amounts for testing depend on their conditions.
( 3) The quality specifications of the drug used for stability
testing should be identical to that for production batch, pre-
clinical and clinical studies. ( 4 ) The packaging of drug
substances for accelerated and long-term testing should be the
same as those for marketing. ( 5) The methods for stability
testing of drugs and related substances ( including
degradation products and the products introduced by other
changes) should be accurate, precise, specific and sensitive,
and should be validated to ensure the reliability of the
results. It is note worthy to pay attention to the
determination of degradation products during stability
Vincrstine Sulfate
Vindesine Sulfate
Vinorelbine Tartrate
1-Vinylpyrrolidin-2-one ( 1-Ethenylpyrrolidin-2-one)
Vitamin A
Vitamin C
Vitamin D3 (CholecalciferoD
Vitamin E (Retinol Acetate)
Vitamin K 1 (Phytomenadione)
Warfarin Sodium
Xanthanoic Acid
Xanthinol Nicotinate
Xanthone
Xylometazoline Hydrochloride
Zinc Gluconate
Zine Citrate
testing. (6) Because the quantity of scale-up batch is less
than that of the production batch, the applicant shall promise
that the accelerated and long-term testing are also carried out
for the first three production batches that pass the production
validation after approval.
This guideline includes t\A/O sections, the first section is fer
drug substance and the second one is for drug preparation.
1 Drug Substance
The following tests should be employed for drug substance.
l. Affecting factor testing
This testing is normally carried out under more severe
conditions than those for accelerated testing. This testing
aims to investigate the intrinsic stability of the drug
substance in order to identify the factors affecting stability
and the possible degradation pathways and degradation
products, so as to provide scientific evidences for
manufacturing, package and storage of the drug
preparations, as well as the developtnent of analytical
method for degradation products. This testing can be
carried out on samples of a single batch. The substance
being examined is usually distributed evenly in a suitable
opened container (e. g. , weighing bottle or petri dish) to
form a thin layer with a thickness of not more than 5 mm
or not more than 1 O mm for the loase substance. If
significant changes occur in degradation products during
testing, qualitative and quantitative analyses should be
conducted due to the potential safety problem when
necessary.
( 1) High temperature testing
Open the loaded container and place in a suitable and clean
facility at 60ºC for 10 days. Take samples on the 5 th and the
10 th days and examine the specified items of stability
testing. If the substance changes beyond the specified limits,
an additional testing at 40ºC should be conducted, otherwise
the testing at 40ºC is not necessary.
(2) High humidity testing
Open the loaded container and place in a closed facility with
constant humidity at 25ºC/90% ± 5% RH for 10 days. Take
samples on the 5 th and the 10 th days and exarrúne the specified
items of humidity testing. The weights of the substance before
and after testing should be weighed accurately to evaluate the
 9001 Guidelines for the Stability Testing of Drug Substances and Preparations

hygroscopicity of the substance. If the difference is not less than
5% of its weight, an additional testing at 25ºC/75% ± 5%
RH should be conducted. If the difference is less than 5 % of
its weight, and the other items are qualified, the testing at
25ºC/75% ± 5% RH is not necessary. Constant humidity
can be obtained by placing a saturated salt solution in the
lower part of the closed container, such as a desiccator.
According to the requirements for relative humidity, saturated
solutions of NaCl (75% ± 1% RH at 15. 5ºC to 60ºC) and
KN03 (92. 5% RH at 25ºC) may be used.
(3) Photostability testing by strong light
Open the loaded container and place in an illumination cabinet
with daylight lamps or other suitable lighting devices. The
substance should be exposed to the light with an intensity of
4500 ±500 lux for 10 days and sampled on the 5 th and the
10 th days to examine the specified items of photostability
testing. Any changes in appearance of the substance should
be noticed.
As to the light emitting device, tunable light chamber is
recommended. An illumination cabinet may also be used, in
which several daylight lamps are placed to achieve the defined
illumination intensity. The height of the sample platform
may be adjusted. An aspirator is installed at the upper part
of the cabinet to eliminate the heat possibly produced. An
illuminometer is fixed on the cabinet to monitor the
illumination. The illumination cabinet should not be
interfered by natural light and constant illumination intensity
should be maintained in a dust-free environment.
the actual storage conditions of the drug. This testing aims
to provide data for the shelf life of the drug. In this testing,
three batches with market packages should be placed for
12 months at 25±2ºC/60% ± 10% RH or 30±2ºC/65% ±
5 % RH, which is determined based on clima te variations
throughout the country. Researchers can choose either of the
above conditions according to the actual conditions. Sampling
is carried out to test the specified items of stability every
3 months, e. g. , at the O, 3rd, 6th, 9th and 12th months.
Examinations of the drug at the 18th, 24th and 36th months
are still required. Shelf life of the drug is then determined by
comparing the results at different periods with that at
O month. Reasonable shelf life of the drug can be obtained by
statistical analysis with 95 % confidence limit when taking the
discreteness of data into account. If the variation of three
batches is small, the average value is chosen as the shelf life.
If the variation is significant, the shortest duration is chosen
as the shelf life. Statistical analysis can be omitted for stable
drug as indicated by small variations.
For extremely temperature-sensitive drugs, long-term testing
may be carried out at 6±2ºC for 12 months according to the
above-mentioned requirements. The examination should also
be continued 12 months later in order to determine the shelf
life at low temperatures.
The conditions for long-term stability testing, e. g. , 25 ±
2ºC/60%± 10% RH and 30 ± 2ºC/65% ± 5% RH, are
determined based on the intemational climatic zone (see table).
International climatic zone . ..

Moreover, the proper experiments may be designed when Calculated data Predicted data
necessary to investigate the influence of pH, oxygen and Climatic zone
other related factors on stability of the drug. In addition, Temp. 1
MKT2 Temp. RH
RH/%
analytical methods should also be developed to study the /ºC /°C /°C !% ..

decomposition products of the drug. For innovative or new I Temperate 20.0 20.0 42
drugs, it is necessary to study the properties of their 21 45
zone
decomposition products.
11 Medi terranean
2. Accelerated testing and subtropical 21. 6 22.0 52 25 60
This testing is carried out under accelerated conditions. To provide zone
necessary data for the design, packaging, transportation and fil Arid
storage of drug, stability is investigated by accelerating the 26.4 27. 9 35 30 35
tropic zone
chemical or physical changes of the drug. In this testing, three
batches with market packages should be placed at 40±2"C/75%± N Wet 26. 7 27.4 76 30 76
5 % RH for 6 months. The equipment must be capable of tropical zone
monitoring the actual temperature and humidity and keeping the l. Measured temperature;
variation of temperature within a range of ± 2ºC and that of 2. MKT= mean kinetic temperature.
relative humidity within ± 5 % RH. The drug substance should The United Kingdom, North Europe, Canada and Russia
be examined for the specific items at the end of the lst, 2nd, 3rd belongto the temperate zone. The United States of America,
and 6 th months during the testing periods. If the examined Japan and West Europe ( Portugal-Greece) belong to the
substance <loes not conform to the quality standard in a period of subtropical zone. lran, Iraq and Sudan belong to the arid
6 months under such a condition, an additional testing at an tropic zone. Brazil, Ghana, Indonesia, Nicaragua, and
intermediate condition, such as 30 ± 2ºC / 65 % ± 5 % RH Philippine belong to the wet tropical zone. The long-term
( saturated solution of NaCr04 may be used, the relative testing condition, e. g. , 25 ± 2ºC /60 % ± 10 % RH or 30 ±
humidity is 64. 8%-61. 5% at 30ºC), should be conducted for 6 2ºC / 65 % ± 5 % RH, is adopted here beca use most regions of
months. The water-separating electric heating constant China belong to the subtropical zone while others belong to
temperature culture box ( 20-60ºC) is recommended to be used the wet tropical zone. These conditions are consistent with
for the accelerated testing. A desiccator containing a saturated those adopted by ICH.
salt solution with a definite relative humidity could be laid at the The drug substance for accelerated testing and long-term
lower part of the box. Temperature should be even and testing should be packaged with simulated small barreis,
controllable as required, and suitable for long-term use. A however, the materials and packaging conditions should be
constant humility and temperature box or other suitable device consistent with those of the big barreis.
can also be used.
For extremely temperature-sensitive substances that can only be
n Drug Preparation
The stability testing for drug preparation should be based on
stored in the refrigerator ( 4-8ºC) , the accelerated testing may be
the knowledge of stability of the drug substance, especially
carried out at 25± 2ºC / 60 % ±5 % RH for 6 months.
regarding to the influence of temperature, humidity and light
3. Long-term testing on the drug substance stability. For formula optimization and
Long-term testing is carried out under the conditions close to process design, methods used for drug substances should be
 9001 Guidelines for the Stability Testing of Drug Substances and Preparations
taken as reference for affecting factor testing, accelerated
stability testing and long-term stability testing based on the
properties of drugs and excipients.
l. Affecting factor testing
This testing aims to investigate the rationality of the
formulation, manufacturing processes and packing
conditions. This testing may be carried out on a single batch.
Remove the externa! packing of tablets, capsules and
injections ( for sterilized powders for injection in vials, do
not open the cap to keep the package sealed) , place in a
suitable container for high temperature testing, high
humidity testing and photostability testing by strong light.
Testing conditions, methods and sampling time points are
identical to those for drug substances. Majar examining
items are shown in the following table.
2. Accelerated testing
This testing is carried out under accelerated conditions to
study the stability of drug preparation through accelerating
the chemical or physical changes, which provides necessary
data for the formula design, improvement, process quality
study, package improvement, transportation and storage. In
this testing, three batches with market packages should be
placed at 40 ± 2ºC/75% ± 5% RH for 6 months. The
equipment must be capable of monitoring the actual
temperature and humidity and keeping vanat1on of
temperature within a range of ± 2ºC and that of relative
humidity within ± 5 % RH. The drug preparation should be
examined for the specific items at the end of the lst, 2nd,
3rd and 6 th month during the testing periods. If the
examined preparation does not conform to the quality
standard in the period of 6 months under such a condition,
additional testing at an intermediate condition, such as 30±
2ºC/65% ± 5% RH, should be conducted for 6 months.
Relative humidity is not required for solutions, suspensions,
creams and injections. The equipments used are the same as
those used for drug substance.
For extremely temperature-sensitive drug preparations that
can only be stored in the refrigerator ( 4-8ºC), the
accelerated testing may be carried out at 25±2ºC/60%±5%
RH for 6 months.
For emulsions, suspensions, creams, ointments, pastes, gels, eye
ointments, suppositories, aerosols, effervescent tablets and
effervescent granules, the accelerated testing could be directly
carried out at 30± 2ºC / 65 % ± 5% RH, while other requirements
are the same as those described above.
For drug preparations in semi-permeable containers such as
infusion bags, plastic ampoules and eye preparation
containers made of low density polyethylene, the accelerated
testing should be carried out at 40 ± 2ºC /25 % ± 5 % RH
(saturated solution of CH3 COOK•l. 5H20 may be used).
3. Long-tenn testing
Long-term testing is carried out under the conditions clase to
the actual storage conditions of the drug preparations. This
testing aims to provide data for the determination of shelf life
of the drug preparation. In this testing, three batches with
market packages should be placed at 25±2ºC/60%±10%
RH or 30 ± 2ºC/65% ± 5% RH for 12 months, which is
determined based on climate variations throughout the
country. Researchers can choose either of the above
conditions according to the actual conditions. Sampling is
carried out every 3 months, e. g. the O, 3rd, 6th, 9th and
12th month, to test the specified items. Examinations are
still required at the 18th, 24th and 36th month. Shelf life of
the preparation is then determined by comparing the results
at different periods with that at O month. Reasonable shelf
life can be obtained by statistical analysis with 95 %
confidence limit when taking the discreteness of data into
account. If the variation of three batches is small, the
average value is chosen as the shelf life. If the variation is
significant, the shortest duration is chosen as the shelf life.
Statistical analysis can be omitted for stable drug
preparationas indicated by small variations.
For extremely temperature-sensitive drug preparations, long-
term testing may be carried out at 6 ± 2ºC for 12 months
according to the above-mentioned requirements. The
examination should also be continued 12 months later to
determine the shelf life at low temperatures.
For drug preparations in semi-permeable containers, long-
term testing should be carried out at 25±2ºC/60%±10%
RH or 30 ± 2ºC/65% ± 5% RH. Researchers can choose
either of the conditions.
In addition, for sorne drug preparations, stability should be
investigated before or during the process of use.
4. lmportant items of drug preparation stability testing
The major items are shown in the following table. For
preparation types and items unlisted in this table, items for
stability testing can be determined according to their dosage
forms and characteristics.
Table The important items of stability testing for new
drug substances and preparations
Type of
lmportant items for drug stability
preparation
Appearance, mehing point, content, related
Drug
substances, hygroscopicity and other items
substance
according to drug property
Appearance, content, related substances,
Tablets
disintegration time or dissolution or drug release
Appearance, content, related substances,
disintegration time or dissolution or drug
Capsules
release, water content, precipitate for soft
capsules
Appearance, content, pH, visible particles,
Injections insoluble particles, related substances,
sterility should be inspected
Appearance, content, softening time and
Suppositories related substances
Appearance, homogeneity, content, particle
Ointments
size and related substances
Appearance, homogeneity, content, particle
Creams
size, related substances and demixing
Appearance, homogeneity, content, particle
Pastes
size and related substances
Appea.rance, hormgeneity, content, particle size,
Gels
related substances, demixing for emlusion
Appearance clarity, visible particles, content,
pH, and related substances for solutions
Appearance, content, pH, related substances,
particles size and redispersibility for suspensions
Ophthalmic
Appearance, clarity, content, pH, related
preparations
substances and sterility for eye lotions
Appearance, clarity, content, pH, related
substances, particles size and sterility for eye
pills
 9011 Guidelines far in viw Bioavailability and Bioequivalence Studies far Drug Preparations

continued usually evaluated by the plasma concentration-time curve.

Type of
lmportant items far drug stability
preparation
Appearance, content, related substances
Pills
and disintegration time
Appearance, content, clarity, relative
Syrups
density, related substances and pH
Oral Appearance, content, clarity and related
solutions substances
Appearance, content, demixing and related
Oral creams
substances
Oral Appearance, content, sedimentation volume
suspensions ratio, related substances and redispersibility
Appearance, content, particle size, related
Powders
substances and unifarmity of appearance
Uniformity of dosage for delivery, dosage of
Inhale microparticles, related substances, number of
aerosols deliveries of each bottle, total dosage for delivery
and delivery rate
Preparations Unifarmity of dosage for delivery and dosage
for inhala tion of microparticles
Number of deliveries in each bottle, volume
in each delivery, dose in each inhale,
Sprays
delivery rate, total dosage far delivery and
dosage of microparticles
Appearance, content, particle size, related
Granules
substances, dissolution or release
Patches
Appearance, content, related substances,
( transdermal
release and adhesion
patches)
Rinsing agents. Appearance. content, related substances,
lotions, demixing ( emulsion type ) , dispersibility
enemas (suspension type), sterility for rinsing agents
Liniments, Appearance, content, related substances,
paints, film demixing ( emulsion type), dispersibility
paints (suspension type), filming far film paints
Appearance, content, related substances. For
Ear ear powders, sprays and semi-solid
preparations preparations, selection of items is based on
requirements of the dosage form respectively
Appearance, pH, content, related
substances. For nasal powders, sprays and
Nasal
semi-solid preparations, selection of items is
preparations
based on requirements of the dosage form
respectively
Note: The changes of number and quantity, including
decomposition products and other products derived from
other changes, should be specified in the item of related
substances. If possible, it should be stated which related
substance is the intermediate of the drug substance or
degradation product. Degradation product is an important
item in stability testing.
9011 Guidelines for in vivo Bioavailability
and Bioequivalence Studies for
Drug Preparations
Bioavailability refers to the usable rate and extent of active
ingredients in the action site after being released from the
drug preparation and being absorbed into the body, which is
Bioavailability data of oral solid preparations can be compared
with those of solutions, suspensions or intravenous injections
and used to estímate the fraction of drug absorbed into the
systemic circulation. Moreover, bioavailability studies can
provide valuable infarmation on the distribution and
elimination, effect of faod on absorption, proportional
relation of different dosages, pharmacokinetic linearity of
active ingredients and non-active ingredients under certain
conditions and sorne other useful pharmacokinetic data.
If two drug preparations with identical active ingredients are
pharmaceutical equivalent or pharmaceutical alternatives, and
their bioavailabilities (rates and extents) are both within the
acceptable limits after equimolar administration, the
preparations are considered as bioquivalent. These limits
should be properly determined to assure that different
preparations display comparable in viw behaviors, which
means that their efficacy and safety profiles are similar.
In bioequivalence studies, bioequivalence is evaluated based
on relative bioavailability comparison between the test and
reference preparations, which is calculated using selected
pharmacokinetic parameters and preset values of acceptable
limits. AUC ( area under the plasma concentration-time
curve) reflects the exposure extent of the active ingredient,
and Cmax (maximum plasma concentration) and tmax (peak
time) reflect the absorption rate.
This guideline aims to propase requirements far design,
implementation and evaluation of bioequivalence studies.
Furthermore, the possibility of substitution of in viw test
with in vitro test is also discussed.
l. Design, implementation and evaluation of bioequivalence
studies for ordinary preparations
1.1 Scope
This part of the guidance establishes specifications fer
design, implementation and evaluation of bioequivalence
studies on ordinary preparations with systemic effect.
Bioequivalence study is the basis of new generic drug
application. The purpose of bioequivalence study is to
confirm the bioequivalence between a generic drug and a
reference one, thus bridging their pre-clinical and clinical
studies. Generic drugs should have the same composition and
content of the active ingredient and the same dosage farm as
those of the reference drug, and the bioequivalence should be
vailidated by proper bioavailability studies. Active
ingredients with different salt forms, isomer mixtures and
clathrates are considered to be an identical active ingredients,
unless there is a significant difference observed in safety or
efficacy. Moreover, different forms of oral preparations are
also considered as an identical pharmaceutical form.
This guidance only applies to chemical drugs. For biologics,
the corresponding guidelines shall apply. Although the
concept of bioequivalence may be applied to chinese
medicine, it is not suitable for those in which the active
ingredients have not been elucidated.
If bioequivalence could not be confirmed by drug
concentrations, pharmacodynamics or clinical trial endpoint
studies may be required for few exceptions, which can refer
to specific guidelines in therapeutic field.
l. 2 Study design
Since the number and design of the study depend on the
physiochemical and pharmacokinetic properties and
compositions of the drug, related reasons should be stated,
particularly for studies requiring linear pharmacokinetics,
fast or postprandial state, enantiomer selectivity and
biowaiver of extra doses.
 9011 Guidelines for in vivo Bioavailability and Bioequivalence Studies for Drug Preparations
Moreover, study design should be a ble to distinguish the
effects derived from preparations from the other affecting
factors.
Standard design
For two preparations, random, two-period and two-order
single-dose cross-over experiments are recommended.
Administration cycle should be determined by washout
period, which should assure that plasma drug concentrations
in subjects is lower than detection limit of the analytical
methods at the beginning of the second cycle. Generally,
seven half lifes are necessary to achieve this requirements.
Alternative design
In sorne cases, if the design and statistical analysis are
sophisticated, alternative design can be considered. For
example, parallel experiments can be used for sorne drugs
with extra long half life, while multiple dosage experiments
can be employed for drugs with significant variations of
pharmacokinetic properties.
For single <lose experiments that could not be performed in
healthy subjects due to tolerance, or single <lose experiments
that are not suitable for subjects, multi-dose experiments
could be carried out.
l. 3 Reference preparations and test preparations
Reference preparations
The data on the reference preparation should be cited and this
drug should be on the market in China or approved for
import and comprehensive data are available. The applicant
should also state the reason for selection of the reference
preparation.
For generic drug applications, the test preparation may
usually be compared with the corresponding reference
preparations obtained from the market. If there are many
forms of preparations of one drug available on the market,
the initially approved preparation ( used for clinical
pharmacodynamics study and safety study) is recommended.
It is the obligation of the applicant that the selection of
reference preparations for bioequivalence study should be
based on the content analysis and dissolution data. The
variation between the contents of active ingredients of the
selected batch of test preparation and reference preparation
should be less than 5% unless otherwise stated.
Test preparations
The test preparation for the study should represent the upcoming
drug. For example, the selection of oral solid preparation for
systemic use should meet the following requirements:
(1) The scale of the test preparation should be not less than
one tenth of the production batch or 100 000 units. The
larger one is chosen unless otherwise stated.
(2) For the selected batch, its product and manufacturing
process should be of proper scale. If the scale of this batch is
less than 100 000 units, samples should be taken from the
whole batch.
(3) The critica! quality property of the selected batch, such
as dissolution, should be demonstrated.
(4) To support the application, samples from an addtional
preliminary experiment or the whole selected batch should be
compared with the samples for bioequivalence study. Their
dissolution curves should be similar under proper testing
conditions.
For other forms of ordinary preparations for systemic use,
representativeness of the batch for test preparation should
also be discussed and confirmed similarly.
Package of testing ~
Reference preparations and test preparations should be
packaged separately for every subject and every test period.
They should be packaged before transport to or at the
experimental sites. Packages and labels should be in
accordance with the GMP requirements that clearly identify
the drugs for various subjects and various test periods.
l. 4 Subjects
The number of subjects
The number of subjects should be determined according to
proper sample size calculation method. Generally, at least 18
assessable subjects should be included in a bioequivalence
study.
Selection of subjects
The subject group for bioequivalence study should be selected
to identify the variations among drugs. The experiment is
generally carried out in healthy volunteers to reduce
variations irrelevant to drugs, unless ethical concerns may be
raised due to sorne safety issues of the drug. The in vivo
model of healthy volunteers can distinguish the variations
between preparations in most cases and the results can be
also extrapolated to other patient groups approved for the
drug, such as the elder, children, those with liver or kidney
inj ury, etc.
Principies of inclusion and exclusion should be clearly listed
in the study plan. The subjects should be at ages of not less
than 18 years with BMis ranging from 19 kg/m 2 to 26 kg/m 2
in general.
Based on therapeutic indication and safety model of the drug,
subjects should be screened through laboratory examination
in the clinic, medical history and medical examination.
Special medical examinations and precaution may be
performed before, during or after the study. Either gender is
acceptable, however, the risk existing in pregnant women
should be concerned. Subjects who are non-smoking, non-
alcoholic and non-drug abusers are recommended. The
subjects can be screened for enzyme phenotype or genotype
due to the concerns on safety and pharmacokinetics.
In parallel experiments, all factors which may influence drug
pharmacokinetics, such as age, weight, gender, race,
smoking habit, and fast/low metabolism, should be
comparable between different groups, which establish the
foundation of effective and convincible results from
experiments.
If side effects of the active ingredient are already known, and
its pharmacologic effect or risk is not acceptable for healthy
volunteers, the experiment should then be carried out in
patients under proper monitoring and precaution.
l. 5 Study implementation
Standardimtion
The examination conditions should be standardized to minimize
variations of other factors than drugs. Therefore, standardized
diets, liquid intake and sports are recommended.
Administration time on the experiment day should be
stipulated. The subjects should fast for at least 8 hours
before administration unless otherwise stated. Because liquid
intake may influence gastric emptying of oral preparations,
both reference and test preparations should be administrated
at standard liquid volumes (200 ml in general). Any drinking
is acceptable one hour before or one hour after
administration. Subject should remain fast for 4 hours after
administration. Compositions and time of diets after
administration should also be standardized and last for an
enough time (for example 12 hours).
Experiments carried out after a meal should be in accordance
with the package inserts. To have the meal 30 minutes
before administration and to finish within 30 minutes is
 9011 Guidelines for in viw Bioavailability and Bioequivalence Studies for Drug Preparations
recommended.
Befare a certain time of the experiment and during the
experiment, subjects should stay away from food that may
influence blood circulation, gastrointestinal tract and
hepatorenal functions. Other drugs including herb medicine
should not be taken during this period.
In the bioequivalence study of endogenous substances, the
factors which may affect the levels of endogenous substances
should be controlled as far as possible. For example, food
intake should be strictly controlled.
Sampling time
Enough samples should be collected to fully describe the plasma
concentration-time curve. There should be enough sampling
points near tmax to reliably evaluate the peak concentration of
drug. The experiment should also be carefully designed to avoid
making Cmax the first point in the curve. The experiment should
also cover enough time of the curve to properly evaluate the
exposure degree of the drug, where AUCco-1) covers at least 80 %
of AUCco-+=) . However, for any bioequivalence study of
ordinary preparations, sampling cycle is no longer than 72 hours
regardless of the half-life period.
In multi-dose experiments, the zero-hour sample should be
collected immediately after administration ( within 5
minutes), and the last sampling point of the whole cycle
should be within 10 minutes of the labeled time in order to
calcula te the AUCco-1) accurately.
If urine is taken as the sample of biological fluids, the entire
sampling time should cover at least three half-life periods of
the drug. Similar to plasma samples, the entire sampling
time is no longer than 72 hours. If excretion rate is to be
determined, sampling interval in absorption phase should be
as short as possible.
For endogenous substance, the baseline level of every subject
in every cycle should be characterized, which is generally
determined by two or three samples befare administration. In
other cases, the baseline can be determined by periodical
sampling 1-2 days befare administration to acquire the
variations of the baseline over time.
Experiments under fast or po.strandial condition
The bioequivalence study is in general carried out under a
fastíng condition, which is the most sensitive condition to
identify the variations of different preparations. If the
administration of reference drug is recommended under a fast
condition ora condition regardless of meals according to the
package insert, the bioequivalence study should be carried
out under a fast condition. If only a postprandial condition is
recommended for reference preparation in the package insert,
the study should be conducted after meal.
However, for special forms of preparations ( e. g. ,
microemulsion or solid dispersion ) , bioequivalence study
should be conducted under both fast and postprandial
conditions unless the preparations are specified to be
administered only under eíther of the conditions.
When the data of both fast and postprandial conditions are
needed, two individual twin cross-over or one quadrivial
cross-over study is acceptable.
In postprandial experiments, the di et should be determined
according to the characteristics of the drug. If there is no
specific recommendation, high-calorie and high-fat diet
should be adopted.
l. 6 Indexes for investigation
Phannacokinetic parameters
Pharmacokinetic parameters should be evaluated using the
actual sampling time. AUC<o-1), AUC<o-=), surplus areas,
e max and t max should be determined in a single-dose
bioequivalence study. In a 72-hour experiment, if the
concentratíon at 72 hour could still be quantified, AUCco--=>
and surplus areas can be removed from the report. Sorne
additional parameters can be reported, including terminal
elimination rate constant 0.z) and t112.
In bioequivalence studies of ordinary preparations under
steady state, AUC(o-1), Cmax.ss and tmax,ss. should be
determined.
For urine samples, Aeco-1) should be determined, and Rmax
should be examined if applicable.
Non-compartment model should be employed to calculate the
parameters in bioequivalence studies.
Parent drugs and metabolites
General principies
Cmax of parent drug is usually more sens1t1ve than that of the
metabolite for comparing the variations of different preparations in
absorption rates. Therefore, the bioequivalence evaluation should
be based on concentrations of parent drugs. For bioavailability
test, if proper analytical methods are available for parent drugs and
metabolites, it is recommended to evaluate both of them
Inactive prodrugs
Even for inactive prodrugs, it is recommended to validate the
bioequivalence using parent drugs rather than metabolites.
However, sorne prodrugs may be at low plasma
concentrations and be eliminated quickly, which makes it
difficult to validate the bioequivalence of parent drugs. In
this case, bioequivalence can be validated using the mam
active metabolites rather than parent drugs.
Data Jrom metabolites instead of active parent drugs
Only in few exceptions, metabolites are used instead of active
parent drugs. In this situation, applicants should submit all
available data to support that the exposure level of
metabolites would reflect the absorption of parent drugs and
the amount of metabolite formed is not saturated at
therapeutic dosage.
Enantiomer
In general it is acceptable to use achiral bioanalytical methods
to evaluate the bioequivalence. However, if all of the
following conditions are met or unknown, single isomer
should be determined: There are pharmacokinetic differences
between enantiomers; The pharmacodynamics of enantiom-
ers are significantly different; Ratios of AUC of enantiomers
vary considerably at different absorption rates.
If one enantiomer is bioactive and the other is inactive or has
little efficacy, the bioequivalence can then be determined only
using the active one.
For bioavailability studies, single isomer is generally
determined.
Endogenous substances
For bioequivalence studies of endogenous substances, excess
dosage can be considered if this dosage is well tolerated, tlie
increased concentrations above the baseline can be reliably
determined, and the pharmacokinetic parameters reflect the
increased concentration after administration.
Usage of orine data
If it is impossible to accurately determine the plasma
concentration-time curve of a parent drug, urine excretion
data can be used to evaluate the exposure level instead of
plasma concentrations. However, if the urine data are used
to evaluate the peak concentration of the exposure, the
reason should be carefully stated.
l. 7 Strengths of test preparations
If the test preparation has several strengths ( the content of
 9011 Guidelines far in vivo Bioavailability and Bioequivalence Studies far Drug Preparations
active ingredient in a preparation unit) , it is enough to
evaluate bioequivalence using one or two strengths, which
depends on the compositions of different strengths and
related items below. The strength used far evaluation
depends on the linearity of pharmacokinetics of the active
ingredients.
In nonlinear pharmacokinetics ( the increase of AUC is not
proportional to the increase of dosage) , the sensitivities may
be different far detecting the variations of preparations at
different strengthes. The linearity is evaluated by whether
the variations of AUC are within 25 % after dosage
normalization.
If a particular strength ( s) has already been confirmed to be
most sensitive far detecting variations, the other strengths
can be exempted far bioequivalence study.
Linear pharmacokinetics
Bioequivalence study is generally conducted in the highest
strength. For preparations with linear pharmacokinetics and
high water solubility, it is still acceptable to select a lower
strength. lt is also reasonable to choose a lower strength due
to the concerns on safety and tolerance issues of the highest
one in healthy volunteers. Moreover, if the analytical method
is not sensitive enough to accurately determine the plasma
concentration of single dose administration of the preparation
in the highest strength, an even higher strength can be then
chosen ( multiple doses with the highest strength is
recommended). The selected dose can be higher than the
highest therapeutic dose as long as this dose could be
tolerated by healthy volunteer and there is no limitation of
absorption and solubility.
Nonlinear pharmacokinetics
For preparations with nonlinear pharmacokinetics, when the
increase of AUC is higher than those of doses within the
therapeutic doses, bioequivalence study is generally
conducted in the highest strength. A lower strength is also
reasonable due to the concerns on safety and tolerance issues
of the highest one in healthy volunteers.
Far preparation in which the increase of AUC is lower than
of dose within the therapeutic doses, bioequivalence study is
generally conducted in the highest and lowest ( or a strength
within the linear range) strengths, i. e. , two tests are
necessary in this situation.
If the analytical method is not sensitive enough far the lowest
strength or there are safety and tolerance issues of the
highest one, other strengths can also be selected.
l. 8 Analytical methods for biological samples
Regarding to requirements of analytical methods far biologic
samples, please refer to Guidance of Quantitative Analysis
Methods and Validations of Biological Samples ( 9012).
l. 9 Evaluation of bioequivalence
In bioequivalence studies, it is generally unnecessary to
calibrate pharmacokinetic parameters because of the content
variations of test and reference preparations. However, when
reference batches cannot be obtained with the variation of
contents within 5 % compared to test batches, calibration is
acceptable. If calibration is to be used, it should be included
in the experimental design and reasons should be specified in
the design by comparing the content results of test and
reference preparations.
lnclusion of subjects
In an ideal situation, all subjects in the study should be
included in statistical analysis. However, the subjects who
provide non-evaluable data far both test and reference
preparations in cross-over experiment or single cycle parallel
experiment should not be included.
Reasons for exclusion
In random experiments identical rules and equal treatment to
all subjects are required far an unbiased assessment. These
regulations should be independent on administration or
results. Therefare, exclusion of subjects in statistical
analysis should be determined befare sample analysis.
In principle, reasons far exclusion are valid only when they
are stipulated in the study design and determined befare
analysis. However, the exclusion far data should be avoided
as muchas possible, because it would reduce the reliability of
the experiment, and at least 18 evaluable subjects should be
included at the end of study.
The reasons far exclusion of subjects in a certain cycle
include vomiting and diarrhea which may lead to unreliable
plasma concentration-time curves. Administration of other
drugs can also be a reason far exclusion.
Reasons far exclusion should be stipulated in the study
design. If one of the reasons occurs in the study, it should be
indicated in the case report farms. Subjects who are excluded
in the study according to previously determined reasons
should be clearly described and listed in the report.
The exclusion based on statistical analysis or pure
pharmacokinetic issues is not allowed because it is impossible
to distinguish pharmaceutical factors affecting pharmaco-
kinetics from other factors.
There are severa! exceptions: subjects haven' t taken the
medicine as prescribed or washout period is not enough, thus
reliability of the experiment can be questioned in this case.
Samples of subjects excluded from statistical analysis should
also be determined and reported.
When the sampling cycle is less than 72 hours, AUCco--t)
should cover at least 80 % of AUC<O-=). If the number of the
subjects whose AUCco--t) is less than 80 % of AUCco--oo)
exceeds 20% of the total number, the reliability of the
experiment should be discussed.
Parameters and acceptable limits
In single-dose bioequivalence study, AUCco--t) [sometimes
AUCco--nh) ] and Cmax should be analyzed. For these
parameters, the geometric mean ratios of test and reference
preparations at 90 % confidence interval should fall in the
range from 80. 00% to 125. 00%. For this reason, the lower
limits reserving two decimal fractions should be equal to or
more than 80. 00 % after rounding off, while the upper limits
should be equal to or less than 125. 00 % .
For bioequivalence study of ordinary preparations at steady
state, above mentioned acceptable limits should be adopted
far analysis of AUCco--t) and Cmax,ss·
For urine samples, the above mentioned acceptable limits of
AUCco--t) should be adopted far Aeco--t) , and the that of Cmax
far Rmax.
Statistical analysis of tmax is unnecessary. However, if rapid
release is claimed to be important far clinic utilization,
therapeutic effects and related to side effects, the median
value and variation of tmax of test preparations should not be
significantly different from those of reference preparations.
If the therapeutic window is narrow, the above mentioned
acceptable limits may be reduced. Moreover, far drugs with
high variability, the acceptable limits of Cmax may be
broadened.
Statistical analysis
Bioequivalence evaluation Is based on the 90 % confidence
interval of geometric means of test and reference
preparations. This method is equal to two one-sided tests
with the null hypothesis of bioinequivalence at 5%
significance level.
Pharmacokinetic parameters should be analyzed by analysis
 9011 Guidelines far in vivo Bioavailability and Bioequivalence Studies far Drug Preparations
of variance. Data should be logarithmically transfarmed
befare analysis. Confidence interval of variations between
preparations on the log coordinate can be obtained from the
analysis of variance model. The confidence interval should be
then transfarmed back to the expected confidence interval on
the original coordinate.
The exact model for analysis should be defined in the study
design in advance. The statistical analysis should be able to
reasonably suppose the source of variation affecting the
variables. Items in analysis of variance include rank,
subjects, cycles and preparations.
Residual Effects
The residual level should be determined by the plasma
concentration befare the second cycle of administration.
If the plasma concentration of any subject befare the second
cycle is higher than 5% of Cmax of this period, data of this
subject should be excluded.
Design of tw&-period experiment
Two-period experiments are acceptable far bioequivalence
study. If bioequivalence cannot be confirmed by the first
group of test subjects, one more group can be recruited and
both of their data are combined in the final analysis. The
proposal of two-period experiment should be stipulated in the
study design and the significant level of each analysis after
adjustment should also be stipulated in the design.
When the combined data are analyzed, stage ítem should also
be included in the analysis of variance model.
Data submis.sion
For each preparation, concentrations and pharmacokinetic
parameters of all the subjects should be listed, as well as
summary on statistical analysis, including geometric mean,
median, arithmetic mean, standard deviation, variable
coefficient, maximum and mínimum values. The plasma
concentration-time curve of each subject should be provided
in both linearity/linearity and log/linearity coordinates.
The methods far calculating parameters from original data
should be described. The number of points far terminal
logarithmic linearity should be stipulated to evaluate the
terminal rate constants, which is necessary far reliable
evaluation of AUC"'.
Far pharmacokinetic parameters used in statistical analysis,
point estimation for ratios of test and reference preparations
and 90 % confidence interval should be submitted.
Analysis of variance table, including proper statistical test
far all factors in the model, should also be submitted.
Reports should be detailed enough to make the pharmacoki-
netics and statistical analysis may be repeated. For example,
actual plasma concentrations after drug administration, drug
concentrations, pharmacokinetic parameters of each subject
in every cycle and accompanied schedules should be provided.
Lost fallow-up and withdrawal of subjects should be fully
documented. If possible, related data and pharmacokinetic
parameters of these subjects should be provided in a separate
farm. However, these data should not be included in the
statistical analysis.
Concise descriptions of bioanalytical methods employed,
results of standard samples far calibration and quality control
samples should be included in the bioanalytical report.
Chromatograms of all the subjects should be provided, as
well as chromatograms of quality control samples and
standard samples far calibration and other original data.
l. 10 Preparations with narrow therapeutic windows
For preparations with narrow therapeutic windows, acceptable
limits of AUC should be narrowed to 90. 00%-111. 11%. If
Cmax is particularly important far safety, therapeutic efficacy or
drug concentration monitoring, the acceptable limit of 90. 00 %-
111. 11% can also be adopted by Cmax • Whether an active
ingredient is a drug with narrow therapeutic windows is
determined based on actual conditions in clinic.
1. 11 Highly variable drugs or preparations
A highly variable drug is a drug of which the intra-individual
variations of pharmacokinetic parameters are more than
30 %. If applicants question that a drug may be highly
variable, a reproducible cross-over experiment may be
conducted.
For highly variabile drugs, if small influences on large
variations of Cmax are observed in clinic, acceptable limits can
be broadened based on sufficient clinical reasons. In this
case, acceptable limit of Cmax can be extended to a range of
69. 84%-143. 19 %. To broaden the limit, the bioequivalence
study should be reproducible to confirm that intra-individual
variations of Cmax far the test drug are more than 30 % .
Applicants should also confirm that these variations in
subjects are calculated based on reliable evaluation rather
than outliers. The request to broaden limits should also be
stated in the study design.
Broadening of limits due to intra-individual variations in
subjects is not applicable to AUC of which the limit should
range from 80 % to 125 % regardless of variations.
Three-period or faur-period cross-over experiments are
acceptable in the design of reproducible studies.
2. Bioequivalence studies of modified-release preparations
The reason far developing modified-release preparations is
the correlations of systemic exposure to pharmacological and
toxicological responses of drugs and metabolites. Therefare
in most cases, the purpose of modified-release preparations is
to achieve similar exposure (AUC) of drug or metabolites in
modified-release preparations compared with ordinary
preparations, which <loes not necessarily mean that same
labeled <lose should be administrated ( bioavailability of
modified-release preparations may be different).
2. 1 Bioovailability experine:lts of imdllied-releR preparations
To characterize in vivo behavior of modified-release
preparations, bioavailability experiments may be used to
investigate the rate and extent of absorption, fluctuation of
drug concentration, pharmacokinetic variations caused by
preparations, dose ratio relationship, factors affecting
modified-release preparations, and accident risks of release
(e. g. burst release).
These tests are mainly applied to determine the concentra-
tions of active ingredients or metabolites. Reference
preparations may be ordinary ones with identical active
ingredients on the market. These studies may be carried out
in healthy volunteers or patients. In multiple dosing
experiments, the steady states should be confirmed to be
reached.
2. l. 1 Rates and extents of absorption and fluctuations of
drug concentrations
Single and multiple dosing pharmacokinetic studies should be
carried out to evaluate rates and extents of absorption of
modified-release preparations compared with ordinary preparati-
ons. Fluctuation studies should be performed in steady states
after multiple dosing. The release features of modified-release
preparations should be validated by comparing with ordinary
preparations. Their peak and trough concentrations should be
similar or have small fluctuations, as well as similar drug
exposure. Pharmacokinetic parameters that should be concerned
in this study include AUC, Cmax, Cmin, Cmax/Cmin and other
parameters reflecting fluctuations of drug concentrations, such as
Cmax/ Cmin.
 9011 Guidelines for in viw Bioavailability and Bioequivalence Studies for Drug Preparations
2. l. 2 Variability of phannacokinetic parameters
Variations of pharrnacokinetic parameters between modified-release
preparations and ordinary preparations can be compared by
analyzing the intra-individual variations of pharmacokinetic
parameters. Variations of pharmacokinetic parameters of
modified-release preparations are generally not larger than
those of ordinary preparations. lntra-individual variations of
pharmacokinetic parameters can also be evaluated by
concentration curves measured repeatedly at steady states or
multiple single-dosing studies.
2. l. 3 Dose-response consistency
Studies of <lose-response consistency should be performed if
there are several strengths of test preparation. All necessary
data should be provided based on pharmacokinetic features of
the drug.
For the drug with linear pharmacokinetics, the total exposure
of the modified-release preparation at one strength after
multiple dosing should be similar to that of ordinary
preparation.
For the drug with nonlinear pharmacokinetics within the
range of therapeutic plasma concentrations, comparisons of
the highest and the lowest strengths after multiple dosing of
the modified-release preparation with those of ordinary
preparation should be performed. Moreover, the dose-
response consistency of modified-release preparations at all
strengths should be fully stated in any cases.
2. 2 Factors affecting modified-release property
Different modified-release preparations with the same active
ingredient may have different interactions with food.
Therefore, for the purpose of safety and efficacy, effects of
food on bioavailability of oral modified-release preparations
should be investigated. The optimal condition for this test is
administration of preparations instantly after taking high-fat
diet. Modified-release property should also be evaluated in
addition to AUC and Cmax. If significant interactions with
food are observed, applicants should provide the recommen-
ded dose after adjustment.
If modified-release preparations are used in combination with
gastrointestinal drugs, modified-release properties should be
studied in this situation. If modified-release preparations are
to be used in patients with gastrointestinal diseases, studies
should be performed in these patients.
Taking circadian rhythms into consideration, it is
recommended to produce a 24-hour plasma concentration
curve in steady state.
If the <lose of modified-release preparation is higher than that
of ordinary preparation, accident release ( e. g. , burst
release) may lead to unacceptably high drug exposure, of
which the possibility should be avoided.
If the modified-release preparation is to be applied in subjects
that are beyond the application of ordinary preparation,
pharmacokinetics study should be performed in this group.
2. 3 Bioequivalence studies of modified-release preparations
Bioequivalence studies of modified-release preparations are
recommended to compare two oral preparations in the same
dosage form (test and reference preparations).
If excipients or mechanisms of release in the two preparations
are different, while their dissolution curves in vitro are
similar or their release behaviors validated by discriminate
testing are similar, these drugs may be considered as one
dosage form. If bioequivalence is established, two prepara-
tions can be considered as basically the same.
If excipients or mechanisms of release as well as dissolution curves
in mtro of the two preparations are different, clinical studies
should be considered except of few rare situations in which their
bioequivalence can be confirmed.
2. 3. 1 Extended-release preparations
Bioequivalence of extended-release preparations can be
confirmed by single and multiple dosing experiments. If the
designed study can confirm that:
Extended-release properties of test and ref erence
preparations are similar.
• Active ingredients in test preparations are not accidently
released.
• Behaviors of test and reference preparations are similar in
single dosing and steady state.
• Under the influence of food, in viw behaviors of test and
reference preparations in single dosing after taking high-
fat dietare similar. This study should be carried out on
the same strength.
When there are several strengths of extended-release
preparations, a single-dose fast study should be conducted on
each strength of the preparations. If extended-release
preparations meet the extrapolation standards of bioequivalence
studies of ordinary preparations (linear pharmacokinetics, same
qualitative compositions, etc. ) , steady state studies could be
performed only on the highest strength.
If preparations with different units of one drug display linear
pharmacokinetics, single-dose fasting studies on the highest
strengths are sufficient when the compositions of lower
strengths are in proportion to those of higher strengths, the
preparations have same units, and the dissolution curves are
acceptable.
Bioequivalence of extended-release preparations are evaluated by
AUC, Cmax and Cmin using similar statistical analysis procedures to
those of ordinary preparations. Any relaxation of acceptable
standards should be preliminarily determined in the study design
and applicants should explain it from the clinical aspect
The following studies are recommended for generic extended-
release preparations: (1) a single-dose, non-replicate, fasting
study to compare the test preparation at the highest <lose with the
listed reference preparation; (2) a non-replicate study under the
influence of food to compare the test preparation at the highest
<lose with reference preparation. Because single-dose studies are
considered more sensitive in addressing the primary questions
about bioequivalence (e. g. , release of the drug substance from the
preparation into the systemic circulation), multiple-dose studies
are generally not recommended.
2. 3. 2 Delayed-release preparations
Bioequivalence of the delayed-release preparation is evaluated
using identical parameters and statistical analysis procedures
to those of ordinary preparations, where the delayed release
should be emphasized.
Because food may aff ect the absorption of active ingredient of
enteric coated preparations, the bioequivalence study should
be conducted after meal.
2. 4 Studies on the influence of f ood on drug absorption
The following studies are recommended to investigate the
influence of food on bioavailability of modified-release
preparations. However, due to the complexity of food-drug
interactions, sorne unusual in viw methods are still
acceptable in sorne cases.
2. 4. 1 Modified-release preparations of new chemical entities
Single-dose, two-stage cross-over studies
Drug administration 1: fasting oral administration of modified-
release preparations
Drug administration 2: fasting oral administration of solutions or
ordinary preparations
Drug administration 3: oral administration of modified-
releasepreparations after high-fat diet
 9012 Guidelines for Validation of Quantitative Analytical Method of Biological Samples
Drug administration 4: oral administration of solutions or
ordinary preparations after high-fat diet
2. 4. 2 Modified-releaie preparations of marketed preparations
Single-dose, three-stage cross-over studies
Drug administration 1: fasting oral administration of modified-
release preparations
Drug administration 2: oral administration of modified-release
preparations after high-fat diet
Drug administration 3: fasting oral administration of ordinary
preparations
Conclusion: No obvious food interactions (AUC, Cmax, t112,
MRT) or significant food interactions
2. 4. 3 Modified-release preparations similar to marketed
preparations
The first situation: There are significant food interactions
according to the references orno data available
Single-dose, dual two-stage cross-over studies
Drug administration 1: fasting oral administration of test
preparations
Drug administration 2: fasting oral administration of
reference preparations
Drug administration 3: oral administration of test
preparations after high-fat diet
Drug administration 4: oral administration of reference
preparations after high-fat diet
The second situation: There are no significant food
interactions according to the references
Single-dese, two-stage cross-over studies
Drug administration 1: oral administration of test
preparations after high-fat diet
Drug administration 2: oral administration of reference
preparations af ter high-fat diet
3. Test reports
The reports of hioavailability or bioequivalence studies should
include the complete records of study design, implementation
and evaluation with signed signatures of researchers.
The name and affiliation of the principal investigators, the
site of operation and time of the study should be provided.
The test report should contain evidence to demonstrate the
selection of reference preparation is acceptable, which should
include the name of drug, strength, dosage form, batch,
manufacturer, expiration date, and the place of purchase.
Analytical reports about batches of test and reference
preparations for this study should be included in the
attachment of the test report.
Complying with data submittal requirements, concentrations
as well as data on pharmacokinetic parameters and statistical
analysis should be submitted.
A statement should be submitted to confirm that the subject
preparation has identical quantitative compositions and
manufacturing process to those of the preparation to be
approved. Evidence for scale-up manufacturing should be
submitted. Comparative dissolution curves should also be
submitted.
Validation reports of bioanalytical methods should be
included in the application materials.
Detailed data to make pharmacokinetics and statistical
analysis reproducible should be provided in proper electronic
data format.
4. In vitro dissolution testing relevant to bioequivalence studies
4. 1 General iterns of the testing
In drug development, the dissolution testing could be
employed as a tool to determine the factors that may
influence bioavailability or even contribute decisively. Once
the compositions and manufacturing process are determined,
the dissolution testing of scale-up manufacturing can be used
for quality control to keep batch to batch consistency and
ensure similar dissolution curve observed for the clinical
batch. Moreover, in sorne situations, dissolution testing may
determine whether a bioequivalence study can be waived.
Sufficient sampling points ( at least every 15 minutes)
should be included to obtain a meaningful dissolution curve.
lt is recommended for more frequent sampling in the section
curve wi th large varia tions.
lf an active ingredient is highly soluble, the preparation is
dissolved rapidly at physiological pH, and the excipient has
no effect on bioavailability, it is reasonable to estimate that
bioavailablity is not an issue in the test. However, if an
active ingredient is poorly soluble, the dissolution could be
the rate-limiting step. The release of the active ingredient
controlled by the excipient is in a similar situation. In these
cases, various testing methods should be employed with
sufficient number of sampling points.
4. 2 Similarity of dissolution curves
Sufficient number of sampling points is the foundation of any
conclusion (e. g. , rationality of biowaiver) derived from the
similarity testing of dissolution curves.
For ordinary preparations, in addition to items mentioned
above, the comparison at 15 minute is necessary in arder to
determine whether the preparation can be completely
dissolved befare gastric emptying.
The f 2 statistics can be used to determine the similarity of
dissolution curves of test and reference preparations.
5. Biopharmaceutics d~cation system(BCS)-based biowaivers
Biopharmaceutics classification system (ff.S)-based biowaivers is
an approach to reduce the number of in viw bioequivalence
studies, which suggests it can be used to replace the in viw
bioequivalence study. If the bioequivalence hypothesis for in viw
behavior can be completely validated by sufficient in vitro
data, the in vivo bioequivalence study can be waived.
BCS-based biowaivers can only be applied to the drug with
high solubility and known information on in vivo absorption.
This drug should not have a narrow therapeutic window.
This concept is applicable to the same dosage form of
ordinary oral preparation for systemic use. However, it
should not be applied to sublingual preparations, buccal
preparations or modified-release preparations.
9012 Guidelines for Validation of
Quantitative Analytical Method of
Biological Samples
Section 1 Scope
Accurate measurement of drug concentrations in biological
matrices ·(e. g. , whole blood, serum, plasma and urine) is
essential far research and development of drugs and drug
preparations. These data support the efficacy and safety of a
drug and are critical to decision making based on results from
toxicokinetic, pharmacokinetic, and bioequivalence studies.
Therefore, bioanalytical methods applied in the studies
should be fully validated and documented in details to yield
reliable results.
This guideline describes the requirements far the validation of
bioanalytical methods, basic requirements of analyzing
samples from non-clinical or clinical trials, as well as the
requirements far using partial validation or cross validation in
replace of the full validation of an analytical method. Sections
2 and 3 of this guideline mainly focus on chromatographic
methods, while section 4 facuses on analytical methods far
 9012 Guidelines for Validation of Quantitative Analytical Method of Biological Samples
ligand binding assays.
The validation of bioanalytical methods and analysis of test
samples should comply with technical requirements specified
in this guideline and the analysis on biosamples should follow
the principles of Good Laboratory Practice ( GLP) or Good
Clinical Practice ( GCP).
Section 2 Validation of bioanalytic.al method
2. 1 Foil validation of an analytic.al method
The main objective of method validation is to demonstrate the
reliability of a particular method for the determination of an
analyte concentration in a specific biological matrix.
Moreover, validation should be conducted using the same
anticoagulant with the test samples. Generally a full
validation study should be performed for each new analytical
method and new analyte. In sorne cases, when it is difficult
to obtain an identical matrix in biological experiments, a
suitable alternative matrix may be used with proper
explanation.
Major characteristics of a bioanalytical method are: selectivity,
lower limit of quantification, the response function and calibration
range ( calibration curve performance ) , accuracy, precision,
matrix effect, stability of the analyte (s) in the biological matrix
and in the solutions for storage and processing procedures.
There are cases that concentrations of more than one analyte
should be determined, such as two different drugs, one
parent drug and its metabolites, or enantiomers or isomers of
one drug. Principles of validation and analysis should be
applicable to all analytes involved.
Reference standards
During method validation, a blank biological matrix will be spiked
with the solution containing reference standards for analytes to be
analyzed. In addition, suitable internal standards (IS) are usually
employed in chromatographic methods.
The reference standards should be obtained from an authentic
and traceable source. The suitability of reference standards
should be demonstrated scientifically. A certificate of
analysis is required to confirm purity of reference standards
and provide information on storage conditions, expiration
date and batch number. Only suitability for use, e. g. , lack
of analytical interferences from the substance itself or any
impurities thereof, should be demonstrated for IS.
When mass-spectrometry ( MS) detection is used in the
bioanalytical method, a stable isotope-labelled IS, with high
enough isotope purity and no isotope exchange reaction to
interfere results, is recommended whenever possible.
2. l. 1 Selectivity
By this analytical method, the analytes of interest and IS
should be differentiated from endogenous components in the
matrix or other components in the sample. Selectivity should
be demonstrated using appropriate blank matrix from at least
6 test subjects (different batches of blank matrix of animals
can be mixed), which are analysed separately and evaluated
for interference. It is generally acceptable that responses of
interfering components are less than 20 % of the lower limit
of quantification for analytes and 5 % for the responses of IS.
It is necessary to investigate the extent of any interference
caused by metabolites of the drugs, degradation products
through sample preparation, and possible co-administrated
drugs. Under certain conditions, the possibility of back-
conversion of a metabolite into parent analyte should be taken
into consideration during the analysis.
2. l. 2 Residues
Residues should be addressed and minimized during
development of analytical methods. Residues may not affect
the accuracy and precision. Residue should be assessed by
blank samples administrated after high concentration samples
or calibration standards. Residue in the blank sample
following the high concentration standard should not be
greater than 20 % of the lower limit of quantification and less
than 5 % for the IS. If it appears that a residue is
unavoidable, specific measures that are validated during
method development should be considered to be applied to
sample analysis in order to prevent possible influences on
accuracy and precision, such as performance analysis of test
sample after the injection of blank samples following
administration of high concentration samples.
2.1. 3 Lower limit of quantification
With an acceptable accuracy and precision, lower limit of
quantification ( LLOQ) is the lowest concentration of
analytes in a sample that can be quantified reliably. The
LLOQ is the lowest point of the calibration curve that should
be suitable for expected concentrations and the aim of the
study.
2. l. 4 Calibration curve
The Calibration curve represents the responses to analytes by
analytical instruments within a defined range of
concentrations. Standard samples for calibration at various
concentrations should be prepared by the mixture of the
analyte (and IS) at known concentrations with blank matrix
that are identical to the matrix used in test samples.
Individual calibration curve should be derived for each analyte
of interest and each batch of samples during method
validation.
It is recommended to know the range of expected concentra-
tions before the validation of analytical methods. The
concentration range in a calibration curve should cover the
range of expected concentrations, which is defined by the
LLOQ and upper limit of quantification ( ULOQ) as the
lowest and highest concentrations of calibration standard,
respectively. The range of calibration curve should
adequately describe the pharmacokinetics of the analytes of
interest.
A mínimum of six calibration concentration levels should be
used in addition to the blank sample ( processed matrix
sample without analyte and IS) and a zero sample
(processed matrix with IS). Each calibration standard can be
processed and analysed for multiple times.
A simple but adequate relationship equation should be derived
to describe the response of the instrument with regard to the
concentration of analyte. Results of the blank and zero
sample should not be used in the calculation of parameters for
calibration curve.
The parameters for calibration curve and the back calculated
concentrations of the calibration standards should be
reported. Of all the available curves obtained during
validation, a minimum number of three curves should be
reported.
The deviation of back calculated concentrations of the
calibration standards should be less than ± 15 % from the
nominal values ( ± 20% at LLOQ) . These criteria should
be fulfilled by at least 75 % of the calibration standards
including a minimum of six concentration levels of calibration
standards. In case a calibration standard <loes not comply
with these criteria, this calibration standard sample should be
rejected and the calibration curve without this calibration
standard should be re-evaluated, including regression
analysis.
Although freshly prepared sample solutions are recommen-
ded for the calibration curve, it is acceptable to use
previously prepared and stored calibration samples, if
appropriate stability data is available.
 9012 Guidelines for Validation of Quantitative Analytical Method of Biological Samples
2. l. 5 Accuracy
The accuracy of an analytical method describes the closeness
between determined concentration obtained by the method
and nominal concentration of the analyte, which is expressed
as ( determined value/nominal value) x 100 % . Accuracy
should be assessed by quality control samples (QC samples)
with known concentrations of the analyte added. Separate
stock solutions are used for the preparations of QC samples
and calibration standards.
The concentrations of QC samples are determined by the
calibration curve, which are then compared with the nominal
value. The accuracy should be reported as percent of the
nominal value. Accuracy should be evaluated using data of
QC samples from both a single run ( the within-run
accuracy) and multiple runs (the between-run accuracy).
To evaluate any trends over time within one batch, it is
recommended to confirm the accuracy by analyzing QC
samples with at least equivalent numbers of samples in a
prospective analytical run of test samples.
Within-run accuracy
Within-run accuracy should be determined by the analysis of
QC samples of a single run at four different concentration
levels: LLOQ, low, medium and high concentrations, at
least five samples for each. The four concentration levels
cover the concentration range of the calibration curve: the
LLOQ, low concentration which is not more than three times
of the LLOQ, medium concentration near the middle of the
calibration curve, and high concentration which is about 75 %
of the upper limit of concentration in calibration curve. The
deviation of mean value for measured concentrations of QC
samples should be within 15 % of their nominal values, while
the accuracy at LLOQ should be within 20%.
Between-run accuracy
The between-run accuracy should be determined on at least two
separate days and by OC samples from at least three runs at four
different concentration levels: LLOQ, low, medium and high
concentrations, at least five samples for each level. The deviation of
mean value for measured concentrations of OC samples should be
within 15 % of their nominal values, while the accuracy at LL0Q
should be within 20%.
Data used to determine accuracy and precision should include
all experimental results except for those experimental errors
that are obvious and documented.
2.1. 6 Precision
The precision of an analytical method describes the closeness
of repeated measurements of one analyte. Precision is
expressed as the coefficient of variation of measured values
(CV). Precision of a single run or multiple runs should be
determined using the data from the same run ( s) for
accuracy determination and QC samples at four different
concentration levels, i. e., LLOQ, low, medium and high
concentrations.
The within-run precision should be determined at four
concentration levels ( LLOQ, low, medium and high
concentrations) with at least five samples for each level in a
single run. The within-run CV should be within 15%, while
that at LLOQ should be within 20 %.
The between-run precision should be determined on at least
two separate days and by QC samples from at least three
runs at four different concentration levels ( LLOQ, low,
medium and high concentrations) with at least five samples
for each level. The between-run CV should be within 15%,
while that at LLOQ should be within 20%.
2. l. 7 Dilution reliability
The accuracy and precision should not be affected by dilution
of samples. Reliability of dilution should be demonstrated by
blank matrix diluted solutions ( at least five measurements
per dilution factor) from the sample prepared by spiking the
matrix with an analyte concentration above the ULOQ.
Accuracy and precision should be ±15%. Dilution reliability
study should cover the dilution factors applied to the test
samples.
Partial method validation is acceptable for dilution reliability.
Use of another matrix is also acceptable if precision and
accuracy are not affected.
2. l. 8 Matrix effect
Matrix effects should be investigated for mass spectrometric
methods using at least 6 batches of blank matrix from various
vendors. Pooled matrix should not be used. If the matrix is
difficult to obtain, it is acceptable to use less than 6 batches
of matrix and the reason should be stated.
For each matrix, the matrix factor ( MF) for each analyte
and IS should be calculated by the ratio of the peak area in
the presence of matrix (measured by the blank matrix spiked
with analyte and IS) to the peak area in absence of matrix (pure
solution of the analyte and IS). The IS-normalized MF should
also be calculated by the division of the MF of the analyte by the
MF of the IS. The CV of the IS normalized MF calculated from
six batches of matrix should not be greater than 15%. The
determination of matrix effect should be performed at a low and a
high concentration level separately.
In sorne cases where the above method is not applicable, for
instance on-line sample pretreatment, the batch-to-batch
variability of the response should be assessed by analyzing at
least six batches of matrix spiked with a low Oess than three
times the concentration of LLOQ) and a high concentration
(close to the ULOQ) level. The validation report should
include the peak areas of the analyte and IS and the calculated
concentration for each individual sample. The overall CV of
calculated concentrations should not be greater than 15%.
In addition to the normal matrix, it is recommended to
investigate matrix effects from other samples, e. g. ,
haemolyzed and hyperlipidaemic plasma samples.
2. l. 9 Stability
Stability should be ensured for each procedure of the
analytical method and the conditions in the stability study,
such as sample matrix, anticoagulant, container materials,
storage and analytical conditions, should be similar to those
applied to the actual test samples. lt is not sufficient to
establish stability using data from published literatures.
Stability of the analyte in the studied matrix should be
evaluated immediately after preparation and storage under the
conditions to be evaluated using QC samples at low and high
concentrations ( blank matrix spiked with the analyte at a
maximum of three times of the concentration of LLOQ and
close to the ULOQ, respectively). Using a calibration curve
obtained from freshly spiked calibration standards, measured
concentrations of QC samples are compared to their nominal
concentrations and the range of deviations should be within
±15%.
Taking into consideration the linearity and measuring range
of the detector, stability of the stock and working solutions
should be tested with an appropriate dilution.
The different storage conditions should be investigated in
stability studies over time periods that are equal to or exceed
those applied to the actual test samples.
The following stability studies should be conducted:
• stability of the stock solution and working solutions of the
analyte and interna! standard.
• freeze-thaw stability of the analyte in the matrix from
freezer storage conditions to room temperature or sample
 9012 Guidelines for Validation of Quantitative Analytical Method of Biological Samples
processing temperature.
• long term stability of the analyte in matrix stored in the
freezer.
In addition, the following studies should be performed if
applicable:
• stability of processed samples at room temperature or
under the storage conditions to be used during the study.
• stability of processed samples at temperature of autosampler.
In case of studies of multiple analytes, especially those in
bioequivalence studies, stability of each analyte in all related
matrices should be determined.
In arder to make sure that the concentration of analyte at the
time of sampling is reflected by the measured concentration
by analytical method, it is of significant importance to
determine stability of the analyte in the sample matrix
directly after blood sampling from the subjects and those
pretreated before storage. Depending on the chemical
structure of the analyte, its stability can be determined under
specific conditions.
2. 2 Partial validation
Depending on the nature of the applied changes, a partial
validation may be necessary when minar changes happen to
an analytical method that has already been validated. These
changes include transfer of the bioanalytical method to
another laboratory, changes in equipments, calibration
concentration range, sample volume, another matrix or
species, anticoagulant, processing procedures for samples,
storage conditions, etc. All of these changes should be
reported and the reasons for revalidation or partial validation
shouid be stated.
2. 3 e~ validation
Cross validation on analytical methods should be performed
when comparisons are needed for data collected by different
methods in one or multiple studies or through one method
conducted at multiple sites of study. If applicable, cross
validation should be performed in advance of test samples
being analyzed. Far the cross validation, two methods should
be applied to analyze QC or test samples from the same
series. Far QC samples, deviation of values measured by two
methods from the nominal values should be less than ±15%
and this margin can be broadened with stated reasons. Far
test samples, the difference in measured values of at least
67% of the samples by two methods should sit in the range
of ±20% of mean value.
Section 3 Analysis of test samples
After validation of the analytical method, analysis of test or
subject samples can be carried out. Before the analysis of test
samples, the performance of the bioanalytical method should
be verified.
Test samples, QC samples and calibration standards should
be processed according to the validated analytical method to
ensure the acceptability of the analytical run.
3.1 Analytical ron
An analytical run consists of a blank sample, a zero sample,
calibration standards at not less than six concentration levels,
QC samples at not less than three concentration levels
(duplicate samples at low, medium and high concentrations
or at least 5 % of the number of test samples, whichever is
higher) , and test samples to be analyzed. All samples
( calibration standards, QC samples, and test samples)
should be processed and extracted from one single batch of
samples in the sequence of submission in an analytical run.
Samples in a single batch are carefully handled by the same
analyst using same reagents under identical conditions at the
same time, i. e. , sequentially processed without interruption.
QC samples should be included in each step of the run to ensure
accuracy and precision of the whole run.
Far bioequivalence studies, it is recommended to analyze all
the samples of one subject in one analytical run to reduce
variations in results.
3. 2 Acceptance criteria of an analytical ron
Criteria for acceptance or rejection of an analytical run should
be defined in the study design of bioanalytical study or a
Standard Operating Procedure (SOP). When the entire run
includes more batches, acceptance criteria should be applied
to the entire run and the individual batches. The following
acceptance criteria should apply:
The deviations of back calculated concentrations of calibration
standards should be within ± 15% of their nominal values
and it should be within ± 20 % at the concentration of
LLOQ. These criteria should be fulfilled by not less than
75 % of at least six calibration stardards. If one of the
calibration standards <loes not meet these criteria, this
standard sample should be rejected and the calibration curve
should be re-evaluated without this calibration standard
followed by regression analysis.
The deviation of measured values of the QC samples should
be less than ± 15 % from the nominal values. At least 67 %
of the QC samples and at least 50 % of samples at each
concentration level should comply with this criterion.
Otherwise this analytical run should be rejected and test
samples should be re-processed and analyzed.
In the case of the simultaneous determination of several
analytes, there should be one calibration curve for each
anaiyte of interest. If an anaiyticai run is accepted far one
analyte and rejected for another analyte, the data for the
accepted analyte can be used, while the rejected analyte
should be re-processed and analyzed for determination.
When multiple calibration standards are used and standard
samples fail to meet the criteria only at one of the LLOQ or
ULOQ, the calibration range remains unchanged.
Far all accepted runs, the mean accuracy and precision
should be calculated for QC samples at each concentration
level and reported in the analytical report. When the overall
mean accuracy beyonds the range of 85%-115%, and the
presion (CV) was more than 15%, additional investigations
should be performed to evaluate this deviation and state the
reasons. This situation may result in the rejection of data for
bioequivalence studies.
3. 3 Calibration range
When a narrow concentration range of the analyte of test
samples is known or anticipated before the analysis, it is
recommended to narrow the calibration curve range, adjust
the concentrations of QC samples, or introduce additional
proper concentrations of QC samples, to adequately reflect
the concentrations of test samples.
When the concentrations of a large number of the analytes in test
samples are above ULOQ, it is recommended to expand the
calibration curve range, introduce additional concentrations of QC
samples or adjust their concentrations, if possible.
Concentrations of not less than two QC samples should fall within
the range of concentrations measured in test samples. When the
calibration curve range is modified, the bioanalytical method
should be re-validated (partial validation) to confirm the response
function as well as accuracy and precision.
3. 4 Reanalysis of test samples and selection of reported data
Possible reasons for reanalysis of test samples and data
selection criteria should be defined at the first place in the
study design ora Standard Operating Procedure (SOP). The
number of samples that have been reanalyzed and their
 9012 Guidelines far Validation of Quantitative Analytical Method of Biological Samples
percentage in the total number of samples should be provided
in the analytical report.
The following are examples of reasons far test sample
reanalysis:
• rejection of an analytical run because this run fails to meet
the acceptance criteria about accuracy of the calibration
standards and/ or the QC samples.
• significant diff erences in the responses observed between
interna! standard, calibration standard and QC samples.
• improper sample injection or malfunction of equipment.
• measured concentration is higher than ULOQ or lower
than LLOQ, and higher LLQQ in runs compared with
other runs since standard sample at the lowest
concentration is rejected from the calibration curve.
• identification of quantifiable levels of analytes in samples
befare dosing or placebo samples.
• poor chromatography.
For bioequivalence studies, it is generally acceptable to
reanalyze test samples because of pharmacokinetic reasons.
When r~analysis is performed because of positive results of
pre-dose samples or a pharmacokinetic reason, the
information should be provided including the identification of
reanalyzed samples, the initial values, the reason far
reanalysis, the val u es af ter reanalyses, accepted val ues and a
justification far the acceptance.
In case of instrument malfunction, reinjection of samples is
acceptable if reproducibility of reinjection and intra-stability
of injector have been demonstrated during method validation.
However, it is necessary to re-process test samples far
rejected analytical runs.
3. 5 Chromatogram integration
Chromatogram integration and re-integration should be
described in the SOP. Any deviation from this SOP should
be discussed in the analytical report. Chromatogram integra-
tion parameters should be documented in the laboratory and
initial and final integration data should be submitted upon
request in case of re-integration.
3. 6 Incurred sample reanalysis for evaluating method
reproducibility
Actual test samples may not properly be represented by the
use of calibration standards and QC samples during method
validation. Accuracy and precision of the analyte in the
samples may be influenced by many factors during processing
and storage, e. g. , protein binding, back-conversion of
known and unknown metabolites, sample homogeneity or co-
administrated medications. Therefore it is recommended to
evaluate accuracy of real samples by incurred sample
reanalysis in separate runs on different days. The extent of
reanalysis depends on the analyte, test samples, as well as
in-depth understanding of the analytical method and analyte.
It is recommended to re-analyze samples around Cmax and in
the elimination phase. Generally, 10 % of the samples
should be re-analyzed. When the total number of samples is
more than 1000, 5 % of the surplus samples should be re-
analyzed.
For at least 67 % of re-analyzed samples, the difference
between the initial concentration and the concentration from
re-analysis should be within 20 % of their mean value.
When incurred sample reanalysis showed deviating results,
adequate steps should be taken to evaluate and optimize
analytical method.
lncurred sample reanalysis should be performed in the
following situations:
• toxicokinetic studies once per species.
• all pivotal bioequivalence studies.
• clinical trial on drugs used in humans far the first time.
• clinical trial on drugs used in patients far the first time.
• clinical trial on drugs used in patients with impaired
hepatic and/ or renal function far the first time.
For animal studies, incurred sample reanalysis may only be
performed in pivoital studies at early stage of development,
such as studies on the relationship between administered dose
and measured in vivo concentrations.
Section 4 Ligand binding assays
Ligand binding assays ( LBA) are mainly used far macro-
molecules. The validation principles and the considerations
with regard to analysis of test samples indicated befare
should also be applied in general to ligand-binding assays.
However, due to the inherent characteristics and complex
structure of the macromolecules, the extraction process is
problematic thus these assays are often performed without
prior separation of the analyte of interest. In addition the
testing endpoints of these assays are not directly related to
responses from the analyte rather than indirect signals from a
binding reaction with other reagents. In ligand binding
assays, each calibration standard, QC sample and test
sample are evaluated in replicates. Unless otherwise
specified, analysis on duplicate is applied here.
4. 1 Considerations before method validation
4. l. 1 Selection of reference standards
Macromolecules are heterogeneous and their potency and
immunoreactivity may vary. The reference standard should
be well characterized and documented. If possible, reference
standard with the highest purity available at the time should
be procured. It is strongly recommended that the batch of
the reference standard used for the preparation of calibration
standards and QC samples is the same as those used far
dosing in the non-clinical and clinical studies. In case of
change of batch, an analytical characterization and
bioanalytical evaluation should be carried out prior to use to
ensure that the performance characteristics of the method are
not altered.
4. l. 2 Selection of matrix
Generally it is not recommended to use extracted matrix (e. g.
charcoal, immuno-affinity) or alternative matrix (e. g. , protein
buffers, dialysed serum) to replace actual sample matrix to
establish analytical method. In sorne cases, the measurement of
sorne macromolecules may not be possible in a complex matrix
due to high interferences with high levels of structurally related
endogenous compounds. When other quantitative methods are
unavailable at that time, it is acceptable to use a surrogate matrix
for establishment of analytical method and its necessity should be
demonstrated.
The calibration standard curve may be established by a
surrogate matrix. QC samples should be prepared in the
actual sample matrix and the absence of matrix effect should
be reflected by the calculated accuracy.
4. 1. 3 Minimum required dilution
To establish and validate analytical methods, it may be
necessary to dilute the matrix to reduce the high background
signal generated by the matrix. In this case, it is necessary
to determine the mínimum required dilution. The mínimum
required dilution is the lowest dilution to which a sample
must be diluted in buffer to enhance signal to noise ration,
minimize the matrix effect and optimize accuracy and
precision in an assay run. Spiked samples should be prepared
in the same matrix as that far test samples far determination
of the mínimum required dilution.
4. l. 4 Reagents
Critica! reagents, including binding proteins, aptamers,
 9012 Guidelines for Validation of Quantitative Analytical Method of Biological Samples
antibodies or conjugated antibodies and those containing
enzymatic moieties etc. , have direct impact on the results of
the assay and therefore their quality must be assured.
Accordingly, when reagent batches are changed during
validation or sample analysis, the analytical performance of
the method must be verified to ensure that it is not altered
compared with the original or previous batch.
In order to ensure that the performance of the method is not
affected over time, it is necessary to document conditions
guaranteeing the maintenance of the stability of both critica!
and non-critica! reagents ( e. g. , buffers, diluents or
acidification reagents).
4. 2 Method validation
4. 2. 1 Full validation
4. 2. 1.1 Calibration curve and quantitative range
Calibration curve reflects the relationship between concentration of
analyte and the response of instrument The response function of
the calibration curve in ligand binding assays is measured
indirectly, which is generally non-linear and often in a sigmoidal
shape.
At least six calibration standards should be employed to
establish the calibration curve. The calibration standards
should be approximately evenly spaced on a logarithmic scale
within the anticipated range. In addition to the calibration
standards, anchor points outside the range of quantification
can be used to facilitate the fitting of the curve.
At least six independent runs should be evaluated during the
validation. The results must be reported in a table to
establish the overall robustness of the regression model of the
calibration curve. A calibration standard may be excluded
from the curve due to a technical error with an assignable
cause. The deviation of target back-calculated concentrations
of the calibration standards should be less than 20 % from the
nominal value (25% at LLOQ and ULOQ) for at least 75%
of calibration standards. The quantitative range is the
interval between the LLQO and ULOQ of the calibration
curve. The anchor point calibration samples, which are the
calibration standards beyond the quantitative range, are used
to facilitate the fitting of non-linear regression standard curve
of ligand binding analysis. They do not require acceptance
criteria since they are beyond the quantitative range of the
curve.
4. 2. l. 2 Specificity
Specificity is the ability to measure the analyte unequivocally in
the presence of other exogenous or endogenous compounds in the
matrix. Ideally the analyte should be specific such that no cross-
reactivity occurs with structurally "related compounds" or with
anticipated concomitant medication. During method development
and validation, these " related molecules" are frequently not
available. Evaluation of specificity may be conducted after the
original validation is completed. Specificity should be tested with
QC samples by adding increasing concentrations of available
"related molecules" or drugs expected to be concomitantly
administered, into drug-naive sample matrix ( matrix obtained
from animals or subjects never exposed to the analyte) and
measuring the accuracy of the macromolecule of interest at both
LLOQ and ULOQ. Matrix without analyte should also be
measured concurrently. The deviation of measured values from
the nominal values of at least 80 % of the QC samples should be
less than 20% (25% at LLOQ). In addition, measurement of
matrix without analyte should be less than LLOQ.
4. 2. l. 3 Selectivity
Selectivity is the ability to precisely measure the analyte of
interest in the presence of unrelated compounds in the
matrix. Generally there is no extraction due to the inherent
characteristics of macromolecules. Then, unrelated compounds
presented in matrix may interfere with the measurement of the
analyte of interest. Selectivity is tested by spiking at least 10
sources of sample matrix at or near the LLOQ and ULOQ,
and the matrices without analyte should also be measured.
The deviation of measured value should be less than 20 %
(25% at the LLOQ) from the nominal spiked concentration
in at least 80 % of the matrix evaluated. In addition,
measurement of matrix without analyte should be less than
LLOQ. In cases where interference is concentration
dependent, it is essential to determine the minimum
concentration where interference occurs. It may be necessary
to adjust the lower level of quantification accordingly, befare
assay validation. Depending on project requirements, it may
be necessary to test the selectivity of matrix of relevant
disease population or sorne special matrices (e. g. , lipemic
and haemolyzed samples).
4. 2. l. 4 Precision and accuracy
At least five QC samples [anticipated LLOQ, less than 3 times
of the LLOQ, mid (medium of calibration curve), high (more
than 75% of the ULOQ) and anticipated ULOQ] should be
used to assess accuracy, precision and the total error of the
method. Nominal values of low, mid and high concentration QC
samples should not be the same as the nominal value of
calibration sample concentration. For the estimation of precision
and accuracy, QC samples should not be freshly prepared, but
should be frozen and treated in the same way as those for the
analysis of study samples. Measurements should be made across
at least 6 independent assay runs over severa! days. Each run
shouid contain at least 3 QC samples ( at least 5 different
concentrations for each QC sample). Regarding within-run and
between-run accuracies, the deviation of mean concentration of
each QC sample should be less than 20 % from the nominal value
at each concentration level ( 25 % at the LLOQ and ULOQ).
The within-run and between-run precisions ( CV) should not
exceed 20% (25% at LLOQ and ULOQ). Furthermore the
total error (i. e. , sum of absolute value of the % relative error
and % coefficient of variation) should not exceed 30 %
(40% at LLOQ and ULOQ).
4. 2. l. 5 Dilution Iinearity
Because of the narrow range of the calibration standard
curve, it is necessary to demonstrate with QC samples that
the analyte of interest, when present in concentrations
exceeding the range of quantification (above ULOQ), can be
accurately measured by the assay after dilution in blank
matrix to bring the analyte concentrations into the validated
range for analysis. An additional reason for conducting
dilution experiments is to detecta possible prozone or "hook
effect" i. e. , a signal suppression caused by high concentra-
tions of analyte.
The deviation of back-calculated concentration for each dilution
should be less than 20 % from the nominal concentration after
correction for dilution and the precision of the final concentrations
across all the dilutions should not beyond the range of 80%-
120%.
4. 2. l. 6 Parallelism
If test samples are available, parallelism between the
calibration standard curve and serially diluted test samples
should be assessed to detect possible matrix effects or
differing affinities for metabolites. A high concentration
study sample ( preferably exceeding ULOQ) should be
diluted to at least three concentrations with blank matrix.
The precision ( CV) between samples in a dilution series
should not exceed 30 %. In case the sample is not diluted
linearly ( i. e. , in a non parallel manner) , a procedure for
 9013 Guidelines for Sustained, Controlled and Delayed Release Preparations
reporting a result should be defined a priori. If test samples
are not available during the validation of the method,
parallelism should be evaluated as soon as test samples
become available.
4. 2. l. 7 Stability of the samples
Stability of the analyte is evaluated using the low and high
concentration QC samples. The investigation of stability
should cover short-term stability at room temperature or
sample processing temperature and freeze-thaw stability. In
addition, long-term freezer stability should be studied at each
temperature at which test samples will be stored. The
deviation of mean concentration of at least 67 % of QC
samples at each concentration should be less than 20 % from
the nominal value.
4. 2. l. 8 Commercial kits
Commercial kits may be used for sample analysis, however,
they must be validated following the requirements of this
guideline befare use.
4. 2. 2 Partial validation and cross-validation
All the validation aspects reported in previous sections 2. 2
and 2. 3 are applicable to ligand binding assays.
4. 3 Analysis of study samples
4. 3. 1 Analytical ron
Most often microtiter plates are used for LEA. An analytical
run generally corresponds to one individual plate. Each plate
should contain an individual set of calibration standards and
QC samples to compensate far difference in plate
performance. The sample capacity in sorne platforms may be
limited. Then, it may be acceptable that a set of calibration
standards be placed in the first and the last platforms and QC
samples on every single platform. It is recommended to
assay a study sample in replicate.
4. 3. 2 Acceptance criteria for test sample analysis
The deviation of back calculated concentrations of the
calibration standards should be less than 20 % from nominal
value, except for LLOQ and the ULOQ for which it should
be less than 25%. At least 75% of the calibration standards,
with a minimum number of six, must fulfill this criterion.
Each plate should contain at least three ( low, medium and
high) concentrations of QC samples at least in duplicate.
Also during study validation, the QCs should mimic the
analysis of the test sample with regard to the number of wells
used per test sample. The deviations of at least 67 % QC
samples on each plate and 50 % at each concentration should
be less than 20 % from the nominal values.
4. 3. 3 Actual sample reanalysis
All the considerations regarding the incurred sample reanalysis
reported in previous section 3. 6 are applicable to ligand binding
assays. The deviations of concentration obtained for the initial
analysis and reanalysis should be less than 30% of their mean for
at least 67 % of the repeats.
Section 5 Reports
S. 1 Validation report
Depending on the level of detail of the information provided
in the validation report, reference to the SOPs for relevant
analysis specific procedures may be sufficient. Otherwise
these SOPs should be appended to the report.
All source data should be available in its original format and
available on request.
Any deviation from the validation protocol should be recorded.
The validation report should include at least the following
informati o ns:
• summary of the validation performances;
• details of the applied analytical method and where appropriate,
the source of the analytical method;
• details of the assay procedure (analyte, IS, sample pre-
treatment, extraction and analysis) ;
• reference standards (origin, batch number, certificate of
analysis, stability and storage conditions);
• calibration standards and OC samples (matrix, anticoagulant if
applicable, preparation, preparation dates, and storage
conditions);
• run acceptance criteria;
• analytical run: table of all analytical runs including calibration
range, res¡x:>nse function, back-calculated concentrations, and
accuracy; table of OC results of all accepted analytical runs;
stability data of stock solution, working solution, OC, covering
the applied storage conditions; data on selectivity, um, rany-
over, matrix effect if applicable, dilution integrity;
• unexpected results obtained during validation with full
justification of the action taken;
• deviations from method and/ or SOPs.
All measurements with the individual calculated concentrations
have to be presented in the validation report.
S. 2 Analytical report
The analytical report should include a reference to the
validation report applicable to the analysis of the study
samples. Furthermore it should include a detailed description
of the analysis of the study samples.
All source data should be available in its original format and
available on request.
Any deviation from the protocol, analytical procedure or
SOPs should also be discussed in the analytical report.
The analytical report should include at least the following
informati o ns:
• ref erence standards;
• calibration standards and OC samples (storage conditions);
• run acceptance criteria ( short description, reference to
specific protocol or SOPs) ;
• sample tracking (dates of receipt and contents, sample
conditions on receipt, storage location and conditions, if
applicable) ;
• study sample analysis: table identifying all analytical runs
and study samples, with run dates and results; table of
calibration results of all (previous) analytical runs; table
of QC results of all ( previous) analytical runs; values
outside acceptance criteria should be clearly marked;
• failed analytical runs ( identity, assay date, reason for
failure);
• deviations from method and/or SOPs;
• reanalysis results.
The results of incurred sample reanalysis may be supplied
either in the validation report, in the analytical report or in a
standa lone report.
Far bioequivalence studies, all chromatograms from the runs
of the subjects, including the corresponding QC samples and
calibration standards should be appended to the analytical
study report.
9013 Guidelines for Sustained,
Controlled and Delayed
Release Preparations
Compared with ordinary preparations, sustained and controlled
release preparations have long therapeutic effects, low adverse
effects and decreased dosing frequency. According to the
requirements of the design of the preparations, drugs in these
 394 9013 Guidelines for Sustained, Controlled and Delayed Release Preparations
preparations can release in mw slowly, and plasma drug
concentrations show less peak-valley fluctuation, thus the
adverse effects caused by extra amount of drugs beyond the
therapeutic range ( therapeutic window) can be avoided and
efficacy can be maintained by the effective concentration in the
therapeutic range. Sustained and controlled release preparations
also include ocular, nasal, ear, vaginal, rectal, oral cavity or
dental, transdermal or subdermal, intramuscular and subdermal
implant preparations, which can slow the release and absorption
of drugs and avoid the " first pass effect" of portahepatic
system. Delayed release preparations are those which do not
release drug rapidly after administration, such as intestinal-
specific drug delivery systems or colon-specific drug delivery
systems that can avoid deactivation in stomach or irritations of
drugs to stomach. Delayed release preparations also include
pulsatile release systems that burst release their drugs under
certain conditions.
Release theories of sustained, controlled and delayed release
preparations mainly include controlling dissolution, diffusion,
corrossion or a combination of dissolution and diff usion,
sometimes also include osmotically controlled and ion-
exchange mechanism. Releasing processes can be fitted using
different equations, such as first-order kinetic, Higuchi and
zero-order kinetic equations, etc. The main difference
between sustained release and controlled release is that
sustained release preparations do not release their drugs at a
steady rate, i. e. its release rate is not constant but declines
with time; the controlled release preparations release their
drugs display zero-order kinetic pattern of which the release
ratc maintains constant ovcr time. i\..dministration of thcsc
controlled preparations results in a considerably steady
plasma drug concentration with little peak-valley fluctuation
till the complete absorption. Usually sustained and controlled
release preparations include the larger amount of drugs than
the corresponding ordinary single dose preparations and
involve complex processes for manufacture. In arder to
generate reliable therapeutic effects and reduce adverse
effects caused by burst releasing, measures should be taken
to avoid or reduce burst releasing in the process of
preparation design, pilot production and manufacturing. In
vitro and in vivo release profiles and performances of these
preparations must accord with clinical requests and be devoid
of effects by few, if any, physiological or food factors. On
the other hand, in order to control the quality and ensure the
safety and efficacy of the preparations, release profile testing
in vitro must reflect the basic conditions in vivo.
While the following guidelines will focus on sustained, controlled
and delayed release oral preparations, the principies may be
applicable to other routes of drug administration.
Definition of sustained, controlled and delayed release
preparations
l. Sustained release preparations They are a kind of
preparations which slowly release drug or drugs at a non-
steady rate in a stipulated medium, and the dosing frequency
should be reduced to half of or less than that of the
corresponding ordinary preparations, thus can evidently
improve the patient' s compliance.
2. Controlled release preparations They are a kind of
preparations which slowly release drug or drugs at a steady
rate in a stipulated medium, and the dosing frequency should
be reduced to half of or less than that of the corresponding
ordinary preparations, the plasma drug concentration should
be more steady than that of sustained release preparations,
thus can evidently improve the patient' s compliance.
3. Delayed release preparations They are a kind of
preparations which do not release drugs rapidly after
administration, including intestinal dissolution preparations,
colon-specific preparations and pulsatile release preparations.
Enteric-coated preparations are a kind of intestinal specific
preparations which do not or almost do not release drugs in a
certain acidic solution within a fixed duration, but can release
drugs completely or almost completely in phosphate buffer
solution pH 6. 8.
Conlon-specific preparations are a kind of preparations which
do not release drugs at the upper part of the gastrointestinal
tract, but can release most or all of drugs in the colon.
Therefare they basically can not release the drugs in an acidic
medium or a phosphate buffered solution of pH 6. 8 within a
fixed duration, but can release most or all of the drugs in a
phosphate buffer solution pH 7. 5-8. O.
Pulsatile release preparations are a kind of preparations which
do not release drugs rapidly after administration but can
burst release once or several times under certain conditions
(such as in body fluid far a period of time, certain pH value
or the effect of sorne enzyme).
Drug release test in vitro
This test is conducted under the imitative condition of
gastrointestinal environment in mvo (such as temperature,
pH value of the medium and stirring speed, etc. ) to
investigate the drug release rate of the preparations. And
then finally, on the basis of the testing, to establish a
rationalized release profile in vitro which can be used for
supervision of the manufacturing process and quality control.
L A..pparatus Unless otherwise specified, release test in 7..ntro
of sustained, controlled and delayed release preparations can be
carried out with dissolution determination apparatuses.
Patches can be tested by release test methods ( 0931 , method
3) and the results should comply with the requirements.
2. Temperature cantrol The release testing of sustained,
controlled and delayed release preparations should be conducted
at 37 ± O. 5ºC approximating to the body temperature, but
patches at 32 ± O. 5ºC approximating to the temperature of
epidenn.
3. Release medium Deaerate freshly prepared purified
water is regarded as the commonly used release medium; if
substantiated by the solubility characteristic of the drug
substance, the formulation or the absorption site, dilute
hydrochloric acid ( O. 001 mol/L to O. 1 mol/L) or
phosphate buffer solution pH 3 to 8 may be used; far sorne
drugs with limited solubility use of organic solvents is not
favored, but addition of a small amount of surfactants (such
as sodium lauryl sulfate) is allowed.
The volume of the release medium should conform to the
sink condition.
4. Time points of sampling far release test Except that for
delayed release preparations, release profile testing in vitro
should reflect the characteristics of the variation of the
release profile of the tested preparations and meet the needs
of statistic analysis. The complete release time duration
should not be less than the interval time of dosing frequency
and the generated cumulative release rate should be more
than 90 %. Unless otherwise specified, usually the data of
the entire releasing process are utilized to make a curve of
cumulative release rate versus time, thus the rational release
rates are worked out.
From the release rate curve at least three test time points are
chosen to characterize the in mtro drug release profile for
sustained release preparations. An early time point, usually
O. 5-2 hours is chosen to show that the potential burst release
 9013 Guidelines for Sustained, Controlled and Delayed Release Preparations
is not happened. An intermediate time point is chosen to
define the character in vitro release profile of the dosage
form, and a final time point is chosen to convey essentially
complete release of the drug. These three points can be used
to convey the drug release profile in vitro as a whole.
For controlled release preparations two additional test time
points are needed, and these five points can be used to convey
the drug release profile of controlled release preparations in
vitro. The range of percentage of drug release amount should
be smaller than that of sustained release preparations. If
necessary, more test time points can be added.
The test time points of delayed release preparations are
chosen according to the clinical requirements.
Far products containing more than a single active ingredient,
every main active ingredient should be tested according to the
above requirements.
5. Test on reproducibility and uniformity of manufacturing
process Batch-to-batch reproducibility for the release profile
in vitro of over three batches ( six tablets or pills for each
batch) should be examined. The uniformity for the release
profile in vitro of the same batch product (six tablets or pills
for each batch) should be examined.
6. Model fitting for drug release The data of drug release
can be fitted using first-order equation and Higuchi equation:
ln (1-M1 /Moo) = - kt (first-order equation)
Mi/Moo = kt 112 (Higuchi equation)
The data of drug release of controlled release preparations
can be fitted using zero--order equation:
M 1 /Moo = kt (zero--order equation)
Where M 1 is the cumulative release amount at time t;
Moo is the cumulative release amount while time
tends to=;
M 1 /Moo is the percentage of cumulative drug release
amount at time t. The fitted results of the
largest regression coefficient ( r ) and the
smallest mean square error ( MSE ) are
regarded to be the best.
Tests on sustained, controlled and delayed release preparations
in vivo
In vivo pharmacodynamic and pharmacokinetic tests should be
used to evaluate the safety and efficacy of sustained, controlled
and delayed release preparations. First of all, the special
physicochemical characters of the drugs in sustained, controlled
and delayed release preparations should be studied completely,
which include polymorphism, particle size and distribution,
solubility, dissolution rate, stability and variables controlling the
drug release under extreme conditions of physiological environ-
ment. Sorne physicochemical characters of drugs in preparations
(such as solubility) may be affected by the prescription, so
solubility of drugs under related conditions should be tested. For
the drugs with limited solubility, if any surfactant ( such as
sodium lauryl sulfate) is used in their prescription, it is
necessary to understand their solubility.
For the pharmacokinetic characters of drug, the ordinary
preparations of the drug (vascular or oral solution, or other
approved preparation) are recommended as reference to recognize
and evaluate the release and absorption of sustained, controlled
and delayed release preparations. During the designing course of
oral sustained, controlled and delayed release preparations, it
is significant to test the drug absorption in each section of
gastrointestinal tract ( especially for absorption in colon
section of colon-specific drug delivery system). lnfluence of
food should also be considered.
The pharmacodynamic characters of drug should reflect the
relationship in sufficiently extensive dosage range between
395
the plasma drug concentration and clinical response ( therapeutic
effect or side effect). In addition, the characteristics of
equilibrium time between plasma drug concentration and clinical
response should be studied. If there is a well-defined relationship
between the plasma concentrations of drug or metabolite and
clinical response, the clinical perfonnance of sustained,
controlled and delayed release preparations could be characterized
by plasma concentration-time data, otherwise, clinical tests and
pharmacokinetics-pharmacodynamics tests should be carried out.
Details of bioavailability and bioequivalence testing of
sustained, controlled and delayed preparations can be seen in
Guidelines for in vivo Bioavailability and Bioequivalence
Studies for Drug Preparations <9011 > under the guidelines
on bioavailability in vivo and bioequivalence of preparations.
Stimulation and/ or allergy at their action sites should be
examined for non-oral sustained, controlled and delayed
release preparations.
In vivo-in vitro correlations
Methods for establishment of in vivo-in vitro correlations
In vivo-in vitro correlations refer to the establishment of a
rational quantitative relationship between a biological property
produced by a dosage form, or the parameters (e. g. ' t max ' emax
or AUC ) derived from the biological property, and the
physicochemical property or characteristic ( such as release
profile in vitro) of the same dosage form.
For sustained, controlled and delayed release preparations, a
test on correlation between drug release in vitro and
absorption in vivo should be carried out. It should reflect the
relationship between the whole release curve in vivo and the
whole plasma concentration-time curve which determines the
correlation. Only when the correlation between release in
vitro and absorption in vivo exists, the situation in vivo can
be predicted by the release curve in vitro.
There are three situations involved in the in vitro--in vivo
correlation: CD Each corresponding time point on release in
vitro curve and absorption in vivo curve (cumulated by drug
plasma concentration data) is correlated, respectively. This
is called the point-to--point correlation, showing that the two
curves are coincident. CZ)The correlation between the average
release time in vitro and the average delay time in vivo is
established by using the statistical moment analysis
principie. Because the average delay time of close values may
be produced by many different curves in vivo, the average
delay time in vivo can not indicate the whole plasma
concentration-time curve in vivo. ®Single point correlation
between a release time point ( tso% , t 9a% etc. ) and a
pharmacokinetic parameter (e. g. , AUC, e max or t max) only
indicates the partial correlation.
Methods adopted by these guidelines
These guidelines on the in vitro-in vivo correlation of
sustained, controlled and delayed release preparations admit
that, when each time point on the absorption curve of the
absorbing phase in vivo and each corresponding time point on
the release curve in vitro are regressed, if the correlation
coefficient of the obtained linear regression equation meets
the corresponding requirements, correlation can be
concluded.
l. Establishment of in vitro-in vivo correlation
( 1) In vitro release curve of cumulative release percentage in
vitro versus time If the release profile of sustained, controlled
and delayed release preparations changes with external
conditions, two types of samples should be prepared ( one
with slower release and the other one with faster release than
the original preparation). The external conditions affecting
the release speed should be investigated first. Then,
 9014 Guidelines for Microparticle Preparations
according to the optima! conditions determined by the release
test in vitro, in vitro release curve of cumulative release
percentage in vitro versus time can be drawn.
(2) In vivo absorption curve of absorption percentage in
vivo versus time Based on plasma concentration-time curve
generated from the single dose crossover test, in vivo
absorption curve of absorption percentage in vivo versus time
can be determined for the drug of which the in vivo
absorption fits with one-compartment model. The in vivo
absorption percentage CF.) of the drug at any time can be
calculated by the following Wangner-Nelson equation:
F.= (et +kAUCo-1) /(kAUCo-oo) X 100%
Where ci is the plasma concentration at time t.
k is the elimination rate constant.
The absorption percentage at each time point of drug with
two-compartment model can be calculated by simplified Loo-
Rigelman equation.
2. Examination of in vi.w-in vi.tro correlation When the release
of drug in vi.tro is the rate-determining step of the absorption
process in vi.w , the release percentage on the release curve in
vi.tro and the absorption percentage of each corresponding time
point on the absorption curve of the absorbing phase in vi.w of the
same batch samples are regressed by the method of linear least
square regression principle, thus the linear regression equation can
be obtained.
If the correlation coefficient of the line is larger than the
critica! correlation coefficient ( P <O. 001 ) , the in vivo-in
vitro correlation is confirmed.
9014 Guidelines for Microparticle
Preparations
Microparticle preparations, also named microparticle drug
delivery system ( MDDS), are salid, liquid, or gaseous
preparations which consist of particles made from drugs or/
and fine carriers (commonly are biodegradable materials) in
certain particle size ( micron scale or nano scale ) using
specific dispersion and embedding techniques. They belong to
a large category of new drug preparations designed to cover
up unpleasant odor and taste, solidify liquid drugs, reduce
incompatible changes of compound preparations, increase
solubility of insoluble drugs or drug bioavailability, improve
drug stability, reduce drug adverse reactions, delay drug
releas e, or increase drug targeting releas e, etc.
According to the classification principle of pharmaceutical
dispersion system, the dispersion systems with unit diameters
ranged from 10- 4 m to 10- 9 m are classified as particle
dispersion systems. Among them, the dispersion with units
diameters from 1 µm to 500 µm are MDDS of thick ( micron)
dispersion systems, including microcapsules, microspheres,
and sub-microemulsions, etc. ; dispersion systems with unit
diameters less than 1000 nm are MDDS of nano-dispersion
systems, including liposomes, nano-emulsions, nanoparticles,
and polymeric micelles, etc. Microcapsules, microspheres, sub-
microemulsions, liposomes, nano-emulsions, nanoparticles, and
polymeric micelles can be used as drug carriers.
With the development of modem preparation techniques,
preparations of microparticle carriers are increasingly used in
clinics. The drug administration routes include externa! use,
oral, and injection. Microparticle preparations for externa! use
and oral dosing generally increase drug permeability across
biomembranes, such as skin, mucosa, etc. Microparticle
preparations for injections generally possess delayed, controlled,
or targeted release characteristics. Drug preparations with
targeted characteristics are usually named targeted preparations.
Targeted preparations belong to a category of new prepara-
tions that use carrier to concentrate the drug from circulatory
system in or near the targeted organs, tissues, cells and
intracellular structures. They can enhance the therapeutic
effects and significantly decrease the adverse drug reaction on
other tissues, organs and the whole body. Targeted
preparations can be divided into three types: CD Targeted
preparations of the first class are those which deliver the drug
in the capillary vessel bed of the targeted organs; @Targeted
preparations of the second class are those which deliver the
drug in specific cells ( e. g. , tumor cells) of the targeted
area without influence on normal cells; @ T argeted
preparations of the third class are those which act on a certain
area in the cells.
Types of drug carriers
l. Microcapsules Microcapsules are extra fine capsules
produced by coating the solid or liquid drug with a thin layer
of carrier excipients. Usually their size ranges from 1 µm to
250 µm, while those of which the size ranged from O. 1 µm
to 1 µm are known as sub-microcapsules, and those from
10 nm to 100 nm as nanocapsules.
2. Microspheres Microspheres are extra fine spherical solid
entity in which the drug dispersed or dissolved in the carrier
excipients. Usually their size ranges from 1 µm to 250 µm,
while those of which the size ranges from O. 1 µm to 1 µm are
known as sub-microspheres, and those from 10 nm to
100 nm as nanospheres.
3. Liposomes Liposomes are extra fine vesicles formed by
entrapping drug in the molecular bilayers of lipids. They are
divided into unilamellar and multilamellar vesicles. Usually
small unilamellar vesicles are O. 02-0. 08 µm in size and large
unilamellar vescicles are O. 1-1 µm in size; multilamellar are
1-5 µm in size. Usually small unilamellar vescicles are known
as nano-liposomes. Pro-liposomes are the precursor form of
liposomes. Phospholipid is generally, in a form of thin film,
absorbed onto the surface of skeleton to form powders.
When dispersed in an appropriate solvent, phospholipid can
exists as the molecular state and liposomes can be reformed
by hydrating with diluents befare use.
4. Sub-microemulsions Sub-microemulsions are dispersion
systems of oil-in-water microparticle drug carriers a sizes
ranging from 100-600 nm, in which drug emulsified by fatty
oil/vegetable oíl is dispersed by phospholipids in the water
phase. Size ranges of nano-emulsions are from 50-100 nm.
Dry emulsions are solid freeze-dried preparations made from
sub-microemulsions or nano-emulsions by lyophilization
techniques. This category of product can produce uniform
sub-microemulsions or nano-emulsions by hydrating and
dispersing with appropriate diluents.
5. Nanoparticles Nanoparticles are salid particles of drugs
or/ and carrier excipients at size less than O. 5 µm that are
dispersed by nanostructure techniques. Nanocrystals or nano-
drugs are nanoparticles with only drug molecules, albumin
nanoparticles are those formed by albumin as a drug carrier, and
lipo-nano-particles are formed by lipomaterial as a drug carrier.
6. Polymeric micelles Polymeric micelles, also called
high molecular micelles, are micelle solutions at size less
than O. 5 µm formed by amphiphilic block polymer carrier
excipients after self-assembling and embedding insoluble
drugs in water, which are systems with thermodynamic
stability.
Excipients in common use
Usually excipients can be divided into three types:
 9015 Guidelines for Studies and Quality Control of Drug Polyrnorphism
l. Natural materials Bioconsistent and biodegradable
natural materials include gelatin, protein ee. g. ' albumin) '
starch, chitosan, alginate, phospholipid and cholesterol,
fatty oil, vegetable oil, etc.
2. Semisynthesized materials They are divided into two
types: biodegradable and nonbiodegradable materials in
vivo. The biodegradable materials include hydrosoybean
phospholipid, polyethylene glycol distearic phospholipo
ethanolamine, etc. Nonbiodegradable materials include
methylcellulose, ethylcellulose, carboxymethylcellulose salt,
hydro:xypropylmethyl cellulose, cellulose acetate phthalate, etc.
3. Synthesized materials They are divided into two types:
biodegradable and nonbiodegradable materials in vivo. The
biodegradable materials that are considerably widely used
include poly-lactic acid, poly-amino acid, poly-hydroxybuturate,
poly-lactide-glycolide, etc. Nonbiodegradable materials include
polyamide, polyvinyl alcohol, Eudragin, and silicon rubber, etc.
In addition, wetting agents, emulsifiers, antioxidants or
surfactants can be added for preparing microcapsules,
microspheres and liposomes.
Testing items for controlling manufacturing processes and
during storage
l. Limit test of harmful organic solvents When harmful
solvents are introduced to the manufacturing processes, the
residual solvent should be determined according to the
Determination of Residual Solvents ( 0861 ) . lf no limits have
been stipulated, relevant ICH guidelines can be consulted, or
a method for determining the solvent and the limit should be
established.
2. Tests for shape, particle size and its distribution
C1 ) Shape observation Microcapsules, microspheres and
liposomes can be observed under a optical, scanning or
electric microscope. Photos should be provided in all the
cases.
(2) Particle size and its distribution The average value of
particle size and the data or graphs of its distribution should
be provided. There are several methods to determine the
particle size, such as optical microscopy, electrical induction
method, light induction method or laser scattering method,
etc.
The data of particle size distribution of rnicroparticle preparations
are often expressed by the partide numbers or percentage in
different particle size ranges; sometimes span is employed
instead. The smaller the span is, the narrower the distribution
is, 1. e. , the particles are more uniformed in size.
Span= (Dgo -Dio) /Dso
Where Dio, D 5o, Dgo are diameters corresponding to the
positions of 10 %, 50 %, 90 % in the accumulated distribution
graph, respectively.
If graphs are necessary, the partid e size is plotted as abscissa
against the frequency ( the percentage of particle numbers in
different size ranges in the total number of the particles) as
the ordinate to produce the histogram of size distribution.
The particle size distribution curve can be obtained by
plotting the frequencies of diff erent particle size ranges
against the mean values of different particle size ranges.
3. Examination of drug content or entrapment rate The
data of drug content or entrapment rate must be provided for
microparticle preparations.
Drug content is the percentage of drug by weight in the
microparticle preparations:
drug wt. in the system % suspensions, etc. To ensure the controllability of efficacy,
D rug content= X 100 o
total wt. of the system
Where wt. is the weight of the drug.
If microparticle preparations are dispersed m the liquid
medium, determination should be performed after using
proper separation methods esuch as gel column chromatography'
centrifugation or dialysis) , then the following equation is used to
calculate the entrapment rate:
trapped drug wt. in the system X %
Entrapment rate ld . h 100 o
tota rug wt. m t e system
= (l untrapped drug wt. i~ the liquid medium) X 100
%
total drug wt. m the system
Entrapment rate should not be less than 80 % in general.
4. Examination of burst effect or leakage rate Drug m
microparticle preparations are commonly trapped by
adsorption, entrapment or implantation. The drug absorbed
on the surface of microcapsules, microspheres and liposomes
releases rapidly when tested in vitro, which is called burst
effect. Released amount should be less than 40 % in the first
o. 5 h.
If the product of microparticle preparations are dispersed and
stored in the liquid medium, the leakage rate should be
calculated according to the following equation:
Leakage rate = drug amount leaked into the medium
after a period of storage X lOO %
trapped drug amount befare storage
5. Examination of oxidation degree The oxidation degree
of microparticle preparations with carrier excipients such as
phosphatide, vegetable oil etc. which may be easily oxidated
need to be examined. There are three stages for the oxidation
of phosphatide in the lipid mixed with unsaturated fatty acid:
the coupling of single double bond, the formation of
oxidation products, and the formation of aldehyde and the
rupture of bond. The oxidation degree is difficult to be
evaluated by just one test, because the products formed in
various stages are different.
The oxidation degree of phosphatide, vegetable oil or other
carrier excipients that may easily be oxidized need to be
examinedby appropriate methods and a control index should
be proposed.
6. Other requirements Besides the requirements in this
guideline, microparticle preparations should also comply with
general requirements described for guidelines of the
corresponding preparations (e. g. , tablets, capsules, injections,
ophthalmic preparations, nasal preparations, patches, aerosols,
etc.).
Sustained, controlled and delayed release preparations made
from microparticle preparations should comply with the
Guidelines of Sustained, Controlled and Delayed Release
Pre parations ( 9013 ) .
7. Evaluation of targeted drug delivery systems For
microparticle preparations with targeting characteristics,
related data on targeted release, such as drug distribution
data in vivo and pharmacokinetic parameters of distribution
in vivo, should be provided.
9015 Guidelines for Studies and
Quality Control of Drug Polymorphism
When a solid drug shows polymorphism that can affect
efficacy, safety, and quality of the drug, it is necessary to
perform qualitative and quantitative controls on crystalline
states of drug in salid preparations, semisolid preparations,
safety, and quality, the preponderant crystalline state should
be selected as crystalline state of drug in the clinic, which
should also be preserved in drug preparations.
 9015 Gu,idelines for Studies and Quality Control of Drug Polymorphism
When multiple crystalline states exist for a drug, the
preponderant crystalline state is the form with good clinic
efficacy, nice safety profiles, and high stability, which is
also suitable for drug development.
l. Basic concepts of drug polymorphism
In so lid state chemistry, drug polymorphism is used to
describe the state of material by an array of parameters (e. g. ,
lattice parameters, molecular symmetry, arrangement rules,
molecular force, molecular conformation, crystal water or crystal
solvent). When the change of one or sorne of these parameters
leads to the co-existence of two or more states of the solid
substance, this phenomenon is named polymorphism or
allomorphism. In general, polymorphism is more easily
observed for drugs with poor solubility.
A solid substance is formed by molecular stacking. Due to
the different ways of molecular stacking, there are crystalline
ecrystal ) and noncrystalline eamorphous ) forms in the
substance. Molecular stacking in the crystalline material is
sequential, symmetrical and periodic, while molecular
stacking in the amorphous material is disordered. Crystal
form of material covers both crystalline (ordered molecules)
and noncrystalline forms ( disordered molecules) of solid
compounds.
A preponderant polymorph may be one form or several
forms. One of theses or a mixture of two or more crystal
forms with a certain proportion can be selected as the
polymorph of the drug.
2. Preparation of polymorphic samples
Solid crystalline samples in different polymorphs can be
obtained by changing the crystallization conditions through
chemical or physical methods. Common chemical methods
include recrystallization, rapid solvent removal method,
precipitation method, and seeding with diamond particles,
etc. ; common physical methods include melt crystallization,
lattice destruction and physical crystal transformation, etc.
Direct and indirect methods can be used to prepare
polymorphic samples. Sorne important technical parameters
aff ecting the formation of polymorphic substances include
solvents e e. g. ' category, compos1t1on, proportion)'
concentration, nucleation rate, growth rate, temperature,
humidity, luminosity, particle size and so on. Due to
structural diversity of drug structures, parameters for the
formation of polymorphic substances are also different.
Preparation methods and conditions should be properly
selected according to the properties of the drug.
3. Stability of polymorphic fonns
There are three states of solid substances in nature, stable,
meta-stable, and unstable states, which can be applied to
polymorphic substances. Crystal transformation is the
change of the polymorphic form to another dueto alterations
of environmental conditions (e. g. , temperature, humidity,
luminosity and pressure).
Since the stability of a polymorph of the drug can influence
drug efficacy and safety in the clinic, it is necessary to
perform a stability study on the polymorph of the drug in the
preparation showing polymorphism. The study should
include: stability of crystal forms of chemical ingredients;
compatibility of crystalline chemical ingredients with
pharmaceutic excipients of the preparation; the effects of the
processing techniques Ce. g. , granulation, moulding and drying)
for drug preparations on crystal form of the substance.
Stability studies on crystalline substance can provide
scientific evidence for the selection of preponderant polymorph of
drug, formulation of the preparation, control of manufacturing
process and storage conditions of drug, etc. Only the substances
showing stable or metastable crystal forms are drugcandidates.
According to testing conditions and methods for affecting
factors in the stability study, the stability of crystalline
substances should be investigated under high temperature,
high humidity and illumination. The pressure method is used
to evaluate the stability of crystalline substance under pressure
and determine whether crystal transformation occurs.
4. Biological evaluation of crystalline drugs
Proper experimental methods should be applied to perform
biological evaluation on crystalline substances. The actual
biological characteristics of salid crystalline substances can
not be reflected by in vitro cell-based method with aqueous
solution or in vivo administration of a suspension where
crystal transformation has occurred. Therefore, an in vivo
animal study administrated with solid drug should be used
to acquire actual biological evaluation data of crystalline
drug.
5. Solubility or dissolution evaluation of crystalline drugs
This method is an auxiliary approach for the in vitro
evaluation of crystalline substances.
When differences in crystal forms are observed for crystalline
drug substances and preparations, solubility or dissolution of
crystalline substances and solid preparations may show significant
variations. Therefore, studies should be conducted to investigate
the relationship between drug polymorphism and solubility or
dissolution using solubility rate or dissolution rate as the
evaluation index. The solubility curve is used for the
chemical ingredients and the dissolution curve for the solid
pharmaceutical preparation.
6. Quality control of drug polymorphism
Different crystal forms in diverse drugs may display identical
or altered specificity to qualitative or quantitative methods.
Effective methods for quality control can be absolute and
relative methods.
(1) ldentification of polymorph-Qualitative methods
Absolute identification methods: these methods may be used
for identification independently, which are only applied to
crystalline drug substances.
Single crystal X-ray diffraction ( SXRD): it belongs to
absolute identification methods. Many parameters, including
compositions ( compounds, crystal water and solvent) , cell
parameters, molecular symmetry ( crystal system, space
groups)' molecular bonding ehydrogen bond, salt bridge,
coordination bond), and molecular conformations, can be
used for identification. The method is applicable to identify
crystal form of crystalline substances.
Relative identification methods: these methods may be used
for identification with the help of available information on
polymorphs of drug substances. These methods are applied
to polymorph identification where there are differences shown
in the spectrogram data. The polymorph of the test
substance is identified by comparing with the spectrogram
data of known polymorph of samples. These methods are
only applied to crystalline drug substances.
Method 1: Powder crystal X-ray diffraction (PXRD)
Sharp peaks are observed in the PXRD spectrum of the
crystalline substance, while dispersing peaks in those of the
amorphous substance. The identification of the polymorph is
based on the number of diffraction peaks, their positions (2()
or d ) and intensity ratios. This method is suitable for
comparison between multiple forms of crystalline substances,
such as crystalline to crystalline forms, crystalline to amorphous
forms, amorphous to amorphous forms. If two crystal forms
 9015 Guidelines for Studies and Quality Control of Drug Polymorphism
of crystalline drugs are determined to be identical, the
following criteria are fulfilled: they should have same
number of diffraction peaks, the difference between 2fJ
positions of diffraction peaks within ± O. 2º , the difference
between relative intensity of diffraction peaks at the same
position with in ± 5 %, and an identical sequence in peak
intensities. If two crystal forms of amorphous samples are
determined to be identical, their geometric topological shapes
of dispersing diffraction peaks should be identical.
Method 2: lnfrared spectroscopy (IR)
Crystal form of test substances can be determined by specific
changes of dipole moments induced by in molecular
vibrations, which is reflected in alterations on absorption
peaks in IR spectrum at the predefined range of wavelength,
including positions, intensities, and geometric topological
parameters, etc. This method is applicable to identify crystal
form of substances under the control of molecular force
changes. The attenuated total reflection sampling method is
recommended. Grinding and tableting should be avoided to
prevent crystal transformation.
Method 3: Raman spectroscopy (RM)
The crystal form of test substances can be determined by
specific changes of molecular polarizability, which is reflected
by alterationson peaks in Raman spectrum at the predefined
range of wavelength, including positions, intensities, and
geometric topological shape parameters, etc.
Method 4: Differential scanning calorimetry (DSC)
The crystal form of test substances can be determined by specific
changes of thermodynamic properties, which is reflected by
alterations on endothermic or exothermic peak in term of
number, position, shape, and endothermic amount (endothermic
enthalpy), etc. This method is applicable to identify crystal form
of substances showing large variations in molten endothermic peak
values or with different number and category of crystal solvents
(water).
Method 5: Thermogravimetry (TGA)
The crystal form of test substances can be determined by specific
changes of weights, which is reflected by alterations on parameters
of the relationship between weightlessness percentage and
temperature. This method is applicable to identify crystal form of
substances with different number and category of crystal solvents
(water).
Method 6: Capillary melting point (MP)
The crystal form of test substances can be determined by
phase transition and light transmittance during heating. This
method is applicable to identify crystal form of substances
with large variations in melting point. The melting range can
reflect the purity of crystal form. If the melting range is less
than 1 ºC, it indica tes that the polymorph of test substance is
highly pure. Grinding should be avoided to prevent crystal
transforma tion.
Method 7: Light microscope (LM)
When different crystal forms exhibit different characteristics
of salid appearances, these solid appearances can be used to
identify crystal form of test substances.
Method 8: Polarizing microscope ( PM)
The crystal form of test substances can be determined by the
changes of polarized effect related parameters between
crystalline and amorphous forms.
Identification of polymorph
When chemical constituent of drug substance is defined and
the same analytical method is applied, altered spectrum or
data suggests the change of types or composition of
crystalline material in the samples, which results from
transition between two polymorph or category or proportion
changes in mixed polymorphs.
( 2) Content analysis of drug polymorph-Quantitative methods
Content analysis of drug polymorph is to determine the
content of substance with certain polymorphs in the test
sample, which is manifested by percentage. The analytical
method of drug polymorph is the quantitative or limit
analysis on crystalline substance.
Quantitative methods are recommended for quality control of
drug polymorph. Quantitative methods include single crystal
X-ray diffraction, powder crystal X-ray diffraction,
diff erential scanning calorimetry and infrared spectroscopy,
etc.
Method validation
The quantitative analysis or limit detection of crystalline
substance should meet the requirements of accuracy,
repeatability, specifícity, limit of detection, linearity, linear
range and robustness defined in Guidelines for Validation of
Analytical Method Adopted in Pharmaceutical Qu,ality
S pecification ( 9101 ) .
Because of different basic principies employed by quantitative
analysis or limit detection methods, one to three
characteristic parameters should be selected to define the
linear relationship of crystalline substance and content.
Analytical method to quantify drug polymorph
Method 1: Quantitative single crystal X-ray diffraction method
to determine the content of the polymorph in 100 % pare drug
substance
The analytical target of SXRD is a single crystal. SXRD is
based on the diffraction effect of X-ray on the crystal that
reflects data from one crystal form in one crystal. SXRD can
reveal information on crystal formation and provide
quantitative crystallographic data on crystalline substance.
Based on analysis result from SXRD, PXRD spectrum and
related data can be calculated for the polymorph in 100%
purity, which can be employed as the standard spectrum of
crystalline substance.
Method 2: Quantitative powder crystal X-ray diffraction
CPXRD) method to determine the content of the polymorph in
test drug
PXRD is based on the diff raction effect of X-ray on the test
sample, of which the spectrum reflects the relationship
between the position of the diffraction peak ( 2() or d) and
diffraction intensity. The amount of diffraction peaks of the
test sample polymorph correlates with the symmetry and
periodicity. The position of diffraction peak is expressed as d
CÁ) or 2() (º); the intensity of diffraction peak is expressed
as the height of peak or peak area, its absolute intensity is
defined by CPS units of accounting points per second, while
its relative intensity is equal to ( the absolute intensity of a
certain peak) / ( the absolute intensity of the highest
peak) X 100 % . The ratio of peak intensity reflects relative
intensity among diffraction peaks and the change in geometric
topology of peak shapes.
(a) Analysis of crystalline chemical ingredient: To
accomplish quantitative control of crystalline drug substance,
CD one to three characteristic diffraction peaks should be
selected to reflect the content variation of crystalline drug
substance and the intensities of these peaks should display a
linear relation to the content; ®standard curves of mixed
crystalline drug substance: Mixed crystalline samples are
prepared by combination of two or more crystalline forms in
various ratios of content and standard curves are established
for the content of crystalline form and intensities of
 9101 Guidelines for Validation of Analytical Method Adopted in Pharmaceutical Quality Specification
characteristic diff raction peaks, which then are used to
quantify category and content of specific crystalline drug
substance; ®to complete the determination of crystalline
drug at different time points, an accompanying standard
curve or standard curve with externa! standard method can be
established for the quantification of crystalline forms in test
sample at different time points.
( b) Analysis of crystalline drugs in the pharmaceutical
preparations: To accomplish the purpose of quantitative control
of crystalline drugs in pharmaceutical preparations, CD it is
necessary to include the salid preparation, crystalline drugs and
blank pharmaceutical excipients; ® one to three characteristic
diffraction peaks should be selected to reflect the content
variations of crystalline drugs in the pharmaceutical preparations
and the intensities of these peaks should display a linear relation;
® standard curves of the content of crystalline drugs in
pharmaceutical preparations: Mixed samples are prepared by
combining various ratios of blank pharmaceutical excipients with
crystalline drugs and standard curves are established for the
content of crystalline drugs and intensities of characteristic
diffraction peaks, which are then used to quantify the content
of crystalline drugs in the pharmaceutic preparations; @to
complete the determination of crystalline drugs at different
time points, an accompanying standard curve or standard
curve with externa! standard method can be established for
the quantification of crystalline drugs in test sample at
diff erent time points.
(c) Instructions of the method: CD the quantitative method
relies on the data and PXRD spectrums of drug substance
of 100 % purity that are theoretically calculated based on
SXRD data or standard spectrums produced by reference
standards of crystalline substance; ® the pretreatment of
samples are required. The organic sample should be sifted
through a 100 mesh screen and the inorganic sample should
be sifted through a 200 mesh screen. The samples for
quantification should be accurately weighed; ®the content
of crystalline drug substance in solid preparations should be
within the linear range of the standard curve; @ An
external standard Alz Ü3 should be used to calibrate the
instrument and data.
Method 3: Quantitative differential scanning calorimetry
(DSC) method to determine the content of the polymorph in
test sample
Quantitative DSC method is applicable to crystalline
substances with different molten endothermic peak values,
and the content of the polymorph in samples is linearly
correlated to the heat absorded.
(a) Analysis of crystalline drug substance: several quantifies
of crystalline samples are accurately measured. With the
developed linear relation of the mass and the endothermic
enthalpy, the standard curve is established to quantify the
content of crystalline substance in the sample.
(b) Analysis of rnixed polymorphs drug: when the contents of
different polymorphs are proportional to the endotherrnic
enthalpy and a linear relation is developed, standard curves are
established to quantify the content of polymorph in rnixed
polymorphs samples in which accurately weighted polymorphs
are combined with different ratios.
( c) lnstructions of the method: CD this method is only
applicable to the crystalline drug substances. ® for mixed
polymorphs samples with large variations in molten
endothermic peak values, the linear range of the standard
curve should be broadened, while the linear range of the
standard curve can be narrowed for those samples with small
variations. ®sometimes DSC method is only used for the
limit detection.
Method 4: Quantitative infrared spectroscopy (IR) method to
determine the content of the polymorph in test sample
Quantitative IR method can be used for the quantification of
crystalline drug substances and pharmaceutical preparations.
The common method is relative peak intensity method.
The selection principie of characteristic peaks of polymorph
includes: CDselect the characteristic peaks specific for each
crystalline form. ® the characteristic peaks of the two
polymorphic forms are independent without any interference.
® the intensities of the characteristic peaks should be
proportional to the content of crystalline forms.
For crystalline drug substances and pharmaceutical preparations,
tableting should be avoided if crystal transformation would
occur.
(a) Analysis of crystalline drug subsbances: When the
relative strength method is used, the positions Cb1 and bz)
of characteristic peaks of two crystalline forms are selected,
the photometric values CA1 and Az) of characteristic peaks
of two crystalline forms are recorded in one IR spectrum and
the ratio ( r) of photometric values of characteristic peaks
are calculated for two crystalline forms. A serial of mixed
crystalline samples with different proportions of two crystalline
forms are prepared and the linear relation between the ratio of
photometric values and the content of crystalline sample is
established to develop the standard curve, which is then used
to quantify crystalline form in mixed sample.
(b) Analysis of crystalline drug substances in the pharma-
ceutical preparations: When the relative strength method is
used, the positions ( b1 and bz) of characteristic peaks of
crystalline drug substances and the pharmaceutical excipients
are selected, the photometric values ( A 1 and A 2 ) of
characteristic peaks of crystalline drug substances and the
pharmaceutical excipient in one IR spectrum are recorded and
the ratio (r) of photometric values are calculated. A serial
of mixed samples containing different contents of crystalline
drug substances and pharmaceutical excipients are prepared
and the linear relation between the ratio of photometric
values and the content of polymorphic drug substance is
established to develop the standard curve, which is then used
to quantify crystalline drug substances in the pharmaceutical
preparati o ns.
Notes: Other internationally recognized methods for phase
analysis also can be employed for quantitative or qualitative
analysis of drug polymorphism.
9101 Guidelines for Validation of
Analytical Method Adopted in
Phannaceutical Quality Specification
The purpose of validation of an analytical method is to ensure
that the adopted method meets the requirements for the
intended analytical applications. In the course of drafting of
the drug quality specification, the analytical method must be
validated. In case of changing of pharmaceutical synthetic
processes or the components of preparation, or revising of
the original analytical method, the analytical method of the
specification must also be validated. Both process and results
of the method validation must be recorded in the description
of draft and revision of a pharmaceutical quality specification.
For biological product quality control, physico-chemical
analytical method and biological determination method can be
employed. Because the principie of physico-chernical analytical
method is applicable for both chernical and biological products,
this guideline is suitable for corresponding method validation on
biological product after paying special attention to their unique
9101 Guidelines for Validation of Analytical Method Adopted in Pharmaceutical Quality Specification

characteristics in the validation study. Compared to physico- known quantity of reference substance. If it is not possible to
chemical analytical method, more influencing factors are obtain all the components of excipient, the accuracy may be
present in biological determination method that is out of determined by adding known amounts of analyte to the
scope of this guideline. preparation, or by comparing the result obtained by this
The analytical items that should be validated include identification, established method with the result obtained by another
limit or quantification test, content determination of the active method with known accuracy.
ingredient in drug substance or preparation, as well as other The accuracy can be calculated by the precision, linearity and
components in the preparation, such as antiseptic, residues in specificity of the method.
traditional Chinese medicine, additive etc. In the dissolution
2. Accuracy of quantitative determination of impurity for
test and drug release test of pharmaceuticals, the analytical
chemical medicine
method of dissolution amount must also be validated.
The accuracy may be deterrnined by spiking the drug substance
The valida tion indexes incl ude accuracy, precision ( incl uding
or blank excipient of prescription dosage with known quantity of
repeatability, intermediate-precision and reproducibility) ,
impurity. When the reference substance is not available for
specificity, detection limit, quantitation limit, linearity,
impurity or degradation product, the accuracy may be deterrnined
range and robustness. Standard substance must be utilized in
by comparing the result obtained by this established method with
the validation of analytical method. Because of intrisic
the result obtained by another matured method, such as
characteristics of validation method and possible influences
pharmacopoeia method or validated method. lmpurity content,
from analytes, the parameters to be validated should be
which is clearly represented as relative ratio of a single impurity
decided depending on specific analytical method involved.
or total impurities to principle constituent in weight and peak
The analytical items and the corresponding parameters to be
area, can be calculated by principle constituent self-contrast
validated are listed in Table 1, which can be used as a
method without correction factor if the correction factor or the
reference.
relative correction factor of the impurity or degradation product
Table 1 List of validation characteristics to the drug substance can not be deterrnined.
required to be evaluated in test of each type
3. Accuracy of determination of ingredients for traditional
lmpurity test Determi- Chinese medicine
Corree- Ref erence substances can be used for the deterrnination of the
Identifi- nation of
Parameters Quanti- Lirnit of tion recovery of added sample, e. g. certain amount of the reference
cation content or
tation test factor substance of test substance is precisely added into the test sample
relea se with known content of analyte to be examined. The recovery
ratio is calculated by margin of the deterrnined value and the
Accuracy - + - + + amount of the substance being examined divided by the amount
Precision of added reference substance. In the test of the recovery of added
Repeatability - + - + + samples, the sum of the added amount of the reference substance
and the amount of the analyte in the substance being
Intermedia te
- +<D - +<D + examined must be in the linearity range of the standard
precision curve. The amount of the added reference substance should
be proper. A very low amount of reference substance will
Specificity® + + + + + cause a large relative error while a high amount of reference
Detection limit - _@
+ - -
substance will reduce the relative amount of the interference
Qauntification substances, so the authenticity is poor.

limit
- + - -
Linearity - + -
+ +
Range - + -
+ +
Robustness + + + +
CI) It is not necessary to valida te the intermedia te precision
when the reproducibility has been developed.
(Z)Lack of specificity of an individual analytical method may
be compensated by other supporting analytical methods.
@It depends on the specific condition.
Accuracy
The accuracy of an analytical method is the closeness of test
results obtained by that method to the true value or the
reference value. Accuracy is often represented as percent
recovery and should be determined in the specified range.
l. Accurac y o f the method f or content determination o f
chemical medicine
The accuracy for drug substance may be determined with a
reference substance, or by comparing the result obtained by
this method with the result obtained by another method of
which the accuracy has been established. For drug
preparation, its accuracy may be determined by spiking the
exact amount of blank excipient in prescription dosage with
+ Recovery ( %) = ( C- A)/ B X 100 %
where:
A is the amount of the analyte in the substance being
examined;
B is the amount of the added reference substance;
+ e is the determined value.
4. Accuracy of correction factor
For a chromatography method, the absolute ( or quantitative)
correction factor refers to the quotient of the content of analyte
divided by the chromatographic peak area. Relative correction
factor is the ratio between absolute correction factor of analyte
and reference substance. Relative correction factor is normally
used in the deterrnination of related substance for chemical drug,
or multiple-index substance in crude drugs and compound
preparations. Correction factor has a lot of expressions, in this
guideline it refers to the relative weight correction factor in gas
chromatography and high performance liquid chromatography.
The relative correction factor can be obtained by the ratio
of slopes of standard curves of the substitute ( reference
substance) and the substance (test substance). When
using UV-absorbance detector, relative correction factor
can be calculated by comparing the absorption
coefficients of the substitute ( reference substance) and
the substance ( test substance ) under specified
wavelength and solvent.
 9101 Guidelines for Validation of Analytical Method Adopted in Pharmaceutical Quality Specification
S. Requirement for the data
In specified range, the accuracy should be evaluated by using
results from at least 6 samples of test substance at the same
concentration ( equivalent to 100 % concentration level), or 9
samples with 3 different concentrations of test substance and
3 test solutions at each concentration. For chemical drug, the
ratio between the amount of reference substance added and
test substance to be determined in the sample is recommended to
be controlled around l. 2 : 1, 1 : 1 and O. 8 : 1 for the high,
middle and low concentration, respectively. The percent recovery
of the added amount, or the difference between the average value
of testing results and nominal value and its relative standard
deviation or confidence interval (normally at 95% confidence
level) should be reported. For traditional Chinese medicine,
the ratio between the amount of reference substance added
and test substance to be determined in the sample is
recommended to be controlled around l. 5 : 1, 1 : 1 and
O. 5 : 1 for the high, middle and low concentrations,
respectively. The amount of sample used, the content of test
substance in the sample, the amount of reference substance
added, the testing result and calculated percent recovery, and
the relative standard deviation CRSD, %) or confidence interval
of percent recovery should be reported. For correction factor, the
testing method, testing result and RSD should be reported.
Table 2 can be used as a reference for the relation between the
content of test substance in sample and the limit of percent
recovery. The limit of percent recovery can be broadened in sorne
conditions, such as complex matrix, the content of
component lower than O. 01% and multi-components
analysis.
Table 2 The content of test substance in sample and the
limit of percent recovery
Content of test
Limit of percent recovery
substance in sample
100% 98-101
10% 95-102
1% 92-105
0.1% 90-108
o. 01% 85-110
lOµg/g (ppm) 80-115
lµg/g 75-120
lOµg/kg (ppb) 70-125
Precision
The precision of an analytical method is the closeness of
agreement between a series of measurements obtained from
multiple sampling of the same homogeneous sample under
the prescribed conditions. The precision of an analytical
method is usually expressed as deviation, standard deviation
or relative standard deviation.
Repeatability is the precision obtained by the same analyst
within a laboratory overa short period of time with the same
equipment. Intermediate-precision is the precision obtained
by different analysts within the same laboratory on different
days with different equipment. Reproducibility is the
precision obtained by different analysts in different
laboratories using the same analytical procedure.
Precision of the method should be considered when the
content of the active ingredient or impurity is determined.
l. Repeatability
In specified range, the repeatability of the precision study
should be evaluated using results from at least 6 samples of
test substance at the same concentration ( equivalent to
100 % concentration leve}), or 9 samples with 3 different
concentrations of test substance and 3 test solutions at each
concentration. When the repeatability is evaluated by
9 testing results, the ratio between the amount of reference
substance added and test substance to be determined in the
sample is recommended to be controlled around l. 2 : 1, 1 :
1 and O. 8 : 1 for ehemical drugs and l. 5 : 1 , 1 : 1 and
O. 5 : 1 for traditional Chinese medicine, for the high,
middle, and low concentrations, respectively.
2. Intermediate-precision
A scheme should be designed to inspect the effect of random
variable factors on the precision. The variable factors include
different dates, different analysts and different equipments.
3. Reproducibility
Reproducibility should be tested when an analytical method is
adopted as the national drug quality standard, for example,
reproducibility should be inspected by different laboratory
studies. Both the process of the collaborative study and
result of the reproducibility should be recorded in the
description of draft file. Where a reproducibility testing is to
be conducted, the sample should be uniform, properly stored
and transported to obtain reliable result.
4. Requirements f or data
Deviation, standard deviation, relative standard deviation
and confidence interval should be reported. Table 3 can be
used as a reference for the content of test substance in
sample and acceptable range of the RSD for the precision.
Acceptable range of the RSD for the precision can be
broadened in sorne conditions, such as complex matrix,
the content of component lower than O. 01 % and multi-
components analysis.
Table 3 The content of test substance in sample and
acceptable range of the RSD for precision
Content of test Repeatability Reproducibility
substance in sample CRSD%) CRSD%)
100% 1 2
10% l. 5 3
1% 2 4
0.1% 3 6
o. 01% 4 8
lOµg/ g (ppm) 6 11
lµg/g 8 16
lOµg/kg (ppb) 15 32
Specificity
The specificity of an analytical method is the ability to
measure the analyte accurately and specifically in the
presence of components that may be expected to be present in
the sample matrix, such as impurities, degradation products
and excipients. Specificity concerns should be investigated on
identification, impurity test and content determination. If the
specificity of the method is not enough, other methods with
9101 Guidelines far Validation of Analytical Method Adopted in Pharmaceutical Quality Specification
diff erent principies should be adopted far supplementation.
l. ldentification
The compounds that may coexist or have close related
structures should be distinguished from the active ingredient. All
the samples without the tested ingredients, compounds with
closely related structures and related chemical compounds
should produce a negative response.
2. Assay and test for impurity
The representative graphs should be recorded far verifying
specificity when chromatography or other separation methods
are used. The position of each component should be marked
in the graph. The resolution of the chromatographic method
should meet the requirements.
If the reference substances of impurities are available, the
impurities or excipients may be added to the sample far assay
to inspect whether the result is interfered, and the result can
be compared with that from the sample without adding
impurities or excipients. As to test far impurity, a certain
amount of the impurity may be added to the sample to
inspect whether all ingredients including the impurity can be
separated from other ingredients.
If the impurities or degradation products are not available,
the sample with impurities or degradation products may be
used far determination, and the result may be compared with
that obtained by the Pharmacopoeia method or other validated
methods. Accelerating decomposition may be done far studying
degradation products, such as irradiation with strong light, high
temperature, high humidity, acidic or alkaline hydrolysis,
oxidation etc. The results of two methods should be
compared far content determination and the number of
impurities should be compared with that obtained in test far
impurity. Diode array detector and mass spectrometer may
be used far purity test when necessary.
Detection Limit
Detection limit is the lowest concentration of the analyte in a
sample that can be detected. The detection limit far
identification and impurity test should be determined by
experiment. The detection limit can only be used as the
reference far limit test and qualitative identification, which is
not applicable to quantitative analysis. The methods in
common use are as follows.
l. Noninstrumental method
The detection limit is generally determined by the analysis of
samples with known concentrations of analyte and by
establishing the mínimum level at which the analyte can be
reliably detected.
2. Signal-to-noise ratio method
For the instrumental method recording the noise at the
baseline, the lowest concentration or content of test substance
that is reliably detected can be calculated by comparing the signal
of sample at a known low concentration and the signal of the
blank. The concentration or the amount injected into the
instrument corresponding to the signal-to-noise ratio of 2 : 1
or 3 : 1 is generally accepted.
3. Method of standard deviation and slope of standard
curve based on response value
The detection limit is calculated by the following formula:
LOD=3. 3a/S. In the formula, LOD is the detection limit, a
is the standard deviation of response value, and Sis the slope
of standard curve.
a can be determined by following method: CI)determining the
standard deviation of blank; @substituting by the residue
standard deviation or the standard deviation of intercept of
standard curve.
4. Requirement f or data
Detection limit obtained by above methods must be validated
by samples with similar content of test substance. The test
graphs should be attached and the test procedures and the
results of detection limit should be reported.
Quantitation Limit
Quantitation Limit is the lowest concentration of the analyte
in a sample that can be determined with acceptable precision
and accuracy under the stated experimental conditions. The
quantitation limit should be determined far the analytical
method of micro or trace substance or the quantitative
determination far impurities and degraded products. The
methods in common use are as following.
l. Noninstrumental method
Quantitation limit is generally determined by the mm1mum
concentration or content of the analyte in a known
concentration of sample that can be reliably detected.
2. Signal-to-noise ratio method
For the instrumental method recording the noise at the
baseline, the lowest concentration or content of test substance
that is reliably determined can be calculated by comparing signal
of sample at a known low concentration and signal of the blank.
The concentration or amount injected into the instrument
corresponding to the signal-to-noise ratio of 10 : 1 is generally
accepted.
3. Method of standard deviation and slope of standard
curve based on response value
The quantitation limit is calculated by the following formula:
LOQ = lOa/S. In the formula, LOQ is the quantitation
limit, a is the standard deviation of response value, s is the
slope of standard curve.
a can be determined by following method: CI)determining the
standard deviation of blank; @substituting by the residue
standard deviation or the standard deviation of intercept of
standard curve.
4. Requirement f or data
Quantitation limit obtained by above methods must be
validated by samples with similar content of test substance.
The test graphs should be attached and the test procedures
and the results of quantitation limit, including validation data
of precision and accuracy, should be reported.
Linearity
The linearity of an analytical method is its ability to elicit test
results that are directly proportional to the concentration of
analyte in samples within a given range.
Linear relationship should be determined over the claimed
range of the method. The samples with varying concentrations of
analyte for linearity determination are prepared by diluting
accurately a stock solution, or by measuring accurately an
amount of analyte separately. At least 5 portions of samples
should be prepared. The treatment is normally a calculation
of a regression line by the method of least squares of test
results versus analyte concentrations. In sorne cases, the test
data may have to be subjected to mathematical transformation
prior to the linearity regression analysis. It is acceptable to use a
non-linear model for concentration-response relation.
Requirement far data: regression equation, correlation
coefficient and the linear graph should be listed eor other
mathematical models).
Range
The range of an analytical method is the concentration or
quantity interval between the upper and lower levels of
analyte eincluding these levels) that have been demonstrated
to be determined with precision, accuracy, and linearity
 9102 Guidelines far the Analysis of Impurities in Drugs
using the method as written.
The range of the analytical method should be determined
based on specific application of the method, its linearity,
accuracy and precision, and related requirement. For content
determination of drug substance and preparation, the range
should be 80 % to 120 % of the test concentration. For
content unifarmity of preparation, the range should be 70 %
to 130 % of test concentration and this range may be widened
appropriately far special dosage farms, such as aerosols and
sprays. For dissolution test and drug release test, the range
should be ± 30 % of the limi t. If the range of limi t is
provided, it should be - 20% of lower limit to + 20% of
upper limit. For impurity determination, the range should be
stipulated from -20% to +20% of the provided limit on the
basis of preliminary actual determination. If the content
determination and impurities test are performed simultaneously
with the peak area normalization method, the linear range should
be -20%of the provided limit of impurity to +20% of the
provided limit of content Cor upper limit).
For traditional Chinese medicine, the range of analytical
method should be determined based on specific application,
linearity, accuracy and precision of the method, and related
requirement. For toxic ingredients or those with unique
efficacy or pharmacological effect, the range to be validated
should be wider than the range of content.
For correction factor, its range should be determined on the
basis of the range of test object.
Robustness
Robustness of an analytical method is the degree of tolerance
that the determination result is not aífected when there is
small change in the operational condition. The robustness of
the method should be taken into account at the beginning to
develop an analytical method. If the requirement far test
condition is strict, it should be recorded clearly in the method
and the acceptable range of variations should be indicated.
Unifarm design can be used far determination of primary
influencing factor then the changing range can be confirmed
by single factor analysis. The typical variable factors are
stability of the test solution, times and duration of sample
extraction, and so on. The variable factors of liquid
chromatography are composition and pH value of the mobile
phase, same type of chromatographic column from different
manufacturers or batches, column temperature, flow rate,
etc. The variable factors of GC are column and stationary
phase with different brands or batches, different types of
support, carrier gas flow rate, column temperature,
temperature of injection port and detector, etc.
To ensure reliability of the method, the testing conditions with
slight change should be confirmed to meet the requirements for
system suitability.
9102 Guidelines for the Analysis of
lmpurities in Drugs
This guideline is used to provide guidance on the quality
specifications about the analysis of impurities in chemically
synthesized or semi-synthesized organic drug substances and
their preparations, which can also be used as reference far
the drug research, manufacture, drafting or revising of
quality specifications.
Any substance that affects the purity of a drug is considered
as an impurity. Impurities included in pharmaceutical quality
specification are those introduced from production process,
starting materials or excipients of a drug produced by defined
process and materials approved officially by the state drug
regulatory authority. The impurities may be the degradation
products arising in the storage period, which could be
validated by stability testing. Impurities in the drug specifications
should not include new impurities introduced by change of
production process or materials, or foreign substances from
adulteration or contamination. Request for revision of
pharmaceutical quality specification due to new impurities
introduced by alterations in production process or materials
shall be submitted to the state drug regulatory authority far
approval according to law. Drugs shall not be adulterated or
contaminated by fareign substances. Non-official methods
may be adopted to monitor the counterfeit drugs on the basis
of individual case when necessary.
The classification of impurities
According to their chemical category and characteristics,
impurities are classified as organic impurities, inorganic
impurities and organic volatile impurities. According to their
originating source, impurities are classified as ordinary
impurities and special impurities. Ordinary impurities, widely
distributed in the nature, are easily introduced during the
production and storage of drugs, such as molysite and
ammonium salt, etc. Special impurities are those substances
introduced by specific drug manufacturing and storage.
According to their toxicity, impurities also are classified as
toxic impurities (e. g. , heavy metal, arsenic salt) and
signaling impurities (e. g. , chloride or sulfate salt) that are
generally nontoxic but their content reflects purity of the
drug or the problems in processing techniques or
manufacturing processes. Due to the different ways to
classify impurities, the item title for an impurity test in a
drug specification shall be normalized based on the
requirements of the Manual Jor Revising to National Drug
Standards published by the Commission of the Chinese
Pharmacopoeia. The item title far organic impurity may be
selected according to the fallowing principies:
(1) When the impurity is a known substance, the chemical
name of the impurity can be used as the item title, such as
"Morphine" in the monograph of Codeine Phosphate,
"p-Chlorophenol" in the monograph of Clofibrate,
"Piperidylpropiophenone" in the monograph of Benzhexol
Hydrochloride, "Lincomycin B" in the monograph of
Lincomycin Hydrochloride, and "Chymotrypsin" in the
monograph of Trypsin. If the chemical name of the impurity is
too long to describe and no corresponding adopted abbreviation is
available, the following principie may be used far the selection of
item title, such as "Mercapto compounds" in the monograph of
Spironolactone, " Phenones " in the monograph of
Adrenaline, and "Alkene" in the monograph of Difenidol
Hydrochloride. The impurities with confirmed structure
should be provided in the description of the draft of the
quality specification.
(2) When the category of the impurity is known and the
impurity cannot be justified to be a single substance, the item
title of such impurity may be described as "other steroids",
"other alkaloids", "other amino acids", "reducing sugars",
"fatty acids ", " primary aroma tic amines", " chlorinated
compounds", "residual solvents", or "related substance",
and so on.
(3) For sorne unknown impurities, the selection of item title
may be based upon the testing method, such as "light absorption
of impurity ", " oxidizable substances ", " carbonisable
substances", "non-volatile substances", "volatile impurities",
and so on.
Selection of impurity testing items for quality specification
Study on impurities in new drug substances and new
preparations shall be conducted in accord with the relevant
 9102 Guidelines for the Analysis of Impurities in Drugs
requirements far registration application of new drugs in
China, or referring to the ICH Guidelines Q3A or Q3B. The
safety evaluation far impurities and degradation products
should be perfarmed. Effective separation and analytical
methods shall be adopted by the new drug research
institutions for determining actually present and potential
impurities arising from synthesis, purification and storage.
Characterization or structure confirmation shall be carried out
for the impurity that its apparent level is O. 1 % or above
O. 1 %, and far the toxic impurity or impurity with significant
biological action that its apparent level is even less than
O. 1 %. The degradation products faund in the stability testing
( accelerated and long-term testing ) shall be studied
according to the above requirements. Impurity testing items
in the new drug specification should include the impurities
and degradation products detected in the quality study and
stability testing, and appeared in the batches produced in an
industrial scale. The corresponding acceptance criteria shall
be provided. Such impurities with or without known
structure are specified impurities. Impurities controlled in the
drug substance specification will generally not be controlled in its
preparation specification except far degradation products and
toxic impurities. Testing items far inorganic impurity in drug
substances and preparations shall be selected according to the
manufacturing process and starting materials. Testing items
far toxic inorganic impurities shall be established far the drug
specification.
When the impurity profile found in the study or production of
a generic drug is different from that of the innovated one or
the existing official standard, the new impurities shall be
studied according to the above requirements, and an
application far revising to the original specification or
establishing a new specification shall be submitted to the
relevant drug regulatory authority for review and approval.
The concomitant isomers and the antibiotic:s rnulti-
components are generally not the testing items of impurities.
For concomitants, their proportion shall be specified in the
specification to ensure the consistency of drug substances
used far production and those far registration application
when necessary. The toxic concomitants will be considered
as impurities instead of concomitants. When the drug is a
single enantiomer, other possible co-existent enantiomers are
impurities and the specific optical rotation should be tested.
The testing ítem of optical rotation should be set far the
specification of the corresponding racemic drug if necessary.
Testing items far residual organic volatile impurities may be
established according to the organic solvents used in the production
process and the residual amount. The requirements far testing
organic volatile impurities in Pharmacopoeia or the Q3C of the ICH
document may be used as a reference. The testing items far the
residual toxic solvents shall be established.
Analytical methods for impurity testing and acceptance criteria
of impurity
The analytical methods far impurity testing should be specific and
sensitive. Mxlem analytical methods far separation should be used
far impurity testing. The substance to be examined should be well
resolved with impurities and degradation products, the lirnit of
detection should meet the requirement far the lirnit tests. For the
impurities to be quantified, the lirnit of quantification should meet
the corresponding requirement.
The analytical methods far impurity testing should be validated
according to the requirements of the Chinese Pharmacopoeia.
Severa! different methods or conditions far separation and
deterrnination may be adopted in the research stage, results
obtained should be compared to select a suitable method as the
compendium method. The suitability of an established method
should be considered, and the equipment and materials selected
should be available. The special materials used far the testing
should be specified in the specification when necessary. In the
research stage far the analysis of impurity, testing solutions can be
prepared far chromatographic deterrnination using the possible
impurities and products from farced degradation, or by adding
such impurities into the substance to be examined. Suitability
requirements should be established by necessary adjustment of
chromatographic parameters to ensure the method to be specific
and sensitive.
Impurities should be prepared by isolation and purification, or by
synthesis far the safety evaluation and quality study. When the
impurities or degradation products could not be obtained, reason
should be provided in the application dossier submitted and in
the description of the draft of the quality specification by the
research institution.
Where modem chromatographic method is used for the
separation of impurities, known impurities of the specified
impurities and toxic impurities may be identified using
reference substances of impurity. Relative retention values
can be used for the identification when the reference
substances are not available. The unknown impurities of the
specified impurities can be identified using relative retention
values. Content of impurities can be measured using general
principie of thin layer chromatography < 0502 ) and High
Performance Liquid Chromatography <0512).
Chiral chromatography and high-perfarmance capillary
electrophoresis are widely used far stereomers test. Chiral
HPLC includes chiral stationary phase method, chiral mobile
phase additives method (direct approach) and chiral reagent
derivatization method ( indirect approach). Chiral stationary
phase method is the most widely used method in stereomer
testing because of its high-accuracy, and ease to conduct and
derivatization free. However, there is limit in the usage of
chiral stationary phase method for the narro\v applicable
scope of each chiral stationary phase. So the stereo selectivity
and chiral conversion test are the key points in the method
validation of stereomer testing. It is good for resolution and
sensitivity if the peak of the stereomers exhibits a shorter
retention time than the parent drug. Chiral chromatography
should be complementary to optical rotation test or specific
optical rotation test far effectively controlling the chiral drug
quality because it can' t reflect the optical activity of the chiral
drug directly.
The results of the impurity limit test obtained by using
chromatography may be influenced by the chromatographic
parameters, and relevant operation precautions should be
provided in the description of the draft of the specification.
For critica! parameters the adjustments should be defined
clearly in the specification to ensure system suitability when
necessary.
Several factors should be considered far establishing the
acceptance criteria of impurities, such as the toxicology study
of the impurity or the substance containing limited quantity
of the impurity, the route of administration, daily <lose,
patient population, possible pharmacology research on the
impurity, source of drug substance, treatment cycle, the
acceptability of the cost far the manufacturers to produce safe
and high quality drugs and acceptability of the drug price for
the consumers.
Strict limits for toxic impurities or toxic residual organic
solvents should be reinforced in the drug quality
specification. The limit of residual organic solvents may be
established according to the requirements of relevant
general chapter of the Pharmacopoeia or relevant ICH
documents.
 9103 Guidelines for Hygroscopicity
9103 Guidelines for
Hygroscopicity
Hygroscopicity represents the capability and degree of
drugs absorbing moisture at certain temperature and
humidity. The test substance should be a bulk solid that
complies with the test of its quality standard. The results of
the test can also be used as a reference for selecting suitable
package and storage conditions of the drugs.
Method:
l. Use a dry glass weighing vessel ( 50 mm in external
diameter and 15 mm in height) with a stopper. One day
before the test, place the vessel in a isothermal desiccator at
25±1 ºC , which contains a saturated solution of ammonium
chloride or ammonium sulfate. Alternatively, place it in a
factitious clima tic cabinet set at 25 ± 1ºC and 80 % ± 2 %
relative humidity. And then, weigh accurately Cm1 ).
2. Place a moderate quantity of the substance being examined
in the above vessel and weigh accurately ( m2 ). The
substance being examined should be evenly distributed to
form a layer of no more than 1 mm in thickness.
3. Open the weighing vessel, maintain it and its stopper for
24 hours at the above conditions.
4. Cover the weighing vessel with its stopper and weigh
accurately (m3 ). Calculate the percentage increase in mass
using the following equation:
m3 -m2 ¿
0 particular attention to the suitability of the sampling system
Percentage increase in mass X 100 /o
m2-m1
5. The result is interpreted as follows:
deliquescent : sufficient water is absorbed to form liquid.
extremely hygroscopic: the increase in mass is not less than
15%.
hygroscopic: the increase in mass is less than 15 % but not
less than 2 %.
slightly hygroscopic: the increase in mass is less than 2%
but not less than O. 2 %.
practical nonhygroscopic: the increase in mass is less than
0.2%.
9104 Guideline for Near-infrared
( NIR) Spectrophotometry
Near-infrared ( NIR) spectrophotometry is an analytic
technique used in qualitative and quantitative analyses in
which characteristic spectrum of the substance to be
examined is measured among near-infrared region ( from
about 780 nm to about 2500 nm or from about 12 800 cm- 1
to about 4000 cm- 1 ) and the correlation information is
extracted by suitable chemometric algorithms. NIR spectra
are dominated by C-H, N-H, (}-H and S-H overtone
resonances and combinations of fundamental vibrational
modes. NIR bands are much weaker than the fundamental
mid-IR vibrations from which they originate. NIR bands are
always overlapping. Direct comparison of the spectrum of the
substance to be examined with a reference spectrum of a
chemical reference substance is not appropriate. Suitable
validated mathematical treatment of the data is required.
Application range
Near-infrared spectrophotometry is advantageous because
quick and accurate measurement can be often made without
destroying the sample. This method has a wide variety of
applications for both chemical and physical analyses of
pharmaceutical substances. It not only can be used for
quantification of active substances, excipients, water content,
hydroxyl value, iodine value and acid value, but also can be used
for the identification and qualification of raw materials,
intermediates and packaging materials. Moreover, NIR technique
provides an interesting platform for monitoring and control of
pharmaceutical process.
Apparatus and control of instrument performance
l. Apparatus
NIR spectrophotometers consist of a suitable light source, a
monochromator or interferometer, sampling device,
detector and suitable data processing and evaluation
system. Based on different operating principles, there are
several kinds of NIR spectrophotometers, such as filters,
grating-based dispersive, acousto-optical tunable filter
CAOTF), Fourier-transform NIR CFT-NIR), etc. High
intensity light sources such as tungsten lamp with shell of
quartz or similar are used usually. The tungsten lamp light
source can be highly stabilized. Silicon, lead sulphide,
indium arsenide, indium gallium arsenide, mercury cadmium
telluride ( MCT ) and deuterated triglycine sulphate are
commonly used detector materials. Conventional cuvette sample
holders, fiber-optic probes, transmission clip cells and
spinning or traversing sample holders are a few common
sampling devices. The selection of detector and sampling
device are based on the intended application, paying
for the type of sample to be analysed.
2. Qualification and verification of NIR instruments
In order to ensure that an instrument is suitable for its intended
application, periodic instrument operational qualifications using
SRM ( standard reference material ) must be made. The
objective is to ensure that no sudden wavelength calibration shift
or change in sensitivity occurs during the analysis. The
instrument qualification and verification include wavelength
uncertainty, absorbance-scale, linearity, and high-and low-
light flux noise tests. Performance is verified by matching
the current spectra to those collected from previous
instrument qualifications.
Instrument calibration should be performed regularly or after
a repair or optical reconfiguration. Table 1 shows
recommended specifications on wavelength uncertainty,
photometric linearity, and spectrophotometric noise level for
pharmaceutical analysis applications.
Measurement methods
Two different measurements commonly performed in the
NIR spectral range are transmittance and reflectance.
l. Diffuse reflection mode This method is usually suitable
for solid sample. The diffuse reflection mode gives a measure
of reflectance (R), the ratio of intensity of light reflected
from the sample (J) to that reflected from a background or
reference reflective surface (Ir). The sample is placed in a
suitable sampling device. NIR radiation can penetrate a
substantial distance (1 to 3 mm) into the sample, where it
can be absorbed by vibrational combinations and overtone
resonances of the analyte species present in the sample. Non-
absorbed radiation is reflected back from the sample to the
detector. A typical NIR reflectance spectrum is obtained by
calculating and plotting log (1/R) versus the wavelength or
wavenumbers.
R=I!Ir,
AR= lg(l/R) = lg(L/D
Where: I is the intensity of light diffusively reflected from
 9104 Guideline for Near-infrared (NIR) Spectrophotometry

the sample. Affecting factors of spectral response

Ir is the intensity of light reflected from the The main factors affecting the NIR spectral response are
background or reference reflective surface. sample temperature, humidity and solvent residues, sample
thickness, sample optical properties, polymorphism and time
Table 1 Recommended NIR instrument specifications® for storage of samples. The NIR spectra of liquid samples
are sensitive to ambient temperature. Samples with different
Wavelength SRM 1920a® peaks occur at 1261, 1681 crystalline structure have different NIR spectra.
uncertainty and 1935 nm Basic requirement of qualitative analysis and quantitative
analysis by near-infrared (NIR) spectrophotometry
l. Qualitative analysis
±1 nm at 1200 nm or ±8 cm- 1 Qualitative models construction contains the following steps:
at 8300 cm- 1 Record the NIR spectra of representative samples first, then
±1 nm at 1600 nm or ±4 cm- 1 pre-treat data and reduce it dimensionality by selected
Tolerances
at 6250 cm- 1 chemometric methods, establish the qualitative model and
±1. 5 nm at 2000 nm or ±4 cm- 1 finally validate it.
at 5000 cm- 1
( 1) Collection of representative samples
Record the spectra of a suitable number of batches of the substance
which have been fully tested according to established specifications
AoaslAREFa> at 1200, 1600, and and which exhibit the variation typical for the substance to be
Photometric 2000 nm; analysed (for example, manufacturer, morphology, particle size).
linearity slope = l. O± O. 05; intercept=O. O± The set of spectra represents the information for identification and
0.05 charactemation that defines the sirnilarity border for that substance
and is the entry for that substance in the spectral library used to
identify the substance.
measured for 100-nm ( 300 cm- 1 )
Spectrophoto- ( 2) Pretreatment of data
segments between 1200 and 2200 nm
metric noise In many cases, sorne form of mathematical pretreatment of
(8300 and 4500 cm- 1 )
the spectrum must be performed prior to the development of
a classification or calibration model. Typical methods are
multiplicative scatter correction (MSC), the Kubelka-Munk
Average Rl\15 for
less than O. 3 X 10- 3 ; no RMS nmse transforms, spectral compression techniques that may
measurements
greater than O. 8 X 10- 3 include windowing and noise reduction and the numerical
at high-light flux
calculation of the first-or second-order derivative of the
spectrum. In sorne cases spectra may also be normalized.
Caution must be excercised when performing any rnathernatical
Average Rl\15 for transformation, as artifacts can be introduced or essential
less than 1 X 10- 3 ; no RMS nmse
measurements at information can be lost In all cases the rationale for the use of
greater than 2. OX 10- 3
low-light flux transform must be docurnented.
Since multivariate NIR spectral data contain a huge number
CDA maximum nominal instrument band width of 10 nm at of correlated variables ( = colinearity) , there is a need for
2500 nm or 16 cm- 1 at 4000 cm- 1 is appropriate for most reduction of variables, i. e. , to describe data variability by a
applications. few uncorrelated variables containing the relevant information
®SRM 1920a is a standard substance provided by NIST for for calibration modeling. The best known and most widely
NIR wavelength calibration. To determine wavelength used variable-reduction method is principal component
uncertainty, the wavelength value of 1935 nm peak in a analysis CPCA).
spectrum is calibrated by SRM 1920a.
( 3) Model construction
®Aoas is the observed absorbance, and AREF is the tabulated
In qualitative analysis, sample properties that have to be
absorbance of the reference reflectors at each of the three
related to spectral variations have discrete values that
specified wavelengths.
represent a product identity ora product quality. In general,
2. Transmission mode This method is usually suitable for pattern-recognition methods are used for grouping samples
liquid sample. Transmittance ( T) is a measure of the with similar characteristics. These methods are subdivided
decrease in radiation intensity at given wavelengths when into discriminative analysis and cluster analysis. Discriminant
radiation is passed through the sample. The sample is placed analysis is used to build classification rules for a number of
in the optical beam between the source and detector. The pre-specified subgroups, i. e. , the group structure of the
result can be represented directly in terms of transmittance training set is known. Cluster analysis is suitable for the
(T) or/and absorbance (A) in both cases. grouping of sample without any requirements on apriori
T=I/Io, knowledge about the group structure in the data.
A =-lg T= lg(l/T)= lgCio/D ( 4) Model validation
Where I 0 is the intensity of incident radiation. In qualitative analysis, sample properties that have to be
I is the intensity of transmitted radiation. related to spectral variations have discrete values that
Transflection mode is a combination mode of transrnission and represent a product identity or a product quality.
reflection. The detector and light source are placed on the same a. Specificity
side of the sample. In the measurement of transflectance, a rnirror The validation of selectivity involves of the classification
or a diffuse reflectance surface is used to reflect the radiation using database spectra for positive identification of a given
transrnitted through the sample a second time. material and adequate discrimination against other materials
 9105 Guidelines for Bioactive Assays of Traditional Chinese Medicine
in the database is to be established during the validation
procedure. Potential challenges must be addressed to the
spectral database. These can be materials received on site
that are similar to database members in chemical structure.
This challenge must fail identification.
b. Robustness
The robustness of the qualitative procedure must also be
challenged to test the effect of minar changes to normal
operating conditions on the analysis. There must be no changes
to pre-processing and calibration algorithm parameters. Typical
challenges are:
CDEffect of differences operators;
@Environmental conditions ( for example, temperature and
humidity in the laboratory);
@Effect of sample temperature, sample positioning on the
optical window and pro be depth and compression/ packing of
material;
@Replacement of instrument parts.
2. Quantitative analysis
Quantitative model construction also contains the following
steps: Record the NIR spectra of representative samples
first, then pre-treat data and reduce its dimensionality by
selected chemometric methods, establish the qualitative
model and finally validate it.
( 1) Collection of representative samples
Record the spectra of a suitable number of samples with
known values of the content within the range to be
measured. The content range of calibration sets should be
broader than that of validation sets, the samples can be
attained by accelerated testing or special preparation if
necessary.
(2) Pre-treatment of data
See Qualitative analysis.
(3) Model construction
Sample preparation is generally not required for NIR
applications. However, NIR measurements can be affected
by many factors. So it is difficult to gain an accurate quantitative
result with a single wavelength. Multivariate calibration methods
are commonly used for model construction, such as multiple
linear regression ( MLR ) , partial least square regression
(PLSR) , principal component regression ( PCR) and artificial
nerve network (ANN).
(4) Validation parameters
To validate NIR methods, analytical performance
characteristics to be considered are similar to those required
for any other analytical procedures. Specific acceptance
criteria for each validation parameter must be consistent with
the intended use of the method. The validation parameters
include specificity, linearity, accuracy, precision and
reproducibility.
Ongoing model evaluation
Revalidation of a qualitative model will be necessary when
changes in the physical properties of the material occur and when
changes in the source of supply take place. Revalidation of a
quantitative model is required due to changes in the composition
of the finished product, in the manufacturing process and in
sources/ grades of raw materials.
Model transfer
When models are transferred to another instrument, type of
the instrument, format of the data, spectral range,
number of data points, spectral resolution and other
parameters have to be taken into consideration. Far
example, to establish a model on one instrument by
representative samples ( the number of which is decided by
the model) , the spectrum of these samples on another
instrument are measured. The spectra on both instruments
are analyzed by the model established on the first
instrument. Two results must be tested statistically to
demonstrate whether the model is valid on the second
instrument or not, otherwise a separate model must be
rebuilt on the second instrument.
9105 Guidelines for Bioactive Assays of
Traditional Chinese Medicine
As the method for determining the efficacy of the medicine
based on its biological effect, bioactive assay uses
biostatistics analysis as tools and implements a special test
design to ensure quality control of medicine. The methods
include potencytest and bioactive limit test.
Since the source of Chinese materia medica is numerous and
varied, and the manufacturing process is complex, it is
difficult to control the quality of products. Additionally,
since traditional Chinese medicine comprises of multiple
active ingredients with m ultiplex pharmacological effects,
it is impossible to ensure the quality completely and reflect
clinical efficacy by only monitoring a few of chemical
ingredients. In arder to better ensure safety and efficacy of
every batch product in the clinical use by the quality
specifications of traditional Chinese patent medicine, it is
necessary to add bioactive assays to the current
specifications.
The purpose of the guideline is to standardize the study of
bioactive assays of traditional Chinese medicine and provide
principle requirements for test design, methodology study
and the application of bioassays, etc.
General Principie
Compliance with the basic principies of pharmacological
research The established method of bioactive assays should
comply with the basic pharmacological principles of randomness,
comparability and reproducibility. It should be simple and
precise, and have clear criteria.
Incarnate the characteristic of Traditional Chinese Medicine
It is encouraged to research the quality control of
Traditional Chinese patent Medicine with bioactive
assays. The determination index of the method to be
established should be relevant with " the actions and
indications" of the traditional Chinese medicine according
to traditional theory.
Selecting product reasonably The Chinese material medica,
prepared slices, extractions and finished products of
Traditional Chinese patent Medicine to be studied should be
clear in actions and indications. The products with well-
defined indications should be considered as a top-priority.
The injections and the drugs for emergencies and severe case
are researchedwith emphasis.
Scientific and reliable method Potency bioassays are preferred.
If potency assay can not be established for the product, bioactive
limit test can be considered. When the condition is ready,
potency assay should be taken in the future.
Primary Content
l. Experimental Condition
Test System Pharmacological experimental systems used for
bioactive assays include animal, isolated organ, blood or serum,
microorganism, tissues, cells, subcellular organelles, receptors,
ionic channels and enzymes. The selection of experimental
 9106 Guidelines far Pharmaceutical Evaluation based on Microarrays
system is closely related to the experimental principies and the
determination index. The test system should have sufficient
and clear background infarmation, fewer affecting factor,
sensitive and inexpensive index. It should investigate all
possible affecting factors in the test system, and control
these factors if necessary.
lf the test systems are animal-based, mice or rats are
recommended because of their ample resources and low
cost. It is required to describe the species, strain, sex and
age of the animals. The application of animals should
comply with the '3R' theory of Refinement, Reduction
and Replacement.
Selection of Sample The manufacture processes of samples
should be stable, the quality should meet the specifications,
and at least three batches should be provided. Far prepared
slices, the origin species should be clear.
Selection of Reference Standard If potency testing will be
used far bioactive assays, a homogenized reference standard
or reference materials should be used to measure relative
biological activity of the sample. Reference standards or
reference materials should be manufactured with Traditional
Chinese patent Medicine as far as possible. However,
Chemical medicine can be used as the ref erence standard. In
bioactive limit tests, Chinese Medicine ingredient or chemical
drug can be used to validate the test as positive reference
materials. These materials should be selected according to
experimental data and theoretical basis. Reference standards
or reference materials should meet the requirements of
relevant official standard.
2. Experimental Design
Design principies The principie of test should be clear, the
determinate index should be objective and specific. It should
reflect the action or indications or pharmacologic actions of
the product.
Design type The experiment design of potency test should
fallow " Statistical Methods far Biological Assay "
( 1431 ) . In the case of bioactive limit test, it can be taken
into consideration to set samples group, negative and
positive control group. When animal model is used, it
should be considered to set up model comparison group. If
the test has good repeatability, positive control can be
omitted or is set up only in retest.
Dosage design In the case of potency test, samples and
standards may be advisable far multi-dosage groups, which
should comply the requirements of bioassay statistical analysis,
moreover the measurement among various dosages should be
marked differently. In bioactive limit test, it is recommended to
set one liminal dosage, and bioactive effect should be observed
under that dosage, however, in the research of methodology, a
multi-dose study should be carried out to clarify the reason of the
liminal dosage set in specifications.
Administration Route It is recommended to choose the same
administration route as that in clinic, or it is required to state
the reasons.
Administration Frequency Advisable administration frequency
should be selected on the basis of pharmacodynamic study. One-
time or multiple administration is adoptable.
Determination index The selected index should be objective,
clear and accuracy and reflect the action or indica tions of the
product. The rationality, suitability, and representativeness
of the index should be fully explained.
3. Results and statistics
The evaluation of test results should meet the requirements
of bioassay statistical analysis. In a potency study, it is
necessary to comply with "Statistical Methods far Biological
Assays" ( 1431 ) and potency range and fiducial limit CFL %)
are determined by the variability of bioassays data of the
product. In bioactive limit test, it is necessary to explain
deviation control, provide unambiguous criterion for the
acceptability of the bioassay, perform statistical analysis of
the results, and describe detailed method and selected reason
clearly.
4. Evaluation
In the case of potency study, the results of test should be
evaluated according to the definitive potency range and the
fiducial limit ( FL%). In bioactive limit test, it is
recommended to judge the results below the limit dosage of a
drug. If the result is significant in preliminary examination,
the requirements are met. If it is not significant in
preliminary examination, a retest can be carried out by
including more specimens anda positive control group. If the
results of retestare statistically significant, the requirements
are met, otherwise not.
Validation of Bioassay Method
l. Investigation on affecting factors
To ensure the accuracy and specificity of the test, all
affecting factors should be investigated and the optimum
experimental conditions are determined accordingly. According
to the results of investigation, the control limit of experimental
error or reliability of statistic analysis is provided. For
experiments on isolated organs, the in vitrcrin viw correlation
should be examined.
2. Precision investigation
The precision includes repeatability, intermediate-precision
and reproducibility.
Repeatability For a gíven method, test resuits from at ieast
3 batches (3 measurement per test) or 6 measurements far
one batch should be evaluated. The results from hioassay
experiments should be similar in general.
Intennediate-precision The internal changes of conditions in
one laboratory should be evaluated on their effects on results,
such as changes in experimental time, persons, instrument.
At least the effect of different examiner m one laboratory
should be examined.
Reproducibility The reproducibility test should be carried
out by at least three different laboratories and the results
should be consistent.
3. Suitability Study
To accumulate infarmation and evaluate the suitability of the
bioactive assay, at least 10 batches product should be tested
using the method and dosage intended far use in bioactive
assay.
9106 Guidelines for
Pharmaceutical Evaluation
based on Microarrays
This guideline stipulates the principie, definition and primary
technical parameters of microarrays when applying to safety
and efficacy evaluation of pharmaceutical products, as well as
the requirements of test sample, the preparation of graphs
and analysis methods. The purpose is to standardize safety
and efficacy evaluation of pharmaceutical products using
microarrays and provide instructions on fundamental
requirements far experiment design, methodology establishment
 9106 Guidelines for Pharmaceutical Evaluation based on Microarrays
and adaptability of determination methods.
l. Definition and Principie
Pharmacogenomics, which is also referred as pharmaceutical
genomics or pharmacology genomics and belongs to the
research field of pharmacology, studies altered effects
exerted by medicines due to genetic variationsby correlating
efficacy or toxicity of medicines with fluctuations of gene
expression or single nucleotide polymorphism from genomic
study.
Toxicogenomics is to study the interactions between toxic
effects of drugs and gene expression from a multi-gene or
whole genome level, which mainly aims at promoting the
understanding of the relations between environment stressors
and disease susceptibilities, elucidating the mechanism of
toxic effects, screening and confirming the biomarkers for
disease or toxin exposure.
DNA microarray, also referred as DNA array or DNA chip,
is commonly known as gene chip. DNA microarray is a
special glass or silicon chip with a dimension of square
centimeters, which is loaded with thousands or tens of
thousands of nucleic acid probes. When the DNA, cDNA or
RNA of the sample are hybridized with probes, the
information about number or sequence of sample can be
obtained from intensity of hybridization signal using
fluorescence or electricity detection. Massive information
about gene sequence is provided by one microarray test, thus
it is known for high throughput, multi-factor, microscale
and sensitivity.
Rationale: Specific recogrütions between biological molecules are
utilized for biological inforrnation processing.
Depending on biological samples used in the experiment,
microarray can be categorized into cDNA chip, single
nucleotide polymorphism chip, miRNA chip, siRNA chip,
chromatin immunoprecipitation-chip, and MeDIP-chip, etc.
2. General Principie
Genomics methods adopted in safety and efficacy evaluation
of pharmaceutical products should comply with general
principle of in pharmcogenomic research, e. g. , randomness,
comparability and reproducibility, simplicity and precision,
as well as clear criteria for decision making.
3. Primary content
( 1) Acquirement of biological sample
Experimental systems
Experimental systems used in microarray adopted in safety
and efficacy evaluation of pharmaceutical products include
animal, isolated organ, blood or serum, tissues, cells, etc.
The selection of the systemis closely related to the
experimental principies and determination index. Selected
experimental system should possess sufficient and clear
background information, fewer affecting factors, sensitive
determination index and inexpensive cost. If possible, all
affecting factors in the experimental system should be
investigated and controlled by necessary methods.
If animals are used in experimental system, mice or rats are
recommended due to multiple sources for supply and low
cost. It is required to describe the species, strain, sex and
age of the animals. The use of animals should comply with
the' 3R' theory of Refinement, Reduction and Replacement.
Selection of Sample
The testing samples should be manufactured by stable processes
and their qualities should be tested and meet required
specifications. At least three batches of samples should be
evaluated. If samples are decoction pieces of traditional chinese
medicine, their origin species should be clear.
Selection of standard or reference substances
Selection of standard or reference substances should base on
scientific theory and/ or experimental data. The applications
of standard or reference substances should comply with
relevant regulatory requirements.
(2) Biological experimental design
Design Principies
The principie of test method should be clear with sensitive,
objective and specific determination index.
Design Type
Experimental design should consider involving sample group,
negative or positive control group. It should be considered to
include model group when animal model is used. For study
with good repeatability, positive control can be omitted or
only included for retest.
Dosage design
According to requirements on bioassay, rational dosage
design should be performed according to clinical dosage of the
drug. The dosage that produces detectable bioactivity in
microarrays should be selected as experimental dosage. To
set up quality standard, multi-dose study should be carried
out to clarify the reason of dosage limit in specifications.
Administration route
It is recommended to choose the administration route used in
the clinic, otherwise the reasons should be stated for using
different administration routes.
Administration frequency
Administration frequency should be decided on the basis of
pharmacodynamic study. Once or muitipie administration is
acceptable and the frequency used in the study should be kept
the same as clinical administration frequency if possible.
Determination index
The selected index should be objective, clear and accurate
and reflect efficacy and safety of the drug.
Preparation of Biological Sample
If possible, aseptic, disposable plastic products or RNase-free
labeled, unopened plastic products should be used in the study.
Proper amount of RNA stabilizer can be added to the reagent.
Maximum number of samples that can be handled every run
should be determined and the influences of preparation
procedures to RNA integrity should be minimized. The affecting
factor of RNA quality should be evaluated, such as sample
size, etc. When biological samples have been collected,
RNA preparation should be carried out immediately. When
temporary storage is needed, the sample should be quickly-
frozen with liquid nitrogen and stored in - 80ºC freezers. In
RNA preparation, material stored in freezer should be
ground in liquid nitrogen to break up cells after taken out.
Thawing is forbidden to avoid the effect of RNase.
( 3) Microarrays Technology
To ensure precision, stability and reliability of microarrays
data, the standard operating procedure (SQP) of microarrays
should be established and strictly followed. microarrays
adopted in safety and efficacy evaluation of pharmaceutical
products mainly include: microarray preparation, sample
acquirement, total RNA extraction, amplification, fluorescence
labeling hybridization, wash, differential expressed gene
detection, analysis and validation.
The general rationale and notes for each procedure are
describedin details below.
Extraction, separation and preparation of RNA
The extraction of RNA mainly includes RNA extraction from
tissue, cell, whole blood or peripheral blood mononuclear
cells CPBMCs) and commonly used RNA extraction methods
 9107 Guidelines for Molecular DNA Barcoding of Chinese Materia Medica

are TRizol method, phenol method, guanidinium/~-sulfur Reproducibility

based ethanol method, etc. Extracted total RNA should be
purified by RNA purification kit, which removes compounds
inhibiting reverse transcriptase activity, avoids pollution
from organic solution and metal ions, and prevents
contamination of protein, polysaccharide and lipid molecules.
Preparation of microarrays
Using glass or slicon chip as carrier, oligonucleotide
fragments or cDNA are ordered on the carrier as probes by
in-situ synthesis and microarray method.
Fluorescence labeling
During the amplification step of DNA, dUTP or dCTP with
Cy3 or Cy5 fluorescinare added to produce newly synthesized
DNA strand with fluorescence labeling.
Hybridization and wash
Hybridization is the step that fluorescence labeled gDNA/
cDNA is complementarily combined with the probes in chip
with high specificity.
Microarray scanning
Hybridized gene chip is submitted to the microarray scanner
for general graph of intensity of hybridization signal from
different probes.
Data analysis
Chip graph is analyzed by image analysis software in arder to
transform the image signal into digital signal, locally
weighted scatter plot smoothing method CLOWESS or
LOESS) is applied to normalize the data and the differential
expressed genes are determined on the basis of intensity or
ratio of Cy3 or Cy5 signal.
Real time fluorescence quantitative PCR is used for
quantification and validation of differential expressed genes.
( 4) Validation of Microarrays
Cl)Affecting facíors study
All affecting factors should be investigated to determine the
optimum condition, which should ensure the accuracy and
specificity of the test. Based on the results of affecting factor
study, the control limit of experimental error can be determined or
the reliability of statistical analysis can be evaluated.
@Precision
The prec1s1on study should determine repeatability,
intermediate-precision and reproducibility of the method.
Repeatability
Far a given test method, results comparison should be
performed on at least three tests on three batches or 6 tests
for the same batch of sample. All the results from
microarrays should be similar in general.
Intennediate-precision
The influence of internal factors of the study Ce. g. , different
persons, different instruments, different dates or experimental
time, etc. ) on test results should be evaluated.
The results of microarrays should be reproduced by at least
three independent laboratories.
@Suitability Study
Using proposed microarrays and dosage, at least 10 batches
of product should be tested to accumulate data and evaluate
the suitability of these methods in quality specifications.
9107 Guidelines for Molecular DNA
Barcoding of Chinese
Materia Medica
This guideline is applicable to authentication of Chinese
Materia Medica (crude drug and sorne decoction pieces) and
origin at species.
As an effective method to complement with traditional
morphology-based approaches, molecular DNA barcoding
refers to a molecular biological technique for species
identification using a commonly recognized, relatively short
DNA sequences of the genome. The DNA sequences of
various species are composed of four repeating nucleotide
bases [ adenine CA) , guanine CG ) , cytosine CC ) , and
thymine (T) J in unique orders. Therefore a specific DNA
sequence of one species could be used to discriminate it from
other species.
Molecular DNA barcoding of Chinese Materia Medica refers to a
methodological system to identify Chinese Materia Medica through
the second internal transcribed spacer of nuclear ribosomal DNA
CITS2) O as the main barcode sequence for identification. With
ITS2/ITS as the main sequence, psbA-trnH 8of chloroplast can
be used as the complementary sequence for identification of
herbal Chinese Materia Medica. For identification of Chinese
Materia Medica from animal origin, Cytochrome C oxidase
subunit I CCQI)C. is used as the main sequence and ITS2 is
used as the complementary sequence.
l. General Requirements for lnstruments
The instruments involved in the guideline include electronic
scales, centrifuge, polymerase chain reaction C PCR)
instrument, electrophoresis meter and DNA sequencer, etc.
DNA sequencer is a fully automatic computer-controlled
instrument to determine the nucleotide sequence or size through gel
pouring, sampling, data collection and analysis, which also is an
accurate instrument far the quantification of DNA fragments.
Dideoxy chain termination method, also known as Smger method,
is the main method used far sequencing. Four types of dideoxy
nucleotide CddNfP) bases are labeled with different fluorescents.
When running through the capillary, these four fluorophores of
DNA fragments with different lengths will emit different colours of
fluorescence after excited by laser. The fluorescence is recognized

O ITS: ITS (interna! transcribed spacer of nuclear ribosomal DNA) is interna! transcribed spacer, which is a part of the non-
transcribed region of the ribosomal RNA CrRNA) genes. Located between the 18S rRNA genes and the 28S rRNA genes, ITS can be
divided into ITSl ( the first interna! transcribed spacer) and ITS2 ( the second interna! transcribed spacer) by the intermediate 5. 8S rRNA.
The evolutionary rate of 5. 8S, 18S and 28S is relatively slow. Thus they are commonly used to explore the phylogenetic problems at the
family level and above. While the evolutionary rate of ITS spacer ( including ITSl and ITS2) is generally faster, this spacer is usually used
to study the phylogenetic relationship at relatively low taxonomic ranks, such as inter-genus, inter-species and inter-population
classifica tions.
8 psbA-trnH: psbA-trnH gene region is a non-coding region located between psbA and trnH gene of chloroplast with a fast evolutionary
rate, which is usually used to study the phylogenetic anlysis of plants from different genus or species.
C. COI: COI represents cytochrome C oxidase subunit l that is a protein coding gene in mitochondrial genome. Since evolutionary rate
of this gene is fast, it is commonly used to analyze phylogenetic relationships of closely related species, subspecies or geographic populations.
 9107 Guidelines far Molecular DNA Barcoding of Chinese Materia Medica
by the image sensor detection system of the charge-coupled device
(CCD) and translated into DNA sequence to obtain the peak
charts and sequence files of test substance.
2. Procedure
This method mainly includes sample preparation, DNA extraction,
PCR amplification on DNA barcode sequence, electrophoresis
detection and sequencing, sequence assembly and result
determination. The main steps are described as fallows:
( 1) Sarnple preparation
Sampling of the crude drug and decoction pieces should be
carried out by methods described in Sampling of Crude Drugs
and Decoction Pieces ( 0211 ) . In order to prevent
contamination by external microorganisms, crude drug and
decoction pieces generally should be wiped by 75 % ethanol
then dried, or using other effective methods to remove
microbial contamination. A total of 10-100 mg of crude drug
and decoction pieces need to be weighted far further use. The
sampling sites on crude drug or decoction pieces are
dependent on the characteristics of corresponding Chinese
medicine, which should be stipulated accordingly.
(2) DNA extraction
The procedures of DNA extraction include breaking and crushing
cells into fine powder by a mortar or beveller and DNA isolation
and purification by commercial kits. Currently, the commonly
used kits include plant genomic DNA extraction kit and animal
tissue/ cell genomic DNA extraction kit. The kit used in
experiment should be able to extract template DNA that meet the
requirements for further experiments.
Because herbal chinese medicines display a great variety; the
extraction method can be improved accordingly. For example,
during the process of DNA extraction, a large amount of
polysaccharides, polyphenols and other secondary metabolites in
the plant cells can co-precipitate with DNAs to form sticky
gelatinous material that is difficult to dissolve or easy to be
browned by oxidation, which would seriously influence the yield
and quality of DNA extraction and disrupt subsequent PCR
amplification experiments. The antioxidant ¡t-mercaptoethanol
can be added during the process of DNA extraction to prevent
browning by inhibiting oxidation reactions. Also polyvinyl-
pyrrolidone (PVP) is a phenol chelator that can farm an
insoluble complex compound with polyphenols, thus
effectively removing polyphenols and reducing the contamina-
tion of polyphenols during DNA extraction. Furthermore, it
can chelate with polysaccharides and effectively remove these
compounds. Therefare, the contamination by polyphenols
and polysaccharides in the process of DNA extraction can be
effectively prevented when PVP and ~-mercaptoethanol are
used cooperatively. Moreover, Ethylene Diamine T etraacetic
Acid ( EDTA) can chelate Mgz+ or Mnz+ to inhibit the
activity of DNA enzyme ( DNase) and prevent the
degradation of DNA by DNase. In natural conditions, the
DNA and protein exist in the farm of DNA-protein complex
(DNP). Cetyltrimethyl ammonium bromide (CTAB) is a
type of cationic detergent to release the DNP from cells by
dissolving the cell membrane and farming a complex with
DNA. This complex can be fully dissolved in the liquid phase
by concentrated salt solution (>O. 7 mol/L NaCD. Through
organic solvent extraction and addition of ethanol,
impurities, such as proteins, polysaccharides and polyphe-
nols, etc. , are precipitated and DNA can be separated from
the complex Tris ( hydroxymethyl) methyl aminomethane
( Tris-HCl) ( pH 8. O ) solution can provide a buffer
environment to prevent the degradation of DNA.
Materials from root, rhizome, stem and peel: Usually a high
content of polyphenols and polysaccharides are faund in
tissues of root and rhizome. The polyphenols are easily
oxidized to quinones when grinding, which results in
coloured DNA that is difficult to remove during the
purification process and affects the subsequent PCR
reactions. Special attentions should be paid to the removal of
polysaccharides and polyphenols when extracting DNA from
root and rhizome of herbal chinese medicines. It generally
takes 90 mins to extract DNAs of these materials using water
bath. For those chinese medicines from solid root, rhizome
and stem, it is recommended to prolong the duration of
incubation and simultaneously reduce the temperature of
water bath, such as 56ºC water bath far 8-12 hours, to make
sure of a full release of DNA to the buffer solution. In
addition, rhizome is enriched with storage materials of plants
such as fiber and starch, etc. Therefare, to obtain adequate
amount of extracted DNA, it is necessary to increase the
amount of sample used and centrifuge tubes with large
volume (5 ml or 15 mD. The peel, where parenchyma tissue
and fibers are enriched, is not easy to be ground into fine
powders with liquid nitrogen. Thus the amount of samples,
{3-mercaptoethanol, and PVPshould be increased accordingly.
Materials from leaves, flowers and whole herb: Generally,
using an appropriate kit, DNA can be successfully extracted
far chinese medicines derived from those materials. For
leaves, flowers and whole herb that have been in the storage
far a long time, it is recommended to properly increase the
bath time and reduce the temperature of water bath
simultaneously, such as 56ºC water bath far 8-12 hours.
Materials from fruits and seeds: Chinese Materia Medica that
are derived from fruits and seeds are enriched with oils, thus
they are easy to be oxidized during grinding and adhere to the
wall of the mortar, then leading to a great loss. The amount
of samples used far DNA extraction should be increased. In
addition, the materials after grinding can be immersed with
acetone in order to remove the fat-soluble phenol
compounds.
Materials from animal origin: Chinese Materia Medica from
animal origin enriched with muscle tissues, such as
syngnathus, snake, gecko, etc. , should be wiped by 75%
ethanol solution to eliminate exogenous contamination, and
then completely ground after the evaporation of ethanol.
Those animal organs enriched with lipids, such as toad oil,
should first be soaked in the buffer solution without protease
K and twelve alkyl sodium sulfate (SDS). SDS is an anionic
surfactant, which can lyse cells and release the nucleic acid
under 55-65ºC. It is beneficia! far the removal of lipids by
increasing the content of SDS in the digestive buffer solution
of the kit. Dueto their low DNA content, increased amount
of samples and a large volume of centrifugal tube should be
used to extract DNA from Chinese Materia Medica derived
from horn and carapace, such as tortoise, turtle shell, and
antier, etc. The symbiotic or parasitic organisms faund in
Shell materials, such as Concha Haliotidis, Concha Arcae,
and Concha MeretricisseuCyclinae, etc. , should be removed
befare extraction.
( 3) PCR arnplification
ITS2 or psbA-trnH sequence is used far amplification far
herbal chinese medicine and their original species. COI
sequence is used far amplification far Chinese Materia Medica
farm animal origin and their original species. Common
primers and amplification conditions are shown as fallows.
Special requirements are shown in the monograph of
corresponding Chinese Materia Medica.
The farward primer of ITS2 amplified sequence, ITS2F, is 5'-
iITGCGfil'ACfTGGTGTGAAr -3'; reverse primer ITS3R:
 9201 Guidelines for Validation of Alternative Microbiological Methods for Pharmaceutical Products
5'-GAa;crrcrccAGACTACAfil -3'. The forward primer of
psbA-trnH amplified sequence, psbAF, is 5'-GTTATGCA-
1GA!í:GIAAITrIC-3;~ ¡:rirrer t:rnI-IR:5'-cn:ID'IT-
GGTGGATTCACNITCC -3' . The forward primer of OOI
amplified sequence, Ha:>2198, is 5'-TAAAGITICAGGGT-
GACCAAAAAfrrCA-3', reverse primer lill1490: 5'-
GGTCAACAAXrCATI\AAGKDTITGG -3'.
A 25 µl PCR reaction system is taken as the reference,
including 1 X PCR buffer Cwithout MgClz ) , 2. O mmol/L
MgCl 2 , O. 2 mmol/L dNTPs, O. 1 µmol/L primers in pairs,
template DNA, l. O U Taq DNA polymerase then double
distilled sterile water is added to 25 µl. The PCR reaction
without template DNA is used as a negative control.
The sequence amplification procedure of ITS2: 94 ºC, 5 min;
94 ºC, 30 s, 56ºC, 30 s, 72ºC, 45 s, 35-40 cycles; 72ºC, 10
min. The sequence amplification procedure of psbA-trnH:
94ºC, 5 min; 94ºC, 1 min, 55ºC, 1 min, 72ºC, l. 5 min, 30
cycles; 72ºC, 7 min. The sequence amplification procedure
of COI: 94ºC, 1 min; 94ºC, 1 min, 45ºC, l. 5 min, 72ºC,
l. 5 min, 5 cycles; 94ºC, 1 min, 50ºC, l. 5 min, 72ºC,
1 min, 35 cycles; 72ºC, 5 min.
( 4) Detection of PCR products
PCR products are detected by agarose gel electrophoresis
method. After electrophoresis, the target strip of the PCR
product is expected to appear in the position of corresponding
length of the DNA barcode. (For details, refer to monograph
of the material). There is no strip expected for the negative
control group.
e5) Sequencing
The target strip should be isolated immediately to make gel
under the ultraviolet light then purified by the agarose gel
DNA recycling kit. The target strip is then sequenced by
bi-directional sequencing. PCR amplification primers are
used as sequencing primers and the sequencing principle was
the same as the Sanger method. Samples with a target strip
will be sequenced in DNA sequencer instrument by bi-
directional sequencing.
( 6 ) The acquisition of DNA barcode sequences of Chinese
Materia Medica
CD Sequence assembly: The sequence assembly of the
bi-directional sequencing map can be performed using
professional software with sequence assembly function. The
primer area should be excluded.
@ Quality and direction of sequence: To ensure the
reliability of the DNA barcode sequence, the corresponding
DNA sequence can be obtained by excluding the weak-signal
or overlapped areas at both ends of the result and keeping the
same direction of the forward primer used in PCR
amplifica tion.
(7) Determination of results
The obtained sequence should be aligned with the standard
sequence of DNA barcode of Chinese Materia Medica approved by
the state department of pharmaceutical administration.
3. Method Valiclation
The validation should comply with the requirements listed in
Guideline for Validation of Analytical Methed Adopted in
Pharmaceutical Quality Specification (9101 ).
( 1) Investigations of affecting factors
Molecular DNA barcoding method should investigate these
affecting factors, including DNA extraction C sample
amount, bath temperature and time), PCR conditions
(degeneration time, annealing temperature and time as well
as extension time) and product purification e different
purification kits), to ensure the accuracy of the method.
(2) Investigation of method suitability
At least 20 batches of Chinese Materia Medica or origin
species should be determined by the molecular DNA
barcoding method. The da ta are collected to determine the
intra-species sequence variations to ensure the suitability of
the method.
C3) Comparison validation of origin species
Leaves of origin species identified by taxonomists are selected
as samples and their DNA barcodes data are obtained by the
method mentioned above, which are aligned and compared
with sequences of corresponding Chinese Materia Medica.
The contamination of the endophytic fungi should be avoided
to ensure the accuracy of the result.
4. Notes
(1) The testing laboratory should fulfill basic requirements
of a molecular biology laboratory.
C2) This method is currently unsuitable for mixtures,
processed or sulphur fumigated chinese medicine.
C3 ) To prevent the contamination by the exogenous
microorganism, the laboratory appliances should be
sterilized by high pressure before the experiment and wiped
by 7 5 % ethanol. Far chinese medicine containing the
endophytic fungi, the interna! tissue is selected for testing if
the fungi exist in the externa! tissue. If the fungi are
distributed all around Chinese Materia Medica, psbA-trnH
barcode enot existed in fungus) should be used instead of
the ITS2 sequence. BLAST method of GenBank can be
used to validate the obtained ITS2 sequence to ensure the
accuracy of the result.
(4) This method is suitable far the authentication of the
original species of Chinese Materia Medica, however it is not
applicable far the authentication of medicinal parts of Chinese
Materia Medica.
(5) Sorne other methods can be combined with this method
to give comprehensive determination when necessary.
(6) Determination of intra-species thresholds. Variations of
sequences from different samples of one species are often
observed, which is known as intra-species thresholds. These
variations are species-dependent and related to barcode
sequenceused. The variations Cintra-species genetic distance
thresholds) of each origin species should be defined in the
monograph of corresponding Chinese Materia Medica.
9201 Guidelines f or Validation of
Alternative Microbiological
Methods f or Pharmaceutical
Producís
The objective of this chapter is to provide guidance far
validating methods for use as alternatives to the compendia!
microbiological methods for pharmaceutical products.
Wi th the rapid development of rnicrobiology, sorne new methods
far pharmaceutical rnicrobiological control have been introduced,
which are roughly divided into three types: e1) growth-based
detection methods, where a detectable signal is usually
achieved by a period of subculture, such as bioluminescence,
electrochernical methods and turbidimetry; (2) direct detection
measurement of viable rnicroorganisms, such as salid phase
cytometry and flow cytometry; e3) specific cell component
analysis, such as fatty acid analysis, nucleic acid amplification
techniques and genetic fingerprinting. Compared with compendia!
methods, these technologies have shown more convenience and
rapidity. Sorne techniques have potential for real-time or
near-real-time monitoring, which will be possible to take
 9201 Guidelines for Validation of Alternative Microbiological Methods for Pharmaceutical Products
corrective measures early in the production and to guide good
manufacturing practice. Moreover, use of new technologies
also contributes to reducing costs and improving the ability of
inspection.
For controlling pharmaceutical microbiological quality,
microbiological testing laboratories sometimes use non-
compendial test methods ( i. e. , alternative methods) for a
variety of reasons, such as economics, throughput,
convenience and requiring better quality for pharmaceutical
products. Validation of these methods is required. The
alternative method will yield results equivalent to, or better
than, the results generated by the compendial method.
Types and Validation Parameters of
Microbiological Tests
There are two major types of microbiological tests for
pharmaceutical products: qualitative test and quantitative
test. Qualitative test is to determine whether viable
microorganisms are present in a sample, such as sterility test
and specific microorganism test. Quantitative test is used to
quantify the number of viable microorganisms in a sample,
such as plate count method.
The test results are subject to several factors of
microbiological method, due to the unique nature of
biological assays, for example, the sampling error, dilution
error, operator error, incubation error and enumeration
error. The guidelines far validatian af analytical methad
adapted in pharmaceutical quality specificatian ( 9101 )
may not be suitable for validation of alternative
microbiological methods. Vaiidation parameters of
alternative pharmaceutical microbiological methods are listed
in Table l.
Table 1 Validation Parameters by
Type of Microbiological Test
Qualitative Quantitative
Parameter
Tests Tests
Accuracy + promotion of the media for qualitative methods that rely upon
Precision + microorganisms growth to demonstrate presence or absence of
Specificity + +
Limit of detection + consider the effects for the test articles. However, for those
Limit of quantification + presence, the specificity of the assay for microbes assures that
Linearity + extraneous matter in the test system <loes not interfere with
Range + the test. While testing specified microorganism by alternative
Robustness + +
Reproducibility + +
Note: +: the parameters need to be validated
- : the parameters needn' t to be validated
Though validation parameters of alternative methods share
sorne of the same concerns with the analytical methods
adopted in pharmaceutical quality specification, the validation
parameters should be established by the unique nature of
microbiological assay. Statistical analysis is required to
validate the alternative microbiological methods. In general,
the qualitative test should be evaluated by nonparametric
statistical methods. In contrast, quantitative test may require
to be evaluated by parametric statistical methods.
When validating the alternative microbiological methods, it is
critical to the validation effort to identify the portian of the
test addressed by the alternative method rather than the
entire test method. For example, a sterility test by
membrane filtration may be performed according to the
compendia procedure up to the point of combining the
processed filter with the recovery media, and after that the
presence of viable cells might then be demonstrated by use of
sorne of the available technologies. Validation of this
application would, therefore, require validation of the
microbial recovery system employed rather than the entire
test.
General Requirements
Before validating an alternative method, it is necessary to
have a comprehensive understanding of the method. First of
all, the alternative method should contain necessary
evidences to show its specificity, precision, limit of detection
and other parameters are suitable for different types of
microbiological tests when samples are absent. Such evidence
would be provided by the suppliers of the alternative method,
or by the users.
The parameters in Table 1 should be validated in the presence
of sample after the initial verification of the alternative
methods to confirm whether the alternative methods are
suitable. At least two batches of samples should be used and
3 replicates should be assayed for each batch.
The bacteria involved in validation parameters should include
the strains under the growth promotion and suitability test of
media in Microbialogical Examinatian af Nan-sterile
Product: Micrabial Enumeratian Tests ( 1105 ) ,
Micrabialagical Examinatian af Nan-sterile Praducts:
Tests far Specified Micraarganisms ( 1106) and Sterility
Test <1101 ) , as well as the additional strains according to
the nature of alternative methods and the characteristics of
the samples. The bacteria should be validated separately.
Validation of Qualitative Tests of
Microorganisms in A Sample
l. Specificity
The specificity of an alternative qualitative microbiological
method is its ability to detect a range of microorganisms that
may be present in the test article. Besides confirming growth
microorganisms, the specificity of the method should also
methods do not require growth as an indicator of microbial
method, the strains with similar characteristics to the specified
microorganism should be selected and validated.
2. Lirnit of detection
The limit of detection is the lowest number of microorganisms in
a sample that can be detected under the stated experimental
conditions. Due to the nature of microorganisms, the limit of
detection refers to the number of organisms present in the
original sample before any dilution or incubation steps; it <loes
not refer to the number of organisms present at the point of
assay. For example, the absence of Salmonella spp. , the
limit of detection is defined as the lowest number of
Salmonella spp. can be detected in 10 g.
The method to demonstrate the limit of detection would be to
evaluate the two methods ( alternative and compendia!) by
inoculation with a low number of challenge microorganisms
(not more than 5 cfu per unit) followed by a measurement of
recovery. The level of inoculation should be adjusted until at
least 50 % of the samples show growth in the compendial
test. It is necessary to repeat this determination for at least
five times. The ability of the two methods to detect the
 9201 Guidelines for Validation of Alternative Microbiological Methods for Pharmaceutical Products
presence of low numbers of microorganisms can be
demonstrated using Chi square test (X2 ).
3. Reproducibility
The reproducibility of a qualitative microbiological method is
the degree of precision of test results obtained by analysis of
the same samples under a variety of normal test conditions,
such as different laboratories, analysts, instruments, reagent
lots. Reproducibility can be defined as the intrinsic resistance
to the influences exerted by operational and environmental
variables on the results of the microbiological method.
Reproducibility is a validation parameter best suited to
determination by the supplier of the test method. Samples
inoculated with a certain number of microorganisms
( inoculum size should be more than the limit of detection)
would be analyzed by compendial methods and alternative
methods respectively, by different people, at different times,
or using different reagents ( or equipment). The differences
of the reproducibility between the two methods are evaluated
by Chi square test ( X2 ) • The consistency of the sample
should be concerned in the validation process.
4. Robustness
The robustness of a qualitative microbiological method is a
measure of its capacity to remain unaffected by small but
deliberate variations in method parameters, and provides an
indication of its reliability during normal usage. Robustness
is a validation parameter best suited for determination by the
supplier of the test method. Compared with the compendial
method, the test conditions of the alternative method should be
described in the method if it is more critical. The measure of
robustness is not necessarily a comparison between the alternative
method and the compendial method, but rather a necessary
component of validation of the alternative method so that the user
knows the operating parameters of the method.
Validation of Quantitative Tests of
Microorganisms in A Sample
The plate count method is the more common assay of
quantitative microbiological method. The data processing of
the enumeration results often requires the use of statistical
methods. As colony-forming units follow a Poisson distribution,
the use of statistical tools appropriate to the Poisson rather than
those used to analyze normal distributions is encouraged. If the
user is more comfortable using tools geared towards normally
distributed data. Raw counts can be transformed to normally
distributed data either by taking the loglü unit value for that
count, or by taking the square root of count + l. These two
techniques are all suitable far the statistical analysis of the
enumeration results.
l. Accuracy
The accuracy of this type of microbiological method is the
closeness of the test results obtained by the alternative
method to the value obtained by the compendial method. It
should be demonstrated across the operational range of the
test. Accuracy is usually expressed as the percentage of
recovery ( %) of microorganisms by the assay method.
The accuracy within the range of the detection should meet the
requirements. Accuracy may be shown by preparing a suspension
of rnicroorganisms at the upper end of the range of the test that
can be able to count accurately, that has been serially diluted clown
to the lower end of the range of the test ( such as less than
10 CFU per mD. For example, if the alternative method is meant
to replace the compendial plate count method far viable counts,
then the suspension of the rnicroorganisms at the upper
concentration can be 103 CFU per ml, which has been serially
diluted clown to 10° CFU per ml at least 5 suspensions across the
415
range of the test should be analyzed far each challenge organism.
The altemative method should provide an estimate of viable rnicro-
organisms not less than 70 % of then estimate provided by the
compendial method, or the new method should be shown to
recover at least as many organisms as the compendial method by
appropriate statistical analysis. Note that the possibility exists that
an alternative method may recover an apparent higher number of
rnicro-organisms than the compendial method. It is necessary to be
deterrnined in the specificity evaluation.
2. Precision
The precision of a quantitative microbiological method is the
degree of agreement among individual test results when the
procedure is applied repeatedly to multiple samplings of
suspensions of laboratory microorganisms across the range of
the test. The precision of a quantitative microbiological
method is usually expressed as the standard deviation or
relative standard deviation. However, other appropriate
measures may be applied.
One method to demonstrate precision uses a suspension of
microorganisms at the upper end of the range of the test that
can be able to count accurately, that has been serially diluted
clown to lower end of the range of the test (such as less than
10 CFU per mD. At least 5 suspensions across the range of
the test should be analyzed for each organism. Far each
suspension at least 10 replicates should be assayed in arder to
be able to calculate statistically significant estimates of the
standard deviation or relative standard deviation. Generally,
an acceptable relative standard deviation CRSD) would be no
more than 35 % . Irrespective of the specific results; the
alternative method should have a relative standard deviation
( RSD) that is not larger than that of the compendial
method. For example, the relationship between the
acceptable RSD of compendial plate count method and the
concentrations of microorganisms is shown in Table 2.
Tabie 2 Expecied RSD as a iunciion oi CFlJ per plate
CFU per plate Expected RSD
<10 <35%
10-30 <25%
30-300 <15%
3. Specificity
The specificity of a quantitative microbiological method is its
ability to detect a panel of microorganism suitable to
demonstrate that the method is fit for its intended purpose.
For example, the purpose of the plate count method is to
detect a certain number of microorganisms, and the
specificity validation should demonstrate the detection of
these microorganisms by plate count method, while the test
results will not be affected by the presence of samples. It is
important to challenge the alternative technology in a manner
that would encourage false positive results in alternative
technology to demonstrate the suitability of the alternative
method. This is especially important with those alternative
methods that do not require growth or turbidity for microbial
enumeration ( for example, any that do not require
enrichment or can enumerate microorganisms in the range of
1-50 CFU).
4. Limit of quantification
The limit of quantification is the lowest number of
microorganisms that can be accurately counted. As it is not
possible to obtain a reliable sample containing a known
number of micro-organisms, it is essential that the limit of
quantification of an assay is determined from a number of
replicates Cn>5) at each of at least 5 different points across
 416 9202 The Guidelines for Microbiological Examination of Non-sterile Products
the operational range of the assay. The limit of quantification
should not be a number greater than that of the compendial
method. Note that this may have an inherent limit due to the
nature of bacterial counts and the Poisson distribution of
bacterial counts. Therefore, the alternative method need only
to demonstrate that it is at least as sensitive as the
compendial method to similar lower limits.
One method to demonstrate the limit of quantification uses 5
suspensions at the lower end of the range of the test. For
each suspension at least 5 replicates should be assayed by
alternative method and compendial method. The differences
in results between the alternative method and the compendial
method have been statistically evaluated to estimate the limit
of quantification of the alternative method.
5. Linearity
The linearity of a quantitative microbiological test is its
ability to produce results that are proportional to the
concentration of microorganisms present in the sample within
a given range. The linearity should be determined over the
range of the test. A method to determine this would be to
select at least 5 concentrations of each standard challenge
microorganism and conduct at least 5 replicate readings of
each concentration. An appropriate measure would be to
calculate the correlation coefficient "r ", from a linear
regression analysis of the data generated above, taking the
test results as dependentvariable and the expected number of
microorganisms in the sample as independent variable. While
the correlation coefficient does not provide an estimate of
linearity, it is a convenient and commonly applied measure to
approximate the relationship. The correlation coefficient of
alternative method should not less than O. 95.
6. Range
The operational range of a quantitative microbiological
method is the interval between the upper and lower levels of
microorganisms that have been demonstrated to be
determined with precision, accuracy, and linearity.
7. Reproducibility
The reproducibility of a quantitative rnicrobiological method is the
degree of precision of test results obtained by analysis of the same
samples under a variety of normal test conditions, such as different
laboratories, analysts, instruments, reagent lots. Reproducibility
can be defined as the intrinsic resistance to the influences exerted
by operational and environmental variables on the results of the
rnicrobiological method. Reproducibility is a validation parameter
best suited for deterrnination by the supplier of the test method.
Smiples inoculated with a certain number of rnicro-organisms
Cinoculum size should be more than the lirnit of quantification)
would be analyzed by compendial methods and alternative methods
respectively, by different people, at different times, using different
reagents Cor equipment). The differences in results between the
two methods have been statistically evaluated by the relative
standard deviation (RSD). The consistency of the sample should
be concemed in the validation process.
8. Robustness
The robustness of a quantitative rnicrobiological method is a
measure of its capacity to remain unaffected by small but
deliberate variations in method parameters, and provides an
indication of its reliability during normal usage. Robustness is a
validation parameter best suited to deterrnination by the supplier
of the test method. Compared with the compendial method, the
test conditions of the alternative method should be described in
the method if it is more critical. The measure of robustness is
not necessarily a comparison between the alternative method and
the compendia!, but rather a necessary component of validation
of the alternative method so that the user knows the operating
parameters of the method.
9202 The Guidelines for Microbiological
Examination of Non-sterile Products
The objective of this chapter is to provide guidance for
further applications of Microbiological Examination of
Non-sterile Products: Microbial Enumeration Tests
( 1105 ) , Microbiological Examination of Non-sterile
Products: Tests far Specified Micro-organisms ( 1106), and
Microbiological Acceptance Criteria of Non-sterile
Pharmaceutical Products ( 1107).
Microbiological contamination in non-sterile products may
decrease drug activities, eliminate therapeutic effects, which
will bring potential danger to patient health. Thus,
pharmaceutical manufactures should decrease the
microorganisms contamination by critically following the
guidelines of Good Manufacturing Practice in manufacture,
storage and circulation. Microbial enumeration tests, tests
for specified micro-organisms and microbiological quality of
non-sterile pharmaceutical preparations and substances can
not only be used to examine whether the acceptance criterion
in non-sterile preparations, pharmaceutical raw materials and
excipients is consistent with the compendia! standard, but
also can be used to guide the draf ting standards in the area of
pharmaceutical preparations, raw materials and excipients.
And they can monitor the microbial qualifications of
intermediates. This chapter provides detailed explanation on
the method of microbiological examination for non-sterile
products, acceptance criterion and guidelines for its
application.
l. If surface-active substances, inactivators or neutralizing
agents need to be used in microbiological examination of
non-sterile products, their efficacy and their absence of
toxicity for microorganisms m ust be demonstrated.
Thus, strains used for the demonstration are not limited
to those test strains listed in pharmacopeia, other
potential microorganisms of contamination need to be
considered as well.
2. In Microbial Enumeration Tests, it is preferred to choose
simple and rapid methods in sample preparation, antimicro-
bial elimination and microbial enumeration of bacteria, fungi
and yeasts. The method must not affect any microorganisms
that are to be revealed in the test. When it comes to the
sample with high antimicrobial efficiency, it is recommended
to choose membrane filtration method if properties permit.
3. The reference media especially prepared with high quality is
used for the suitability test of the media to ensure the quality of
media in rnicrobiological examination. The reference media are
prepared and delivered by National Institutes for Food and Drug
Control.
4. When applying the suitability test of the enumeration
tests, if there is no method suitable to eliminate antimicrobial
activity, and causing a recovery failure of tested micro-
organisms, the higher dilution of validation test need be
evaluated to pursuit a good recovery. The suitable and
validated dilution should be consistent with acceptance
criterion and enumeration principie in compendial request.
For example, if the acceptance criterion of total aerobic
bacteria is 10 3 CFU per gram, the highest dilution is 1 : 10 3 •
If the antimicrobial activity still could not be eliminated at the
highest dilution, where lead to the failure recovery for one or
more micro-organisms, the method with the best recovery
results should be employed for examination. Far example, a
sample is hypothetically sensitive to an antimicrobial activity
 9203 Guidelines for Quality Management of Microbiology Laboratory for Phannaceutical Products
to sorne micro-organisms, the recovery of the membrane
filtration method is 40 %, and that of media volume increase
method is 30 %, then the membrane filtration method should
be adopted for sample examination. In this case, the
manufacture or the R&D department should carry out
microbiological risk assessment according to microbial
contamination of the raw material, excipients, manufacture
process and the product properties, ensuring reliability of
examination methods and quality of products.
5. There is no detail description for further confirmation of
pathogenic bacteria detected in Microbiological Examination
of Non-sterile Products: Tests for Specified Microorganisms.
If the suspect specified microorganism is detected, it is
recommended to choose approved or recognized identification
methods, for example, Bergey' s Manual of Systematic
Bacteriology.
6. In microbiological examination, if it has been shown that
none of prescribed tests will allow valid enumeration of
microorganisms at the level prescribed, an evaluated method
with a limit of the detection as close as possible to the
indicated acceptance criterion is used.
7. Local delivery preparations for surgery, burn and severe
wounds should meet the requirements of sterility. The
severity of wound is usually difficult to judge, preparations
supposed for those wound should also meet the requirements
of sterility, unless there is confidence evidence to prove its
safety of application.
8. In acceptance criterion of microbiological examination,
total aerobic microbial count CTAMC) and total yeasts and
molds count ( TYMC) are necessary tests to valida te the
quality of pharmaceutical raw materials, excipients and crude
extracts. When it comes to developing microbial acceptance
criterion, it should be considered with either influenced
factors, such as the use of product, the nature of raw
materials and excipients; the method of applications,
manufacture process and properties of preparations, or
quality control of potential pathogenic bacteria.
9. The tests of microbiological examination is obligatory, if
the preparations embodied in the general principles of 2015
edition Pharmacopoeia of People' s Republic of China is
required. For other preparations ( such as pills, tablets,
capsules and granules) , which the microbiological require-
ment is only illustrated on principle, the risk assessment of
microorganism contamination should be taken for
systematical evaluation. Under the precondition of patient
safety, microorganism contamination data should meet the
acceptance criteria of non-sterile products approved by
retrospective validation or on-line validation, then micro-
biological examination carried out batch by batch is not
necessary, but the results of final products must meet the
acceptance criteria for microbiological examination of non-
sterile products. Microbiological examination should be taken for
the solid preparation mentioned above, which is easily
contaminated by microorganisms by reason of its nature or the
manufacture process. The acceptance criteria of microbiological
examination should be set for those preparations as well.
10. Microbial acceptance criteria of non-sterile products
should be established systematically on the base of reasonable
safety, considering with Microbiological Acceptance Criteria
of Non-sterile pharmaceutical Products (1107), as well as
potential danger for patients from the source and nature of
raw materials, manufacture process, route of administration
and microbial contamination. For example, microbial
acceptance criteria for special preparations can be set based on
the minimal sample package units. If necessary, sorne
preparations should be regulated by a restricted acceptance
417
criterion considering their therapeutics, stability, potential
danger for patients, and it also should be illustrated in
monographs.
9203 Guidelines for Quality Management
of Microbiology Laboratory for
Pharmaceutical Products
Guidelines for quality management of microbiology
laboratory for pharmaceutical products are used to guide the
quality control activities in the pharmaceutical microbiology
laboratory.
The microbiological testing results are affected by many factors,
for example, microorganisms may be unevenly distributed within
a sample and microbiological assays are subject to considerable
variability of outcome, etc. Therefore, in order to ensure the
reliability of the testing results, pharmaceutical microbiology
laboratories should use validated methods and strictly follow the
guidelines for quality management of microbiology laboratory for
pharmaceutical products.
Guidelines for quality management of microbiology laboratory
for pharmaceutical products consist of the following aspects:
personnel, media, reagents, microbiological cultures,
environment, equipment, sample, test method, disposal of
hazardous waste, quality assurance and quality control, laboratory
records, interpretation of assay results, test report, documentation
et al.
Personnel
The demands of microbiological testing require that the core
educational background of the staff be in microbiology or a
closely related biological science.
Training curricula should be established for each laboratory
staff member specific for his or her job function. They
should not independently conduct a microbial test until they
are qualified to run the test. Laboratory staff should be
trained on the subjects of essential instrument operation and
microbiology testing skill, such as sterile operation, media
preparation, sterilization, plate pouring, colony counting,
spice passage and preservation, micro-test method and
microbial identification, etc. , and passed the appraisal
process for qualifying the job.
Laboratory staff should be trained about laboratory bio-
safety, to assure the safety of oneself, and to prevent the
micro-contamination in the laboratory.
Meanwhile, further education should be given to all-level
laboratorystaff periodically.
Laboratory staff must be familiar with relevant test methods,
procedures, purposes and evaluation of the results.
Microbiologists with supervisory or managerial responsibilities
should have appropriate education and in-house training in
supervisory skills, laboratory safety, scheduling, budget,
researches, evaluation of results and investigations of data
deviation, technical report writing and so on.
Laboratory staff ability can be assessed by internal quality
control, proficiency testing or standard strain operation and
re-training and re-evaluation can be applied if necessary. When an
unusual method or technique is used, it' s necessary to confirm
the operational skill of the microbiologists before testing.
All the training, appraisal contents and results should be
documented and archived.
Media
Culture media are the basis for microbiological tests. &feguarding
 418 9203 Guidelines for Quality Management of Microbiology Laboratory for Pharmaceutical Products
the quality of the media is therefore critical to the success of the
microbiology laboratory. Appropriate media preparation, proper
storage and quality control testing can assure a consistent supply of
high quality media.
l. Media preparation
The media can be prepared in a laboratory from individual
components or use dehydrated media which comply with the
formula tion.
It is important to choose the correct media or components in
making media based on the use of accepted sources or
references for formulas. The manufacturer' s formula and
instructions for preparation routinely accompany dehydrated
media. It is important to follow these instructions to ensure
preparation of acceptable media quality. lli not use dehydrated
media with lumps or colour change.
Dehydrated media or components should be stored under
proper conditions ( i. e. , low temperature, dry and
darkness). All containers should be sealed, in particular load
with dehydrated media. A certificate of analysis describing
formula and instructions accompanies commercial ready-made
media, as well as expiry date, recommended storage
conditions, and the quality control organisms used in growth-
promotion and selectivity testing of that media.
Consistent preparation of media requires accurate weighing of
dehydrated media or media constituents, and appropriate
weight precision. Purified water is most often used for media
preparation. The weight of the components and the volume
of the water used should be recorded.
The container for preparation should not affect the quality of
media. A glass container is normaHy used. Clean containers
and tools for media preparation should be used to prevent
detergent and foreign substances that may alter the
composition of the finished media from entering the
formulation. Be sure that the residues of detergent and
foreign substances are thoroughly rinsed out with purified
water. Safeguarding the sterility of heat-sensitive media, the
dispensing container should be sterilized previously, such as
sugar f ermentation media.
Dehydrated media should be thoroughly dissolved in water
prior to dispensing and sterilization. If heating is necessary to
help dissolve media, care should be taken not to overheat
media. Darkening of media is a general indication of
overheating. When adding required supplements to media,
adequate mixing of the medium after adding the supplement
should be performed.
Sterilization of media should be performed within the
parameters provided by the manufacturer or validated by the
user. Commercially prepared media should provide documenta-
tion of the sterilization method that was used. Autoclaving by
moist heat is the preferred sterilization technique, except in
instances, where sterilization by filtration may also be
appropriate for sorne formulations.
lmproper heating or sterilizing conditions for media may
result in a difference in colour change, loss of clarity, altered
gel strength, or pH drift from the recommended range.
Therefore, media should be sterilized in the light of validated
sterilization method. The effects of the sterilization method
and conditions on the media should be validated by sterility
and growth-promotion testing of the media. In addition, if
sterilized by moist heat, the autoclave cycle should be
validated to ensure proper heat distribution for selected loads
and volumes. Sterilization cycles in which the autoclave is
slow to come up to temperature may result in overheating of
the media. Media growth-promotion capacity may be
destroyed by oversterilization. As the media volume and the
load configuration of the autoclave will influence the rate of
heating, comprehensive sterilization cycle validations should
be performed based on diff erent media property.
The pH of each batch of sterilized medium should be
confirmed after it has cooled to room temperature (25ºC).
The pH of media should be in a range of ±O. 2 of the value
indicated by the manufacturer, unless a wider range is
acceptable by the validated method.
Prepared media should be checked by appropriate inspection
of plates and tubes for: cracked containers or lids, unequal
filling of containers, excessive number of bubbles,
dehydration resulting in cracks or dimpled surfaces on solid
medium, crystal formation from possible freezing, microbial
contamination, lot number and expiry date checked and
recorded, sterility of the media.
2. Media storage
Media prepared in-house should be labelled properly with
media name, batch or lot number, preparation date,
preparation person, etc. , and should be stored under
validated conditions. Commercial ready-made media should
be labelled properly with name, batch or lot numbers,
preparation and expiration dates, and media identification.
Media should be stored according to the manufacturer' s
instructions. Manufacturers and users of media should use
storage and transport conditions that minimize the loss of
moisture, and provide mechanical protection to the prepared
media.
The media can' t be stored in the autoclave after sterilization.
[)o not store agar at or below üºC, as freezing could damage
the gel structure. Protect stored media from exposure to
light and loss of moisture. Agar plates should be prepared
befare use, and if stored, sealed in refrigerator no more than
1 week. Before prolonged storage of agar plates, shelf life
should be validated.
Re-melting of an original container of solid media should be
performed only once to avoid media whose quality is
compromised by overheating or potential contamination. It is
recommended that re-melting be performed in a heated water
bath or by using free-flowing steam. If other method is used,
it should be evaluated as not effecting media quality. The
molten medium should be held at a temperature of 45ºC to
SüºC for not more than 8 hours. Disposal of used cultured
media (as well as expired media) should follow national
biological hazard safety procedures.
3. Quality control testing
Microbiological laboratory should perform quality control
testing and establish procedures for media to assure a
consistent compliance with the requirements for relevant
testings.
The quality of media prepared in a laboratory or by a
manufacturer is highly dependent on preparation. lmproper
media preparation can cause unsatisfactory conditions for
microbial growth or recovery and unreliable results.
Quality control tests should be performed on all prepared
media. Tests routinely performed on in-house prepared
media are pH, growth promotion, and periodic stability
checks to confirm the expiry date. Expiration dates on media
should be supported by growth-promotion testing to indicate
that the performance of the media still meets acceptance
criteria up to and including the expiration date. The length of
shelf life of a batch of media will depend on the stability of
the ingredients and formulation under specified condition, as
well as the type of container and closure.
When in-house prepared media are properly prepared and
sterilized using a validated method, the growth-promotion
testing of each incoming lot of dehydrated mediamay be
undertaken only once, unless otherwise instructed by the
 9203 Guidelines far Quality Management of Microbiology Laboratory far Pharmaceutical Products
relevant compendial method. If the media preparation was
not validated, then every batch of media would be subjected
to growth-promotion testing. Test organisms may be
selected from the appropriate compendial test chapter or may
include representative isolates from the environment and
products.
When a batch of media <loes not meet the requirements of
quality control tests, an investigation should be initiated to
identify the cause. This investigation should include a
corrective action plan to prevent the recurrence of the
problem. Any nonconfarming lot should not be used.
Special care should be taken with media that is used in
environmental monitoring. Media used far environmental
monitoring of critica! areas should preferably be double-
wrapped and terminally sterilized. If terminal sterilization is
not perfarmed, media should be subjected to pre-incubation
and 100 % inspection prior to use. This will prevent
extraneous contamination from being carried into controlled
environments and will prevent false-positive results.
Reagents
Procedures shall exist far the reception, check and storage of
reagents in microbiological laboratory. These reagents shall
be verified to comply with specified requirements befare use.
Laboratories should verify the suitability of each batch of
reagents critical far the test, in the process of opening and
storage. The laboratory should manage reagents and achieve
allrelevant records.
Laboratories should label the name, preparation method,
applicability, concentration, biological value, storage
condition, preparation date, expiry date and preparation
person of all reagents and test solution.
Microbiological Cultures
!11olog1cal specimens can be the most del1cate standards to
handle because their viability and characteristics are
dependent on adequate handling and storage. Standardizing
the handling and storage of cultures by the user laboratory
should be done in a way that will minimize the opportunity of
contamination or alteration of growth characteristics. The
careful and consistent treatment of stock cultures is critically
important to the consistency of microbiological test results.
Cultures for use in rnicrobial tests should be acquired from inside
or outside the national culture collection center, or commercial
cultures derived from the reference cultures/ strains.
Preparation and resuscitation of cultures should fallow the
instructions of the supplier or a validated, established
method. The ref erence culture/ strain from inside or outside
the national culture collection center is resuscitated and grown in
an appropriate medium to make "stock culture". Confirmation of
the purity and the identity should be performed far stock
cultures. Aliquots of this stock culture are suspended in a
cryoprotective medium, transferred to vials and stored freeze-
dried, in liquid nitrogen or frozen below - 30ºC . If stored
below -70ºC, or in lyophilized form, strains may be prolonged.
These frozen stocks can then be used to inoculate monthly or
weekly working cultures. If stock cultures have been thawed,
they must not be refrozen and reused.
The generations from the original reference cultures should
be tracked strictly to prevent the increase of the risk of
phenotypic alteration. No working culture should be used for
testing af ter five passages. Ref erence cultures/ strains from
culture collection is defined as zero generation. Generation 1
is defined as the transfer of organisms from a viable culture
to a fresh medium with growth of the microorganisms. Any
farm of subculturing is considered to be a transfer/passage.
419
Working cultures may require confirmation of purity and
identity if necessary.
Working culture could not supersede ref erence culture/
strain, and commercial culture derived from the reference
culture/ strain is only used as working culture. The cultures
should be sterilized and disposed lf they are useless or fail to
pass confirmation testing, since it is already perished,
degenerated, contaminated.
Records of all cultures should be established and reserved in
microbiology laboratory, including turnover, collection,
storage, confirmation testing and disposal. Procedure of
culture management from reference culture/ strain to working
culture should be in place. These include purchase arder far
reference culture/ strain, operation and record from reference
culture to working culture, periodic transfer/passage,
confirmation and record of the purity and identity, culture
information as name, reference number, inoculation date
andpassage number, medium to grow and incubation condition,
storage location and condition, others (if necessary).
Environment
Laboratories should be equipped with proper and sufficient
facilities far microbial test and ensure that the environmental
conditions do not invalidate the results. There should be
effective separation between testing area and office.
Microbiology laboratories should be dedicated and separated
from other areas, especially from production areas.
l. Laboratory layout and operations
Laboratory layout and design should carefully consider the
requirement of instrument installation, the good
microbiological practices and laboratory safety. It is essential
that cross-contamination of microbial cultures should be
minimized, and it is also important to protect staff and
environment from micro-contamination. Appropriate layout
and division of the laboratory will improve the reliability of
the microbiological test.
Laboratories should be appropriately designed and should
take into account the suitability of construction materials to
enable appropriate cleaning, disinfection and minimize the
risks of contamination. There should be separate a air supply
to clean room to meet the requirements of testing, including
temperature and humidity controls.
Environment condition, such as air pressure, illumination
and sound, should comply with the testing methods as well.
The microbial laboratory should be divided into clean areas
and live culture areas. Separate the incompatible test
minimize in time and space according to different testing
purposes to the cross-contamination of microbial cultures be.
In general, microbiological laboratory must include
independent clean room or isolator system complying with
the requirements of Sterility Test ( 1101 ) and Microbiological
Exarnination ( 1105, 1106) far sterility test, rnicrobial lirnit test
and sterile sampling. And design the positive bacterial
laboratory, incubation room, observation room, media and test
tools preparation ( including sterilization) area, sample
receiving and stock area, standard strains stock area, disposal
area of contarnination substance and documentation area as
supporting area and label these areas clearly.
Laboratory activities should be segregated to minimize risks
of cross-contamination, false-positive and false-negative
results. The sterility testing should be carried out within a
Class A clean zone wi th an unidirectional airflow protected
zone which should be located within a clean room with a
Class B background. Alternatively, the testing can be carried
out within an isolator system. Microbial examination tests
should be carried out within a no less than Class B clean zone
 9203 Guidelines for Quality Management of Microbiology Laboratory for Pharmaceutical Products
wi th unidirectional airflow protected zone which should be
located within a clean room with a Grade D background. Air
supplied to clean zones with Class A or B should be via
terminal high efficiency particulate air ( HEP A) filters.
Microbial growth demonstrating further analysis, such as
subculture, stammg, identification, or characterization
should be undertaken in a restricted laboratory section for
live culture. No micro-organism culture could be opened in
clean zone of laboratory. Careful segregation of contaminated
samples and materials will reduce false-positive results.
Isolation and identification of pathogenic microorganisms
should be carried out in biosafety level-2 laboratory.
Control procedure and SOP for personnel and material
entering/leaving clean room should be established. The work
which may affect the test results shall be controlled,
monitored and recorded ( such as validation and monitoring
of cleanliness, disinfection, cleaning and maintenance for
clean rooms). Only authorized personnel can access to the
microbiological laboratory restrictively. Personnel should be
made aware of: the appropriate entry and e:xit procedures
including gowning, the intended use of a particular area; the
restrictions imposed on working within such areas; the
reasons for imposing such restrictions; and the appropriate
containment levels.
2. Environmental monitoring
The laboratory should establish SOPs for validation and
monitoring of clean room ( zone) /isolator system by
national standards. The environmental monitoring items,
frequency and the occurrence of the excessive results should
be specmea m wrinen procedures. Where necessary and
appropriate, an environmental monitoring program should be
in place which covers, for example, airborne particles,
airborne microbe, settling microbe, surface microbe and
physical parameters ( temperature, relative humidity, air
changes, air velocity, pressure differentials and noise, etc.).
Environmental monitoring should be performed in accordance
with guidelines for microbiological monitoring and control of
clean rooms for pharmaceutical products <9205).
3. Cleaning, disinfection and sanitization
There should be a documented cleaning and disinf ection
program. Results of environmental monitoring should be
considered where relevant. The laboratory should be
disinfected befare and after use, and the disinfection effect
should be monitored regularly. Adequate hand-washing and
hand-disinfection facilities should be available. There should
be a procedure for dealing with spillages of microbial
contamination.
The disinfectant used in microbiology laboratory should meet
the requirements of specified cleanliness standards and its
types should be changed at periodic time. The ideal disinfectant
can kill a wide range of microorganisms, harmless to human, no
corrosion or pollution to equipment, but also play the role of
cleaning agent with stable performance, quick acting, less
residual and reasonable price. The contamination of disinfectants
and detergents shall be monitored and be used befare expire date.
The disinfectants and detergents used in Class A and Class B
clean zone should be sterile or sterile treated.
Equipment
Laboratory should be equipped with suitable and sufficient
instrument/ equipment with its test capability and workload. The
testing type, range and accuracy of instruments or equipment
should meet the requirements of specific standard, and the
installation and layout must facilitate operation, maintenance,
cleaning and calibration and keep as clean and good working
condition. Ea.ch equipment or instrument used far testing,
verification and calibration should be uniquely identified.
Equipment should have a certificate of conformity.
Laboratory instrument/ equipment should not operate until it
has been verified, calibrated, validated, qualified and
established relevant SOPs for operation and maintenance.
Daily use and monitoring shall be recorded and documented.
l. Maintenance of equipment
Regular maintenance and periodic verification should be performed
to ensure the function of the equipment and keep the records
available. Laboratory equipment should be checked or calibrated
when it is out of the laboratory or the maintenance.
Important equipment, such as incubators and refrigerators
should be monitored by specified person to ensure it' s
running well and under control. Meanwhile, backup
equipment should be ready to ensure the continuity of
incubation. The laboratory management should ensure that
all personnel have received adequate training and been certificated
far operation of special equipment, such as, autoclaves, isolators,
biosafety cabinets. Far the critical equipment, far example,
incubators, refrigerators and autoclaves which may affect the test
accuracy should continuously observe and record critica!
parameters (e. g. , tempera- ture and pressure) , when they
are in operation as critical, and automatic recording
apparatus is needed if necessary. When deviation occurs,
assessment of impact to the earlier test and corrective action
shall be taken.
In proper, regular cleaning and disinfection of equipment,
such as, water baths, incubators, refrigerators, and
biosafety cabinets should be performed to minimize the
potential contamination.
Sterile tools, apparatus and vessels must be cleaned and
sterilized by correct means and the relevant SOPs should be
established. Proper labels must be set to distinguish sterile
ones and non-sterile ones.
Sorne devices (e. g. , incubator, autoclave and glassware)
should be dedicated, unless there is a proper measure to
prevent cross-contamination.
2. Calibration, performance verification and monitoring of use
Instruments used in a microbiology laboratory should be
calibrated on a regular schedule and tested to verify
performance on a routine basis and recorded. The frequency
and content of calibration and performance verification will
vary based on the type of instrument and the importance to
the laboratory. The dates for calibration and recalibration
should be clearly indicated on the instrument.
Temperature measurement device
Where temperature has a direct effect on the result of an
analysis or is critical for the correct performance of
equipment, temperature measuring devices should be of
appropriate quality to achieve the required accuracy (e. g. ,
liquid-in-glass thermometers, thermocouples and platinum
resistance thermometers ( PRTs) used in incubators and
autoclaves). Calibration of devices should be traceable to
national or international standards for temperature.
Temperature measuring devices can be used to monitor thc
temperature of equipment, far example, refrigerators, ultra-
low temperature refrigerators, incubators and water baths.
The performance of such devices should be verified befare
use.
Weights and balances
Weights and balances shall be calibrated traceably at regular
intervals using appropriate standard weights traceable to
certified standard weights. Cleaning should be performed
every time af ter using. Non-corrosive disinfection can be
performed, if necessary.
 9203 Guidelines for Quality Management of Microbiology Laboratory for Pharmaceutical Products
Volumetric equipment
Microbiology laboratories should carry out initial verification
of volumetric equipment (automatic dispensers, pipettes and
dispenser) to ensure the precision. Equipment should be
checked for the accuracy of the delivered volume as well as
the precision and reproducibility.
F or disposable volumetric equipment, lalx>ratories should procure
from well-known companies with a recognized quality. After initial
validation on the suitability of the equipment, random checks on
precision are recommended. Lalx>ratories should check each batch of
equipment for suitability, if necessary.
Biosafety cabinet, laminar flow clean benches, high efficiency
particulate air CHEPA) filters
lnstallation and replacement of biosafety cabinet, laminar
flow clean benches and HEP A filters should be carried out by
a certificated person. Biological and physical tests should
follow verified methods and revalidate regularly.
Ventilation of biosafety cabinet and laminar flow clean bench
should meet the requirements of biosafety level and laboratory
safety. Monitoring of biosafety cabinet and laminar flow clean
benches may be achieved to ensure the performance comply with
relevant requirements. All documents on inspection should be
recorded and archived.
Other equipment
Airborne particulate counter and active air sampler should be
regularly calibrated. The performance of conductivity
meters, pH meters and other similar instruments should be
verified regularly or before each use. Where humidity is
important to the test, hygrometers should be calibrated, the
calibration being traceable to national or international
standards. Where time is important to the test, timers
should be calibrated. When centrifuges are used in testing,
an assessment of the rotations per minute should be
evaluated. When it is critica!, the centrifuge should be
calibrated.
Sample
l. Sampling
Sampling should be carried out according to randomization
principle in controlled conditions. If possible, all samples
should be taken under aseptic environment in specialized
sampling areas. During sampling, aseptic techniques should
be performed to avoid contaminations and false positive
reBults._ Any disinfection processes used in sampling (e. g. ,
disinfection of sample points) should not adversely affect the
ability of microbial examination.
Containers for sampling should be uniquely identified, including
sample name, batch number, sampling date, sampling container
and sampling personnel. 53.mpling should be performed by trained
personnel aseptically. Environmental conditions and dates of
sampling should be monitored and recorded.
2. Storage and transport of samples
Samples waiting for testing should be stored under suitable
conditions and maintain their integrity to avoid any microbial
contamination. In transport of samples, original storage
conditions should be maintained or appropriate measures
should be taken (e. g. , chilled or frozen). The conditions of
storage and transport should be clearly documented and
recorded.
3. Check and handling of samples
The laboratory should have procedures of sample delivery,
receipt, storage, and check.
Testing of samples should be performed according to
requirements as soon as possible after sample receipt. And
all relevant information of samples should be recorded, e. g. ,
421
date and the time of receipt, nature of the sample on receipt,
sampling information (including sampling date and sampling
status) , and status of storage.
If there are insufficient quantities of sample or any damage of
packaging, deficient labeling, or incorrect storage
temperature, the laboratory should communicate with the
clients before deciding whether to accept or not. The
packaging and labels of samples may be contaminated and
should be handled and stored with care to avoid dispersa! of
contamination. Disinfection to the outside of package should
not affect the integrity of the sample. Any suspicion about
samples should be noted in the final test report.
Representative samples are required and tested as soon as
possible according to national or international standards, or
by other validated methods.
Written procedures for reservation and disposal of samples
are required. Samples known to be contaminated should be
decontaminated before disposal.
Test Method
l. Selection of test methods
The suitability of test method for a specific product needs to
be validated.
2. Validation of test methods
Standard or compendia! test methods have been validated.
Their suitability should be confirmed when applying in
laboratory.
Test methods other than compendia! or standard methods
should be validated before use to confirm that they are better
or equal to the compendia! method. The validation of the
alternative method should be performed according to
Guidelines far Validatian af Alternative Microbialagical
Metheds far Pharmaceutical Praducts <9201 ).
Laboratory should archive the confirmation data of the
commercial test system or test kits provided by manufacture
or evaluated by a third party. If necessary, the laboratory
should verify the commercial test system.
Disposal of Contaminated Waste
There should be facilities and procedures for disposing
laboratory test samples, expired or invalid media and
hazardous wastes from microbiology laboratory to minimize
contamination of test environment or materials. It should
conform to national and international standards of environ-
mental, health and safety regulations.
Emergency and handling procedures for dealing with
accidents should be established, such as culture spills, to
avoid microbial contamination diffusion.
Quality Assurance of Results and
Quality Control of Process
l. Internal quality control
The laboratory should establish procedures of continuous
performance evaluation to ensure the consistency of results
from day to day and their conformity with defined criteria.
The laboratory should monitor and record detail of the
cleanliness of environment, suitability of media, sterilized
method, purity and viability ( including characteristics),
quality of reagents, etc. at regular intervals.
The competence of personnel involved in tests should be
verified periodically. The consistency of results should be
ensured by use of standard addition, replicate testing and
proficiency-testing.
Significant equipment, e. g. , automation testing equipment,
shall be capable of achieving the accuracy.
 422 9204 Guidelines far Microbial Characterization, Identification, and Strain Typing
2. Externa) quality evaluation
The laboratory should participate in relevant proficiency-testing or
interlaboratory comparison to evaluate the competence.
Laboratory Records
Proper recording of data is critical to the microbiology
laboratory. The principie is that the test should be performed
as written in the SOPs, which should be written to reflect
how the test is actually performed. The laboratory records
should provide all critical details needed to reconstruct the
details of the testing and confirm the integrity of the data.
At a minimum, the laboratory records should include the
following: date; sample specification; name of microbiolo-
gist; procedure number or method; document test results;
deviations ( if any); documented parameters ( equipment,
microbial stock cultures, media lots, temperature);
signature of reviewer or authority.
The test standard should be clearly noted in records. If it is
pharmacopeia standard, it should be currently effective.
Every critical piece of equipment used in testing should be
noted in records. Appropriate logbooks or forms should be
available and supportive of the laboratory records. Equipment
temperatures (water baths, incubators, autoclaves) should be
recorded and traceable.
Changes in the data should be crossed off with a single line
and initialed. Original data should not be erased or covered.
All laboratory records should be archived and protected
against accidental loss. A formal record retention and
retrieval program should be in place.
Interpretation of Results and Test Reports
Due to the nature of microbiological analysis, when
interpreting the results, a comprehensive evaluation of the
results should be undertaken. Even if the results show great
deviations to the recommended limits or standards, including
the possibility of that organisms surviving in the pharmaceu-
tical ingredient, excipient, or environment of testing and the
nature of specific microorganisms. It is critica! to know
whether the finding is statistically significant to the
standards. When results are observed that do not conform to
a compendia! monograph or other established acceptance
criteria, an investigation of deviation is required. There are
generally two distinct reasons far the observation of microbial
contamination that do not comply with requirements: There
may be either a laboratory error or laboratory environmental
conditions that produced an invalid result, or the product
contains a level of contamination or specific microorganisms
outside established levels or limits.
A deviation investigation is required when there are anornalous
results. The laboratory environment, the protective conditions in
place far sampling, historical findings concerning the material
under test, and the nature of the material, particularly with regard
to rnicrobial survival or proliferation in contact with the material,
should be considered in the investigation. In addition, interviews
with the laboratory analyst (s) may provide information regarding
the actual conduct of the assay that can be valuable in determining
the reliability of the result and in determining an appropriate course
of action. If laboratory operations are identified as the cause of the
nonconforming test outcome, then a corrective action plan should
be developed to address the problem (s). Following the approval
and implementation of the corrective action plan, the situation
should be carefully monitored.
If assay results are invalidated on the basis of the discovery of
an attributable error, this action must be documented.
Laboratories also should have approved procedures far
confirmatory testing ( retesting ) , and if necessary, re-
sampling where specific regulatory or compendia! guidance
<loes not govern the conduct of an assay investigation.
The results of each test carried out by the laboratory shall be
reported accurately, clearly, unambiguously and objectively,
and in accordance with any specific instructions.
All the information related to the testing shall be included.
Documentation
Documentation should be sufficient to demonstrate that the
testing was performed in a laboratory and by methods that were
under control. This includes, but is not lirnited to the following:
training and verification of proficiency; equipment validation,
calibration and maintenance; equipment performance during test
(e. g. , key parameters of the equipment); media preparation,
storage and control testing; rnicrobiological culture management;
critical aspects of tests conducted as specified by a procedure;
data and calculations verification; reports reviewed by quality
assurance manager or a qualified responsible manager;
investigation of data deviations ( when required).
9204 Guidelines for Microbial
Characterization, ldentification,
and Strain Typing
This chapter provides guidelines far the identification of
suspected strains from microbiological examination of non-
sterile products: tests far specified microorganisms, and
microorganisms detected in drug substances, excipients,
water for pharmaceuticai use, intermediates, finished drug
products and the manufacturing environment. If controver-
sial microbial identifications arise, the results of
identification should conform to the current edition of
Bergey's Manual of Systematic Bacteriology.
Referring to current microbial taxonomy systems, mjcrobial
identification is based on the process of measuring the
characterization of unknown microorganisms to differentiate
the categories of bacteria, yeast, and mold or to confirm the
genus-level, species-level and strain-level of isolates.
Microbial identification is a critical step in microbiological
examination of pharmaceutical products. The need far
detected microbial identification is specifically cited in
Microbiological Examination of Non-sterile Products:
Tests for Specified Micro-organisms <1106). This chapter
indicates a requirement far confirmatory identification tests
far organisms that grow on selective or diagnostic media.
Also, Sterility Tests ( 1101 ) allows far invalidation of the
test, if after identification of the microorganisms isolated
from positive tests. Guidelines for Microbiological Control
and Monitoring of Clean rooms for pharmaceutical
products <9205) recommends that microbial isolated from
clean rooms or other controlled environments should be
identified at a rate sufficient to support the environmental
monitoring program. Additionally, microorganisms detected
in drug substances, excipients, water far pharmaceutical
use, the manufacturing environment, intermediates, and
finished drug products, typically should be identified at
appropriate level.
Microbial identification may yield genus-level, species-level
and strain-level identification. Routine characterization of
micro-organisms may include the determination of colony
morphology, cellular morphology (rods, cocci, cell groupings,
modes of sporulation, etc. ). Gram reaction or other differential
staining techniques, and certain key biochernical reactions (e. g. ,
oxidase, catalase, and coagulase activity) that can be diagnostic.
Microbial characterization to this level is sufficient far many risk-
 9204 Guidelines for Microbial Characterization, Identification, and Strain Typing
assessment purposes in non-sterile pharmaceutical manufacturing
operations and in sorne sterile product manufacturing
environments. For specified microorganisms tested from
non-sterile drugs, genus and species-level identification is
generally required. Genus-level, species-level or even strain-
level identification is necessary, where positive results in
sterility tests have occurred, and in the assessment of
contamination recovered from failed aseptic process
simulations, i. e. , media fills.
l. Microbial ldentification Procedures
The basic procedures of microbial identification include
isolation of pure cultures and identification. Generally, the
preliminary characterization of testing microorganisms
should be performed first. Phenotypic and genotypic
methods of microbial identification may be selected
according to the level of identification required. Micro-
biological identification systems are based on different
analytical methodologies, and limitations may be inherent
to the method and/or arise from database limitations.
Identification is accomplished by matching characteristics
( genotypic and/ or phenotypic) to an established standard
( reference) organism such as a type strain. If a
microorganism is not included in the database, it will not
be identified. Bearing in mind both these limitations and
the level of identification required ( genus, species and
strain) , users must select the appropriate technology to use
in routine microbiological identification testing. Multiple
identification technologies should be used if necessary.
( 1) lsolation and purification of testing cultures
The first step in identification is to isolate a pure culture for
analysis. This is typically accomplished by successive
streaking of the colony of interest on appropriate general
microbiological solid media with the objective of obtaining
discreet colonies that usually yield pure cultures. Repeat this
step if necessary. This technique also allows phenotypic
expression and growth of sufficient inoculums for succeeding
identification procedures. Additionally, microorganisms isolated
from pharmaceutical ingredients, water for pharmaceutical use,
the manufacturing enviromnent, intermediates, and finished
products may be physiologically stressed. Through the procedure
of isolation and purification, the microorganisms will pass
from a metabolic state suitable for survival under adverse
ambient conditions to culture conditions that are far richer
nutritionally and are at an optimal incubation temperature,
which is important for accuracy of microbial identification.
(2) Preliminary screening and characterization
In routine microbial identification, the first step is to
determine the basic characteristics of microbial isolates by
preliminary screening. Common preliminary screening tests
include Gram staining, spore stammg, observation of
staining results under a microscope, cellular morphology,
key biochemical tests, etc.
Key biochemical screening tests are described below:
Oxidase Test: to separate Gram-negative, rod-shaped bacteria
into nonfermenters (oxidase positive) and enteric (oxidase
negative) bacteria;
Catalase Test: to separate Staphylococci (catalase positive)
from Streptococci (ca talase negative) ;
Coagulase Test: to separate coagulase negative Staphylococci
( presumptively nonpathogenic) from coagulase positive
Staphylococci (more likely pathogenic) .
Valuable information can be provided by preliminary screening
tests for evaluation processes. This is a critical step for many
phenotypic identification schemes. If the wrong characteristics
are assigned to an isolate, subsequent testing may be conducted
using the wrong microbial identification kit and PCR primers,
resulting in an incorrect result.
(3) Microbial ldentification by Phenotypic Methods
Phenotypic methods use expressed gene products to
distinguish among different micro-organisms. lt is classic
microbial classification and identification method. Phenotypic
methods use cell morphology and phenotypic characteristics
as the main indicators. The result of identification is
concluded by comparing the difference of colony morpho-
logies, physical and chemical characteristics, and typical
chemical composition with a standard organism such as a
type stain. Phenotypic characteristics used in microbial
taxonomy are listed in Table l.
Table 1 Phenotypic characteristics
used in microbial taxonomy
Categories Characteristics
Colony morphology, colony colour, shape
Culture
and size, pigment production
Cellular morphology, cell size, cell shape,
flag ella type, reserve material, Gram
Morphological
reaction, spore and acid-fast staining, mode
of sporulation
Oxygen tolerance, pH range, temperature
Physiological
optimum and range, salinity tolerance
Carbon utilization, carbohydrate oxidation
Biochemical
or fermentation, enzyme patterns
Bile salt-tolerance, antibiotic susceptibility,
lnhibition
dye tolerance
Serological .i\gglutination, fluorescent antibody
Chemo- Fatty acid profile, microbial toxins, whole
taxonomic cell composition
Ecological Origin of the organism
Expression of the microbial phenotype controlled by genes
(i. e. , cell size and shape, sporulation, cellular composition,
antigemc1ty, biochemical activity, and sens1t1v1ty to
antimicrobial agents) may be affected by isolate origins,
media selection, and growth conditions. Generally, phenoty-
pic methods require a relatively large number of cells in pure,
monoclonal culture. Recovery and growth methods for
microbial identification are limited by the length of incubation
and the fact that many organisms present in the environment
are not recovered by general microbiological growth media.
Additionally, freshly isolated, stressed microorganisms by
subculture from primary recovery may not result in a full
expression of phenotypic properties. Therefore, the impact
of the media selection, incubation length, and culture
generations should be considered in identification by
phenotypic methods. At present, methods based on carbon
utilization and biochemical reactions, as well as fatty acid
profiles by gas-liquid chromatography and whole-cell
composition by MALDl-TOF mass spectrometry, are always
based on a specific identification database system. These
systems rely on specified culture media and incubation
conditions to achieve consistent identification.
Phenotypic microbial identification methods are successfully
used in pharmaceutical microbiological testing laboratories.
Phenotypic methods provide information that enables
microbiologists to know the classification of microorganisms
 9204 Guidelines far Microbial Characterization, ldentification, and Strain Typing
in contaminated pharmaceutical products, to make infarmed
decisions regarding product risk and to recognize changes in
environmental microflora. In many quality control investiga-
tions, phenotypic identification alone is sufficient and will
enable scientists to conduct a thorough investigation and to
recommend appropriate corrective actions as needed.
( 4) Microbial identification by genotypic methods
In contrast, the microbial genotype generally is unaffected by
growth media or the viability of the isolate. Therefore, once the
isolation of a pure, monoclonal colony is assured, the
microorganism may be analyzed without concem over the culture
condition. Genotypic microbial identification methods are
theoretically more reliable because nucleic acid sequences are
highly conserved in most microbial species. Applicable genotypic
methods include DNA-DNA hybridization, PCR, 16S and 18S
rRNA sequencing, multilocus sequence typing ( MLST),
pyrosequencing, DNA probes, and analytical ribotyping. These
methods can be technically challenging for microbiologists. They
also require more expensive analytical equipment and supplies.
Therefore, the use of genotypic identification methods is typically
limited to critica! microbiological investigations such as product
failure investigations. Further, if applied, analysts must ensure
that the method is appropriate.
Bacteria! taxonomy as described in Bergey' s Manual of
systematic bacteriology is at present accomplished by
comparative analysis of genetic material. When the DNA
from an unknownorganism is compared to the DNA from a
known organism, the degree of relatedness can be deter-
mined. Genotypic identification is accomplished through the
use of DN.A... hybridization, restriction fragment pattern
comparisons, and/ or DNA pro bes. For example, greater
than 70 % relatedness with DNA-DNA hybridization indica tes
the organisms are the same genus. Phylogenetic analysis
(Table 2) is typically perfarmed by comparing the base
sequence of a portian of the 16S ribosomal RNA gene far
bacteria, or the 18S ribosomal RNA gene far fungi.
Polymerase chain reaction ( PCR) is used to amplify these
genes, and the amplified region is then isolated and base
sequenced using an electrophoretic and dideoxy chain
termination method seperately. Comparisons can be made
using validated proprietary databases or those that are
publicly available. [CAUTION: Publicly available databases
may not be validated].
Table 2 Genotypic/ phylogenetic characteristics
that can be used in microbial taxonomy
Categories Characteristics
DNA-DNA hybridization, DNA base ratio
Genotypic ( G + C content) , restriction fragment
patterns, and DNA pro bes
Phylogenetic 16S and 18S rRNA sequences
Nucleic acid-based methods can be used to screen far
damaged microorganisms in transition. The problem of
amplif ying DNA from nonviable bacteria! cells can be
overcome by using reverse transcription to convert rRNA
that is transitional, hence related to viability, to DNA far
PCR amplification. The steps associated with this activity are
sample collection, nucleic acid extraction, target amplifica-
tion, hybridization, and detection. Issues include the
detection of microbial variants, limits of detection, matrix
effects, positive cutoff verification, instrument and system
carry-over, diagnostic accuracy, and reproducibility.
The evolutionary history of micro-organisms is recorded in
their rRNAs, so that the analysis of rRNA sequences can be
used in microbial systematic classification and identification.
2. Verification of Microbial ldentification Methods
The verification of a microbial identification test system can
include one of the fallowing: (1) using an existing system far
parallel testing of microbial isolates obtained from routine
testing ( the number of isolates tested may be as high as 50,
and any discrepancies in identification can be arbitrated using
a referee method); (2) testing 12-15 known representative
stock cultures of different commonly isolated species far a
total of 50 tests; or ( 3) confirming that 20-50 organism
identifications, including 15-20 different species, agree with
the results of a reference laboratory testing of split sample.
In each case the appropriate quality control organisms, as
recommended by the supplier and the compendia, should be
included in the verification process.
With identification systems, verification of the identity of the
species should be evaluated and the level of agreement should
be considered. Typically greater than 90 % agreement can be
achieved with samples of microorganisms that are appropriate
far the identification system. Groups of organisms that are
challenging to identify (e. g. , nonfermenting bacteria,
corynebacteria, and coagulase-negative Staphylococci) may
be included, when appropriate, in the verification process but
may yield lower levels of agreement.
A microbial identification system may not be able to identify
an isolate because the organism is not included in the
database, the system parameters are not sufficiently
comprehensive to identify the organism, the isolate may be
nonreactive in the system, or the species may not have been
taxonomically described. Misidentification is more difficult to
determine, but any microbial identification should be
reviewed far reasonableness in terms of the microorganism' s
morphology, physiological requirements, and source of
isolation. Misidentification could lead to inappropriate
corrective and preventive actions and product disposition.
The verification of microbial identification method includes
accuracy, specificity, reproducibility, sensitivity, and
positive and negative predictive value.
The most important verification tests are accuracy and
reproducibility. These measurements can be defined as follows:
Accuracy (%) =(Number of correct results/
Total number of results) X 100
Reproducibility ( %) = (Number of correct results in agreement/
Total number of results) X 100.
The user should establish suitable acceptance criteria for accuracy
and reproducibility, taking into account method capability.
Other measurements, such as sensitivity, specificity, and
positive and negative predictive value, are best illustrated by
an example. A clinical microbiology laboratory tested 100
split samples using a DNA hybridization probe and a culture
method. The positive result by DNA hybridization probe was
10 % higher than that by culture method. The results are
presented in Table 3.
Table 3 Comparison of the distribution of negative and
positive results for the DNA probe and culture methods
Culture Results
Positive Negative
Positive 9 2
DNA Probe
Results Negative 1 88
Analytical accuracy=[ (9+88) /100] X 100=97%
lnclusivity= [9/(9+ 1) JX 100=90%
Exclusivity=[88/(88+2)] X 100=97. 7 %
Positive Predictive Value (PPV)=[9/(9+2)]Xl00=81. 8%
Negative Predictive Value (NPV)=[88/(88+D]Xl00=98. 9%
 9205 Guidelines far Microbiological Monitoring and Control of Clean Rooms far Pharmaceutical Products
Note that the positive predictive value CPPV) is not intrinsic
to the test; it also depends on the prevalence of the
microorganism in clinical samples. PPV is directly propor-
tional to the prevalence of the disease or condition. If the
group of people tested had included a higher proportion of
people with infection, then the PPV would probably be
higher and the negative predictive value CNPV) lower. If all
persons in the group had infection, the PPV would be 100%
and the NPV 0%. The mathematical derivation of these
functions is outlined in Table 4.
Table 4 A two-row by two-column contingency
table with respect to the reference
Culture Method and the Altemate PCR Method
PCR
Positive Negative Sum
a b
Positive a+b
True positive False negative
Culture
c d
method Negative c+d
False positive True negative
Sum a+c b+d
lnclusivityC %) =[a/ Ca+ b)] X 100
ExclusivityC %) = [d/ Cc+d)] X 100
Positive predictivityC %) =[a/Ca+c)] X 100
Negative predictivityC %) = [d/ Cb+d)] X 100
Analytical accuracyC % ) = [Ca+ d) /Ca+ b+ c+ d)] X 100
Kappa lndex= 2Cad-bc) /[ Ca+c) X Cc+d) +Ca+ b) X Cb+d)]
3. Phylogenetic Considerations
The organization of content in the second edition of Bergey' s
Manual of systematic bacteriology follows a phylogenetic
framework, based on analysis of the nucleotide sequence of
the ribosomal small subunit 16S RNA, rather than a
phenotypic structure.
Phylogenetic trees or dendrograms show the closest genetically
related organisms. The application of this technology has
resulted in taxonomic revisions and the renaming of sorne
well-known microorganisms; e. g. , the fungus A. niger
ATCC 16404 was renamed A. brasiliensis. In general,
organisms with relatedness less than or equal to 97 % are
considered different genera and those with relatedness less
than or equal to 99 % are considered different species, but
there are many exceptions to this generalization.
Differences in genotype and phenotype are relatively
uncommon, e. g. , same or very similar genotype shared by
microorganisms with different phenotypes, similar phenotypes but
different genotypes, and microorganisms that are genotypically too
distant to be the same species or genus. The concept of
polyphasic taxonomy that refers to assembly and use of many
levels of information, including molecular biology,
physiology, morphology, serology or ecology, C e. g. ,
microbial characterization, phenotypic and genotypic data,
and origin of the microorganisms), can be successfully
applied to microbial identification. This avoids decisions
made solely using identification method that make no sense.
4. Traceability Analysis
Traceability analysis is the process of determining the origins
of contamination through homologous analysis of the micro-
organisms isolated from contaminated products and those
isolated from related monitoring environment.
Strain level identification can be useful in an investigation to
determine the source of the microorganisms, and is especially
common when organisms are recovered at atypically high
rates or in numbers that exceed recommended levels for
specific categories of products. Additionally, strain level
identification is useful in aseptic processing and is necessary
where sterility test positives have occurred and in the
assessment of contamination recovered from failed aseptic
process simulations, i. e. , media fills.
Phenotype and genotype of the same microorganisms from
the same place are mostly identical. Phenotype of the same
micro-organisms from different places may be identical, but
the conserved and variable regions of genes may be variant.
Therefore, investigation of microbial contamination should
be based on the genotypic identification, and supplemented
by the information from phenotypic identification.
DNA sequences of the conservative regions of the 16S rRNA
gene for bacteria and the 18S rRNA gene for fungi is useful
for identification to the species level, but may not provide
sufficient power to resolve among closely related species or
strains of the same species. In contrast, southern hybridization of
restriction endonuclease digests is powerful and can be effective in
dermnstrating differences between two strains. H the banding
patterns appear identical, this shows only that restriction
endonuclease has similar cleavage sites in that region of the two
organisrns. llimnstration that the two organisms are the same
should include two or rmre different restriction endonuclease digests,
each of which yields bands in the area of interest. All bands from the
two organisms must be identical. For example, pulsed field
electrophoresis distinguishes different strains based on this principle.
In practice, a positive sterility test allows for invalidation, if
after traceability analysis and identification of the
microorganisms isolated from the test, the growth of this Cor
these) species are unequivocally ascribed to faults with
respect to the material and/ or the technique used in
conducting the sterility test procedure. Otherwise, the
product to be examined does not comply with the test for
sterility. Microbial isolates frum clean rooms and other
controlled environments may be identified at a rate sufficient
to support the investigation of microbial contamination.
Analysis of more gene sequences, specific gene fragments, or
even the entire genome should be assigned to demonstrate that
two organisms are identical strains belonging to the sarne
species. This information can not only achieve identification and
traceability of the targeted microorganisms, but also ensure the
accuracy of results. Additionally, traceability of sorne micro-
organisms needs the information of phenotypic characteristics,
such as serological test of Salmonella.
9205 Guidelines for Microbiological
Monitoring and Control of Clean Rooms for
Pharmaceutical Products
The objective of this chapter is to monitor and control microbial
contamination in clean rooms and other controlled environments
used for pharmaceutical microbiological examinations.
Pharmaceutical clean zones refer to clean rooms, isolators
and other controlled environments for sterility testing or
microbiological examinations for pharmaceutical products.
Cleanliness applicable to clean rooms is classified into four
classes, A, B, C and D, based on the particulate size and
concentration of airborne particles according to the current
edition of Good Manufacturing Practice far Drugs. In order
to maintain the stability of the operating environment, uphold
quality and safety far pharmaceutical products, and ensure
accuracy of detection, microbial monitoring and control should be
performed to maintain the risk of microbial contamination in
controlled environments at an acceptable level.
 9205 Guidelines far Microbiological Monitoring and Control of Clean Rooms far Pharmaceutical Products

This chapter includes requirements far personnel, parameter
validation far as-built clean rooms, microbial monitoring
methods, monitoring frequency and programs, monitoring
criteria, alert and action levels, data analysis and deviation
handling, and microbial identification and control.
Personnel
The personnel engaged in rnicrobial monitoring and control of
pharmaceutical clean rooms should meet the requirements of the
general chapter, Guidelines far quality management of
micromological lahoratory for pharmaceutical products ( 9203) in
current edition of Pharmacopoeia of People' s Republic of China.
Qualification
The parameters of as-built clean rooms should be validated at the
initial use. Qualification parameters include physical parameters,
airbome particles, and rnicro-organisrns. When significant changes
occur to critica! facilities, such as clean benches and air-
conditioning systems, parameters should be re-verified.
Physical parameters of clean rooms should be tested prior to
microbial monitoring programs in order to ensure proper
facility perfarmances and reliabilities. Major physical
Table 1 The recommended criteria of physical parameters for classified clean environments
Physical parameters
Cleanliness
classes Filters Airflow velocity
Airflow patterns
integrity (average air velocity)
parameters include integrity of high-efficiency air filters,
airflow patterns, airflow velocity (average air velocity), air
changes, air pressure difference, temperature, relative
humidity, etc. Testing of physical parameters should be
perfarmed under conditions that simulate normal detection.
Tablel shows the recommended criteria and maximal time
interval far monitoring physical parameters far classified
clean environments. If necessary, appropria te parameter
criteria far individual clean rooms should be set up based on
utility of clean rooms and nature of pharmaceutical products.
Test methods far physical parameters are perfarmed
according to the current national standard of "Code for
construction and acceptance of clean room", Appendix D3
"In-situ scanning leak test method for HEPA filters ",
Appendix E12 "Test method far airflow pattern test",
Appendix El "Test method for airflow volume and airflow
velocity", Appendix E2 "Test method for static air pressure
dij ference ", and Appendix ES "Test method far
temperature and relative humidity" .
For the initial use of clean rooms, qualification and monitoring of
airbome particles and rnicroorganisms should be performed
according to the following "monitoring'' prograrn.
Pressure Relative
Air changes Temperature
difference humidity

O. 25-0. 50m/ s
(equipment)
Time interval far
O. 36-0. 54m/ s
A unidirectional -
(facility)
airflow: 24 months
Time interval: 12
months No less than 10
pa between clean
1) Unidirectional 1) Unidirectional
Time zone and non-
1) Time interval far airflow ( at-rest ) airflow
interval clean zone; 18-26ºC 45%-65%
unidirectional O. 25-0. 50 m/ s -
far No less than 10 Time Time
airflow (at-rest): Time interval: 12 2) Turbulent
B leakage pa between clean interval: interval:
24 months months airflow
test: zones with diffe- each ea ch
2) Turbulent airflow 2) Non-unidirectional 40-60 h- 1
24 rent cleanliness operation operation
- airflow Time interval:
months -
levels Tlffie
12 months
interval:
20-40 h- 1 one week
T urbulentairflow
e -
- Time interval:
12 months

Turbulent airflow 6-20 h- 1 Time

D -
-
interval: 12 months

Monitoring
Microbial monitoring should be performed periodically in
pharmaceutical clean rooms, including nonviable airborne
particles and viable micro-organisms. Microbial monitoring
includes airborne microbes and settling microbes, and
microbes on critical operational surfaces, such as clean
benches, surfaces garment and gloves.
Monitoring should be re-perfarmed when major changes
occur to critical facilities, such as clean benches and air-
conditioning systems. If microbial monitoring or sample
detection results reveal any significant deviations or
contamination may arise in clean rooms after evaluation, the
clean rooms should be cleaned and decontaminated, and then
monitoring should be re-perfarmed.
l. Monitoring Methods
Monitoring of airborne particles in pharmaceutical clean
rooms is performed according to the current national standard
of "Test method Jor airborne particles in clean room (zone)
of the pharmaceutical industry" . Monitoring of settling
microbes is based on the current national standard of "Test
method for settling microbe in clean room ( zone) of the
pharmaceutical industry " . Monitoring of airborne
microbes is perfarmed according to the current national
standard of "Test method for airborne microbe in clean room
(zone) of the pharmaceutical industry" .
Surface microbial monitoring refers to monitoring surfaces of
environment, facilities, and personnel; surface sampling can
be accomplished by the use of contact plates or swabbing
method. Contact plates filled with indicated agar are used for
 9205 Guidelines for Microbiological Monitoring and Control of Clean Rooms for Pharmaceutical Products

sampling regular or flat surfaces and are directly incubated Monitoring frequency should be revised in cases of
for the appropriate time and temperature for recovery of monitoring results continuously exceeding action levels and
viable organisms. Sampling area of per contact plate is about alert levels, microbial contamination in critical zones,
25 cm2 . The microbial estimates are reported in CFU per concernful maintenance of air ventilation systems,
plate. Swabbing method can be used to supplement contact- amendment of samtization procedures, concernful
plate method for sampling of irregular surfaces, especially maintenance or addition of equipment, significant change in
irregular surfaces of equipment. The area that will be the structure or distribution of clean rooms, accident of
swabbed is defined with a sterile template or a ruler of microbial contamination, or tendency of data indicated by
appropriate size. After sample collection the swab is placed routine operation records.
in an appropriate buffer solution or culture medium, and 2. Monitoring Criteria
shaken thoroughly. Then, microbe counting is performed Airborne particulate cleanliness classes of clean rooms are
with a suitable method. Sampling area per swab is about 25
shown in Table 3. The operational criteria of microbial
cm2. The microbial estimates are reported in CFU per swab. monitoring are shown in Table 4.
The culture medium, temperature and time used in contact-
plate and swabbing methods are identical with those for airborne Table 3 The criteria of airborne particulate
microbes or settling microbes. Surface sampling for micro- cleanliness el~ for clean roorns
organisms should be done only at the end of operations.
Maximum concentration limit for
The culture medium used for monitoring of airborne
airborne particles/ m 3
microbes, settling microbes and surface microbes is typically Cleanline.::cs.::.s_____________________
soybean-casein digest agar. Appropriate neutralizing reagent class At-rest Operational
could be added to the medium if necessary. When fungi are
present or factors of season are considered, sabouraud- ~o. 5µm ~o. 5µm ~o. 5µm ~o. 5µm
dextrose agar ( SDA) medium can be used as supplementary.
In the monitoring of pharmaceutical clean rooms, monitoring A 3520 20 3520 20
frequency and parameters are recommended in Table 2.
B 3520 20 352 000 2900
Table 2 The recommended rnonitoring frequency and
pararneters for phannaceutical clean roorns e 352 000 2900 3 520 000 29 000
Controlled Sampling Monitoring D 3 520 000 29 000 Not required Not required
zone frequency program
Table 4 The operational criteria of environrnental
Isolators airborne particles3 ,
rnicrobial rnonitoring for clean roorns1
system airborne microbes3 ,
F.ach
for settling microbes2 , Settling Surface micro-organisms
operation Cl l" Airbome microbes
sterility surface micro-organisms
e~n mess microhes (di Contact olate ( <!>55mm) gloves
testing Cinciuding gioves) classes CFU/m3 90 m~)2---·---'--------
CFU/4 h CFU/plate CFU/glove
airborne particles3 ,
airborne microbes1 , A <1 <1 <1 <1
Each settling microbes 2 ,
A B 10 5 5 5
operation surface micro-organisms
( including gloves and e 100 50 25
Clean room
operational clothing)
for
D 200 100 50
microbiological
examination airborne particles4 , Note: l. average values; 2. Exposure time of each settling plate can
airborne microbes3 , be less than 4 hour, several plates can be used serially for settling
Once per settling micro bes, microbes monitoring and counts are accumulated.
B
week surface micro-organisms
(including gloves and 3. Alert levels and action levels
operational clothing) Proper alert and action levels should be established based on
historical data, combining the classification of various clean
airborne particles4 , zones and suitable methods. Alert and action levels should be
Once per
airborne microbe4 , periodically reviewed once they are set. If historical data
e three
settling microbe, indicate any environmental improvement, these levels should
Clean room months
surface micro-organisms be adjusted to reflect the actual environmental quality. Table
for
5 shows the recommended action levels for environmental
microbiologica
airborne particles, micro-organisms of cleanliness classes.
examination
Once per airborne micro bes, 4. Data analysis and deviation handling
D
six months settling micro bes,
surface micro-organisms Data analysis
Routine environmental monitoring data should be analyzed
Note: l. once per month; 2. no less than three locations and reviewed. Clean rooms are evaluated on compliance
should be tested for routine monitoring of settling microbe on through data collection and tendency analysis. Alert levels,
operational surfaces, and no less than one plate should be action levels and corrective actions are evaluated on
used per sampling location; 3. once per · three months; appropriateness.
4. once per six months.
 9206 Guidelines for Validation of Isolator Systems for Use in Sterility Testing
Table 5 The recommended action levels for
environmental micro-organisms of
cleanliness das.ses.
Action levels for Action levels for settling
Cleanliness
airborne microbes1 microbes 2
class 3 Disinfectants and detergents used in class A and B cleaning
(CFU/m ) (cf>90 mm, CFU/4 h)
A <13 <13
B 7 3 periodically to avoid emergence of resistant strains. Chemical
e 10 5 If necessary, fumigation sterilization can be applied to reduce
D 100 50
Note: l. data refer to the recommended levels of
environmental quality. Action levels can be set based on
detection and analysis methods. 2. The number for settling
plates can be added based on utility of clean zones and natures
of tested drugs. 3. Samples from clean rooms classified as A
should not have any microbial contamination normally.
Microbial contamination should be evaluated not only on
microbe counts but also on microbial contamination recovery
rates. Microbial contamination observed at multiple sites in
an environment within a single sampling period may indicate
increased risk and should be carefully evaluated. Simultaneous
appearance of contamination at multiple sites could also arise
from poor sampling technique procedures, so careful review on
sampling process must be carried out before drawing conclusions
on potential loss of control. Resampling an environment
several days after contamination is of little value, because
sampling is irreproducible.
Deviation handling
If results of microbial monitoring reach the action level,
deviations should be reported, recorded, investigated artd
handled according to approved procedures. Corrective actions
and related evaluation should be taken in within a reasonable
time frame.
S. Identification of microbial isolates
It is recommended that micro-organisms obtained from
controlled environments should be identified at an appropriate
level. A knowledge of the flora in controlled environments
aids in determining the usual microbial flora anticipated for
the facility and in evaluating the effectiveness of the cleaning
and samt1zation procedures, methods, detergents or
disinfectants, and microbial monitoring methods. The
information gathered by an identification program can be
useful in the investigation of the source of contamination,
especially when recommended contamination levels are
exceeded. Identification of isolates from critica! areas should
take precedence over identification of micro-organisms from
noncritical areas. Identification of microbial isolates is
performed according to the general chapter Guidelines far
Microbial Characterization, Identification, and Strain
Typing <9204 >.
Microbial Control
In order to ensure the environment of pharmaceutical clean
rooms maintain an adequate level of cleanliness and under
controlled status, air conditioning systems and facilities must
be well maintained in good operational conditions strictly
following Good Manufacturing Practice. Environment
should be monitored periodically. Reduction of intervention
by personnel is more effective for contamination prevention
than monitoring programs. Temperature and humidity can
be adequately controlled by effective control of personnel and
items movements. Microbial control measures also include
good cleaning and sanitization; therefore pharmaceutical
clean rooms should be cleaned and sanitized periodically.
Disinfectants and detergents should also be monitored on
microbial contamination and used befare expiration dates.
zone should be aseptic or sterilized. Chemical disinfectants to
be used must be certified or proven their effectiveness in
disinfection. Multiple types must be used and altered
sanitization shouldn' t be replaced by ultraviolet sanitization.
the microbial contamination where hard to access. Residual
fumigation agent should be tested.
9206 Guidelines for Validation of
Isolator Systems for Use in
Sterility Testing
This chapter provides guidelines for the validation of isolator
systems for use in sterility testing of compendia! articles,
including biological products, raw materials, preparations
and other articles, which are required to be sterile according
to the Pharmacopoeia.
Isolator systems for use in sterility testing are devices that
create controlled environments in which to conduct
Pharmacopeial sterility tests. Closed isolators, which are
systems with no direct opening to the external environment,
use only decontaminated interfaces or rapid transfer ports for
the transfer of materiais, are normaiiy used for steriiity
testing, although open isolators which allow the egress of
materials through a defined opening that precludes the entry
of contamination by means of air overpressure may be used.
All transfers of material into and out of the isolator are
accomplished in an aseptic fashion while maintaining
complete environment separation. The interior of the isolator
is able to be reproducibly decontaminated and normally is
treated with sterilants that result in the elimination of all
viable bioburden on exposed surfaces. After the sterilization,
the isolator is supplied with air through a HEPA or better air
filter to maintain internal aseptic environment. Isolator
systems protect the test article and supplies from
contamination during handling by essentially elimination
direct contact between the analyst and the test articles.
Aseptic manipulations within the isolator are made with half-
suits or by gloves and sleeves, which are flexible components
of the isolator wall that allow the operator a full range of
motion within the isolator and compatible with sterilants.
Operators are not required to wear special clean-room
clothing for conducting sterility tests within isolators;
standard laboratory clothing is adequate. Therefore the
results in sterility testing are with a high degree of assurance
through isolator systems to protect test articles and auxiliary
facilities from contamination.
1. lsolator design and construction
Isolators used for sterility testing are generally constructed of
stainless steel, glass, rigid plastics or flexible plastics (such
as polyvinyl chloride). The configuration of an insulator
generally includes:
1) Air handling system
An isolator used for sterility testing is equipped with
microbial retentive filters ( HEPA filters or better are
required). At rest, the isolator meets the particular air-
quality requirements for a "Class A" area as defined in Good
Manufacturing Practice for drugs ( GMP ) . When direct
 9206 Guidelines far Validation of Isolator Systems far Use in Sterility Testing
openings to the outside environment exist, constant air
overpressure conditions maintain sterile conditions within the
isolator.
2) Transfer ports and doors
Isolators may be attached to a "pass-through" decontaminator to
enable the direct transfer of sterile media, sterile dilution fluids,
and sterile supplies far the decontaminator into isolator
system. Rapid transfer ports CRTPs) enable two isolators or
more to be connected to one another, so that supplies can be
moved aseptically far one isolator to another. The non-sterile
surfaces of the RTP are connected using locking rings or
flanges. A compressed gasket assembly provides an airtight
seal, thereby preventing the ingress of microorganisms.
3) Decontamination equipment
The connections between gas generator and isolator should be
sealed well enough. The pass-through pipes for decontamination
gas are controlled by valves at the side of the isolator. Those
valves need to be closed while connecting or disconnecting gas
generator and isolator or maintaining asepsis within the
isolator environment.
Decontamination gas need to be supplied into isolator through
HEP A filters and need to be removed from the isolator after
the exposure period. The residual vapors or gases in the
isolator are kept to appropriately low levels befare sterility
testing, which <loes not adversely affect the ability of the
sterility test.
4) Utility service and equipment
Utility service and equipment used in sterility testing, such
as peristaltic pump, vacuum pump and continuous
monitoring devices far asepsis environment should not
adversely affect the internal environment of the isolator.
The design of motor and air-vent parts needs to be at external
environment of the isolator that prevents any adverse effect
from disturbing the air flow of isolator while operating and
gas exhausting. And safety of chemical corrosion is also a
concern of this design.
2. Selection of a Iocation for the isolator
Isolators for sterility testing are suggested to be installed in a
classified clean room meeting the particular air-quality
requirements far a Class D area as defined in GMP far drugs.
It is important to place the isolator in an area that provides
limited access to nonessential staff. The appropriate location
provides adequate space around the isolator far moving
transfer isolators, staging of materials and general
maintenance.
Temperature and humidity control in the room is important
to operator safety and comfort and is critical for the effective
utilization of certain decontamination technologies. Care
should be taken in locating the isolator so that cold spots are avoided
that might result in excessive condensation when condensing
vapors are used far decontamination. Uniform temperature
conditions in the room are desirable when temperature-sensitive
decontamination methods are employed.
3. Validation of the isolator system
The isolator system must be validated befare its use in
sterility testing. The following performance must be
validated regularly after installation performance.
1) Operational qualification
This test verifies that all alert and alarm functions comply with
their functional specifications. The system' s ability to comply
with all set points and adjustable parameters is verified.
2) lsolator integrity check
The integrity of the isolator is maintained during all normal
operating conditions. A leak test is performed to verify the
compliance with the manufacturer' s functional specifications.
To safeguard against adventitious contamination, isolators
are operated at a suitable positive pressure during normal
operation ( differential pressure range is 20-50 Pa ) .
Validation studies must show that the air pressure set point
can be maintained and controlled during operation. The
integrity of HEPA filter of the isolator also needs to be
validated regularly.
3) Decontamination validation
The interior surfaces of the isolator, the equipment within
the isolator, and the materials brought into the isolator are
treated to eliminate all bioburden. The decontamination
methods used to treat isolators, test articles, and sterility
testing supplies are capable of reproducibly yielding greater
than a six-log reduction against highly resistant biological
indicators. Total kill analysis studies are suitable for Bls with
a population of 106 spores per unit, while fraction negative
studies are suitable for Bls with a population of 106 or
greater. A sufficient number of Bls are used to prove
statistical reproducibility and adequate distribution of the
decontaminating agent. Particular attention is given to areas
that pose problems relative to the concentration of the agent.
A larger number of Bls may be required in isolators that are
heavily loaded with equipment and materials. The ability of
the process to reproducibly deliver a greater than six-log kill
is confirmed in three consecutive validation studies.
4) Decontamination cycle verification
A decontamination cycle is run thoroughly to verify the
operational parameters conform to the set point.
5) Cleaning verification
Cleaning of internal environment should be verified, the
airborne particles ( at rest) and viable particles meet the
particular air-quality requirernents for a "Class A" area as
dcfincd in G~1P for drugs.
Decontamination agents need to be removed from the isolator
after exposure period, and it is important to monitor the
concentration of the residual decontamination agent to
acceptable levels in the isolator.
6) Instrument verification
The instruments of the isolator system such as H 2 0 2 sensor,
temperature and humidity sensor, pressure sensor need to be
calibrated regularly.
To verify that the isolator systems are suitable for sterility
tests, validation studies such as operational performance
check and isolator integrity check are performed routinely.
The re-verification is performed according to documentary
procedures and acceptable standards, such as abnormal
operation or internal environment monitoring, operational
program or parameters changing or isolator systems
relocating. All data are adequately summarized and archived.
4. Application of the isolator system
1) Package integrity verification
Screw-capped tubes, ampoules, bottles, or vials sealed with
rubber stoppers and crimp overseals have proven very
resistant to the penetration of cornmonly used decontaminating
agents. Sorne materials, such as filter sets are adversely affected
by decontaminating agents, which can result in inhibition of
microbial growth. It is the responsibility of the operator to
demonstrate, via validation studies, that exposure of product
containers, dilution fluid and media to the decontaminating
agent does not adversely affect the ability of the sterility test
to detect low levels of contamination within these test
articles. If there is potential risk of decontaminating agents
penetrating into product container, supplies, media and
 9301 Guidelines far Application of Safety Tests far Injections
dilution fluid, wrapping them in resistant packages to
decontaminating agents or placing them in a sealed container
will prevent penetration of decontaminating agents.
However, these procedures shall not adversely aff ect the
decontamination.
In sorne cases, the use of shorter duration decontamination
cycles and reduced concentrations may be necessary to
minimize penetration of decontaminating agents into the
package or container.
The operator will choose to treat the surfaces of product
containers under test with the decontaminating agent in order
to minimize the likelihood of bioburden entering the isolator
in many cases. It is the responsibility of the operator to
demonstrate, via validation studies, that exposure of product
containers to the decontaminating agent <loes not adversely
affect the ability of the sterility test to detect low levels of
contamination within these test articles. It is suggested that
the ability of the package to resist contamination be examined
using both chemical and microbiological test procedures.
Bacteriostasis and fungistasis validation tests must be
performed using actual test articles that have been exposed to
all phases of the decontamination process. ( Sterility Test
(1101 > ).
2) Maintenance of asepsis within the isolator environrnent
The ability of the isolator system to maintain an aseptic
environment throughout the defined operational period must
be validated. Microbiological monitoring usually involves a
routine sampling program, which may include, far instance,
sampling fallowing decontamination on the first day of
operation and sampling on thc last day the projected
maintenance of asepsis period. Periodic sampling throughout
the use period can be performed to demonstrate maintenance of
asepsis within the isolator. In addition, a microbiological
monitoring program must be implemented to detect malfunctions
of the isolator system or the presence of adventitious contamination
within the isolator.
The surfaces within the isolator can be monitored using
either contact plates for flat surfaces or swabs far irregular
surfaces. However, because media residues could impose a
risk on isolator asepsis, these tests are generally best done at
the end of the test period. If perfarmed concurrently with
testing, care is used to ensure that any residual medium is
removed from isolator surfaces, and that those surfaces are
carefully cleaned and disinfected.
A potential route for contamination to enter the isolator is
during the introduction of supplies and samples into the
enclosure. Validating that all materials taken into the isolator
enclosure are free of microbial contamination is critica!, as is
periodic inspection of gaskets to detect imperfections that
could allow ingress of micro-organisms. Gloves and half-suit
assemblies are another potential source of microbial
contamination. Gloves are of particular concern because they
are used to handle both sterility testing materials and test
articles. Resistance to puncture and abrasion, and an
adequate tactile feel provision should be considered in the
selection of gloves and sleeves while maintaining their
integrity.
Very small leaks in gloves are difficult to detect until the
glove is stretched during use. There are several commercially
available glove leak detectors; the operator ensures that the
detectors test the glove under conditions as close as possible
to actual use conditions. Microbiological tests are used to
supplement or substitute physical tests. Submersion of the
gloves in O. 1 % peptone water followed by filtration of the
diluents and plating on growth media can detect loss of
integrity in the gloves that would otherwise go unnoticed.
Continuous nonviable particulate monitoring within the
isolator' s enclosure is ideal, because it can quickly detect
filter failure. A second choice is periodic monitoring using a
portable particle counter. Sampling for particles must be
done in a manner that poses no risk to the maintenance of
asepsis within the isolator.
3) lnterpretation of sterility test results
A sterility test resulting in a false positive in a properly
functioning and validated isolator is very unlikely if bioburden
is eliminated from the isolator interior with a high degree of
assurance and the operator has no direct contact with the
sterility testing environment. Nevertheless, isolators are
mechanical devices and good aseptic techniques are still
required. A decision to invalidate a false positive is made only
after fully complying with the requirements of Observation
and Interpretation of Results under Sterility Tests.
4) Safety and training
Operators are trained in operation, maintenance and safety
procedures that are specific to their isolator befare the
conduct of sterility tests in isolators. All training sessions
and the evaluation of the operator' s performance are
documented in the individual' s training record.
All storage and safety precautions of the decontaminating
agentare fallowed by operators. Material Safety Data Sheets
( MSDS) are available in the immedia te area where the
decontaminating agent is being used. All associated
equipment is performed and documented prior to placing the
unit in service.
9301 Guidelines for Application of
Safety Tests for lnjections
This guideline is used for quality control and clinic safety of
injection of chemical medicines and traditional Chinese
medicine.
The guidelines include tests far abnormal toxicity, bacteria!
endotoxins ( or pyrogens), depressor substances ( including
histamine), allergen, haemolysis and agglomeration, etc.
These testing items and their suitability to safety testing
should be established according to ingredients, procedures,
usages and dosage. Depending on the results of suitability
study, bacteria! endotoxins and pyrogens are interchangeable
testing items and only one of them should be selected, which
is also applicable to depressor substances and histamine.
l. Establishment of the safety tests for injection
(1) lnjections for vein
For the injections for vein, the test far bacteria! endotoxins Cor
pyrogens) should be carried out. For injections of chemical
medicines, generally, the test far bacteria! endotoxins is first
recommended. For injections of traditional Chinese medicine, the
test far pyrogens is generally first recommended unless the
results of the the test for pyrogens are interfered by
pharmacological effects or toxic reactions to rabbits, thus the
test far bacteria endotoxins should be chosen for these
injections.
For the injections, which raw materials are derived from
animal, extracted from the microbial fermentation broth,
possess unclear components, or likely contaminated with
toxic impurities without effective analytical methods, test far
abnormal toxicity should be carried out.
For the injections, which raw materials are derived from
animal, extracted from the microbial fermentation broth,
possess unclear components, or likely to contaminated with
heterogeneous protein, and if there is lack of relevant
 9301 Guidelines for Application of Safety Tests for Injections
analytical methods or found allergic in clinical, tests for
allergen should be carried out.
Far the injections, which raw materials are derived from
animal, extracted from the microbial fermentation broth, to
possess unclear components, or likely to contaminated with
depressor impurities, such as histamine and histamine-like
substances, especially for injections of traditional Chinese
medicine, and if there is lack of relevant analytical methods
or found allergic in clinical, tests for depressor substances or
histamine substances should be carried out. Normally, the test
far depressor substances is firstly preferred. The test for
histamine substances can be chosen when the pharmacological
effects of depressor are relevant with the action and the
indications of the injection, or the test far blood pressure is
interfered by injections because of its reaction to cats.
Far injections of traditional Chinese medicine, tests far
haemolysis and agglomeration should be carried out.
(2) lnjections for muscle
Far the injections produced by raw materials derived from
animal, extracted from the microbial fermentation broth,
possessing unclear components, or likely to be contaminated
with toxic impurities, tests far abnormal toxicity should be
carried out when effective analytical methods are unavailable.
Far the injections produced by raw materials derived from
animal, extracted from the microbial fermentation broth,
possessing unclear components, or likely to be contaminated
with heterogeneous protein and unknown allergic substances,
tests for allergen should be carried out if relevant analytical
methods are unavailable and allergic reactions are observed in
the clinic.
Far the injections for muscle which clinical doses are much
larger, or production engineering are vulnerable to
contaminated by bacteria! endotoxins, tests for bacteria!
endotoxins should be carried out.
(3) Injections for special routes
Far the injections for special routes such as spinal canal,
abdominal cavity, eyes, the safety tests should generally be
in line with the requirements of injections for vein. If
necessary, additional safety tests such as test for stimulation
or cytotoxicity should be operated.
( 4) Exipients of injections
Exipients of injections are frequently used with large quantity
and acquired from complex source, however they are directly
related to the safety of the drugs. Necessary safety items should
be established to control the quality of exip1ents of
inj ections based on the source, property, purpose, usage, dosage
as well as the physical and chemical analysis methods.
(5) Others
Far the injections made from raw materials and by special
procedures, additional special safety items should be carried
out, such as test for virus or cytotoxicity, if necessary.
2. Establishment of methods and limits of the safety tests
The methods and limits are established according to the
requirements of various items. After the limit is established,
the validation tests should be carried out far at least 3 batches
of preparations.
( 1) Test for abnormal toxicity
The abnormal toxicity is determined by observing the death
of mice within a specified period after injecting an amount of
the test preparation. The failure of the test showed that toxic
impurities with different toxicity from that of drug itself are
mixed in the test preparation, which may increase acute
adverse reactions in the clinic.
Procedure Refer to Test for Abnormal Toxicity ( 1141 ).
Research before limit establishing Acute systemic toxic1ty
such as LDso or LD1 and the fiducial limits (FL) should be
determined according to reference literatures and single
injection in the vein. If available, the data of LD50 and LD1
should be determined by multiple laboratories using animals
from various sources. The speed of injection is O. 1 ml/ s and
the observation duration is 72 h. If other species of animals
are used, administration routes and frequency are changed or
the observation duration and observation indexare extended,
and another acute toxicity test should be performed using the
corresponding animals, administration methods or
observation index and observation duration.
Establishment of limit The limit <lose of abnormal toxicity
of injections should be less than that of the minimum lethal
dose. Considering the differences in labs, reactivity of
animals and preparations into consideration, the limit should
be at least less than 1/3 of lower FL of LD1 ( 1/3-1/6 is
recommended). If LD1 is difficult to obtain, the limit should
be less than 1/ 4 of lower FL of LD5o ( 1/ 4-1/8 is
recommended). If the ratio of LDso to the clinical dose
measured by weight is less than 20, limit should be
determined as 1/ 4 of lower FL of LDso or 1/3 of the lower
FL of LD1.
Special requirements such as animal, administration routes,
administration frequency, observation items, duration and
limit should be specified in the monograph.
(2) Test for bacteria endotoxins or test for pytogens
The bacteria endotoxins or pytogens of the test preparation
are determined by the limulus amebocyte or rabbits. The
failure of the test showed that the test preparation might
cause the pyrogenic reaction and lead to serious adverse effect
in the clinic.
Procedure Refer to Test for Bacteria Endotoxins ( 1143 )
or "Test for Pyrogens" ( 1142).
Research before limit established For bacteria endotoxins,
interfering test should be carried out for the test to determine
the maximal non-interfering dose. Suitability tests should be
carried out for the test for pyrogens to determine the <lose on
rabbit without toxic reactions, disrupting the normal
temperature and displaying antipyretic effect.
Establishment of the limit Limit of the bacteria endotoxins
and pyrogens should be estimated on the basis of the
maximum recommended human <lose ( per kg of body
weight) of the product in a single hour period. Limit of the
bacteria endotoxins should comply with the requirement
specified in the monograph. Taking the differences of the
drug and indications into consideration, the limit is adjusted to
1/3 to 1/2 of the calculated value in order to ensure the safety.
Limit of the pyrogens should be estimated on the basis of the
above mentioned maximal dose. It is set up to 3-5 times of the
maximum human dose ( per kg of body weight) in a single
hour. Volume of the test injection should be no less than O. 5 ml
and no more than 10 ml per kg weight.
No interference reactions appear in the test for bacteria
endotoxins and limit dose of test for pyrogens should not
affect the normal temperature. If the above disturbance
occurs in the test, special requirements should be added in
the monograph in order to eliminate the interfering, such as
injecting slowly, decreasing concentration or regulating the
pH value or osmotic pressure.
(3) Test for depressor substances
The depressor substances of the test preparation are
determined by comparing the depressor effect on anesthetized
cat with histamine reference standard. The failure of the test
 9302 Guidelines for Establishment of Limit for Harmful Residue of Traditional Chinese Medicine
indicates the amount of the impurities which may influence
the blood pressure is beyond the limit, leading to the
decrease of the blood pressure in the clinic.
Procedure See Test Jor Depressor Suhstances <1145).
Research before limit established After injecting intravenously a
series dose of solution at a uniform rate ( the volume of the test
solution should be equal to that of reference standard solution,
usually O. 2-1. O ml/kg), the <lose-response relationships of the
blood pressure caused by tested solutions are observed on cats to
determine the maximal dose that complies with the dose
specified in the Test Jor Depressor Suhstances ( namely the
maximal non-depressor effect dose).
Establishment of the lirnit Generally, the limit dose should
be determined as 1/5 to 5 times of one single dose in the
clinic, however, for the acute and severe disease, the upper
limit should be determined as much as possible.
In special circumstance, such as a certain degree of
antihypertensive effect was observed, the limit dose should
be determined as 1/2 to 1/ 4 of the maximal non-depressor
effect dose on cats. If no depressor effect was observed at the
dose of 1 ml/kg by injecting into the vein, this dose can be
determined as the limit administration.
( 4) Test for histamine
The test is determined by comparing the degree of contractive
effect of the test preparation on isolated intestine of guinea
pig with that of the histamine reference standard. The failure
of the test indicates that there are histamine or histamine-like
substances in the test preparation, which might decrease the
blood pressure and cause serious adverse reactions such as
allergic-like reaction in the clinic.
Procedure Refer to Testfor Histamine <1146).
Research before lirnit established Befare establishment of the
limit, the interfering effect (inhibitory or enhancing) of the
test preparation on contraction of isolated guinea pig intestine
induced by histamine ref erence standard should be carefully
estimated in arder to obtain a maximal non-interfering dose.
If any result-affecting changes occur on test preparation,
such as the ingredient, procedure or any other factors, a new
interfering experiment should be carried out.
Interfering test According to the Test Jor Histamine, a series
of the dilute solutions are accurately injected in the following
arder, diluted solution of test preparation plus low dose reference
standard, diluted solution of low dose reference standard, diluted
solution of test preparation plus high dose reference standard,
diluted solution of high dose reference standard (ds1 + T, ds1 ,
dSz+T, dSz ). Repeat the test again. If the response induced by
dSL+Tand dsH+T were similar to the corresponding response caused
by ~ and dsH , the test preparation displays no interfering effect
on the test of histamine. Otherwise, the test preparation is not
suitable for the test for histamine and test for depressor substances
should be carried out. Meanwhile, research on the
compliance between this test and the test for depressor
substances should be carried out.
Establishment of lirnit Unless otherwise specifed, the limit
is in agreement with the limit of test for depressor substances
in principie. Limit should be calculated at 1/5 to 5 times of
the one single clinical dosage and histamine dose of O. 1 µg
per kg weight. Calculation formula is shown as follows: L =
K /M, where K represents the amount of histamine per kg
weight of human being (O. 1 µg/kg) and M represents the
limit of test for depressor substances ( mg/kg, ml/kg,
IU/kg). The dose of the test preparation should be less than
the maximal non-interfering dose, however the upper limit
should be determined for drugs for the acute and severe
disease such as antineoplastic agents, cardiovascular drugs.
(5) Test for allergens
The allergens of the test preparation is determined by observing
the allergic response of guinea pigs injected for sensitization
intraperitoneally or subcutaneously then challenged by
intravenous administration after a period of time. The failure of
the test showed that there are allergic substances in the test
preparation that might sensitize patient or cause allergic reaction
in clinic and lead to serious adverse effect.
Procedure Refer to Test Jor Allergen <1147>.
Research before lirnit established Determine the maximal
non-toxic dose of the guinea pig for the intraperital ( or the
subcutaneous) and intravenously injection. If necessary,
semi-product can be used in the preliminary test for
sensitization and challenging test to determine allergy
inducing manner and frequency of sensitization. Optimal
challenging time point should be carried out among the 14 h,
21h, 28 h after sensitized first.
Establishment the limit Dose for sensitization and the
challenging test should be less than the acute toxic dose of
the injection by the same route and clinical dosage can be
used appropriately as a reference. Generally, the challenging
dose is larger than the sensitization dose. Sensitization
usually is induced by inject intraperitoneally or subcutaneously at
the groin area into O. 5 ml of test solution each animal, and
challenging test is performed by injection of l. O ml of test
solution intravenously. If the sensitization dose is smaller,
frequency of the sensitization should be increased accordingly
and special requirements of methods and limit should be
specified in the monograph.
( 6) Haemolysis and agglomeration
The test is determined by observing the haemolysis and
agglomeration on erythrocytes of rabbits by mixing and
incubating 2 % erythrocytes suspension with test solution for
a period of time.
Procedure Refer to Test for Haemolysis and
Agglomeration <1148).
Research before lirnit established In the research of the
haemolysis and agglomeration of the preparation solution or
diluent, visual inspection should be used. Microscope and
colourimetric determination could be added, if necessary.
Haemolysis and agglomeration are observed at the same time
to determine the maximal concentration which does not cause
haemolysis and agglomeration.
Establishment of the lirnit Limit concentration should be
determined as 1/2 times of that which does not cause
haemolysis and agglomeration and generally should not be
lower than that of the maximal clinical dosage. The original
concentration of the preparation should be determined as the
limit concentration, if that does not give rise to haemolysis
and agglomeration.
9302 Guidelines for Establishment of
Limit for Harmful Residue of
Traditional Chinese Medicine
The guideline provides relevant scientific theory for the
establishment of maximal limit of harmful residue and
calculation of theoretical value of maximal limit and specifies
related affecting of the establishment of limit for traditional
Chinese medicine. The guideline is suitable for the establishment
 9302 Guidelines far Establishment of Limit far Harmful Residue of Traditional Chinese Medicine
of harmful residue limit for the materia medica and prepared
slices of traditional Chinese medicine. This guideline may refer to
establishing maximal limit of harmful residue far other products.
The harmful residue in this guideline can be pesticide
residue, heavy metals, harmful element and biotoxins, etc.
l. Overview
The raw material of traditional Chinese medicine sourced
from naturally growing plants, animals and minerals is very
likely to contain harmful residues or contaminants. Harmful
residues or contaminants in traditional Chinese medicine are
mainly pesticide residues, heavy metals and biotoxins, etc.
Heavy metals and harmful elements are mainly lead (Pb),
mercury ( Hg) , cadmium ( Cd) , copper ( Cu) , silver
(Ag), bismuth (Bi), antimony (Sb), stannum (Sn), and
arsenic (As), etc. Biotoxins are mainly aflatoxin (AF), a
type of metabolites produced by Aspergillus flavus and
Aspergillus parasiticus that are widely distributed in nature.
The establishment of limits for harmful residues mainly relies on
the result of risk assessment. Using data on harmful residues
from toxicology, epidemiology or others resources, human
exposure risk to harmful residues can be estimated based on the
properties of the risk by a group of assessment methods that
perform comprehensive analysis and evaluation on the exposure
of contaminant and possible dietary intake. Risk assessment
provides important information for the establishment of limit for
harmful residue.
Acceptable daily intake ( ADD is a commonly used term by
many countries and international organizations. ADI values
far a majority of pesticides and heavy metals have been
published by World Health Organization (WHQ), Food and
Agriculture Organization of the United Nations CFAO) and
other countries or organizations, which can be used as
references. Normally ADI is derived from the no observed
adverse effect level ( NOAEL) from long-term toxicity
testing on sensitive animals. ADI is equal to NOAEL divided
by suitable safety factor, which ( usually is set to 10 X 10
(interspecies difference between human and animals is 10
folds and individual difference between human is 10 folds). In
addition to interspecies and intraspecies differences, the
selection of safety factor should take in to consideration other
factors including toxicity level and exposure routes of
harmful residues, then the safety factor should be increased
or decreased properly. Compared with general toxic
contamination, the safety factor of harmful residues with
genotoxicity and carcinogenicity can be extended to the level
of 1000 to 10 000 and the establishment of the limit should be
stricter far these residues.
Reference <lose ( RfD) is also a common term used in risk
assessment, which is an estimated value of human exposure
risk and shares similar meaning to ADJ.
Under a specified testing condition, NOAEL is the maximal
<lose of test sample when no toxic effect can be observed by
current techniques or detection index. NOAEL is an
important parameter determined by in vivo toxicological test
and plays an important role in the establishment of safety
limit of chemical substances. For the same chemical
substance, the values of NOAEL may be different when
different species of animals, exposure routes, duration of
exposure and detection index are used. Therefore, testing
conditions should be specified when presenting this
toxicological parameter. Along with the progress of testing
method and the discovery of more sensitive detection index,
NOAEL will be updated constantly.
2. Calculation formula of theoretical value of maximal limit
The establishment of a harmful residue limit is based on
analysis and evaluation on toxicological data of harmful
residue combined with its exposure and human daily dietary
intake. Toxicity of harmful residues should be considered as the
primary factor in limit control. Theoretical values of maximal
limit of a harmful residue are calculated mainly according to in
mw toxicological data, the administration dosage of traditional
Chinese medicine and human daily dietary intake. It is a
theoretical value of harmful residue calculated by the following
eguation. During the establishment of the maximal limit of a
harmful residue, other affecting factors should be included to
ensure comprehensive evaluation on the final limit standard
( 1) Pesticide residue
When establishing the limit standard of pesticides residues,
the theoretical value of maximal limit of residue may be
calculated by the following equation.
L=AW/lOOM
Where: L is the theoretical value of maximal limit (rng/kg);
A is acceptable daily intake ( mg/kg) ;
w is the human average body weight (kg), generally
60 kg;
M is the maximal <lose of the materia medica or
prepared slices of traditional Chinese medicine
that can be taken daily per person (kg).
The safety factor is set to 100, suggesting that pesticides
taken daily from the Chinese material medica and its products
is not more than 1 % of total daily exposure <lose including
food and drinking water.
(2) Heavy metals and harmful elements
When establishing the limit standard of heavy metals and
harmful elements, the theoretical value of maximal limit of
the residue may be calculated by the following equation.
L=AW/lOM
Where: L is the theoretical value of maximal limit (rng/kg);
A is acceptable daily intake ( mg/kg) ;
W is the hurnan average body weight (kg), generally
60 kg;
M is the maximal <lose of the material medica or
prepared slices of traditional Chinese medicines
that can be taken daily per person (kg).
The safety factor is set to 10, suggesting the daily heavy
metals taken from the Chinese material medica and its
products is not more than 10 % of total daily exposure <lose
including food and drinking water.
Due to rhe long half-life and trace daily intake or weekly
intake during long-term exposure, sometimes WHO and
F AO use alternative provisional tolerable weekly intake
(PTWI) ( units: µg/kg b. w. ) or provisional tolerable
monthly intake CPTMD ( units: µg/kg b. w. ) rather than
establishing ADJ. ADI can be calculated from PTWI or
PTMI. ADI=PTWI/7 /1000; ADI= PTMl/30/1000.
(3) Aflatoxins
Dueto strong toxicity of aflatoxins, it is now recommended
not to establish its safety tolerance <lose and non-toxic effect
<lose, therefore calculation formula of theoretical value of
maximal limit is not available. The level of aflatoxins should
be kept as low as possible. Depending on specific products
and contamination conditions, the establishment of the limit
standard of aflatoxins should refer to corresponding limit
standard in foreign pharmacopeia or guidelines from related
countries and international organizations and its limit should
be kept as low as possible in arder to reduce safety risk. It is
generally required to determine the limit standard of both
aflatoxin Bi and the total amount of aflatoxin B1 , aflatoxin
Bz , aflatoxin G1 and aflatoxin Gz.
3. Factors affecting of the establishment of limit
During the establishment of limit of harmful residue, m
 9303 Guidelines for Determination of Pigments
addition to theoretical maximal limit calculated by the
formula above, other affecting factors should be included to
ensure comprehensive evaluation on establishing its limit
standard. The maximal residual limit of pesticides and heavy
metals that have been determined by all countries and
intemational organizations should be adjusted regularly according
to the changes of affecting factors. Affecting factors are
including, but not limited to, the fallowing aspects.
Influence of toxicity leve) The risk to human posed by the
residue is increased with its toxicity. Therefare far the
calculation of theoretical value of maximal limit, the value of
safety factor should be increased far the residue with stronger
toxicity and tighter control on the limit should be also placed for
it. Harmful residues with genotoxicity and carcinogenicity are
more harmful than general toxic contaminations, therefore in
theory it is generally recommended not to establish ADI far these
substances. Risk always exists no matter how low the amount of
harmful substance is taken. However in practice it is impossible
to eliminate these substances completely in many cases. Hence,
it is recommended to keep the limitas low as possible. The value
of limit represents the minimum amount of residue to guarantee
the production and supply of raw material, rather than the safe
<lose of the residue.
Influence of expnsure leve) From the perspective of toxicology,
the risk is positively correlated with the exposure <lose, duration
of exposure and the frequency of exposure of harmful residues,
therefore the maximal residual limit of harmful residues should
be controlled more strictly. Far material medica, prepared slices
aml preparations o[ traditional Chinese medicine, large variations
are shown on their administration doses and daily exposure
doses. Additionally, the establishment of a limit is influenced by
routes of administration of traditional aúnese medicine and
corresp:mding patient groups. The standard linút of residues should
be controlled strictly far drugs enterirlg systemic circulation directly,
used far serious diseases or long-term administration, and used far
children, pregnant women or the elders, etc.
Influence of residual leve) To ensure safety, the harmful
residue limit should be determined scientifically on the basis
of the specific conditions of residues.
Influence of production procedure The quality of traditional
Chinese medicine is influenced by many factors, including
agricultura! production, processing method of Chinese
material medica, manufacture process, storage, etc, which
may introduce or eliminate sorne harmful residues.
4. General procedure of establishment of maximal limit
e1) Determination of acceptable daily intake
To determine the limit of a specific harmful residue, the
information of animal long-term toxicity assessment or
human epidemiology data must be first obtained. NOAEL of
the harmful residue is determined from the data of animal
long-term toxicity testing then the ADI calculated by
NOAEL. If the ADI of this residue has been determined in
other countries or by sorne international organizations, those
values can be directly used as reference.
(2) Calculation of theoretical value of maximal limit
With determination ADI, the theoretical value of maximal
limit could be calculated by the formula recommended above
for pesticide residues or heavy metals and harmful elements.
(3) Revision of theoretical value of maximal limit
To determine the limit standard of a harmful residue, in
addition to use of its theoretical value as a reference,
toxicological properties of the harmful residue and its toxicity
should be taken into consideration adequately, as well as the
administration routes and administration <lose and the
duration of treatment, the possible contact between residues
and raw material of traditional Chinese medicine, the
contamination level of traditional Chinese medicine, the
subsequent processing method of traditional Chinese
medicine, current detecting techniques. The revision of
theoretical value should base on comprehensive analysis and
risk evaluation.
9303 Guidelines for
Determination of Pigments
Pigments could be generally divided into natural and artificial
synthetic pigments. The use of pigment in drugs should be in
accordance with relevant regulations of the state department of
pharmaceutical administration. Pigments should not be used in
traditional chinese medicine (crude drugs and decoction pieces,
here in after ) . This guideline provides guidance on the
determination of pigments in drugs, including traditional Chinese
medicine, chemical drugs and excipients.
General Principie
Different matrixes are present in excipients, chemical drugs
and traditional Chinese medicine. Therefore, the effect of the
matrix should be taken into consideration when determining
pigments, and specific methods towards different matrixes
should be used correspondingly. Far traditional chinese
medicine, the pigment with similar colour to that of the drug
should be screened and its content should be accurately
determined qualitatively and quantitatively.
General Content
l. Qualitative Analysis
Common methods include: thin-layer chromatography, high
performance liquid chromatography, high performance liquid
chromatography-mass spectrometry, etc.
(1) Thin-layer chromatography
Preparation of reference solution
Based on physical and chemical properties of the pigments,
proper solvents with suitable concentrations should be
selected and prepared to maintain good solubility and stability
of the pigments. When multiple pigments are mixed in the
reference solution, reasonable grouping of these pigments is
necessary to avoid decomposition or interactions.
Far lipophilic pigments, such as Sudan l , Sudan Il , Sudan
III , Sudan N, Sudan Red G, Disperse Red 9, and 808
Scarlet, acetonitrile can be selected as the solvent. Far
hydrophilic pigments, such as Malachite Green, New l\.1agenta,
Auramine O, Rhodamine B, Orange I, Acid Black I, Brilliant
Blue G, Brilliant Blue, Green S, Tartrazine, Sunset Yellow,
Brilliant Yellow, OrangeI[, Orange G, Erythrosine B, Acid Red
73, Amaranth, Acid Red 18, Allura Red, and Eosin, water can be
used as the solvent.
Preparation of test solution
Considering the matrixes of test samples and the
physicochemical characteristics of pigments, the solvent used
in reference solution can be used for test solution. A fast,
simple and efficient pretreatment technique can be used for
extraction. Purification can be carried out to avoid the
interference of the matrix when necessary.
Far the determination of basic hydrophilic pigments such as
Malachite Green, New l\.1agenta, Auramine O and Rhodamine B,
O. 1% farmic acid methanol solution can be used for extraction.
Far acidic hydrophilic pigments such as OrangeI[, Erythrosine B,
 9303 Guidelines far Determination of Pigments

Acid Red 73 and Amaranth, O. 1 % formic acid methanol solution
can be used. After ultrasonic treatment far 30 minutes, the
supernatant is taken as the test solution.
Thin-layer plates and mobile phase
Appropriate common or high performance thin-layer plates should
be chosen according to the amount of pigments to be determined
and their chromatographic results after developing. Ea.sed on the
R and resolution of adjacent spots after developing, proper
developing solvents can be selected far the pigment.
For instance, the upper solution of cyclohexylamine-ethyl
acetate-ammonia ( 80 : 20 : 1) can be selected as the mobile
phase far lipophilic pigments like Sudan I ; the upper
solution of ethyl acetate-ethanol-water-ammonia ( 6 : 4 : 2 :
O. 5) can be selected as the mobile phase for hydrophilic
pigments such as Malachite Green and Orange II .
Non-instrunmental method
Most pigments can be detected under the sunlight.
Ultraviolet light can be used if necessary.
Announcements
When the content of the pigment is low or the matrix is complex,
false positive and false negative results should be excluded during
qualitative analysis of the thin layer chromatography.
(2) High performance liquid chromatography (HPLC)
Chromatographic conditions and system suitability test
Proper mobile and stationary phases should be chosen
according to the physicochemical properties of the pigments,
and a gradient elution suitable far multiple pigments. In
general, octadecylsilane chemically bonded silica is used as
the stationary phase, and the column with small particle size
or long column length can be employed when necessary.
For example, when determining lipophilic pigments such as
Sudan I , use methanol as mobile phase A and O. 1 % formic
acid solution as mobile phase B with gradient elution: 0-12
min, A: 80% - 100%; 12-20 min, A: 100%. When
determining basic hydrophilic pigments such as Malachite
Green, gradient elution: 0-27 min, A: 40%-72%; 27-27. 1
min, A: 72% - 95%; 27. 1-32minutes, A: 95%; When
determining acidic hydrophilic pigments such as Orange II ,
use methanol as mobile phase A and 20 mmol/L ammonium
acetate as mobile phase B with gradient elution: 0-7 min, A:
5%-45%; 7-17 min, A: 45%-50%; 17-20 min, A: 50%
-65%; 20-30 min, A: 65%-90%; 30-32 min, A: 95%.
The detection wavelength should be determined based on
maximum absorption wavelength of the pigment m
ultraviolet-visible region. The detection wavelength of
yellow, orange, red and bl u e-green pigment can be set to 400
nm, 440 nm, 520 nm, 610 nm, respectively.
To improve reliability of qualitative results, ultraviolet and
visible spectrometry of the reference standard and the test
sample at different wavelengths can be compared to see
whether they are identical.
Malachite Green, O. 1% formic acid methanol solution can be
used far extraction and the polyamide filler is used far
purification. The eluent collected from methanol-0. 1 %
formic acid solution (3 : 2) can be used as the test solution.
When determining acidic hydrophilic pigments such as
Orange II, Erythrosine B, Acid Red 73 and Amaranth,
methanol-0. 1% formic acid methanol solution (3 : 2) can be
used far extraction and the polyamide filler is used for
purification. The eluent collected from methanol-ammonia-
water (7 : 2 : 1) is used as the test solution after adjusting
pH to weak acidity.
Result determination
A specific pigment is determined to be poslt1ve in a test
solution when the same peak appears in ultraviolet-visible
spectrometry of ref erence solution with same retention time.
Announcements
For complex matrixes, it is recommended to wash the
column by high ratio of organic phase after each injection of
test samples, and pre-column or guard column can be used
with chromatographic column to prolong its life.
If there are false positive results or interference is observed in
the chromatogram, other suitable methods can be used far
determina tion.
False negative results should be taken into consideration even
though the pigment is not detected. Limits of detection
should be taken into consideration and more sensitive
methods can be employed when necessary.
(3) High peñonnance liquid chmmtography-~ spectronrtry
Chromatographic conditions and system suitability test
The mobile phase and stationary phase should be selected
according to the physicochemical properties of the pigments,
and gradient elution can be used far multiple pigments.
Proper separations should also be achieved far isomers of
pigments. In generai, octadecyisiiane chemically bonded
silica is used as the stationary phase.
The selection of mobile phases should consider the acquisition
mode of mass spectrometry to improve the efficiency of
ionization. For example, in the positive ionization mode,
acetonitrile-0. 1 % formic acid solution ( ammonium aceta te
20 mmol/L) can be used as the mobile phase. In the negative
ionization mode, acetonitrile-2 mmol/L ammonium acetate
can be employed.
The acquisition mode and the mass spectrometry parameters
should be optimized far each pigment to achieve optimum
performance. When triple quadrupole mass spectrometry is
employed, multiple ion pairs should be selected far each
pigment and the collision energy (CE) should be optimized as
well (Table 1, Table 2).
Table 1 Ion pairs for detection and recommended
collision energy (CE) of selected pigments
(positive ionization mode)

Preparation of reference solution Pigment

Number Parention Production CE
Considering solubility and stability of the pigment, an appropriate Name
solvent with a proper concentration should be prepared.
249. 1 93.0 33
Corresponding item ( 1 ) Thin-layer Chromatography in this 1 Sudan I
249. 1 156. 1 21
guideline can be taken as a reference.
277. 1 121. 1 25
Preparation of test solution 2 Sudan II 277. 1 106. 1 55
Appropriate solvents and fast Simple and efficient pretreatment 353. 1 197. 1 27
technique should be used for extraction. Further purification is 3 Sudan III
353. 1 128. 1 51
normally needed to reduce the contarnination of chromatographic
381. 1 224. 1 31
column and avoid the effect of complex matrixes. In purification, 4 Sudan N
381. 1 225. 1 25
proper purification fillers and elution programs should be used to
avoid the loss of test pigments. 279. 1 123. 1 23
5 Sudan Red G
When determining basic hydrophilic pigments such as 279. 1 108. 1 47
 9303 Guidelines for Determination of Pigments

continued chromatography in this guideline can be taken as a reference.

The solution should be diluted to desirable concentration to
Pigment
Number Parention Production CE prepare the reference solution.
Name
Preparation of test solution
238.2 223. 1 33 The corresponding item under high performance liquid
6 Disperse Red 9
238.2 165. 1 38 chromatography in this guideline can be taken as a reference.
The solution should be diluted to desirable concentration to
368.2 275. 1 25
7 808 Scarlet prepare the test solution.
368.2 219. 1 47
Result determination
329 3 313.2 53 A specific pigment is determined to be pos1t1ve in a test
8 Malachite Green ·
329.3 208.0 46 solution when the peak with the same retention time appears
in spectrum of reference solution and the miz values of ion
330.0 223.3 46
9 New Magenta pairs are identical. In addition, the relative deviation between
330.0 300.3 52
the abundance of qualitative ion pairs in test and reference
267.9 147. 1 40 solutions should not exceed the limits: if the relative ratio>
10 Auramine O
50 %, allowable deviation should be within 20 %; if relative
267.9 252.2 44
ratio is 20 %-50 % , allowable deviation should be within
443.2 399.2 49 25%; if relative ratio 1s 10%-20%, allowable deviation
11 Rhodamine B
443.2 355.2 69 should be within 30%; if relative ratio is ::::;;;10%, allowable
deviation should be within 50%.

Table 2 Ion pairs for detection and recommended Notes

collision energy (CE) of selected When liquid chromatography-mass spectrometry is used as a
pigments (negative ionization mode) qualitative method, different mass spectrometry detectors
can be employed when the accuracy and reliability of the
Pigment
Number Parent ion Product ion CE results is ensured.
Name
The concentration of the pigment in the test solution should
327.1 170.8 -30 be considered, cross-over contamination or residual in the
1 Orange I
327. 1 247.0 -30 system should be avoided. Blank samples should be
1 t::A
.1.V~•
f\
V -24 employed for quality control of the analytical process.
2 Acid Blackl Limits of detection should be taken into consideration when
285.4 218.6 -18
the pigment is not detected.
830 4 170.0 -94
3 Brilliant Blue G ·
830.4 644. 1 -64 2. Quantitative Analysis
-45 Common methods far quantitative analysis include: high
373.3 169.9
4 Brilliant Blue performance liquid chromatography, high performance liquid
373.3 333.2 -24
chromatography--mass spectrometry, etc. After the qualitative
553.2 510.9 -40 results are confirmed by liquid chromatography--mass
5 Green S
553.2 496. 1 -51 spectrometry, HPLC can be used far quantitative analysis. The
232.8 211. o -10 establishment of a quantitative method should be in accordance
6 Tartrazine
232.8 197.8 -20 with Guidelines far Validation of Analytical Method Adopted in
203. 1 170.9 -19 Pharmaceutical Quality Specification (9101 ).
7 Sunset Y ellow
203. 1 206.9 -20 (1) High performance liquid chromatography (HPLC)
289 236.0 -20 Chromatographic conditions and system suitability test
8 Brilliant Y ellow ·O The corresponding ítem under high performance liquid
289.0 249.0 -22
chromatography for qualitative analysis in this guideline can
326. 7 170.8 -35 be taken as a reference.
9 Orangell
326. 7 155. 7 -46
Calculation method
203.0 150.4 -12 The content of the pigment can be calculated by one-point
10 Orange G
203.0 189. 1 -8 external standard method, standard curve method, interna!
834.8 536.8 -52 standard method, etc.
11 Erythrosine B
834.8 662.8 -52
Preparation of reference solution
255.2 150.5 -17 The reference solution should be diluted to proper
12 Acid Red 73
-11
255.2 240.8 concentration that is similar to the concentration of the
268.0 228. 1 -21 pigment to be measured in test solution. Also different
13 Amaranth concentration levels can be prepared for the standard curve of
268.0 220.8 -26
reference solution.
268.0 301. 7 -18
14 Acid Red 18 Preparation of test solution
268.0 206.0 -18
To ensure complete extraction of the pigments, a suitable
225.0 207.0 -22
15 Allura Red method with multiple extractions can be used. The loss of
225.0 199.8 -23
pigment should be avoided during purification. When
647.0 520.8 -38 sampling traditional chinese medicine, representativeness and
16 Eosin
647.0 522. 7 -40 uniformity should be considered. The sample should be
properly ground, however over-grinding should be avoided to
Preparation of reference solution reduce the interference from the matrixes.
The corresponding item under high performance liquid
 9305 Guidelines for Determination of Mycotoxins in Traditional Chinese Medicine
(2) High perfonnance liquid chromatography-~ spectrometry
Chromatographic conditions and system suitability test
The corresponding ítem under high performance liquid
chromatography for qualitative analysis in this guideline can
be taken as a reference.
Calculation method, Preparation of reference solution,
Preparation of test solution
The corresponding items under high performance liquid
chromatography for qualitative analysis in this guideline can
be taken as a reference.
Notes
lf there is matrix effect, the standard curve by blank matrix
solutions (i. e. , the solution prepared by identical method of
test solution without the test pigment) can be used to
eliminate the matrix effect.
Since mass spectrometry responses of sorne pigments, such
as Brilliant Blue G, Tartrazine, Sunset Yellow, Amaranth
and Acid Red 18, are not high enough, the range of the
standard curve can be narrowed according to the instrumental
response.
9304 Guidelines for Determination of
Aluminium (Al) , Chromium ( Cr) ,
lron (Fe) and Barium ( Ba) in Traditional
Chinese Medicine
The metal elements, such as aluminium, chromium, ferrum
and barium, can be introduced during planting, production or
processing of traditional chinese medicine. Residual metals
do not provide any therapeutic benefit and should be
evaluated and restricted on the foundation of safety and
quality based criteria. The guidelines are used for determination
of alu..111irüu..rn., chrorrüu..rn., Iron and bariu..rn. in traditional chinese
medicine.
General Principie
This guideline is suitable for determination of aluminium,
chromium, lron and barium in traditional chinese medicine
except mineral medicine and preparations contammg
minerals. It can be used in combination with the guidelines
for determination of lead ( Pb) , cadmium ( Cd) , arsemc
(As), mercury (Hg) and copper (Cu) (2321 ).
Basic Methods
Selection of methods:
inductively coupled plasma mass spectrometry ( ICP-MS)
( 0412) is first recommended for simultaneous determination
of multiple elements. Other methods with comparable
sensitivity as ICP-MS can be adopted.
Selection of instrument parameters
The instrument parameters should be properly set up depending
on characteristics of ICP-MS. The application of interference
equation calibration or collision reaction cell technique is
recommended to elirninate interference from different types of
rnass spectrometry. General reference conditions of instrument
are radio-frequency (RF) power 1250-1550 W, sampling depth
6. 0-10. O mm, carrier gas flow rate O. 65-1. 20 L/rnin, carrier
gas complementary air flow rate 0-0. 55 L/rnin, sample uptake
flow rate O. 1 ml/rnin, integration time O. 3-3 s, and triplicate
measurements.
Selection of analytical method
In arder to reduce the influence of work conditions on results
and improve the accuracy of quantitative analysis, it is
recommended to use the interna! standard calibrated curve
method.
Selection of target isotope
For elements to be measured and those for intemal standard,
isotopes with least interference and highest abundance should
be selected as target isotopes. Multiple isotopes could be
selected to valida te and compare the results. Generally, the
target isotopes of aluminium (Al) , chromium ( Cr) , iron
(Fe) and barium (Ba) should be 27 Al, 53 Cr, 57 Fe and 137 Ba,
and intemal standard isotopes are 7 Li, 45 Se, 45 Se and 115 In.
Preparation of the standard solution
Under specified instrumental conditions, at least five
different concentrations of standard solution (including blank
solution) ranged from O to 200. O /lg should be tested. The
preparation of standard and test solution should use the same
medium and same acidity should be kept for them. The
concentrations of standard solutions should be adjusted based
on the concentration of element to be measured. A regression
curve is obtained by plotting the responses of target isotope
peak versus corresponding concentrations. Unless otherwise
specified, the correlation coefficient should be not less than
o. 99.
Preparation of test solution
The matrix of traditional chinese medicine samples is
complex and sample pre-treatment technologies have direct
effects on the precision and accuracy of results. Currently
pre-treatment technologies for element analysis are dry
ashing, wet digestion and microwave digestion. In this
guideline, microwave digestion is the pre-treatment technique
recommended to reduce the loss of elements. Microwave
digestion procedure should be properly set up depending on
specific models of microwave digestion systems. Appropriate
digestive reagents should be used to achieve complete
digestion of organic matrix in traditional chinese medicine.
Nitric acid or the mixture of nitric acid and hydrochloric acid
is generally recommended.
After digested solution is cool, the digester should be open
with care and the solution is transferred to a 50-mL teflon
volumetric flask. The cap and wall of digester should be
rinsed with water several times and the washing solution
should be collected in the same flask. Finally, the solution is
diluted to a final volume of 50 mL with water and mixed
properly. The blank solution was obtained with the same
preparation method as test solution.
Notes
Laboratory environment, labware and reagents should be
carefully monitored to prevent contamination of elements to
be measured. Laboratory environment should be kept clean.
Labware should be soaked in high concentrations of acid
solution and high purity reagent should be used.
If the concentration of the element in test solution is too high,
proper dilution should be carried out to ensure accuracy of results.
lt is recommended to start the concentration from low to high and
contaminations of instruments should be avoided.
In arder to ensure accuracy and reliability, traceable standard
substance or recovery rate tests should be applied for each
test to validate the results.
9305 Guidelines for Determination of
Mycotoxins in Traditional Chinese Medicine
Mycotoxins are secondary metabolites produced by fungi.
Several traditional Chinese medicines are prone to contarnination
of mycotoxins produced during the process of cultivation and
 9305 Guidelines for Determination of Mycotoxins in Traditional Chinese Medicine
storage, such as aflatoxin, ochratoxin, deoxynivalenol,
zearalenone and patulin. It is necessary to engage tight control
on relevant mycotoxins because of its serious implications for
human health.
This guideline is used to determine mycotoxins in traditional
Chinese medicine.
General Principie
l. Major kinds of mycotoxins in traditional Chinese medicine
Mycotoxins are produced by various kinds of fungí. Fungí of the
genus Aspergillus, Fusariwn and Penicilliwn are the
predominant mycotoxins producers. Aflatoxin and ochratoxin A
are relevant to the genus Aspergillus. Zearalenone, T-2 toxin,
deoxynivalenol and fumonisin are relevant to the genus Fusariwn.
Patulin and citrinin are relevant to the genus Penicilliwn.
2. Traditional Chinese medicine to be monitored
Depending on toxicological mechanisms of mycotoxins, different
kinds of traditional Chinese medicine are prone to contaminations
from various mycotoxins. Thus traditional chinese medicine that is
susceptible to the mycotoxins contamination should be selected to
develop detection methods for these mycotoxins. Aflatoxin should
be monitored in grain-type and seed-type traditional Chinese
medicine, as well as those rich in oily components._ Ochratoxin,
deoxynivalenol and zearalenone should be monitored in traditional
chinese medicine with analogous matrix to grains, such as Semen
Sojae Preparatwn, Semen Gneis and Semen Lblichoris Albwn.
Patulin should be monitored in acidic fruit-type traditional chinese
medicine, such as FructusLycii, FructusMone and Semen
Zizi phiSpirwsae. Mycotoxin contamination should be monitored in
formula with susceptible crude drugs and Chinese patent medicine
directly made from crude drugs.
3. Detection methods
Classical analytical methods for mycotoxins include thin layer
chromatography, enzyme-linked immunosorbent assays,
gold immunochromatographic assay, high performance liquid
chromatography and liquid chromatography/mass spectrometry.
Among them, thin layer chromatography is suitable for
preliminary screening of samples; enzyme-linked immunosorbent
assays is suitable for centralized detection of large numbers of
samples; gold immunochromatographic assay is appropriate for
instant detection of single sample ora small amount of samples;
high performance liquid chromatography possesses characteristics
of specificity, reproducibility, and low false positives. Liquid
chromatography coupled with mass spectrometry is capable to
determine a variety of mycotoxins simultaneously, which
could solve the problems of incomplete separation and false
positive of chromatography. This guideline provides guidance
to determine mycotoxins using high performance liquid
chromatography and high performance liquid chromatography/
mass spectrometry.
The methods established to determine mycotoxins contamination
should comply with requirements of Guidelines for Validation of
Analytical Method Adopted m Pharmaceutical Quality
Specification ( 9101).
4. &tablishment of rnycotoxins lirnits
The establishment of Mycotoxin limits can incorporate
toxicological data, related limit standards from domestic and
foreign industry, and clinical usage and dosage of traditional
chinese medicine.
General Content
1. High performance liquid chrornatography
This method is normally used to determine the content of
single mycotoxin or multiple mycotoxins of the same type.
( 1) Chrornatographic conditions and system suitability test
Appropriate stationary phase and mobile phase should be
selected according to physicochemical properties of
mycotoxins to be detected. In order to achieve separation
efficiency, gradient elution can be used to detect multi-
mycotoxins. Chromatographic column is usually packed with
octadecylsilane bonded silica gel. To enhance separation, a
chromatographic column with smaller particle size and longer
length can be used. Methanol aqueous solution and
acetonitrile aqueous solution with different ratios is
commonly used as mobile phase. Mycotoxins are detected
with a fluorescence detector or a diode array detector.
For example, in detection of ochratoxin A, acetonitrile-2%
glacial acetic acid ( 49 : 51) is used as mobile phase and a
fluorescence detector ( excitation wavelength: 333 nm,
emission wavelength: 4 77 nm) is used as the detector. In
detection of deoxynivalenol, methanol-water ( 20 : 80) is
used as mobile phase and a UV detector ( detection
wavelength: 220 nm) is used as the detector. In detection of
zearalenone, acetonitrile-water ( 50 : 50) is used as mobile
phase and a fluorescence detector ( excitation wavelength:
232 nm, emission wavelength: 460 nm) is used as the
detector.
(2) Preparation of reference solution
According to physicochemical properties of mycotoxins to be
detected, appropriate solvent should be selected to prepare
stock solution in a suitable concentration, which should
ensure solubility and stability. Methanol and acetonitrile are
the commonly used solvents. Reference stock solution
usuaiiy couid be kept in refrigerator for 1-3 month.
Before use, the stock solution is diluted to working solution
in a suitable concentration by an appropriate solvent. In
general, the organic solvent used for dilution has similar
proportion with test solution.
For example, methanol could be used in dilution of
ochratoxin A and zearalenone. 50 % methanol could be used
in dilution of deoxynivalenol. 2% acetonitrile ( the pH value
is adjusted to 2 by acetic acid) could be used in the dilution of
patulin.
(3) Preparation of test solution
Rapid, simple and efficient methods are adopted for the
extraction of mycotoxins, such as shaking, ultrasonic and
high-speed homogenization. Generally, further purification
and enrichment are needed for the extract. Immunoaffinity
columns and HLB solid phase extraction columns are
recommended in adsorption and elution of mycotoxins.
Immunoaffinity columns are effective to purify mycotoxins
due to specific adsorption of mycotoxins. HLB solid phase
extraction columns are useful in adsorption and purification
of multi-mycotoxins through gradient elution that is achieved
by adjusting polarity of eluent solvents. Despite easy
operations, other mycotoxin clean-up columns can be used
for adsorption of impurities, such as lipids and proteins,
with relatively poor purification performance.
80 % methanol aqueous solution, water and 90 % acetonitrile
are adopted in extraction of ochratoxin A, deoxynivalenol and
zearalenone. Then the extract is purified by corresponding
immunoaffinity columns. For patulin, acetonitrile aqueous
solution is used for its extraction and mycotoxin clean-up
columns are used for its purification.
( 4) Results Determination
Standard curve method is usually used in determination of
mycotoxins. Mycotoxins are determined to be present in the
test solution when the chromatographic peak with same
retention time appears in spectrumof reference solution ( if a
 9305 Guidelines for Determination of Mycotoxins in Traditional Chinese Medicine 439
diode array detector is used, the ultraviolet/visible gradient: 0-5 min, A: 55%-90%; 5-7 min, A: 90%; 7-
(UV/Vis) absorption spectrometry of test and reference 7. 1 min, A: 90%-55%; 7. 1-10 min, A: 55%. Ion pairs
solution should match well ) . And the mycotoxin at the mass-to-charge ratio ( m/z) of 317. 1-174. 9 are
concentration is calculated by the standard curve. selected as quantitative ion pairs and ion pairs at the mass-to-
Recovery and sensitivity tests usually should be performed charge ratio ( m/ z) of 317. 1 - 130. 8 . are selected as
when determining the test samples. If possible, certified qualitative ion pairs in the negative mode of electrospray
standard materials should be used for quality control. Due to ionisation (ESD. For patulin, HPLC gradient elution method
complex matrix in traditional Chinese medicine, extraction is used with acetonitrile as mobile phase A and water as
efficiencies may be varied and it is necessary to include mobile phase B according to the following gradient: 0-4 min,
isotope internal standard to correct the matrix effect when A: 3%; 4-4. 2 min, A: 3%-40%; 4. 2-9 min, A: 40%; 9-
necessary. 9. 5 min, A: 40%-3%; 9. 5-15 min, A: 3%. Ion pairs at
the mass-to-charge ratio (m/z) of 153. 1-80. 9 are selected
(5) Notes
as quantitative ion pairs and ion pairs at the mass-to-charge
It is recommended to wash the column by high ratio of
ratio (m/z) of 153.1-109. O are selected as qualitative ion
organic phase after each injection of test samples, and pre-
pairs in the negative mode of electrospray ionisation (ESD.
column or guard column can be used with chromatographic
To determine the contents of aflatoxin G2, G1, B.i, B1,
column to prolong its life. ochratoxin A, deoxynivalenol, zearalenone, fumonisin B1 ,
Noteworthy, high performance liquid chromatography coupled B.i and T-2 toxin at the same time, HPLC gradient elution
with mass spectrometry should be employed as confirmation method is used with acetonitrile methanol ( 1: 1) as mobile
when false positives or chromatogram interferences occur in this phase A and O. 01% formic acid as mobile phase B according
method. to the following gradient: 0-2 min, A: 5%; 2-2. 01 min, A:
False negative results should be taken into consideration even 5%-40%; 2. 01-5 min, A: 40%-50%; 5-7 min, A: 50%
though the mycotoxin is not detected. Limits of detection -55%; 7-10 min, A: 55%-90%; 10-10. 01 min, A: 90%
should be taken into consideration and more sensitive -5%; 10. 01-13 min, A: 5%. Triple-quadrupole mass
methods can be employed when necessary. spectrometer are employed as the detector with electrospray
2. High performance liquid chromatography coupled with ~ ionisation CESD, positive mode is used for aflatoxin G2, G1,
spectrometry B.i, B1 , fumonisin B1 , B.i and T-2 toxin and negative mode is
High performance liquid chromatography coupled with mass used for ochratoxin A, deoxynivalenol and zearalenone. Mass
spectrometry should be employed when the matrix interference spectrum parameters of selected mycotoxins are present in
occurs or the contents of mycotoxins are too low to be accurately the table below.
determined by high performance liquid chromatography. The Reference values of collision energy (CE) of monitoring ion
method of high performance liquid chromatography coupled with pairs of mycotoxin reference substances
mass spectrometry is also suitable for determining different types
of mycotoxins simultaneously, which is capable of high Mycotoxin Parent Daughter
Number CE (V)
throughput screening and content determination of mycotoxins. Na me ro ns ion
( 1) Chromatographic conditions and system suitability test
331. 1 313. 1 33
To improve the ionization efficiency, acquisition mode of 1 Aflatoxin G2
331. 1 245. 1 40
mycotoxins in the mass spectrometry should be taken into
consideration when deciding composition of mobile phase. 329. 1 243. 1 35
2 Aflatoxin G1,
According to the instrument, mass spectrometry detection 329. 1 311. 1 30
parameters are optimized to achieve the optima! data acquisition
315. 1 259. 1 35
mode. Using triple-quadrupole mass spectrometer, multiple 3 Aflatoxin B.i
315. 1 287. 1 40
characteristic ion pairs should be selected and the collision energy
(CE) should be optimized as well. 313. 1 241. o 50
4 Aflatoxin B1
For example, to determine the content of ochratoxin A, 313. 1 285. 1 40
HPLC gradient elution method is used with methanol as
722.3 352.4 49
mobile phase A and O. 01 % formic acid as mobile phase B 5 Fumonisin B1
722. 3 334.4 53
according to the following gradient: 0-5 min, A: 55% -
90%; 5-7 min, A: 90%; 7-7. 1 min, A: 90%-55%; 7. 1- 706.4 336. 1 49
6 Fumonisin B.i
10 min, A: 55%. Ion pairs at the mass-to-charge ratio 706.4 318.4 52
(m/z) of 402. 1-358. 1 are selected as quantitative ion pairs
489.2 245.3 36
and ion pairs at the mass-to-charge ratio ( m/ z) of 402. 1- 7 T-2 toxin
489.2 387.2 29
211. 1 are selected as qualitative ion pairs in the negative
mode of electrospray ionisation ( ESI). To determine the 402. 1 358. 1 -28
8 Ochratoxin A
content of deoxynivalenol, HPLC gradient elution method is 402. 1 211. o -38
used with methanol as mobile phase A and water as mobile
295. 1 265. 1 -15
phase B according to the following gradient: 0-5 min, A: 9 Deoxynivalenol
295. 1 138.0 -25
10%-40%; 5-6 min, A: 40%-90%; 6-7 min, A: 90%;
7-7.1 min, A: 90%-10%; 7.J-10 min, A: 10%. lonpairs 317.2 175. 1 -32
at the mass-to-charge ratio ( m/ z) of 295. 1 - 265. 1 are 10 Zearalenone
317.2 131. 2 -38
selected as quantitative ion pairs and ion pairs at the mass-to-
charge ratio ( m/z) of 295. 1 - 138. O are selected as (2) Preparation of reference solution
qualitative ion pairs in the negative mode of electrospray Referring to the corresponding item about preparation of
ionisation ( ESD. For zearalenone, HPLC gradient elution reference solution by HPLC method in this guideline, a
method is used with methanol as mobile phase A and O. 01% series of working solutions with different concentrations are
formic acid as mobile phase B according to the following prepared. 50 % acetonitrile aqueous solution is recommended
 9501 Guidelines far the Quality Control of Positron Emission Tomographic Radiopharmaceutical Preparation
to determine aflatoxin G2, Gl, B2, Bl, ochratoxinA,
deoxynivalenol, zearalenone, fumonisin Bl, B2 and T-2
toxin simultaneously.
According to the samples, blank matrix solution ( using
preparation method for test solution, the solution is prepared
by test sample without mycotoxin to be determined) is used
to prepare reference solution.
(3) Preparation of test solutions
Referring to the corresponding ítem by HPLC method in this
guideline, the test solution is prepared after dilution to
appropriate concentration. When determining aflatoxin G2,
Gl, B2, Bl, ochratoxinA, deoxynivalenol, zearalenone,
fumonisin Bl, B2 and T-2 toxin simultaneously, the samples
are extracted with 70 % methanol solution by ultrasonic
extraction then purified by HLB column.
( 4) Intemal reference
Due to complex matrix of traditional Chinese medicine,
extraction efficiencies may be varied between different sample
matrixes. The matrix effect should be corrected by isotope
ínternal standard when necessary. Meanwhile, the
concentration of isotope internal standard should be
ínvestigated.
(5) Detennination
The mycotoxins are determíned to be present in samples
when the peak at the same retention time appears in
spectrum of reference solution and the m/ z values of ion pairs
are identical. In addition, the relative deviation between the
abundance of qualitative ion pairs in test and reference
solutions should not exceed the limits: if the relative ratio>
50 % , allowable deviation should be within 20 % ; if relative
ratio is 20 %-50 % , allowable deviation should be within
25%; if relative ratio is 10%-20%, allowable deviation
should be within 30%; if relative ratio is ::::;:;10%, allowable
deviation should be within 50%.
Standard curve method is normally applied in determining the
contents of multiple mycotoxins.
(6) Notes
When liquid chromatography-mass spectrometry is used as a
qualitative analysis method, different mass spectrometry detectors
can be employed when the accuracy and reliability of the results is
ensured.
&ritable concentrations of the mycotoxins in the test solution should be
used to avoid cross--over contarnination or residues in the systern.
Elank solvent, blank rnatrix and standard substances can be employed
for quality control of the analytical process.
9501 Guidelines for the Quality Control of
Positron Emission Tomographic
Radiopharmaceutical Preparation
Positron emission tomographic ( PET) radiopharmaceutical
preparations refer to radiopharmaceuticals contammg
positron-emitting radionuclides. They are generally prepared
at the time of clinical use by medical institutions or
radiopharmaceutical manufacturers. There are two main
sources of positron-em1ttmg radionuclides: cyclotron
produced and generator produced. The guideline is only
suitable for the quality control of the PET
radiopharmaceutical preparations produced through
cyclotron. The quality control of PET radiopharmaceutical
preparations produced through generator should refer to
Guidelines for the Quality Control of Technetium [ 99 m Te]
Radiopharmaceutical Preparations <9502).
In order to assure the safety and efficacy of PET
radiopharmaceutical preparation administration, quality
control tests must be carried out accordíng to the
corresponding national standard for each batch of PET
radiopharmaceutical preparation produced. If there is no
national standard for a PET radiopharmaceutical preparation,
the acceptance criteria should be established by the
manufacturer, but such an acceptance críteria must be
validated by the National lnstitute for the Food and Drug
Control before íts practical use.
The characteristics of preparatíon and quality control of PET
radiopharmaceutical preparations:
(1) Because of a very short physical half-life of the positron-
emitting radíonuclide, the preparation of PET
radiopharmaceutícal preparations must be rapid. Automated
radiochemical synthesis devices are usually used to protect
personnel involved from excessive radiation exposure.
( 2) PET radiopharmaceutical preparations are generally
prepared locally, typically at sites located within, or
affiliated with, medical institutions at the time of use. With
a relatively longer half-life of fluorine [ 18 F], fluorine [ 18 F]
PET radiopharmaceuticals can be prepared and supplied by
other qualified medical institutions or radiopharmaceutical
manufacturers nearby.
( 3) With the exception of fluorine [ 18 F] PET radiopharmaceutical
preparations, each batch of PET radiopharmaceutical
preparation generally leads to only a single or several <lose
administration.
( 4) Quality control tests must be rapid and practica!.
Being subject to the characteristics of preparation and qualíty
control of PET radiopharmaceutical preparations described
above, it is impossible to carry out all quality control tests
for every batch of PET radiopharmaceutical preparation
before admínistration. According to the Drug Administration
Law and the Regulations for the Administration of
Radiopharmaceutical Preparation, this guidelines is drawn up
to guarantee the quality of PET radiopharmaceutical
preparations, assure the safety and the efficacy of PET
radiopharmaceutical preparation administration, and
standardize the quality control for PET radiopharmaceutical
prepara tions.
l. For PEf radiopharmaceutical preparatiom Iabelled with a
nuclide having a physical half-lüe longer than 20. O minutes (such
as fluorine [ 18 F] PET radiopharmaceutical preparations)
The following quality control tests should be performed on
each batch prior to release or administration:
( 1) Description.
(2) pH value.
(3) Radiochemical purity.
( 4) Radioactivity or radioactive concentration
Other quality control tests can be performed retrospectively
after release or administration ( retrospective quality control
tests).
2. For PET radiophannaceutical preparations labelled with a
nuclide of a physical half-life equal to or shorter than 20. O
minutes (such as carbon [ 11 C], nitrogen [ 13 N] and oxygen
15
[ O] PET radiopharmaceutical preparations)
A batch is defined as all related preparations ( i. e. , sub-
batches ) of the PET radiopharmaceutical preparation
prepared under the same condition during a given day. The
following quality control tests should be performed on an
initial sub-batch from such PET radiopharmaceutical
preparations prior to preparation of subsequent sub-batches:
(1) Description.
(2) pH value.
(3) Radiochemical purity.
( 4) Radioactivity or radioactive concentration.
 9502 Guidelines for the Quality Control of Technetium [ 99 mTc] Radiopharmaceutical Preparations
Other quality control tests can be performed retrospectively.
3. Retrospective quality control tests
lf all the quality control test results of the PET radiopharma-
ceutical preparations prepared under the same compounding
procedure (at least 6 consecutive batches) consistently conform
to the established minimal acceptance criteria, the retrospective
quality control tests can be performed at a defined periodic
interval, but all quality control tests must be performed on a
batch of PET radiopharmaceutical preparation ata minimum
of once a month.
4. Quality control tests results
Accept or reject the individual batch of the PET
pharmaceutical based on the conformity of quality control
test results with established minimal acceptance criteria. If a
batch of PET radiopharmaceutical preparation <loes not meet
the established minimal acceptance criteria, the distribution or
administration must be stopped immediately, and the subsequent
preparation must be stopped too. The preparation of PET radio-
pharmaceutical preparations for human use can begin again only
after having investigated what resulted in the unacceptable quality
control test results, having solved the problem, and three
consecutive validation studies that meet minimum acceptance
criteria having been performed. If the unacceptable PET
radiopharmaceutical preparation had been used clinically, it is
necessary to observe the related patients carefully and follow
them periodically, and take appropriate measures if necessary.
The manufacturer or medical institution that prepared the
unacceptable PET radiopharmaceutical preparation should submit
a report immediately to the local drug adrninistration bureau and
health bureau in case serious adverse reaction has occurred.
5. Quality as.surance.
( 1 ) A PET radiopharmaceutical manufacturer or medical
institution should have adequate facilities, instruments and
equipment adaptable to the scale of PET radiopharmaceutical
preparation and quality control. The instruments and
equipment should be calibrated periodically to ensure the
correct performance. Written operation and calibration
procedures for the instruments and equipment should be
established. Each use, calibration and maintenance should be
documented.
( 2 ) A PET radiopharmaceutical manufacturer or medical
institution should have an adequate number of corresponding
technical personnel with the appropriate education and
training. Each person performing an activity ora function in
the quality control of PET radiopharmaceutical preparations
should be trained in quality control of radiopharmaceutical
preparations by the National Institute for the Food and Drug
Control or other institutions authorized by State Food and
Drug Administration.
(3) The standard operation procedures for preparation and
quality control of PET radiopharmaceutical preparations
should be established and performed strictly. The
preparation and quality control of PET radiopharmaceutical
preparations should be documented, and the documents
should be maintained at least one year.
(4) Drug substances, materials and reagents used for the
preparation and quality control of PET radiopharmaceutical
preparations must be in compliance with established written
specifications. Regulation procedures for the order, storage,
testing and use of these materials should be established.
( 5) To ensure the stability of the preparation process of PET
radiopharmaceutical preparations, the computer and related
automated equipment for preparation of PET radiopharmaceutical
preparations must be controlled appropriately to ensure that
changes in compounding software are instituted only by
authorized personnel, that such changes are documented and
verified.
( 6) The standard operation procedures and computer
software program that control the preparation process should
be validated periodically, at a minimum of once a year.
Whenever there is a change in the compounding procedures
or computer software program that has the potential to alter the
identity, quality, or purity of the PET radiopharmaceutical
preparation, revalidation must be performed. Verification studies
on a rninimum of three consecutive batches, which show that the
final product meets the acceptance criteria, are to be
performed prior to the approval, for human use, of the
revised standard operation procedures or computer software
program for a given PET radiopharmaceutical preparation.
(7) The cleaning room or the aseptic workstation should be
validated periodically to ensure proper performance.
(8) Each PET radiopharmaceutical preparation prepared by
a medical institution can not be used clinically until the
quality control testing of three consecutive batches of PET
radiopharmaceutical preparations have been performed by the
National Institute for Food and Drug Control or other
institutions authorized by China Food and Drug
Administration, and the quality control tests results meet the
acceptance criteria.
9502 Guidelines for the Quality Control of
Technetium [ 99mTc]
Radiopharmaceutical Preparations
Technetium [ 99 mTe] radiopharmaceutical preparations refer
to radiopharmaceuticals containing technetium [ 99 m Te J
radionuclide for clinical diagnostic use, which include Sodium
Pertechnetate [ 99 m Te] Injection eluted from 99 Mo- 99 m Te
generators and nthPr radiopharmaceutical preparations
prepared with Sodium Pertechnetate [ 99 mTe] Injection and
corresponding freeze-dried cold kits.
Technetium [ 99 m Te J radiopharmaceutical preparations are
generally prepared through reconstituting ready-to-use freeze-
dried cold kits with Sodium Pertechnetate [ 99 m Te J Injection
under aseptic condition by instant-labeling radiopharmaceutical
manufacturers ( radiopharmacy) or medical institutions that
have the third or higher class license for Radiopharmaceutical
Use. As the preparation of Technetium [ 99 mTc] radiopharmaceu-
tical preparations involves complicated chemical reactions
between technetium- 99 m and non-radioactive compound,
quality control tests for final products must be performed in
addition that Sodium Pertechnetate [ 99 mTc] Injection and the
ready-to-use freeze-dried cold kits must meet corresponding
acceptance criteria. Because the half-life of Technetium [ 99 m
Te] is only 6. 02 hour, Technetium [ 99 mTc] radiopharma-
ceutical preparations must be administered within dozens of
minutes to several hours after preparation. It is impossible
for a Technetium [ 99 mTc] radiopharmaceutical preparation to
have completed all quality control tests befare its release or
administration. Technetium [ 99 m Te] radiopharmaceutical
preparations can be released and administered while quality
control tests are being performed pursuant to article 16 of the
Regulations for the Administration of Radiopharmaceutical
Preparation. As one batch of Technetium [ 99 m Te J
radiopharmaceutical preparation generally leads to only a
single or severa! <lose administration, the volume of which is
usually only several milliliters, it is impracticable to perform
all quality control tests for each batch of Technetium [ 99 mTc]
radiopharmaceutical preparation.
Considering the above mentioned specialty of Technetium
 442 9502 Guidelines for the Quality Control of Technetium [ 99 mTc] Radiopharmaceutical Preparations
[
99
mTe J radiopharmaceutical preparations, according to the
Drug Administration Law and the Regulations far the
Administration of Radiopharmaceutical Preparation, this
guideline is drawn up to guarantee the quality of Technetium
99
[ mTc] radiopharmaceutical preparations and to assure the
safety and the efficacy of Technetium [ 99 mTc] radiopharmac-
eutical preparation administration. The guideline is suitable
for quality control of Technetium [ 99 mTe J radiopharmaceu- tical
preparations prepared by instant-labeling radiopharmaceutical
manufacturers ( radiopharmacy) or medical institutions with the
third or higher class license for radiopharmaceutical use.
l. The obligatory quality control tests performed prior to
release or administration
(1) Description Visually examined through lead glass, physical
appearance of a Technetium [ 99 m Te J radiopharmaceutical
preparation should not significantly differ from the description
described in the monograph. For a clear colourless solution
Technetium [ 99 mTe J radiopharmaceutical preparation, if particles
appearance of turbidity or a change in colour is observed, it must
not be released or administered.
(2) pH value The pH value of a technetium [ 99 m Te J
radiopharmaceutical preparation can be determined with a
calibrated precise pH test paper, and must be in the defined
range as described in the monograph.
(3) Radiochemical purity Radiochemical purity should be
determined according to the method described in the corresponding
official monographs or other acceptance criteria. In case it takes a
long time to carry out the methods described in the official
monographs, instant-labeling radiopharmaceutical manufacturers
(radiophartnacy) or medical institutions can establish their ow11
instant validated methods. The validation must be performed on at
least three batches of technetium [ 99 m Te ] radiopharmaceutical
preparations at three different periods ( at time immediately after
preparation, middle, and at the point of expiration time). The
requirement described in the method established by instant-labeling
radiophannaceutical manufacturers ( radiopharmacy) or medical
institutions can not be lower than that described in the official
monograph. Far routine use, the method should be revalidated
periodically, at a minimum of once a year, to ensure its
applicability.
( 4) Radioactivity Radioactivity should be measured according to
the Test of Radiophannaceutical Preparations <1401 ).
( 5) Particle size The Technetium [ 99 mTe J radiopharmaceutical
preparation particle size distribution described in the monograph
should be tested according to Test far particle size distribution
under the Test of Radiopharmaceutical Preparations ( 1401 )
prior to release or administration and should comply with the
requirements.
2. The quality control tests that can be performed while or
after release or administration
( 1) Bacterial endotoxin Carry out the bacterial endotoxins
test on a Technetium [ 99 mTc] radiopharmaceutical
preparation as described under the corresponding monograph
or the Test for Bacterial Endotoxin ( 1143 ) , the result
should meet the minimal acceptance criteria.
(2) Sterility Complies with the Test far Sterility (1101 >.
(3) Biodistribution Far a Technetium [ 99 mTc] radiopharmaceu-
tical preparation of which, test for biodistribution is described in
the monograph, the test should be performed as described under
corresponding monograph. The experiment animals should meet
relevant regulations.
( 4) If the result of any quality control tests described above
does not meet the minimal acceptance criteria, the
production, distribution and administration of related batch
of preparation should be stopped immediately, and the
reasons should be investigated. If the unacceptable
Technetium [ 99 mTc] radiopharmaceutical preparation has
been used clinically, it is necessary to monitor the related
patients carefully and follow up them periodically, and
appropriate measures need to be taken, if necessary. The
manufacturer or medical institution that prepared the
unacceptable technetium [ 99 m Te] radiopharmaceutical
preparation should submit a report immediately to the local
drug administration bureau and health bureau.
(5) If all results of the quality control tests described above
for Technetium [ 99 m Te] radiopharmaceutical preparations
prepared under the same compounding procedure (at least 6
consecutive batches) consistently conform to the established
minimal acceptance criteria, the test for bacterial endotoxins,
sterility and biodistribution can be perfarmed at a defined
periodic interval, which depends on the quality control
testing results.
3. Corresponding quality assurance
( 1) A T echnetium [ 99 mTe] radiopharmaceutical manufacturer
(radiopharmacy) or medical institution should have adequate
facilities, instruments and equipment adaptable to the scale of
Technetium [ 99 mTc] radiopharmaceutical preparation and quality
control. The instruments and equipment should be calibrated
periodically to ensure the proper performance. Written operation
and calibration procedures for the instruments and equipment
should be established. Each use, calibration and maintenance
should be documented.
( 2) A Technetium [ 99 mTe] radiopharmaceutical manufacturer
(radiopharmacy) or medical institution should have an adequate
number of corresponding technical personnel with the appropriate
education and training. Each person performing an activity
or a function in the quality control of Technetium [ 99 mTe]
radiopharmaceutical preparations should be trained in quality
control of radiopharmaceuticals by the National Institutes for
Food and Drug Control or other institutions authorized by the
State Food and Drug Administration.
(3) The standard operation procedures far preparation and
quality control of Technetium [ 99 mTe] radiopharmaceutical
preparations should be established and performed strictly.
The preparation and quality control of Technetium [ 99 mTe]
radiopharmaceutical preparations should be documented, and
the documents should be maintained far at least one year.
( 4 ) Drug substances, materials and reagents used far the
preparation and quality control of Technetium [ 99 m Te J
radiopharmaceutical preparations must be in compliance with
established written specifications. Regulation procedures far the
arder, storage, testing and use of these materials should be
established.
(5) The cleaning room or the aseptic workstation should be
validated periodically to ensure its proper performance.
( 6) F or a Technetium [ 99mTe] radiopharmaceutical manufacturer
(radiopharmacy) , all quality control tests for the first batch of
&xlium Pertechnetate [ 99m Te J Injection eluted from newly
purchased 99 Mo- 99mTc generators should be carried out before
subsequent batches of Sodium Pertechnetate [ 99 m Te]
Injection eluted from the 99 Mo- 99 mTe generators is used to
prepare other technetium [ 99 mTc] radiopharmaceutical
preparations (measurement of Molybdenum [ 99 Mo] content
is only required for radionuclidic purity test). If all bacteria!
endotoxin and sterility tests of Sodium Pertechnetate [ 99 mTc]
Injections eluted from many batches of 99 Mo- 99 mTc generator
( at least 6 consecutive batches produced by the same
manufacturer) meet the minimal acceptance criteria, tests
for bacteria! endotoxin and sterility of Sodium Pertechnetate
[
99
mTc] Injections eluted from 99 Mo- 99mTc generators produced
by' the manufacturer can be performed periodically. But overalt
quality control tests for &xlium Pertechnetate [ 99mTe ] Injection
 9601 Guidelines for Research and Evaluation on Functionality-related Characteristics of Pharmaceutical Excipients
must be carried out at least once a month. Complete quality
control tests for the first batch of Technetium [ 99m Te J
radiopharmaceutical preparation should be carried out when
the batch of ready-to-use freeze-dried cold kit changes.
9601 Guidelines for Research and
Evaluation on Functionality-related
Characteristics of Pharmaceutical Excipients
Pharmaceutical excipients are additives of the pharmaceutical
preparations, playing an important role in drug safety, efficacy,
stability, controllability and compliance. The pharmaceutical
auxiliary rnaterials can be divided into several categories according
to use (see "Pharmaceutical excipients" <0251) ). To ensure
function and quality of the pharmaceutical excipients, setting
appropriate functionality-related characteristics ( FRCs) in the
monographs is necessary. The setting of the FRCs is for a
particular purpose and the sarne material can be classified into
different specifications according to the FRCs. The user can
select suitable excipients to ensure quality of the preparations.
This guidance will introduce the research methods and
establishment ways for the FRCs according to the use. This
guidance for FRCs mainly serves the pharmaceutical
excipients of twelve categories whose FRCs can not be
determined by conventioal chemical methods, including the
diluents; this chapter will not desribe those excipients whose
FRCs can be assessed by chemical ways, such as sorne pure
chemicals, including pH adjusting agents, osmotic pressure
adjusting agents, preservatives, chelating agents, complexing
agents, · flavoring agents, colouring agents, plasticizers,
antioxidants and propellants.
( 1 ) Diluents
Diluents, also known as fillers, are ingredients used to
increase the volume or weight of the preparations, often
including starch, sucrose, lactase, pregelatinized starch,
microcrystalline cellulose, inorganic salts, and sugar
alcohols. Diluents usually take a large proportion in the
preparations, ensuring the size and reducing dose deviation of
the main active ingredient, and improving the
compressibility, and the compactivity. Selection of the type
and amount of the diluents usually depends on their physical
and chemical properties, especially the FRCS.
The diluents can affect the compactability and preparing properties
(such as the powder fluidity, the moldability of wet or dry
granules, the content uniformity, disintegration, dissolution, the
appearance of the tablets, the tablet hardness and friability,
physical and chemical stability, etc. ). Sorne diluents, like
rnicrocrystalline cellulose, are often used as dry binders for their
high chardness during final tableting.
FRCs of the diluents include: (a) particle size and particle
size distribution <0982 ) ; ( 2) the particle shape <0982 ) ;
(3) bulk density/tap density/true density; ( 4) specific
surface area; (5) crystallinity <0981); (6) moisture content
<0832); (7) fluidity; (8) solubility; (9) compressibility and
compactivity; (10) hygroscopicity <9103), etc.
( l[ ) Adhesives
The adhesives are sticky powders or solutions which can
promote the powder with none or lava viscosity to aggregate
into particles. The adhesives dissolve or disperse in the
granulation solution, and sorne are dry powders, and as the
volatile component of the granulation solution they can help the
particles meet the standard, ( such as particle size and its
distribution, form, content uniformity, etc. ). Wet granulation
can facilitate the further process of the particles by improving one
or more characteristics of the particles, such as fluidity,
443
workability, strength, resistance to separation, dust content,
appearance, solubility, drug release or compressibility and
compactivity.
Adhesives can be divided into: (a) natural polymer
materials; ( 2) synthetic polymer; ( 3) sugars. Chemical
properties of the polymer including the structure, the nature
of the monomers and the polymerization sequence, functional
groups, the degree of polymerization and substitution and the
degree of cross-linking will aff ect the interaction of the
granulation process. Since the same type of polymer may be
different in origin or synthetic methods, their properties may
vary significantly. Common adhesives include starch,
cellulose derivatives, povidone, gelatin, and sorne other
adhesives. The adhesives produce wet particles ( aggregates)
by changing binding force inside the granules. They may also
alter the interfacial properties, viscosity or other properties.
During the drying process, they may produce a salid bridge,
which gives certain mechanical strength to dry granules.
The FRCs of the adhesives include: Q)surface tension; @particle
size, particle size distribution ( 0982); ®solubility; @viscosity
< 0633); @bulk density and tap density; @specific surface area,
etc.
( ][ ) Disintegrants
Disintegrants are additives to promote the rapid
disintegration into small units and to quicken the dissolution
velocity of the prescription. When contacting water, gastric
or intestinal fluid, they swell or form a gel by absorbing the
liquid, causing the destruction and disintegration of the
structure of the preparation and promoting the dissolution of
the drug. Disintegrants have four main disintegrating
mechanisms: swelling, deformation, capillary action and
repulsion. In the tablet formula, the functions of the
disintegrants are preferable with two or more. Disintegrants
performance depends on severa! factors, including chemical
properties, particle s1ze distribution and particle
morphology, and other important factor such as hardness
and porosity.
Disintegrants can be natural, synthetic or chernically modified
natural polyrners. Common disintegrants include: dry starch,
sodium carboxymethyl starch, low-substituted hydroxypropyl
cellulose, cross-linked sodium carboxyrnethyl cellulose, cross-
linked povidone, and effervescent tablets. Disintegrants can
be classified as nonionic and anionic. Nonionic polymers are
mainly polysaccharides, such as starch, cellulose,
amylopectin or crosslinked povidone. Anionic polymers are
mainly chemically modified products of cellulose. Ionic
polymers should be considered for their chemical properties.
Gastrointestinal pH alterations or forming complexes with
APis will affect the disintegration performance.
The FRCS of the disintegration include: CI> particle size and
distribution <0982); @Water absorption rate; ®swelling or
swelling index; @ powder fluidity; ® moisture content;
@effervescent amount.
( N ) Lubricants
The lubricants are used to reduce friction between particles
and between particles and solid preparation manufacturing
equipment, such as the friction between time tableting metal
contact of punch and die.
Lubricants can be classified into interface lubricants, fluid
film lubricants and liquid lubricants. Interface lubricants are
amphiphilic long chain fatty acid salts (e. g. , magnesium
stearate), or fatty acid esters ( e. g. sodium stearyl
fumara te), which can attach to solid surfaces ( particles and
machine parts) and reduce the friction between the particles
or between the particles and metals. The surface adhesion is
affected by the substrate surface and the interface lubricants
 9601 Guidelines for Research and Evaluation on Functionality-related Characteristics of Pharmaceutical Excipients
are preferably small plate crystal for the best attaching
effect. The fluid film lubricants are solid fat ( such as
hydrogenated vegetable oil, type I ) , glycerates ( glycerin
dialkyl esters and glyceryl distearate) , or fatty acids ( such as
stearic acid) , which will melt and form a film around the punch
of the tableting under pressure, that will help to reduce friction.
After removal of the pressure, the fluid film lubricant
resolidifies. The liquid lubricant can be absorbed by the particles
befare compression, while the liquid material released from the
particles under pressure can be used to reduce friction between
the metals in the manufacturing apparatus.
Common lubricants include: magnesium stearate, aerosil,
tale, hydrogenated vegetable oils, polyethylene glycols,
sodium lauryl sulfate.
Key FRCs of lubricants include: ( 1) particle size and
distribution <0982) ; ( 2) specific surface area; ( 3) moisture
conternt <0831 and 0832); ( 4) polycrystalline type ( 0981
and 0451 ) ; ( 5) purity (e. g. stearate: palmitate ratio);
(6) melting point or melting range; (7) powder fluidity.
( V) Glidants and anti-caking agents
Glidants and anti-caking agents can increase powder flow rate
and reduce powder aggregation. They are usually inorganic
material powders and are insoluble in water but are not
hydrophobic. Sorne of the materials are complex hydrates.
Glidants and anti-caking agents commonly include fine
inorganic powders of magnesium stearate and aerosil.
Glidants can be adsorbed on the surface of larger particles to
reduce the adhesion and cohesion between the particles and to
improve the fluidity. Furthermore, glidants may be
dispersed between the large particles to reduce friction. Anti-
caking agents are capable of absorbing moisture to prevent
formation of particle bridge in the agglomeration.
FRCs of glidants and anti-caking agents include: (1) particle
size and distribution <0982); (2) surface area; (3) powder
fluidity; ( 4) absorption rate, etc.
( VI) Vacant capsules
As a carrier for liquid or drug powder, capsules can ensure
accuracy of the dose and convenience of the delivery. The
vacant capsules should be compatible with the contents.
Vacant capsules typically include two portions ( i. e. the
capsule cap and the capsule body) , both cylindrical, the
longer one is the capsule body and the other one is the
capsule cap. The cap and body closely integrate to make the
closed capsule. Soft capsule shell is made up of a monolithic,
stitched or not stitched along the shaft.
According to the difference of raw materials, the vacant capsules
fall part into gelatin capsules and others. The former is prepared
with gel of origin of pig, beef, or fish, while other capsules is
prepared with non-animal sourced cellulose and polysaccharides.
Vacant capsules also contain other additives such as elasticizers,
colourants, opacifier and preservatives. Preservatives should be
used sparingly, if it is used. The type and amount of the
additives in vacant capsules should meet the national official
standards and requirements.
Vacant capsules can be filled with solid, serni-solid or liquid
preparations. The traditional vacant capsules should be quickly
dissolved or disintegrated in biological fluids such as
gastrointestinal fluids at 37°C. And enteric material or releasing
controllable polymers can be used in the vacant capsules, in arder
to adjust the release of the contents in capsules.
The moisture content varíes with the types of the vacant
capsules. Moisture has a significant influence on the capsule
brittleness. Balancing moisture plays a key role in the
stability of the dosage form because water molecules can
migrate between the capsule content and the capsule shell.
Air permeability is an important character, as HPMC vacant
capsules have open structure and have greater permeability than
others. Gelatin vacant capsules stored at a high temperature and
humidity (e. g. 40ºC/75% RH) can farm cross-linking but the
HPMC vacant capsules do not The disintegration time of the
acetaldehyde content in the powder may increase on account of
the gelatin cross-linking. Gelatin vacant capsules can be
disintegrated in 15 minutes under conditions of O. 5 %
hydrochloric acid at 36ºC-38ºC, not less than 30ºC . HPMC
vacant capsules can be disintegrated below 30ºC.
The FRCs of the vacant capsule include: ( 1) moisture
content < 0832 and 0831 ) ; ( 2 ) air permeability;
(3) disintegration < 0921 and 0931 ) ; ( 4 ) brittleness;
(5) hardness; (6) bloom strength; (7) tightness, etc.
( "\I ) Coating material
The coating can be used to cover the odor of drugs, improve the
appearance, protect the active ingredients, and adjust drug
release. Coating materials include natural, serni-synthetic and
synthetic materials. Coating materials can be powder or colloidal
dispersion system ( latex or pseudo-latex), which are usually
prepared as a solution or dispersion of aqueous or non-aqueous
phase. Waxes and lipids of molten state can be directly used far
coating without using any solvent.
Research on FRCs of the coating material should address:
(1) solubility, e. g. , enteric coating material is insoluble in
acidic media but soluble in neutral media; ( 2) film-forming
property; ( 3) viscosity; ( 4) substituents and degree of
substitution; ( 5) tensile strength; ( 6) air permeability;
(7) particle size, etc.
(W) Moisteniy agents and/ or solubilizer
Solubilizers comprise a variety of different chemical
structures and levels. Typical solubilizers are nonionizing
surfactants, which spontaneously form micellar structures in
water to solubilize. Solubilizing is related to interaction
between the insoluble drug and the self-assembly micelles.
Sorne other solubilizers help insoluble drug dissolved in the
polymer chain with the charge of polymer chain to increase
the solubility of the drug including hydrophobic interactions.
Solubilizers can be solid, liquid or wax. The chemical
structures of surfactants determine the physical properties.
Physical characteristics and functions of the solubilizing agents
depend on the surfactant properties and hydrophilic-lipophilic
balancevalue(HLB) (0713). e.g., sodiumlaurylsulfate(HLB
value of 40) is hydrophilic, soluble in water. Once it is dispersed
in water, it spontaneously forms rnicelles. The hydrophilic and
lipophilic characteristics can also be characterized by the critical
rnicelle concentration ( CMC).
FRCs of the moistening agents/solubilizers include:
(1) HLB value; (2) viscosity; (3) composition, see test
methods <0301 , 0601 , 0633, 0631 , 0713, 0661 , 0982
etc); (4) critical micelle concentration; (5) surface tension.
( 1X ) Suppository bases
Suppository bases are used for manufacturing rectal supposito-
ries and vaginal suppositories as matrix. Conventional
suppository bases comprise: oleaginous bases such as cocoa
butter, serni-synthetic esters of coconut oil, serni-synthetic or
wholly synthetic fatty acid glycerides; soluble matrix, such as
glycerinated gelatin, polyethylene glycol, poloxamer, etc.
The suppositories should be melted and release drug just
below the body temperature ( 37°C). The mechanism of
release is through corosion and distribution. High melting
point fat suppository base should be melted at body
temperature. Water-soluble base can be dissolved or
dispersed in an aqueous medium and the drug release
mechanism is by corosion and distribution.
The most important physical index of suppository base is the
 9621 Guidelines for General Requirements for Drug Packaging Materials and Containers
melting range, generally during 27-45ºC. However, a single
suppository base has a narrow range between 2-3ºC . The
impacts of other ingredients of the final product should be
considered in selection of the bases.
High-melting lipophilic suppository base is a mixture of
semi-synthetic long chain fatty acid triglycerides, including
monoglycerides, diglycerides, and perhaps ethoxylated fatty
acids. The base can be classified into diff erent grades
according to melting range, hydroxyl value, acid value,
iodine value, coagulating point and saponification value.
Hydrophilic suppository base is usually a mixture of
hydrophilic semi-solid materials, being solid at room
temperature, and releasing drugs by melting, erosion and
dissolution when used in human body. Compared to the high
melting suppository base, hydrophilic suppository base has
more hydroxyl and hydrophilic groups. Polyethylene glycol is
a hydrophilic matrix with a suitable melting behavior.
For FRCs of the suppository bases, please refer to ( 0107,
0612, 0613, 0713, etc. )
( X ) Suspending aids and / or thickening agents
Suspending aids and/ or thickening agents are used for
stabilizing the dispersion system ( e. g. suspensions or
emulsions) by reducing the rate of movement of the particles
or salutes, or the fluidily of liquid preparations.
A variety of mechanisms are involved in stabilizing the
dispersion system or achieving the thickening effect. Firstly,
common fine clay or macromolecular chain bonding solvents
lead to increased viscosity or interrupt the laminar flow.
Secondly, excipient molecules or particles of the suspending
agents form the gel of three-dimensional structure. Thirdly,
minerals and macromolecules can be adsorbed on the surface
of the dispersed particles or droplets and generate three-
dimensional effect. The mechanisms ( viscosi ty increase, gel
formation, or three-dimensional stability) can be resulted
from the rheological properties of materials. Due to the large
molecular weight and big particle size, sorne suspending
agents exhibit the rheology of non-Newtonian fluid. Such
materials exhibit a certain thixotropy in their dispersions.
Suspending aids or thickening agents can be low molecular,
as well as large molecules or minerals. Low molecular
suspending aids or thickening agents contain glycerin and
syrup. Macromolecular suspending aids or thickening agents
include (a) hydrophilic carbohydrate polymer (acacia gum,
agar, alginic acid, carboxymethyl cellulose, carrageenan
gum, dextrin, gellan gum, guar gum, hydroxyethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methyl
cellulose, maltodextrin, methylcellulose, pectin, propylene
glycol alginate, sodium alginate, starch, tragacanth, and
xanthan gum); and ( b) hydrophilic non-carbohydrate
macromolecules, including gelatin, povidone, carbomer, poly
( ethylene oxide) and polyvinyl alcohol. Mineral suspending aids
or thickening agents include attapulgite, bentonite, magnesium
aluminum silicate, silicon dioxide etc. Aluminum monostearate,
classified by function as neither macromolecules nor minerals
suspending agents or thickeners, mainly consists of aluminum
monostearate and aluminum monopalmitate of different
component proportions.
The FRC of the suspending aids and thickening agents are
viscosities ( 0633).
( :xI ) Ointment base
Ointment is a viscous semi-salid external preparation applied
on different parts of the body surface. Ointment base, which
determines its physical properties, is the main component of
the ointment. Ointment bases may be used as a carrier for
topical pharmaceuticals as well as wetting agent or skin
protectant.
Ointment base is a stable mixture with a relatively high
viscous liquid which contains a suspended salid.
Ointment bases are divided into (a) oily base: water-
insoluble, anhydrous, water repellent, difficult to be
removed with water (e. g. , petrolatum) ; ( b) absorbent
ointment base: anhydrous, a ble to absorb a certain amount
of water, water-insoluble and difficult to be removed by
water ( such as lanolin); ( c) emulsion matrix: typically
water-in-oil or oil-in-water type, wherein the water can
absorb moisture and can not be dissolved in water (e. g. ,
cream); (d) water-soluble ointment base: anhydrous itself,
but can absorb water and can be dissolved in water, and can
be removed by water ( such as polyethylene glycoD.
The ointment base should be inert and chemically stable.
Viscosities and melting ranges are important FRCS of the
cream base ( 0633, 0613).
9621 Guidelines for General
Requirements for Drug Packaging
Materials and Containers
Drug packaging materials and drug containers (container
closure systems) refer to the containers, closures and tube
lines that are in direct contact with the drug and used to
contain, protect, and deliver the drug, including drug made
by pharmaceutical companies and formulation dosage
prepared by medical institutions. As part of the approved
drug product, the quality, safety, performance and
compatibility of the DPMC are important factors affecting the
quality of the drug product. DPMC consists of one or several
packaging components, which should ha ve desired safety,
compatibility, stability, function and convenience of usage.
It is essential for ensuring the quality, safety, effectiveness
and proper administration of the drug by meeting the needs
of packaging, storage, transportation and administration.
Drug container closure systems can be classified by packaging
materials, by physical shapes or purposes of use.
Drug container closure systems can be classified based on
packaging materials, such as plastic, metal, glass, ceramic,
rubber, paper and desiccant. It could also be a composite or
combination packaging consisting of two or more above-
mentioned materials, such as laminated film and polymer
aluminum composite foil. Commonly used plastic container
closure systems include low-density polyethylene bottles for
eye drops, high-density polyethylene bottles for tablets and
capsules, polypropylene bottles for infusion; commonly used
glass container closure systems include soda lime glass
bottles for infusion, borosilicate glass ampoules with low
boron oxide content, borosilicate glass tubular bottles for
injection with medium boron oxide content; commonly used
rubber container closure systems include chlorobutyl rubber
stoppers, synthetic polyisoprene pads, silicone rubber
gaskets far oral liquid pharmaceuticals; commonly used
metal container closure systems include aluminum foils for
drug packaging, steel boxes for menthocamphorate.
Drug container closure systems can be classified by purpose
of use and physical shapes as infusion bottles ( bags, films
and accessories), ampoules, pharmaceutical ( injection, oral
or topical farmulations) bottles ( pipes, covers) , rubber
stoppers far drug containers, pharmaceutical pre-filled syringes,
drop bottles far ophthalmic, nasal and otic drug products,
pharmaceutical sheets ( films ) , pharmaceutical fails,
pharmaceutical ointment tubes ( boxes) , pharmaceutical spray
(gas) aerosol pumps ( valves, tanks, tubes), pharmaceutical
desiccants, etc.
 446 9621 Guidelines for General Requirements for Drug Packaging Materials and Containers

The name of a drug container closure system should be in the product on container closure system including the
arder of purpose of use, packaging material, and physical investigation of integrity, functionality and quality of the
shape. The style of name should be concise. Exaggerated drug container closure system after the packaging of drug
language or modification should be avoided. Using of product. Far examples, delamination of glass container,
abbreviation of fareign language is not encouraged. Far stopper deformation, etc. 3 ). The quality changes of the
example, oral liquid pharmaceutical polypropylene bottle. packaged drug products ( drug stability) , including quality
The drug container closure systems should meet the changes in accelerated testing and long-term testing.
following requirements during manufacture and application. Table 1 Cl~ification of risk degree with the
Raw materials for drug container closure systems should be container cl<>Sure systems
subject to physical, chemical and biological safety
assessments, and should have a certain mechanical strength, Degree of concern Likelihood of container closure
chemical stability, biologically non-toxic to humans. The associated with systems-dosage form interaction
manufacturing condition for drug container closure system the route of
should be compatible with that of packed dosage. The administration High Medium Low
manufacturing environments and processes for drug container
closure systems should be rationally designed and in l. lnhalation l. Sterile
accordance with the required air cleanliness level. Far the Aerosols and Powders for
manufacture of container closure system that does not require Sprays 2. Injections
washing prior to use, the cleanliness requirement of product
prototyping and each subsequent process should be the same Highest 2. Injections and
as the cleanliness requirement of the drug manufacturing. lnhalation
Injectable
Depending on the manufacturing process and purpose of use, Powders
3. Suspensions,
container closure systems should meet the requirements of 3. Implants
Rinses
sterility or microbial limit test. The pyrogenicity, bacterial
endotoxin, or sterility requirements of container closure
systems far injection should be consistent with the demands l. Ophthalmic
of the drug. The sterility requirements of container closure Solutions
systems far ophthalmic drug products should be compatible and Suspensions
with the requirements of drug products. 2. Nasal
Only the drug container dosure systems that are approved by Aerosois and
the state and meet the manufacturing quality standard can be Sprays
used far the drugs made by pharmaceutical companies and
High
formulation dosages prepared by medical institutions. The 3. Transdermal
application scopes of drug container closure systems should Ointments,
be compatible with the routes of administration and types of Crearos,
formulation. The drug container closure systems should be Cataplasms,
quality-assured and should have stable quality within the drug Gels, Paste
shelf life. The drug container closure systems for multi- ointments,
dosage should ensure drug quality and stability during use. Film agents
The use of a drug container closure system that unable to
ensure the quality of drugs on the list has been banned by the
state., or has possible safety hazards, is firmly prohibited. Oral
l. Topical
The compatibility studies of a drug container closure system Powders, Tablets
Solutions and
with the drug product are the foundation to select drug Granules, Pills and Oral
Suspensions
container closure systems. Therefore, it is required to Capsules
conduct the compatibility studies of drug container closure
systems with the drug product in the selection of container
Low 2. Topical and
closure systems. ,
Lingual
Compatibility studies should include the degree of concern
Aerosols
regarding the route of administration and the likelihood of
packaging component-dosage farm interactions ( see Table 3. Suppositories
1). In general, compatibility studies should include the
4. Oral
following parts: 1). Studies investigating the effect of a
Solutions and
container closure system on the quality of drug product,
Suspensions
including the extraction of packaging components ( such as
inks, adhesives, additives, residual monomers, and small The standards for drug container closure systems are the
molecule compounds, decomposition products generated technical requirements to ensure the quality of the packed
during processing and use of the products), the migration of drug products. National Standards far drug container closure
packaging component and it' s extraction studies, the systems consist of standards far drug container closure
toxicological evaluation of component' s migration, the systems promulgated by the State Food and Drug Administration
likelihood of packaging component-dosage form interactions, ( pharmaceutical packaging YBB standards ) and product
the adsorption or absorption of the active drug substance or registration standards. The quality standards far drug container
functional excipients by container closure systems, the closure systems are divided into methodology standards and
migration of drug products into the container closure system, product standards. The quality standards should be based on
and the leaching of the fareign objects into the drug drug packaging production conditions and processes that
products, etc. 2). Studies investigating the effect of a drug
 9622 Guidelines far Pharmaceutical Glass Materials and Containers

approved by the competent authorities, as well as the raw
material grades and sources, etc. The development of testing
methods and selection of technical criteria should be in
accordance with the properties of materials, the structural
characteristics of the product, the packaging and clinical
requirements. If any of these factors is changed, the quality
standards should be re-enacted, and the applicability of drug
packaging quality standards should be verified in order to
ensure the controllability of drug packaging quality. The
development of a drug container closure system should meet
the criteria of drug safety, adaptability, stability,
functionality and convenience of use. Drug container closure
systems far different routes of administration have different
specifications and quality standards. The specifications far
drug packaging should be based on the actual situation of
formulated dosage. The quality control program should be in
accordance with the requirements of preparation and using
methods. The development of quality standards far drug
container closure systems should take in to account the safety
and compatibility of drug packaging, and the effect of
container closure system on the transportation, quality,
safety, and efficacy of drug product. Drug container closure
systems should abide by the YBB standards promulgated by
the state. If the development of the product registration
standards is needed, the technical requirements and project
scopes should not be lower than the YBB standard of similar
products. The standards of drug container closure systems
mainly includes the following three parts: 1 ) Physical
properties: physical properties focus on characteristics that
aff ect the performance of products, including physical
parameters, mechanical properties, and functional
indicators. For examples, puncture resistance and fragment
of rubber products, sealing and barrier properties of plastic
and composite film products. The testing of physical
properties and the subsequent evaluation shall be in
accordance with standard inspection rules and sampling
methods. 2) Chemical properties: study the criteria that
affect product performance, quality and purpose of use, such
as extractable and leachable testing, residual solvent. 3)
Biological properties: the inspection of the container closure
system should be based on the requirements of
pharmaceutical products. For examples, inspection of
container closure system far injection drugs includes
cytotoxicity, acute systemic toxicity, and hemolysis test;
inspection of container closure system far eye-drop bottles
should include abnormal toxicity and eye irritation test.
The packaging of a drug container closure system should be
labeled with scope of application, dimensions, storage
requirements, as well as the shelf life.
9622 Guidelines f or Phannaceutical
Glass Materials and Containers
Pharmaceutical Glass Materials and Containers CPGMC) is
an amorphous and transparent solid material, formed by
cooling clown of high temperature fusion, which is one of the
most chemically stable materials. Glass is widely used in
packaging of various pharmaceuticals because it possesses a
number of advantages, such as excellent hydrolytic resistance
and acid resistance, average alkali resistance, optimal
thermal stability, desired mechanical strength, smooth and
transparent, easy to clean and sterilize, superior barrier
property, and easy to seal.
PGMC can be classified by the chemical compositions and
properties, hydrolytic resistance, molding methods, etc.
According to the national drug packaging standards far glass
far pharmaceutical use ( YBB standards), glass far
pharmaceutical use is classified as four types, borosilicate
glass with high boron oxide content, borosilicate glass with
medium boron oxide content, borosilicate glass with low
boron oxide content, and soda-lime glass, based on their
chemical compositions, coefficients of linear thermal
expansion. and performance requirements of glass far
pharmaceutical use. The following is a summary of the
compositions and performance requirements of these four
types of glasses:

Glass types

Chemical compositions and properties Borosilicate glass Borosilicate glass Borosilicate glass
with high boron with medium boron with low boron Soda-lime glass
oxide content oxide content oxide content

B2Ü3 C%) ~12 ~8 ~5 ~5

Si02 * C%) ,.._,31 ,.._,75 ,.._,71 ,.._,70

Na20 + KzO * C%) ,.._,4 4-8 "'11. 5 12-16

Mgü+Caü+ Baü+ (Srü) * (%) / ,.._,5 "'5. 5 ,.._, 12

Alz03 * C%) 2-3 2-7 3-6 0-3. 5

Mean coefficient of linear thermal

3. 2-3. 4 3. 5-6. 1 6. 2-7. 5 7. 6-9. o
Expansion 1 : X 10- 6 K- 1 (20-300ºC)

Hydrolytic resistance of glass grains at 121 ºC 2 Grade 1 Grade 1 Grade 1 Grade 2

Grade HGBl or Grade HGB2 or

Hydrolytic resistance of glass grains at 121ºC 3 Grade HGBl Grade HGBl
HGB2 HGB3
 9622 Guidelines for Pharmaceutical Glass Materials and Containers

continued
Glass types

Chemical compositions and properties &rosilicate glass &rosilicate glass &rosilicate glass
with high boron with medium boron with low boron Soda-lime glass
oxide content oxide content oxide content

Hydrolytic resistance of the interior

Grade HCl Grade HCl Grade HCl or HCB Grade HC2 or HC3
surfaces of glass containers 4

Gravimetric method Grade 1 Grade 1 Grade 1 Grade 1 or 2

Acid resistance Atomic absorption

spectrophotometry 100 µg/dm 2 100 µg/dm 2 / /

Alkali resistance Grade 2 Grade 2 Grade 2 Grade 2

Note: The chemical compositions of different types of glasses are not stoichiometric but are expressed overa range of compositions. Therefore,
there is allowable variation within a glass type, and the chemical compositions of a same type of glasses may vary slightly among glass
producers.
Note 1: referring to "Standard test method for the determination of mean coefficient of linear thermal expansion."
Note 2: referring to "Hydrolytic resistance of glass grains at 121 ºC-Method of test and Classification. "
Note 3: referring to "Hydrolytic resistance of glass grains at 98ºC-Method of test and Classification. "
Note 4: referring to "Hydrolytic resistance of the interior surfaces of glass containers-Methods of test. "

Glass for pharmaceutical use is classified based on the
hydrolytic resistance of glass grains as Type I and Type fil.
Type I is borosiiicate giass, having a high hydroiytic
resistance. Type fil is the soda-lime type glass with a
moderate hydrolytic resistance. Neutralization treatment of
the inner surface of Type fil soda-lime glass containers will
raise their hydrolytic resistance from a moderate to a high
level, changing the classification of the glass to Type II.
Glass containers for pharmaceutical use can be divided into
molded and tubular glass containers according to the
manufacturing process. The main varieties of molded glass
containers are large volume bottles for injection, small
volume bottles for injection ( or Penicillin bottles), and
bottles for oral formulations. Tubular glass containers
include small volume ampoules for injection, tubing
ampoules for injection (or Penicillin bottles), pre-filled glass
syringes and needles, pen-type injectors with glass cylinder
Cor cartridges), liquid bottles for oral formulation, medicine
bottles, etc. Different ha ve molding production processes
different effects on the quality of glass containers. The
chemical resistance of the inner surface of heat processing
site in a tubular bottle is lower than that of unheated sites.
The quality of tubular bottles processed by the same tubing
formation process may be different.
Glass containers for pharmaceutical use should meet the
following basic requirements m the production and
application.
The chernical composition of glass containers for pharmaceutical
use should meet the performance requirements. The formula for
production of glass should be strictly controlled to ensure the
stability of the glass components and control the introduction of
toxic and hazardous substances. In the case of toxic and
hazardous substances used in the production, it should
comply with state regulations and should not affect the drug'
s safety.
To facilitate the inspection of visible foreign matters,
impurities, and deterioration in pharmaceutical liquids, glass
containers for pharmaceuticals use should be clean and
transparent. Common drugs should be packaged in colourless
glass containers. Light-sensitive drugs should be protected
by using amber glass containers rather than other colour
containers. Giass containers for pharmaceutical use should
have good thermal stability to ensure the suitability of high-
temperature sterilization or freeze-drying; glass containers
for pharmaceutical use should possess sufficient mechanical
strength to withstand higher pressure generated by autoclaving,
and to avoid darnage during production, transportation and
storage; glass containers for pharmaceutical use should have
desired clinical effects, such as the bending force to open
ampoule should meet the standard requirements; glass
containers for pharmaceutical use should have a certain
chemical stability to prevent the adverse effects on the drug
quality, such as the leaching and migration between drug and
glass container, the delamination of glass containers, pH
shifting of the drug solution.
Drug manufacturer should select an appropriate pharmaceutical
glass container based on the physical and chernical properties of a
drug and the results of compatibility studies. The selected
pharmaceutical glass container for biological products, slightly
alkaline and acidic and pH-sensitive injections should be
hydrolytic resistance grade I by glass grains at 121 ºC method and
hydrolytic resistance of the inner surface should meet with
grade HCl or other appropriate packaging materials.
Compatibility studies of the glass container with the drug
product should be focused on the migration of metal ions
from glass components to the drug solution, the leaching of
harmful substances from glass container should not exceed a
safe threshold, the overall leaching of ions should not affect
the quality of drug. For examples, the leaching of alkali ions
should not lead to pH shifting in the drug solution. The
investigation of effect of drug on glass surface should
examine the extent of erosion on the glass surface, glass
flakes and particles, and assess the degree of risk for
delamination and formation of sub-visible and visible glass
particles. Glass containers should be durable and resistant to
the delamination effect of the drug product, and the inner
surface of the glass container should not be damaged during
the drug storage.
 9901 Guideline for Preparation of Pharmaceutical Standard Substances of China
There are a number of factors affecting the durability of the
inner surfaces of glass containers including the chemical
composition of glass, the converting temperature and
converting speed of tubular containers, the treatments and
coatings for the inner surface of the glass containers
(ammonium sulfate treatment), storage temperature and
humidity, terminal sterilization processes, etc. In addition,
chemicals also adversely affect the durability of the inner
surface of the glass containers, such as active pharmaceutical
ingredients, formulation buffers (e. g. , acetate, citrate, and
phosphate buffers) , organic acid salts ( gluconate, mala te,
succinate, tartrate, etc. ) , alkali salts with high ionic
strength, complexing agent disodium edetate. Therefore,
the impact of all these factors on the durability of the inner
surface of the glass containers should be considered in
compatibility studies.
9901 Guideline for Preparation of
Pharmaceutical Standard
Substances of China
This guideline is used to standardize and guide the
preparation of pharmaceutical standard substances to ensure
the execution of pharmaceutical specifications of China.
Detennination of the types of standard substances
To satisfy the need of establishment and emendation of
pharmaceutical specifications of China, the types of standard
substances are determined.
The selection of raw material to prepare candidate standard
substances
Raw material should be selected under the guidance of
applicability, representativeness and availability.
The properties of raw material should meet the need for
usage.
The homogeneity, stability and corresponding characteristic
properties range should be suitable for the application of
standard substances.
Preparation of standard substances
According to physical and chemical properties of candidate
standard substances, suitable preparation methods and
processing procedures are selected to prevent modifications of
characteristic properties and contaminations.
For candidate standard substances that are lacking of uniformity,
uniform measurements and uniformity test should be performed
during the preparation.
For candidate standard substances with unstable characteristic
properties, the affecting factors of stability should be investigated
during preparation, necessary means should be placed to ensure
their stability, and proper storage conditions should be selected.
When the amount of candidate standard substances to be
prepared is large, subpackaging is acceptable for the purpose
of storage.
The supplier of candidate standard substances should equip
with good experimental conditions and capacity, in addition,
the information below should be provided:
experimental method, measured values, the number of
experimental repeats, necessary materials about spectrum
and chromatogram;
storage condition that is suitable for the requirements of
stability ( temperature, humidity, and lightening time,
etc.);
hygroscopicity test and description of candidate standard
substances;
results of accelerated stability test;
identification of related substances and its content, detailed
information about the relative response factors of the
principal component in pharmaceutical specifications of
China;
updated safety information regarding to human health.
Standardization of Candidate Pharmaceutical Standard
Substances of China
Candidate standard substances should be standardized by the
following requirements, comparison may be performed with
international standard substances if necessary.
Certification of chemical composition and component
Physical and chemical parameters, spectrum data, related
references and comparison results are needed to certificate
substances with known chemical composition. Without
published referenecs, it is necessary to provide detailed
information about structure elucidation.
If pharmaceutical standard substances can not be certified by
modern physicochemical methods, the component should be
certificated with appropriate methods.
Physicochemical property test
ltems in physicochemical property tests should be certificated
according to the properties of pharmaceutical standard
substances, such as morphology, melting point, specific
rotation, crystalline form, loss on drying, hygroscopicity,
etc.
Purity and related substance test
According to the requirements of utilization for
pharmaceutical standard substances purity and related
substances, such as reaction intermediate, co-product,
related impurities, should be certificated.
Homogeneity test
It is necessary to conduct homogeneity tests for candidate
standard substances that are prepared in batches then
packaged to the minimum packaging unit. It is necessary to
conduct homogeneity tests for candidate standard substances
thats are subpackaged from a large package to the minimum
packaging unit.
Quantitation
Once all the requirements mentioned above are satisfied,
quantitation is carried out.
The methods used for quantitation should be validated by
methodological study to confirm accuracy and reliability. First,
systematic error or accidental error occurring in measuring
method, process of measuring and sample treatment, for
example, pollution and loss during dissolution and separation,
should be investigated. Measuring instruments should be
calibrated regularly and substances for calibration should be
traceable. Valid quality control systems should be established to
ensure traceability of measured values.
Principie of quantitation
When measuring the content of a candidate chemical standard
or reference substance, the total amount of water, organic
solvent, inorganic impurities and organic component should
be 100%.
Methods for quantitation of candidate pharmaceutical
standard substance
CDUse absolute measuring method or authoritative measuring
method that is highly accurate. During testing, experiments
should be conducted independently by more than two
experimentalists using different experimental systems.
@Quantitation should be carried out by more than two
reliable methods with known accuracies and different working
mechanisms. The precision of methods with different
working mechanisms should be investigated, the systematic
 9901 Guideline far Preparation of Pharmaceutical Standard Substances of China
error should be estimated, and the accuracy of the method
should be validated by necessary approaches.
® Cooperation on quantitation among different certified
laboratories. Necessary conditions and laboratory qualifications
required by quantitation of candidate standard substances should
be satisfied by each laboratory participating in the cooperation.
Each laboratory should employ the specified measuring
method. The number of participating laboratories or the
number of independent groups far quantitation should be in
accordance with the statistical requirements.
Stability test of candidate standard substances
Under defined storage and usage condition, stability tests on
corresponding characteristic properties should be regularly
perfarmed far candidate standard substances.
The time intervals far stability test should be defined from
small values to large ones. During the testing period,
monitoring data should be collected at multiple time
intervals.
Far candidate standard substances with multiple characteristic
properties, the property with the most flexibility and
representativeness should be selected.
Stability test should be perfarmed by the method with
precision not less than that of the quantitation method and
enough sensitivity;
The samples used far stability test should be obtained by
random sampling from overall samples. The number of
samples selected should ensure the representativeness of
overall samples.
Results from time-dependent tests should fluctuate within the
limits of uncertain region of measuring method.
 Annex table I·.·. 45J

Annex table

Atomic Weights of Elements

C=12. 00
Element Symbol Atomic weight Element Symbol Atomic weight
Aluminium Al 26.981538 (2) Magnesium Mg 24. 3050 (6)
Antimony (Stibium) Sb 121. 760 (1) Manganese Mn 54. 938049 (9)
Argon Ar 39. 948 (1) Mercury (Hydrargyrum) Hg 200.59 (2)
Arsenic As 74.92160 (2) Molybdenum Mo 95. 94 (2)
Barium Ba 137.327 (7) Nickel Ni 58.6934 (2)
Bismuth Bi 208. 98038 (2) Nitrogen N 14.0067 (2)
Boron B 10. 811 (7) Oxygen o 15. 9994 (3)
Bromine Br 79. 904 (1) Palladium Pd 106. 42 (1)

Cadmium Ca 112. 411 (8) Phosphorus p 30. 973761 (2)

Calcium Ca 40.078 (4) Platinum Pt 195.078 (2)
Carbon c 12.0107 (8) Potassium (Kalium) K 39. 0983 (1)

Cerium Ce 140. 116 (1) Selenium Se 78. 9fl en

Chlorine Cl 35.453 (2) Silicon Si 28.0855 (3)
Chromium Cr 51. 9961 (6) Silver (Argentum) Ag 107. 8682 (2)
Cobalt Ca 58.933200 (9) Sodium (Natrium) Na 22. 989770 (2)
Copper ( Cuprum) Cu 63.546 (3) Strontium Sr 87. 62 (1)

Fluorine F 18.9984032 (5) Sulfur s 32.065 (5)

Gallium Ga 69. 723 (1) Technetium Te [99]
Germanium Ge 72. 64 (1) Tellurium Te 127.60 (3)
Gold ( Aurum) Au 196. 96655 (2) Thorium Th 232. 0381 (1)

Helium He 4.002602 (2) Tin (Stannum) Sn 118. 710 (7)

Holmium Ho 164. 93032 (2) Titanium Ti 47. 867 (1)

Hydrogen H l. 00794 (7) Tungsten (Wolfram) w 183. 84 (1)

Indium In 114. 818 (3) Uranium u 238.02891 (3)

Iodine I 126.90447 (3) Vanadium V 50. 9415 (1)

!ron (Ferrum) Fe 55.845 (2) Xenon Xe 131. 293 (6)

Lanthanum La 138. 9055 (2) Ytterbium Yb 173.04 (3)
Lead (Plumbum) Pb 207. 2 (1) Zinc Zn 65.409 (4)
Lithium Li 6. 941 (2) Zirconium Zr 91. 224 (2)
Notes l. The last digit of an atomic mass is shown in parentheses.
2. The figure in square brackets is the atomic mass of an isotope which exists with the longest half-life.
 Ingredients of Patent Medicines not Included in the Pharmacopoeia
Ingredients of Patent Medicines not
Included in the Pharmacopoeia
Achyranthes Asperae Radix et Rhizoma Dried root and
rhizome of Achyranthes aspera L. (Fam. Amaranthaceae).
Aconiti Balfourii Radix Dried root of Aconitum balfourii
Stapf or Aconitum szechenyianum Gay ( Fam.
Ranunculaceae).
Aconiti Brachypodi Radix Dried root of Aconitum
brachypodum Diels (Fam. Ranunculaceae).
Aconiti Coreani Radix Dried root of Aconitum coreanum
(Lévl.) Rapaics. (Fam. Ranunculaceae).
Aconiti Herba Dried herb of Aconitum naviculare Stapf or
Aconitum tanguticum (Maxim.) Stapf (Faro. Ranunculaceae).
Aconiti Kosnezoffii Germinatos Dried seedling of Aconitum
kusnezoffii Reichb. (Fam. Ranunculaceae).
Aconiti Sttchenyiani Folimn Leaf of Aconitum szechenyianum
Gay. (Fam. Ranunculaceae).
Aconiti Tangutici Herba Dried herb of Aconitum tanguticum
(Maxim.) Stapf and Aconitum naviculare ( Bruhl.) Stapf
(Fam. Ranunculaceae).
Agastaches Herba Dried aerial part of Agastache rugosus
(Fisch. et Mey.) O. Ktze (Fam. Lamiaceae).
Alangii Radix Dried rootlet and fibrous root of Alangium
chinense (Lour.) Harms (Fam. Alangiaceae).
Aleuritopteritis Argenteae Herba Dried herb of
Aleuritopteris argentea (Gmel.) Fee. (Faro. Pteridiaceae).
Allii Cepae Bulbos Recens Fresh bulbs of Allium cepa L.
(Fam. Liliaceae).
Anemones Altaicae Rhizoma Dried rhizome of Anemone
altaica Fisch. ex C. A Mey (Fam. Ranunculaceae).
Angelicae Sinensis Radi Rami Dried branch root of Angelica
sinensis (Oliv.) Diets (Fam. Umbelliferae).
Anisochili Rhizoma et Radix Dried rhizome and root
attached with old stem of Anisochilus carnosus (L.) Wall.
(Fam. Labiatae).
Anser Fel Pulvis Dried power of the hile of Anser cygnoides
dommerstica Brisson. ( Fam. Anatidae).
Alpiniae Japonicae Radix et Rhizoma Dried root and rhizome
of Alpinia japonica (Thunb.) Miq. (Fam. Zingiberaceae).
Arachidis Hypogaeae Testa Testa of ripe seed of Arachis
hypogaea L. (Fam. Leguminosae).
Araliae Radix Dried root of Aralia armata (Wall.) Seem.
or Aralia decaisneana Hance (Fam. Araliaceae).
Aretii Herba Recens Herb of Arctium lappa L. (Fam.
Compositae).
Armadillidimn Dried body of Armadillidium vulgare (Lat-
reille) (Fam. Armadillididae).
Artemisiae Anomalae Herba Dried aerial part of Artemisia
anomala S. Moore or Artemisia actiflora Wall. ex DC.
(Fam. Compositae)
Artocarpi Radix Dried root of Artocarpus styracifolius Pie-
rre (Fam. Moraceae).
Asparagi Pseudofilicini Radix Dried root of Asparagus
pseudofilicinus Wang et Tang (Fam. Liliaceae).
Asparagi Subscandi Radix Dried root of Asparagus
subscandens F. T. Wang et S. C. Chen (Fam. Liliaceae).
Asterias Dried body of Asteri asamurensis Lütken or
Asterias rollestoni Bell (Fam. Asteriidae).
Auriculariae Dried fruiting body of Auricularia auricula
(L. ex Hook) Underw (Faro. Auriculariacaee).
Bambusae Recens Suecos Liquid in fresh stem of
Phyllostachys nuda Mcclure. and other species of the same
genus (Fam. Gramineae).
Begoniae Rhizoma Dried rhizome of Begonia crassirostris
Irmsch., Begonia palmate D. Don or Begonia pedatifida
Lévl. (Fam. Begoniaceae)
Benincasae Fructos Dried fruit of Benincasa hispida
( Thunb.) Cogn. ( F am. Cucurbitaceae).
Benineasae Semen Dried ripe seed of Benincasa hispida
(Fam. Cucurbitaceae).
Berberidis Cortex Dried root bark of Berberis soulieana
Schneid. and other species of the same genus ( Fam.
Berberidaceae).
Berchemiae Herba Dried herb of Berchemia floribunda
Brongn. (Fam. Rhamnaceae).
Boehmeriae Niveae Rhizoma et Radix Dried root and rhizome
of Boehmeria nivea (L.) Gaud. (Fam. Urticaceae).
Boehmeriae Rhizoma et Radix Dried rhizome and root of
Boehmeria nivea (L.) Gaud. (Fam. Urticaceae).
Bombycis Feculae Dried excrements of Bombyx mori
Linnaeus (Fam. Bombycidae).
Borax Crystal refined from natural borax.
Boris Cor Heart of Bos taurus domesticus Gmelin or
Bubalus bubalis Linnaeus (Fam. Bovidae).
Bovis Fel Bile of Bos taurus domesticus Gmelin ( Fam.
Bovidae).
Bovis Lac Milk of Bos taurus domesticus Gmelin or
Bubalus bubalis Linnaeus (Fam. Bovidae).
Bovis seo Bubali Medalla Marrow of Bos taurus domesticus
Gmelin or Bubalus bubalis L. (Fam. Bovidae).
Brassicae Campestidis Polleo Dried pallen of Brassica
campestis Linn. (Fam. Cruciferae) collection by Apis cerana
Fabricius (Fam. Apidae) and other spado.
Breyniae Herba Dried herb of Breyniafruticosa (L.) Hook.
f. (Fam. Euphorbiaceae).
Bubali Como Pulvis A fine powder of dried horns of
 lngredients of Patent Medicines not lncluded in the Pharmacopoeia
Bubalus buba lis Linnaeus (Faro. Bovidae). The horn was
rem-oved from horn plug, the salid core part of horn tip
obtained by sawing, planed to pieces, and pulverized to fine
powder. (Fam. Bovidae).
Bufo Suecys Dried body of Bufo bufo gargarizans Cantor
or Bufo melanostictus Schneider.
Bufonis Corium Dried skin of Bufo bufo gargarizans
Cantor or Bufo melanostictus Schneider. (Fam. Bufanidae).
Bupleuri Marginati Herba Dried herb of Bupleurum
marginatum Wall. ex OC. (Umbelliferae).
Bupleuri Marginati Radix Dried root of Bupleurum
marginatum Wall. ex OC. (Fam. Umbelliferae).
Buxi Ramulun et Folium Branches and leaves of Buxussinica
(Rehd. et Wils.) Cheng and other species of the same genus
(Fam. Buxaceae).
Calcitum Carbonates of calcite group mineral, mainly
containing calcium carbonate.
Calcitum (proc~ with milk) Break 1000 g of drug to
pieces, add 10 g of potassium nitrate anda quantity of water,
and boil far 3 hours. Decant the liquid, wash with water far
10-15 times until the washing clear, and dry in air. Pulverize
to fine powder, add a quantity of milk, stir to dough shaped,
make into round cakes of about 10 cm in diameter and less
than 3 cm thick, dry in the shade.
Caicitum (processed with skim milk) A processed product
by calcining the clean Calcitum to white colour by carrying
out the method far calcining and quenching ( Appendix 11
D), put into "lada" ( skim milk) until it crisp, taken out
and pul verized.
Calc-tufa A powered lump mainly composed of calcium
carbonate.
Calophylli Herba Dried herb of Calophyllum membranaceum
Gardn et Champ. (Fam. Hypercaceae).
Camelliae Assamicae Folium Dried leaf of Camellia sinensis
O. Ktze. var. assamica Kitamura (Fam. Theaceae).
Camphora Crystals prepared from the dried branch, leaf
and root of Cinnamomum camphora (L.) Sieb. ( Fam.
Lauraceae).
Camphorae Oleum The volatile oil obtained by steam
distillation from the fresh young branch and leaf of
Cinnamomum. camphora (L.) Presl (Fam. Lauraceae).
Campylotropis Hirtellae Radix Dried root of Campylotropis
hirtella (Ftanch.) Schindl. (Fam. Leguminosae).
Canis Os Skeleton of Canisfamiliaris L. (Faro. Canidae).
Canis Penis et Testis Dried penis and testis of
Canisfamiliaris L. (Fam. Canidae).
Caprae Corno Horns of Capra hircus L. (Fam. Bovidae).
Caprae seo Ovis Musculus, Fei et Jecur Meat, gallbladder
and fresh liver of Capra hircus Linnaeus or Ovis aries
Linnaeus (Fam. Bovidae).
Caprae sen Ovis Os Dried skeleton, removal of head, of
Capra hircus L. or Ovis aries L. (Fam. Bovidae).
Carpesii Herba Dried herb of Carpesium abrotanoides L.
(Fam. Compositae).
Castaneae Mollissimae Periclinium Dried involucre of
Castanea mollisslma Bl. (Fam. Fagaceae).
Catharsius Dried insect of Catharsius molossus Linnaeus
(Fam. Scarabaeidae).
Celastri Fructus Dried fruit of Celastrus articulates Thunb.
(Fam. Celastraceae).
Cervi Sanguis Dried blood of Cervus ni p pon Temminck or
Cervus elaphus Linnaeus (Fam. Cervidae).
Cervus Siccus A processed product of the dried entire body
of Cervus nippon Temminck (Fam. Cervidae).
Cervus Cardiac Pulvis Dried powder of heart of Cervus
nippon Temminck or Cervus elaphus Linnaeus. ( Fam.
Cervidae). The drug is collected annually, the healthy dear is
heart is taken out from the killed, baked to dry and
pulverized to powder.
Chalcanthitum Mineral of chalcanthite, mainly containing
hydrated copper sulphate.
Chloranthi Radix et Rhizoma Dried root and rhizome of
Chloranthus henryi Hemsl. or Chloranthus multistachys Pei.
( F am. Chloranthaceae).
Cinnamomi Radix Dried root of Cinnamomum camphora
(L.).
Cinnamomi Radix et Rhizoma Dried root and rhizome of
Cinnamomum parthenoxylon Oack.) Nees or Cinnamomum
camphora (L.) Presl (Fam. Lauraceae).
Cinnamomi Migaonis Fructus Dried fruit of Cinnamomum
migao H. W. Li (Fam. Lauraceae).
Cissi Assamicae Caulis Dried rattan of Cissus assamica
(Laws.) Craib (Fam. Vitaceae).
Claoxyli Cacumen Dried young branch with leaves of
Claoxylon polot (Burm.) Merr. (Fam. Euphorbiaceae).
Cleistocalycis Cortex Dried bark of Cleistocalyx operculatus
(Roxb.) Merr. et Ferry.
Composita Medicata Fermentata A dried processed product
by fermentation of Polygoni Criopolitani Herba, Xanthii
Sibirici Semen and other twenty one crude drugs.
Corydalis Strictae Herba Dried herb of Corydalis stricata
Steph. (Fam. Papaveraceae).
Crataegi Cuneatae Fructus (stir-baked) Dried mature fruit
of Crataegus cuneata Sieb. et Zucc. (Fam. Rosaceae).
Crataegi Semen Extraetum Extract prepared by
carbonization and distillation of the kernel of Crataegus
pinnatifida Bnge var. majar N. E. Br. (Fam. Rosaceae).
Crotonis Caudati Radix, Caulis et Folium Dried root, stem
and leaf of Croton caudatus Geisel. var. tomentosa Hook.
(Fam. Euphorbiaceae).
Crotonis Crassifolii Radix Dried root of Croton crassifolius
Geisel. ( Fam. Euphorbiaceae). The drug is collected m
autumn and winter, washed clean and dried in the sun.
Crotonis Tiglii Caulis et Radix Dried root and stem of
Croton tiglium L. (Fam. Euphorbiaceae).
Cucumis Sativi Fructus Dried mature fruit of Cucumis
sativus L. (Fam. Cucurbitaceae).
Cudraniae Radix Dried root of Cudrania cochinchensis
(Lour.) Kudo. et Masam. or Cudrania tricuspidata (Carr.)
Bur. (Fam. Moraceae).
Cudraniae Radix et Ramulus Dried root and branch of
Cudrania tricuspidata (Carr.) Bur. (Fam. Moraceae).
Cumini Cymini Fructus Dried mature fruit of Cuminum
cyminum L. (Fam. Umbelliferae).
Cynanchi Thesioidis Semen Dried seed of Cynanchum
thesioides (Frcyn) K. Schum ( Fam. Asclepiadaceae).
 lngredients of Patent Medicines not Included in the Pharmacopoeia
Daphne Giraldii Cortex Dried stem and root bark of Daphne
giraldii Nitsche (Fam. Thymelaeaceae).
Decoctum Herbae A processed product by fermentation of
Galla Chinensis and Folium Camelliae Sinensis, etc.
Dioscoreae bulbiferae Rhizoma Dried rhizome of Dioscorea
bulbifera L. ( Fam. Dioscoreaceae).
Distiller's Yeast A processed product fermented by Hordei
Fructus and Pisi Fructus etc.
Distiller' s Yeast Praeparata A processed product of Distiller'
s Yeast, fermented by Saccharomyces Cerevisiae Fermentata.
Dracaenae Combodianae Resina Dried resin of Dracaena
combodiana (Fam. Agavaceae).
Dracocephali Tangutici Herba Dried aerial part
Dracocephalum tanguticum Maxim. (Fam. Labiatae).
Draconis Dens Tooth fassils of ancient mammals, such as
Hipparion, Rhinoceros, Bos, Corvus, Elephants, etc.
Draconis Os Bone fassils of ancient mammals, such as
Hipparion, Rhinoceros, Bos, Cervus, Elephants, etc, or
incisor fassils of Elephants.
Dried hile of Selenaretos thibetanus prepared by surgical
drainage of gallbladder.
Droserae Mulpisepalae Herba Dried herb of Drosera peltata
Smith var. mulpisepala Y. Z. Ruan (Fam. Droseraceae).
Duchesneae lndicae Herba Dried herb of Duchesnea indica
(Andr.) Focke (Fam. Rosaceae). The drug is collected in
summer and autumn, washed clean and dried in the sun.
Ecliptae Caulis Liquidum J uice obtained by pressing the
fresh stem of Eclipta Prostrata L. (Fam. Compositae),
mixed with small quantity of water, and filtered.
Elaeagni Folium Dried leaf of Elaeagnus pungens Tbunb.
(Fam. Elaeagnaceae).
Eleocharidis Amylum Starch of the dried corm of Eleocharis
tuberosa (Roxb.) Roem. Et Schult. (Fam. Cyperaceae).
Elephantopi Scaberis Herba Dried herb of Elephantopus
scaber L. ( Fam. Compositae). The drug is collected in
summer and autumn befare flowering, washed clean, and
dried in the sun.
Emiliae Herba Dried herb of Emilia sonchifolia (L.) OC.
(Fam. Compositae).
Entadae Caulis Dried rattan of Entada phaseotoides (L.)
Merr. (Fam. Leguminosae).
Eretmochelydis Carapax Shell of Eretmochelys imbricata
(Linnaeus) (Fam. Cheloniidae).
Erinacei Corium Dried outer skin of Erinaceus europaeus L.
or Hemichianus dauricus Sundevall (Fam. Erinaceidae).
Eriocheir et Potamon Dried body of crab [Eriocheir sinensis
H. Miline-Edwalds, Potamon ( Potamo) denticulate or
Potamon (Potamon) yunanensis (Fam. Catametopa) ].
Erythrinae Cortex Dried stem bark of Erythrina eariegata
L. var. oriental is ( L.) Merr. or Erythrina arborescens
Roxb. (Fam. Leguminosae).
Eumeces Chinensis Dried body of Eumeces chinensis (Gray)
(Fam. Scincidae).
Euonymi Alati Pterocaulis Wing part of dried stem of
Euonymus alatus CThunb.) Sieb. (Fam. Celastraceae).
Euonymi Fortunei Herba Dried stem and branch with leaf
of Euonymus Jortunei ( Turcz.) Hand. -Mazz., Euonymus
japonicus L. or Euonymus subsessilis Sprague ( Fam.
Celastraceae).
Eupatorii Chinensis Radix Dried root of Eupatorium
chinense L. (Fam. Compositae).
Ferri Pulvis (processed with Chebulae Fructus) Boil 130 g
of Tamaricis Cacumen in 100 ml of water far 3 hours, filter,
add 500 g of fine iron powder to the filtrate anda quantity of
water to macerate it, boil far 3 hours, discard the liquid,
wash the residue with water far 3 times, add 50 g of table
salt and 1 000 ml of water, boil far 2 hours, discard the
liquid, wash the residue again with water far 4 times, add 2
500 g of fine powder of Chebulae Fructus, mix well, add 1
800 ml of hot water, stir, stand far 3 days, stir far 3 times a
day, take out on the faurth day, spread and dry in the shade,
draw out the unreacted iron powder with magnet, pulverize
of
to fine powder, and sift. The drug can not be prepared in
summer.
Ficus Receptaculum Dried hypanthium receptacle of Ficus
pumila L. (Fam. Moraceae).
Ficus Simplicissimae Herba Dried herb of Ficus
sim plicissima Lour. ( Fam. Moraceae ) . The drug is
collected all the year round, removed from rootlets, washed
clean, and dried.
Fissistigmae Radix et Caulis Dried root and rattan of
Fissistigma oldhamii (Hemsl.) Merr. (Fam. Annonaceae).
Fragariae Herba Dried herb of Fragaria orientalis Lozinsk
and other species of the same genus (Fam. Rosaceae).
Galli Ovi Chorion ( Stir-baked) Chorionic shell of Gaollus
gallus domesticus Brissum. (Fam. Phasianidae).
Galli Os Skeleton of Gallus gallus domesticus Brisson
(Fam. Phasianidae).
Gallos Nigroris Fresh body of Silkie ( Fam. Phasianidae) ,
removed from feather, viscera and subcutaneous fat. The
Silkie was killed, blanched slightly with boiling water,
removed from feather, washed clean, the abdomen cut
opened, removed from viscera and subcutaneous fat, and
washed clean again. Use freshly or store in low temperature
far the later use.
Gallos Ovi Follicularis Membrana Dried inner shell
membrane of egg of Gallus gallus domesticus Brisson (Fam.
Phasianidae).
Gaultheriae Herba Dried herb of Gaultheria yunnanensis
(Franch.) Rehd. (Fam. Ericaceae).
Gentianae Straminiae Flos Dried flower of Gentiana
straminia Maxim. (Fam. Gentianaceae).
Glyeosmis Folium Dried leaf of Glycosmis parvifLora
(Sims) Kurz (Fam. Rutaceae).
Gneti Caulis Dried rattan of Gnetum montanum Markgr. or
Gnetum parvifolium (Warb.) C. Y. Cheng ex Chun (Fam.
Genetaceae).
Gueldenstaedtiae Herba Dried herb of Gueldenstaedtia verna
(Georgi) A Bar. (Fam. Leguminosae).
Gynoacemmae Herba Dried aerial part of Gynoacemma
pentaphyllum (Thunb) Mak (Faro. Cucurbitaceae). The
drug is collected in autumn, removed from fareign matter
and dried in the sun.
Gypsum Rubrum Sulfate mineral red gypsum of anhydrite
family, mainly composed of hydrated calcium sulfate
(CaS04. 2H2m.
Halloysitum album Take fine powder of Halloysitum album
in to the vinegar, mix well, rub, cut in to chunks and dry.
 lngredients of Patent Medicines not Included in the Pharmacopoeia
Calcine it to thoroughly red by carrying out the method for
calcining openly ( Appendix I1 D). Mash or grind into fine
powder in use. Use 25 kg vinegar per 100 kg of Halloysitum
al bum.
Hedychii Venusti Rhizoma Rhizome of Hedychium
venustum Wight (Fam. Zingiberaceae).
Hedyotidis Chrysotrichae Herba Dried herb of Hedyotis
chrysotricha (Palib.) Merr. (Fam. Rubiaceae).
Hedyotidis Hedyotideae Herba Dried herb of H ed yotis
hedyotidea DC. (Fam. Rubiaceae).
Hericii Mycelli A dried mixture of the mycelium of
Hericium erinaceus (Bull ex Fr.) Pers (Fam. Hydnaceae)
and the Epiphytic salid culture medium.
Herpe~ Caudigeri Semen Dried seed of Herpetospennum
caudigerum WalL CFam. Cucurbitaceae).
Hippophae Fructus Extractum Wash mature Hippophae
Fructus and eliminate foreign matter. Place the fruit in a
copper or aluminum pot, add a quantity, of water, about 6-
10 cm over the drug, heat with vapour or fire to boil for 1-2
hours, decant the decoction. Decoct the residue with water
again, combine the decoctions, allow to stand for 12 hours to
precipitate, decant the supematant and filter the solution at
the bottom, then concentrate the solution at a high
temperature at the beginning, decrease the temperature with
the solution concentrated gradually. keep it boil slightly and
stir constantly to prevent charring until the concentrated
decoction can be raised like sliver or can not seep through
paper.
Hirsuteila Mycelii Pulvis Dried powder of the mycelium of
Hirsutella sinensis obtained by submerged fermentation.
Holotrichiae Dried larva of Holotrichia diom phalia Bates
or other species of the same genus (Fam. Scarabacidae).
Hordei Fruetus Dried fruit of Hordeum vulgare L. (Fam.
Gramineae)
Humuli Herba Dried aerial part of Humulus scandens
(Lour.) Merr. (Fam. Moraceae).
Hydnocarpi Semen Dried seed of Hydnocarpus
anthelmintica Pierre. (Fam. Flacourtiaceae).
Hygrophilae Megalantae Semen Dried and mature seed of
Hygrophila salicifolia ( Vahl.) Nees var. meglantha
(Merr.) H. S. Lo et L. D. Chou (Fam. Acamhaceae).
Hypecoi Leptocarpi Herba Dried herb of Hypecoum
leptocarpum Hook. f. et Thorns. (Fam. Papaveraceae).
Hyperici Japonici Herba Dried herb of Hypericum
japonicum Thunb. (Fam. Guttiferae).
Hyptidis Herba Dried herb of Hyptis suaveolens (L.) Poit.
(Fam. Labiatae).
Inulae Cappae Radix et Rhizoma Dried root and rhizome of
Inula cappa CBuch. Ham.) DC. (Fam. Compositae).
Ilicis Asprellae Radix Dried root of llex asprella ( Hook. et
Arn.) Champ. ex Benth. (Fam. Aquifoliaceae).
Ilicis hainanensis Folium Processed dried leaf of I lex
hainanensis Merr. (Fam. Aquifoliaceae).
llicis Pubescns Radix Dried root of I lex puhescens Hook. et
Arn. (Fam. Aquifoliaceae).
lllicii Lanceolati Radix Dried root of I llicium lanceolatum
A C. Smith (Fam. Magnoliaceae).
Impateintis Caulis Recens Dried stem of Impateins
balsamina L. (Fam. Balsam in aceae).
lnulae Cappae Herba Dried herb of Inula cappa (Buch.
Ham. ) DC. (Fam. Compositae).
lnulae Cappae Radix Dried root of Inula cappa DC. (Fam.
Compositae).
Inulae Racemosae Radix Dried root of I nula racemosa
Hook. f. (Fam. Compositae).
Isodonis Herba Dried aerial part of Isodon striatus (Benth.)
Kudo or Isodon sorra (Maxim.) Kudo. (Fam. Labiatae).
lxeritis Chinensis Herba Dried herb of Ixeris chinensis
CThunb.) Nakai (Fam. Compositae).
Kadsurae Heteroclitae Caulis Dried rattan of Kadsura
heteroclita (Roxb.) Craib. (Fam. Magnoliaceae).
Kadsurae Radix sen Caulis Dried root of Kadsura coccinea
(Lem.) A C. Smith or dried rattan of Kadsura heteroclite
(Roxb.) Craib. (Fam. Magnoliaceae).
Kaki Folium Dried leaf of Diospyros kaki Thunb. (Fam.
Ebenaceae). The drug is collected in autumn, removed from
the foreign matter and dried in the sun.
Kalimeridis Herba Dried herb of Kalimeris indica (L.)
Sch. Bip. (Fam. Compositae).
Lagerstroemiae Cortex Dried stem bark of Lagerstroemia
indica L. (Fam. Lythraceae).
Lespedezae Virgatae Herba Dried herb of Lespedeza virgata
(Thunb.) DC. (Fam. Leguminosae).
Lingnaniae seo Barmbusae Folium Dried crimpy tender leaf
of Lingnania chungii ( McClure) McClure or Barmbusa
pervariabilis McClure (Fam. Gramineae).
Liquidambaris Folium Dried leaf of Liquidambar
taiuxmiana Hance ( Fam. Hamamelidaceae). The drug is
collected in summer, washed clean, dried in the sun or used
in fresh.
Litseae Radix et Rhizoma Dried root and rhizome of Litsea
cuheba (Lour.) Pers. (Fam. Lauraceae).
Lygodii Herba Dried aerial part of Lygodium japonicum
(Thunb.) Sw. (Fam. Lygodiaceae).
Lysimaehiae Herba Dried herb of lysimachia
foenumgraecum Hance. (Fam. Primulaceae).
Malloti Apeltae Radix et Rhizoma Dried root and rhizome of
Mallotus apelta (Lour.) Muell-Arg. (Fam. Euphorbiaceae).
Margaritiperae Pulvis Powder prepared from the inner layer
of pearl cells.
Massa Medicata Cantonia Rectangular massive prepared by
Peucedani Radix, Glycyrrhizae Radixet Rhizoma, Rhei Radix
et Rhizoma, and other fifty nine crude drugs.
Massa Medicata Composita Rectangular massive prepared by
Massa Medica ta Fermenta ta ( stir-baked) , Hordei Fructus
Germinatus, Crataegi Fructus ( stir-baked) and other
fourteen crude drugs.
Massa Medicata Fermentata A fermented product by
fermentation of a mixture of Polygoni Hydropiperis Herba,
Artemisiae Annuae Herba, Armeniacae Semen and wheat flour.
Massa Medicata Fermentata ( stir-baked ) Cut Massa
Medicata Fermentata to small pieces, stir-bake as described
under the method for simple stir-baking (Appendix l D)
until a dark yellow colour develops on the surface.
Melastomae Herba Dried herb of Melastoma dodecandrum
Lour. (Fam. melastomaceae).
Melicopes Pteleifoliae Caulis et Cacumen Dried stem and
 Ingredients of Patent Medicines not Included in the Pharmacopoeia
young foliferous branch of Melicopeptelei folia ( Champ. ex
Benth.) T. G. Hartlry (Fam. Rutaceae).
Melastomae Normale Radix Dried root of Melastoma
normale D. Don (Fam. Melastomataceae).
Millettiea Pulchrae Radix Dried root of Millettia pulchra
Kurz var. laxior (Dunn) Z. Wei (Fam. Papilionaceae).
Mineralium Viridianum Green rust on the surface of
copper, formedby the effect of carbon dioxide or acetic acid,
mainly containing basic copper carbonate.
Millettiae Caulis Dried rattan of Millettia nítida Benth.
var. hirsutissima Z. Wei (Fam. Leguminosae).
Moghaniae Radix Dried root of Moghania philippinensis
(Merr. et Rolfe) Li., Moghania macrophylla CWilld.) O.
Kuntze or Moghaniaferruginea ( Wall. ex Benth.) Li.
(Fam. Leguminosae).
Monochasmae Herba Dried herb of Monochasma savatieri
Franch. Ex Maxim. ( Fam. Scrophulariaceae).
Nigellae Sativae Semen Dried seed of Nigella sativa L.
(Fam. Ranunculaceae).
Oldenlandiae Diffusae Herba Dried herb of Oldenlandia
diffusa (Willd.) Roxb. (Fam. Rubiaceae)
Onocrotalum The mother liquor after salt prepared by
subterranean yellow brine as raw material, processed
products by evaporation and concentration. It contains not be
less than 70% of Calcium Chloride dihydrate CCaCb•2H2Ü).
Onosmae Cortex Dried root bark of Onosma paniculatum
Bur. et Franch. (Fam. Boraginaceae).
Origani Vulgaris Herba Dried herb of Origanum vulgare L.
( Fam. Labiatae). The drug is collected in summer and
autumn at flowering, removed from foreign matter, and
dried in the sun.
Orpimentum A mineral of sulfides of Orpiment group,
containing mainly arsenic disulfide ( As2 Sz ) .
Oryzae Sativae Semen Dried seed of Oryza sativa L. (Fam.
Gramineae).
Oxalitis Herba Dried herb of Oxalis comiculata L. ( Fam.
Oxalidaceae).
Oxytropis Chiliopgyilae Herba Dried herb of Oxytropis
chiliophylla Royle or Oxytropts falcata Bge. ( Fam.
Leguminosae).
Oxytropis Myriophyllae Herba Dried herb of Oxytropis
myriophylla (Pall.) DC. (Fam. Leguminosae ).
Paederiae Scandentis Herba Dried aerial part of Paederia
scandens (Lour.) Merr. (Fam. Rubiaceae).
Parabarii seu Ecdysantherae Cortex Dried stem bark of
Parabarium chunianum Tsiang, Parabarium micranthun
CA DC. ) Pierre or Ecdysanthera utilis Hay. Et Kaw.
(Fam. Apocynaceae).
Passeris Encephaion Brains of Passer montanus saturates
Stejneger (Fam. Ploceidae).
Passeris Montani Dried body of Passer montanus saturates
Stejneger (Fam. Ploceidae). The drug is collected annually,
removed from hair and Víscera, wiped clean and dried.
Patriniae Scabiosaefoliae Herba Dried herb of Patrinia
scabiosaefolia Fisch. (Fam. Valerianaceae).
Patriniae Herba Dried herb of Patrinia scabiosaefolia Fisch.
or Patrinia villosa Juss. (Fam. Valerianaceae).
Persicae Semen Pulveratum A defatted powder of Persicae
Semen. Triturate Persicae Semen to a paste, wraps with abs-
orbent paper and press, rip paper day interval for several
times until almost no oil exist and texture is soft, pulverize
to fine powder.
Phaseoli Radiati Semen Dried seed of Phaseolus radiates L.
(Fam. Leguminosae).
Phlomis Radix Dried root of Phlomis younghusbandii
Mukerjee. (Fam. Lamiaceae).
Phlomis Tuberosae Radix Dried root tuber of Phlomis
tuberosa L. CFam. Labiatae).
Pimpinellae Herba Dried herb of Pimpinella thellungiana
Wolff (Fam. Umbelliferae).
Pinelliae Fermentata A product prepared from pinellia
tuber, ginger juice, alum, Massa Medicata Fermentata and
wheat powder.
Pini Folium Recens Fresh leaf of Pinus massoniana Lamb.
(Fam. Pinaceae).
Pini Poria Cure Radixa White part of dried sclerotium of
Paria cocos (Schw.) Wolf (Fam. Polyporaceae), embedded
with twig and root of pine in the middle of the sclerotium.
Piperis Puberuli Ramulus et Folium Dried branch with leaves
of Piper wallichii ( Miq.) Hand. -Mazz or Piper
puberulum (Benth.) Maxim. (Fam. Piperaceae).
Piperis Sarmentosi Herba Dried aerial part of Pi per
sarmentosum Roxb. CFam. Piperaceae).
Polygoni Aubertii C.aulis Dried stem of Polygonum aubertii
Henry (Fam. Polygonaceae).
Polygoni Capitati Herba Dried herb or aerial part of
Polygonum capitatum Buch. -Ham. ex D. Don ( Fam.
Polygonaceae).
Pteridis Multifidae Herba Dried herb of Pteris multifida
Poir. ex Lam. (Fam. Pteridaceae).
Pterocarpi Lignum Wood of Pterocarpus santalinus L.
(Fam. Leguminosae).
Pulvis Fumi Carbonisatus Soot of ruderal process from
combustion in vest bottom of kettleor, inside of chimney.
Leviter scrape, and using a sieve removed from foreign
matter.
Pumex Dried skeleton of Costazia aculeate Canu et Bassler
(Fam. Celleporidae).
Punicae Granati Fructus Dried fruit of Punica granatum L.
(Fam. Punicaceae).
Pyrrosiae Calvatae Folium Dried leaf of Pyrrosia calva.ta
(Bak. ) Ching (Fam. Polypodiaceae).
Querci Folium Dried leaf of Quercus dentate Thunb. (Fam.
Fagaceae).
Rabdosiae Herba sen Rhizoma Dried aerial part or rhizome
of Rabdosia amethystoides ( Benth) Hara, Rabdosia
macrocalyx (Dunn) Hara, and other species of the same
genus (Fam. Labiatae).
Rhodiolae Sacrae Radix et Rhizoma Dried root and rhizome
of Rhodiola sacrae ( Prain ex Hamet) S. H. Fu ( Fam.
Crassulaceae).
Rhodomyrti Tomentosae Radix Dried root of Rhodomyrtus
tomentosa CAit.) Hassk. (Fam. Myrtaceae).
Coptidis Fibra Dried rootlet of Coptis chinensis Franch.,
Coptis deltoidea C. Y. Cheng et Hsiao or Coptis teeta Wall.
(Fam. Ranunculaceae).
 lngredients of Patent Medicines not Included in the Pharmacopoeia
Rosae Davuiicae Fructus Dried mature fruit of Rosa
davuiica Pall. (Fam. Rosaceae).
Rosae Radix Dried root of Rosa laevigata Michx. , Rosa
cymosa Tratt. or Rosa multiflora Thunb. var. cathayensis
Rehd. et Wils. (Fam. Rosaceae).
Rubi ldaei Caulis Dried stem of Ruhus idaeus L. ( Fam.
Rosaceae).
Rubi Lignum Xylem of Ruhus sp. (Fam. Rosaceae).
Rubi Parvifolii Radix Dried root of Ruhus parvifolius Linn.
(Fam. Rosaceae). it is also called She Pao Le.
Rubi Sachalinensis Ramulus Dries stem and branch of Ruhus
sachalinensis Leveille (Fam. Rosaceae).
Sai Ammoniacus Mineral of Halitum violaceum mainly
containing ammonium chloride.
Saitpetre Crystals refined from natural potassium nitrate.
Sambuci Racemosae Ramulus Dried stem with leaf of
Sambucus racemosa L. (Fam. Caprifoliaceae).
Sapindi Mukorossi Fructus Dried fruit of Sapindus
mukorossi Gaertn. (Fam. Sapindaceae)
Saxifragae Tanguticae Herba Dried herb of Saxijraga
tangutica Engl. (Fam. Saxifragaceae).
Schefflerae Ramios Dried branch with leaf of Schejjlera
kuxmgsiensis Merr. ex Li. (Fam. Araliaceae).
Schoepfiae Herba Dried herb of schoep jia chinensis Gardn.
et Champ. Or Schoepjia jasminodora Sieb. Et Zucc. (Fam.
Olacaceae).
Securidacae HerbaDried herb of Securidaca inappendiculata
Hassk (Fam. Polygalaceae).
Selaginellae Doedeleinii Herba Herb of Selaginella doedeleinii
(Gen. Cupressues, Fam. Selaginellaceae).
Selenitum Small plate sulfates mineral gypsum formed in
long period of time, mainly composed of hydrated calcium
sulfate.
Senecionis Cannabifolii Herba Dried aerial part of Senecio
cannabijolius Less. and Senecio cannahijolius Less. var.
integrijolius (Koidz) Kitag. (Fam. Compositae).
Serpentis Fel Liquidum Bile of many species of snakes
(Fam. Elapidae, Fam. Colubridae, and Fam. Crotalidae).
The gallbladder was taken out from the killed snake and
preserved in equal quantity ( g/ g ) of Chinese Liquor
containing more than 50 % of alcohol. The membrane of
gallbladder is removed befare use, and both the hile liquid
and the Chinese Liquor used for medicine.
Serpentis Succys Dried body of Bungarus multicinctus Blyth
( Fam. Elapidae) , Agkistrodon strauchii Bedriaga ( Fam.
Crotalidae) or Opheodrys majar ( Guenther) ( Fam.
Colubridae), removed from the head, tail and skin.
Smilacis Ripariae Herba Herb of Smilax riparia A OC.
(Fam. Liliaceae).
Smilacis Sieboidii Radix et Rhizoma Dried root and rhizome
of Smilax scobinicaulis C. H. Wright, Smilax stans Maxim
or Smilax siebold Miq. (Fam. Liliaceae).
Smilacis Sieboldii Radix et Rhizoma (processed with wine)
Dried root and rhiwme of Smilax stans Maxim ( Fam.
Liliaceae).
Solani lyrati Herba Dried herb of Solanum lyratum Thunb.
(Fam. Solanaceae). The drug is collected in summer or aut-
umn, washed clean, and dried in the sun.
Solani Nigri Herba Dried aerial part of Solanum nigrum L.
(Fam. Solanaceae).
Solani Radix et Caulis Dried root and old stem of Solanum
indicum L. , Solanum surattense Burm. f. , Solanum torvum
Swartz. or Solarium xanthocarpum Schrad. et Wendl. (Fam.
Solanaceae).
Solani Radix et Caulis Root and stem of Solanwn melongena L
(Fam. Solanaceae).
Sonchi Herba Dried herb of Sonchus arvensis L. ( Fam.
Compositae)
Sophorae Ramulus Dried twig of sophorajaponica L. (Fam.
Leguminosae).
Speranskiae Tuberculatae Herba Dried herb of Speranskia
tuberculata (Bge.) Baill. (Fam. Euphorbiaceae). The drug
is collected in summer and autumn, removed from foreign
matters and dried.
Spiriferis Fossilia Fossil of Cyrtiospirijer sinensis
(Graban) or CyrtiosPirijer sp. (Fam. Spiriferidae).
Steviae Folium Dried leaf of Stevia rebaudiana ( Bertoni)
Hemsl.
Stiga Maydis Dried style and stigma of Zea mays L. (Fam.
Gramineae).
Streptocauli Radix Dried root of Streptocaulon Grijfibhii
Hook. f. (Fam. Asclepiadaceae).
Succinum Succinum is a kind of fossil resin of Pinus plants,
formed by undergoing a certain chemical changes when
buried in the ground for millions of years.
Suillus Os Dried skeleton of Sus seroja domestica Brisson
(Fam. Suidae).
Suillus Unguis Dried hoof nail of Sus seroja domestiea
Brisson (Fam. Suidae). The nails are collected from pigs,
washed clean and dried.
Sumi Sinensis Chinese ink prepared from pine soot.
mucilage, borneol as well as perfume.
Susis Cerebri Pulvis Dried power of encephala of Sus seroja
domestica Brisson (Fam. Suidae).
Susis Fel Liquidum Bile of Sus seroja domestica Brisson
(Fam. Suidae).
Susis Medulla Spinalis Spinal cord of Sus seroja domestica
Brisson (Fam. Suidae). The Spinal column bone was taken
out from the killed healthy pig and the fresh Spinal cord is
used.
Swertiae Mus.sotii Herba Dried herb of Sv.Jertia mussotii
Franch. (Fam. Gentianaceae).
Syringae Folium Dried leaf of Syringa vulgaris L.,
Syringa dilatata Nakai or Syringa oblate Lindl. ( Fam.
Oleaceae).
Syringae Pinnatifoliae Radix Dried root of Syringa pinnatijolia
Hemsl. (Fam. Oleaceae).
Tabanus Female insect of Tabanus bivittatus Matsumura
(Fam. Tabanidae).
Taccae Rhizoma Dried rhizome of Tacca esquirolii (LevU.
Terra Flavausta The scorched clay formed in the old wood
stove, using the red and hard sintered center part.
Thalictri Radix et Rhi7Ama Dried root and rhizome of
Thalictnon glandulosissimum (Fin et Gagn) W. T. Wang et S.
H. Wang, Thalictnon cultratum Wall, Thalictnonjolioloswn
OC. or Thalictrum aguilegilolium L. var. sibiricum Regel. et
 Ingredients of Patent Medicines not Included in the Pharmacopoeia
Tiling (Fam. Ranunculaceae).
Theae Folium A dried processed product of tender leaf or
shout of Camellia sinensis (L.) O. Ktze (Fam. Theaceae).
Tinosporae Caulis Dried stem of Tinospora sinensis (Lour.)
Merr. or Tinospora cordifolia ( Willd.) Miers. ( Fam.
Menispermaceae).
Thlpae Bispimsae Fructus Dried fruit of Trapa bispinosa Roxb.
or Trapa maximowiczii Korsch. (Fam. Claricepitaceae).
Tremolitum Tremolite belonging to Amphibole group of
silicates mineral, mainly containing hydrated calcium silicate.
Trionycis Carapax Colla Salid glue prepared from Trionycis
Carapax by decoction and concentration.
Tripterygii Hypoglauci Radix Dried root of Tripterygium
hypoglaucum (Levl.) Hutch. (Fam. Celastraceae).
Tritici Fructus Dried mature fruit of Triticum aestivum L.
(Fam. Gramineae).
Tritici Fructus Levis Dried light and thin fruit of Triticum
aestivum L. (Faro. Gramineae).
Trogopteri Faeces Dried faeces of Trogopterus xanthipes
Miline-Edwards (Fam. Sciuridae).
Trollii Chinensis Flos Dried flower of T rollius chinensis
Bge. (Fam. Ranunculaceae).
Trona Ramified crystals occurring around the salt lake,
mainly containing sodium carbonate.
Typhonii Flagelliformis Rhizoma Dried rhizome of
Typhonium flagelliforme (Lodd.) Blume (Fam. Araceae).
Ultivarietas Oryzae Sativae et Monasci Hyphae and spores of
Monascus purpureus Went ( Fam. Eurotiaceae) cultivated
artificially in Oryzae Sativae seed until the grains tum red.
Urenae Herba Dried aerial part or herb of Urena lobata L.
(Fam. Malvaceae).
Uris Fel Pulvis
Vespae Polypide of Vespa manifica Smith (Fam. Vespidae).
Vespertilionis Faeces Dried excrements of Vespertilio
superans Thomas (Fam. Vespertilionidae).
Viciae Herba Dried aerial part of of Vicia amoena Fisch,
Vicia cracca L. , Vicia pseudo-orobus Fisch. et Mey, Vicia
amoena Fisch var. sericea Kitag. or Vicia amoena Fisch var.
angusta Freyn. (Fam. Leguminosae).
Viticis Foliwn Dried leaf of mtex trifolia L. (Fam. Rosaceae).
Viticis Negundo Fructus Dried mature fruit of Vitexnegundo
Linnaeus or Vitexnegundo var. cannabi folia
(Siebold&.Zuccarini) Handel-Mazzetti (Fam. Verbenaceae).
Viticis Trifoliae Radix Dried root of Vitex trifolia L. var.
simplicifolia Cham. or vitex Trifolia L. (Fam. Rosaceae).
Vitis Viniferae Fructus Dried fruit of Vitis mnifera L. (Fam.
Vitaceae).
Wikstroemiae Radix seu Rhizoma Dried root or rhizome of
Wikstroemia indica C. A Mey (Fam. Thymelaeaceae).
Zanthoxyli Dissiti Radix et Caulis Dried root and stem of
Zanthoxylum dissitum Hemsl. (Fam. Rutaceae).
Zanthoxyli Semen Dried seed of Zanthoxylum bungeanum
Maxim. or Zanthoxylum schinifoliam Sieb. et Zucc. ( Fam.
Rutaceae).
Zingiberis Corallini Rhizoma Fresh or dried rhizome of
Zingiber corallinum Hance (Fam. Zingiberaceae).
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

The list of the code number of the general chapters in

Chp 2015 compared with that of the appendix in Chp 2010

The ccx:le The volume

Naroe of the general chapters in Chp 2015 Naroe of the appendices in Chp 2010
number in Chp 2010

0100 General Requireroents for Preparations

I I D Tablets
0101 Tablets II I A Tablets

m I E Tablets

I I u lnjections
0102 Injections II I B Injections

m I A Injections

I I L Capsules
0103 Capsules II I E Capsules

m I F Capsules

I I c Granules
0104 Granules II I N Granules

m I J Granules

I I y Eye Preparations
0105 Eye Preparations II I G Eye Preparations

m I c Eye Preparations

I I X Nasal Preparations
0106 Nasal Preparations II I R Nasal Preparations

m I L Nasal Preparations

I I w Suppositories
0107 Suppositories II I D Supposi tories

m I B Suppositories

I I A Pills
0108 Pills II I K Syrups

II I H Pills

I I R Ointroents

0109 Ointroents, Crearos II I F Ointroents, Crearos and Pastes

II I G Ointroents anf Crearos

0110 Pastes II I F Ointroents, Crearos and Pastes

0111 Preparations for lnhalation II I L Aerosols, Powders for Spray and Sprays

I I z Aerosols and Sprays

0112 Sprays II I L Aerosols, Powders for Spray and Sprays

m I H Sprays
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The code The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

I I z Aerosols and Sprays

0113 Aerosols
Il I L Aerosols, Powders for Spray and Sprays

I I Q Gels

0114 Gels Il I u Gels

III I M Gels

I I B Powders

0115 Powders Il I p Powders

III I K Powders

I I H Syrups
0116 Syrups
Il I K Syrups

I I V Liniments, Lotions, Smeared films

0117 Liniments
II I T Liniments, Paints, Pigments

II I T Liniments, Paints, Pigments

0118 Paints
III I D Preparations for Externa! Application

I I V Liniments, Lotions, Smeared films

0119 Pigments
II I T Liniments, Paints, Pigments

I I N Tinctures
0120 Tinctures
Il I e Tinctures

I I 1 Cataplasms
0121 Patches
II I V Patches

0122 Cataplasms I I 1 Cataplasms

Oral Solutions, Oral Suspensions, Oral Oral Solutions, Oral Suspensions, Oral
0123 II I o
Emulsions Emulsions

0124 lmplants II I J lmplants

0125 Pellicles II I M Pellicles

0126 Ear Preparations II I Q Ear Preparations

I I V Liniments, Lotions, Smeared films

0127 Lotions
II I s Lotions, lrrigants, Enemas

I I V Liniments, Lotions, Smeared films

0128 lrrigants
II I s Lotions, lrrigants, Enemas

0129 Enemas II I s Lotions, Irrigants, Enemas

0181 Mixtures I I J Mixtures

0182 Troches I I E Troches

0183 Concentrated Decoctions I I F Concentrated Decoctions

0184 Glues I I G Glues

0185 Medicinal Wines I I M Medicinal Wines

 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The code The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

0186 Plasters I I p Plasters

0187 Medicinal Distillates I I s Medicinal Distillates

0188 Medicinal Teas I I T Medicinal Teas

0189 Liquid Extracts and Extracts I I o Liquid Extracts and Extracts

General Requirements for Other Phannaceutical

0200
l\1aterials

Sampling of Crude Drugs and Decoction Sampling of Crude Drugs and Pre pared
0211 I lI A
Pieces Slices

General Principie for lnspection of Crude General Quality Control Methods for Crude
0212 I lI B
Drugs and Decoction Pieces Drugs and Prepared Slices

0213 The Processing of Crude Drugs I lI D The Processing of Crude Drugs

General Requirements for Pharmaceutical

0251 lI lI Pharmaceutical Excipients
Excipients

I XIV Water for Pharmaceutical Purposes

0261 Water for Pharmaceutical Purposes
lI XVII Water for Pharmaceutical Purposes

General Requirements for National

0291
Pharmaceutical Reference Standards

0300

I N General Identification Tests

0301 General Identification Tests
lI m General Identification Tests

I V Spectrophotometry

0400 Spectrometry lI N Spectrophotometry

m lI Spectrophotometry

I V A Ultraviolet-visible spectrophotometry

0401 Ultraviolet-Visible Spectrophotometry lI N A Ultraviolet-visible spectrophotometry

m lI A Ultraviolet-visible spectrophotometry

I V e Infrared spectrophotometry
0402 Infrared Spectrophotometry
lI N e Infrared spectrophotometry

lI N E Fluorimetry
0405 Fluorescence Spectrophotometry
m lI e Fluorimetry

I V D Atomic Absorption Spectrophotometry

0406 Atomic Absorption Spectrophotometry lI N D Atomic Absorption Spectrophotometry

m lI B Atomic Absorption Spectrophotometry

lI N F Flame Photometry
0407 Flame Photometry
m lI D Flame Photometry
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued

The code The volume

Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

Inductively Coupled Plasma-Atornic Ernission Inductively Coupled Plasma-Atornic Ernission

0411 I XI E
Spectrometry Spectrometry

0412 Inductively Coupled Plasma-Mass Spectrometry I XI D Inductively Coupled Plasma-l\1ass spectrometry

0421 Raman Spectrometry II XX L Raman Spectrometry

0431 Mass Spectrometry II IX J Mass spectrometry

0441 Nuclear Magnetic Resonance Spectrometry II IX K Nuclear Magnetic Resonance Spectrometry

0451 X-ray Diffraction II IX F X-ray powder diffraction

0500 Chromatography

I VI A Paper chromatography
0501 Paper Chromatography II V A Paper chromatography

III III A Paper chromatography

I VI B Thin-layer chromatography
0502 Thin-Layer Chromatography
II V B Thin-layer chromatography

I VI e Column chromatography
0511 Column Chromatography
II V e Column chromatography

I VI D High performance liquid chromatographyhy

0512 High Performance Liquid Chromatography II V D High performance liquid chromatographyhy

III III B High performance liquid chromatographyhy

I VI G Ion Chromatography

0513 Ion Chromatography II V J Ion Chromatography

III III E Ion Chromatography

II V H Size-exclusion chromatography
0514 Size-Exclusion Chromatography
III III D Size-exclusion chromatography

I VI E Gas chromatography

0521 Gas Chromatography II V E Gas chromatography

III III e Gas chromatography

0531 Supercritical Fluid Chromatography
0532 Liquid Chromatography at Critica! Condition

II V F Electrophoresis

III N A Cellulose Acetate Film Electrophoresis

0541 Electrophoresis III N B Agarose Electrophoresis

III N e SDS-Polyacrylamide Gel Electrophoresis

III N D Isoelectric Focusing Electrophoresis

I VI F Capillary Electrophoresis
0542 Capillary Electrophoresis
II V G Capillary Electrophoresis

0600 Determination of Physical Constant

0601 Determination of Relative Density

I w A Determination of Relative Density
II VI A Determination of Relative Density
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued

The code The volume

Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

I VIl B Determination of Distilling Range

0611 Determination of Distilling Range
Il VI B Determination of Distilling Range
I VIl e Determination of Melting Point
0612 Determination of Melting Point
Il VI e Determination of Melting Point

I VIl D Determination of Congealing Point

0613 Determination of Congealing Point
Il VI D Determination of Congealing Point

I VIl E Determination of Optical Rotation

0621 Determination of Optical Rotation
Il VI E Determination of Optical Rotation

I VIl F Determination of Refractive Index

0622 Determination of Refractive Index
Il VI F Determination of Refractive Index

I VIl G Determination of pH Value

0631 Determination of pH Value Il VI H Determination of pH Value

m V A Determination of pH Value

I XI F Determination of Osmolality

0632 Determination of Osmolality Il IX G Determination of Osmolality

m V H Determination of Osmolality

0633 Determination of Viscosity Il VI G Determination of Viscosity

0661 Thermal Analysis Il VIII Q Thermal Analysis

Determination of Conductivity of Water for Determination of Conductivity of Water for

0681 Il VIII s
Pharmaceutical Use Pharmaceutical Use

Determination of Total Organic Carbon m Determination of Total Organic Carbon in

0682 Il VIII R
Water for Pharmaceutical Use Water for Pharmaceutical Use

0700 Other Determination Methods

I VIII A Potentiometric Titration and ~d-stop Titration

0701 Potentiometric Titration and Ikad-stop Titration
Il VIl A Potentiometric Titration and ~d-stop Titration

I VIII B Non-aqueous Titration

0702 Non-aqueous Titration
Il VIl B Non-aqueous Titration

0703 Oxygen-Flask Combustion Il VIl e Oxygen Flask Combustion

I IX L Determination of Nitrogen

0704 Determination of Nitrogen Il VIl D Determination of Nitrogen

m VI A Determination of Nitrogen

I IX M Determination of Ethanol
0711 Determination of Ethanol
Il VIl E Determination of Ethanol

Determination of Methoxy, Ethoxy and Determination of Methoxy, Ethoxy and

0712 Il VIl F
Hydroxypropoxy Groups Hydroxypropoxy

I IX N Tests of Fats and Fatty Oils

0713 Tests of Fats and Fatty Oils
Il VIl H Tests of Fats and Fatty Oils
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The axl.e The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

0721 Vitamin A Assay JI VIl J Assay of Vitamin A

0722 Vitamin D Assay JI VIl K Assay of Vitamin D

JI VIl M Determination of Protein Content

0731 Determination of Protein Content
IlI VI B Determination of Protein Content

0800 Limit tests

I IX e Limit Test for Chlorides

0801 Limit Test for Chlorides
JI VIII A Limit Test for Chlorides

0802 Limit Test for Sulfates JI VIII B Limit Test for Sulfates

0803 Limit Test for Sulfides JI VIII e Limit Test for Sulfides

0804 Limit Test for Selenium JI VIII D Limit Test for Selenium

0805 Limit Test for Fluorides JI VIII E Limit Test for Fluorides

JI VIII F Determination of Cyanide

0806 Determination of Cyanide
IlI VI X Determination of Residual Cyanide Content

I IX D Limit Test for Iron

0807 Limit Test for Iron
JI VIII G Limit Test for Iron

0808 Limit Test for Ammonium JI VIII K Limit Test for Ammonium

I IX E Limit Test for Heavy Metals

0821 Limit Test for Heavy Metals
JI VIII H Limit Test for Heavy Metals

I IX F Limit Test for Arsenic

0822 Limit Test for Arsenic
JI VIII J Limit Test for Arsenic

I IX G Determination of Loss on Drying

0831 Determination of Loss on Drying JI VIII L Determination of Loss on Drying

IlI VIl L Determination of Loss on Drying

I IX H Determination of Water

0832 Determination of Water JI VIII M Determination of Water

IlI VIl D Determination of Water

I IX J Determination of Residue on lgnition

0841 Determination of Residue on lgnition
JI VIII N Determination of Residue on lgnition

0842 Limit Test for Readily Carboniz.able Substances JI VIII o Limit Test for Readily Carboniz.able Substances

JI VIII p Determination of Residual Solvents

0861 Determination of Residual Solvents
IlI VI V Determination of Residual Solvents
0871 Determination of Methanol I IX T Determination of Methanol
0872 Acetic Acid in Synthetic Peptides JI VIl N Acetic Acid in Synthetic Peptides
0873 Determination of 2-Ethylhexanoic Acid JI V1I L Determination of 2-Ethylhexanoic Acid
0900 Tests for Special Properties or Other Indicators

I XI A Colour of Solution
0901 Colour of Solution
JI IX A Colour of Solution
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The code The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

0902 Clarity of Solution 11 IX B Clarity of Solution

I IX R Test for Particulate Matter in lnjections

0903 Test for Particulate Matter in lnjections 11 IX e Test for Particulate Matter in lnjections

III V 1 Test for Particulate Matter in Injections

I XI e Test for Visible Particles

0904 Test for Visible Particles 11 IX H Test for Visible Particles in Injections

III V B Test for Visible Particles

I XII A Determination of Disintegration

0921 Determination of Disintegration 11 X A Determination of Disintegration

III V e Determination of Disintegration

Disintegration Test for Suppositories and

I XII B
Vaginal Tablets

Disintegration Test for Suppositories and Disintegration Test for Suppositories and
0922 11 X B
Vaginal Tablets Vaginal Tablets

Disintegration Test for Suppositories and

III V D
Vaginal Tablets

11 X G Test for Tablet Friability

0923 Test for Tablet Friability
III V E Test for Tablet Friability

11 X e Dissolution Test
0931 Dissolution and Drug Release Test
11 X D Drug Release Test

0941 Test for Content Uniformity 11 X E Test for Content Uniformity

I XII e Mínimum Fill

0942 Mínimum Fill 11 X F Mínimum Fill

III V F Mínimum Fill

Preparations For Inhalation: Aerodynamic Determination of Particle or Droplet Size

0951 11 X H
Assessment of Fine Particles Distribution of Inhalation Preparations

I XII E Determination of Cataplasms Adhesion

0952 Determination of Adhesion
11 X J Determination of Patchs Adhesion

0981 Crystallini ty 11 IX D Crystallinity

I XI B Determination of Particle Size

Determination of Particle Size and Particle Determination of Particle Size and Particle
0982 11 IX E
Size Distribution Size Distribution

III V G Determination of Particle Size

0983 Measurement of Consistency by Penetrometry 11 X K Measurement of Consistency by Penetrometry

1100 Biological Tests

I XIII B Test for Sterility

1101 Sterility Tests 11 XI H Test for Sterility

III XII A Sterility Test

 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The code The volurne
Name of the general chapters in Chp 2015 Name of the appendices in Chp 201 O
number in Chp 2010

I XIII e Microbial Limit Test

Microbiological Examination of Non-sterile
ll05 Il XI J Microbial Limit Test
Products: Microbial Enumeration Tests
III Xll G Microbial Limit Test
I XIII e Microbial Limit Test
Microbiological Examination of Non-sterile
ll06 Il XI J Microbial Limit Test
Products: Tests for Specified Microorganisms
III Xll G Microbial Limit Test
I XIII e Microbial Limit Test
Microbiological Acceptance Criteria of Non-
ll07 Il XI J Microbial Limit Test
sterile Pharmaceutical Products
III Xll G Microbial Limit Test

Guidelines for Antimicrobial Effectiveness

I XVIll D
Testing

Guidelines for Antimicrobial Effectiveness

ll21 Antimicrobial Effectiveness Testing Il XX N
Testing

Guidelines for Antimicrobial Effectiveness

III XVIl A
Testing

I XIII E Test for Abnormal ToY...icity

ll41 Test for Abnormal Toxicity Il XI e Test for Undue Toxicity

III Xll F Test for Abnormal Toxicity

I XIII A Test for Pyrogens

ll42 Test for Pyrogens Il XI D Test for Pyrogen

III Xll D Pyrogen Test

I XIII D Test for Bacteria! Endotoxin

ll43 Test for Bacteria! Endotoxin Il XI E Test for Bacteria! Endotoxin

III Xll E Test for Bacteria! Endotoxin

ll44 Test for Vasopressor Substance Il XI F Test for Vasopressor Substance

I XIII F Test for Depressor Substances

ll45 Test for Depressor Substances
Il XI G Test for Depressor Substances

ll46 Test for Histamine

I XIII G Test for Allergen

ll47 Test for Allergen
Il XI K Test for Allergen

I XIII H Test for Hemolysis and Agglomeration

ll48 Test for Hemolysis and Agglomeration
Il XI L Test for Hemolysis and Agglomeration

1200 Biological Activity Assays

1201 Microbiological Assay of Antibiotics Il XI A Microbiological Assay of Antibiotics

Preparation of Penicillinase and Iktermination of Preparation of Penicillinase and Iktermination

1202
Its Activity
Il XI B
of Its Activity

1205 Biological Assay of Vasopressin Il Xll A Biological Assay of Vasopressin

 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The code The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

1206 Determination of Cytochrome C Activity II XII B Determination of Cytochrome C Activity

1207 Assay of Hyaluronidase II XII c Assay of Hyaluronidase

1208 Biological Assay of Heparin II XII D Biological Assay of Heparin

1209 Biological Assay of Chorionic Gonadotrophin II XII E Biological Assay of Chorionic Gonadotrophin

1210 Biological Assay of Oxytocin II XII F Biological Assay of Oxytocin

1211 Biological Assay of lnsulin II XII G Biological Assay of lnsulin

Test for the Prolongation Effect of

1212 II XII H Test for the Prolongation of lnsulin Effect
Protamine Zinc lnsulin

1213 Biological Assay of Protamine Sulfate II XII J Biological Assay of Protamine Sulfate

1214 Biological Assay of Digitalis II XII K Biological Assay of Digitalis

1215 Toxicity Test of Sodium Stiboglu-conate II XII L Toxicity Test of Sodium Stibogluconate

Biological Assay of Follicle Stimulating Biological Assay of Follicle Stimulating

1216 II XII M
Hormone Hormone

1217 Biological Assay of Luteinising Hormone II XII N Biological Assay of Luteinising Hormone

1218 Biological Assay of Calcitonin II XII o Biological Assay of Calcitonin

1219 Biological Assay of Growth Hormone II XII p Biological Assay of Growth Hormone

1401 The Test of Radiopharmaceutical Preparations II XIII The Test of Radiopharmaceutical Preparations

I XVI Methods of Sterilisation

1421 Methods of Sterilization II XVII Methods of Sterilisation

III XV Methods of Sterilisation

1431 Statistical Methods for Biological Assays II XIV Statistical Methods for Biological Assays

Special Methods for Traditional Chinease

2000
Medicines

2001 Microscopical ldentification I II c Microscopical Identification

2101 Determination of Swelling Capacity I IX o Determination of Swelling Capacity

2102 Determination of Plaster Softening Point I XII D Determination of Plaster Softening Point

2201 Determination of Extractives I X A Determination of Extractives

2202 Determination of Tannins I X B Determination of Tanninoids

2203 Determina tion of Cineol I X c Determination of Cineol

2204 Determination of Volatile Oíl I X D Determination of Volatile Oil

2301 Determination of Foreign Matter I IX A Determination of Foreign Matter

2302 Determination of Ash I IX K Determination of Ash

2303 Limit Tests of Rancidity I IX p Limit Tests of Rancidity

Determination of Lead ( Pb), Cadmium Determination of Lead ( Pb), Cadmium

2321 ( Cd) , Arsenic ( As) , Mercury ( Hg) and I IX B ( Cd) , Arsenic (As), Mercury (Hg) and
Copper (Cu) Copper (Cu)
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The ccx:l.e The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

Determination of Mercury and Arsenic

2322
Speciation and Their Valence States

2331 Determination of Residue of SulfurDioxide I IX u Determination of Residue of Sulfur Dioxide

2341 Determination of Pesticide Residues I IX Q Determination of Pesticides Residue

2351 Determination of Aflatoxins I IX V Determination of Aflatoxins

2400 Determination of Materials in Injections I IX s Determination of Materials in Injection

3000 Special Mothods for Biological Products

3100 The Mothods for Content Determination

3101 Determination of Total Salid m vn M Determination of Total Salid

Determination of Sialic Acid Content

3102
(Resorcinol Colourimetry)
m VI e Determination of Sialic Acid Content

3103 Determination of Phosphorus Content m vn A Determination of Phosphorus Content

3104 ~termination of Ammonium Sulfate Content m vn e ~termination of Ammonium Sulfate Content

3105 Determination of Sodium Bisulfite Content m vn E Determination of Sodium Bisulfite Content

Determination of Aluminium Hydroxide (or Determination of Aluminium Hydroxide

3106
Aluminium Phosphate) Content
m wl1L F
( or Aluminium Phosphate) Content

3107 Determination of Sodium Chloride Content m vn G Determination of Sodium Chloride Content

3108 Determination of Citrate Content m vn H Determination of Citrate Content

3109 Determination of Potassium Content m vn I Determination of Potassium Content

3110 Determination of Sodium Content m vn J Determination of Sodium Content

3111 Determination of Sodium Caprylate m VI K Determination of Sodium Caprylate Content

3112 Determination of Acetyltryptophan m VI w Determination of Acetyltryptophan

3113 Determination of Phenol Content m VI M Determination of Phenol Content

3114 Determination of Meta Cresol Content m VI N Determination of Metacresol Content

3115 Determination of Thimerosal Content m vn B Determination of Thimerosal Content

~termination of Methyl Parahydroxybenzoate ~termination of Methyl Parahydroxybenzoate

3116
and Propyl p- Hydroxybenwate Contents
m VI T
and Propyl p- Hydroxybenzoate Contents

3117 Determination of 0-Acetyl Content m VI F Determination of 0-Acetyl Content

3118 Determination of Adipic Dihydrazide Content m VIII K ~termination of Adipic Dihydrazide Content

Determination of Content in High Molecular Determination of Content m High

3119 III VIII L
W eight Conjugate Molecular W eight Conjugate

Determination of Saccharide and Sugar Determination of Saccharide and Sugar

3120
Alcohol Contents in Human Blood Products
m VI p
Alcohol Contents in Human Blood Products

Determination of Polymer Content m Determination of Polymer Content m

3121
Human Albumin
m VI Q
Human Albumin

Determination of lgG Monomer and Dimer Determination of lgG Monomer and Dimer
3122
in Human lmmunoglobulins
m VI R
in Human Immunoglobulins
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The code The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

Determination of Glycine Contentin Human Determination of Glycine Content m

3123 III VI s
lmmunoglobulin Human Immunoglobulin

Determination of Protein Content m Determination of Protein Content m

3124 Recombinant Human Granulocyte Colony- III VI u Recombinant Human Granulocyte Colony-
stimulating Factor stimulating Factor

Determination of Free Histamine Phosphate Deterrnination of Free Histamine Phosphate

3125 III VI E
in Human Histamine Immunoglobulin in Human Histamine Immunoglobulin

Determination of lgG Content ( Ultraviolet-

3126 III XI K Deterrnination of lgG Content
visible Spectrophotometry)

Deterrnination of M>lecular Size Heterogeneities

3127
in Mmoclonal Antibodies

3200 Determination of Chemical Residues

Determination of Residual Ethanol Content

3201 III VI D Determination of Residual Ethanol Content
( Conway Diffusion Method)

Determination of Residual Polyethylene Glycol Determination of Residual Polyethylene Glycol

3202 III VI G
Content Content

Determination of Residual Polysorbate 80 Determination of Residual Polysorbate 80

3203 III VI H
Content Content

Determination of Residual Glutaraldehyde Determination of Residual Glutaraldehyde

3204 III VI 1
Content Content

Determination of Residual Tributyl- Determination of Residual Tributylphosphate

3205 III VI J
phosphate Content Content

Determination of Residual Carbodiimide Determination of Residual Carbodiimide

3206 III VI y
Content Content

Determina tion of Free Formaldehyde Determination of Free Formaldehyde

3207
Content
m VI L
Content

Deterrnination of Residual Aluminium Determination of Residual Aluminium

3208
Content in Human Albumin
m VIl K
Content in Human Albumin

3209 Determination of Residual Hydroxylamine

3300 Microbiological Test

3301 Test far Mycoplasma m XI[ B Test far Mycoplasma

3302 Test far Adventitious Viruses III XI[ e Test far Adventitious Viruses

3303 Test far Murine Viruses III XI[ H Test far Murine Virus

3304 Test far Nucleotide Sequence of SV40 III IX H Test far Nucleotide Sequence of SV40

3305 Neurovirulence Test in Monkeys III XI L Neurovirulence Test in Monkeys

 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The code The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

Requirements for Virus Nucleic Acid Testing on

3306
&Jurce Plasma used for Blood Products

3400 Bioassay Method

3401 lmmunoblot III VIII A lmmunoblot

3402 Immunodot III VIII B lmmunoblot

3403 Double lmmunodiffusion III VIII e Double Immunodiffusion

3404 Immunoelectrophoresis III VIII D Immunoelectrophoresis

3405 Peptide Mapping III VIII E Peptide Mapping

3406 Test for Losing Rate of Plasmid III IX G Test for Losing Rate of Plasmid

3407 Determination of Residual Extraneous DNA III IX B Determination of Residual Extraneous DNA

Determination of Residual Antibiotics

3408
(Culture Method)
III IX A Determination of Residual Antibiotics

Determination of Prekallikrein Activator Determination of Prekallikrein Activator

3409 III IX F
Content Content

3110 Test for .A.nticomplement Activity m TV

lA
T.T
I'\. Test for Anticomplement Activity

Determination of Residual Bovine Serum Determina tion of Residual Bovine Serum

3411 III VIII 1
Albumin Content Albumin Content

Determination of Residual Host Bacterial Determination of Residual Host Bacteria!

3412
Protein (E. coli)
III IX e Protein (E. coli)

Determination of Residual Host Bacteria! Determination of Residual Host Bacterial

3413 III IX D
Protein (Pseudomonas) Protein (Pseudomonas)

3414 Iktermination of Residual Host Yeast Protein III IX E Iktermination of Residual Host Yeast Protein

Test for Blood Group A-Like Substance

3415 III IX 1 Test for Blood Group A-like Substance
(Hemagglutination lnhibition Assay)

3416 Determination of Residual Murine lgG III IX L Determination of Residual Murine lgG

Identity Test for Acellular Pertussis Vaccines Identity Test for Acellular Pertussis Vaccines
3417 III IX s
(Enzyme Linked Immunosorbent Assay) (Enzyme Linked lmmunosorbent Assay)

Identity Test for Antitoxin and Antiserum Identity Test for Antitoxin and Antiserum
3418 III IX T
(Enzyme Linked lmmunosorbent Assay) (Enzyme Linked lmmunosorbent Assay)

Determination of Molecular Size Distribution Determination of Molecular Size Distribution

3419 III VIII G
for Group A Meningococcal Polysaccharide for Group A Meningococcal Polysaccharide

Determination of Molecular Size Distribution Determination of Molecular Size

3420 III VIII H
far Typhoid Vi Polysaccharide Distribution far Typhoid Vi Polysaccharide

Determination of Polysaccharide Content in Determination of Polysaccharide Content in

3421 Haemophilus influenzae Type b Conjugate III VIII J Haemophilus influenzae Type b Conjugate
Vaccine Vaccine
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The code The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

3422 Test for Human Thrombin Activity III IX N Test for Human Thrombin Activity

3423 Test for Activated Coagulation Factor Activity III IX o Test for Activated Coagulation Factor Activity

Iktermination of Heparin Content ( Coagulation

3424 III IX p Determination of Heparin Content
Method)

Test for Anti-A and Anti-B Hemagglutinins

3425 III IX J Test for Anti-A and Anti-B Hemagglutinins
(lndirect Anti-human Globulin Test)

Determination of Human Erythrocyte Determination of Human Erythrocyte

3426 III IX Q
Antibody (Microtitre Plate Method) Antibody

3427 Determination of Human Platelet Antibody III IX R Determination of Human Platelet Antibody

3500 Test for Biological Activity/Potency

In vitro Test for Relative Potency of In vitro Test for Relative Potency of
3501 III X A
Recombinant Hepatitis B Vaccine (Yeast)
Recombinant Hepatitis B Vaccine (Yeast)

In vitro Test for Relative Potency In vitro Test for Relative Potency of
3502 III X s
ofHepatitis A Vaccine, Inactivated Hepatitis A Vaccine, lnactivated

Potency Test on Rabies Vaccine for Human Potency Test on Rabies Vaccine for Human
3503 III XI A
Use (NIH Method) Use

3504 Potency Test on Adsorbed Tetanus Vaccine III XI B Potency Test on Adsorbed Tetanus Vaccine

3505 Potency Test on Adsorbed Diphtheria Vaccine m Xl e Potency Test on Adsorbed Diphtheria Vaccine

3506 Determination of Flocculation Unit of Toxoid III XI D Determination of Flocculation Unit of Toxoid

Potency Test on Diphtheria Antitoxin

3507 III XI E Potency Test on Diphtheria Antitoxin
(Rabbit Skin Test)

Potency Test on Tetanus Antitoxin (Mouse

3508 III XI F Potency Test on Tetanus Antitoxin
Bioassay)

Potency Test for Gas-gangrene Antitoxins

3509 III XI G Potency Test for Gas-gangrene Antitoxins
(Mouse Bioassay)

Potency Test for Botulinum Antitoxin

3510 III XI H Potency Test for Botulinum Antitoxin
(Mouse Bioassay)

3511 Potency Test for Antivenins (Mouse Bioassay) III XI 1 Potency Test for Antivenins

3512 Potency Test for Rabies lmmunoglobulin III XI J Potency Test for Rabies Antiserum

Potency Test for Diphtheria Antitoxin m Potency Test for Diphtheria Antitoxin m
3513 III X o
Human lmmunoglobulin Human lmmunoglobulin

3514 Test for Fe Function in Human Immunoglobulin III X p Test for Fe Function in Human lmmuooglobulin

Potency Test for Anti-human T Potency Test for Anti-human T

3515 Lymphocyte lmmunoglobulin (E Rosette III X Q Lymphocyte lmmunoglobulin (E Rosette
Formation-inhibition Test) Formation-inhibition Test)

Potency Test for Anti-human T Lymphocyte Potency Test for Anti-human T Lymphocyte
3516 III X R
lmmunoglobulin (Lymphocytotoxicity Test) lmmunoglobulin (Lymphocytotoxicity Test)
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The code The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

Potency Test for Human Coagulation Factor Potency Test for Human Coagulation
3517
11 ( One-step Method)
m X J
Factor 11 ( One-step Method)

Potency Test for Human Coagulation Factor Potency Test for Human Coagula tion
3518 III X K
VH ( One-step Method) Factor VI[

Potency Test for Human Coagulation Factor Potency Test for Human Coagula tion
3519 III X L
IX ( One-step Method) Factor IX

Potency Test for Human Coagulation Factor Potency Test for Human Coagulation
3520
X ( One-step Method)
m X M
Factor X

Potency Test for Human Coagulation Factor Potency Test for Human Coagulation
3521
VIIl ( One-step Method)
m X N
Factor VIII

In viw Test for Biological Activity of

In vivo Test for Biological Activity of
3522 Recombinant H.nnan Erythro¡x>ietin (Reticulocyte m X B
Recombinant Human Erythropoietin
l\.1etln.l)

3523 Biological Activity Test on Interferon III X e Biological Activity Test on Interferon

Biological Activity Test for Recombinant

Biological Activity Test for Recombinant
3524 Human lnterieukin-2 ( CTLL-2 Ceii /MTT fil X D
Human lnterleukin-2
Colorimetric Method)

Biological Activity Test for Recombinant

Biological Activity Test for Recombinant
Human Granulocyte Colony-stimulating
3525
Factor ( NFS-60 Cell Strain/MTT
m X E Human Granulocyte Colony-stimulating
Factor
Colorimetric Method)
Biological Activity Test on Recombinant Biological Activity Test on Recombinant
Human Granulocyte/Macrophage Colony- Human Granulocyte/ Macrophage Colony-
3526 III X F
Factor ( TF-1
stimulating Cell/MTT stimulatingFactor ( TF-1 Cell/MTT
Colorimetric Method) Colorimetric Method)

Biological Activity Test for Recombinant fuvine

Biological Activity Test for Recombinant
3527 Basic Fibroblast Growth Factor ( Cell m X G
Bovine Basic Fibroblast Growth Factor
Proliferation/MIT C.OlorimetricMethod)

Biological Activity Test for Recombinant

Biological Activity Test for Recombinant
3528 Epidermal Growth Factor ( Cell Proliferation/ III X H
Epidermal Growth Factor
MTT ColorimetricMethod)

Biological Activity Test for Recombinant Biological Activity Test for Recombinant
3529
Streptokinase
m X 1
Streptokinase

Biological Activity Test for Mouse Nerve

3530
Growth Factor

Biological Activity Test for Nimotuzumab

3531
Injection

Biological Activity Test for Recombinant

3532 Human lnterleukin-11 ( lnterleukin B9-11
Cell/MTT Colorimetric Method)
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
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Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

Potency Test far Botulinum Toxin Type A

3533
(Method of Parallel Lines)

3600 Specific Biologica Raw Materials/ Animals

Test Requirements far Monitoring SPF Test Requirements far Monitoring SPF
3601
Chick Embryos
III XIII A
Chick Embryos

Requirements far Microbiological Test on Test Requirements of Micro bes far

3602
Laboratory Animals
III XIII B
Laboratory Animals

Requirements far Parasitological Test on Test Requirements of Parasites far

3603
Laboratory Animals
III XIII c
Laboratory Animals

3604 Test Requirements far Newborn Calf Serum III XIII D Test Requirements far Newbom Calf Serum

Culture Media for Biochemical Reactions of Culture Media far Biochemical Reactions of
3605
Bacteria and Test Method
III XIV
Bacteria and Test Method

3700

List of National Standards far Biologics (far

3701 Potency, Ti ter, Activity, Concentration and
Content Tests)

8000 Reagents and Reference Substance

I XV A Reagents
8001 Reagents
Il XV A Reagents

I XV B Test Solutions
8002 Test Solutions
Il XV B Test Solutions

I XV c Test Papers
8003 Test Papers
Il XV c Test Papers

I XV D Buffer Solutions
8004 Buffer Solutions
Il XV D Buffer Solutions

I XV E Indicator Solutions
8005 Indicator Solutions
Il XV E Indicator Solutions

I XV F Volumetric Solutions
8006 Volumetric Solutions
Il XV F Volumetric Solutions

Chemical Reference Substances, Reference

Reference Standards, Reference Drugs and I XV G
Drugs and Reference Extractives
8061
Reference Extractives
Il XV G Reference Standards

8062 Reference Standards

9000

Guidelines far the Stability Testing of Drug Guidelines far the Stability Testing of Drug
9001
Substances and Preparations
Il XIX c
Substances and Preparations
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The cocle The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
number in Chp 2010

Guidelines for in vivo Bioavailability and Guidances m VlVO Bioavailability and

9011 Il XIX B
Bioequivalence Studies for Drug Preparations Bioequivalence Studies for Drug Preparations

Guidelines for Validation of Quantitative

9012
Analytical Method of Biological Samples

Guidelines for Sustained, Controlled and Guidelines for Sustained, Controlled and
9013 II XX D
Delayed Release Preparations Delayed Release Preparations

Guidelines for Preparations of Microcapsules,

9014 Guidelines for Microparticle Preparations II XX E
Microspheres and Liposomes

Guidelines for Studies and Quality Control of

9015
Drug Polymorphism

Guidelines for Validation of Analytical

I XVlll A Method Adopted m Traditional Chinese
Guidelines for Validation of Analytical
Medicine Quality Specification
9101 Method Adopted in Pharmaceutical Quality
Specifica tion Guidelines for Validation of Analytical
Il XIX A Method Adopted in Pharmaceutical Quality
Specifica tion

Guidelines for the Analysis of lmpurities in Guidelines far the Analysis of Impurities in
9102 II XIX F
Drugs Drugs

9103 Guidelines for Hygroscopicity II XIX J Guidelines far Hygroscopicity

Guideline for Near-infrared ( NIR ) Guideline for Near-infrared ( NIR )

9104 Il XIX K
Spectrophotometry spectrophotometry

Guidelines for Bioactive Assays of Guidelines far Bioactive Assays of

9105 I XVlll e
Traditional Chinese Medicine Traditional Chinese Medicine

Guidelines for Pharmaceutical Evaluation

9106
based on Microarrays

Guidelines for Molecular DNA Barcoding of

9107
Chinese Materia Medica

Guidelines for Validation of Alterna ti ve

I XVlll E Microbiological Methods for Pharmaceutical
Products

Guidelines for Validation of Alterna ti ve Guidelines for Validation of Alterna ti ve

9201 Microbiological Methods for Pharmaceutical II XIX o Microbiological Methods for Pharmaceutical
Products Products

Guidelines far Validation of Alterna ti ve

III XVIl B Microbiological Methods far Pharmaceutical
Products

The Guidelines for Microbiological I XVlll F The Guidelines of the Microbial Limit Tests
9202
Examination of Non-sterile Products II XIX p The Guidelines of the Microbial Limit Tests
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The cod.e The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
nurnber in Chp 2010

Guidelines for Pharmaceutical Microbiology

Guidelines for Quality Management of I XVHI G
Laboratory Practices
9203 Microbiology Laboratory for Pharmaceutical
Products Guidelines for Pharmaceutical Microbiology
II XX Q
Laboratory Practices

Guidelines for Microbial Characterization,

9204
Identification, and Strain Typing

Guidelines for l\.1icrobiological Monitoring and

9205 Control of Clean Rooms for Pharmaceutical
Products

Guidelines for Validation of Isolator Systems

9206
for Use in Sterility Testing

Guidelines for Application of Safety Tests for

I XVHI B
Injection of Traditional Chinese Medicines
Guidelines for Application of Safety Tests
9301
for Inj ections
Guidelines for Application of Safety Tests
II XIX M
for Injection of Chemical Medicines

Guidelines for Establishment of Limit for

9302 Harmful Residue of Traditional Chinese
Medicine
-

9303 Guidelines for Determination of Pigments

Guidelines for Iktermination of Aluminium

9304 (Al), Chromium (Cr), Iron (Fe) and Barium
(Ba) in Traditional Chinese Medicine

Guidelines for Determination of Mycotoxins

9305
in Traditional Chinese Medicine

Guidelines for the Quality Control of Positron Guidance for the Quality Control of
9501 Emission Tomographic Radiopharmaceutical II XIX G Positron Emission Tomographic Radiophar-
Preparation maceutical Preparation

Guidelines for the Quality Control of Technetium Guidance for the Quality Control of Technetium
9502 II XIX H
[
99
m Te J Radiopharmaceutical Preparations [99 mTc] Radiopharmaceutical Preparations

Guidelines for Research and Evaluation on

9601 F unctionali ty-rela ted Characteris tics of
Pharmaceutical Excipients

Guidelines for General Requirements for

9621
Drug Packaging Materials and Containers

Guidelines for Pharmaceutical Glass Materials

9622
and Containers
 The list of the code number of the general chapters in Chp 2015 compared with that of the appendix in Chp 2010

continued
The code The volume
Name of the general chapters in Chp 2015 Name of the appendices in Chp 2010
mnnber in Chp 2010

Guideline for Preparation of Pharmaceutical

9901
Standard Substances of China

Names, Symbols and Atomic Weights of

I XVJ[
Elements

Names, Symbols and Atomic Weights of Names, Symbols and Atomic Weights of
JI XVlll
Elements Elements

Names, Symbols and Atomic Weights of

III XVlll
Elements

lngredients of Patent Medicines not lncluded lngredients of Patent Medicines not

I III
in the Pharmacopoeia Included in the Pharmacopoeia
MONOGRAPHS
PART ll
 Contents of the Appendices

Acacia •·····••·•···•····•·••······••····•••···•••·····••·•·····••· 482 Croscarmellose Sodium ·····•·················••·•·····•······ 520

Acetic Acid · · · · · · · · · •••· · · · · •· · · ••••· · · · •· •· · · · · · •· · · · · ••· •· · · •· · 482 Crospovidone •· •··· · · · •· •· · · ••• ··• · •• · •· · · · •• · ·· · ••• ••· ·· ••· •· · · 522
Activated Charcoal (For Injection) ···•···•···•·••··••••··· 483 Cyclomethicone · · •••• · ·· ·•• •·• ... ••• ••• ••• ·•• ... •·· ••• ••• •·· ... 522
Adenine · •••••· .. · •••••· · · ••· •· · · · · · ••· · •· · · · · ••· •••· · ••· •· · · · •· ••· · 484 Dextrates ......................................................... 523
Agar •·••·•······•·······•··•···•·•·••·••···•·•••····•··•··••·•··•·· 485 Dextrin · · · · · · ••· · · · · · · · · · · · · ••· · · •· •· · · · · · · •· · · •· · •· · · · · ••· · · · · •.. • 523
Alanine · · · · · · · · · · · · · · · · ••· · •· · · · · •••· ••· •· · · · •· •· · · •· · •· · · · · •· · · · · · 486 Diammonium Hydrogen Phosphate ........................ 524
Alginic Acid ········•••····•·••···•·•·•··········•······••·••····• 486 Dilute Acetic Acid ............................................. 524
Aluminum Magnesium Silicate · · •· ·· •· •· · · •· · ••· · ·• · •• ··· · •· 487 Dilute Phosphoric Acid ·····•······•·······•·········•·····•·· 525
Aluminum Sulfate ·····•••···••••···••••··•••······••·•·····••· 488 Diluted Hydrochloric Acid •· •· ·· •· · · · · ·• · · · · · · · · · •· · · · · · · · · · · · 525
Ammonium Sulfate ·••··•···•·•••··••·····•·••··•··•·····•······ 488 Dimethicone •····•••······••··•·•·••······••··••••··•··••··•····•• 526
Anhydrous Calcium Hydrogen Dimethyl Sulfoxide · · · · · •· · · •· · · ·• · ·· · ·• · · · ·· · · · · •·· · ·• ••• ·· · .. · 527
Phosphate ··· · · •••· ··• · · · · · · ••• •· · · •· · · •· ·· •· •· · ••• · ••· · •• · •· ··• · •• 489 Dipotassium Hydrogen Phosphate •• · · · · · · •· · · · · · •· · · · · · .. •• · 527
Anhydrous Citric Acid •··•····•··•···•·••··•··••····•••····•· 489 Dipotassium Hydrogen Phosphate Trihydrate ............ 528
Anhydrous Sodium Carbonate •·· •·• · •· ·• · · •· · · · •· ••· · · · · •· · 490 Disodium Edetate ................................................ 529
Anhydrous Sodium Sulfite •··•··•·•···••·····••····••··•··•··• 491 Disodium Hydrogen Phosphate Anhydrous •·• ••• · · ... · · · · 530
Arabino Galactan •••··••·••··•·•·····•······•·•···•••·•····•••••· 491 Disodium Hydrogen Phosphate Dodecahydrate 530
Arginine · ·· ••• •· · · · · · · ••· · •·• •·· ··• · •• ·· · •· ••·· · · · •· · · · · •· •· · · •• · 492 Egg Yolk Lecithin ............................................. 531
As paragine · · · · · · · · · · ••· · •· · •· · ••· •••· •· · · ••••· · · •· · •· · · · · •· · · •· · 492 Egg Yolk Lecithin (For Injection) ........................ 533
Aspartame · · · · ·· · •• · · · · · · •· ••· · · · · •· · · · · •• · · · · ••• ••· · •• · •• ·· •· •· 492 Enterosoluble Vacant Gelatin Capsules · · ••· · •·• ••· •·• •• .. •· 535
Aspartic Acid •···········••••·•··••·····•·····•··•······•···•·•· 492 Ethanol ·" •• · · ·· ••• .. · ··• · •• · · · •.. •·· · ·• ••· · ·· .. · •· · · .. •·• · ·• •• · •• · 536
Benzalkonium Bromide •·•··•••···•••·•·•·•••···•·•····•···•·· 492 Ethyl Acetate •.. · · •••· · · · .. •· .. · .. •· · · · · · •· · · · · ••.. •.. · .. •.. •· •• 536
Benzalkonium Chloride · · •· ·• ••• •· · •·• · · · •• · · •· · · •· · •· · · •· ••· · 493 Ethyl Acrylate and Methyl Methacrylate Copolymer
Benzyl Alcohol · •· · · · ·• · · •· ·· ••·· •· · •• •· · · •· · •· · ··• · · •· ·· · •• ••· · ·· 493 Dispersion · · · ··· .. •· · · · .. •.. · · · ••· · ·· "· •.. · •• •• · •·· ·•• ••• ··· ·.. 536
Benzyl Hydroxybenzoate ••• •· · · · •· · · · · · •· · •· ••· ••·• ••• · · · ··• •• · 493 Ethyl Olea te ··· ••· ··· ·•· ·•· ...... ··· ··• ••· ...... ··· ... •·• •·· ... ••• 537
Betacyclodextrin ····•••··••••····•·••·•••·••··•·••····••·····•·· 494 Ethylcellulose · · · · •• ·• ••• · · ·· ......... ••• ... ·•• ......... ••· ... ••• 538
Black Ferric Oxide ............................................. 495 Ethylcellulose Aqueous Dispersion •• .. •.. ·• · ...... ·" .... • 539
I3orax ............................................................... 496 Ethylcellulose Aqueous Dispersion Type B .................. 539
I3oric Acid ••• · ·· ••• •• · · · · •• · •· · ·· ••· · · · •· · •••· ••• · · · •• ••· · •• ••• · ·· · 497 Ethylparaben ................................................... 541
Brown Ferric Oxide ·•• ··••·· ··• •·• ·•· •·· ··• ••••··•·••····• ··· •·· 497 Eugenol · •· •· · · .... · · · ••.. ·· · · •• .. · .. · •.. ·· · · •· · · · · •• · · •· · · .. •.. · · · • 542
Butyl Alcohol · · •· · · •• · · ·· · · ••·• ··• •• •··· •· · · · · ··· · · · · ·• · · ••· · · · · 498 Fructose * '!''!''!'•····················································· 543
Butylated Hydroxytoluene ·· · .. · ·· · · · · •· · •· ••• · ·· · · .. · ·· ·•• · •• 498 Fumaric Acid ••• ·· · · · •· · · ·• · · · •... ·· •· · · · · · ••· ·· · •· •· •· · · ••· •· ·• 543
Butylparaben ·•• •• · · · •· · · ••• ·· •· · •· ·• · · · •· · •· · •· · ·· · •·• · · •·•• •• · 499 Gallic Acid · · •••· · · · · ••· •· · · · ••••· · •••· •· · · · ••••· · •••••· · · •••· · · • 543
Calcium Chloride ••···••·••··•·••••··••··••••••···••••···••······ 500 Gelatin For Capsules · · · · •• •· · · .. •.. ·•· ... •· · · · ••• · · ·• •• · •· · ·• • 544
Calcium Glycerophosphate ··••••··•••·•·••••·····••····•··•··· 501 Glacial Acetic Acid •.. · · · · •· .. · · .. •.. · .. · •· · · · · ••.. · •· · •.... · · · • 545
Calcium Oxide ······••·•····•······•••··•••·•···••·····•·•••···•· 501 Glycerol · ••· · •· · ••••.. · .. · ••· · · •.. · · · · •.. ••· · · · · •· .. · .. •.. · · · •.. • 546
Calcium Phosphate ·• · •· · ·· ••· •· · · •· •· · · •· · •· · ·• · · •• ·· · · •· ••· ••• 502 Glycerol (For Injection) ··• ••· •·· ·•• •.. · .. •• · •·· ..... •··· ••• 54 7
Calcium Stearate ••·······••·•·•··•·•····•·•···•·····•·•••····••· 503 Glyceryl Behenate ... · •· · ·· ... •·• ··· ... · ·· ••· · · · ·· · ... ·· · ·• · · •· 548
Calcium Sulfate · · · · · ••· · •· •·· · ·•• · · · ·· •••• · ·• · · ••· · · · ••· · · · · •· · 503 Glycine · · · •· · •· · · · · · •· · · · · •· · .. · · •· · · · · · ••· · · · · •· · · •· · · .. •••· .. · .. • 550
Caprylic Acid · · · · •· •· · .. •· · · · .... · · · •.. ••· · ••· ••· · · ••· •· · · · · •••· 504 Hard Fat ..... •........ · •..... "· .. · •.. •....... · ..... •.. · · · •.. •·.. 550
Caramel · •· · · · · ••••· .. · ••· .. ••· · •· · •· •· · •••· •· · .. •· ••· · •· •· •••·.. 505 Histidine ......... · · •· .. •· •.. · · ·· ..... · · •· •· · · •· · .. · · · · · •· · •· · · ·" 550
Carbomer •· · · · · •.. ••· · · •••· · · ••· •· · •· ••· · · •· · •· · · · · .. · · •· .. · .. · · · • 506 Hydrochloric Acid ............................................. 550
Carbomer Copolymer · · .. •· •· · · .. · •· · · ••· · •.. •· •••· ••••· · •· · •· · 507 Hydrogenated Castor Oil .................................... 551
Carbon Dioxide · · · · •· ••••· •· · •· ••· •••••· ••· •••.. •· ••· · •· ••· · · · ••· · 508 Hydrogenated Soybean Oil .................................... 551
Carboxymethylcellulose Calcium ••••·" •· •· •· .. •••· · ••· •· · · 509 Hydroxyethyl Cellulose ..... •.. " .. •.............. •.... " .. ·· • 552
Carboxymethylcellulose Sodium •· •· · ••·" · · •· · · •••••· · ••••••· 509 Hydroxypropyl Betadex · ........... " ......... · .. •.. · · .... •.. · 554
Carnauba Wax ................................................... 510 Hydroxypropyl Cellulose •· .... ••• ......... ·• ...... •...... ·.. 555
Cellacefate ......................................................... 511 Hypromellose · · · •• · •· · ·• · · ·· · · •.. · •· · ·" ... · •· · · •........... · .. · 556
Cellulose Acetate ................................................ 512 Hypromellose Acetate Succinate ........................... 557
Cetostearyl Alcohol ··· ••· ... ··••·· ··• ... •·· ••· ••• ......... •·• ·•· 513 Hypromellose Phthalate ....................................... 558
Cetyl Alcohol ••··•··•···•••••·•··•·•····•····••···••••···•·••••• 513 Isopropyl Alcohol ... · .. •· · ... ••• ...... · · · ·" ·" · · •· · · · · •· .. ·.. 558
Chitosan ......................................................... 514 Kaolin ............................................................ 559
Chlorobutanol ................................................... 515 Lactose •••.. · · .. · .... ••· · ••· · · •.. ••· · •.. •· · .. · •.. •· · .. · .. •.. •.. · .. · 560
Chlorocresol ...................................................... 515 Lanolin · · •.. •.. ••.... ••· •.. •.. ••· · .. ••· ••· · .. •••· •.. · · · .. · •· · · •· ••• 561
Cholesterol ......... ·•· ··· ••• ··• ·•• ... ·•· ••· •·•••• ... •••••• ... •·· 516 Laurocapram ................................................... 561
Citric Acid ••••···•• •••·•••••··· ••• ··· ............ ·•• ••• ··· ••• ••· 517 Lauroyl Macrogolglycerides (6) ........................... 562
Clove Leaf Oil · .. · · •••· •••· · · · .. ••· · ••••· · · •· •••· · ••· •· · "· •· · ·.. 517 Lauroyl Macrogolglycerides (8) ........................... 564
Clove Oil ......................................................... 518 Lauroyl Macrogolglycerides 02) ........................... 565
Cocoa Butter ...................................................... 519 Lauroyl Macrogolglycerides (32) ........................... 567
Colloidal Silicon Dioxide ••· •· ••· •.. •· · · · .. •· · · .. · •••· .. ••· · •••· 519 Leucine ............................................................ 568
Compressible Sugar · .. · •· ·· •••• •·• •• · ·· •· •• · ·· · · •· •· ... •• · ... ••· 520 Light Liquid Paraffin •·· •.. ••• ............... •• · "· ... · •· ••• · •· 568
 Light Magnesium Oxide · ·· "· •• •··· · •.... · ·· · ·• · •· •• · · · •· .. ·.. 569 Pregelatinized Starch .......................................... 635
Liquid Paraffin ••· ···•·· ••· ··• ·•• ··• ···•··•·• ·•· ··· ••• ··· ··· ·•••·• 570 Propionic Acid ••• •· · · · · · · •••• · · •· · · · · · · · ••· · · •• •·· •· · •• · · ·• ·• · · · • 636
Low-substituted Hydroxypropyl Cellulose ··• ••• ·····• · ·• 571 Propylene Carbonate ··• · •••• ••· · · · · ··• •·• · · · · · · · · · · · •·· · · ·• · · • 636
Macrogol 300 (For Injection) .............................. 571 Propylene Glycol ··· •·· •·· •·• ··· ··• ·•• ··• ··• •··••· ...... ··· ••• ... 637
Macrogol 400 ··· ... ·•· ··• ··• ... ··• ·•· ·•• ... ··• ··••·• ··· ··••·• ·•· 573 Propylene Glycol (For Injection) ••• •····· ··••·· ··• ··· ·•• ··• 637
Macrogol 400 (For Injection) .............................. 574 Propylparaben · · · · ·· •· · •· · ••• · · · · · •·• · · · •· · · ··• ··• ••• ... · ·· •· ••• · 639
Macrogol 600 · · · •· · · · •· · •· · · · · · · · •· · · · · · · ••••· •· · · · · · · ••· · •· •· •• 576 Purified Water* ··•••••······•••·•··•····•·•····•·····••··•·•··· 639
Macrogol 1000 ··• ··· ··· ·•· ... ··· ·•• ... ·•· ··· •·· ·•· ··· ••· ... ··· ··· 577 Purple Ferric Oxide ............................................. 639
Macrogol 1500 •·• ............ ··· ··• ......... ·•· ... ··· ...... •·• ··• 578 Red Ferric Oxide ••· ••· · · · · · · •· ••· ••· •·· · ·•· •· · · •• •· · · · · •· · •· •· · · 640
Macrogol 4000 ................................................... 579 Refined Com Oil •· · · •· ••· •· •· •· · · · •••· · · •· •· •· · •••••••· · · · · ••••• 641
Macrogol 6000 · · · •· · .. •·· · · · · ·•• •· · · · · •· •... · ·· •· · •• · •· · · · · ••• ·.. 580 Silicified Microcrystalline Cellulose •·•······•····••·· ··•··• 641
Magnesium Chloride · · · · · •...... •·· · •• · .. •·· · •• · · •· · · · •• · · · •· · 582 Silicon Dioxide · · · •· · ••· ••· •· •· · · · · ••••· · · · · · · · · · •· •••· •· •· · · ••••· 645
Magnesium Oxide · · · ••· •.. · · · · · · •· · · · •· · ••· · .. · · · · · · •••· •· · •· · 582 Simple Syrup · •· · ••· •· •· · · · · · •••· •· · ••· · · •••· · · •••· · · · · ••· •· •· · · 646
Magnesium Stearate .......................................... 583 Soda Lime ••· •· · · · · •· •••· ••· · · · · · · · · · .. · · · · · · · •••.. · •· · · •••· ••· · •· · 646
Magnesium Trisilicate ••· ... •·· •·· ...... ··· •·· ·•· ··· ··· •·· ······ 585 Sodium Aceta te · · · •· · •· · · · · · · •· •• · · ••·· ••· •· · · •• · •· · · •••· ••• · · · 64 7
Maize Starch · · · · · · •••· .. · · · · · •·" · · •· · · · · •.. ••· · · · •· · •.. · · · ••· · · •· 586 Sodium Alginate •·· · · · · •· · ••·• · ·· · ··• · · · •· · •·· ·· · ••• ••• · ·• ·· •••• 64 7
Maleic Acid ... · · · · •· · •· ··· ••• •· · •.. ·· · •• •· · · •· · · •• •• · •· · · · · · •• •· · 586 Sodium Benzoate · •· •· · •· ••••· · · · · · · · ••· •· •.. · · · · ••· · · · •.. · · · •· · • 648
DL-Malic Acid ··· •••·•···· ··• ... ······ ··• ... ·····• ··• •·· ··•••· ·•· 587 Sodium Bicarbonate · · · · •• •• ••·· ··• ••• · · · •· · ··· ·· · •·• •• · · · · ·• · ••• 649
L-Malic Acid ··••···•· ·•• ... ··· ••· ...... ··· •·· ·•• ··· ··· ••• ·•· ··· 588 Sodium Bisulfite · · · ••· •· · · · · · · ••· •· · · ·· •··• •· · · •• · · · · · · ••• •· •· · · 649
Maltodextrin ...................................................... 589 Sodium Caprylate · •• · •· · •· ·· ••• · •·• ••• · ·• · · · · •• •• · · ·· ·• · · ·• •• · •·· 650
Maltol ............................................................ 590 Sodium Chloride (For Injection) ··• ·•••·· ··••·· ··•••· •·• ··· 650
Maltose · · •••· •· · · · •· ••· ••· · · •· · •· · •· · •· · · •••· · · · · ••· •· · •· · · · · ••· · •• 590 Sodium Citrate · · · •·· · · · · •· · •••·· · ·· ••• · · · •· · · ·· ·• · ••• ·•• •· · ·•• ••• 652
l-Menthol ......................................................... 591 Sodium Cyclamate ............................................. 652
Methacrylic Acid Copolymer I ······························ 592 Sodium Deoxycholate ••• •• · •· •··· ••• · ·• •· · · ·· ·• · •• · ••• •· · ·• •••• 653
Methacrylic Acid Copolymer 11 .............................. 592 Sodium Glutamate •· · · · · ·•• · · · •· · · · · •· · · •• •• · · · •· · · · · •••• ••• · · • 654
Methylcellulose ··· ·•· ... ••· ··· ••· ·•·••• ··· •·· ·•· ··· •·· ... ·•··•• 593 Sodium Hydroxide · •· •· · · •· ·•• ... •·• · ·• · · · · · ••• · ••· ·· · •· · ... ··· 654
Methylparaben •·• ·· · · · •· · ••· · · ·• •· · •· · ·· •·•• ••• •· · · · •••• · •• · · · · •• 594 Sodium Lauryl Sulfate ·· · ·• •· · •· · ••· · ·· · ••• •• · · · · •· · ·· · •·· •· •· · · 654
Microcrystalline Cellulose .................................... 595 Sodium Metabisulfite · •• · •· ••· •·• ••• · ·• •· · · ·· ·• · •• · ·• · ••· •·• ••• 655
Microcrystalline Wax .......................................... 598 Sodium Methyl Parahydroxybenzoate •· · · · ••· ••· ••·· •· •· · • 655
I'--~icotinamide ............................................................. 599 Sodium Oieate •.. · ·· · · ••· •... •· · ............ · .... · .. · .. · .. · ... ... 656
Nicotinic Acid ................................................... 599 Sodium Propyl Parahydroxybenzoate ••• •• ......... •....... ·• 658
Oleoyl Macrogolglycerides •· •••••· •· · · · · •· •••· •· · · ••· •••· · · · · · 600 Sodium Starch Glycolate · · · ••• ......... •• ......... · ... ... .. .... 658
Olive Oil •····••••····•·••••·•···••··•··•····•••••·····•••··••··•• 601 Sodium Starch Phosphate ........ · .. •... •.... " ·· •· ••.. •.. · · • 659
Oxyquinoline Sulfate · · •· · · •·• ••• · · · · · ••·• •·· •· •· · ••• · ··• · · ••• · 603 Sodium Tartrate ·••···•·••·• •·· ......... ••· ···••• ••• ··· ••· ••• ••• 660
Paraffin · •••· · •· · · · •· ••· ••· · · •••••· •· · · · •· •••· · •· · · •· ••· •· · · · · ••· · · • 603 Soluble Starch ..... · ·· · · •· •• •· ·· · ·• •· · •· · •.... · · · ••· ••· •· ·· · · •· · · 660
Pectin ............................................................ 603 Sorbic Acid ...................................................... 661
Phosphoric Acid · · •· · · •· ••••· · · · ••· •••· · · · •••· · ••· · · •· •••· •· · · · • 604 Sorbitan Laurate (Span 20) ................................. 661
Poloxamer 188 ................................................... 604 Sorbitan Monostearate (Span 60) ........................ 662
Poloxamer 407 · · · · · •· · •· · •· · · · •· · ••· · · · · ••· · · •· · •· · · •••••.. · •· · •• 606 Sorbitan Oleate (Span 80) .................................... 663
Poly Oactide-co--glycolide) (5050) (For Injection) 608 Sorbitan Palmitate (Span 40) .............................. 664
Poly Oactide-co--glycolide) (7525) (For Injection) •••··• 609 Sorbitan Trioleate (Span 85) .............................. 665
Poly Oactide-co--glycolide) (8515) (For Injection) ···••• 611 Soya Lecithin •· · ··• •· •· · •· · · ··• ••• ••• •·• · · · · •• ·• · •• · · · · ••• •• · ... 665
Polyacrylic Resin 11 ••••••••••••••••••••• •••••••••••••••••••••••• 612 Soya Lecithin (For Injection) ··· •·· •·· ••·•·· ... ·•• ·•• ...... ·•• 667
Polyacrylic Resin ill ............................................. 612 Soybean Oil ...................................................... 669
Polyacrylic Resin N ............................................. 613 Soybean Oil (For Injection) ··· ............ ··• ... ...... ...... 670
Polyethylene Oxide ............................................. 613 Stearic Acid ...................................................... 671
Polyoxyl (35) Castor Oil .................................... 615 Stearyl Alcohol · · •· · •· •· · •· · · ••· · •· · •· •· · · •••· ••· · •· · •· · ••· · · •••· • 672
Polyoxyl (40) Stearate ....................................... 616 Steviosin •·•••·····•••·•·•···••••••••····•·····•··•···•··•·•••···· 672
Polyoxyl Oleate •••··•···•··•··•···•···•••·••····•·••••··•·••••• 617 Strong Ammonia Solution .................................... 673
Polysorbate 20 ··•··•··•··•·••••······••··•···•···•·•••·····•····• 618 Succinic Acid •···••··•·•••·•··••·•••·•··•····•··•••••·•··•••••·· 673
Polysorbate 40 ................................................... 619 Sucralose ·······•·····••·•••·•···•··•··••······••••·••···•···••••· 673
Polysorbate 60 ................................................... 620 Sucrose · •· · · · · · · •· · · ••· · · · · · · · •· •· •· · · · · •· •· •· · ••· · · · •••· ••· •· · · ·.. 674
Polysorbate 80 •·· •• •·· · ·•· ·· · · •• ... · ·· ··• · •· .. •.. · ·· ••· •· •· · · · ·• · 622 Sucrose Octaacetate ·····•·•••·•····•··•·•••·····•··•··•••···•••• 675
Polysorbate 80 (For Injection) ·· · ··• •... · •· · · · •• •• · •· · ··· ·• • 623 Sucrose Stearate ·••·····•··•·····•••· ····••··•···••••••···•··•·• 676
Polyvinyl Alcohol · •· ··• ••• ••• · ·· ·•· · · •·•• •·· · · · ••• ••• · · •••• •· · ·•· 625 Sulfuric Acid ·•·····•··••·····••··•······••·••••······••••·•••·· 677
Potassium Bicarbonate ··•••····•·•••·•·•·····•··••·•···••··••··· 626 Sugar Spheres •····•··•·••···••••••······••••••··•·····••····•··· 678
Potassium Chloride ···••••····•·••····••····•·••••····•·•••·•··· 627 Tale ............................................................... 678
Potassium Dihydrogen Phosphate ........................... 627 Tapioca Starch ................................................... 679
Potassium Hydroxide ····•••••·••······••··••·····••·••·•···•·• 627 DL-Tartaric Acid ................................................ 680
Potassium Nitra te · ·· · · · •· · · •· .. · ··• · ·• ..... •·•· •· •· •• ·· · · ·• · · • 628 Taurine * ......................................................... 681
Potassium Sorba te •·• ••• · •· · ·• · · •·• •.. · · · · · •• •· ••·• · · •· · ••· •··· 629 Thymol ............................................................ 681
Pota to Starch •· · ·· · ·•• · · · •· · ·• · •• · ••• •·· •·· •• •· · · •· •· •· •• · · •· · · • 630 Titanium Dioxide ••• · · •· · · · · · ••• •·· · ·· •· · · ••·•• ··· · · •· ·• · •· · · · •· · 682
Povidone K30 · · · · · ••· ••.. · · · · ••· · •· · •· · · · · · · · •••••· · · · •· · ••· · · · · 630 Tragacanth ...................................................... 683
Powdered Cellulose ............................................. 631 Trehalose · ·· ••• · · •··· · · •· · •· · · ·•• ••• ••• •·· · · · ·• •••• · •· · ·• ••• · · •••· 683
Pregelatinized Hydroxypropyl Starch ·····•·••••···•··•••• 635 Triacetin ·············•·••••··•··••··•·····•···•·•·••···•····•••·· 684
Tributyl Citrate .. •·• · · ·• ••· ••· •••••• · · · · · •· · •••• · · •••• ••• ••• ·• · 685 Wheat Starch ............................................. ••• ... 691
Triethyl Citrate •· •·· ••· · •· · · •· ••• •· · •· •••• ·•• ••• · •• · •••• ••• ••· · 686 White Beeswax · · · · · •••• •·· .. •· · ••· · · · •· · · •·· · · •· •• · · •· · · .. · •· · •• · 692
Trolamine •·• •·• •• •·•• ·· •••• · · · ••• ••· · · · ••• •• ••·· •· · · •• ·• ••· ••· •••· 686 White Vaselin ................................................... 692
Trometamol ···•••·•·••••••••·•·•••·····•••••···•·•••···••·••••·•· 687 Xanthan Gum ... ••• .. · •• · · · •... · .. ••• ••• •.. ·• ••• · •• · ... · · •"· ·.. 693
Tryptophan ·• · · •• ·· •·· · · ·• ••· · •· •• •••• · · •· · •· · · ••• •· ••· · ••• ••• •· · 688 Xylitol · · ............... · .... •• •• · .. · •.. ••• ... ·• ••· ••.... · ... •· •... 694
Tyrosine * ··· ................................................... ··· 688 Xylose ............................................................ 695
Urea* ............................................................ 688 Yellow Ferric Oxide .......................................... 696
Vacant Capsules from Hydroxypropyl Starch ••••· ••· •· · • 688 Yellow Vaselin .. · · · ••••••.. ••.. •.. •· ••· .. •.. · ••••· ••· .. · .. •.. · ·.. 696
Vacant Gelatin Capsules · •· •· · ••· · · · · · · •· •· · · · · ••· •· · •· ••· ••· · • 689 Zein ............................................................... 697
Valine* ............................................................ 690 Zinc Oxide •· · •• · .. •.. · •• · · •• ••• •· · ••• ••• · •· .. ••· •· · ••· · ... ••• ••• 697
Vitamin E Polyethylene Glycol Succinate •• .. •• ...... •• · ·.. 690 Zinc Stearate ••• •· •.. •... •.. · •• •· •... •· · .. · ••· .. •· •• •.. · · · ...... ••• 698
 482 Acacia
Acacia
[900-01-5]
Acacia is the dried gummy exudates from the stems and
velated branches of Acacia Senegal (Linné) Willdenow or
other species of Acacia (Fam. Leguminosae).
Description A white to slightly yellow flakes, granules or
power.
Sparingly soluble in water, insoluble in ethanol.
ldentification The position and colour of the principal spots
in the chromatogram obtained with the test solution in the
test for Glucose and fructose correspond to the principal
spots obtained with reference solutions of galactose CRS,
arabinose CRS and rhamnose CRS.
Insoluble matter Dissolve 5. O g in 100 ml of water, add
10 ml of 3 mol/L hydrochloric acid solution, boil gently
for 15 minutes, fil ter through a sintered-glass crucible
(No. 4) previously dried to constant weight at 105ºC.
Wash filter with hot water for several times, and dry to
constant weight at 105ºC. The residue is not more than
1. O% of its weight.
Starch or dextrin Boil a solution ( 1-+ 50) , cool, and drop
iodine TS in the solution, no bluish or reddish colour is
produced.
Glucose and fructose Place O. 1 g in a thick-walled centrifuge
tube, add 2 ml of 1% trifluoroacetic acid solution, shake
vigorously to dissolve, stopper the tube and heat at 120ºC for
1 hour . Centrifuge and transfer the clear supernatant
carefully into a 50 ml flask, add 10 ml of water and evaporate
the solution to dryness under reduced pressure. To the
resulting clear film add O. 1 ml of water and O. 9 ml of
methanol. Centrifuge to separate the amorphous precipitate.
Dilute the supernatant, if necessary, with 1 ml of methanol.
Dissolve 10 mg of arabinose CRS, 10 mg of galactose CRS,
10 mg of glucose CRS, 10 mg of rhamnose and 10 mg of
xylose CRS in 1 ml of water and dilute to 10 ml with
methanol as reference solution. Carry out the method for
thin-layer chromatography ( 0502), using silica gel G as the
coating substance, and a mixture of l. 6 % solution of sodium
dihydrogen phosphate -butanol-acetone (10 : 40 : 50) as the
mobile phase. Apply separately to the plate 10 µl both of the
above solutions. After developing and removal of the plate,
dry it in air and then spray with p-anisaldehyde solution (to
O. 5 ml of p-anisaldehyde, add 10 ml of glacial acetic acid, 85
ml of methanol and 5 ml of sulfuric acid mix welD heat at
llOºC for 10 minutes. The chromatogram obtained with the
reference solution shows 5 clearly separated coloured zones
due to galactose ( greyish-green or green) , glucose (grey) ,
arabinose ( yellowish-green ) , xylose ( greenish-grey or
yellowish-grey ) or rhamnose ( yellowish-green ) . The
chromatogram obtained with the test solution shows no grey
zone orno greyish-green zone between the zones corresponding to
galactose and arabinose in the chromatogram obtained with the
reference solution.
Tragacanth The chromatograms with the test solution
obtained in the test for Glucose and fructose shows no
greenish-grey or yellowish-grey zone corresponding to the
zone of xylose in the chromatogram obtained with the
reference solution.
Tannin-bearing gum To 10 ml of a solution (l-+50), add
O. 1 ml of ferric chloride TS, neither the precipitate nor the
liquid shows black colour.
Sterculia gum Place O. 2 g in a ground-glass-stoppered
cylinder graduated in O. 1 ml. Add 10 ml of 60% ethanol
stopper and shake. Any gel formed occupies a maximum of
l. 5 ml. To l. O g add 100 ml of water and shake. Add O. 1
ml of methyl red IS. Not more than 5. O ml of sodium
hydroxide( O. O1 mol/L) VS is required to change the colour
of the solution.
Loss on drying When dried at 105ºC for 5 hours, loses not
more than 15. O% of its weight ( 0831).
Total ash Not more than 4. O% ( 2302 >.
Acid-insoluble ash Not more than O. 5 % ( 2302).
Heavy metal Carry out the limit test for heavy metals( 0821
method 2) , using O. 5 g: not more than O. 004 % .
Arsenic Mix O. 67 g with l. O g of calcium hydroxide, add
2 ml of water and stir well. After dried at lOOºC, heat gently
until it is thoroughly charred and then ignite at 480ºC until
the incineration is completed. Dissolve the cooled residue in a
mixture of 5 ml of hydrochloric acid and 21 ml of water.
Carry out the limit test for arsenic ( 0822 method 1 ) : not
more than O. 0003%.
Category Pharmaceutical excipients, suspending agent and
thickening agent, etc.
Storage Preserve in tightly closed containers and stored in a
cool and dry place.
Acetic Acid
C2 H4 02
60. 05
[64-19-7]
Acetic acid contains not less than 36% (g/g) and not more
than 37 % (g/ g) of Cz H4 02 .
Description A colourless clear liquid; odour, strong and
characteristic; taste, extremely sour.
Miscible with water, ethanol and glycerol.
Relative density l. 04-1. 05, at 25ºC (0601 >.
Identification (1) lt changes the blue litmus TP to red.
(2) When neutralized with sodium hydroxide TS, yields the
reactions characteristic of acetates ( 0301 ) .
Chloride Carry out the limit test for chlorides ( 0801 ) ,
using l. O ml. Any opalescence produced is not more
pronounced than that of a reference solution prepared by
using 7. O ml of sodium chloride standard solution
(0. 007%).
Sulfate Dilute 2. 5 ml with water to 20 ml. Carry out the
limit test for sulfates ( 0802) , using 5. O ml of the resulting
solution. Any opalescence produced is not more pronounced
than that of a reference solution using l. 5 ml of potassium
sulfate standard solution (O. 024%).
Formic acid and readily oxidizable substances Dilute 5. O ml
in 6 ml of sulfuric acid, mix well, cool to 20ºC, add 2. O ml
of potassium dichromate (O. 016 67 mol/L) VS, and allow to
stand for 1 minute. Add 25 ml of water, 1 ml of potassium
iodide TS and 1 ml of starch IS; not less than l. O ml of sodium
thiosulfate (O. 1 mol/L) VS is required to change the colour of
the solution.
Reducing substances Dilute 5. O ml with 20 ml of water, add

O. 2 ml of potassium permanganate (O. 02 mol/L) VS, mix
well, and allow to stand far 1 minute; the pink colour <loes
not disappear completely.
Aldehydes Dilute 5. O ml, accurately weighed, with water to
10 ml, mix well, transfer 2. 5 ml into a headspace vial, add
2. 5 ml of 3. 2 mol/L sodium hydroxide solution,
immediately seal, shake well and use as the test solution.
Dilute a quantity of acetaldehyde CRS, accurately weighed,
with l. 6 mol/L sodium acetate solution to produce a solution
containing O. 05 mg per ml, accurately measure 5 ml in a
headspace vial, seal and use as the reference solution. Carry
out the method far residual solvent( 0861 method 2), using a
capillary column packed with polyethyleneglycol polysiloxane
as the stationary phase, maintain the column temperature at
35ºC far 5 minutes, then raise the temperature to 120ºC by
30ºC per minute, maintain far 2 minutes. The temperature
of injection is 200ºC, the temperature of detection is 250ºC,
the equilibrium temperature of the headspace vial is SOºC and
maintain far 30 minutes. lnject each of the gases from
headspace vials of test solution and the reference solution
onto the column. Calculate the content of acetaldehyde with
respect to the area obtained in the chromatogram by the
external standard method: not more than O. 02%.
Non-volatile matter Measure 20 ml in an evaporating dish
previously dried to constant weight at 105ºC, evaporate to
dryness on a water bath, and dry to constant weight at
105ºC. The residue is not more than 1 mg.
Heavy metals Evaporate 10 ml to dryness on a water bath,
add 20 ml of water to dissolve the residue. To 15 ml of the
resulting solution add l. 5 ml of aceta te BS ( pH 3. 5) and
then sufficient water to produce 25 ml. Carry out the limit
test far heavy metals < 0821 method 1 ) : not more than
o. 0002%.
~y Accurately measure about 4 ml in a conical flask,
dilute with 40 ml of freshly boiled and cooled water, add
3 drops of phenolphthalein IS, and titrate with sodium
hydroxide ( 1 mol/L) VS. Each ml of sodium hydroxide
( 1 mol/L) is equivalent to 60. 05 mg of Cz H4 02.
Category Pharmaceutical excipients, acidif ying agent and
buffer, etc.
Storage Preserve in tightly closed glass bottles.
Activated Charcoal ( For lnjection)
[ 7440-44-0 J
Activated Charcoal is a loase porous substance with high
adsorption power, obtained from the charcoal, various fruit
shells and super coal by physical and chemical methods
including crushing, sieving, catalyst activation, rinsing,
drying and screening.
Description Black powder, odorless, tasteless; free from
grittiness.
ldentification Transfer O. 1 g to a heat-resistant glass tube,
while slowly feeding compressed air, burning the place where
the sample resides with alcohol burner (note: no visible fire
allowable), resulting gas passes through the solution of
calcium hydroxide, a white precipitate is farmed.
Acidity or alkalinity Take 2. 5 g, add 50 ml of water, boíl
for 5 minutes, allow to cool, filter, wash the residue with
water, combine filtrate and the washings to 50 ml, the
Activated Charcoal (Far lnjection)
filtrate is colourless and is neutral to litmus.
Chloride Take 10 ml of the filtrate obtained in the test far
acidity or alkalinity, dilute to 200 ml with water, shake
well; dispense 20 ml, carry out the limit far chlorides
<0801), any opalescence produced is not more pronounced
than that of a reference solution prepared by using 5. O ml of
sodium chloride standard solution (O. 1 %).
Sulfate Take 20 ml of the remaining filtrate obtained in the
test far acidity or alkalinity, carry out the limit far sulfate
<0802) , any opalescence produced is not more pronounced
than that of a reference solution prepared by using 5. O ml of
potassium sulfate standard solution (O. 05 %).
Non-carbonated substances Take O. 25 g, add 10 ml of sodium
hydroxide TS, boíl and then filter. Any colour produced is
not more intense than that of the reference solution (measure
O. 3 ml of colourimetric cobalt chloride solution, O. 2 ml of
colourimetric potassium dichromate solution, and 9. 5 ml of
water, mix welD.
Sulfide Take O. 5 g, add 20 ml of water and 5 ml of
hydrochloric acid, boíl, the steam can not turn the humid
lead acetate test paper to black.
Cyanide Transfer 5 g to a distillation flask, add 50 ml of
water and 2 g of tartaric acid, distill, absorb the distillate in
an ice water bath with an absorbing liquid, the absorption
liquid is 2 ml of sodium hydroxide solution and 10 ml of
water, produce 25 ml of distillate, dilute with water to
50 ml, add 12 drops of ferrous sulfate TS, heat to almost
boiling, allow to cool, add 1 ml of hydrochloric acid TS, no
blue colour is produced.
Ethanol-soluble substances Take 2. O g, add 50 ml of
ethanol, boíl and reflux far 10 minutes, immediately filter,
dilute the filtrate to 50 ml with ethanol, take 40 ml of the
íiltrate, dry to constant weight at 105ºC, residue is not more
than 8 mg.
Fluorescent substances Transfer 10. O g to a distillation
flask, add 100 ml of cyclohexane, distill far 2 hours, dilute
the distillate with cyclohexane to 100 ml as the test solution.
Take a quantity of quinine, accurately weighed, add O. 005
mol/L sulfuric acid solution to dissolve and produce a
solution containing 83 ng per ml as the reference solution.
Carry out the method far ultravioletvisible spectrophotometry
<0401 ) , measure the absorbance at 365 nm. The absorbance
of the test solution is not more than that of the reference
solution.
Acid-soluble substances Take l. O g, add 20 ml of water and
5 ml of hydrochloric acid, boíl far 5 minutes, filter, wash
the residue with hot water, combine the filtrate and
washings, add 1 ml of sulfuric acid, after being evaporated,
bum to constant weight, the residue is not more than 8 mg.
Loss on drying When dried to constant weight at 120ºC,
loses not more than 10. O% <0831 ) .
Residue on ignition Take about O. 50 g, and Take about
O. 50 g, and add 2 to 3 drops of ethanol to wet. Carry out
add 2 to 3 drops of ethanol to wet. Carry out the limit test
far the residue <0841 ) : not more than 3. O%.
lron Take l. O g, add 25 ml of 1 mol/L hydrochloric acid
solution, boíl far 5 minutes, allow to cool, filter, wash
several times with 30 ml of hot water, combine filtrate and
washings, add water to 100 ml, shake well; transfer
accurately 5 ml to a 50 ml Nessler tube. Carry out the limit
test for iron <0807), any colour produced is not more intense
than that of a reference solution prepared by using l. O ml of
 Adenine
iron standard solution (0. 02%).
Zinc Take l. O g into 25 ml of water, boil for 5 minutes,
allow to cool, filter, wash the residual several times with 30
ml of hot water, combine filtrate and washings, add water to
100 ml, shake well; transfer accurately 10 ml to a 50 ml
Nessler tube, add O. 5 g of ascorbic acid, add 4 ml of
hydrochloric acid solution ( 1 - 2 ) , and 3 ml of potassium
ferrocyanide, dilute to the volume with water, shake well. If
there is turbidity produced, compare with the reference solution
prepared with O. 5 ml of standard zinc solution ( transfer
accurately 44 mg of zinc sulfate CZnS0 4 • 7H 2 0) into a 100
ml volumetric flask, dissolve and dilute to the volume with
water, shake well, transfer accurately 10 mi to a 100 ml
volumetric flask, dilute to volume with water, shake well.
Each ml of the solution is equivalent to 1 O µg of zinc). Any
colour produced is not more intense than that of the reference
solution prepared with the same method (0. 005%).
Heavy metals Take l. O g, add 10 ml of dilute hydrochloric
acid and 5 ml of bromine TS, boil for 5 minutes, filter, wash
the residual with 35 ml of boiling water, combine filtrate and
washings, add water to 50 ml, shake well; dispense 20 ml,
add 1 drop of phenolphthalein IS, drip ammonia solution
until the solution turns pale red, add 2 ml of acetate salt
buffer (pH 3. 5) and a quantity of water to 25 ml, dissolve
O. 5 g of ascorbic acid. Carry out the limit test for heavy
metals( 0821 method 1): not more than O. 003%.
Sorptivity ( 1) Take l. O g dried to constant weight, add
100 ml of O. 12% quinine sulfate solution, shake vigorously
for 5 minutes at the room temperature which is not less than
20ºC, fílter with dried filter paper of medium speed, take
10 ml of subsequent fíltrate, add 1 drop of hydrochloric acid
and 5 drops of potassium mercuric iodide TS, no turbidity is
produced.
( 2) Take two 100 ml stopper graduated cylinders,
transfer O. 25 g of the substance being examined dried to
constant weight into one. Then add respectively and
accurately 50 ml of O. 1 % methylene blue solution, cap the
plug, shake vigorously for 5 minutes at the room
temperature which is not less than 20ºC, filter the solutions
in two cylinders through dried filter paper at medium speed,
transfer accurately 25 ml of the subsequent filtrate
respectively to two 250 ml volumetric flasks, respectively
add 50 ml of 10 % sodium acetate solution, shake well,
while constantly rotating, respectively add accurately 35 ml
of iodine titration (O. 05 mol/L) VS, cap the plug, shake
well, allow to stand, shake vigorously every 10 minutes,
dilute with water to volume after 50 minutes, shake well, stand
for 10 minutes, filter respectively with a dried fílter paper,
transfer accurately 100 ml of the fíltrate, respectively, titrate
with sodium thiosulfate (O. 1 mol/U VS. The difference
between the consumed volumes of iodine titration (O. 05 mol/L)
VS is not more than l. 4 ml.
l\.1icrobial limits Comply with the requirements for microbiolo-
gical limit( 1105 and 1106), the total aerobic bacteria count
is not more than 1000 cfu per g and the total combined yeast/
mold count is not more than 100 cfu per g of the substance
being examined, Escherichia coli should not be detected per
g, and salmonella species should not be detected per 10 g.
Bacteria! endotoxin Background endotoxin value of the
activated charcoal Take about 75 mg, add about 5 ml of
water for BET, produce a mixture solution containing
activated carbon l. 5 % (l. 5 g/100 mD, vortex mix for 9
minutes, centrifuge at 1500 rpm for 5 minutes, fílter the
super nataut with O. 22 µm microfíltration pyrogen free
membrane af ter centrifugation. Carry out the limit test for
bacterial endotoxin ( 1143 ) : not more than 2 EU/ g.
Activated Charcoal capacity adsorbing bacteria! endotoxin
Take a vial of national standard bacteria! endotoxin, prepare
standard endotoxin solutions ata concentration of 200 EU per
ml and 20 EU per ml according to the instruction manual.
Weigh two samples of 75 mg of the substance being e:xamined,
add about 5 ml of the standard solutions mentioned above
respectively to produce the mixture a solution containing
l. 5% activated charcoal. Vortex mixing for 9 minutes,
centrifuge at 1500 rpm for 5 minutes, filter the supernatant
with O. 22 µm microfíltration pyrogen free membrane after
centrifugation. Carry out the limit test for bacteria! endotoxin
( 1143 ) : the activated charcoal descend the bacteria!
endotoxin levels by two orders of magnitude ( an adsorption
rate of 99 %).
Sterility ( applicable to preparations without terminal
sterilization process or products without bacterial removal
process). Carry out the method for sterility test ( 1101), it
complies with the requirement.
Category Pharmaceutical excipients, adsorbent.
Storage Preserve in well-closed containers.
Adenine
Cs Hs Ns 135. 13
[73-24-5]
Adenine is 7H -purin-6-amine. It contains not less than
98. 5 % of Cs Hs Ns , calculated on the dried basis.
Description A white or almost white powder or crystal or
crystalline powder; odourless and tasteless.
Sparingly soluble in hot water, slightly soluble in ethanol and
very slightly soluble in water.
ldentification (1) Dilute a quantity with dilute acetic acid
to produce a solution containing 1 mg of adenine per ml as
test solution. Prepare a reference solution of 1 mg of adenine
CRS per ml in the same manner. Dissolve 10 mg of adenine
CRS and 10 mg of adenosine CRS with dilute acetic acid
Cheat if necessary) in a 10 ml volumetric flask as the system
suitability solution. Carry out the method for thin-layer
chromatography ( 0502 ) , using silica gel GF 254 as the
coating substance anda mixture of strong ammonia solution-
ethyl acetate- propyl alcohol ( 20 : 40 : 40) as the mobile
phase. Apply separately to the plate 5 µl each of the above
solutions. After developing and removal of the plate, dry it
in air and observe under a ultra-violet lamp at 254 nm. There
are two clear and separated spots for the system suitability
solution. The position and colour of the principal spot in the
chromatogram obtained with the test solution correspond to
the principal spot obtained with the reference solution.
(2) The infrared absorption spectrum is concordant with that
of adenine CRS ( 0402).
Acidity or alkalinity Dissolve 2. 5 g in 50 mi of water and
boíl for 3 minutes, cool, dilute with water to 50 ml, filter.
Measure 10 ml of the filtrate ( the residue filtrate is
reserved).

Add O. 1 ml of bromothymol blue IS and O. 2 ml of sodium
hydroxide solution (O. O1 mol/L) , a blue colour is produced.
Add O. 4 ml of hydrochloric acid solution (O. 01 mol/L), a
yellow colour is produced.
Clarity and colour of solution Dissolve O. 5 g in 50 ml of
dilute hydrochloric acid and the solution is clear and
colourless( 0901 and 0902).
Chlorides lgnite O. 5 g to carbonize gently and then at 500-
600ºC until completely carbonized, cool. Carry out the limit
test far chlorides ( 0801 ) . Any opalescence produced is not
more pronounced than that of a reference solution using
5. O ml of sodium chloride standard solution (O. 01%).
Sulfates Measure 10 ml of the filtrate obtained in the test
far Acidity or alkalinity, carry out the limit test far sulfates
( 0802). Any opalescence produced is not more pronounced
than that of a reference solution using l. 5 ml of potassium
sulfate standard solution (O. 03 %) .
Organic impurities Reference solution Accurately weigh a
quantity of the adenine CRS, add a quantity of hot water,
cool, quantitatively dilute with water to produce a solution
containing about O. 19 mg per ml. Transfer 3 ml, accurately
measured, to three 100 ml volumetric flasks separately,
dilute with O. 1 mol/L hydrochloric acid solution, O. 1 mol/L
sodium hydroxide solution and pH 7. O phosphate buffer
solution [dissolve 4. 54 g of potassium dihydrogen phosphate
with water, dilute to 500 ml as the solution A; dissolve
4. 73 g of anhydrous disodium hydrogen phosphate with
water, dilute to 500 ml as the solution B. Measure 38. 9 ml
of the solution A and 61. 1 ml of the solution B, mix well
( adjust pH to 7. O by dropwise adding solution B if
necessary) J to the volume and mix well.
Test solutions Prepare three kinds of test solutions as
described for the reference solution.
Procedure Transfer the corresponding two solutions respect-
ively, using water as the blank solution and in the range of
220-230 nm ( 0401), record the maximum absorbance value
and the absorbance A; of the test solution comply with the
requirements of the following formula:
M; X (1-C) XVs X As X O ::::::::A-:::::::::
98
MsXV;XCs ' ':::::: '~
M; X (1-C) XVs X As X l.
02
M, XV; XC,
A;: Absorbance of the test solution;
A,: Absorbance of the reference solution;
M; : Sampling weight of the substance being examined, mg;
M,: Sampling weight of the reference substance, mg;
V;: Dilution volume of the test solution, mi;
V,: Dilution volume of the reference solution, ml;
C: Result of loss on drying of the substance being examined;
C,: Labelled content of the reference substance, %.
Nitrogen content Carry out the method far determination of
nitrogen ( 0704 method 1 ) , using 50 mg. The content of
nitrogen is between 50. 2% and 53. 4%, calculated on the
dried basis.
Los.s on drying When dried to constant weight at 105ºC,
loses not more than O. 5 % of its weight ( 0831 ) , using 1 g.
Residue on ignition Not more than O. 1 % ( 0841 ) , using 1 g.
Ammonium Carry out the limit test far ammonium ( 0808) ,
using 2 g. Any opalescence produced is not more pronounced
than that of a reference solution using 2. O ml of ammonium
Agar
chloride standard solution (0. 001%).
Heavy metals Carry out the limit test far heavy metals
( 0821 method 2), using the residue obtained in the test far
Residue on ignition: not more than O. 001%.
Assay Dissolve O. 1 g, accurately weighed, in 20 ml of
acetic anhydride and 30 ml of anhydrous acetic acid. Carry
out the method far potentiometric titration ( 0701 ) , titrate
with perchloric acid (0. 1 mol/L) VS. Each mi of perchloric
acid (O. 1 mol/L) VS is equivalent to 13. 51 mg of Cs Hs Os.
Category Pharmaceutical excipients, cryoprotectant.
Storage Preserve in well closed containers.
Agar
[9002-18-0]
Agar is a dehydrated mucilaginous substance extracted from
Gelidium amansii Lamx or other related red algae.
Description Slender strips, almost white or pale yellow;
translucent, surface crumpled and slightly lustrous. Light,
flexible and difficult to break; brittle and easy to break when
dry; odourless; taste, slight.
Agar powder is particle or scaly, colourless or pale yellow;
irregular polygonal mucilaginous fragments are observed
under a microscope with cold water; odourless; taste,
slight.
Soluble in boiling water; insoluble in cold water but swelled
into gelatinous mass. Its aqueous solution is neutral.
ldentification ( 1) Dissolve 1 g in 65 mi of water, heat to
boiling with stirring, and supply with hot water. It
coagulates into translucent eiastic gelations substance when
cooled to 32-39ºC and remelts when heated to 85ºC.
(2) When immersed in O. 02 mol/L iodine solution far a few
minutes, it is dyed brownish black. The fragments gradually
become purple after macerating in water.
(3) Dissolve about O. 1 g in 20 mi of water by heating. To
4 ml of the solution add O. 5 ml of hydrochloric acid and heat
on water bath far 30 minutes. Add 3 mi of sodium hydroxide
TS and 6 mi of alkaline cupric tartrate TS, heat in water
bath. A red colour precipitate is produced.
Water absorbability Place 5. O g in a 100 ml measuring
cylinder and add water to volume, mix well. Allow to stand
at 25ºC far 24 hours, filter through a layer of moistened
glass wool into another measuring cylinder. Total volume of
the filtrate is not more than 75 ml.
Starch Dissolve O. 10 gin 100 ml of water by boiling, Allow to
cool. Add 2 drops of iodine TS, no blue colour is produced.
Gelatin To l. O g add 100 mi of water and heat on a water
bath until dissolved. Allow to cool to 50ºC. To 5 mi of the
solution add 2-3 drops of a mixture of O. 2 mol/L potassium
dichromate solution and 3 mol/mi hydrochloric acid (4 : 1):
no yellow precipitate is farmed.
Water-insoluble substances Dissolve l. 5 gin 200 mi of water
in a beaker by boiling, filter while hot through a No. 3
sintered glass crucible, previously dried to constant weight at
lOSºC. Wash the beaker with hot water far several times,
filter the washings. Dry the residue to constant weight at
lOSºC. Not more than 15 mg (l. 0%).
Foreign matter Weigh 250 g, spread out in a thin layer.
Detect the foreign matter by inspection with eye or with lens
 Alanine
( 5-1 OX ) , separa te the foreign ma tter: not more than l. O%.
Acid-insoluble ash Boil the residue obtained from the test
for ash with 25 ml of 3 mol/L hydrochloric acid solution for
5 minutes in a crucible, filter through an ashless filter paper,
transfer the residue with water to the filter paper, put the
paper and the residue in a same tared crucible, gently raise
the temperature, ignite to constant weight using the method
described under the test for ash. The residual acid insoluble
ash is not more than O. 5 % .
Loss on drying When dried at 105ºC for 5 hours, loses not
more than 20. O% of its weight <0831 ) . Strip agar must be
reduced to small fragments before drying.
Ash lgnite slowly about 1 g, accurately weighted, in a
crucible, previously ignited to constant weight, until the
substance being examined is completely carbonized, raise the
temperature gradually to 650±25ºC, incinerate completely to
constant weight, The residual ash is not more than 5. O% of
its weight.
Heavy metals Carry out the limit test for heavy metals
<0821 method 2) , using O. 50 g; not more than O. 004 % .
Arsenic To l. O g, add 5 ml of sulfuric acid anda few glass
beads, and digest in a fume hood, preferably on a hot plate
and at a temperature not exceeding 120ºC, until charring
begins ( Additional sulfuric acid may be necessary to wet
sorne specimens completely, but the total volume added
should not exceed 10 mD. Cautiously add, dropwise, 30%
hydrogen peroxide solution, allowing the reaction to subside
and again heating between drops. Add the first few drops
very slowly with sufficient mixing, in order to prevent a
rapid reaction. Discontinue heating if foaming becomes
excessive. When the reaction has abated, heat cautiously,
rotating the flask occasionally to prevent the specimen from
caking on glass exposed to the heating unit. Maintain
oxidizing conditions at ali times during the digestion by
adding small quantities of the hydrogen peroxide solution
whenever the mixture tums brown or darkens. Continue the
digestion until the organic matter is destroyed, gradually
raising the temperature of the hot plate until fumes of sulfur
trioxide are copiously evolved, and the solution becomes
colourless or retains only a light straw colour, Cool, add
cautiously 10 ml of water, mix, and again evaporate to
strong fuming, repeating this procedure to remove the trace
of hydrogen peroxide. Cool, add cautiously 10 ml of water,
and dilute with water to 35 ml. Pipet 3. O ml of Standard
Arsenic solution, carry out the procedure above, Carry out
the limit test for asenic <0822 methord 2): not more than
o. 0003%.
Microbial limit Comply with test for microbial limit <1105
and 1106 ) , the total of aerobic bacteria! count is not more
than 1000 cfu per g and total combined yeast/mold count is
not more than 100 cfu per g, Escherichia coli is not detected.
Category Pharmaceutical excipients, suspending agent and
release retardant.
Storage Preserve in well closed containers, stored in a dry palee.
Alanine
See the same monograph in Volume 1I .
Category Pharmaceutical excipients, pH adjuster and
bulking agent.
Alginic Acid
[9005-32-7]
Alginic Acid is a hydrophilic colloidal carbohydrate extracted
with dilute alkali from various species of brown seaweeds
(Phaeophyceae), the final products is refined by treating
with inorganic acid. It contains not less than 19. 0% and not
more than 25. O% of carboxyl groups (-COOH), calculated
on the dried basis.
Description A white to pale yellowish powder, odorless and
almost tasteless.
Insoluble in water, methanol, ethanol, acetone, chloroform,
soluble in Sodium hydroxide TS.
ldentification ( 1) Dissolve about 30 mg of alginic acid in
5 ml of O. 1 mol/L sodium hydroxide solution, add 1 ml of
calcium chloride TS, a gelatinous precipitate is formed.
(2) Dissolve about of 30 mg in 5 ml of O. 1 mol/L sodium
hydroxide solution, add 1 ml of diluted sulfuric acid. A
gelatinous precipitate is formed.
(3) To 10 mg add 5 ml of water, 1 ml of freshly prepared
1 % solution of 1, 3-dihydroxynaphthalene in ethanol and
5 ml of hydrochloric acid, shake well. Boil gently for 5 minutes,
cool, shake with 15 ml of isopropyl ether. Allow to stand for
severa! minutes and divide the ether layer. Carry out a blank
test. The ether layer exhibits a deeper bluish-red colour than
that obtained with the blank.
Acidity Dissolve l. 5 g in 50 ml of water, and shake for
5 minutes, pH 1.5-3.5 (0631).
Starch Dissolve O. 10 g in 100 ml of sodium hydroxide
solution0-2500), take 5 ml, add 1 drop of iodine TS, no
instant blue colour is produced.
Chloride Transfer 2. 5 g, accurately weighed, to a 100 ml
volumetric flask, add 50 ml of dilute nitric acid, shake for
1 hour, and add with dilute nitric acid to volume. Shake and
filter. To 50 ml of the successive filtrate, add 10 ml of silver
nitrate(O. 1 mol/L) VS, accurately measured, add 5 ml of
tolueneand, 2 ml of ferric ammonium sulfate IS. Titrate with
ammonium thiocyanate (0. 1 mol/L) VS, shake vigorously
towards the end point. Perform a blank determination and
make any necessary correction. Each ml of silver nitrate (O. 1
mol/L) VS is equivalent to 3. 545 mg of chloride( l. O%) : not
more than l. O%.
Loss on drying when dried at 105ºC for 4 hours, loses not
more than 15. O% of its weight ( 0803).
Residue on ignition Not more than 5. O% <0841 ) , using
o. 5 g.
Iron Take l. O g, heat gently until it becomes carbonized,
then ignite at 500-600ºC until the incineration is complete.
Dissolve the cooled residue in 3 ml of hydrochloride acid,
transfer it to the 50 ml volumetric flask, dilute to the volume
with water, shake well, transfer accurately 5 ml of this
solution to Nessler cylinder, dilute with water to 25 ml. Carry
out the limit test for iron using the resulting solution ( 0807) ,
the colour produced is not more intense than that of a reference
solution prepared with 5. O ml of iron standard solution
(0. 05%).
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), using the residue obtained in the test for

residue on ignition: not more than O. 002%.
Arsenic Mix O. 5 g with O. 5 g of anhydrous sodium
carbonate, oisten with little of water, heat gently until it is
thoroughly charred, ignite at 500-600ºC until the incineration
is complete, allow to cool. Dissolve the residue in a small
amount of hydrochloric acid until no air bubble is produced.
Add 5 ml of hydrochloric acid and 23 ml of water. Carry out
the limit test far arsenic ( 0822 method 1 ) : not more than
o. 0004%.
Microbial limit Comply with the requirements far
microbiological limit ( 1105 and 1106), the total aerobic
bacteria count is not more than 1000 cfu per g and the total
combined yeast/ mold count is not more than 100 cfu per g of the
substance being examined. Escherichia coli should not be detected
per g, and salmonella species should not be detected per 10 g.
As.say Dissolve about O. 25 g, accurately weighed, in 25 ml
of water. Add accurately 25 ml of O. 1 mol/L sodium
hydroxide VS, add O. 2 ml of phenolphthalein IS, titrate with
hydrochloric acid ( O. 1 mol/L) VS. Each ml of sodium
hydroxide (O. 1 mol/L) VS is equivalent to 4. 502 mg of
-COOH.
Category Pharmaceutical excipients, adhesives and disintegrating
agent.
Storage Preserve in well closed containers.
Aluminum Magnesium Silicate
[12511-31-8]
Aluminum Magnesium Silicate is obtained from refining,
drying, crushing a thin mortar made by mixing a silicon of
high amount of magnesium by the kaolinite class and slag
with water. The ratio of aluminum content and magnesium
content is O. 5-1. 2.
Description A white or almost white, soft and smooth plate
or micronized powder, odorless, tasteless, hygroscopic.
Swells in water to produce a colloidal dispersion.
Practically insoluble in water and in ethanol.
Viscosity An aqueous solution of 5 %, carry out the method
far determination of viscosity( 0633 method 2), using NDJ-1
rotating viscosimeter and No. 2 rotator with the rotation
speed of 30 rpm, the dynamic viscosity at 20±0. 1ºC is O. 3-
0. 6 Pa•s.
Identification (1) Dissolve about O. 5 g in 10 ml of dilute
hydrochloric acid, filter with gentle warming, add sodium
hydroxide TS dropwise into the successive filtrate to
alkaline, a white colloidal precipitate is produced. Add a few
drops of O. 1 % sodium alizarinsulfanate solution, the
precipitate shows cherry red.
(2) Add sodium hydroxide TS to a filtrate being examined of
Identification ( 1) to alkaline, a white colloidal precipitate is
produced. Continue to add 3 ml of sodium hydroxide TS, the
precipitate was dissolved partially. Filter, wash precipitate
with water completely. To the precipitate add iodine TS, a
red-brown appears.
(3) Take a ring made of platinum wire, clip the
crystallization of sodium ammonium phosphate particles and
melt sodium ammonium particles into a small transparent
ball in the flame. Dip the substances being examined when
the ring is hot, and melt it, then silicon dioxide will
gradually floating on the surface of the ball. Cool, the ball
Aluminum Magnesium Silicate J ·; ' '4/IJ}
becomes network nontransparent ball.
Alkalinity Dissolve 1 g in 20 ml of water, mix well, pH
9. 0-10. o <0631 >.
Acid demand To 5. O g add 500 ml of water. Stirring at a
constant rate, add each 3. O ml of O. 1 mol/L hydrochloric acid
solution exactly at 5, 65, 125, 185, 245, 305, 365, 425, 485,
545, 605, 665 and 725 seconds separately, and add l. O ml of
O. 1 mol/L hydrochloric acid solution at 785 seconds. Determine
the pH at 840 seconds: not more than 4. O ( 0631 ) .
Loss on drying When dried to constant weight at 105ºC,
loses not more than 8. O% of its weight ( 0831 ) .
Loss on ignition When ignition to constant weight at 700-
800ºC, loses not more than 17. 0% of its weight, using l. O g.
Heavy metal To 4. O g of the substance being examined, add
6 ml of hydrochloride and 30 ml of water, heated to boiling.
After cooling, add 2 drops of phenolphthalein IS and
concentrated ammonia solution until the colour of solution
changes to light pink. Filter, rinse the filtrate with water and
combine the filtrate. Add O. 5 g of as corbic acid, dilute to 50
ml with water and shake well. Take 12. 5 ml into a Nessler
tube, add 2 ml of acetale BS ( pH 3. 5) and dilute to 25 ml
with water. Carry out the limit test far heavy metals ( 0821
methol 1), not more than O. 001 5%.
Arsenic Dissolve l. O g of the substance being examined in
10 ml of dilute hydrochloric acid, boil, allow to cool and
filter, evaporate the filtrate to dryness on a water-bath, add
5 ml of hydrochloric acid and 23 ml of water, carry out the
limit test far arsenic ( 0822 method 1 ) , not more than
o. 0002%.
Microbial limit Comply with the requirements far
microbiological limit ( 1105 and 1106), the total aerobic
bacteria count is not more than 1000 cfu per g and the total
combined yeast/mold count is not more than 100 cfu per g of
the substance being examined. Escherichia coli should not be
detected.
As.say Aluminum Dissolve O. 5 g of the substance being
examined accurately weighed, in 2 ml of hydrochloric acid
and 50 ml of water by boiling far 3 minutes, allow to cool,
filter, wash the residue and the container with 25 ml of water
in several portions. Combine the washings and filtrate, add
ammonia TS dropwise until a precipitate is just produced,
add dilute hydrochloric acid dropwise until the precipitate is
just dissolved, add 10 ml of acetic acid-ammonium acetate BS
(pH 6. 0) ,add 20 ml of disodium edetate (0. 05 mol/U VS,
accurately measured, boil far 10 minutes, cool, add 1 ml of
xylenol orange IS, titrate with zinc (O. 05 mol/L) VS until
the colour of solution changes from yellow to red. Perform a
blank determination and make any necessary correction.
Each ml of disodium edetate (O. 05 mol/L) VS is equivalent
to l. 349 mg of Al.
Magnesium Dissolve O. 36 g of the sabstance being examined
accurately weighed, in 3 ml of hydrochloric acid and 50 ml of
:water by boiling, allow to cool, add 1 drop of methyl red IS.
Add ammonia TS dropwise until the colour changes from red
to yellow, continue to boil far 5 minutes. Filter the hot
solution, wash the residue and container with 30 ml of 2 %
ammonium chloride solution in several portions. Combine the
washings and filtrate, cool, add 10 ml of ammonia TS and 5
ml of triethanolamine solution ( 1 - 2 ) , add O. 3 g of
eriochrome black T IS, titrate with disodium edetate
(O. 05 mol/L) VS untill the colour of solution changes blue.
Each ml of disodium edetate (O. 05 mol/L) VS is equivalent
to l. 215 mg of Mg.
 Aluminum Sulfate
Calculate the ratio of aluminum content to magnesium
conten t.
Category Pharmaceutical exipients, suspending agents and
absorbent.
Storage Preserve in well containers, stored in a cool and dry
place.
Aluminum Sulfate
Alz CS04 )3•xH2 O
[17927-65-0]
Aluminum Sulfate is produced by reaction between bauxite
and sulfuric acid under pressure, or reaction between
aluminum hydroxide and sulfuric acid. Containing diff erent
amounts of water of crystallization. Aluminum Sulfate
contains not less than 54. O% and not more than 59. O% of
Alz cso4 )3.
Description A colourless or white crystal or crystalline
powder.
Soluble in water, practically insoluble in ethanol.
ldentification Yields the reactions characteristic of
aluminum and sulfate ( 0301 ) .
Acidity Dissolve O. 5 g in 25 ml of water, pH 2. 5-4. O
(0631 ).
Clarity and colour of solution A solution of 2. 5 g in 50 ml of
water is colourless ( 0901 method 1 ) ; any opalescence
produced is not more pronounced than that of reference
suspension 3 ( 0902).
Ammonium Dissolve O. 4 g in 100 ml of water. Carry out
the limit test far ammonium ( 0808) , using 1O ml: not more
than O. 05%.
Water 41. O%-46. O% ( 0832 method 1 ( 1)).
Alkali metals and alkaline earth metal salts To a boiling solution
of l. O gin 150 ml of water add 2 drops of methyl red IS and
then add ammonia TS just until the colour of the solution
changes to a distinct yellow. Dilute with hot water to 150 ml
and filter while hot. Evaporate 75 ml of the filtrate to
dryness, and ignite at 600ºC to constant weight: not more
than 2 mg of the residue remains (O. 4 %) .
Iron Carry out the limit test far iron ( 0807) , using O. 1 g.
Any colour produced is not more intense than that of a
reference solution using l. O ml of iron standard solution
(0. 01 %).
Heavy metals Dissolve l. O gin 23 ml of water, add 2 ml of
acetate BS ( pH 3. 5). Carry out the limit test far heavy
metals( 0821 method 1): not more than O. 002%.
Assay Transfer about l. 5 g, accurately weighed, to a 50 ml
volumetric flask, and dissolve in water. Dilute with water to
the volume, mix, and pipet 10 ml of the solution into a 250
ml cornic flask, and precisely add 25 ml of disodium edetate
(0. 05 mol/L) VS, add 20 ml of acetic acid-ammonium
acetate buffer ( pH 4. 5) , heat to near boiling and maintain 5
minutes, allow to cool, add 50 ml of ethanol, add 2 ml of
dithizone IS ( dissolve 25. 6 mg of dithizone in ethanol and
dilute with ethanol to 100 ml, store two months in cold
place), titrate with zinc (0. 05 mol/L) VS to a bright pink,
perfarm a blank determination and make any necessary
correction. Each ml of disodium edetate( O. 05 mol/L) VS is
equivalent to 8. 554 mg of Alz CS04) 3.
Category Pharmaceutical excipients, suspending agents.
Storage Preserve in well-closed containers.
Ammonium Sulfate
CNH4 )zS04 132. 14
[7783-20-2]
Ammonium Sulfate contains not less than 99. O% and not
more than 100. 5% of CNH4)2SÜ4.
Description A colourless or white crystal or granule.
Freely soluble in water; insoluble in ethanol.
Identification A solution ( 1 - 20) yields the reactions
characteristic of ammonium salt and sulfate ( 0301 ) .
Acidity Dissolve l. O g in 20 ml of water, pH 5. 0-6. O
(0631 ).
Chloride Carry out the limit test far chloride ( 0801), using
2. O g. Any opalescence produced is not more than that of a
reference solution using l. O ml of chloride sodium standard
solution (O. 0005 % ) .
Phosphate Dissolve 4. O g in 25 ml of O. 5 mol/L sulfuric
acid solution, add 1 ml of ammonium molybdate sulfate
solution ( transfer 5 g of ammonium molybdate to a 100 ml
volumetric flask, add O. 5 mol/L sulfuric acid, dissolve and
dilute to volume, mix welD and 1 ml of methylaminophenol
sulfate solution ( transfer O. 2 g of methylaminophenol
sulfate, add 100 ml of water and 20 g of sodium bisulfite,
dissolve and mix well. Store one month in well-closed
container), put on room temperature far 2 hours, the solution
is not more intensely coloured than a reference solution
pre pared by 2. O ml of phosphate standard solution ( transfer
O. 1433 g of potassium dihydrogen phosphate, previously
dried far two hours at 105ºC, accurately weighed, to a 1000
ml volumetric flask, dissolve in water and dilute to volume,
mix well. Pipet 10 ml of the solution to a 100 ml volumetric
flask, dilute with water to volume, mix well. Each ml is
equivalent to 10 µg of P0 4) in same method (O. 0005%).
Nitrate Transfer l. O g to a test tube, dissolve in 5 ml of
water, cool in ice bath, add O. 4 ml of 10% potassium
chloride solution and O. 1 ml of O. 1 % diphenylamine sulfuric
solution, mix well, slowly drip 5 ml of sulfuric acid, mix
well, place the tube at 50ºC water bath far 15 minutes, the
blue colour produced by this solution is not more intense than
a reference solution prepared by mixing 1 ml of the standard
nitrate solution [ dissolve O. 163 g of potassium nitrate, in
water and dilute to 100 ml, mix well, pipet 1 ml, dilute with
water to 100 ml, mix well (each ml is equivalent to 10 µg of
N0 3)], and 4 ml of water without nitrate, in same method
(O. 001%).
Water insoluble substance Transfer 20 g to a beaker,
dissolve with 200 ml of water, heat on a water bath far 1
hour. Filter through a sintered-glass crucible ( No. 2 )
previously dried to constant weight, wash the beaker and
funnel with hot water, dry at 105ºC to constant weight, the
residue is not more than O. 005 % .
Residue on ignition Not more than O. 005 % (0841), using 20 g.
lron Dissolve 2. O g of in 40 ml of water, add 2 ml of
hydrochloric acid Carry out the limit test far iron ( 0807).
Any colour produced is not more intense than that of a
reference solution using l. O ml of iron standard solution
(O. 0005%).

Assay Transfer l. 25 g to a 250 ml cornic flask, accurately
weighed, dissolve with 50 ml of water, precisely add 25 ml
of sodium hydroxide ( 1 mol/L) VS, place a glass funnel on
the bottle mouth, boil 15 to 20 minutes until ammonia gas
completely escapes (neutral to litmus paper), allow to cool,
add 3 drops of thymol blue IS, titrate with sulfuric acid
(0. 5 mol/L) VS, and correct the result with blank test.
Each ml of sulfuric acid (O. 5 mol/L) VS is equivalent to
66. 07 mg of CNH4) 2 S04.
Category Pharmaceutical excipients, buffers.
Storage Preserve in well closed containers.
Anhydrous Calcium Hydrogen
Phosphate
CaHP04 136. 06
[7757-93-9]
Anhydrous Calcium Hydrogen Phosphate contains not less
than 98. O% and not more than 103. O% of CaHP04.
Description A white or almost white powder; odourless.
Practically insoluble in water and in ethanol, freely soluble in
dilute hydrochloric acid and in dilute nitric acid.
Identification The acidic aqueous solution yields the
reactions characteristic of calcium salts and the characteristic
(2) and (3) of phosphates ( 0301).
Chloride Dissolve O. 20 g in 10 ml of water and 2 ml of nitric
acid with gentle heating, cool, carry out the limit test far
chloride ( 0801 ) . Any opalescence produced is not more
pronounced than that of a reference solution using 10. O ml of
sodium chloride standard solution (0. 05%).
Sulfate Dissolve exactly l. O g in a quantity of dilute
hydrochloric acid, dilute with water to 100 ml, mix well and
filter. To 20 ml of filtrate add 5 ml of water, carry out the
limit test far suflate ( 0802 ) . Any opalescence produced is
not more pronounced than that of a reference solution using
4. O ml of potassium suflates standard solution (0. 2%).
Fluoride Plastic container should be used in operation.
Dissolve 221 mg of sodium fluoride previously dried at 105ºC
far 4 hours, accurately weighted, in a quantity of water in a
100 ml volumetric flask, add 50. O ml of sodium citrate BS
(Dissolve 73. 5 g of sodium citrate in 250 ml of water),
dilute with water to volume, mix well as the fluoride
standard stock solution (Each ml is equivalent to 1 mg of F).
Accurately dilute a quantity of the fluoride standard stock
solution with sodium citrate BS to produce solution of O. 1,
O. 2, O. 5 and l. O µg F per ml as the standard solutions.
Dissolve about 2. O g, accurately weighted, in 20 ml of water
and 2. O ml of hydrochloric acid in a 100 ml volumetric flask
with swing, add 50 ml of sodium citrate BS and dilute with
water to volume, mix well as the test solution. The indicator
electrode is a fluoride selective electrode and the reference
electrode is a saturated calomel electrode, measure the
electric potential ( m V) of the standard solutions and the test
solution . A calibration curve is produced, using the
logarithm ClogC) of fluoride standard solution concentration
(µg/ml) as X axis and the electric potential as Y axis.
Measure the electric potential of the test solution, the
content of fluoride: not more than O. 005%.
Carbonate Mix l. O g with 5 ml of water and add 2 ml of
hydrochloric acid, no effervescence is produced.
Anhydrous Citric Acid
Insoluble substances in hydrochloric acid Heat to dissolve
about 5. O g, accurately weighted, in a mixture of 10 ml of
hydrochloric acid and 40 ml of water and dilute with water to
100 ml. Filter and wash any residue with water until the
washing <loes not give a reaction far chloride, and dry the
residue at 105ºC far 1 hour. The weight of the residue: less
than 5 mg.
~ on ignition Weight accurately l. O g, ignite to constant
weight at 800ºC, loses 6. 6%-8. 5% of its weight.
Bariurn Add O. 50 g with 10 ml of water and heat, add
hydrochloric acid dropwise with stirring until no more
dissolves. Filter and to the filtrate add 2 ml of potassium
sulfate TS, no turbidity is produced within 10 minutes.
Iron Dissolve 2. 5 gin 20 ml of dilute hydrochloric acid by
heating and dilute with water to 50 ml. Using l. O ml of
dilution, carry out the limit test far iron ( 0807). Any colour
produced is not more intense than that of a reference solution
using 2. O ml of iron standard solution (O. 04 % ) .
Lead Weight accurately about O. 2 g in a 50 ml volumetric
flask, add nitric acid solution ( 1-100) and dilute with the
same solution to volume, mix well as the test solution. Dilute
a quantity of lead standard solution ( Each ml is equivalent to
10 µg of Pb) with nitric acid solution Cl-100) to produce
solution of O, 10, 20, 30, 40 and 50 ng per ml as the
reference solutions. Carry out the method far atomic
absorption apectrophotometry( 0406 method 1), measure the
absorbance at 283. 3 nm with a graphite fumace atomizer.
The content of lead: not more than O. 0005 % .
Arsenic Dissolve l. O gin 5 ml of hydrochloric acid and 23 ml
of water. Carry out the limit test far arsenic ( 0822 method
1): not more than O. 0002%.
Assay Dissolve about O. 6 g, accurately weighted, in 10 ml
of dilute hydrochloric acid with heating, cool, transfer to a
100 ml volumetric flask, dilute with water to volume and
mix well. To 10 ml of the solution, accurately measured,
add 50 ml of water, adjusted to neutral by ammonia TS, and
25 ml, accurately measured, of disodium edetate (O. 05 mol/L)
VS, heat far a few minutes. Allow it to cool, add 10 ml of
ammonia-ammonium chloride BS ( pH 10. O) and a small
amount of eriochrome black T IS. Titrate with zinc
(O. 05 mol/L) VS until the solution tums to reddish-violet.
Perfarm a blank determination and make any necessary
correction. Each ml of disodium edetate (O. 05 mol/L) VS is
equivalent to 6. 803 mg of CaHP04.
Category Pharmaceutical excipients, diluent.
Storage Preserve in tightly closed containers.
Anhydrous Citric Acid
º~º
OH HO
Cs Hs 01 192. 12
[77-92-9]
Anhydrous Citric Acid is 2-hydroxy-l, 2, 3-propanetricarboxylic
acid. It contains not less than 99. 5% and not rmre than 100. 5%
of Cs Hs Ü? , calculated on the anhydrous basis.
 Anhydrous Sodium Carbonate
Description Colourless translucent crystals, white powder
ora white crystalline powder; odourless, taste, strongly
sour; slightly efflorescent in dry air; the aqueous solution
exhibits an acidic reaction.
Very soluble in water, freely soluble in ethanol, sparingly
soluble in ether.
Melting point 152-154 ºC, with decomposition ( 0612).
Identification ( 1 ) Yields the reaction characteristic of
citrates ( 0301 ) .
(2) The infrared absorption spectrum is concordant with the
reference spectrum (IR Album No. 263).
Clarity and colour of solution Dissolve 2. O g in 10 ml of
water, the solution is clear and colourless ( 0901 and 0902
method 1 ) ; any colour produced is not more intense than
that of reference solution Y2 or YG2 ( 0901 method 1 ) .
Chloride Carry out the limit test for chloride ( 0801), using
10. O g. Any opalescence produced is not more pronounced
than that of a reference solution using 5. O ml of sodium
chloride standard solution (O. 0005 % ) .
Sulfate Carry out the limit test for sulfate ( 0802) , using
l. O g. Any opalescence produced is not more pronounced
than that of a reference solution using l. 5 ml of potassium
sulfate standard solution (0. 015%).
Oxalate Dissolve l. O g in 10 ml of water, neutralize the
solution with ammonia TS, add 2 ml of calcium chloride TS,
allow to stand for 30 minutes at room temperature. No
opalescence is produced.
Readily carbonizable substances Transfer l. O g to a Nessler
cylinder, add 10 ml of 95% (g/g) sulphuric acid, heat for
1 hour at 90 ± 1ºC , allow to cool immediately. Any colour
produced is not more intense than that of a reference (a
mixture of o. 9 ml of standard cobaltous chloride es, 8. 9 ml
of standard potassium dichromate es ando. 2 ml of standard
copper sulfate CS.
Water Not more than O. 5 % ( 0832 method 1 ( 1 ) ) .
Residue on ignition Not more than O. 1% ( 0841 ) .
Calcium Dissolve l. O g in 10 ml of water, neutralize with
ammonia TS, add few drops of ammonium oxalate TS. No
opalescence is produced.
Heavy metals Dissolve 4. O gin 10 ml of water, add 1 drop
of phenolphthalein IS, add dropwise a quantity of ammonia
TS until a pink colour is produced. Add 2 ml of acetate BS
(pH 3. 5) and sufficient water to produce 25 ml. Carry out
the limit test for heavy metals ( 0821 method 1): not more
than O. 0005 % .
Arsenic Dissolve 2. O g in 23 ml of water, add 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822
method 1): not more than O. 0001%.
Assay Dissolve about l. 5 g, accurately weighed, in 40 ml
of freshly boiled and cooled water. Add 3 drops of
phenolphthalein IS, ti trate with sodium hydroxide ( 1 mol/L)
VS. Each ml of sodium hydroxide ( 1 mol/L) VS is
equivalent to 64. 04 mg of Cs Hs 01.
Category Pharmaceutical excipients, pH regulator, stabilizer
and acidifying agent.
Storage Preserve in tightly closed containers.
Anhydrous Sodium Carbonate
Na2CÜ3 105. 99
[ 497-19-8]
Anhydrous Sodium Carbonate is prepared from ammonia
soda process (Sol va y' s ammonia soda process). It contains
not less than 99. O% of Na2 C0 3 , calculated on the dried
basis.
Description A white or almost white crystalline powder;
hygroscopic.
Freely soluble in water; insoluble in ethanol.
Identification Yields the reaction characteristic of sodium
salts and carbonates ( 0301).
Clarity and colour of solution Dissolve 2. O g in 10 ml of
water, the solution is clear and colourless ( 0901 and 0902);
any opalescence produced is not more pronounced than that
of reference suspension 1 ( 0902) ; any colour produced is not
more intense than that of reference solution Y1 ( 0901
method 1 ).
Chloride Carry out the limit test for chloride ( 0801) using
O. 4 g. Any opalescence produced is not more pronounced
than that of a reference solution prepared using 5. O ml of
sodium chloride standard solution (O. 0125%).
Sulfate Carrj out the lirnit test for sulfate <0802) using l. O g.
Any opalescence produced is not more pronounced than that
of a reference solution prepared using 2. 5 ml of potassium
sulfate standard solution (O. 025%).
Ammonium Mix l. O g with 10 ml of sodium hydroxide TS,
heat and the vapours formed could not change the colour of
moistened red litmus paper blue.
Sodium bicarbonate Dissolve O. 4 g in 20 ml of water, add
20 ml of barium chloride TS and filter. Mix 10 ml of
successive filtrate with O. 1 ml of phenolphthalein IS, no red
colour is produced. fuil the rest of the successive filtrate for 2
minutes and the solution is clear.
Loss on drying When dried for 4 hours at 105ºC, loss not
more than 2. O% of its weight ( 0831).
Iron Dissolve l. O g in a quantity of water, make the
solution weak acid with dilute hydrochloric acid, boil to
remove the C0 2 , allow to cool and dilute to 25 ml with
water. Carry out the limit test for iron ( 0807) , any colour
produced is not more pronounced than that of a reference
solution using 5. O ml of iron standard solution (O. 005 %) .
Heavy metals Carry out the method ( 0821 method 1) with
5 ml of the solution as described under the Arsenic: not more
than O. 005 %.
Arsenic Mix 2. O g with 5 ml of hydrochloric acid and 25 ml
of water, boil to remove the C02, allow to cool and add
5 mol/L sodium hydroxide solution until the solution is
neutral and dilute to 50 ml with water. Carry out the limit
test for arsenic ( 0822 method 1 ) , using 10 ml: not more
than O. 0005 %.
Assay Dissolve about l. 5 g, accurately weighed, in 50 ml
of water, add 10 drops of methyl red-bromocresol green IS,
ti trate with hydrochloric acid ( l. O mol/L) VS until the
solution becomes purple red, boil for 2 minutes and cool to
room temperature, continue titrating with hydrochloric acid
(l. O mol/U VS until the solution becomes dark purple.

Perform a blank determination and make any necessary
correction. Each ml of hydrochloric acid (l. O mol/L) VS is
equivalent to 53. 00 mg of Na2 C03.
Category Pharmaceutic excipients pH regulator.
Storage Preserve in tightly closed containers.
Anhydrous Sodium Sulfite
Na2 S03 126. 04
[7757-83-7]
Anhydrous Sodium Sulfite contains not less than 97. 0% and
not more than 1OO. 5 % of Na 2S0 3.
Description White crystals ora powder.
Freely soluble in water; very slightly soluble m ethanol;
practically insoluble in ether.
ldentification (1) The aqueous solution 0 -1 O) is alkaline
and yields the characteristic of sulfite ( 0301 ) .
(2) Yields the reaction characteristic ( 1) of sodium salts
< 0301).
Clarity and colour of solution A solution of l. O g in 20 ml of
water is clear and colourless ( 0901 and 0902).
Thiosulfate To 2. O g add 100 ml of water, shake to
dissolve, add 10 ml of formaldehyde solution and 10 ml
acetic acid and mix well. Allow to stand for 5 minutes,
transfer 100 ml of water, carry out the procedure beginning
at the words "add 10 ml of formaldehyde solution... " as the
blank. Add O. 5 ml of starch IS and titrate with iodine
(O. 05 mol/L) VS. Carry out the blank titration. The
difference between the volumes used in the titrations is not
more than O. 15 ml.
lron To l. O g add 2 ml of hydrochloric acid, evaporate to
dryness on a water bath, dissolve the residue in a volume of
water. Carry out the limit test for iron ( 0807). Any colour
produced is not more intense than that of a reference solution
using l. O ml of iron standard solution (0. 001%).
Zinc Dissolve about 10. O g, accurately weighed, in 25 ml
of water in a 250 ml conical flask with shaking, add 15 ml of
hydrochloric acid gently, heat until boiling. Cool and transfer
quantitatively to a 100 ml volumetric flask, dilute with water
to volume, mix well, transfer accurately a quantity of
solution, and dilute quantitatively with water to produce a
solution of 20 mg per ml as the test solution. Transfer
accurately 5 ml of standard zinc solution ( containing Zn
lOOOµg per mD to a 200 ml volumetric flask, dilute with
water to volume, mix well, transfer accurately 2 ml to a 100
ml volumetric flask, add 3 ml of hydrochloric acid, and dilute
with water to volume, mix well as the reference solution. Carry
out the method for atomic absorption spectophotometry <0406
method 1 ) , measure the absorbance of the test solution and
reference solution at 213. 9 nm, the absorbance of the test
solution is not more than that of the reference solution
(O. 0025%).
Heavy metals Carry out the limit test for heavy metals
( 0821 method 1), using l. O g: not more than O. 001%.
Selenium To 3. O g add 10 ml of formaldehyde solution, add
2 ml of hydrochloric acid gently and heat on a water bath for
20 minutes. Any pink colour produced in the solution is not
more intense than that of a ref erence solution pre pared by
using l. O g of the substance being examined, add accurately
measured O. 2 ml of selenium standard solution (Accurately
Arabino Galactan 1 .
weighed O. 100 g of selenium, add 2 ml of mtnc acid,
evaporate to dryness, add 2 ml of water to dissolve the
residue and evaporate to dryness, repeat procedure three
times. Dissolve the residue in dilute hydrochloric acid,
transfer the solution into a lOOOml volumetric flask, dilute to
volume with the same solvent and shake welD, then add 10
ml of formaldehyde solution, add 2 ml of hydrochloric acid
gently and heat on a water bath for 20 minutes (O. 001%).
Arsenic Dissolve O. 5 g in 10 ml of water, add 1 ml of
sulfuric acid, evaporate on a sand bath until white fumes are
evolved. Allow to cool, add 21 ml of water and 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822
method 2) : not more than O. 0004 % .
Assay Dissolve 2. O g, accurately weighted, in a quantity of
water with shaking, add 50 ml of iodine (0. 05 mol/L) VS,
accurately measured, stopper. Allow to stand in dark for
5 minutes. Titrate with sodium thiosulfate (0. 1 mol/L) VS,
add 1 ml of starch IS towards the end point of the titration
and continue the titration until the blue colour disappears.
Perform a blank determination and make any necessary
correction. Each ml of iodine (O. 05 mol/L) VS is equivalent
to 6. 302 mg of Na2 S03.
Category Pharmaceutical excipients, antioxidant.
Storage Preserve in tightly closed containers.
Arabino Galactan
Arabino Galactan is water-soluble polysaccharide obtained
from the xylem of Larch (Larix gmelinii).
Description A white to pale yellow powder.
Freely soluble in water; insoluble in ethanol.
ldentification ( 1) Stir about 6 g with 10 ml of water,
viscous liquid is produced which colour is amber.
(2) Transfer about O. 1 g to centrifuge tube, add 2 ml of
trifluoroacetic acid solution ( 6. 7-100), shake, insert the
stopper, allow to stand at 120ºC for 1 hour. Centrifuge,
transfer the supernatant carefully into a 50 ml round
bottomed flask, add 10 ml of water and evaporate under
reduced pressure at 60ºC, using rotary evaporator. Dissolve
the residue in 2 ml of 90% methanol solution, filtrate, and
use the successive filtrate as test solution. Dissolve 10 mg of
arabinose CRS, galactose CRS with 5 ml of 90% methanol
solution, as reference solution. Carry out the method for
thin-layer chromatography ( 0502), using silica gel G as the
coating substance and a mixture of l. 6 % sodium dihydrogen
phosphate -butanol -acetone ( 10 : 40 : 50) as the mobile
phase. Apply separately to the plate 5 µl of the test solution
and 10 µl of the reterence solution, developing about a path
of 10 cm, dry in a current of warm air for a few minutes.
And developing again about a path of 15 cm using the same
mobile phase. Dry and spray with anisaldehyde solution
(Mix O. 5 ml of anisaldehyde, 10 ml of glacial acetic acid, 85
ml of methanol and 5 ml of sulfuric acid. ) , heat at llOºC
until clear spots are produced. The colour and position of the
spots in the chromatogram obtained with the test solution
correspond to the spot of arabinose and galactose in the
chromatogram obtained with reference solution.
Loss on drying When dried at 105ºC for 5 hours, loses not
more than 12. O% of its weight ( 0831 ) . Using l. O g.
Ash Not more than 4. O% ( 2302). Using l. O g.
 Arginine

Heavy metals Carry out the limit test for heavy metals sample solution, accurately measured, into a 100 ml
<0821 method 2), using the residue obtained in the test for volumetric flask, dilute with mobile phase to volume, mix
Ash: not more than O. 002%. well as reference solution. Carry out the method for high
Arsenic Dissolve O. 67 g in 5 ml of hydrochloric acid and performance liquid chromatography <0512), using a column
packed with octadecylsilane bonded silica gel and a mixture of
23 ml of water. Carry out the limit test for arsenic <0822
citrate BS ( dissolve 9. 6 g citric acid in about 800 ml of
method 1 ) : not more than O. 0003 % .
water, adjust to pH 4. 7 with 1 mol/L sodium hydroxide and
Category Pharmaceutical excipients, suspending agent and dilute with water to lOOOml.) - methanol (67 : 33) as the
adhesive. mobile phase. Detection wavelength is 254 nm. Dissolve a
Storage Preserve in well closed containers. quantity of L-aspartyl-L-phenylalanine and phenylalanine in
mobile phase to produce a solution containing about 15 µg per
ml, inject 20 µl of the solution onto the column and record
the chromatograram. The resolution factor between the
peaks of L-aspartyl-L-phenylalanine and phenylalanine should
Arginine comply with the related requirements. Inject 20 µl each of
sample and test soution onto the column separately, and
See the same monograph in Volume Il. record the chromatogram for twice the retention time of the
principal peak. The sum of impurity peak areas of sample
Category Pharmaceutical excipients, solubilizer and
solution is not greater than the principal peak area of
cryoprotectant.
reference solution (2. 0%), if any impurity peak observed in
the sample chromatogram.
Los.son drying When dried at 105ºC for 4 hours, loses not
more than 4. 5 % of its weight <0831 ) .
Asparagine Residue on ignition Not more than O. 2% <0841 ) , using
l. o g.
See the monograph of asparagine in volume 1I .
Heavy metals Carry out the limit test for heavy metals
Category Pharmacutical excipient, solubilizer and freeze- <0821 method 2) , using the residue obtained in the test for
dried protecting agent. Residue on ignition: not more than O. 001%.
Arsenic Mix O. 67 g of sampie to be examined with l. O g oí
calcium hydroxide, triturate with 2 ml of water, and dry at
40ºC. lgnite gently to carbonize and then ignite at 500-600ºC
Aspartame until the incineration is complete. Cool, add 8 ml of
hydrochloride acid and 23 ml of water. Carry out the limit
test for arsenic <0822 method 1 ) : not more than O. 0003 % .
Assay Dissolve about O. 25 g, accurately weighed, in a

ºº
mixture of 3 ml of formic acid and 50 ml of glacial acetic
acid. Carry out the method for potentiometric titration

HOY::C~~OCH, <0701 ). Titrate immediately with perchloric acid (O. 1 mol/U

VS. Perform a blank determination and make any necessary
O H NH2 O correction. Each ml of perchloric acid (O. 1 mol/L) VS is
equivalent to 29. 43 mg C14 H1s N2 Os.
C14 H1s N2 Os 294. 31
[22839-47-0] Category Pharmaceutical excipients, sweetening agent and
Aspartameis 3-amino-N- ( a-carboxyphenethyl) succinamic taste-masking agent.
acid N-methyl ester. It contains notless than 98. 0% and not Store Preserve in tightly closed containers.
more than 102. O% of C14 H 18 Nz Os , calculated on the dried
basis.
Description white crystalline powder; taste sweet.
Very slightly soluble in water, insoluble in ethanol, m
hexane and in dichloromethane.
Aspartic Acid
Specific optical rotation + 14. 5º to + 16. 5°, in a solution of
See the monograph of aspartic acid in volume Il .
40 mg per ml in 15 mol/L formic acid solution < 0621 ) .
Perform the determination immediately. Category Pharmacutical excipient, solubilizer and freeze-
dried protecting agent.
ldentification The infrared absorption spectrum <0402) is
concordant with the reference spectrum (IR Album No. 768).
Absorbance The absorbance of a solution of 1 % in 2 mol/L
hydrochloric acid solution at 430 nm <0401) is not more than
o. 022. Benzalkonium Bromide
Acidity Dissolve l. O g in 125 ml of water, pH 4. 0-6. O
<0631>. See the same monograph in Volume 1I.
Related substance Dissolve a quantity of the substance being Category Pharmaceutical excipients, preservative.
examined, accurately weighed, in mobile phase to produce a
solution of 6 mg per ml as test solution. Transfer 2 ml of the

Benzalkonium Chloride
See the same monograph in Volume Il .
Category Pharmaceutical excipients, preservative.
Benzyl Alcohol
()OH
GHaO 108.14
[100-51-6]
Benzyl alcohol contains not less than 98. O% of G Ha O,
calculated on the anhydrous basis.
Description A colourless liquid; odour, slightly characteristic;
taste, burning; hygroscopic.
Soluble in water, miscible with ethanol, chloroform or
ether.
Relative density l. 043-1. 050 <0601 ) .
Boiling range Carry out the determination of boiling range
( 0611 ) , not less than 95 % ( ml/ml) is distilled at 203-
206ºC.
Refractive index l. 538-1. 541 <0622).
Identification ( 1) Add 2-3 drops to 2 ml of potassium
permanganate TS and 2 mi oí dilute sulfuric acid, mix well,
the benzylaldehyde characteristic odour is perceptible.
(2) The infrared absorption spectrum is concordant with the
reference spectrum (IR Album No. 236).
Acidity To 10 ml add 10 ml of ethanol and 1 ml of
phenolphthalein IS. Titrate with sodium hydroxide (O. 1
mol/U VS until a pink colour is produced, the volume of
sodium hydroxide (O. 1 mol/L) VS required is not more than
0.2 ml.
Clarity and colour of solution To 2 ml add 58 ml of water
and shake, the solution is clear and colourless ( 0901 and
0902).
Chloride Carry out the limit test for chlorides ( 0801 ) ,
using 1 g. Any opalescence produced is not more pronounced
than that of a reference solution using 3. O ml of sodium
chloride standard solution (O. 003 % ) .
Related substances Use the substance being examined as test
solution. Dissolve a quantity of benzaldehyde CRS, accurately
weighed, in acetone to produce the reference solution of O. 5
mg per ml. Carry out the method for gas chromatography
( 0521 ) , using polyethylene glycol 20 M as the stationary
phase. Split injection and the split ratio is 20 : l. The
temperature of the column is 50ºC, then raise the
temperature at a rate of 5ºC per minute to 220ºC and maintain
for 35 minutes. Maintain the temperature of injection port at
200ºC and the detector temperature at 310ºC . Inject
accurately 1 µl each of the test solution and the reference
solution into the column respectively and record the
chromatograms. Disregard any peak with area less than
O. 0001 % of the area of the principal peak in the chromatogram
obtained with the test solution. The content of benzaldehyde is
Benzyl Hydroxybenzoate
not more than O. 1 % , calculated by the externa! standard
method. The content of any other impurity is not more than
O. 02%, calculated by the peak area normalization method,
and the total amount of all other impurities is not more than
O. 1 % . lntended use for injection, the content of
benzaldehyde is not more than O. 05 % , calculated by the
externa! standard method. The content of any other impurity
is not more than O. 01 % , calculated by the peak area
normalization method, and the total amount of all other
impurities is not more than O. 05%.
Water Not more than O. 5 % ( 0832 method 1 ( 2) ) .
Bacterial endotoxin ( Entended use for injection) Carry out
the test for bacterial endotoxin ( 1143 ) : not more than O. 1 EU
per g of benzyl alcohol.
Assay Carry out the method for gas chromatography
( 0521 ) , using a column packed with polyethylene glycol
20 M as the stationary phase. The temperature of the
injection port is 200ºC and that of the detector is 250ºC, the
column temperature is 130ºC.
Procedure Accurately weigh a quantity of the substance
being examined, dilute with methanol to produce a solution
containing 1 mg per ml. Accurately inject 1 µl of the test
solution onto the column and record the chromatogram.
Repeat the operation, using benzyl alcohol CRS instead of the
substance being examined. Calculate the content of G Ha O
with respect to the area obtained in the chromatogram by
external standard method.
Category Pharmaceutic excipients, preservative.
Storage Preserve in well closed containers, protected from
light.
Benzyl Hydroxybenzoate
HO~
rJlº'10
~
Cl4 H12 03
228. 25
[94-18-8]
Benzyl Hydroxybenzoate is benzyl-4-hydroxybenzoic acid. It
contains not less than 98. O% and not more than 102. O% of
C14 H12 Ü3 , calculated on the dried basis.
Description A white or almost white crystalline powder.
Soluble in methanol and in ethanol; practically insoluble in
water.
Melting point 11 l-113ºC <0612 >.
ldentification (1) The retention time of the principal peak
of the substance being examined in the chromatogram is
identical with that of the principal peak of benzyl hydroxy
benzoate CRS in the chromatogram obtained in the Assay.
(2) Weigh a quantity of the substance being examined to
produce a solution of 5 µg per ml in ethanol, the light
absorption of the solution exhibits a maximum at 260 nm
(0401).
(3) The infrared absorption spectrum is concordant with that
of benzyl hydroxy benzoate CRS ( 0402).
Acidity Dissolve O. 2 g in 5 ml of 50% ethanol, mix well,
add 2 drops of methyl red IS: not more than O. 1 ml of
sodium hydroxide (O. 1 mol/L) VS is required to change the
 Betacyclodextrin

colour of the solution to orange. Heavy metals Carry out the limit test for heavy metals
Chloride Heat 2. O g of the substance being examined with ( 0821 method 2), using the residue obtained in the test for
50 ml of water at 80ºC water bath for 5 minutes, cool and residue on ignition: not more than O. 002%.
filter. Carry out the limit test for chlorides ( 0801 ) , using ~y Carry out the method for high performance liquid
5 ml of the filtrate. Any opalescence produced is not more chromatography ( 0512 ) , using a column packed with
pronounced than that of a reference solution using 7. O ml of phenylsilane bonded silica gel, 1 % glacial acetic acid solution
sodium chloride standard solution (O. 035 % ) . as the mobile phase A and methanol as the mobile phase B,
Sulfate Carry out the limit test for sulfates ( 0802) , using elute with gradient elution as the following table. Detection
25 ml of the filtrate obtained in the test for chloride. Any wavelength is 254 nm. lnject 20 µI of the system suitability
opalescence produced is not more pronounced than that of a solution in the test for related substances into the column.
reference solution using 2. 4 ml of potassium sulfate standard The resolution factor between peaks of butylparaben and
solution (O. 024%). benzyl hydroxybenzoate is not less than 3. O.
Related substances Dissolve a quantity of the substance Procedure Dissolve a quantity, accurately weighed, in the
being examined, accurately weighed, in solvent [1% acetic mixture of 1% glacial acetic acid solution-methanol (40 : 60)
acid solution -methanol ( 40 : 60) ] to produce a solution of to produce the test solution of O. 1 mg benzyl hydroxybenzoate
1 mg per ml as the test solution. Measure accurately 1 ml of per ml. lnject 20 µl of the resulting solution, accurately
the test solution to a 100 ml volumetric flask, dilute to measured, into the column and record the peak areas
volume with solvent, mix well, as the reference solution corresponding obtained in the chromatogram. Accurately
(1). Dissolve a quantity of p-hydroxybenzoic acid CRS in weigh benzyl hydroxybenzoate CRS and repeat the operation.
solvent to produre a solution of 10 µg per ml as the reference Calculate the contents of benzyl hydroxybenzoate with
solution ( 2). Carry out the method for high performance respect to the peak area obtained in the chromatogram by the
liquid chromatography ( 0512), using a column packed with externa} standard method.
phenyl bonded silica, 1 % glacial acetic acid solution as
mobile phase A and methanol as mobile phase B, elute with Time The mobile The mobile
gradient elution as the following table. Detection wavelength (minutes) phase A C%) phase B (%)
is 254 nm. Dissolve a quantity of system suitability solution of
butylparaben CRS and benzyl hydroxybenzoate CRS in solvent o 40 60
to produce a mix solution containing 10 µg each of the two 17 40 60
substances pcr ml. Inject 20 µ.l of the resulting solution into
the column, the resolution between butylparaben and benzyl 18 o 100
hydroxybenzoate should not less than 3. O. Inject 20 µl of the
reference solution (1) into the column. Adjust the 23 o 100
attenuation so that the principal peak height in the
24 40 60
chromatography is about 25 % of the full scale of the chart.
Inject separately 20 µl of the test solution, reference solution 30 40 60
( 1) and the reference solution ( 2), accurately measured,
into the column and record the chromatogram for twice the
retention time of the principal peak. Calculate the content of Category Pharmaceutical excipients, preservative.
p-hydroxybenzoic acid with respect to the peak area obtained
Storage Preserve in well closed containers.
in the chromatogram by externa! standard method, not great
than l. O% . The area of any other impurity peak is not
greater than O. 5 times the area of the principal peak in the
chromatogram obtained with the reference solution ( 1)
(O. 5 %) , the sum of the areas of any other impurity peaks is Betacyclodextrin
not greater than the area of the principal peak in the
chromatogram obtained with the reference solution ( 1)
(l. 0%). HO

HO~~/~~d,
Time The mobile The mobile
(minutes) phase A (%) phase B (%)

o 40 60

17 40 60 ~'OH
OH t;__)
O HO O
40 o 100 OH OH

u
45 o 100

46 40 60

52 40 60 HO O OH
O HO
o
OH
Loss on drying When dried in vacuum in a desiccator
containing silica gel to constant weight, loses not more than OH
O. 5 % of its weight ( 0831). CC6 H10 Os )1 1134. 99
Residue on ignition Not more than O. 1 % <0841 ) , using l. O g. [7585-39-9]

Betacyclodextrin is a cyclic oligosaccharide compound
composed of seven a-1, 4 linked D-glucopyranosyl units and
is obtained from the action of cyclodextrin glucosyltransferase on
starch. It contains not less than 98. O% and not more than
102. o% of CC6H100s)1, calculated on the dried basis.
Description White crystals or a crystalline powder;
odourless; tas te slightly sweet.
Sparingly soluble in water; practically insoluble in methanol,
ethanol, acetone or in ether.
Specific optical rotation +
159º to + 164º, in a solution of
10 mg per ml in water <0621 ) .
Identification ( 1) Dissolve about O. 20 g in 2 ml of iodine
TS by warming in a water bath, and cool to room
temperature. A yellowish-brown precipitate is produced.
( 2) The retention time of the principal peak in the
chromatogram of the test solution obtained in the Assay is
identical with that of the principal peak in the chromatogram
of the reference solution.
(3) The infrared absorption spectrum ( 0402) is concordant
with that of the reference standard.
Light-absorbing impurities Dissolve about 1 g of the
substance being examined, accurately weighed, in 100 ml of
water. The absorbance of the solution in the range of 230-
350 nm (0401) is not more than O. 10, and that in the range
of 350-750 nm is not more than O. 05.
Acidity or alkalinity Dissolve O. 20 gin 20 ml of water, add
O. 2 ml of a saturated solution of potassium chloride, pH
5. 0-8. o <0631 >.
Clarity and colour of solution Dissolve O. 50 g in 50 ml of
water, comply with the test for clarity and colour of solution
( 0901 and 0902), the solution is clear and colourless; any
opalescence produced is not more pronounced than that of
reference suspension 2 ( 0902 method 1 ) .
Chloride Carry out the limit test for chlorides ( 0801 ) ,
using O. 39 g. Any opalescence produced is not more
pronounced than that of a reference solution using 7. O ml of
sodium chloride standard solution (0. 018%).
Reducing sugars Dissolve about l. O g, accurately weighed,
in 25 ml water, add 40 ml of alkaline cupric tartrate TS. Boíl
gently for 3 minutes and allow to stand overnight at room
temperature. Filter with a G4 sintered glass filter, wash the
precipitate with warm water until the washings are neutral.
Discard the filtrates and washings. Dissolve the precipitate in
20 ml of hot ferric sulfate TS and filter. Wash the filter with
100 ml of water. Combine the filtrates and washings, and
heat to 60ºC . Titrate the hot solution immediately with
potassium permanganate (O. 02 mol/L) VS. The potassium
permanganate (O. 02 mol/L) VS consumed is not more than
3. 2 ml (l. O%) for ea ch g of substance taken, calculated on
the dried basis.
Cyclohexane Dissolve about O. 2 g of the substance being
examined, accurately weighed, in 10. O ml of internal
standard solution ( Dissolve a quantity of 1, 2-
dichloroethylene in 20 % dimethyl suifoxide solution and
dilute to produce a solution of O. 04 µg per ml) into a
headspace vial as the test solution. Dilute a quantity of
cyclohexane, accurately weighed, with internal standard
solution to produce a solution containing O. 078 mg of
cyclohexane per ml, measure 1O. O ml in to a headspace vial as
the reference solution. Carry out the method for residual
solvent ( 0861 ) , using a capillary column packed with 100 %
dimethyl polysiloxane as the stationary phase; a flame
Black Ferric Oxide 1 \4Sf./ '
ionization detector is used. The temperature of column is
90ºC; the temperature of injection is 200ºC; the temperature
of detection is 250ºC; maintain the headspace vial at 70ºC for
20 minutes. lnject each of the gases from headspace vials into
the column and record the chromatograms. Calculate the
contents of cyclohexane with respect to the area obtained in
the chromatogram by the internal standard method. The
result complies with the requirements.
Loss on drying When dried to constant weight at 105ºC,
loses not more than 14. O% of its weight ( 0831 ) .
Residue on ignition Not more than O. 1 % ( 0841 ) , using
l. o g.
Heavy metals Carry out the limit test for heavy metals
<0821 method 2) , using the residue obtained in the test for
Residue on ignition: not more than O. 001%.
Microbial limit Comply with the requirements for
microbiological limit ( 1105 and 1106), the total aerobic
bacteria count is not more than 1000 cfu per g and the total
combined yeast/mold count is not more than 100 cfu per g of
the substance being examined. Escherichia coli should not be
detected.
Assay Carry out the method for high performance liquid
chromatography ( 0512 ) , using a column packed with
octadecylsilane bonded silica gel, a mixture of water-
methanol (85 : 15) as the mobile phase and a differential
refractometer as detector. The number of the theoretical
plates of the column is not less than 1500, calculated with
reference to the peak of Betacyclodextrin.
Procedure Shake about 50 mg, accurately weighed, with a
quantity of water in a 10 ml volumetric flask, dilute with
water to volume and mix well as the test solution. Inject 10
µl of the test solution into the column, record the figure of
chromatograph. Repeat the operation, using 50 mg of
Betacyclodextrin CRS instead of the substance being
examined, calculated the content of Betacyclodextrin with the
peak area obtained on the chromatogram by the external
standard method.
Category Pharmaceutical excipients, inclusion agent and
stabilizer, etc.
Storage Preserve in well closed containers, stored in a dry
place.
Black Ferric Oxide
Fe2 Ü3• FeO 231. 53
[1317-61-9]
Black Ferric Oxide contains not less than 96. O% of Fe2 0 3 ,
calculated with reference to the substance ignited to constant
weight.
Description A black powder; odorless; tasteless.
Insoluble in water; freely soluble in boiling hydrochloric
acid.
ldentification To O. 1 g add 5 ml of dilute hydrochloric acid,
boil and allow to cool, the solution yields the reactions
characteristic of ferric salts ( 0301 ) .
Water soluble substances To 2. O g add 100 ml of water,
reflux on a water bath for 2 hours and filter. Wash the
residue with a small amount of water, evaporate the combined
 :f¡fg&:•.···· . •.·..
·······.··.·.·.'..•.·.·.·.·.¡ Borax
filtrate and washings to dryness in an evaporating dish previously
dried to constant weight at 105ºC, dry the residue to constant
weight at 105ºC: not more than 10 mg (O. 5%).
Acid insoluble substances Dissolve 2. O g in 25 ml of
hydrochloric acid by heating on a water bath, add 100 ml of
water, filter through a sintered glass crucible (No. 4)
previously dried to constant weight at 105ºC, wash the
residue with hydrochloric acid solution ( 1-100) until the
washing colourless, then wash the residue with water until
the washing yields no reaction of chlorides, dry the residue to
constant weight at 105ºC: not more than 6 mg (0. 3%).
Loss on ignition Take about l. O g ( accurately weighted),
when ignited to constant weight at 800ºC, loses not more
than 4. O% ( 0831 ) .
Barium To O. 2 g add 5 ml of hydrochloric acid, heat to
dissolve. Add 1 drop of hydrogen peroxide TS and 20 ml of
10 % sodium hydroxide solution, fil ter and wash the residue
with 10 ml of water. Combine the filtrate and washings, add
10 ml of sulfuric acid solution (2-10), no opalescence is
produced.
Lead Transfer 2. 5 g of the substance into a 100 ml concial
flask with stopper, add 35 ml of O. 1 mol/L hydrochloric acid
solution, and stir for 1 hours. Filter and wash the residue
with O. 1 mol/L hydrochloric acid solution, combine the
filtrate and washings in a 50 ml volumetric flask, dilute with
O. 1 mol/L hydrochloric acid solution to volume. Shake well
and use as the test solution. Carry out the method for atomic
absorbance spectrophotometry ( 0406 ) , measure the
absorbance at 217. O nm. To 2. 5 ml of lead standard solution
in a 50 ml volumetric flask, add 5 ml of 1 mol/L
hydrochloric acid solution, dilute with water to volume,
shake well and use as the standard solution. Repeat the
operation, using the standard solution instead of the test
solution. The absorbance of the test solution is not greater
than that of the standard solution (O. 001 %).
Arsenic To O. 67 g add 7 ml of hydrochloric acid, heat to
dissolve. Add 21 ml of water and acidic stannous chloride TS
dropwise until the yellow colour disappears. Carry out the
limit test for arsenic ( 0822 method 1 ) : not more than
o. 0003%.
Assay Place about O. 15 g the substance which is ignited to
constant weight at 800ºC, accurately weighted, into a concial
flask with stopper, add 5 ml hydrochloric acid, heat to
dissolve with water bath, add 2 ml H 2 0 2 TS. Continue to
heat until the solution begins to boil. Cool. Add l. 5 g
potassium iodide and 2. 5 ml hydrochloride acid, plug, mix
well, allow to stand in dark place for 15 minutes, titrate
with sodium thiosulfate (O. 1 mol/L) VS, add 2. 5 ml of
starch IS towards the end of titration, titrate until the blue
colour disappear. Each ml of sodium thiosulfate
(0. 1 mol/L) VS is equivalent to 7. 985 mg of Fe2Ü3.
Category Pharmaceutical excipients, colourant and coating
material.
Storage Preserve in tightly closed containers.
Borax
Na2B4Ü1•lOH20 381. 37
[1303-96-4]
Borax is sodium tetraborate. lt contains not less than 99. O%
and not more than 103. 0% of Na2B4Ü1•lOH20.
Description Colourless, translucent crystals or a white
crystalline powder; odourless; efflorescent.
Freely soluble in boiling water, soluble in water, insoluble in
ethanol.
ldentification Yields the reactions characteristic of sodium
salts and borates ( 0301).
Alkalinity Dissolve l. O g in 25 ml of water, pH 9. 0-9. 6
<0631>.
Clarity and colour of solution Dissolve O. 5 g in 10 ml of
water, the solution is clear and colourless ( 0901 method 1
and 0902) ; any opalescence produced is not more pronounced
than that of reference suspension 2 ( 0902 method 1 ) .
Chloride Carry out the limit test for chlorides ( 0801 ) , using
O. 25 g. Any opalescence produced is not more pronounced
than that of the reference solution using 5. O ml of sodium
chloride standard solution (0. 02%).
Sulfate Carry out the limit test for sulfates ( 0802) , using
O. 50 g. Any opalescence produced is not more pronounced
than that of the reference solution using 2. O ml of potassium
sulfate standard solution (O. 04 % ) .
Carbonates and bicarbonates Dissolve O. 25 g in 5 ml of
water, add 3 ml of dilute hydrochloric acid, no effervescence
is produced.
Calcium Dissolve O. 25 g in 10 ml of water, acidify with
acetic acid, add l. O ml of ammonium exalate TS, allow to
stand for 1 minute, add 5 ml of ethanol, mix well. Any
opalescence produced after 15 minutes is not more pronounced
than that of the reference solution prepared in the same
manner, using 2. 5 ml of calcium standard solution (Place O. 125
g of calcium carbonate, previously dried to constant weight at
105-llOºC, weigh accurately, in a 500 ml volumetric flask.
Add 5 ml of water and O. 5 ml of hydrochloric acid to
dissolve, dilute to volume with water, mix well. Measure
accurately 10 ml to a 100 ml volumetric flask, dilute to
volume with water, and mix well. Each ml is equivalent to
10 µg of Ca) (0. 01%).
Magnesium Dissolve O. 50 g in 8 ml of water, neutralize
with dilute hydrochloric acid, add water to 10 ml, add 5 ml
of 8 % sodium hydroxide solution and O. 2 ml of O. 05 % titan
yellow IS, mix well. Any colour produced is not more
intense than that of the reference solution prepared in the
same manner, using 5. O ml of magnesium standard solution
(dissolve 16. 6 mg of magnesium oxide, previously ignited to
constant weight at 800ºC, in 2. 5 ml of hydrochloric acid and
sufficient water to produce lOOOml, mix well. Each ml is
equivalent to 10 µg of Mg) (0. 01%).
Iron Dissolve l. O g in 25 ml of water. Carry out the limit
test for iron ( 0807 ) . Any colour produced is not more
intense than that of the reference solution using 3. O ml of
iron standard solution (O. 003 % ) .
Ammonium Carry out the limit test for ammonium
( 0808 ) , using 2. O g. Any colour produced is not more
intense than that of the reference solution using 2. O ml of
ammonium chloride standard solution (O. 001%).
Heavy metals Dissolve l. O g in 16 ml of water, add
dropwise dilute hydrochloric acid to neutral, add 2 ml of
acetate BS (pH 3. 5), add water to produce 25 ml. Carry out
the limit test for heavy metals ( 0821 method 1 ) : not more
than O. 001%.
Arsenic Dissolve O. 4 g in 23 ml of water, add 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822

method 1 ) : not more than O. 0005 % .
Assay Dissolve O. 15 g, accurately weighed, in 25 ml of
water, add 1 drop of O. 05% methyl orange IS and titrate
with hydrochloric acid (O. 1 mol/U VS until the solution
becomes reddish-orange, boil far 2 minutes, allow to cool. If
the solution is yellow, continue the titration until the colour
just changes to reddish-orange, add 5 g of mannitol and
dissolve, add 3 drops of phenolphthalein indicator, titrate
with sodium hydroxide (O. 1 mol/U VS until the solution
becomes pink. Perform a blank determination and make any
necessary correction. Each ml of sodium hydroxide (O. 1
mol/U VS is equivalent to 9. 534 mg of Na2B401•lOH20.
Category Pharmaceutical excipients, preservative
buffering agent.
Storage Preservein tightly closed containers.
Boric Acid
H3B03 61. 83
[10043-35-3]
Boric acid contains not less than 99. 5% of H3B03, calculated
on the dried basis.
Description Colourless with little glossy pearl crystals, or
loase white powder, creamy feeling; odorless.
Soluble in ethanol or water; very soluble in boiling water or
boiling ethanol.
ldentiflcation Aqueous solution yields the reactions characteristic
of })()Tate (0301 ).
Acidity Dissolve l. O g in 30 ml of water, pH 3. 5-4. 8
<0631>.
Oarity and color of solution Dissolve l. O g in 30 ml of water,
the solution is clear and colourless <0901 method 1 and
0902). Any opalescence produced is not more pronounced
that of reference suspension 1 <0902 method 1 ) .
Clarity of ethanol solution A solution of l. O g in 25 ml of
ethanol is clear.
Chloride Carry out the limit test far chlorides < 0801 ) ,
using O. 50 g. Any opalescence produced is not more
pronounced than that of a reference solution using 5. O ml of
sodium chloride standard solution (0. 01 %).
Sulfate Carry out the limit test far sulfate <0802) , using
O. 50 g. Any opalescence produced is not more pronounced
than that of a reference solution using 2. O ml of potassium
sulfate standard solution (0. 04%).
Phosphate Dissolve O. 50 g in 15 ml of water, add 2 drops
of 2, 4-dinitrophenol saturated solution, add dropwise
sulfuric acid solution ( 12 - 100) until the yellow colour
disappears, dilute with water to 20 ml, add 4 ml of sulfuric
acid solution (12-100), 1 ml of 5% ammonium molybdate
solution and phosphate solution, shake well and incubate at
60ºC water bath far 10 minutes, any colour produced should
be not more intense than that of the reference solution
prepared in the same manner, using 5. O ml of phosphate
standard solution ( take O. 1430 g of potassium dihydrogen
phosphate, accurately weighed, to lOOOml volumetric flask,
dissolve in water and dilute to the volume, shake well,
precisely transfer 10 ml to 100 ml volumetric flask, dilute
with water to the volume, shake well. Each ml is equivalent
to 10 µg of P04) (0. 01%).
Brown F erric Oxide
Calcium Dissolve O. 50 g in 10 ml of water, add ammonia
TS turning alkaline, add O. 5 ml of ammonium oxalate TS, 5
ml of ethanol, add water to 20 ml, shake well, any
opalescence produced should be not more pronounced than
that of the reference solution prepared in the same manner,
using 5. O ml of standard calcium solution (accurately weigh
O. 125 g of calcium carbonate that has been dried to constant
weight at 105ºC, transfer to 500 ml volumetria flask, add
5 ml of water and O. 5 ml of hydrochloric acid, dilute with
water to the volume, shake well. Each ml is equivalent to
10 µg of Ca) (0. 01 %).
Magnesium Dissolve O. 50 g in 8 ml of water, neutralize
with 8% sodium hydroxide solution to be neutral, add water
and
to 10 ml, add 5 ml of 8% sodium hydroxide solution and
O. 2 ml of O. 05% tanzania yellow solution, shake well; any
colour produced is not more intense than that of the reference
solution prepared by 5. O ml of magnesium standard solution
CAccurately weigh 16. 6 mg of magnesium oxide that has
been ignited to constant weight at 800ºC, add 2. 5 ml of
hydrochloric acid and water to dissolve into lOOOml, mix
well. Each ml is equivalent to 10 µg of Mg) (O. 01%).
Iron Carry out the limit test far iron <0807), using l. O g
dissolved in 25 ml of water. Any colour produced is not more
intense than that of a reference solution using l. O ml of
standard iron solution (0. 001%).
Ammonium Carry out the limit test far ammonium <0808 ) ,
using 2 g: not more than O. 001%.
Loss on drying Place 1 g in silica gel drier far 5 hours, loses
not more than O. 5 % of its weight <0831 ) .
Heavy metals Dissolve l. O gin 23 ml of water, add 2 ml of
acetate salt BS (pH 3. 5), carry out the limit test far heavy
metals (0821 method 1): not more than O. 001%.
Arsenic Dissolve O. 40 g in 23 m1 of water, add 5 m1 of
hydrochloric acid. Carry out the limit test far arsenic <0822
method 1 ) : not more than O. 0005 % .
Assay Accurately weigh O. 1 g, add 25 ml of 20% neutral
mannitol solution (neutral to phenolphthalein IS), slightly
heat to dissolve, cool immediately, add 3 drops of
phenolphthalein indicator solution, titrate with sodium
hydroxide (O. 1 mol/U VS. Each ml of sodium hydroxide (O. 1
mol/U VS is equivalent to 6. 183 mg of H 3B03.
Category Pharmaceutical excipients, preservative and
buffering agent.
Storage Preserve in tightly closed containers.
Brown Ferric Oxide
Brown Ferric Oxide is a mixture of a fixed proportion of red
ferric oxide, yellow ferric oxide and black ferric oxide. It
contains not less than 98. O% of Fez 03 , calculated with
reference to substance ignitated to constant weight.
Description A red-brown powder; odorless; tasteless.
Insoluble in water; freely soluble in boiling hydrochloric
a cid.
ldentification Boil about O. 1 g with 5 ml of dilute
hydrochloric acid, cool, the solution yields the reactions of
ferric salts <0301 ) .
Water soluble substances To 2. O g add 100 ml of water, reflux
on a water bath far 2 hours and filter. Wash the residue with a
 Butyl Alcohol
quantity of water, evaporate the combined filtra te and
washings to dryness in an evaporating dish previously dried
to constant weight at 105ºC, dry the residue to constant
weight at 105ºC: not more than 10 mg (0. 5%).
Acid insoluble substances Dissolve 2. O g in 25 ml of
hydrochloric acid by heating on a water bath, add 100 ml of
water, filter through a sintered glass crucible ( No. 4 )
previously dried to constant weight at 105ºC, wash the
residue with hydrochloric acid solution ( 1-100) until the
washing become colourless, then wash the residue with
water until the washings give no reaction of chlorides, dry
the residue to constant weight at 105ºC: not more than 6 mg
(0. 3%).
Barium To O. 2 g add 5 ml of hydrochloric acid, heat to
dissolve. Add 1 drop of hydrogen peroxide TS and 20 ml of
10 % sodium hydroxide solution, fil ter and wash the residue
with 10 ml of water. Combine the filtrate and washings, add
10 ml of sulfuric acid solution ( 2-10), no opalescence is
produced.
Lead Transfer 2. 5 g of the substance being examined to a
100 ml conical flask with stopper, add 35 ml of O. 1 mol/L
hydrochloric acid solution, and stir for 1 hour. Filter and
wash the residue with O. 1 mol/L hydrochloric acid solution,
combine the filtrate and washings in a 50 ml volumetric
flask, dilute with O. 1 mol/L hydrochloric acid solution to
volume, shake well and use as the test solution. Carry out
the method for atomic absorbance spectrophotometry
( 0406), measure the absorbance at 217. O nm. Place 2. 5 ml
of lead standard solution in a 50 ml volumetric flask, add
5 ml of 1 mol/L hydrochloric acid solution, dilute with water
to volume, shake well and use as the standard solution.
Repeat the operation, using the standard solution instead of
the test solution. The absorbance of the test solution is not
greater than that of the standard solution (0. 001%).
Arsenic To O. 67 g add 7 ml of hydrochloric acid, heat to
dissolve. Add 21 ml of water and acidic stannous chloride TS
dropwise until the yellow colour disappears. Carry out the limit
test for arsenic (0822 method 1 ) : not more than O. 0003 % .
Assay Place about O. 15 g of the substance being examined,
previously ignited to constant weight at 800ºC, accurately
weighed, in a conical flask with stopper, add 5 ml of
hydrochloric acid and heat to dissolve on a water bath. Add 2
ml of hydrogen peroxide TS, heat to boil for a few minutes,
add 25 ml of water, allow to cool. Add l. 5 g of potassium
iodide and 2. 5 ml of hydrochloric acid, stopper and shake
thoroughly, allow to stand in a dark place for 15 minutes.
Titrate with sodium thiosulfate (O. 1 mol/L) VS, add 2. 5 ml
of starch IS towards the end of titration, continue the
titration until the blue colour disappears. Each ml of sodium
thiosulfate (0.1 mol/L) VS is equivalent to 7. 985 mg of Fei(h.
Category Pharmaceutical excipients, colourant, coating
material, etc.
Storage Preserve in tightly closed containers.
Butyl Alcohol
CH100 74.12
[71-36-3]
Butyl alcohol 1s produced by carboxyl synthesis method,
acetaldehyde synthesis method or fermentation.
Description A colourless and clear liquid, with a characteristic
penetrating vinous odour.
Soluble in water, miscible with ethanol and with ether.
Relative density O. 807-0. 809 at 25ºC (0612).
Distilling Range 116-119 ºC , distils wi thin a range of l. 5ºC ,
<0611 >.
ldentification The infrared absorption spectrum ( 0402) is
concordant with the reference spectrum of butyl alcohol
CRS.
Acidity Add 2 drops of phenolphthalein IS to 74 ml of the
substance being examined. The solution should be colourless.
Titrate with ethanolic potassium hydroxide (O. 02 mol/L) VS
until a pink colour persists 15 seconds. Not more than 2. 5 ml
of ethanolic potassium hydroxide ( O. 02 mol/L ) VS 1s
required.
Aldehyde Add 10 ml of ammoniated sliver nitrate TS to
10. O ml of the substance being examined, stopper and mix.
Allow to stand in a dark place for 30 minutes, the solution is
colourless.
Non-volatile matter Evaporate 100 ml in an evaporating
dish, previously dried to constant weight at 105ºC, to
dryness on a water bath, dry at 105ºC for 30 minutes: the
residue is not more than 4 mg.
Butyl ether and related substances Carry out the method for
gas chromatography ( 0521 ) , using a capillary column coated
polyethylene glycol (PEC'.-20 M) (or stationary phase with
similar polarity), maintaining the column temperature at
75ºC; maintaining the temperature of the injection port at
260ºC and that of the detector at 280ºC . Mix well butyl
ether, 2-butanol, isobutyl alcohol and butyl alcohol by the
same volume, Inject 1 µl of the mixture into the column and
record the chromatogram. The resolution factor between any
two peaks complies with the requirements.
Procedure Inject 1 µl of the substance being examined into
the column and record the chromatogram. Calculate the
contents by the peak area normalization method. The content
of butyl ether is not more than O. 2 % . The sum of the areas
of all impurity peaks is not more than O. 5 % times of the
areas of the total peaks.
Water Not more than O. 1 % , ( 0832 method 1 ( 2) ) .
Category Pharmaceutical excipients, solvent and defoaming
agent.
Storage Preserve in well closed containers, protected from
light, and prevent exposure to excessive heat.
Butylated Hydroxytoluene
C1 s H24 O 220. 35
[128-37-0]
Butylated Hydroxytoluene is 2, 6-bis ( 1, 1-dimethylethyl) -
4-methylphenol. It contains not less than 98. 5 % of C1 s H24 O,
calculated on the anhydrous basis.

Description Colourless, white or almost white crystals or a
crystalline powder.
Very soluble in acetone, freely soluble in ethanol, insoluble
in water and propylene glycol.
Congealing point 69-70ºC (0613).
Specific absorbance Measure the absorbance of a solution of
50 p.g per ml in ethanol at 278 nm <0401), the value of A
CE~!) is 80. 0-90. o.
Identification C1) The retention time of the principal peak
of the test solution in the chromatogram obtained in the
Assay is identical with that of the principal peak in the
chromatogram of the reference solution.
(2) The infrared absorption spectrum <0402) is concordant
with that of butylated hydroxy to luene CRS.
Clarity and colour of solution Dissolve l. O g in 10 ml of
methanol, the solution is clear and colourless < 0901 and
902) ; any colour produced is not more intense than that of
reference solution Y3 <0901 method 1 ) .
Sulfate Dissolve 10. O g in about 40 ml of water, shake
thoroughly and filter. Carry out the limit test for sulfates
<0802) , using the filtra te. Any opalescence produced is not
more pronounced than that of a reference solution using
2. O ml of potassium sulfate standard solution (0. 002%).
Free phenol Dissolve about 10 g, accurately weighed, in
50 ml of O. 25% sodium hydroxide solution, heat and shake
in a water bath at 65ºC for 5 minutes. Cool and filter,
transfer the filtrate to iodine flask, wash the residue with 30
ml of water for several times, then transfer the washings to
the same flask. Accurately add 10 ml of bromine eo. 05 mol/L)
VS and 5 ml of hydrochloric acid, insert the stopper
immediately, shake thoroughly, clase the stopper with 5 ml
of 10% potassium iodide solution, allow to stand below 15ºC
in dark for 15 minutes, slightly open the stopper to let the
potassium iodide solution flow into the flask. Insert the
stopper immediately, shake thoroughly, clase the stopper
with water, allow to stand in dark for 5 minutes. Titrate
with sodium thiosulfate (O. 1 mol/L) VS, add 5 ml of
starch TS when the end point is nearly approached, continue
the titration until the blue colour disappears. Perform a
blank determination and make any necessary correction.
Each ml of sodium thiosulfate (0. 1 mol/L) VS is equivalent
to 10. 81 mg of C1 H 8 O. It contains not more than O. 02% of
free phenol calculated as~ H 8 O.
Related substances Dissolve O. 2 g in methanol to produce a
solution containing 20 mg per ml as the test solution.
Transfer accurately 1 ml of the test solution to a 200 ml
volumetric flask, dilute to volume with methanol and mix
well as the ref erence solution. Carry out the method for thin-
layer chromatography < 0502), using silica gel G as the
coating substance and methylene chloride as the mobile
phase. Apply separately to the plate 10 p.l each of above two
solutions, after developing overa path of 15 cm and removal
of the plate, dry it in air and spray with a freshly prepared
mixture of 5 % potassium ferricyanide solution-10 % ferric
chloride solution-water C10 : 20 : 70 ). Any spot in the
chromatogram obtained with the test solution, other than the
principal spot, is not more intense than the principal spot in
the chromatogram obtained with the reference solution
(0. 5%).
Water Not more than O. 1 % < 0832 method 1 ( 1 ) ) ,
using 5 g.
Residue on ignition Not more than O. 1 % <0841 ) , using
l. o g.
Butylparaben
Heavy metals Not more than O. 0004% <0821 method 2),
using 2. 5 g.
Arsenic Mix 2. O g with 2. O g of calcium hydroxide, add
small amount of water, stir well. After drying, heat gently
until charred, then ignite at 600ºC until carbonized, allow to
cool. Dissolve the residue in 5 ml of hydrochloric acid and
23 ml of water. Carry out the limit test for arsenic <0822
method 1 ) : not more than O. 0001 % .
Assay Carry out the method for high performance liquid
chromatography < 0512 ) , using a column packed with
octadecylsilane bonded silica gel and a mixture of methanol-
water C9 : 1) as the mobile phase. Detection wavelength is
278 nm. The number of the theoretical plates of the column
is not less than 3000, calculated with reference to the peak of
butylated hydroxytoluene.
Procedure Dissolve about 20 mg of the substanée being
examined, accurately weighed, in a 100 ml volumetric flask,
dilute to volume with methanol and mix well. Inject
accurately 10 p.l of the resulting solution into the column and
record the chromatogram. Repeat the operation, using
butylated hydroxytoluene CRS instead of the substance being
examined. Calculate the content of C1 5 H 24 O with respect to
the peak area obtained in the chromatogram by the extemal
standard method.
Category Pharmaceutical excipients, antioxidant.
Storage Preserve in tightly closed containers, stored in a dry
and cool place.
Butylparaben
o
~O~CH3
HO AJ
Cn Hl4 03194. 23
[94-26-8]
Butylparaben is butyl 4-hydroxybenzoate. It is prepared by
the esterification of butanol and p-hydroxybenzoic acid. It
contains not less than 98. O% and not more than 102. O% of
Cn Hl4 0 3 , calculated on the dried basis.
Description White or almost white crystals or a crystalline
powder.
Very soluble in ethanol, in acetone and in ether; slightly
soluble in hot water; practically insoluble in water.
Melting point 68-71 ºC <0612).
ldentification ( 1) The retention time of the principal peak
of the substance being examined in the chromatogram is
identilcal with that of the priacipal peak of butylparaben CRS
in the chromatogram obtained in the Assay.
(2) Weigh a quantity of the substance being examined to
produce a solution of 5 p.g per ml in ethanol, the light
absorption of the solution exhibits a maximum at 258 nm
<0401>.
(3) The infrared absorption spectrum is concordant with the
reference spectrum (IR Album No. 851).
Acidity To 2 ml of the solution prepared under clarity and
colour of solution add 2 ml of ethanol and 5 ml of water, mix
well, add 2 drops of bromoeresol green IS, not more than
 Calcium Chloride
O. 1 ml of sodium hydroxide (O. 1 mol/L) VS is required to
change the colour of the solution to blue.
Clarity and colour of solution Dissolve l. O g of the
substance being examined in 10 ml of ethanol. Carry out the
limit test for clarity and colour of solution ( 0901 and 0902),
the solution is clear and colourless; any colour produced is
not more intense than that of reference solution Y1 or YGr
( 0901 method 1 ) .
Chloride Heat 2. O g of the substance being examined with
50 ml of water at 80ºC water bath far 5 minutes, cool and
filter. Carry out the limit test for chlorides ( 0801 ) , using
5. O ml of the filtrate. Any opalescence produced is not more
pronounced than that of a reference solution using 7. O ml of
sodium chloride standard solution (O. 035 %) .
Sulfate Carry out the limit test for sulfates ( 0802) , using
25 ml of the filtrate obtained in the test for chloride. Any
opalescence produced is not more pronounced than that of a
reference solution using 2. 4 ml of potassium sulfate standard
solution (O. 024%).
Related substances Dissolve a quantity of the substance
being examined in the mobile phase to produce a solution of
1 mg per ml as test solution; measure accurately 1 ml, into a
100 ml volumetric flask, dilute to volume with the mobile
phase, mix well, as reference solution. Carry out the method
as described under Assay. Inject 20 µl of the reference
solution into the column and adjust the attenuation so that
the principal peak height in the chromatogram is about 25 %
of full scale of the chart, then inject separately 20 µl of the
test solution and reference solution into the column, record
the chromatogram for 4 times the retention time of the
principal peak. The area of any impurity peaks in the
chromatogram is not greater than O. 4 times the area of the
principal peak in the chromatogram obtained with the
reference solution ( O. 4 % ) , the sum of the areas of all
impurity peaks is not greater than O. 8 times area of the
principal peak in the chromatogram obtained with the
reference solution (O. 8%).
Loss on drying When dried in vacuum in a desicca tor
containing silica gel to constant weight, loses not more than
O. 5% of its weight (0831 ).
Residue on ignition Not more than O. 1 % <0841 ) , using l. O g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), using the residue obtained in the test far
residue on ignition: not more than O. 002%.
Arsenic Mix l. O g of the substance being examined with
l. O g of calcium hydroxide, add a small volume of water and
mix well. Dry, heat gently until it is thoroughly charred,
then ignite at 500-600ºC until the incineration is complete,
cool, add 5 ml of hydrochloric acid and 23 ml of water.
Carry out the limit test for arsenic ( 0822 method 1): not
more than O. 0002%.
Assay Carry out the method for high performance liquid
chromatography < 0512 ) , using a column packed with
octadecylsilane bonded silica gel and a mixture of methanol-
1 % glacial acetic acid solution ( 60 : 40) as the mobile
phase. Detection wavelength is 254 nm. Dissolve a quantity
of methylparaben CRS and ethylparaben CRS in mobile phase
to produce a mixed solution containing 1 O µg each of the
two substances per ml. Inject 20 µl of the resulting
solution onto the column. The resolution factor between
peaks of methylparaben and ethylparaben complies with the
requirement.
Procedure Dissolve a quantity, accurately weighed, in the
mobile phase to produce the test solution of O. 1 mg butylparaben
per ml. Inject 20 µl of the resulting solution, accurately
measured, onto the column and record the peak areas
corresponding obtained in the chromatogram. Accurately weigh
butylparaben CRS and repeat the operation. Calculate the
contents of butylparaben with respect to the peak area
obtained in the chromatogram by the external standard
method.
Category pharmaceutical excipients, preservative.
Storage Preserve in well closed containers.
Calcium Chloride
CaClz•2H20 147. 02
[10035-04-8]
Calcium Chloride contains not less than 97. 0% and not more
than 103. o% of CaClz•2H 20.
Description White, hard solids or granules or crystalline
powder; odourless; very hygroscopic.
Very soluble in water; freely soluble in ethanol.
ldentification The aqueous solution yields the reactions
characteristic of calcium salts and chlorides ( 0301 ) .
Acidity or alkalinity Dissolve l. O g in 20 ml of water, pH
6. 0-9. 2 (0631 ).
Clarity ami colourof soltttion A solution of l. O gin 10 ml of
water is clear and colourless ( 0901 and 0902 ) . Any
opalescence produced is not more pronounced than that of
reference solution 1 ( 0902).
Sulfate Carry out the limit test far sulfate ( 0802) , using
l. O g. Any opalescence produced is not more pronounced
than that of a reference using 2. O ml of potassium sulfate
standard solution (O. 02 % ) .
Barium Dissolve 2. O g in 20 ml of water and filter. Divide
the filtrate into 2 equal parts. To one part add 5 ml of freshly
prepared calcium sulfate TS, to the other part add 5 ml of
water, allow to stand for 1 hour. The solutions are equal in
clarity.
Aluminum, Iron and Phosphate Dissolve l. O g in 20 ml of
water, add 2 drops of dilute hydrochloric acid and 1 drop of
phenolphthalein IS. Add dropwise ammoniated ammonium
chloride TS until a pink colour is produced. No turbidity or
precipitate is produced when boiling.
Magnesium and Alkali metals Dissolve l. O g in 40 ml of
water, add O. 5 g of ammonium chloride and heat the solution
to boíl. Add an excess of ammonium oxalate TS to
precipitate calcium completely and heat on a water bath far 1
hour. Cool to room temperature, dilute with water to 100
ml, stir well and filter. To 50 ml of the filtrate add O. 5 ml of
sulfuric acid, evaporate to dryness and ignite to constant
weight. The residue does not exceed 5 mg.
Heavy metals Dissolve 2. O gin a 23 ml of water, add 2 ml
of acetate BS (pH 3. 5). Carry out the limit test far heavy
metals <0821 method 1 ) : not more than O. 001 % .
Arsenic Dissolve l. O g in 5 ml of hydrochloric acid and
23 ml of water. Carry out the limit test for arsenic ( 0822
method 1): not more than O. 0002%.
Assay Weigh accurately about l. 5 g of the substance being
examined in a 100 ml volumetric flask containing 10 ml of
water. Dilute with water to volume and mix well. Measure

accurately 10 ml of the solution to a conical flask, add 90 ml
of water, 15 ml of sodium hydroxide TS and about O. 1 g of
calcon indicator mixture, titrate with disodium edentate
(O. 05 mol/L) VS until the colour changes from purplish-red
to pure blue. Each ml of disodium edetate (O. 05 mol/L) VS
is equivalent to 7. 351 mg of CaCl2• 2H2 O.
Category Pharmaceutical excipients, osmotic pressure
regulator.
Storage Preserve in tightly closed containers, stored in a dry
place.
Calcium Glycerophosphate
C3 H1Ca05P 210. 14
[27214-00-2]
Calcium Glycerophosphate is a mixture of ,B-D-and L-a-
glycerol phosphate calcium prepared by heating glycerol and
phosphate, then neutralizing with lime cream, precipitate
with ethanol, collecting the precipitation after washing and
drying. It contains not less than 18. 6 % and not more than
19. 4% of calcium (Ca), calculated on the dried basis.
Description A white to tiny yellow powder, odourless or
slight odour, slightly hygroscopic.
Slightly soluble in water, practically insoluble in ethanol.
ldentification ( 1) Dissolve O. 1 g with 10 ml of water and
10 ml of dilute nitric acid. Add 5 ml of ammonium molybdate
TS, heat to boiling and produce yellow precipitate.
(2) Mix O. 1 g with O. 1 g of monopotassium sulfate in a test
tube, heat, the irritating odour of acrylic acid is produced.
(3) Yields the flame reaction of calcium salts <0301 ).
Acidity or alkalinity Dissolve l. O gin 100 ml of water, add
2 drops of phenolphthalein IS, titrate with sodium hydroxide
(O. 1 mol/L) VS or hydrochloric acid (0. 1 mol/L) VS, not
more than l. 7 ml of sodium hydroxide (O. 1 mol/L) VS or
Hydrochloric acid (O. 1 mol/L) VS is consumed.
Clarity and colour of solution Dissolve l. O g in 100 ml of
water, the solution should be colourless <0901 and 0902 ).
Any opalescent produced is not more pronounced than that of
reference suspension 3 <0902 method 1 ) .
Chloride Carry out the limit test for chlorides < 0801 ) ,
using O. 25 g, any opalescence produced is not more
pronounced than that of a reference solution prepared by
using 5. O ml of sodium chloride standard solution (0. 02%).
Sulfate Carry out the limit test for sulfates <0802), using
O. 25 g. Any opalescence produced is not more pronounced
than that of a reference solution prepared by using 5. O ml of
potassium sulfate standard solution (0. 2%).
Phosphates To l. O g in 25 ml nessler cylinder, dissolve
with 10 ml of dilute nitric acid, add 10 ml of ammonium
molybdate solution, shake well, stand for 10 minutes. Any
colour produced is not more intense than that of 10 ml of
phosphate standard solution (Place O. 192 g of potassium
dihydrogen phosphate, weighed accurately, in a 100 ml
volumetric flask, dilute with water to volume, mix well.
Transfer 3 ml, accurately measured, to a 100 ml volumetric
flask, dilute with water to volume, mix well.) (O. 04%).
Citrate Dissolve 5. O g of the substance being examined into
a beaker with 20 ml of freshly boiled and cooled water and
filter. To the filtrate add O. 15 ml of sulfuric acid, shake and
filter again. To the filtrate add 5 ml of mercuric sulfate TS,
Calcium Oxide
and heat to boil. Add O. 5 ml of potassium permanganate
solution and heat to boil again. No precipitate is formed.
Free glycerol and ethanol-soluble substances To l. O g add
25 ml of dehydrated ethanol, shake for 2 minutes and filter.
Wash the residue with 5 ml of dehydrated ethanol, combine
filtrate and washings into an evaporating dish, previoulsly
dried to constant weight at 70ºC , and evaporate on a water
bath to dryness and dry the residue at 70ºC for 1 hour, the
weight of residue is not more than 5 mg (O. 5 % ) .
Loss on drying When dried at 105ºC for 4 hours, loses not
more than l. O% of its weight <0831).
Iron To l. O g add 23 ml of water and 2 ml of dilute
hydrochloric acid. Carry out the limit test for iron <0807).
Any colour produced is not more intense than that of the
reference solution, prepared in similar manner, using 2. O ml
of iron standard solution (O. 002%).
Heavy metals To l. O g add 2 ml of aceta te BS ( pH 3. 5)
and a volume of water to produce volume of 25 ml. Carry out
the limit test for heavy metals <0821 method 1): not more
than O. 002%.
Arsenic Dissolve O. 67 g with 23 ml of water and 5 ml of
hydrochloric acid. Carry out the limit test for arsenic
<0822) : not more than O. 0003 % .
Assay Dissolve 2. O g of the sabstance being examined,
accurately weighed, with 300 ml of water, shake. Add 6. O
ml of sodium hydroxide solution (1 O mol/L) and 15 mg of
calcon indicator. Titrate with disodium edetate (O. 05 mol/L)
VS until the colour of solution turns from violet to blue. Each
ml of disodium edentate (O. 05 mol/L) VS is equivalent to
2. 004 mg of Ca.
Category Pharmaceutical excipients Diluent and hygroscopic
agent.
Storage Preserve in tightly closed containers, and keep in a
dry place.
Calcium Oxide
CaO 56. 08
[73018-51-6]
Calcium Oxide contains not less than 98. O% of Ca O,
calculated with reference to substance ignited to constant
weight.
Description A white or almost white masses, granules or
powder, odourless.
Practically insoluble in ethanol and in boiling water.
Identification (1) Moisten 1 g with several drops of water,
heat is generated, anda white powder is obtained. Mix with
5 ml of water, a smooth magma of lime forms that is alkaline
to litmus.
(2) Yields the flame reaction characteristic of calcium salts
<0301>.
Acid-insoluble substances Moisten 5. O g with severa! drops
of water, then add 100 ml of water, mix well, add
hydrochloric acid to acidic, then add 1 ml of hydrochloric
acid. Boil the solution for 5 minutes and filter by G4 sintered
glass crucible previously dried to constant weight at 105ºC.
Wash the residue with boiling water until the washing shows
no opacitas by adding silver nitrate solution. Dry the residue
to constant weight at 105ºC. The residue is not more than
 Calcium Phosphate
10. O mg (0. 2%).
Carbonate Moisten l. O g with severa! drops of water, mix
with 50 ml of water, add an excess of dilute nitric acid to the
solution, no any effervescence produced.
Magnesiurn and alkali metals Dissolve l. O g in 75 ml of
water, make acidity by hydrochloric acid, then add 1 ml of
hydrochloric acid. Boíl the solution for 1-2 minutes, then
neutralize the solution with ammonium hydroxide TS, add an
excess of ammonium oxalate TS. Heat the mixture on a
water bath for 2 hours, allow to cool, dilute with water to
200 ml, mix and filter. To 50 ml of the filtrate add O. 5 ml of
sulfuric acid, evaporate to dryness on a water bath, ignite to
constant weight at 600ºC. The weight of the residue is not
more than 15 mg.
Residue on ignition When iguited to constant weight at
900ºC, loses not more than 10. o% of its weight, using l. O g
( 0841>.
Assay Weigh O. 4 g accurately into 250 ml volumetric flask,
add 8 ml of hydrochloric acid 0-3), dissolve by ultrasound
(about 10 minutes). Allow to cool, add water to volume, mix
well. Transfer 10 ml of the solution accurately, add 50 ml of
water and 2 ml of 8 mol/L potassium hydroxide solution. Then
add 5 mg of Calcon-carboxylic acid indicator mixture, titrate with
disodium edetate (O. 02 mol/L) VS until the colour changes
from purple to blue. Ea.ch ml of disodium edetate (O. 02 mol/L)
VS is equivalent to l. 122 mg of CaO.
Category Pharmaceutical excipients, diluent and alkalizing
agenl.
Storage Preserve in tightly closed containers.
Calcium Phosphate
Ca3 (P04 )z 310. 18
[7758-87-4]
Calcium Phosphate contains not less than 34. O% and not
more than 40. O% of Ca, calculated on the ignited basis.
Description White or almost white powder.
Practically insoluble in water, soluble in dilute hydrochloric
acid or dilute nitric acid.
ldentification (1) Dissolve about O. 5 gin dilute nitric acid,
add ammonium molybdate TS, warm, a yellow precipitate is
produced.
(2) Yields the reactions characteristic of calcium salts ( 1)
(0301>.
Chlorides Dissolve O. 25 g in 50 ml of dilute nitric acid,
fil ter if necessary, by a filter paper which is free from
chlorides. Transfer 10 ml of the successive filtrate to a 25 ml
Nessler cylinder, add 1 ml of silver nitrate TS, dilute to
25 ml with water, carry out the limit test for chlorides
( 0801 ) . Any opalescence produced is not more pronounced
than that of a reference solution using 7. O ml of sodium
chloride standard solution (0. 14%).
Sulfates Dissolve O. 40 gin 4 ml of dilute hydrochloric acid,
dilute with water to 100 ml, mix well and filter. Transfer
25 ml of the successive filtrate to a 50 ml Nessler cylinder,
carry out the limit test for sulfates ( 0802 ) . Any opalescence
produced is not more pronounced than that of a reference solution
using 5. O ml of potassium sulfatestandard solution (O. 5 % ) .
Flurides Accurately weighed 221 mg of sodium fluoride,
previously dried for 1 hour at 105ºC, in 100 ml volumetric
flask, dissolve with a quantity of water, add 50. O ml of
Buffer solution (Dissolve 73. 5 g of sodium citrate in 250 ml
of water), dilute to volume with water, mix well, as the
fluoride standard solution. Each ml is equivalent to 1 mg of F.
Transfer 2. O g to a plastic beaker containing a plastic-coated
stirring bar. Add 20 ml of water and 3. O ml of hydrochloric
acid, stir to dissolve. Add 50. O ml of the Buffer solution.
Filter, wash the filter with water far 3 times, each of 15 ml.
Combine the filtrate and washings, dilute with water to
100. O ml. Use a fluoride-specific ion-indicating electrode and
a silver-silver chloride reference electrode ( using 3 mol/L
potassium chloride solution as the salt bridge solution) ,
insert the electrodes into the solution, stir, and at 1 minute
intervals add 150 µl, 200 µl, 250 µl and 300 µl of the fluoride
standard solution, to produce the test solution containing 150
µg, 350 µg, 600 µg and 900 µg of fluoride ion. Reading the
potential after each addition (mV). Plot the logarithms of the
cumulative fluoride ion concentration ( X-axis ) versus
potential (Y-axis) , in m V. The absolute value of the
interception on the X -axis represents the logarithms of the
fluoride ion concentration, e (in µg/mD, in test solution.
Calculate the content of fluoride in the portian of Calcium
Phosphate:
Result =lOOXCXl0- 6 /WXlOO%
in which e is the concentration of fluoride ion in the test
solution (µg/mD;
W is the weight of Calcium Phosphate (g).
The result is not more than O. 0075%.
Reducing substances To 10. O g, add 100. O ml of dilute
sulfuric acid, mix well and filter, to 50. O ml of the successive
filtrate, add O. 10 ml of potassium permanganate (0. 02 mol/L)
VS and heat on a water bath far 5 minutes, the solution remains
a purplish red colour.
Oxidizing substances Protect from light in the procedure.
To l. O g, add 100. O ml of dilute sulfuric acid, mix well and
filter, Transfer 50. O ml of the successive filtrate to a Nessler
cylinder, add O. 2 g of potassium iodide, 2 ml of 1% starch
solution, mix well. The colour produced is not more intense
than that of a reference solution, using l. O ml of freshly
prepared 3-chloroperbenzoic acid solution in ethanol ( 10 µg
per ml) to a Nessler cylinder, add dilute sulfuric acid to 50
ml, beginning at the words "add O. 2 g of potassium
iodide... ", prepared in the same manner.
Acid insoluble substances To 2. O g, accurately weighed, add
25 ml of dilute hydrochloric acid, heat to dissolve, filter
through a No. 4 sintered glass crucible previously dried to
constant weight, wash the residue with hot water until the
washing is free from chloride, dry the residue to constant
weight at 105ºC: not more than O. 2%.
Water soluble substances To 2. O g, accurately weighed, add
100. O ml of water, heat on a water bath for 30 minutes, cool
and add a quantity of water to the same volume, mix well
and filter. Accurately transfer 50 ml of the successive filtrate
to an evaporating dish previously dried to constant weight,
evaporate on water bath and dry the residue to constant
weight at 120ºC: not more than O. 5%.
Bariurn To O. 50 g, add 10 ml of water, heat, and dissolve
by adding hydrochloric acid dropwise, and then add 2 drops
of hydrochloric acid in excess. Filter and add 1 ml of potassium
sulfate TS to the filtrate, no opalescence is produced within
15 minutes.
Loss on ignition When ignite at 800ºC for 30 minutes, loses
not more than 8. O% of its weight, using l. O g.

Lead Accurately transfer O. 2 g to a 50 ml volumetric flask,
dissolve and dilute with nitric acid solution ( 1-- 100) to
volume, mix well and use as the test solution. Transfer
accurately a quantity of standard lead solution [ measure
accurately a volume of lead single-element standard solution
dilute with nitric acid solution 0-100) to produce a solution
of 10 µg Pb per ml], dilute with nitric acid solution ( 1--
100) to produce a successive solutions of O, 10, 20, 30, 40
and 50 ng per ml as reference solutions. Carry out the
method far atomic absorbance spectrophotometry ( 0406
method 1 ) , using furnace atomizer and measure the
absorbance of reference solutions and test solution at 283. 3
nm. Calculate the content of lead: not more than O. 0005%.
Arsenic Dissolve O. 67 g in 5 ml of hydrochloric acid and
23 ml of water. Carry out the limit test far arsenic ( 0822
method 1 ) : not more than O. 0003 % .
Assay Dissolve O. 6 g, accurately weighed, with 10 ml of
dilute hydrochloric acid, heat if necessary to dissolve, cool,
transfer to a 100 ml volumetric flask, dilute with water to
volume and mix well. To 10 ml of the solution, accurately
measured, add 50 ml of water, add ammonia TS dropwise
until the precipitate is just produced, then add dilute
hydrochloric acid dropwise until the precipitate is just
dissolved. Add 25 ml accurately measured, of disodium
edetate (O. 05 mol/L) VS. Boil the solution far 3 minutes,
cool, add 10 ml of ammonia-ammonium chloride BS ( pH
10. 0) and a small amount of eriochrome black T indicator
mixture. Ti trate with zinc (O. 05 mol/L) VS until the
solution turns to violet. Perform a blank determination and
make any necessary correction. Each ml of disodiurn edetate
(O. 05 mol/L) VS is equivalent to 2. 004 rng of Ca.
Category Pharmaceutical excipients, filler.
Storage Preserve in tightly closed containers.
Calcium Stearate
[1592-23-0]
Calciurn stearate is a mixture consisted chiefly of calciurn stearate
( G6 H10 Ü4 Ca ) and calciurn palmitate ( Gz H62 Ü4 Ca ) . It
contains not less than 9. 0% and not more than 10. 5% of
calcium oxide ( CaQ).
Description A white powder.
Insoluble in water, in ethanol and in ether.
ldentification ( 1) Mix 25 g with 200 ml of hot water and
60 ml of dilute sulfuric acid, heat the mixture with frequent
stirring, until the fatty layer is separated. Wash the fatty lay
with boiling water until the washing is free from sulfate,
collect the fatty layer in a small beaker, and warm on a
steam bath until the water is separated and the fatty layer is
clear. Allow to cool, discard the water layer, heat to melt
the fatty layer, filter immediately into a dry beaker, and dry
at 105ºC far 20 minutes. The congealing point is not lower
than 54 ºC ( 0613).
(2) Heat l. O g with a mixture of 25 ml of water and 5 ml of
hydrochloric acid: fatty acids are liberated and appear as an
oily layer floating on the surface of the liquid. Cool, and the
aqueous layer yields the reactions characteristic of calcium
salts ( 0301).
~ on drying When dried to constant weight at 105ºC,
loses not more than 4. O% of its weight ( 0831 ) .
Calci um Sulfate 503··
Heavy metals Place 2. 5 g in a porcelain dish as the test
sample. Place O. 5 gin another dish as the reference sample.
Add separately 5 ml of a solution of magnesium nitrate in
ethanol ( 1 - 4) . Cover the dishs with short-stem funnels.
Heat on a hot plate ata level of low temperature far 30 minutes,
then heat at a level of medium temperature far 30 minutes,
and cool. Remove the funnels, add 2 ml of lead standard
solution to the reference sample, and heat each dish until the
incineration is complete. Cool, add 10 ml of nitric acid, and
transfer the solutions into 250 ml beakers. Add separately 5
ml of the perchloric acid solution ( 7 --10), evaporate to
dryness, add 2 ml of hydrochloric acid to the residues, and
wash the inner wall of the beakers with water. Evaporate to
dryness again, swirling near the dry point. Repeat the
operation from adding 2 ml of hydrochloric acid, then cool,
and dissolve the residues in about 10 ml of water. To each
solution add 1 drop of phenolphthalein IS and add sodium
hydroxide TS until the colour of the solutions changes to
pink, then add dilute hydrochloric acid until the solutions
become colourless. Add 1 ml of dilute acetic acid anda small
amount of charcoal to each solution, and filter into a 50 ml
Nessler cylinder. Wash the residue with water, dilute with
water to 40 ml, add l. 2 ml of thioacetamide-glycerin base
TS and 2 ml of the acetate buffer (pH 3. 5) to each tube, and
allow to stand far 5 minutes. The colour of the test solution
is not more intense than that of the reference solution
(O. 0001%).
Microbial limit Comply with the requirements far
microbiological limit ( 1105 and 1106), the total aerobic
bacteria count is not more than 1000 cf u per g and the total
combined yeast/ mold count is not more than 100 cfu per g of
the substance being examined. Escherichia coli should not be
detected.
Assay Transfer about l. 2 g, weighed accurately, to a
fiask, add 50 ml oí O. 05 mol/L sulfuric acid solution, heat
to boil far about 3 hours using a watch glass cover to avoid
splattering until the separated fatty layer is clear. If
necessary, add water to maintain the original volume. Cool,
filter, and wash the filter and the flask thoroughly with
water until the washing shows not acid to blue litmus test
paper. Neutralize the filtrate with sodium hydroxide TS to
blue litmus test paper. While stirring with a magnetic
stirrer, titrate with disodium edetate (O. 05 mol/U VS. Add
about 30 ml, then add 15 ml of sodium hydroxide TS and 2
mg of hydroxyl naphthol blue indicator, and continue the
titration until the colour of solution changes to blue. Each ml
of disodium edetate (O. 05 mol/L) VS is equivalent to 2. 804
mg of CaO.
Category Pharmaceutical excipients, lubricant and
emulsifier.
Storage Preserve in well-closed containers, stored in a dry
and cool place.
Calcium Sulfate
CaS04•2H20 172. 17
[10101-41-4]
Calcium sulfate is prepared by the reaction of calcium
carbonate with sulfuric acid, or by the reaction of calcium
chloride solution with soluble sulfate. It contains not less
than 99. O% of CaS04 , calculated on the ignited basis.
Description White powder, odourless and tasteless.
 Caprylic Acid
Slightly soluble in water, insoluble in ethanol.
Identification The solution in dilute hydrochloride acid TS
yields the reaction characteristic of calcium salts and sulfates
<0301).
Acidity or alkalinity Add 15 ml of water to l. 5 g and shake
for 5 minutes, stand for 5 minutes, filter; Add O. 25 ml of
sodium hydroxide (O. 01 mol/L) VS to 10 ml of the
successive filtrate, add O. 1 ml of phenolphthalein IS, a red
colour is produced; Add O. 30 ml of hydrochloric acid (0. 01
mol/L) VS, the solution is colourless, then add O. 20 ml of
methyl red IS, a colour of orange red is produced.
Chloride To O. 50 g add 5 ml of nitric acid solution 0-2)
and 40 ml of water, shake to dissolve, carry out the limit
test for chlorides <0801 ) . Any opalescence produced is not
more pronounced than that of a reference solution using
9. O ml of sodium chloride standard solution (O. 018%).
Carbonates Mix l. O g with 5 ml of water, add dropwise
dilute hydrochloric acid; no effervescence is produced.
Loss on ignited When ignited to constant weight at 700-
800ºC, loses not less than 19. O% and not more than 23. O%
of its weight, using l. O g.
Iron To O. 20 g add 50 mg of ammonium persulfate and
10 ml of dilute hydrochloric acid, shake to dissolve, dilute
with water to 50 ml, add 5. O ml of Ammonium Thiocyanate
TS and shake well. Carry out the limit test for iron ( 0807).
Any colour produced is not more intense than that of the
reference solution treated in similar manner using 2. O ml of
iron standard solution (O. 01%).
Heavy metals To 2. 5 g add 2 ml of hydrochloric acid and
15 ml of water, heat to boil. Allow to cool and add 2 drops
of phenolphthalein IS, then add dropwise concentrated
ammonia solution until the colour of the solution turns to
pink. Add O. 5 ml of glacial acetic acid and dilute to 25 ml
with water. Filter, using 12 ml of the filtrate as the test
solution. To 2 ml of the filtrate add l. O ml of lead standard
solution and dilute to 12 ml with water, and use as the
reference solution. To 2 ml of filtrate add 10 ml of water and
use as the blank solution. Transfer each of the above three
solutions to three Nessler cylinders respectively, shake well
and add 2 ml of acetate BS ( pH 3. 5) and l. 2 ml of
thioacceamide TS respectively, shake well and allow to stand for
2 minutes. The test is invalid if the colour produced in the
reference solution is more intense than that produced in the
blank solution. The colour produced in the test solution is
not more intense than that produced in the reference solution
(0. 001%).
Arsenic To O. 20 g add 10 ml of 10 % hydrochloric acid solution,
heat to dissolve on a water bath at 50ºC for 5 minutes. Add 5 ml
of hydrochloric acid and 21 ml of water, carry out the limit
test for arsenic <0822 method 1), not more than O. 001 %.
Assay To O. 2 g of substance being examined, accurately
weighed, add 10 ml of dilute hydrochloric acid and 100 ml of
water, heat and shake to dissolve, allow to cool. Add
accurately measured 20 ml of disodium edetate (O. 05 mol/L)
VS under stirring, shake well, then add 15 ml of sodium
hydroxide solution ( 1 - 5) and O. 1 g of calcon indicator.
Ti trate with disodium edetate (O. 05 mol/L) VS until the
colour turns from violet to blue. Each ml of disodium edetate
(O. 05 mol/L) VS is equivalent to 6. 807 mg of CaS04.
Category Pharmaceutical excipients, dilute agent.
Storage Preserve in tightly closed containers, protected from
light.
Caprylic Acid
Cs H16 02 144. 2
[124-07-2]
Caprylic Acid is an eight-carbon saturated fatty acid, contains
not less than 99. O% of Cs Hl6 02 , calculated on the anhydrous
has is.
Description clear, colourless and slightly yellowish, oily liquid
Very soluble in acetoneand and in ethanol, soluble in diluted
solutions of alkali hydroxides, very slightly soluble in water.
Relative density O. 909-0. 912 ( 0601 ) .
Identification The retention time of principal peak in the
chromatogram of the test solution obtained in the related
substances is identical with the principal peak in the
chromatogram of the standard solution.
Clarity and colour of solution Dissolve 5. O g in 50 ml of
water ( 0901 and 0902): the solution is clear and colourless;
any colour produced is not more intense than that of reference
solution Y3 ( 0901 method 1 ) .
Related substances Dissolve O. 1 g of the substance to be
examined in ethyl acetate and dilute to 10 ml with the same
solvent as test solution. Measure accurateiy quantity oí test
solution, dilute with ethyl acetate to produce a solution of 10
µg/ml as reference solution. Dissolve O. 1 g of caprylic acid
CRS in ethyl acetate and dilute to 10 ml with the same
solvent as standard solution. Carry out the method of Gas
chromatography ( 0521), using capillary column packed with
a layer of nitroterephthalic acid modified polyethylene glycol
20 M (30 mX O. 25 mm IDX O. 25 µm); maintain the initial
temperature of the column at lOOºC for 1 minute, and then
raise the temperature at a rate of 5ºC per minute to 220ºC
and maintain at 220ºC for 20 minutes; the injection port is
maintained at a temperature of 250ºC . The detector is
maintained ata temperature of 250ºC. lnject separately 1 µl
of the test solution and the reference solution onto the
column and record the chromatogram. The signal-to-noise
ratio of the principal peak obtained with the reference
solution is not less than 5. Calculate the content of impurities
by peak area normalized method, the area of any impurity is
not more than O. 3 % of all the peak areas, and the sum of all
the impurities found is not greater than O. 5% (disregard any
peaks from test solution with an area less than half of the
area of the principal peak from the reference solution).
Water Not more than O. 7 % ( 0832 method 1 ( 1 ) ) .
Residue on ignition Not more than O. 1% ( 0841), using l. O g.
Heavy metals Dissolve l. 2 g in ethanol and dilute to 25 ml
with the same solvent as the test solution. Carry out the limit
test for heavy metals ( 0821 method 1 ) : not more than
o. 001%.
Assay Dissolve O. 125 g, accurately weighed, in 25 ml of
ethanol. Carry out the method for potentiometric titration
( 0701 ) . Titrate with sodium hydroxide (O. 1 mol/L) VS,
perform a blank determination and make any necessary
correction. Each ml of sodium hydroxide (O. 1 mol/L) VS is
equivalent to 14. 42 mg of Cs H16 02.
Category Phannaceutical excipients, stabilizer and preservative
 Caramel I•
Storage Preserve in well closed containers and stored in a 5 ml of 2 % boric acid solution, with 5 drops of mixed
cool a dark place. indicator solution ( mix with 5 parts of O. 2% ethanolic
Bromocresol green solution and 1 part of ethanolic methyl red
solution). Stop the distillation when the total collected
distillate is about 100 ml. Titrate the distillate with
hydrochloric acid (O. 1 mol/L) VS until the colour turns
Caramel
grayish red. Perform a blank determination and make any
necessary correction. Each hydrochloric acid (O. 1 mol/L)
[8028-89-5]
VS is equivalent to l. 7 mg of NH3, The content of
Caramel is obtained by heating carbohydrates, such as
Ammonia nitrogen with absorbance at O. 10 calculated with
sucrose or glucose.
the value under Absorbance: not more O. 5%.
Description A dark brown thick liquid with characteristic
odour and almost tasteless.
sulfur dioxide Apparatus The apparatus consists of A: a
500 ml round-bottom boiling flask with three-neck; B: a
Miscible with water, soluble in ethanol solution with
separatory funnel, having a capacity of 100 ml greater; C: a
concentration less than 55% ( ml/ml), immiscible with
ether, chloroform, acetone, benzene and n-hexane. reflux condenser, having a jacket length of 200 mm; and D:
a receiving test tube.
Relative density not less than l. 30 ( 0601 ) . Assemble the apparatus as shown as the following befare
Purity Dilute 1 ml to 20 ml with water, add O. 5 ml of use, and sealed.
phosphoric acid, mix well, no precipitate is produced.
Absorbance Accurately weigh a quantity of the substance
being examined, dilute with water to produce a solution
containing l. O mg per ml and mix well. The absorbance is
not more than O. 600 at 610 nm (0401 ).
4-methylimidazole Place 10. O g, accurately weighed, in a
polypropylene beaker, add 5. O ml of 3 mol/L Sodium
hydroxide solution and mix well, add 20 g of diatomite
(chromatography grade) and mix until a uniform colour is
obtained. Transfer and evenly fill the mixture to a
chromatographic column (250 mmX 25 mm) with polytetra-
fluoroethylene stopcock. Wash polypropylene beaker with
dichloromethane, transfer the washing to the column and
turn off the stopcock when dichloromethane flows to the
stopcock. Allow to stand at least 15 minutes and turn on the
stopcock, add dichloromethane and keep the elution rate of
5 ml per minute until collected eluent is about 300 ml. Fig. Apparatus far sulfur dioxide
Evaporate to dryness at 35ºC by rotary vacuum evaporation.
Accurately measure 10 mi of water to the flask of rotary
Procedure Place 25. O g, accurately weighed, in a 500 ml
evaporator to dissolve the residue, as the test solution.
round-bottom boiling flask CA), add 250 ml of water and
Dissolve an accurately weighed quantity of 4-methylimidazole
mix well. Keep the gas inlet tube above the liquid surface and
CRS in water to produce a solution with certain concentration
clase the stopcock of the separatory funnel (B), add 80 ml
(according to the absorbance value and the limit), as the of 2 mol/L hydrochloric acid solution to the separatory
reference solution. Carry out the method far high funnel. Open the stopcock of the separatory funnel (B) to
performance liquid chromatography ( 0512), using a column permit the 2 mol/L hydrochloric acid solution to flow into
packed with octadecylsilane bonded silica gel anda mixture of the boiling flask, guarding against escape of sulfur dioxide
O. 05 mol/L phosphate BS ( dissolve 6. 8 g of potassium into the separatory funnel by closing the stopcock befare the
phosphate monobasic and 1 g of sodium heptanesulfonate in last few about 5 ml of hydrochloric acid drain out. Place 10
900 ml of water, adjust pH to 3. 5 with phosphoric acid, ml of 3 % hydrogen peroxide solution in the receiving test
dilute to 1000 ml with water) - methanol ( 85 : 15) as tube (D). Start the condenser coolant flow. Heat to boil and
mobile phase. Detective wavelength is 210 nm. The number begin the flow of carbon dioxide at a rate of 100 ± 5 ml per
of theoretical plates calculated far the peak of 4- minute through the apparatus immediately. Boil the mixture
methylimidazole is not less than 3000. The resolution factor far 2 hours. Remove the receiving test tube (D), transfer its
between peak of 4-methylimidazole and adjacent impurity contents to a conical flask, wash the receiving test tube with
peak complies with the requirement. Inject 10 µl of test a small portian of water, combine the washing to the conical
solution and reference solution respectively into the column, flask Heat on a water bath for 15 minutes, and allow to cool.
record the chromatogram, calculate the content of 4- Add 2 drops of bromophenol blue IS ( dissolve a suitable
methylimidazole with respect to the peak areas obtained in quantity of bromophenol blue with ethanol to produce a
solution containing O. 2 mg per mD , and titrate the contents
the chromatogram by external standard method. The content
with sodium hydroxide ( O. 1 mol/L) VS until the colour
of 4-methylimidazole with absorbance at O. 10 calculated with
changes from yellow to violet-blue. Perform a blank
the value under Absorbance: not more O. 02%.
determination and make any necessary correction. Each sodium
Ammonia nitrogen Place 5. O g, accurately weighed, in a hydroxide (O. 1 mol/L) VS is equivalent to 3. 203 mg of sulfur
500 ml distillation flask, add 2 g of magnesium oxide and dioxide. The content of sulfur dioxide with absorbance at O. 10
200 ml of water, heat to distill. The distillate is collected to calculated with the value under Absorbance: not more
 506 Carbomer
0.1%.
Ash Accurately weigh 3. O g of the substance being
examined. Ignite slowly until it is completely carbonized.
Then ignite to constant weight at 600ºC. The remaining ash
is not more than 8. O% of its weight.
Lead Place O. 25 g, accurately weighed, in a PTFE vessel,
add 5-10 ml of nitric acid, mix well, stopper and allow to
stand overnight. Digest in a microwave digestion system.
After digestion has been completed, evaporate the solution
on the electric heating plate until reddish-brown fumes are no
longer evolved and near to dryness by gently heating.
Transfer to a 100 ml volumetric flask with 2% nitric acid
solution, and dilute to volume as test solution. A blank
solution is prepared at the same time and in the same
manner. Transfer accurately a quantity of lead standard
solution, dilute with 2% nitric acid solution to produce a
successive solutions of 0-80 ng per ml as lead reference
solutions. Carry out the method for atomic absorbance
spectrophotometry < 0406 method 1 ) , measure the
absorbances of reference solutions and test solution at 283. 3
nm, using furnace atomizer and 2. O% ammonium
dihydrogen phosphate solution as a matrix modifier solution.
Calculate the content of lead: not more than O. 001%.
Arsenic Place 2. O gin a Kjeldahl flask, add 5 ml of sulfuric
acid and a few glass beads, ignite at a temperature not
exceeding 120ºC ( additional sulfuric acid may be necessary,
but the total volume added should not exceed 1O ml).
Cautiously add, dropwise, concentrated hydrogen peroxide,
when the reaction finish and heat again between drop:s, until
fumes are copiously evolved and the solution turns colourless
or retains only a light yellow colour. Cool, add 10 ml of
water and again heat to remove residual hydrogen peroxide.
Add 5 ml hydrochloric acid and a quantity water, Carry out
the method for arsenic ( 0822 method 2 ) : not more than
o. 0001%.
Microbial limit Comply with test for microbial limit ( 1106 ).
Escherichia coli is not detected per g of the substance being
examined.
Category Pharmaceutical excipients, colourant.
Storage Preserve in well closed containers.
Carbomer
[54182-57-9]
Carbomer is a high molecular weight polymer of acrylic acid
cross-linked with allylsucrose or allylethers of pentaerythritol,
using non-benzene solvent as polymerization solvent. It
contains not less than 56. O% and not more than 68. O% of
carboxylic acid (-COOH) groups, calculated on the dried
basis.
Description A white, fluffy powder; odour, slight and
characteristic; hygroscopic.
ldentification ( 1) Disperse O. 1 g in 20 ml of water and add
O. 4 ml of 10 % sodium hydroxide solution, a gel is produced.
(2) To about O. 1 g, add 10 ml of water, shake well and add
O. 5 ml of thymol blue IS. An orange colour is produced. To
about O. 1 g, add 10 ml of water, shake and add O. 5 ml of
cresol red IS. A yellow colour is produced.
(3) To about O. 1 g, add 10 ml of water and adjust pH to 7. 5
with 1 mol/L sodium hydroxide solution. Add 2 ml of 10 %
calcium chloride solution while stirring. A white precipitate
is produced.
( 4 ) The infrared absorption spectrum ( 0402 ) of the
substance being examined has characteristic absorption at or
near wavenumbers of 1710±5 cm- 1 , 1454±5 cm- 1 , 1414±
5 cm- 1 , 1245±5 cm- 1 , 1172±5 cm- 1 , 1115±5 cm- 1 and
801±5 cm- 1 , with the strongest absorption at 1710 cm- 1 •
Acidity Swell O. 10 g in 10 ml of water till uniformly
dispersed, pH 2. 5-3. 5 ( 0631 ).
Viscosity To l. O g of the substance being examined that has
been dried under 80ºC for 1 hour, add 200 ml of water while
stirring until disperse uniformly, adjust pH to 7. 3-7. 8 with
15 % sodium hydroxide solution and mix well ( avoid air
bubbles), stand in a water bath at 25ºC for 1 hour. Type A
is 4-11 Pa•s, type Bis 25-45 Pa•s, and type C is 40-60 Pa•s
<0633).
Benzene, ethyl acetate and cyclohexane Transfer about O. 2 g
of the substance being examined, accurately weighed, to a
headspace vial, accurately add 5 ml of dimethylsulfoxide,
seal as the test solution. Dilute a quantity of benzene, ethyl
acetate and cyclohexane, accurately weighed, to a mixture
solution containing 4 µg, O. 2 mg and O. 12 mg per ml
respectively with dimethylsulfoxide. Transfer accurately 5 ml
to a headspace vial, seal as the reference solution. Carry out
the method for residual solvent ( 0861 method 2), usíng a
capillary column packed with 100 % dimethyl polysiloxane as
the stationary phase (or stationary phase of similar polarity);
a flame ionization detector is used. Maintain the colurnn
temperature at 40ºC for 3 minutes, raise the temperature to
120ºC by 5ºC per minute, and maintain for 20 minutes, then
raise the temperature to 220ºC by 20ºC per minute and
maintain for 3 minutes, then raise the temperature to 240ºC
by 20ºC per minute and maintain for 8 minutes. The
temperature of injection port is 260ºC and that of detector is
260ºC . The equilibrium temperature is 85ºC and the
equilibrium time is 90 minutes. Inject the test solution and
the reference solution separately, measured accurately, each
of the gases from headspace vials into the column and record
the chromatograms. Calculate the contents of benzene, ethyl
acetate and cyclohexane with respect to the area obtained in
the chromatogram by the externa! standard method. Benzene
should not be detected. Ethyl acetate and cyclohexane is not
more than O. 5 % and O. 3 %, respectively.
Acrylic acid Transfer about 50 mg of the substance being
examined, accurately weighed, to a stoppered centrifuge tube
with stopper, add 5 ml of 2. 5% potassium aluminium
sulfate solution. Seal and shake for 1 hour at '250 rpm at
50ºC. Centrifuge for 10 minutes at 10 000 rpm, filter. Use
the filtrate as test solution. Dissolve and dilute a quantity of
acrylic acid CRS, accurately weighed, to a solution
containing 25 µg per ml with water as the reference solution.
Carry out the method for high performance liquid
chromatography ( 0512 ) , using a column packed with
octadecylsilane bonded silica gel and a mixture of monobasic
potassium phosphate solution (dissolve l. 36 g of monobasic
potassium phosphate in 1000 ml of water and adjust pH with
phosphoric acid to 3. O± O. 1) -methanol ( 80 : 20) as the
mobile phase. The detection wavelength is 200 nm.
Accurately inject 10 µl of the test solution and the reference
solution onto the column and calculate the content of acrylic
acid with the respect to the area obtained in the
chromatogram by the externa! standard method. Acrylic acid

is not more than O. 25%.
Loss on drying When dried in vacuum at 80ºC for 1 hour,
loses not more than 2. O% of its weight <0831 ) .
Residue on ignition Not more than 2. O% <0841 ) , using
l. o g.
Heavy metals Carry out the limit test for heavy metals
<0821 method 2), using the residue obtained in the test for
Residue on ignition: not more than O. 002%.
Assay Disperse uniformly O. 4 g, accurately weighed, in
400 ml of water, stir to dissolve. Carry out the method for
potentiometric titration < 0701 ) . Ti trate with sodium
hydroxide ( O. 25 mol/L ) VS ( towards the end of the
titration, stir the mixture for at least 2 minutes after each
addition). Each ml of sodium hydroxide (0. 25 mol/L) VS is
equivalent to 11. 25 mg of carboxylic acid ( -COOH )
groups.
Category Pharmaceutical excipients, ointment base and
release retardant, etc.
Storage Preserve in well closed containers.
Labeling The labeling states the viscosity type (A, B or
C), the viscosity value, instrument and parameter for used.
Carbomer Copolymer
Carbomer Copolymer is a high molecular weight copolymer
of acrylic acid and a long-chain alkyl methacrylate cross-
linked with allyl ethers of polyalcohols, using non-benzene
solvent as polymerization solvent. The carboxylic acid group
( -COOH) is not less than 52. O% and not more than
62. O%, calculated on the dried basis.
Description A white, fluffy powder; odour, slight and
characteristic, hygroscopic.
ldentification ( 1) Add 5 g of the substance being examined
to 500 ml of water, and stir. A dispersion is formed, with a
foam layer that persists after the dispersion allowed to stand
at room temperature for 1 hour.
( 2) The infrared absorption spectrum <0402) of the substance
being examined has characteristic absorption at or near wave
numbers of 1710 ±5 cm- 1 , 1454 ±5 cm- 1 , 1414 ±5 cm- 1 ,
1245 ±5 cm- 1 , 1172 ±5 cm- 1 , 1115 ±5 cm- 1 and 801 ±
5 cm- 1 , with the strongest absorption at 1710 cm- 1 •
Acidity Swell O. 1 gin 10 m1 of water till uniformly dispersed,
pH 2. 5-3. 5 <0631 ).
Viscosity To 2. O g of the substance being examined that has
been dried under 80ºC for 1 hour, add 200 ml of water while
stirring until disperse uniformly, adjust pH to 7. 3-7. 8 with
15 % sodium hydroxide solution and mix well ( avoid air
bubbles), stand in a water bath at 25ºC for 1 hour. Type A
is 4. 5-13. 5 Pa•s, type Bis 10-29 Pa•s, and type C is 25-
45 Pa • s <0633 >.
Bemene, ethyl acetate and cyclohexane Transfer about O. 2 g of
the substance being examined, accurately weighed, to a
headspace vial, accurately add 5 ml of dimethylsulfoxide,
seal as the test solution. Dilute a quantity of benzene, ethyl
acetate and cyclohexane, accurately weighed, to a mixture
solution containing 4 µg, O. 2 mg and O. 12 mg per ml
respectively with dimethylsulfoxide, Transfer accurately 5 ml
to a headspace vial, seal as the reference solution. Carry out
the method for residual solvent <0861 method 2), using a
Carbomer Copolymer 501
capillary column packed with 100 % dimethyl polysiloxane as
the stationary phase Cor stationary phase of similar polarity);
a flame ionization detector is used. Maintain the column
temperature at 40ºC for 3 minutes, raise the temperature to
120ºC by 5ºC per minute, and maintain for 20 minutes, then
raise the temperature to 220ºC by 20ºC per minute and
maintain for 3 minutes, then raise the temperature to 240ºC
by 20ºC per minute and maintain for 8 minutes. The
temperature of injection port is 260ºC and that of detector is
260ºC . The equilibrium temperature is 85ºC and the
equilibrium time is 90 minutes. Inject the test solution and
the reference solution separately, measured accurately, each
of the gases from headspace vials into the column and record
the chromatograms. Calculate the contents of benzene, ethyl
acetate and cyclohexane with respect to the area obtained in
the chromatogram by the externa} standard method. Benzene
should not be detected. Ethyl acetate and cyclohexane is not
more than O. 5 % and O. 3 %, respectively.
Acroleic acid Transfer about O. 1 g of the substance being
examined, accurately weighed, into a centrifuge tube with
stopper, add 9 ml of water and shake for 2 hours. Add 2
drops of 50 % sodium hydroxide solution and shake. Add
l. O ml of 10 % calcium chloride solution, shake until the gel
disintegrates, centrifuge, filter the supernatant. The filtrate
is used as the test solution. Dissolve and dilute a quantity of
acroleic acid CRS, accurately weighed, with water to produce
a solution containing 25 µg per ml of acroleic acid as the
ref erence solution. Carry out the method for high
performance liquid chromatography <0512), using a column
packed with octadecylsilane bonded silica gel anda mixture of
potassium dihydrogen phosphate solution (dissolve l. 36 g of
potassium dihydrogen phosphate in 1000 ml of water, adjust
pH to 3. 0±0. 1 with phosphoric acid) -methanol (80 : 20)
as the mobile phase. Detection wavelength is 200 nm.
Accurately inject 10 µl of the reference solution and the test
solution into the column. Calculate the content of aroleic acid
with respect to the area obtain in the chromatogram by the
extemal method. Acrylic acid is not more than O. 25%.
Loss on drying When dried in vacuum at 80ºC for 1 hour,
loses not more than 2. O% of its weight <0831 ) .
Residue on ignition Not more than 2. O% <0841 ) , usmg
l. o g.
Heavy metals Carry out the limit test for heavy metals
<0821 method 2), using the residue obtained in the test for
Residue on ignition: not more than O. 002%.
Assay Disperse uniformly O. 4 g, accurately weighed, in
400 m1 of water, and stir to dissolve. Carry out the method for
potentiometric titration <0701 ). Titrate with sodium hydroxide
(O. 25 mol/L) VS ( towards the end of the titration, stir the
mixture for at least 2 minutes after each addition). Each m1 of
sodium hydroxide (0. 25 mol/U VS is equivalent to 11. 25 mg
of carboxylic acid ( -COOH) groups.
Category Pharmaceutical excipients, ointment base, release
retardant, etc.
Storage Preserve in well closed containers.
Labeling The laberling states the viscosity type (A, B or
c) ' the viscosity value' instrument and parameter for
u sed.
 Carbon Dioxide

~y Use a L-shaped carbon dioxide analyzer ( Fig. 2),

open two-way stopcocks C and D, connect the metal cylinder
pressure reducing valve outlet of the gas being examined and
the glass tube at c with a rubber pipe, fully replace the air in
Carbon Dioxide the analyzer and the connecting pipe with the gas being
examined ( more than 1 O times of the volume replaced).
C02 44. 01 Clase stopcock D, then close the bottom stopcock C, take
[124-38-9] off the rubber pipe, and rapidly rotate D several times to
Carbon Dioxide contains not less than 99. 5% of C02 (ml/ml). equilibrate the pressure of the gas in the analyzer with the
atmosphere pressure. Transfer 105 ml of 30 % potassium
Description Colourless gas; odourless, the aqueous solution
hydroxide solution to the dropping funnel, slowly open
exhibits acid reaction.
stopcock D to let the 30 % potassium hydroxide solution
At 20ºC and at normal pressure, 1 volume dissolves in about
flow into the horizontal absorber A. Close stopcock D
1 volume of water.
when carbon dioxide is completely absorbed ( which means
ldentification ( 1) Pass a stream of the substance being that 30 % potassium hydroxide solution no longer flow into
examined through barium hydroxide TS. A white precipitate is the horizontal absorber A, and the volume of the residual
formed which dissolves in acetic acid with effervescence. gas is constant). Read the scale value of the liquid surface
(2) Flame can be put out by the substance being examined. burette of the absorber A.
(3) The infrared absorption spectrum <0402 > is concordant
with the reference spectrum (Fig. 1).
Acidity Add O. 2 ml of methyl orange IS to 100 ml of water,
mix well. Transfer 50 ml each of the solution to nessler
cylinders A and R To cylinder B add l. O ml of hydrochloric
acid (0. 01 mol/L) VS and mix well. Pass 1000 ml of the
substance being examined through cylinder A (ata flow rate of
4000 ml per hour). The red colour produced in cylinder A is
not more intense than that in cylinder R
Carbon monoxide Not more than O. 001 % , determined
using a carbon monoxide detector tube.
Phosphine Not more than O. 00003 % , determined using a
phosphine detector tube.
Hydrogen sulfide Not more than O. 0001%, determined
using a hydrogen sulfide detector tube. Fig. 2 L-shaped carbon dioxide analyzer
A: Absorber ( capacity: 100 ± O. 5 ml, in which the
Hydrocarbon Use the substance being examined as the gas being mínimum scale value from 99 ml to 100 ml is O. 05 mD;
examined and a mixture containing O. 0020 % methane in nitrogen B: Dropping funnel (capacity: 120 ml, there is a scale line at
as the reference gas. Carry out the method for gas chromatography 105 mD;
<0521 >, using a column packed with glass beads (O. 8 m X 4 C, D: Two-way stopcock.
mm, 80 meshes). Maintain the column temperature at llOºC. The Note: Before the test or determination, the metal cylinder of
temperature of injection port is llOºC . The temperature of the gas being examined should be placed at the laboratory
detector is 120ºC. Inject the gas being examined and the reference temperature for more than 6 hours.
gas into the column and consider the peak area measured at the
purification temperature of 360ºC as the blank. Inject the gas Category Pharmaceutical excipients, a1r substitute, pH
being examined and the reference gas into the column and regulator and aerosol propellant.
consider the peak area subtract the blank as the corrected peak
Storage Preserve in a pressure-resistant container.
area. Calculate the content of hydrocarbon with reference to the
corrected peak area obtained in the chromatogram by the external Attached: Gas detector tubes
standard method: not more than O. 0020%. Gas detector tubes are cylindrical, transparent and sealed
tubes in wnich containing inert carrier coated with chemical
1001.--~~-~~~~-. r-r~-- ...........--~~~~---
95 reagent. If necessary, they also contain preliminary layers or
90 adsorbent filters to eliminate substances that interfere with
85
~80 the substance being examined. Cut off the two ends of the
~ 75 tu be when using, the test is carried out by passing the
"---' 70
~ 65 required volume of the gas being examined through the
~ 60 detector tube. The gas being examined immediately react
.s 55
~ with the chemical reagents. The type or concentration of the
·a 5045 gas can be examined by the length or intensity of the colour
~ 40
.s 3035 change from the chemical reagents .
Carbon monoxide detector tube: The minimum value
25
20 indicated is not more than 5 ppm, the relative standard
15
deviation (RSD) is not more than ±15%.
l0'--~~-3000~~--'-2-00_._0~~-1-5-00~~~1-000~~~-5-00-
Phosphine detector tube: The mínimum value indicated is not
W avenumber ( cm- 1 ) more than O. 05 ppm, the relative standard deviation (RSD)
is not more than ±10%.
Fig. 1 Reference spectrum of C0 2 (gas cell method)

Hydrogen sulfide detector tube: The mm1mum value
indicated is not more than O. 2 ppm, the relative standard
deviation (RSD) is not more than ±10%.
Carboxymethylcellulose Calcium
[9050-04-8]
Carboxymethylcellulose Calcium is the calcium salt of
carboxymethylcellulose.
Description A white and yellow white powder; hygroscopic.
Swells in water and forming a suspension, practically
insoluble in acetone, in ethanol and in methylbenzene.
ldentification Dissolve O. 1 g of the substance being
examined with 10 ml of water, shake, and add 2 ml of 1
mol/L sodium hydroxide, and stand for 10 minutes as the
test solution.
(1) To 1 ml of the test solution add water to make 5 ml.
Transfer 1 drop of the resulting solution, add O. 5 ml of
chromotropic acid TS, and heat in a water bath far 10 minutes.
A red-purple colour is produced.
(2) Take 5 ml of the test solution, add 10 ml of acetone and
shake. A white flocculent precipitate is formed.
(3) Transfer 5 ml of the test solution, add 1 ml of ferric
chloride TS and shake. A brown flocculent precipitate is
formed.
( 4) Ignite 1 g of the substance being examined and dissolve
the residue in 10 ml of water and 5 ml of 6 mol/L acetic acid,
and filter if necessary. Boil the filtrate, cool, and neutralize
with 6 mol/L ammonium hydroxide. The solution yields the
reaction characteristic of calcium salts ( 0301).
Acidiíy Shake 1. O g with 100 ml boiled and cooled water,
add 2 drops of phenolphthalein IS and prohibit develops red
colour in solution.
Chloride Dissolve O. 8 g (calculated on the dried basis) with
50 ml of water. Add 10 ml of 1 mol/L sodium hydroxide,
and dilute to 100 ml with water as test stock solution. To
20 ml of test stock solution, add 10 ml of 2 mol/L nitric
acid, heat on a water bath, flocculent precipitation formed,
cool, centrifuge separation and take the supernatant. Wash
the precipitate with water and centrifuge for 3 times, each of
10 ml, combine the supernatant and wash solution, dilute to
100 ml with water and mix well. Transfer 10 ml to carry out
the limit test for chlorides <0801 ) . Any colour produced is
not more intense than that of a reference solution using 5 ml
of sodium chloride standard solution (O. 3 %) .
Sulfate To 10 ml of the test stock solution in Chloride, add
1 ml of hydrochloric acid, and mix well, heat on a water
bath, flocculent precipitation farmed, cool, centrifuge
separation and take the supematant. Transfer the supematant into
a 100 ml volumetric flask, wash the precipitate with water
and centrifuge separation far 3 times, each of 10 ml.
Combine the supernatant and elution into a 100 ml volumetric
flask, dilute to volume with water, and mix well. Transfer
accurately 25 ml of the solution and carry out the limit test
far sulfates ( 0802). Any colour produced is not more intense
than that of a reference solution using 2 ml of potassium
sulfate standard solution (l. 0%).
Loss on drying When dried at 105ºC for 4 hours, loses not
more than 10. O% of its weight ( 0831).
Residue on ignition Not less than 10 % and not more than
20 % ( 0841 ) , using 1. O g, calculated on the dried basis.
Carboxymethylcellulose Sodium
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), not more than O. 002%.
Category Pharmaceutical excipients, disintegrant and filler,
etc.
Storage Preserve in well closed containers.
Carboxymethylcellulose Sodium
OH
[9004-32-4]
Carboxymethylcellulose sodium is the sodium salt of
carboxymethylcellulose reacted by cellulose with sodium
chloroacetic acid under alkaline condition. It contains not less
than 6. 5% and not more than 9. 5% of sodium (Na),
calculated on the dried basis.
Description A white to pale yellow fibrous powder or
granular; odorless; hygroscopic.
Disperses and swells in water forming colloidal solutions,
insoluble in ethanol, in ether and in chloroform.
ldentification Add 1 g to 50 ml of warm water, stir to
produce a uniform dispersion. Continue stirring until a
colloidal solution is obtained. Cool for the following tests.
(1) To 10 ml of the solution, add 1 ml of copper sulfate TS.
A blue, flocculent precipitate is formed.
(2) To 5 ml of the solution, add an equal volume of barium
chloride TS, a white precipitate is formed.
(3) The solution yields the flame reaction characteristic of
sodium salts ( 0301).
Viscosity Weigh accurately 4. O g ( calculated on the dried
basis) in a 250 ml beaker, weighed previously, and add
150 ml of hot water. Keep in a hot water bath for 30
minutes. Stirring rapidly until the powder is well wetted,
cool and add sufficient volume of water to make the mixture' s
weight to 200 g. Allow to stand, stirring occasionally until
the substance is dissolved well. Determine the viscosity at
25ºC ( 0633 method 3), using appropriate single column type
rotary viscometer ( Brookfield type LV model or performance
equivalent viscometer), and carry out determination with the
following test condition. It is not less than 75 % and not
more than 140 % of the labeled viscosity.
Labeled Viscosity (m Pa • s) Rotator Speed (rpm)
1000-2500 No. 3 30
(not include 2500) No. 2 12
2500-8000 No. 4 60
(not include 8000) No. 3 12
8000-12 000 No. 4 30 and 60
(not include 120 00) No. 3 6
 510 Camauba Wax
Acidity or alkalinity To O. 5 g add 50 ml of warm water,
vigorously stirring until a colloidal solution is obtained, cool
to room temperature, pH 6. 5-8. O (0631 ).
Clarity and Colour of solution To 1. O g add 90 ml of 40-
50 ºC boiled and cooled water, vigorously stirring until a
colloidal solution is obtained, cool to room temperature,
dilute to 100 ml with boiled and cooled water. Any
opalescent produced is not more pronounced than that of
reference suspension 3 ( 0902 method 1 ) , any colour
produced is not more intense than that of reference solution
Y3 ( 0901 method 1 ) .
Chloride Dissolve l. O g, calculated on the dried basis,
accurately weighed, with 5 ml of dehydrated ethanol and
150 ml of water in a 250 ml conical flask. Add 5 drops of
30 % hydrogen peroxide solution, heat gently to boiling and
maintain for 10 minutes, cool. Add 1 ml of potassium
chromate IS and titrate with silver nitrate (0. 1 mol/L) VS.
Each ml of sil ver nitra te CO. 1 mol/L) VS is equivalent to
3. 545 mg of Cl: not more than l. 0%.
Sulfate Dissolve O. 5 g Ccalculated on the dried basis) with
50 ml of water. Transfer 10 ml, add 1 ml of hydrochloric
acid, and mix well, heat on a water bath, a flocculent
precipitate is formed, cool, and centrifuge. Transfer the
supematant into a 50 ml volumetric flask, wash the
precipitate and centrifuge with 3 portions of water, each of
10 ml. Combine the supematant and washings into the same
50 ml volumetric flask, dilute to volume with water and mix
well. Transfer accurately 10 ml of the solution into a 50 ml
1'Jessler cylinder, add 10 ml of v1ater. Carry out the limit test
for sulfates ( 0802). Any colour produced is not more intense
than that of a reference solution using l. O ml of Potassium
sulfate standard solution (0. 5%).
Silicate lgnite gently l. O g Ccalculated on the dried basis) ,
in crucible until charred completely, add 20 ml of dilute
hydrochloric acid, put on the glass dishes, heat gently to
boiling and maintain for 30 minutes. Remove the glass
dishes, evaporate to dryness on a water bath, low heat for 1
hour, add 10 ml of hot water, and mix well. Filter with
ashless filter, wash the precipitate with hot water until
precipitate is not formed in the washing water by adding
silver nitrate. Transfer the precipitate and ashless filter to a
constant weight crucible, ignite to constant weight at 500-
600ºC. Remaining residues are not more than O. 5%.
Sodium glycollate Protect from light throughout the
procedure. Weigh accurately O. 5 g Ccalculated on the dried
basis) to a beaker. Add 5 ml of 5 mol/L acetic acid solution
and 5 ml of water. Stir until sodium glycollate is completely
dissolved (at least 30 minutes). Add 80 ml of acetone and 2 g
of sodium chloride, stir until Carboxymethylcellulose
precipitate is complete. Filter and quantitatively transfer the
filtrate with acetone into a 100 ml volumetric flask, dilute to
volume with acetone. Mix well and allow to stand for 24
hours. Use the clear supernatant liquid as test solution.
Dissolve O. 310 g of glycolic acid, previously dried for 12
hours in vacuum, with water in a 1000 ml volumetric flask
and dilute to volume with water. Transfer 5 ml of the
solution, accurately measured, in a 100 ml volumetric flask,
add 5 ml of 5 mol/L acetic acid and allow to stand for 30
minutes. Add 80 ml of acetone and 2 g of sodium chloride,
dilute to volume with acetone. Shake throughly and allow to
stand for 24 hours as reference solution. Transfer separately
2. O ml each of the resulting solution and reference solution
into two 25 ml Nessler cylinders. Heat on a water bath to
evaperate acetone. Cool and accurately add 20 ml of 2, 7-
dihydroxynaphthalene solution edissolve 10 mg of 2' 7-
dihydroxynaphthalene in 100 ml of sulfuric acid, allow to
stand until decolourized, and use within 2 days) to each
Nessler cylinder. Stopper and mix well. Heat on a water
bath for 20 minutes. Cool and examine the solutions viewing
vertically. The colour of the test solution is not more intense
than that of the reference solution. If necessary, measure the
absorbance of the resulting solutions at 540 nm ( 0401 )
within 10 minutes: not more than O. 4%.
Loss on drying When dried at 105ºC for 6 hours, loses not
more than 10. O% of its weight ( 0831 ) , using l. O g.
Iron lgnite gently l. O g ( calculated on the dried basis) , in
crucible until charred completely, cool and moisten the
residue with O. 5 ml of sulfuric acid. Heat at low temperature
to expel the vapor of sulfuric acid, ignite at 550-600ºC until
incineration is complete, cool, add 1 ml of hydrochloric acid
and 3 drops of nitric acid, evaporate to dryness on a water
bath. Stand to cool and dissolve the residue in 16 ml of dilute
hydrochloric acid and a volume of water. Transfer to a
100 ml volumetric flask, dilute to volume with water and
mix well, filter if necessary. Transfer accurately 25 ml of the
solution into a 50 ml Nessler cylinder. Carry out the limit
test for iron ( 0807 ) . Any colour produced is not more
intense than that of a reference solution using 4. O ml of iron
standard solution (0. 016%).
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), not more than O. 001%, using l. O g
(calculated on the dried basis).
Arsenic Mix O. 67 g Ccalculated on the dried basis) with
l. O g of calcium hydroxide, add 2 ml of water, stir well.
After drying, heat gently until completely charred, ignite at
500-600ºC until the incineration is complete. Allow to cool,
add 8 ml of hydrochloric acid and 23 ml of water to dissolve
the residue. Carry out the limit test for arsenic ( 0822
method 1 ) : not more than O. 0003 % .
Assay Weigh accurately O. 25 g of the substance being
examined obtained in the test for Loss on drying into a
150 ml conical flask, add 50 ml of glacial acetic acid, shake
well. Heat under a reflux condenser for 2 hours. Stand to
cool, transfer the solution into a 100 ml beaker. Wash the
conical flask with glacial acetic acid for 3 portions, each of
5 ml and combine the washings. Carry out the method for
potentiometric titration ( 0701 ) , titrate with perchloric acid
CO. 1 mol/L) VS. Perform a blank determination and make
any necessary correction. Each ml of perchloric acid
CO. 1 mol/L) VS is equivalent to 2. 299 mg of Na.
Category Pharmaceutical excipients, disintegrant and filler,
etc.
Storage Preserve in tightly closed container.
Labeling States the viscosity with mPa • s and Pa •s.
Carnauba Wax
[8015-86-9]
Camauba Wax is purified wax, obtained from the leaves of
Copernicia cerifera Mart.
Description A pale yellow or yellow powder, flakes or masses.
Freely soluble in hot xylene; soluble in hot ethyl acetate;
practical insoluble in water and in ethanol.
Melting range 80-86ºC ( 0612 method 2).

Acid value Dissolve 5 g, accurately weighed, with 100 ml of
xylene, in a 250 ml flake by heating. Add 50 ml of ethanol
and 2. 5 ml of bromothymol blue IS, heat until the solution is
clear. Titrate immediately with potassium hydroxide in
ethanol (O. 1 mol/L) VS until the colour of the solution
changes to green. Perform a blank determination and make
any necessary correction. The acid value is not less than 2
and not more than 7 <0713).
Saponification value Transfer 3 g, accurately weighed, in a
500 ml flask, add 50 ml of a mixture of isopropanol-toluene
(5 : 4), add 15 ml, accurately measured of O. 5 mol/L
potassium hydroxide solution in ethanol, heat under reflux
far 3 hours, add 1 ml of phenolphthalein IS, titrate with
hydrochloric acid ( O. 5 mol/L) VS until the red colour
disappears, Heat the solution to boil, continue the titration if
the red colour reappears. Perform a blank determination and
make any necessary correction. The saponification value is
not less than 78 and not more than 95 ( 0713).
lodine value Transfer about l. 8 g, accarately weighed, in a
500 ml drynessiodine flask, add 30 ml of chlorofarm, dissolve
the sample by shaking the flask in water bath at 80±1 ºC. The
Iodine value is not less than 5 and not more than 14 (0713).
ldentification Dissolve O. 1 g of the substance to be
examined in 5 ml of chlorofarm by heating. use the hot
solution as the test solution. Dissolve 10 mg of menthol,
10 mg of thymol and 10 µl of menthyl acetate in a 20 ml
volumetric flask, dilute with toluene to volume and mix well
as the ref erence solution. Carry out the method far thinlayer
chromatography <0502 ) , using silica gel G as the coating
substance anda mixture of ethyl acetate-chlorofarm (2 : 98)
as the mobil phase. Apply separately to the plate 6 µl of the
test solution and 2 µl of the reference solution. After
developing and removal of the plate, dry it in air and spray
v:ith a freshly prepared 20 % phosphomolybdic aicd in
ethanol. Heat at 105ºC for 10-15 minutes. The chromatogram
obtained with the reference solution shows a dark blue spot
(menthoD in the lower part, a reddish spot ( thymol) above
this spot and a dark blue spot ( menthyl acetate) in the upper
part. The chromatogram obtained with the test solution
shows a large blue spot ( triacontanol) many small spots
below it at a level between the positions of thymol and
menthol spots. Many blue spots are visible at levels between
the positions of the menthyl acetate and thymol spots; above
these spots, others are visible in the chromatogram obtained
with the test solution; the spot with the highest R 1 value is
clear. The starting point is coloured blue.
Clarity and colour of solution Dissolve O. 1 g of the
substance to be examined in 10 ml of chlorofarm with
heating, the solution is clear and colour less ( 0901 and
0902). Any colour produced is not more pronounced than
that the reference solution ( using l. O ml of Potassium
Dichromate Standard Solution, add 15 ml of water, mix
well).
Residue on ignition NJt rrore than O. 25 % ( 0841 ) , using l. O g.
Heavy metals Carry out the limit test far heavy metals
( 0821 method 2) , using the residue obtained in the test far
Residue on ignition; not more than O. 002%.
Category Pharmaceutical excipients, coating and release
retardan t.
Storage Preserve in tightly closed containers, protected
from light.
Cellacefate
Cellacefate
[ 9004-38-0 J
Cellacefate is a condensation compound of partially acetylated
cellulose acetate and phthalic anhydride. It contains not less
than 30. O% and not more than 36. O% of phthalyl ( Cs H 5 0 3)
and not less than 21. 5 % and not more than 26. O% of acetyl
(C2 H3 0), calculated on the anhydrous bases after deducting
the amount of free acid.
Description A white or almost white amorphous fibriform
of fine strip, flakes, granules or powder.
Insoluble in water and in ethanol, dissolves in acetone and
swells to produce a clear or slightly opalescent colloidal
solution.
ldentification Triturate to fine powder by grinder, by
potassium bromide tabletting method, the Infrared
absorption spectrum is concordant with the Infrared
absorption spectrum of cellacefate CRS ( 0402).
Viscosity To 15 g, accurately weighed, calculated on the
anhydrous bases, into a conical flask with stopper. Add
accurately 85 g of a mixture of acetone-water ( 249 : 1)
(W/W), and shake until completely dissolved. Determine
the kinetic viscosity ( 0633 method 1 ) , select capillary with
suitable inside diameter to get an appropriate dropping time
greater than 200 seconds: not less than 45 mPa • s and not
more than 90 mPa •s.
Water Dissolve about O. 5 g, accurately weighed, in a
mixture of anhydrous ethanol-methylene chloride ( 3 : 2) ( if
the dissolution is difficult, use dehydrated ethanol instead of
anhydrous ethanol), carry out the determination of '-Nater
( 0832 method 1): not more than 5. 0%.
Free acid Transfer 3. O g, accurately weighed, to an iodine
flask, add 100 ml methanol solution ( 1 - 2), insert the
stopper, shake far 2 hours, filter and wash the flask and the
residue with methanol solution 0-2) far two times, 10 ml
each time, combine the washing and filtrate, add 3 drops of
phenolphthalein IS, ti trate with sodium hydroxide (O. 1 mol/L)
VS. Perform a blank determination and make any necessary
correction. Each ml of sodium hydroxide (O. 1 mol/L) VS is
equivalent to 8. 306 mg of Cs H6 0 4 • The free acid is not
more than 3. O%, calculated as phthalic acid ( C8 H 60 4 ).
Residue on ignition Not more than 0.1% (0841), using 2. O g.
Heavy metals Carry out the limit test far heavy metals
( 0821 method 2) , using the residue obtained in the test far
Residue on ignition: not more than O. 001 %.
Assay Phthaloyl Dissolve about 1 g, accurately weighed,
in 50 ml of ethanol-acetone ( 3 : 2) solution with shaking.
Add 2 drops of phenolphthalein IS, and titrate with sodium
hydroxide (O. 1 mol/L) VS. Perform a blank determination
and make any necessary correction. Each ml of sodium
hydroxide (0. 1 mol/U VS is equivalent to 14. 91 mg of Cs Hs ÜJ,
calculate the content of Phthaloyl as fallows:
14. 91(V - Vo) l. 7955
[lOOO(lOO%-a)(lOO%-S)W] (100%- S)
where W is the weight of sample, g;
V is the volume of sodium hydroxide (O. 1 mol/L)
VS used in sample determination, ml;
Vo is the volume of sodium hydroxide (O. 1 mol/L)
VS used in blank determination, ml;
 Cellulose Acetate
a is the percent content of water;
S is the percent content of free acid.
Acetyl To O. 1 g, accurately weighed, add accurately
measured 25 ml of sodium hydroxide (O. 1 mol/U VS, and
heat on a water-bath under a reflux condenser for 30
minutes. Cool, add 5 drops of phenolphthalein IS, titrate
with hydrochloric acid (O. 1 mol/L) VS. Perform a blank
determination and make any necessary correction. Each ml of
hydrochloric acid (O. 1 mol/U VS is equivalent to 4. 304 mg
of Ci H 3 O, calcula te the content acetyl as follows.
4. 304(Vo -V)
[lOOOClOO% - a ) ClOO% - S ) W]
0.51825
ClOO% _ S> -O. 5773P
Where W is the weight of sample, g;
V is the volume of hydrochloric acid (O. 1 mol/U
VS used in sample determination, ml;
Vo is the volume of sodium hydroxide (O. 1 mol/U
VS used in blank determination, ml;
a is the percent content of water;
S is the percent content of free. acid;
P is the percent content of phthaloyl.
Category Pharmaceutical excipients, coating material and
release retardant.
Storage Preserve in well closed containers and protected
from light.
Note: Preparation of dehydrated ethanol
Place anhydrous ethanol 20 ml in a 500 mlround flask, add
l. 2 g dry and clean magnesium strip, install a reflux
condenser with an anhydrous calcium chloride drying tube.
Heat gently to boiling, then stop heating and add a few
pieces of iodine plates ( without shaking), and there will be
chemical reactions instantly. After the reaction is complete,
add 200 ml anhydrous ethanol and several zeolites, heat
under reflux for 1 hour, then distil and receive in a dry
distilling flask. The dry distilling flask connect with a drying
tube with anhydrous calcium chloride. Heat the distilling
flask and collect the distillate and preserve in a well closed
glass container.
The water of dehydrated ethanol and the mixture of
anhydrous ethanol-methylene chloride (3 : 2): not more than
O. 05 % ( 0832 method 1 ( 1 ) ) for both the dehydrated
ethanol and the mixture, using 1O ml.
Cellulose Acetate
OR
n
R=H,COCH3
[9004-35-7]
Cellulose Acetate is partly or completely acetylated cellulose.
It contains not less than 29. 0% and not more than 44. 8% of
acetyl (C2 H3 0) groups and not less than 90. 0% and not
more than 110. O% of the labelled amount of the acetyl
content, calculated on the dried basis.
Description A white, pale yellowish-white or greyish-white
powder or granules; hygroscopic.
Practically insoluble in water and in ethanol, soluble in
acetone, in formic acid and in a mixture of equal volumes of
methanol and methylene chloride.
Identification Dissolve the substance being examined in
dioxane. Add 1 drop of the solution on a blank potassium
bromide clise. Heat at 105ºC for 1 hour. The infrared
absorption spectrum ( 0402) is concordant with the spectrum of
cellulose acetate CRS.
Viscosity Weigh accurately 10 g of the substance being
examined into a 100 ml of a mixture of equal volumes of
methanol and methylene chloride, and shake to dissolve.
Carry out the method for determination of kinetic viscosity
( 0633 method 2 ) at 20 ± O. 1ºC using a suitable single
cylindrical type rotating viscometer ( Brookfield type LV
model or Effectiveness quite viscometer) with No. 2 rotator
at the speed of 60 rpm, the viscosity is not less than 75%
and not more than 140 % of the labeled.
Free acid Weigh accurately 5. O g, add 150 ml of freshly
boiled and cooled water, shake gently and allow to stand for
3 hours. Filter, wash the residue with freshly boiled and
cooled water. Combine the filtrate and washings, add 2-3
drops of phenolphthalein IS. Titrate with sodium hydroxide
(0. 01 mol/U VS until a pink colour is produced. Each ml
of sodium hydroxide (O. 01 mol/L) VS is equivalent to
O. 6005 mg of free acid. Not more than O. 1 % , calculated on
the dried basis.
Residual solvents Dissolve O. 1 g of the substance being
examined, accurately weighed, in a headspace vial and add
accurately 5 ml of water, and l. O g of anhydrous sodium
sulfate and seal as the test vial. Dissolve a quantity of
methylene chloride, chloroform, 1, 1, 2-trichloroethylene
and dioxane, accurately weighted, in water to produce a
mixed solution containing 12 µg methylene chloride, l. 2 µg
chloroform, l. 6 µg 1, 1, 2-trichloroethylene and 7. 6 µg
dioxane per ml. Measure accurately 5 ml to a headspace vial,
add l. O g of anhydrous sodium sulfate, and seal as the
reference vial. Carry out the method for determination of
Residual solvents ( 0861 method 1 ) . Using a capillary
column packed with 5 % phenyl-methylsiloxane. Maintain
column temperature at 35ºC, detector temperature at 260ºC
and injection port temperature at lOOºC. Equilibration
temperature of headspace vials is 80ºC and equilibration time
is 60 minutes. Inject l. O ml of the gaseous sample in the
reference vial into the column and record the chromatograms.
The resolution factors between the peaks comply with the
requirements. lnject separately the gaseous sample in the test
vial and the standard vial into the column and record the
chromatogram. Calculate the content of methylene chloride,
chloroform, 1, 1, 2-trichloroethylene and dioxane with
respect to the peak area obtained in the chromatogram by
external standard method. The content of methylene chlorid,
chloroform, 1 , 1, 2-trichloro ethylene and dioxane complies
with the requirement.
Loss on drying When dried at 105ºC for 3 hours, loses not
more than 5. O% of its weight <0831).
Residue on ignition Not more than O. 1 % <0841 ) , using
2. o g.
Heavy metals Carry out the limit test for heavy metals
<0821 method 2), using the residue obtained in the test for
Residue on ignition, not more than O. 001%.
Assay For cellulose acetate containing not more than

42. O% of acetyl groups.
Transfer 2. O g, accurately weighed, into a conical flask, add
100 ml of acetone and 10 ml of water. Clase the flask and
stir with a magnetic stirrer to dissolve. Add accurately 30. O
ml of sodium hydroxide (l. O mol/L) VS. Clase the flask
and stir with a magnetic stirrer for 30 minutes. Add 100 ml
of hot water, washing clown the inside of the flask, stir for 2
minutes and cool to room temperature. Add 2-3 drops of
phenolphthalein IS, titrate with sulphuric acid (O. 5 mol/L)
VS. Perform a blank determination and make any necessary
correction. Each ml of sulphuric acid (O. 5 mol/L) VS is
equivalent to 43. 05 mg of C2 H3 O.
Far cellulose acetate containing more than 42. O% of acetyl
groups.
Transfer 2. O g, accurately weighed, into a conical flask, add
30 ml of dimethyl sulphoxide and 100 ml of acetone. Clase
the flask and stir with a magnetic stirrer for 16 hours. Add
30. O ml of sodium hydroxide (l. O mmol/L) VS. Clase the
flask and stir with a magnetic stirrer for 6 minutes. Allow to
stand for 60 minutes. Add 100 ml of hot water, washing
clown the inside of the flask, stir for 2 minutes and cool to
room temperature. Add 4-5 drops of phenolphthalein IS,
titrate with hydrochloric acid (O. 5 mol/L) VS. Add O. 5 ml
of hydrochloric acid (O. 5 mol/L) VS in excess, stir for 5
min and allow to stand for 30 minutes. Titrate with sodium
hydroxide (0. 5 mol/L) VS, until a pink colour is produced.
Perform a blank determination and make any necessary
correction. Each ml of hydrochloric acid (O. 5 mol/L) VS is
equivalent to 21. 525 mg of Cz H3 O.
Category Pharmaceutical excipients, release retardant and
coating material, etc.
Storage Preserve in tightly closed containers.
Labeiiug States the viscosity with mPa • s and Pa •:s.
Cetostearyl Alcohol
Cetostearyl Alcohol is a mixture of cetyl alcohol and stearyl
alcohol. lt contains not less than 40. O% of stearyl alcohol
CC1s H3s 0) and the total content of cetyl alcohol (Cl6 H34 0)
and stearyl alcohol ( Crn H3 8 0) is not less than 90. O%.
Description White granules, flakes or masses.
Freely soluble in ethanol and in ether, practically insoluble in
water.
Melting range 49-56ºC <0612 method 2).
Acid value Not more than l. O (0713).
Saponification value Not more than 2. O < 0713), using
20. o g.
Iodine value Not more than 2. O <0713 >. Dissolve 2. O g in
25 ml of chloroform by shaking.
Hydroxyl value 208-228 <0713 >.
Identification The retention time of the principal peak of the
test solution in the chromatogram obtained in the Assay is
identical with that of the principal peak in the chromatogram
of the reference solution.
Clarity and colour of ethanolic solution To O. 50 .g add 20 ml
of ethanol, heat to dissolve and allow to cool, the solution is
clear and colourless < 0901 and 0902); any opalescence
produced is not more pronounced than that of reference
Cetyl Alcohol
suspension 1 <0902 method 1 ) .
Assay Carry out the method for gas chromatography
<0521 ) , using a capillary column packed with 100 %
polydimethylsiloxane as the stationary phase. A flame
ionization detector is used. The column temperature is
maintained at 205ºC. The temperatures of injection port and
detector are both 250ºC. The number of theoretical plates of
the column is not less than 10 000, calculated for the peak of
cetyl alcohol. The resolution factor between the peaks of
cetyl alcohol and stearyl alcohol complies with the requirements.
Procedure Dissolve 100 mg, accurately weighed, in a
100 ml volumetric flask, dilute to volume with anhydrous
ethanol and mix well. Inject accurately 1 µl of the resulting
solution into the column and record the chromatogram.
Dissolve a quantity of cetyl alcohol CRS and stearyl alcohol
CRS, accurately weighted, in anhydrous ethanol to produce a
solution containing each of O. 5 mg per ml and mix well.
Repeat the operation. Calculate the content of C1 8 H3s O and
the total content of Cl6 H 34 O and C1s H3s O with respect to the
peak area in the chromatogram obtained by the externa!
standard method.
Category Pharmaceutical excipients, retardant and ointment
vehicle.
Storage Preserve in well closed containers, stored in a cool
and dry place.
Cetyl Alcohol
cl6 H34 o 242. 44
[36653-82-4]
Cetyl Alcohol is prepared by methyl esterification,
hydrogenation and purification from natural oil. lt contains
not less than 96. O% of C16 H34 O and not more than l. 5 % of
other alkanes.
Description A white powder, granules, flakes or masses;
odour, greasy. lt turns in to a transparent oily liquid after
melting.
Miscible with ethanol, practically insoluble in water.
Melting range 46-52ºC <0612 method 2).
Acid value Not more than l. O <O713 ) .
Saponification value Not more than l. O ( 0713), using
10 g, accurately weighed.
Hydroxyl value 220-240 <0713).
lodine value Not more than l. 5 (0713).
ldentification The retention time of the principal peak of the
test solution in the chromatogram abtained in the Assay is
identical with that of the principal peak in the chromatogram
of the reference solution.
Clarity and color of ethanolic solution To O. 50 g add 20 ml
of ethanol, heat to dissolve and allow to cool, the solution is
clear and colourless ( 0901 and 0902 ) ; any opalescence
produced is not more pronounced than that of reference
suspension 1 <0902 method 1 ) .
Assay Carry out the method for gas chromatography
 Chitosan

( 0521 ) , using a capillary column packed with 5 % diphenyl
polysiloxane Cor equivalent column) as the stationary phase.
The column temperature is maintained at 120ºC, then raise
the temperature to 240ºC by 5ºC per minute. The number of
theoretical plates of the column is not less than 10 000,
calculated with reference to the peak of cetyl alcohol. The
resolution factor between the peaks of cetyl alcohol and
hexadecane complies with the requirements.
Procedure Dissolve a quantity of the substance being
examined in anhydrous ethanol to produce a solution
containing about 10 mg per ml as the test solution. Dissolve
a quantity of hexadecane CRS and cetyl alcohol CRS in
anhydrous ethanol to produce a solution containing about 10
mg of cetyl alcohol and 1 mg of hexadecane per ml as the reference
solution. Inject separately 1 µl each of the test solution and the
reference solution into the column, and record the chromatogram.
Calculate the content of cetyl alcohol (X) and other alkanes (Y) as
follows:
X AROH XlOO%
ARH
AROH +-f- + L; Ax
A;H + I:Ax
y V HCI : the volume of hydrochloride acid titration (O. 3
A X100%
AROH + ;H + L; Ax
Where ARoH is peak area of cetyl alcohol m the
chromatogram;
A RH is peak area of hexadecane;
Ax is peak area of other unknown impurities;
f is the relative response factor of alkane;
f = SRH XmRoH
SRoH X mRH
Where mRH is weight of hexadecane CRS;
mRottis weight of cetyl alcohol CRS;
S RH is peak area of hexadecane CRS;
S ROH is peak area of cetyl alcohol CRS.
Category Pharmaceutical excipients, ointment vehicle and
emulsifier.
Storage preserve in well closed containers.
Chitosan
(2) Dissolve O. 2 gin 80 ml of water, stir to disperse. add 20
ml of glycolic acid solution (O. 1- 20) , mix gently at room
temperature to make the solution clear ( stir for about 30-60
minutes) , add 5 ml of O. 5 % sodium lauryl sulfate solution,
a gelatinous mass is formed.
Tests Viscosity Weigh accurately l. O g, add 100 ml of 1%
glacial acetic acid, stir to dissolve completely. Carry out the
method for determination of viscosity ( 0633 method 3 ) ,
using a NDJ-1 type rotatory viscometer, the dynamic
viscosity at 20ºC is not less than 80 % and not more than
120 % of the labeled viscosity.
Degree of deacetylation Weigh accurately O. 5 g, add accurately
18 ml of hydrochloride acid titration (O. 3 mol/U VS, and stir
for 2 hours at room temperature to dissolve. Add 3 drops of 1 %
methyl orange IS and titrate with sodium hydroxide (0.15 mol/U
VS until an orange colour is produced. Calculate the degree of
deacetylation by the formula:
D D % = CNttcI X VHCI - NNaOH X VNaOH) X O. 016 X lOO%
0 0
• • G X (100-W) X 9. 94%
Where D. D. % is the degree of deacetylation, % ;
N HCI : the concentration of hydrochloride acid titration
(0. 3 mol/U VS, mol/L;
mol/U VS, ml;
N NaOH : the concentration of sodium hydroxide
titration (0. 15 mol/U VS, mol/L;
VNaOH: the volume of sodium hydroxide titration
(0. 15 mol/U VS, ml;
G: the weight of the test sample, g;
W: percentage of loss on drying in test sample, % ;
O. 016: amounts of amino group equivalent tol mol/L
hydrochloride acid, g;
9. 94%: theoretical content of amino group.
The degree of deacetylation is more than 70%.
Acidity or alkalinity To O. 50 g, add 50 ml of water and stir
for 30 minutes, allow to stand for 30 minutes, pH 6. 5-8. 5
( 0631>.
Protein Take O. 1 g into a 10 ml volumetric flask, dissolve
with 1 % glacial acetic acid, dilute to volume and mix well.
Carry out the method for determination of protein ( 0731
method 5) , not more than O. 2 % .
Loss on drying When dried to constant weight at 105ºC,
loses not more than 10% of its weight ( 0831), using l. O g.
Residue on ignition Not more than l. O% ( 0841 ) , using
l. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2) , using the residue obtained in the test for

HO--S-0- --S-0 ---S-OH

HO NH 2 HO NH HO NH 2
Residue on ignition: not more than O. 001%.
Arsenic Mix 2. O g with l. O g calcium hydroxide, add 2 ml
of water and mix well. After dryness on a water bath, ignite
i n
gently until it is thoroughly charred and then ignite at 500-
600ºC until the incineration is completed. Dissolve the cooled
residue in a mixture of 5 ml of hydrochloride acid and 23 ml of
water. Carry out the limit test for arsenic ( 0822 method 1 ) :
R=H or
not more than O. 0001%.
Category Pharmaceutical excipients, disintegrant and
[9012-76-4]
thickening agent.
Chitosan is an unbranched binary polysaccharide consisting of
N-acetyl-D-glucosamine and D-glucosamine units. Storage Preserve in well-closed and light-resistant containers,
stored in cool and dry place.
Description Almost white powder, odorless and tasteless.
Slightly soluble in water, practically insoluble in ethanol. Labelling States the viscosity in mPa • s or Pa •s.
ldentification ( 1 ) The infrared absorption spectrurn is
concordant with the reference spectrurn of chitosan CRS <0402).

Chlorobutanol
H3CXOH , t H20
H3C CCI 3
1
C4 H1Cbü·-z H20 186. 47
[6001-64-5]
Chlorobutanol is 1, 1, 1-trichloro-2-methyl-2-propanol hemihy-
drate. It contains not less than 98. 5% of C4H1CI3Q, calcu-
lated on the anhydrous basis.
Description White crystals; odour, camphoraceous; readily
volatile.
Freely soluble in ethanol, in chloroform, in ether and in
volatile oil; slightly soluble in water.
Melting point Not lower than 77°C ( 0612), determined on
the undried substance.
Identification ( 1) Dissolve about 25 mg in 5 ml of water,
add 1 ml of sodium hydroxide TS and then add slowly 3 ml of
iodine TS, a yellow precipitate is formed, and the odour of
iodoform is perceptible.
(2) The infrared absorption spectrum ( 0402) is concordant
with the spectrum of Chlorobutanol CRS.
Acidity To 5. O g of the substance being examined add 10 ml
of ethanol, shake to dissolve. To 4 ml of the resulting
solution add 15 ml of ethanol and O. 1 ml of bromothymol
blue IS, shake well; the blue colour of the resulting solution
produced is not more intense than that of the reference
solution (to l. O ml of sodium hydroxide solution add 18 ml
of ethanol and O. 1 ml of bromothymol blue IS, shake welD.
Clarity of solution To 5. O g of the substance being
examined add 10 ml of ethanol to dissolve; any opalescence
produced is not more pronounced than that of the reference
suspension 2 ( 0902).
Chloride To O. 10 g of the substance being examined add
25 ml dilute ethanol, shake to dissolve. Add l. O ml of nitric
acid and a volume of dilute ethanol to produce 50 ml, add
l. O ml silver nitrate TS, shake well and allow to stand in the
dark for 5 minutes. Any opalescence produced is not more
pronounced than that of a reference solution (to 5. O ml of
sodium chloride standard solution add l. O ml of nitric acid
anda volume of dilute ethanol to produce 50 ml, add l. O ml
of silver nitrate TS) (0. 05%).
Water 4. 5 %-5. 5 % <0832 >.
Residue on ignition Not more than O. 1 % ( 0841 ) .
Assay To an accurately weighed O. 1 g of the substance being
examined, add 5 ml of ethanol and 5 ml of 20% sodium
hydroxide solution, heat under reflux for 15 minutes. Allow to
cool, add 20 ml of water and 5 ml of nitric acid, accurately add
30 ml of silver nitrate (O. 1 mol/U VS, add 5 ml of dibutyl
phthalate, stopper and shake vigorously. Add 2 ml of ferric
ammonium sulfate IS, ti trate with ammonium thiocyanate (O. 1
mol/L) VS. Perform a blank determination and make any
necessary correction. Each ml of silver ni trate (O. 1 mol/L) VS
is equivalent to 5. 915 mg of C4 H1 Cb O.
Category Pharmaceutical excipient, preservative and plasticizer.
Storage Preserve in tightly closed containers.
Chlorocresol
Chlorocresol
GH1CIO 142.58
[59-50-7]
Chlorocresol is 4-chloro-3-methylphenol. It contains not less
than 98. O% and not more than 101. O% of G H1 ClO.
Description White or almost white, crystalline powder or
massive crystals, characteristic smell of phenol and the
colour darkens gradually on exposure to light and air.
Very soluble in ethanol, soluble in ether and petroleum ether,
slightly soluble in water and freely soluble in alkaline solution.
Melting point 64-67°C ( 0612).
Identification (1) Dissolve 40 mg in 10 ml of water, shake,
add 1 drop of ferric chloride TS, a bluish violet colour is produced.
(2) Dissolve 30 mg in 10 ml of water, shake, add bromine
TS, a white precipitate is produced.
(3) Mix well 50 mg of the substance being examined and O. 5
g of anhydrous sodium carbonate, heat the mixture until a
dark red colour is produced, continue to heat for 10 minutes,
cool, dissolve with water and the filtrate yields the reactions
characteristic of chlorides ( 0301).
Acidity Dissolve 3. O g, previously powdered, in 60 ml of
water, shake for 2 minutes, filter, to 10 ml of the filtrate, add
O. 1 ml of methyl red IS, the solution is organge or red; not
more than O. 2 ml of (0. 01 mol/L) sodium hydroxide solution is
required to change the colour of the solution to yellow.
Clarity and colour of solution Dissolve l. 25 g in 25 ml of
ethanol, the solution is clear and colourless ( 0901 and
0902). Any colour produced is not more intense than that of
reference solution OL2 ( 0901 method 1 ) .
Related substances Accurately weigh l. O g in a 100 ml
volumetric flask, dissolve and dilute with acetone to volume,
shake well and use as the test solution; accurately transfer
1 ml of the test solution into a 100 mi volumetric flask,
dilute with acetone to the volume and mix well, transfer 10
mi of the solution accurately measured into a 100 mi
volumetric flask, dilute with acetone to the volume, mix
well, use as the reference solution. Dissolve a quantity of m-
cresol CRS, accurately weighed, in acetone to produce a
standard solution containing 50 µg per ml. Carry out the
method for gas chromatography ( 0521), using a capillary
column packed with 35 % phenylmethylpolysiloxane as the
stationary phase ( 30 m X O. 32 mm, O. 5 µm, DB-17). The
injector port temperature is 210ºC, the detector temperature
is 280ºC and the column temperature is 125ºC. Dissolve a
quantity of o-creso! CRS and m-cresol CRS, in acetone to
produce a mixture solution of each of 50 µg per ml. Inject 1
µl of the mixture solution into the column. The resolution
factor between the peaks due to o-creso! and m-cresol
complies with the requirements. Inject 1 µl of the reference
solution into the column, the retention time of peak due to
chlorocresol is about 8 minutes, adjust the sensitivity of the
detector so that the height of the principal peak in the
chromatogram is about 20 % of the full scale of the recorder.
Inject accurately 1 µl each of the reference solution, the
 Cholesterol
standard solution and the test solution into the column
respectively, and record the chromatogram to 3 times of the
retention time of the principal peak. In the chromatogram
obtained with the test solution, the content of m-cresol is not
more than O. 5 % calculated by the externa! standard method;
the area of any other peak is not more than the area of the
principal peak of in the chromatogram obtained with the
ref erence solution (O. 1 % ) . The sum of the areas of all other
peaks is not more than 5 times of the area of the principal
peak in the chromatogram obtained with the reference
solution (O. 5 % ) .
Non-volatile substances Evaporate 2. O g in an evaporating
dish previously dried to constant weight at 105°C to dryness
on a water bath, dry at 105ºC to constant, The residue is not
more than 2 mg (O. 1%).
Assay Place about 70 mg, accurately weighed, in an iodine
flask, dissolve with 30 ml of glacial acetic acid. Add 25 ml of
potassium bromate ( O. 01667 mol/L ) VS, accurately
measured, add 20 ml of 15% potassium bromide solution and
10 ml of hydrochloric acid. Protected from light and allow to
stand for 15 minutes. Add 1 g of potassium iodide and
100 ml of water, titrate with sodium thiosulfate (O. 1 mol/L)
VS, add 1 ml of starch TS towards the end of titration,
continue the titration until the blue colour disappears.
Perform a blank determination and make any necessary
correction. Each ml of potassium brornated (O. 01667 mol/L)
VS is equivalent to 3. 565 mg of G H1 Clü.
Category Pharmaceutical excipients, preservative.
Storage Preserve in tightly closed containers, protected
from light.
Cholesterol
C21 H46 O 386. 7
[57-88-5]
Cholesterol is obtained from animal organ by extracting and
refining. It is cholest-5-en-3,B-ol. It contains not less than
95. o% of Cz1H460, calculated on the dried basis.
Description White, flaky crystals; odourless.
Freely soluble in chloroform, soluble in ether, sparingly
soluble in acetone, in ethyl acetate and in petroleum ether;
slightly soluble in ethanol and practically insoluble in water.
Melting range 14 7-150ºC < 0612).
Specific rotation - 34 º to - 38º, using a solution containing
20 mg per ml in dioxane <0621 ) .
ldentification ( 1) Dissolve about 10 mg in 1 ml of
chloroform, add 1 ml of sulfuric acid, the chloroform layer
acquires a blood-red colour and the sulfuric acid layer shows a
green fluorescence.
(2) Dissolve about 5 mg in 2 ml of chloroform, add 1 ml of
acetic anhydride and 1 drop of sulfuric acid, a pink colour is
produced which rapidly changes to blue, and finally to a
brilliant green.
( 3) Dissolve separately a quantity of the substance being
examined and cholesterol CRS in acetone to prepare the test
solution (2 mg per ml) and reference solution ( 2 mg per
mD. Carry out the method for thin layer chromatography
<0502) , using silica gel G as the coating substance and a
mixture of ethyl acetate -toluene ( 1 : 2) as the mobile
phase. Apply separately to the plate 20 µl each of the test
solution and reference solution. After developing and
removal of the plate, dry it and spray 30% solution of
antimony trichloride in chloroform. Dry at 105ºC for 5
minutes. Examine immediately. The position and colour of
the principal spot in the chromatogram obtained with the test
solution correspoud to the principal spot of the reference
solution.
(4) The infrared absorption spectrum is concordant with the
spectrum of Cholesterol CRS ( 0402).
Acidity Place l. O g in a flask with stopper, add 10 ml of
ether to dissolve, add accurately measured 10 ml of O. 1 mol/L
sodium hydroxide solution and shake for about 1 minute.
Warm gently to expel the ether, boil for 5 minutes. Allow to
cool and add 10 ml of water, add 2 drops of phenolphthalein
IS with magnetic stirring, and titrate with sulfuric acid (0. 05
mol/L) VS until the pink colour is disappeared. Carry out a
blank determination. The difference between the volume of
the sulfuric acid (O. 05 mol/U VS consumed in the blank
sample and that of the test substance is not more than O. 3 ml.
Alcohol-insoluble matter Dissolve O. 5 g in 50 ml of ethanol
under gentle heating, allow to stand for 2 hours, no
precipitate or turbidity is formed.
Loss on drying When dried to constant weight at 105ºC,
loses not more than O. 3 % of its weight <0831 ) .
Residue on ignition Not more than O. 1 % <0841 ) , using 1 g.
Heavy metals Not more than O. 001% <0821 method 2),
using the residue obtained in the test for residue on ignition.
Arsenic To l. O g add l. O g of calcium hydroxide, mix well
with a small volume of water. Allow to dry, ignite gently to
ash, then ignite at 500-600ºC until the incineration it
completed, cool and dissolve the residue in a mixture of 5 ml
of hydrochloric acid and 23 ml of water. Carry out the limit
test for arsenic: not more than O. 0002 % <0822 method 1 ) .
Assay Carry out the method for liquid chrornatography <0512).
Using a column packed with octadecylsilane bonded silica gel
and methanol as the mobile phase, equipped with an
evaporation light scattering detector. lnject 20 µl of the
reference solution ( the cholesterol concentration is O. 1 mg
per mD onto the column. The number of theoretical plates
calculated for the cholesterol peak is not less than 5000. Inject
reference solution five times, relative standard deviation of
the peak area for cholesterol is not more than 2. O%.
Procedure Toan accurately weighed quantity of cholesterol
CRS add absolute ethanol to dissolve, and dilute to produce a
series reference solutions containing O. 05, O. 1, O. 2, O. 3 and
O. 5 mg per ml respectively. lnject 20 µl of each of the
reference solutions onto the column and record the
chromatogram. Plot a standard curve with the logarithm of
peak area as the ordinate and the logarithm of concentration
of the reference solution as the abscissa. The related
coefficient r is at lest O. 99. Dissolve a quantity of the
substance being examined in absolute ethanol to prepare a
solution containing about O. 125 mg per ml, and use as the
test solution. Repeat the operation and calculate the content
of cholesterol in the test substance by the calibration
equation.
Category Pharmaceutical excipients, emulsifier and ointment
base.
Storage Preserve m tightly closed containers, protected
from light.
Citric Acid
C6 Hs 01• H2 O 210. 14
[5949-29-1]
Citric acid is 2-hydroxy-l, 2, 3-propanetricarboxylic acid,
monohydrate. It contains not less than 99. 5 % of C6 Hs 01 ,
calculated on the anhydrous basis.
Description Colourless translucent crystals, white granules
or white crystalline powder; odourless; taste, strongly
sour; slightly efflorescent in dry air; The aqueous solution
exhibits an acidic reaction.
Very soluble in water, freely soluble in ethanol, sparingly
soluble in ether.
ldentification (1) Dried at 105ºC for 2 hours, the infrared
absorption spectrum ( 0402) is concordant with the reference
spectrum CIR Album No. 263).
(2) Yields the reactions characteristic of citrates ( 0301).
Clarity and colour of solution Dissolve 2. O g in 10 ml of
water, the solution is clear and colourless ( 0901 and 0902
method 1 ) ; any colour produced is not more intense than
that of reference solution Y2 or YG2 ( 0901 method 1 ) .
Chloride Carry out the limit test for chlorides ( 0801 ) ,
using 10. O g. Any opalescence produced is not more
pronounced than that of a reference solution using 5. O ml of
sodium chloride standard solution (O. 0005 % ) .
Sulfate Carry out the limit test for sulfates ( 0802) , using
l. O g. Any opalesence produced is not more pronounced than
that of a reference solution using l. 5 ml of potassium sulfate
standard solution (O. 015%).
Oxalate Dissolve l. O g in 10 ml of water, add ammonia
solution to neutralize, add 2 ml of calcium chloride TS, stand
at room temperature for 30 minutes, no turbidity appears.
Readily carbonizable substances Transfer l. O g to a Nessler
tube, add 10 ml of 95% (g/g) of sulfuric acid, heat for
1 hour at 90 ± 1ºC , allow to cool immediately. Any colour
produced is not more intensely coloured than that of a
reference solution prepared by mixing O. 9 ml of cobaltous
chloride es, 8. 9 ml of potassium dichromate es ando. 2 ml
cupric sulfate CS.
Water 7.5%-9.0% (0832method1 (1) ).
Residue on ignition not more than O. 1 % ( 0841 >.
Calcium Dissolve l. O gin 10 ml of water, add ammonia TS
to neutralize, add a few drops of ammonium oxalate TS, no
turbidity appears.
Iron Carry out the limit test for iron ( 0807) , using l. O g.
Add butanol to extract. Any colour produced is not more
intense than that of reference solution using l. O ml of iron
standard solution (0. 001%).
Heavy metals Dissolve 4. O gin 10 ml of water, add 1 drop
of phenolphthalein IS anda few drops of ammonia TS until a
pink colour is produced. Add 2 ml of aceta te BS ( pH 3. 5)
and sufficient water to produce 25 ml. Carry out the limit
test for heavy metals ( 0821 method 1 ) : not more than
o. 0005%.
Arsenic Dissolve 2. O g in 23 ml of water, add 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822
method 1 ) : not more than O. 0001 % .
Assay Dissolve about l. 5 g, accurately weighed, in 40 ml
of water, add 3 drops of phenolphthalein IS, titrate with
potassium hydroxided ( 1 mol/L) VS. Each ml of potassium
hydroxided ( 1 mol/L) VS is equivalent to 64. 04 mg of
C6Hs01.
Category pharmaceutical excipients, pH regulator, stabilizers
and acidifer.
Storage Preserve in tightly closed containers.
Clove Leaf Oil
Clove Leaf Oíl is the volatile oíl obtained by steam distillation
from the stems and leaves of Eugenia caryophyllata
Thumb. (Fam. Myrtaceae). It contains 5. o% to 14. 0% of
,B-caryophyllene ( C1 s H24) and 80. O% to 92. O% of eugenol
CC10 H12 02 ).
Description A clear, slightly yellow to yellow liquid;
odour, aromatic, resembling that of clove; taste, pungent
and numb; deteriorated easily on exposure to air.
Very soluble in ethanol, in ether, in glacial acetic acid and in
toluene, practically insoluble in v1ater.
Relative density l. 038-1. 060 ( 0601 >.
Optical rotation Oº to-2. Oº <0621 >.
Refractive index l. 528-1. 537 ( 0622 >.
ldentification (1) Dissolve about 80 mg in 2 ml of toluene
as the test solution. Dissolve a quantity of eugenol CRS and
acetyleugenol CRS in toluene to produce a solution containing
each of 25 mg per ml as the reference solution. Carry out the
method for thin-layer chromatography ( 0502), using silica
gel GF2 54 as the coating substance and toluene as the mobile
phase. Apply separately to the plate 2 µl each of above two
solutions, after developing and removal of the plate, allow to
stand for 5 minutes and develop for the second time, removal
of the plate, dry it in air and examine under ultra-violet light
(254 nm). The chromatogram obtained with the reference
solution shows two clearly separated spots ( from up to
clown, the spots are eugenol and acetyleugenol in tum). The
position and colour of the principal spot in the chromatogram
obtained with the test solution correspond to the principal
spot of eugenol obtained with the reference solution. Then
spray with anisaldehyde solution ( dissolve O. 5 ml of
anisaldehyde in 10 ml of glacial acetic acid, add 85 ml of
methanol and 5 ml of sulfuric acid, mix well. Prepare befare
use. ) , heat at 105ºC for 5-10 minutes. The position and
colour of the principal spot in the chromatogram obtained
with the test solution correspond to the principal spot of
eugenol obtained with the reference solution. There is a red
spot under the foreland of solvent and the spot of
acetyleugenol respectively, and the spot in the foreland of
solvent is ,B-caryophyllene.
(2) The retention times of the principal peaks of the test
 Clove Oil
solution in the chromatogram obtained in the Assay are
identical with those of the principal peaks of ,B-caryophyllene
CRS and eugenol CRS in the chromatogram of the reference
solution correspondingly.
(1) or (2) may be used alternatively.
Clarity of solution Dissolve 1 ml in 2 ml of 70 % ethanol
solution, the solution is clear <0902).
Water-soluble phenol To 1 ml add 20 ml of hot water, mix
well and allow to cool, filter through moistened filter paper.
Add 1 drop of ferric chloride TS to the filtrate. A blue or
purple colour should not be produced except a transient
greyish-green colour.
Fatty oils and resinified es.sential oils Allow 1 drop to fall
onto filter paper. The drop evaporates completely within
24 hours without leaving any translucent or greasy spot.
Heavy metals Not more than O. 001 % <0821 rnethod 2) , using
l. o g.
Aassay Carry out the method for gas chromatography
( 0521 ) , using a capillary column packed with polyethylene
glycol Cor similar polarity) as the stationary phase. Maintain
the column temperature at 80ºC for 1 minute, then raise the
temperature to 180ºC by 3ºC per minute, maintain for 2
minutes. The temperatures of injection port and detector are
both 250ºC. Inject 1 µl of the reference solution into the
column, the resolution factor between the peaks of ,B-
caryophyllene and eugenol complies with the requirements.
Internal standard solution Dissolve a quantity of ethyl
salicylate in n-hexane to produce a solution of 9 mg per ml.
Procedure Dissolve about O. 1 g, accurately weighed, in a
10 ml volumetric flask and dilute to volume with the interna!
standard solution, mix well as the test solution. Dissolve a
quantity of ,B-caryophyllene CRS and eugenol CRS,
accurately weighed, in interna! standard solution to produce a
solution containing about l. O mg and 8. 8 mg per ml
respectively as the reference solution. Inject separately 1 µl
each of the reference solution and the test solution into the
column, and record the chromatograms. Calculate the
contents of C1s H24 and C10 H12 02 with respect to the peak
area obtained in the chromatogram by the interna! standard
method.
Category Pharmaceutical excipients, aromatic and taste masking
agent.
Storage Preserve in tightly closed containers, stored in a
cool and dark place.
Clove Oil
[8000-34-8]
Clove Oil is the volatile oil obtained by steam distillation from the
dried flower buds of Eugenia caryophyllata Thumb. ( Fam.
Myrtaceae). It contains 5. 0% to 14. 0% of ,B-caryophyllene
CC1s H24), 75. 0% to 88. 0% of eugenol CC10 H12Ü2) and 4. o%
to 15. 0% of acetyleugenol CC12H14Ü3).
Description A clear, slightly yellow to yellow liquid;
odour, aromatic, resembling that of clove; taste, pungent
and numb; deteriorated easily on exposure to air.
Very soluble in ethanol, in ether, in glacial acetic acid and in
toluene, practically insoluble in water.
Relative density l. 038-1. 060 <0601 ) .
Optical rotation Oº to- 2. Oº <0621 ) .
Refractive index l. 528-1. 537 <0622).
ldentification ( 1) Dissolve about 80 mg in 2 ml of toluene
as the test solution. Dissolve a quantity of eugenol CRS and
acetyleugenol CRS in toluene to produce a solution containing
each of 25 mg per ml as the reference solution. Carry out the
method for thin-layer chromatography ( 0502), using silica
gel GF2s4 as the coating substance and toluene as the mobile
phase. Apply separately to the plate 2 µl each of above two
solutions, after developing and removal of the plate, allow to
stand for 5 minutes and develop for the second time, removal
of the plate, dry it in air and examine under ultra-violet light
(254 nm). The chromatogram obtained with the reference
solution shows two clearly separated spots ( from up to
clown, the spots are eugenol and acetyleugenol in turn). The
position and colour of the principal spots in the chromatogram
obtained with the test solution correspond to the principal
spots of eugenol and acetyleugenol obtained with the
reference solution. Then spray with anisaldehyde solution
(dissolve O. 5 ml of anisaldehyde in 10 ml of glacial acetic
acid, add 85 ml of methanol and 5 ml of sulfuric acid, mix
well. Prepare before use), heat at 105ºC for 5-10 minutes.
The position and colour of the principal spots in the chromatogram
obtained with the test solution correspond to the principal
spots of eugenol and acetyleugenol obtained with the
reference solution. There is a red spot under the foreland of
solvent and the spot of acetyleugenol respectively, and the
spot in the foreland of solvent is ,B-caryophyllene.
(2) The retention times of the principal peaks of the test
solution in the chromatogram obtained in the Assay are
identical with those of the principal peaks of ,B-caryophyllene
CRS, eugenol CRS and acetyleugenol CRS in the
chromatogram of the reference solution correspondingly.
(1) or (2) may be used alternatively.
Clarity of solution Dissolve 1 ml in 2 ml of 70 % ethanol
solution, the solution is clear ( 0902).
Water-soluble phenol To 1 ml add 20 ml of hot water, mix
well and allow to cool, filter through moistened filter paper.
Add 1 drop of ferric chloride TS to the filtrate. A blue or
purple colour should not be produced except a transient
greyish-green colour.
Fatty oils and resinified es.sential oils Allow 1 drop to fall
onto filter paper. The drop evaporates completely within
24 hours without leaving any translucent or greasy spot.
Heavy metals Not more than O. 001% <0821 method 2),
using l. O g.
Aassay Carry out the method for gas chromatography
( 0521 ) , using a capillary column packed with polyethylene
glycol (or similar polarity) as the stationary phase. Maintain
the column temperature at 80ºC for 1 minute, then raise the
temperature to 180ºC by 3ºC per minute, maintain for 2 minutes.
The temperatures of injection port and detector are both
250ºC. Inject 1 µl of the reference solution into the column,
the elution order is ,B-caryophyllene, eugenol and acetyleugenol.
Internal standard solution Dissolve a quantity of ethyl
salicylate in n-hexane to produce a solution of 9 mg per ml.
Procedure Dissolve about O. 1 g, accurately weighed, in a
10 ml volumetric flask and dilute to volume with interna!
standard solution, mix well as the test solution. Dissolve a
quantity of ,B-caryophyllene CRS, eugenol CRS and
acetyleugenol CRS, accurately weighed, in interna! standard
solution to produce a solution containing about l. O mg, 8. 8
mg and l. O mg per ml respectively as the reference solution.

Inject separately 1 µl each of the reference solution and the test
solution into the column, and record the chromatograms.
Calculate the contents of C1s H24, C10 H12 02 and C12H14Ü3
with respect to the peak area obtained in the chromatogram
by the interna! standard method.
Category Pharmaceutical excipients, aromatic and taste
masking agent.
Storage Preserve in tightly closed containers, stored in a
cool and dark place.
Cocoa Butter
Cocoa Butter is the solid fat obtained from the seed of
Theobroma cacao L. (Fam. Sterculiaceae).
Description A yellowish-white solid; usually brittle at
temperatures below 25ºC; odour, agreeable, having a slight
chocolate-like taste ( obtained by pressing) or a bland taste
(obtained by extraction).
Freely soluble in ether and in chlorofarm, soluble in boiling
absolute ethanol, slightly soluble in ethanol.
Relative density O. 895-0. 904 at 40ºC with water at 20ºC
<0601 >.
Refractive index l. 456-1. 458 at 40ºC <0622).
Acid value Not more than 2. 8 <0713).
Saponification value 188-19 5 <O713 ) .
lodine value 35-40 <O713 ) .
ldentification The retention time of the principal peaks of
methyl palmitate, methyl stearate, methyl oleate and methyl
iinoieate in the chromatogram oí the test solution obtained in
the test far Composition of fatty acids are identical with those
of faur principal peaks in the chromatogram of the reference
solution correspondingly.
Composition of fatty acids Place O. 10-0. 15 g of the
substance being examined in a 50 ml reflux flask, add 4 ml of
O. 5 mol/L methanolic sodium hydroxide solution, and reflux
the mixture in a water bath until the fat substance being
examined turns to solution. Add 5 ml of 14% boron
trifluoride methanol solution, and reflux the mixture in a
water bath for 2 minutes. Add 2-5 ml of n-heptane and
continue boiling for 1 minute. Cool, add 10 ml of saturated
sodium chloride solution, mix well and allow to separate.
Dry the organic layer with anhydrous sodium sulfate, filter
and use the filtrate is as the test solution. Dissolve a quantity
of methyl palmitate, methyl stearate, methyl oleate,
methyl linoleate, methyl linolenate, and methyl arachidate
CRS in n-heptane to produce a mixture of O. 1 mg per ml
respectively as the reference solution. Carry out the method
for gas chromatography <0521 ) , using a column packed with
25% phenyl-25% cyanopropyl-50% methylpolysiloxane as
the stationary phase. Maintain the column temperature at
120ºC, then rasie the temperature to 240ºC by lOºC per
minute, maintain for 5 minutes (note: the time maintained
at 240ºC depends on the elution time of the last peak). The
temperature of injection port is 250ºC. The temperature of
detector is 250ºC. Inject 1 µl of the reference solution into
the column and record the chromatogram, the resolution
factors between all peaks comply with the requirements.
Inject 1 µl of the test solution into the column and record the
chromatogram, calculate the content of each fatty acid by the
area normalization method. Palmitic acid: 23% to 30%;
Colloidal Silicon Dioxide
Stearic acid: 31 % to 37 % ; oleic acid: 31 % to 38 % ; linoleic
acid: l. 6 % to 4. 8 % ; linolenic acid and arachidic acid: not
more than l. 5 % , respectively.
Category Pharmaceutical excipients, lubricant and ointment
vehicle of suppository.
Storage Preserve in well closed containers.
Colloidal Silicon Dioxide
Si02 60. 08
[7631-86-9]
Colloidal silicon dioxide is prepared by the reaction of silicon
tetrachloride in hydrogen and oxygen flame. It contains not
less than 99. 0% and not more than 100. 5% of SiOz, calculated
on the ignition basis.
Description A white porous powder.
Insoluble in water and dilute hydrochloric acid, soluble in hot
sodium hydroxide TS.
ldentification ( 1 ) W eigh about 5 mg into a platinum
crucible, add 200 mg of potassium carbonate and mix well.
lgnite at 600-700ºC for 10 minutes and then cool. Dissolve
with 2 ml of water, heat gently. Add slowly 2 ml of
ammonium molybdate solution (to 6. 5 g of molybdic acid,
add 14 ml of water and 14. 5 ml of concentrated ammonia
solution, shake to dissolve, allow to cool, add a cooled
mixture of 32 ml of nitric acid and 40 ml of water with
stirring, allow to stand for 48 hours and fil ter, use the
filtrate): a deep yellow colour is produced.
(2) Place 1 drop of the solution obtained in Identification
(1) on a filter paper, evaporate the solvent. Add 1 drop of
saturated solution of o-tolidine in glacial acetic acid, and
place the filter paper overa concentrated ammonia solution: a
blue-green spot is produced.
Apparent volume Weigh 2. 5 g to a 100 ml cylinder, the
volume is not less than 35 ml without shaking.
Acidity Take 1 g and add 25 ml of water, shake to suspend
well, pH 3. 5-5. 5 <0631 ).
Chloride Take O. 5 g, add 50 ml of water. Boil under a
reflux condenser for 2 hours. Allow to cool, dilute to 50 ml
with water, shake well and filter. Carry out the limit test for
chlorides <0801), using 10 ml of the subsequent filtrate, any
opalescence produed is not more pronounced than that of a
reference solution prepared by using l. 1 ml of sodium
chloride standard solution (O. 011 % ) .
Loss on drying When dried at 105ºC for 2 hours, loses not
more than 2. 5 % of its weight <0831 ) .
Loss on ignition Weigh accurately l. O g of residual from
loss on drying, igniting at 1000 ± 25ºC to constant weight.
Loses not more than 2. O% of its weight.
Calcium Dissolve l. O g in 30 ml of sodium hydroxide TS,
heat to boil and allow to cool. Add 20 ml of water and 1 drop
of phenolphthalein IS. Add dilute nitric acid until the colour
disappears. lmmediately add 5 ml of dilute acetic acid and
shake well. Dilute the solution with water to 100 ml,
centrifuge. To 25 ml of supernatant, add 1 ml of oxalic acid
TS, dilute the solution with ethanol to 50 ml, immediately
shake and allow to stand far 10 minutes. Any colour
produced is not more intense than that of a reference solution
prepared by using the same method from 25 ml of standard
calcium solution (Dissolve O. 25 g of calcium carbonate dried
 Compressible Sugar
at 180ºC for 4 hours in 3 ml of dilute hydrochloric acid,
dilute with water to 100 ml, shake well. Tansfer 4 ml, add
5 ml of dilute acetic acid, dilute to 100 ml with water and
shake welD.
Alurninurn Dissolve O. 5 g in 40 ml of sodium hydroxide
TS. Heat to boil and allow to cool. Dilute to 50 ml with
sodium hydroxide TS, filter, take 10 ml of subsequent
fil trate, add 17 ml of 31 % glacial acetic acid solution and
shake well. Add 2 ml of rose bengal aluminon solution
(dissolve O. 1 g of rose bengal aluminon with 100 ml of
water, allow to stand for 24 hours). Dilute the solution with
water to 50 ml and allow to stand for 30 minutes. Any colour
produced is not more intense than that of the reference
solution prepared from 15. 5 ml of standard aluminum
solution ( Dissolve O. 176 g of aluminum potassium sulfate
with water to 1000 mD add 10 ml sodium hydroxide TS,
using the same method as described above but starting from
"adding 17 ml of 31% glacial acetic acid solution".
Iron Dissolve O. 2 g in 25 ml of water, add 2 ml of
hydrochloric acid and 5 drops of nitric acid. Heat to boíl for
5 minutes, allow to cool and filter. Wash the filtrate with
water, combine the filtrate and the washing. Add 50 mg of
ammonium persulfate, and dilute with water to 35 ml. Carry
out the limit test far iron ( 0807): any colour produced is not
more intense than the reference solution prepared from 3. O
ml of standard iron solution (0. 015%).
Heavy metals Dissolve 3. 3 gin 40 ml of water and 5 ml of
hydrochloric acid, heat slowly to boil far 15 minutes, allow
to cool and filter the solution. The filtrate is placed into a
100 ml volumetric flask. Wash the filtrate with an
appropriate amount of water and transfer the washing liquid
into the above volumetric flask. Dilute to the volume with
water and shake well. Measure 20 ml, add 1 drop of
phenolphthalein IS, add ammonia TS until red colour is
produced. Add 2 ml of acetate BS (pH 3. 5) and dilute with
water to 25 ml. Carry out the limit test for heavy metals
( 0821 method 1): not more than O. 0025%.
Arsenic To 20 ml of solution prepared in the test for heavy
metal, add 5 ml of hydrochloric acid. Carry out the limit test for
arsenic ( 0822 method 1 ) : not more than O. 0003 % .
Assay Transfer O. 5 g to a platinum crucible ignited to constant
weight at 1000±25ºC. Ignite at 1000±25ºC for 2 hours. Allow
to cool and weigh accurately. Add 3 drops of sulfuric acid to the
residue and wet with ethanol, add 15 ml of hydrofluoric acid,
evaporate on a water bath to near dryness. Transfer the solution
onto an electric furnace, heat slowly until all the acids have
been volatilized, ignite at 1000 ± 25ºC to constant weight.
Allow to cool and weigh accurately, if the residue remains,
repeat the analysis, beginning with "add 15 ml of hydrofluoric
acid". The weight lost by the assay specimen, represents the
weight of Si02 in the substance being examined.
Category Pharmaceutical excipients, thickening agent, stabilizer,
diluent.
Storage Preserve in well-closed containers.
Compressible Sugar
Compressible Sugar is produced by cocrystallization of sucrose
and other pharmaceutical excipients such as maltodextrin, or
by drying granulation process. It contains not less than
95. O% and not more than 98. O% of sucrose ( C12 H22 Ün ) ,
calculated on the dried basis. It may contain starch,
maltodextrin, invert sugar or suitable lubricants.
Description A white or almost white crystalline powder or
microcrystalline granules; odourless; tas te sweet.
Very soluble in water.
Identification ( 1 ) The specific optical rotation of the
uninverted solution obtained under Assay is not less than +
62. 6° and the acid-inverted solution is laevorotatory.
(2) The infrared absorption spectrum ( 0402) is concordant
with the reference spectrum.
Chloride Dilute O. 20 g to 25 ml with water, carry out the
limit test far chlorides ( 0801). Any opalescence produced is
not more pronounced than that of the reference solution using
2. 5 ml of sodium chloride standard solution (0. 0125%).
Sulfate Carry out the limit test far sulfates ( 0802), using
l. O g. Any opalescence produced is not more pronounced
than that of the reference solution using l. O ml of potassium
sulfate standard solution (O. 01%).
Loss on drying When dried at 105ºC far 4 hours, loses not
more than l. O% of its weight ( 0831 ) .
Residue on ignition Not more than O. 1 % ( 0841 ) .
Calciurn Dissolve l. O g in 5 ml of water, add 1 ml of
ammonium oxalate TS. The solution rernains clear within 1
minute.
Heavy metals Dissolve 4. O gin 20 ml of water, add 1 ml of
O. 1 mol/L hydrochloric acid solution and a volume of water
to 25 ml. Carry out the limit test for hcavy metals ( 0821
method 1 ) : not more than O. 0005 %.
Assay Transfer 26 g of the substance being examined,
previously dried 4 hours at 105ºC and accurately weighed, to
a 100 ml volumetric flask, add O. 3 ml of saturated aqueous
solution of lead acetate and 90 ml of water, shake well,
dilute with water to volume, and mix well. Distribute about
8 g of chromatographic siliceous earth on the surface of a
buchner funnel, and filter the solution with the buchner
funnel by the aid of vacuum, discarding the first 20 ml of the
filtrate. Accurately measure 25 ml of the successive filtrate
into each of two 50 ml volumetric flasks. Slowly add 6 ml of
dilute hydrochloric acid ( 1 - 2) to one of the two flasks,
mix well, place the flask in a water bath at 60ºC,
continuously shake the flask in the bath for about 3 minutes,
and allow the flask to remain in the bath far 7 minutes.
Immediately cool to 20ºC by plunging the flask into a cold
bath, dilute with water to volume at 20ºC, and mix well.
Place the other flask in a cold bath at 20ºC, and dilute with
water to volume, mix well. l\1aintain both flasks at 20ºC for 30
minutes. Carry out the determination of optical rotation ( 0621 ) .
Calculate the percentage of C12 H22 Üu taken by the formula:
lOO(ao -a;) /88. 3
Where, ao anda¡ are the specific rotations of the uninverted
and acid-inverted solutions respectively.
Category Pharmaceutical excipients, diluent, sweetening
agent.
Storage Preserve in tightly closed containers.
Croscarmellose Sodium
[74811-65-7]
Croscarmellose Sodium is the sodium salt of a cross-linked,

partly carboxymethylated cellulose.
Description A white or almost white powder, hygroscopic.
Swells in water, forming a suspension, practically insoluble
in anhydrous ethanol, in ether, in acetone and in toluene.
Identification ( 1) Mix 1 g with 100 ml of O. 0004%
methylene blue solution, stir and allow to settle. A blue,
fibrous precipitate is formed.
(2) Mix 1 g with 50 ml of water. Transfer 1 ml of the
mixture to a test tube, add 1 ml of water and 5 drops of
a-naphthol solution in methanol eto 1 g of a-naphthol, add
25 ml of anhydrous methanol and stir to dissolve, prepared
freshly befare use), incline the test tube, carefully add 2 ml
of sulfuric acid. A reddish-violet colour develops at the
interface.
C3) The solution obtained in Identification C2) yields the
flame reaction characteristic of sodium salts <0301 ) .
Settling volwne To 75 ml of water in a 100 ml graduated
cylinder with stopper and add l. 5 g of the substance to be
examined divide into three portious, O. 5 g of each portian,
shake vigorously after each addition. Dilute to 100 ml with
water and shake again until the substance is homogeneously
distributed. Allow to stand for 4 hours. The settling volume
is not less than 1O. O ml and not more than 30. O ml.
Acidity Shake 1 g with 100 ml of water for 5 minutes, pH
5. 0-7. o (0631 > •
Degree of substitution Weigh accurately l. O into a 500 ml
iodine flask with stopper, add 300 ml of 10% sodium chloride
solution and 25. o ml of sodium hydroxide eo. 1 mol/L) vs,
stopper the flask and allow to stand for 5 minutes. After
shaking, add 15 ml of hydrochloric acid (O. 1 mol/L) VS and
5 drops of m-cresol purple IS (Dissolve O. 1 g of m-cresol
purple in 13 ml of O. 01 mol/L sodium hydroxide, dilute to
100 ml v:ith v:ater ). Stopper and shake. If the solution is
violet, add hydrochloric acid eo. 1 mol/L) vs in l. o ml
portions until the solution becomes yellow. Titrate with
sodium hydroxide eo. 1 mol/L) vs until the colour turns to
violet. Calculate the degree of acid carboxymethyl
substitution CA) using the following equation:
The degree of acid carboxymethyl 1l50M
substitution A (7102 - 412M - 80C)
Where M is the num her of millimoles of sodium
hydroxide required for the neutralization of 1
g of Croscarmellose Sodium, on the dried
basis;
e is the percentage of residue on ignition of the
Croscarmellose Sodium as determined in the
test for residue on ignition.
Calculate the degree of sodium carboxymethyl substitution
(S) using the following equation:
The degree of sodium S= (162 58A) XC+
carboxymethyl substitution (7102- 80C)
The degree of substitution is the sum of A and S. It is not
less than O. 60 and not more than O. 85, calculated on the
dried basis.
Sodiwn chloride and sodiwn glycollate Sodium chloride
Weigh accurately 5. O g into a 250 ml beaker, add 50 ml of
water and 5 ml of 30 % hydrogen peroxide solution, and heat
on a water bath for 20 minutes, stirring constantly. Cool,
and add 100 ml of water and 10 ml of nitric acid; titrate with
silver nitrate (O. 05 mol/L) VS with stirring and determine the
endpoint using silver electrode potentiometric titration. Ea.ch ml
of silver ni trate eo. 05 mol/L) vs is equivalent to 2. 922 mg of
NaCl.
Sodium glycollate Protect from light througout the
Croscarmellose Sodium
procedure. Weigh accurately O. 5 g into a 100 ml beaker, add
5 ml of glacial acetic acid and 5 ml of water, and stir for 15
minutes. Add slowly 50 ml of acetone and 1 g of sodium
chloride, and stir for several minutes. Filter into a 100 ml
volumetric flask and dilute the filtrate with accetone to
volume and mix well as the test solution. Weigh accurately
O. 1 g of glycolic acid, previously dried in vacuum desiccator
at room temperature for 12 hours and into a 100 ml
volumetric flask, dissolve and dilute with water to volume.
Transfer l. O ml, 2. O ml, 3. O ml and 4. O ml of the above
solution, respectively, to separate 100 ml volumetric flasks,
add water to each flask to make 5 ml, and then add 5 ml of
glacial acetic acid, dilute with acetone to volume and mix as
the reference solution (1) (2) (3) and (4). Transfer 2. O ml
of the test solution and 2. O ml of each of the reference
solutions to separate 25 ml volumetric flasks. Heat the flasks
for 20 minutes in a water bath to eliminate acetone. Allowed
to cool and add 5. O ml of 2, 7-dihydroxynaphthalene solution
edissolve 1o mg of 2' 7-dihydroxynaphthalene in 100 ml of
sulphuric acid and allowed to stand until the colour of the
solution disappears, and use the solution within 2 days) to
each flask. Mix, add a further 15. O ml of 2, 7-
dihydroxynaphthalene solution and mix. Cover the mouths of
flasks with aluminium foil and heat on a water bath for 20
minutes. Cool and dilute to volume with sulfuric acid, and
mix. Prepare a blank solution using 2. O ml of a solution
containing 5 % each of water and glacial acetic acid in acetone
and operate in the same manner. Measure the absorbance of
each solution at 540 nm <0401 ) . Prepare a standard curve,
and calculate the percentage of sodium glycoate using the
following equation:
. 129w
Percentage of sodmm glycoate= (l -b)w X 100%
Where w is the weight, in mg, of glycolic acid in the
substance being examined;
l. 29 is a factor converting glycolic acid to sodium
glycolate;
b is the loss on drying in the substance being
examined;
W is the weight, in mg, of the substance being
examined.
The sum of the percentages of sodium chloride and
sodium glycollate is not more than O. 5 % , calculated on
dried basis.
Water-soluble substances Weigh accurately 1 O g, add 800
ml of water, and stir for 1 minute every 10 minutes during
the first 30 minutes. Allow to stand for 1 hour and
centrifuge if necessary. Decant 200 ml of the supernatant
liquid onto a fast filter paper in a vacuum filtration funnel,
apply vacuum, and collect 15 O ml of the filtra te. Pour the
filtrate into a tared 250 ml beaker, and weigh the filtrate
accurately. Evaporate to dryness and dry the residue at 105ºC
for 4 hours, weigh accurately and calculate the weight of the
residue. Calculate the content of water-soluble substances in
the specimen using the following expression:
Content of water- W 1 (800 + W 2 ) %
= X 100 o
soluble substances W2 W3 (l - b)
Where W1 is the weight, in g, of the residue;
W2 is the weight, in g, of the substance being
examined;
w3 is the weight, in g, of the filtrate;
b is loss on drying of the substance being
examined.
The percentage of water-soluble substances is not more than
10. o% on the dried basis.
Loss on drying When dried at 105ºC for 6 hours: loses not
 Crospovidone
more than 10. O% of its weight ( 0831 ) .
Residue on ignition 14. 0%-28. 0% ( 0841 >, using l. O g,
calculated on the dried basis. calculated on the dried basis.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), using the residue obtained in the test for
Residue on ignition: not more than O. 001%.
Category Pharmceutical excipients, disintegrant and filler, etc.
Storage Preserve in fightly closed containers.
Crospovidone
[9003-39-8]
Crospovidone is a water-insoluble synthetic cross-linked
homopolymer of N-vinyl-2-pyrrolidinone. The molecular
formula is CC6 H 9 NO)n, while "n" represents the average
number of 1-vinyl-2-pyrrolidinone. It contains not less than
11. 0% and not more than 12. 8% of nitrogen (N), calculated on
the anhydrous basis.
Description A white or almost white powder, almost
odourless, hygroscopic.
Practicauy insoluble in water, in ethanol, in chloroform and
in ethyl ether.
ldentification ( 1) Suspend 1 g in 10 ml of water, add
O. 1 ml of iodine TS and shake for 30 seconds. Add 1 ml of
starch IS and shake. No blue colour develops.
(2) The infrared absorption spectrum ( 0402) is concordant
with the reference spectrum of crospovidone CRS.
Acidity or alkalinity A suspension of l. O g in 100 ml of
water, pH 5. 0-8. O ( 0631).
Water-soluble substances Transfer 25. O g to a beaker, add
200 ml of water and stir for 1 hour. Transfer the solution to
a 250 ml volumetric flask, wash the beaker with water,
combine the washings to the volumetric flask and dilute to
volume. Allow the bulk of the solids to settle (not more than
24 hours). Centrifugalize the supernatant liquid for 30 minutes at
3500 rpm. Filter the supernatant through a O. 45 µm
membrane filter. Measure accurately 50 ml of the filtrate to a
beaker, previously dried to a constant weight, evaporate to
dryness, and dry at 105ºC for 3 hours. The weight of the
residue is not more than 50 mg (l. O%).
N-vinyl-2-pyrrolidinone Accurately suspend l. 25 g in
50 ml of water, stopper and shake for 1 hour. Allow the
bulk to settle and filter the supernatant. Use the successive
filtrate as the test solution. Dissolve an accurately weighed
quantity of N-vinyl-2-pyrrolidinone CRS in the mobile phase
to produce a solution containing about O. 25 µg of N-vinyl-2
pyrrolidinone per ml, as the reference solution. Dissolve a
quantity of N-vinyl-2-pyrrolidinone CRS and vinyl acetate
CRS in methanol to produce a mixture solution containing
1 µg of N-vinyl-2-pyrrolidinone of and 50 µg of vinyl acetate
per ml, as the system suitability test solution. Carry out the
method for high performance liquid chromatography ( 0512),
using a column packed with octadecylsilane bonded silica gel
and a mixture of acetonitrile--water ( 8 : 92) as the mobile
phase. The detection wavelength is 235 nm. lnject 20 µl of
the system suitability test solution into the column and
record the chromatogram. The resolution factor between
peaks of N-vinyl-2-pyrrolidinone and vinyl acetate complies
with the related requirements. Accurately inject 20 µl of the
test solution and the reference solution into the column and
record the chromatograms. Calculate the content of N-vinyl-
2-pyrrolidinone with respect to the peak area obtained in the
chromatogram by external method: not more than O. 001%.
Peroxides Operate at a temperature of between 20ºC and
25ºC. Suspend 2. O gin 50 ml of water. Divide the solution
into two portions: To one portian add 2 ml of titanium
trichloride solution in sulfuric acid ( carefully mix 20 ml of
15 % titanium trichloride solution with 13 ml of sulfuric acid
in an ice bath. Add sufficient strong hydrogen peroxide
solution until the solution is yellow. Heat until white fumes
evolved. Allow to cool. Repeat to dilute with water and
evaporate until a colourless solution is obtained. Dilute to
100 ml with water and filter), mix well, allow to stand for
30 minutes as the test solution. To the other portian add 2
ml of 13% (V/V) sulfuric acid solution, mix well, allow to
stand for 30 minutes, as the reference solution. The
absorbance of the test solution, measured at 405 nm is not
more than O. 35 ( equivalent to O. 04% H2 02), using the
reference solution as blank ( 0401 ) .
Water Not more than 5. O% ( 0832 >.
Residue on ignition Not more than O. 1 % ( 0841 ) , usmg
2. o g.
Heavy metals Carry out the limit test far heavy metals
( 0821 method 2) , using the residue obtained in Residue on
ignition, not more than O. 001%.
Asenic Transfer l. O g to a kjeldahl flask, add 5 ml of
sulfuric acid, ignite gently until the substance being
examined is thoroughly carbonized (add sulfuric acid with a
maximum of 10 ml if necessary), add strong hydrogen
peroxide solution dropwise until the solution is colourless.
Allow to cool, add cautiously 10 ml of water, heat again
until hydrogen peroxide is evoled, cool and add 5 ml of
hydrochloride acid anda volume of water. Carry out the limit
test for arsenic ( 0822) , not more than O. 0002 % .
Assay Weigh accurately about O. 2 g, carry out the method
for determination of nitrogen ( 0704 method 2).
Category Pharmaceutical excipients, disintegrant and filling
agent, etc.
Storage Preserve in tightly closed containers, protected
from light and stored in a cool place.
Cyclomethicone
(Cz H60Si)n n=4-6
[69430-24-6]
Cyclomethicone is methyl cyclosiloxane with repeating unit
[ - CCH3)2SiO-Jn, n=4, 5 or 6, ora mixture of them.
( Cz H 6OSi ) n contains not less than 98. O% of total
cyclomethicone 4, cyclomethicone 5 and cyclomethicone 6. It
contains not less than 95. O% and no more than 105. O% of
the labeled amount of cyclomethicone unit.

Description A transparent, colourless oily liquid.
ldentification ( 1) The retention time of the each principal
peak of the test solution in the chromatogram obtained in the
assay is identical with that of the principal peaks in the
chromatogram of reference solution.
(2) The infrared absorption spectrum ( 0402) is concordant
with the spectrum of cyclomethicone CRS between the range
of 4000-1000 cm- 1 •
Acid content Accurately weigh 30 g, add 60 ml of freshly
boiled and cooled water, reflux for 30 miuntes, cool to
room temperature, wash condenser inner wall with small
amout of freshly boiled and cooled water, transfer the final
solution to a separating funnel, allow it to stand until both
layers are clear. Separate the aqueous layer and add 3 drops
of phenolphthalein IS, titrate with sodium hydroxide
(0. 01 mol/L) VS. Each ml of sodium hydroxide (0. 01 mol/L)
VS is equivalent to O. 365 mg of hydrochloric acid. The acid
content of cyclomethicone is not more than O. 001 % of HCl.
Non-volatile residue Accurately weigh 2. O g in an
evaporating dish previously dried at 150ºC for 2 hours,
evaporate to dryness on water bath, and then dry at 105ºC
for 2 hours. The residue is not more than 3. O mg (O. 15 %) .
Assay Carry out the method for gas chromatography
( 0521 ) , using a column packed with dimethylpolysiloxane
as the stationary phase, the initial temperature of the column
is 120ºC, maintain the temperature for 2 minutes and raise
the temperature to l 90ºC at a rate of lOºC per minute, the
temperature of the injection port is maintained at 260ºC and
that of the FID detector is maintained at 280ºC.
Procedure Dissolve a quantity of the substance being examined,
accurately weighed, in absolute ethanol to produce a solution
containing 1 mg per ml as the test solution. lnject 1 µl into the
column and record the chromatogram. Repeat the operation, using
cyclomethicone 4 CRS, cyclomethicone 5 CRS and cyclomethicone
6 CRS instead of the substance being examined, calculate the
content of cyclomethicone with respect to the peak area obtained in
the chromatogram by the extemal standard method.
Category Pharmaceutical excipients, waterproof agent.
Storage Preserve in tightly closed containers.
Dextrates
[39404-33-6]
Dextrates is a purified mixture of saccharides resulting from
the controlled enzymatic hydrolysis of starch. It is either
anhydrous or hydrated. lts dextrose equivalent is not less
than 93. O% and not more than 99. O%, calculated on the
dried basis.
Description White, free-flowing, porous crystalline spherical
granules; odourless; taste sweet.
Freely soluble in water, soluble in dilute acid solution,
insoluble in ethanol and propylene glycol.
Acidity 20% aqueous solution, pH 3. 8-5. 8 (0631 ).
Loss on drying When dried at 105ºC for 16 hours, the
anhydrous form loses not more than 2. O% of its weight; the
hydrated form loses between 7. 8%-9. 2% of its weight
(0831 >.
Residue on ignition Not more than O. 1 % <0841 ) , using
2. o g.
Dextrin
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2) , using the residue obtained in the test for
Residue on ignition: not more than O. 0005%.
Assay Dextrose equivalent Dissolve about 5 g, accurately
weighed, with a quantity of hot water in a 500 ml volumetric
flask, cool, dilute to volume with water and mix well as the
test solution. Dissolve an accurately weighed quantity of
dextrose CRS in water, to produce a solution of about 10 mg
per ml as the reference solution. Accurately transfer 25 ml of
alkaline cupric tartrate TS to a conical flask, heat to boiling
and titrate with the reference solution approach to the
anticipated endpoint, again heat the conical flask, with
swirling, boíl moderately for 2 minutes, add 2 drops of 1%
methylene blue solution, and titrate with the reference
solution until the hule colour disappear. Complete the titration
within 3 minutes. Accurately transfer 25 ml of alkaline cupric
tartrate TS to another conical flask, similarly titrate with
test solution. Calculate the dextrose equivalent with the
following formula.
CCs/Cu) X CVs/Vu) XlOO%
In which Cu is the concentration of the test solution,
mg/ml;
Cs is the concentration of the reference solution,
mg/ml;
V 5 and Vu are the titrated volumes of the reference
solution and the test solution respectively, ml.
Category Pharmaceutical excipients, sweetening agent.
Storage Preserve in tightly closed containers, in a dry place
at 8-15ºC.
Dextrin
[9004-53-9]
Dextrin is starch, or partially hydrolyzed starch, modified by
heating in a dry state.
Description A white or almost white amorphous powder,
odourless, taste, slightly sweet.
Freely soluble in boiling water, insoluble in ethanol and in
ether.
Identification To 1 ml of 10 % aqueous solution add 1 drop
of iodine TS, a violet-red colour is produced.
Acidity Dissolve 5. O g in 50 ml of water by heating, cool,
add 2 drops of phenolphthalein IS and 2. O ml of sodium
hydroxide (0. 1 mol/L) VS, a pink colour is produced.
Reducing sugars To 2. O g add 100 ml of water, shake for
5 minutes, allow to stand, filter. To 50 ml of filtrate add 50
ml of alkaline cupric tartrate TS and boíl for 3 minutes.
Filter with a sintered glass crucible previously dried to
constant weight at 105ºC. Wash the residue successively with
water, ethanol and ether, then dry at 105ºC for 2 hours. The
residual cuprous oxide is not more than O. 20 g.
Loss on drying When dried to constant weight at 105ºC,
loses not more than 10. O% of its weight ( 0831 ) .
Residue on ignition Not more than O. 5% ( 0841), using
l. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), using the residue obtained in the test for
residue on ignition: not more than O. 002%.
Iron lgnite and incinerate 2. O g. Add 1 ml of hydrochloric
acid and 3 drops of nitric acid to the residue and evaporate on
 Diammonium Hydrogen Phosphate
a water bath to almost dryness, cool. Dissolve the residue in
1 ml of hydrochloric acid, transfer the solution with water to
a 50 ml volumetric flask and dilute to volume, mix well.
Measure accurately 10 ml and carry out the limit test for iron
( 0807). Any colour produced is not more intense than that of
a reference solution using 2. O ml of iron standard solution
(O. 005%).
Microbial limit Comply with the requirements for
microbiological limit ( 1105 and 1106), the total aerobic
bacteria count is not more than 1000 cfu per g and the total
combined yeast/mold count is not more than 100 cfu per g of
the substance being examined. Escherichia coli should not be
detected.
Category Pharmaceutical excipients, filling agent and
binder, etc.
Storage Preserve in well closed containers, stored in a dry
place.
Diammonium Hydrogen Phosphate
(NH4 )z HP04 132. 06
[7783-28-0]
Diammonium hydrogen phosphate is obtained by neutrallzation
reaction of ammonium carbonate or liquid ammonia with
phosphoric acid, and processed by concentration,
crystallization and drying. It contains not iess than 96. O%
and not more than 102. 0% of (NH4)2HPQ4.
Description Colourless or white crystals or crystalline powder.
Freely solution in water, insolution in acetone and in ethanol.
ldentification The aqueous solution yields the reactions
characteristic of ammonium salts and phosphate ( 0301 ) .
Alkalinity Dissolve O. 10 g in 10 ml of water, pH 7. 6-8. 2
<0631).
Water-insoluble substances Dissolve 20. O gin 100 ml of hot
water. Filter through a tared No. 4 sintered glass crucible,
previously dried to constant weight at 105ºC . Wash the
residue with 200 ml of hot water in 10 portions, the
substance of residue, when dry at 105ºC for 2 hours, not
more than 1 mg (O. 005%).
Chloride Carry out the limit test for chlorides ( 0801 ) ,
using l. O g. Any opalescence produced is not more pronounced
than that of a reference solution prepared by using 4. O ml of
sodium chloride standard solution (O. 004%).
Sulfate Carry out the limit test for sulfate ( 0802) , using
O. 20 g. Any opalescence produced is not more pronounced
than that of a reference solutionprepared by using 2. O ml of
potassium sulfate standard solution (O. 1 %) .
Iron Dissolve 1. O g with 15 ml of water, adjust pH to
2. O with hydrochloric acid solution Cl -2), add 1 ml of
2 % ascorbic acid solution, 5 ml of acetate-sodium aceta te
BS ( pH 4. 5 ) and 1 ml of O. 2 % o-phenanthroline
solution, dilute with water to 5 O ml and mix well, allow to
stand for 15 minutes. Any colour produced is not more
intense than that of the reference solution prepared by using
2. O ml of iron standard solution in the same manner
( 0807) (O. 002%).
Lead Take two portions, each of O. 20 g, place each portion
in a 50 ml volumetric flask, separately. To one portion
dissolve and dilute with nitric acid solution ( 1 - 100) to
volume, mix well as the test solution. To another portian
add accurately 2 ml of reference lead solution ( measure
accurately a volume of lead standard solution, dilute with
nitric acid solution Cl-100) to produce a solution of O. 5 µg
Pb per mD, dilute with nitric acid solution ( 1-100) to
volume, mix well as the reference solution. Carry out the
method for atomic absorption spectrophotometry ( 0406
method 2 ) , using furnace atomizer and measure the
absorbance of each of the two solutions at 283. 3 nm. Use the
absorbance of the test solution as a, and that of the reference
solution as b; a is not greater than (b-a) (O. 0005%).
Arsenic Dissolve O. 67 g in 23 ml of water, and then add
5 ml of hydrochloric acid. Carry out the limit test for arsenic
( 0822 method 1): not more than O. 0003%.
Assay Dissolve about O. 6 g, accurately weighed, in 40 ml
of freshly boiled and cooled water. Carry out the method for
potentiometric titration ( 0701 ) , titrate with sulfuric acid
(O. 1 mol/L) VS. Each ml of sulfuric acid (O. 1 mol/L) VS
is equivalent to 26. 42 mg of (NH 4) 2HP04.
Category Pharmaceutical excipients, buffer and effervescent
agent.
Storage Preserve in tightly closed containers.
Dilute Acetic Acid
Dilute acetic acid is prepared by diluting appropriate amount
of acetic acid or glacial acetic acid with water. It contains not
less than 5. 7% (g/g) and not more than 6. 3% (g/g) of
acetic acid ( C2 H4 02 ) .
Description A colourless, clear liquid; odour, strong and
characteristic; taste, extremely sour.
Miscible with water, ethanol and glycerol.
ldentification ( 1) It changes the blue litmus TP to red.
(2) When neutralized with sodium hydroxide TS, yields the
reactions characteristic of aceta tes ( 0301 ) .
Chloride Carry out the limit test for chlorides ( 0301 ) ,
using l. O ml. Any opalescence produced is not more
pronounced than that of a reference solution prepared by
using 7. O ml of sodium chloride standard solution (O. 007 % ) .
Sulfate Dilute 2. 5 ml with water to 20 ml. Carry out the
limit test for sulfates ( 0802), using 5. O ml of the resulting
solution. Any opalescence produced is not more pronounced
than that of a reference solution prepared by using l. 5 ml of
potassium sulfate standard solution (O. 024 % ) .
Formic acid and readily oxidizable substances Mix 5. O ml
with 6 ml of sulfuric acid and cool to 20ºC. Add O. 4 ml of
potassium dichromate (0. 016 67 mol/L) VS, allow to stand
for 1 minutes. Add 25 ml of water, 1 ml of potassium iodide
TS and 1 ml of starch IS, titrate with sodium thiosulfate
(O. 1 mol/L) VS. Perform a blank determination and make
any necessary correction. Not more than O. 2 ml of sodium
thiosulfate (O. 1 mol/L) VS is required to change the colour
of the solution.
Potassium permanganate reducing substances Mix 25 ml with
O. 2 ml of potassium permanganate (O. 02 mol/L) VS, allow
to stand for 1 minute and the pink colour can not completely
disappear.
Aldehydes Distil 75 ml. Add 10 ml of O. 5% mercury
chloride solution to the first 5 ml of the distillate, make

alkaline with 5 mol/L sodium hydroxide solution, allow to
stand far 5 minutes and make acidic with 1 mol/L sulfuric
acid solution: no turbidity is produced.
Non-volatile matter Place 20 ml in an evaporating dish,
previously dried to constant weight at 105ºC, evaporate on a
water bath to dryness and dry at 105ºC to constant weight.
The residue is not more than 1 mg.
Heavy metals Mix 10 ml with 2 ml of acetate BS (pH 3. 5),
dilute with water to 25 ml. Carry out the limit test far heavy
metals (0821 method 1): not more than O. 0001%.
Assay Accurately weigh 20 g in a conical flask, dilute with
30 ml of freshly boiled and cooled water, add 1-3 drops of
phenolphthalein IS and titrate with sodium hydroxide
(1 mol/L) VS. Each ml of sodium hydroxide (1 mol/L) VS
is equivalent to 60. 05 mg of C2 H4 02.
Category Pharmaceutical excipients, pH regulator and buffering
agent
Storage Preserve in tightly closed glass bottles.
Dilute Phosphoric Acid
H3P04 98. 00
Dilute Phosphoric Acid is prepared by diluting 69 ml of
phosphoric acid with water to make 1000 ml. lt contains not
less than 9. 5% and not more than 10. 5% of H3 P04
Cg/mD.
Description A clear, colourless liquid; strongly acidic.
Identification Yields the reactions characteristic of phosphates
I l'V)l"\1 \
\V.JV.l /.
Clarity and colour of solution Dilute 86 g with 150 ml of
water. The solution is clear and colourless ( 0901 and
0902).
Substances precipitated with ammonia To 15 ml of solution
prepared far the test far clarity and colour of solution add
12 ml of ammonia TS. No opalescence is produced.
Hypophosphorous acid and phosphorous acid To 15 ml of
solution prepared far the test far clarity and colour of
solution add 6 ml of silver nitrate TS, heat on a water-bath
far 5 minutes. No opalescence is produced.
Alkaline phosphate Evaporate 20 ml to 5 g on a water bath,
cool. To 2 ml add 6 ml of diethyl ether and 2 ml of ethyl
alcohol, no opalescence is produced.
Nitrate To 5 ml add O. 1 ml of indigo carmine TS and 5 ml
of sulfuric acid. The blue colour of the solution should not
disappear within 1 minute.
Chloride Carry out the limit test far chlorides ( 0801 ) ,
using 10 ml. Any opalescence produced is not more
pronounced than that of a reference solution using 6. O ml of
sodium chloride standard solution (O. 0006%).
Sulfate Carry out the limit test far sulfates ( 0802) , using
20 ml. Any opalescence produced is not more pronounced
than that of a reference solution using 2. O ml of potassium
sulfate standard solution (O. 001%).
Iron Carry out the limit test far iron ( 0807), using 10 ml.
Any colour produced is not more intense than that of a
reference solution using 6. O ml of iron standard solution
(0. 006%).
Diluted Hydrochloric Acid
Heavy metals To 20 ml add 4 ml of ammonia TS and
sufficient water to produce 25 ml. Carry out the limit test far
heavy metals ( 0821 method 1): not more than O. 0001%.
Arsenic To 10 ml add 5 ml of hydrochloric acid and 13 ml of
water. Carry out the limit test far arsenic ( 0822 method 2):
no more than O. 00002 % .
Assay Measure accurately 10 ml, dilute with 50 ml of
water, add O. 5 ml of thymolphthalein IS and titrate with
sodium hydroxide ( 1 mol/L) VS. Each ml of sodium
hydroxide ( 1 mol/L) VS is equivalent to 49. 00 mg of
H3PQ4.
Category Pharmaceutical excipient, pH regulator.
Storage Preserve in tightly closed containers.
Diluted Hydrochloric Acid
Diluted Hydrochloric Acid is prepared by diluting 234 ml of
hydrochloric acid with water to make 1000 ml. lt contains
not less than 9. 5% and not more than 10. 5% of HCL
Description A clear, colourless liquid; strongly acidic.
ldentification ( 1 ) Yields the reactions characteristic of
chlorides ( 0301 ) .
(2) Turns the colour of blue litmus TP to red.
Free chlorine or bromine To 20 ml, add O. 2 ml of zinc
potassium iodide IS. No blue colour is produced within 10
minutes.
Bromide or iodide To 10 ml add 1 ml of chlorofarm and
1 drop of O. 002 mol/L potassium permanganate solution,
shakc, thc chloroform layer is colourlcss.
Sulfate To 100 ml, add 2 drops of sodium carbonate TS,
evaporate to dryness on a water bath. To the residue, add
20 ml of water and carry out the limit test far sulfates
( 0802) , any colour produced is not more intense than that of
a reference solution using l. 25 ml of potassium sulfate
standard solution (0. 000125%).
Sulfite Dilute 15 ml with 40 ml of freshly boiled and cooled
water, and add to the solution prepared with 50 ml of freshly
boiled and cooled water, l. O g of potassium iodide, O. 15 ml
of (O. 005 mol/L) iodine solution and l. 5 ml of starch IS.
The blue colour of the solution should not disappear
completely.
Residue on ignition To 20 ml, add 2 drops of sulfuric acid,
evaporate to dryness. Carry out the test far residue on
ignition ( 0841 ) : the residue of the substance is not more
than 2 mg (0. 01%).
Iron Evaporate 100 ml to dryness on a water bath. To the
residue, add 25 ml of water, carry out the limit test far iron
( 0807). Any colour produced is not more intense than that of
a reference solution using 3. O ml of iron standard solution
(0. 00003%).
Heavy metals Evaporate 10 ml to dryness on a water bath,
add 2 ml of acetate BS (pH 3. 5) and a quantity of water to
produce a solution of 25 ml. Carry out the limit test far
heavy metals ( 0821 method 1): not more than O. 0002%.
Arsenic Dilute 2. O ml with 22 ml of water, add 5 ml of
hydrochloric acid. Carry out the limit test far arsenic ( 0822
method 1) (0. 0001%).
Assay Accurately measure 10 ml, dilute with 20 ml of
 ::¡~-· Dimethicone
'··<·

water, add 2 drops of methyl red IS and titrate with
sodium hydroxide ( 1 mol/L) VS. Each ml of sodium
hydroxide ( 1 mol/L) VS is equivalent to 36. 46 mg of
HCL
Category Pharmaceutical excipient, pH regulator.
Storage Preserve in tightly closed glass bottles.
Dimethicone
[ 9006-65-9 J
Dimethicone is a linear polymer of dimethylsiloxane. It
contains not less than 97. 0% and not more than 103. 0% of
polydimethylsiloxane. It has different viscosity due to the
different degree of polymerization and is classified according
to viscosity into ten types: 20, 50, 100, 200, 350, 500,
750, 1000, 12500 and 30000.
solution is not greater than O. 2.
Loss on drying When dried at 150ºC far 2 hours, it loses not
more than the percentage of its weight specified in the
accompanying table ( 0831 ) .
Heavy metals Mix · l. O g with chlorofarm in a Nessler
cylinder and dilute to 20 ml with the same solvent. Add l. O
ml of a freshly prepared O. 002% solution of dithizone in
chlorofarm, O. 5 ml of water and O. 5 ml of a mixture of 1
volume of ammonia TS and 9 volumes of a O. 2%
hydroxylamine hydrochloride solution, use as the tast
solution. At the same time, prepare a reference solution as
fallows: to 20 ml of chlorofarm add l. O ml of a freshly
prepared O. 002% solution of dithizone in chlorofarm, O. 5 ml
of lead standard solution and O. 5 ml of a mixture of 1 volume
of ammonia TS and 9 volumes of a O. 2% hydroxylamine
hydrochloride solution. Immediately shake each solution
vigorously far 1 minute. Any red colour in the test solution is not
more intense than that in the reference solution (O. 0005 % ).
Arsenic Mix l. O g with l. O g of calcium hydroxide, add a
little water and mix well. After dryness, heat gently until it is
thoroughly charred, and then ignite at 500-600ºC until the
incineration is completed. Dissolve the cooled residue in 5 ml of
hydrochloride acid and 23 ml of water. Carry out the limit test far
arsenic <0822 method 1 ) : not more than O. 0002 % .

Description A clear, colourless oily liquid; odourless or Assay Carry out the method far attenuated total infrared
almost odourless. spectrophotometry, record the infrared spectra of sample and
Very soluble in chlorofarm, in ether, in toluene, in ethyl reference over the range of 4000-700 cm- 1 , determine the
acetate and in methyl ethyl ketone, insoluble in water and in absorbance of the peak ( based on the height ) in each
ethanol. spectrum at about 1259 cm- 1 , calculate the content of
polydimethylsiloxane in the dimethicone as fallows:
Relative density Within the limits specified in the accompanying The content of polydimethylsiloxane ( %) = 100 (Au! A.)
table, at 25ºC <0601 ) . (D./Du)
Refractive index Within the limits specified m the Where Au is absorbance of sample;
accompanying table, at 25ºC <0622). A. is absorbance of reference;
Du is relative density of sample at 25ºC;
Viscosity Within the limits specified in the accompanying
table, at 25ºC <0633 method 1, using a capillary tube with
D. is relative density of reference at 25ºC.
2 mm in internal diameter; using method 2 if the nominal Category Pharmaceutical excipient, antifaaming agent and
viscosity is 1000 mm 2 /sor greater). lubricants.
ldentification (1) Ignite gently O. 5 g with O. 5 ml each of Storage Preserve in tightly closed containers.
sulfuric acid and nitric acid in a crucible. A white fibrous Table Requirements far viscosity, relative density,
matter is farmed and finally a white residue is produced. refractive index and loss on drying.
(2) Heat O. 5 g in a test-tube over a small flame until white
fumes begin to appear. lnvert the tube over another tube Nominal Loss on
Viscosity Relative Refractive
containing 1 ml of a O. 1 % solution of chromotropic acid viscosity drying
(mm2 /s) density index
sodium salt in sulfuric acid so that the fumes reach the (mm2 /s) (%)
solution. Shake the 2nd tube far about 10 seconds and heat on
a water bath far 5 minutes. A violet colour is produced. 20 18-22
o. 946- l. 3980-
20.0
(3) The infrared absorption spectrum ( 0402), is concordant 0.954 l. 4020
with the reference spectrum (IR Album No. 10).
50
47. 5- o. 955- l. 4005-
2.0
Acidity or alkalinity Mix 5 ml each of ethanol and 52.5 0.965 1.4045
chlorofarm, add 1 drop of phenolphthalein IS, and sodium 95- o. 962- l. 4005-
hydroxide (O. 02 mol/L) VS dropwise until a slight pink 100 0.3
105 o. 970 1.4045
colour is produced. Add l. O g of the substance being
examined and mix well. If the solution is colourless, add 200
190- o. 964- l. 4013-
0.3
O. 15 ml of sodium hydroxide (O. 02 mol/L) VS, a pink colour is 220 0.972 l. 4053
produced; if the colour of the solution is pink, add O. 15 ml of
sulfuric acid (0. 01 mol/L) VS, the colour disappears. 350
332. 5- o. 965- l. 4013-
0.3
367.5 0.973 l. 4053
Mineral oils The fluorescence examined in ultraviolet light at
365 nm is not more intense than that of a solution containing 500
475- o. 967- l. 4013-
0.3
O. 1 µg of quinine sulfate per ml in O. 005 mol/L sulfuric acid. 525 o. 975 l. 4053

Phenylated compounds Dissolve 5. O g with shaking in 10 ml 750

712. 5- o. 967- l. 4013-
0.3
of cyclohexane in a test-tube with stopper. At wavelengths 787.5 o. 975 l. 4053
from 250 nm to 270 nm ( 0401 ) , the absorbance of the
 Dipotassium Hydrogen Phosphate 1•

continued solution (O. 025 % diphenyl methane solution in acetone) to

produce a solution containing 50 % of the substance being
Nominal Loss on
Viscosity Relative Refractive examined as the test solution. Dilute a volume of the test
viscosity drying
(mm2 /s) density index solution with the interna! standard solution to produce a
(mm2 /s) C%) solution containing O. 050 % of the substance being examined

1000
950- o. 967- l. 4013-
0.3
as the reference solution. Dissolve a quantity of dimethyl
1050 0. 975 l. 4053 sulfone CRS, accurately weighed, in the interna} standard
solution to produce a solution containing O. 050 % dimethyl
11875- l. 4015- sulfone, as dimethyl sulfone reference solution. Carry out
12500 2.0
13125 l. 4055 the method for gas chromatography ( 0521), using a column
27000- o. 969- l. 4010- packed with 10 % polyethyleneglycol 20 M as the stationary
30000 2.0 phase. The column temperature is maintained at 150ºC,
33000 0. 977 l. 4100
using FID detector. The number of theoretical plates of the
column is not less than 1500, calculated with reference to the
peak of dimethyl sulfone. The resolution factor between the
peaks of dimethyl sulfone and interna! standard is more than
Dimethyl Sulfoxide 2. O. lnject separately 2 µl of the test solution, the reference
solution and dimethyl sulfone reference solution into the
column, record the chromatograms. The ratio for the peak
area of dimethyl sulfone to that of diphenyl methane in the
chromatogram obtained with the test solution is not more
than the ratio of the peak area of dimethyl sulfone to that of
C2 Hs OS 78. 13 diphenyl methane in the chromatogram obtained with
[67-68-5] dimethyl sulfone reference solution (O. 1 %) . The ratio for
Dimethyl Sulfoxide is prepared by air oxidation of dimethyl the sum of the areas of all impurity peaks other than the
sulfide in the presence of nitric oxide, it may be obtained principal peak and interna! standard peak to that of diphenyl
from by-product in the manufacture of paper pulp. methane in the chromatogram obtained with the test solution
It contains not less than 99. 5 % of Cz Hs OS, calculated on is not more than the ratio for the peak area of dimethyl
the anhydrous basis. sulfoxide to that of diphenyl methane in the chromatogram
Description A colourless liquid; odourless or almost obtained with the reference solution (O. 1 %) .
odourless, hygroscopic. Nonvolatile residue Transfer 100 g, accurately weighed, to
Miscible with water, ethanol and ether, insoluble in alkane. an evaporating dish previously dried to constant weight at
Congealing point 17. 0-18. 3ºC ( 0613). 105ºC, evaporate to dryness slowly ( not boiling) on an
electric heating plate in a fume hood, dry at 105ºC for 3
Refractive index l. 478-1. 479 <0622).
hours and weigh the dish. The residue is not more than
Relative density l. 095-1. 105 ( 0601). 0.01%.
ldentification (1) Take 5 ml in a test-tube, add 50 mg of Assay Calculate the content of the substance being examined
nickel chloride and shake to dissolve, a yellowish-green as follows:
colour is produced, warm on a water bath at 50ºC, a green or Assay= 1- nonvolatile residue- related substances X lOO%
a greenish-blue colour is produced, allow to cool, a 1-water
yellowish-green colour is produced.
Category Pharmaceutical excipients, absorpticm enhancer
(2) The infrared absorption spectrum ( 0402) is concordant
and solvent (for externa} use only).
with that of Dimethyl Sulfoxide CRS.
Acidity Dissolve 50. O gin a 100 ml of water, add O. 1 ml of Storage Preserve in well closed containers, stored in a cool
phenolphthalein IS, not more than 5. O ml of sodium and dry place.
hydroxide (0. 01 mol/L) VS is required to change the colour
of solution to pink.
Absorbance To a volume of the substance being examined,
purge with dry nitrogen for 15 minutes, and then measure Dipotassium Hydrogen Phosphate
the absorbance at 275 nm immediately ( 0401 ) , using water
as the blank solution, the absorbance is not more than O. 30. KzHP04 174.18
The ratio of the absorbance at 285 nm and at 295 nm to that [7758-11-4]
of at 275 nm are not more than O. 65 and O. 45, respectively. Dipotassium hydrogen phosphate contains not less than
The substance being examined shows no absorption rnaximum 99. O% of Kz HP04, calculated on the dried basis.
between 270 and 350 nm.
Description A colourless or white crystalline powder or
Suhstances darkened by potassium hydroxide T ransfer
granule or mass; odourless; hygroscopic.
accurately 25 ml into a 50 ml volumetric flask, add O. 5 ml of
Very soluble in water; practically insoluble in ethanol.
water and l. O g of potassium hydroxide, stopper and heat on
a water bath for 20 minutes, allow to cool, the absorbance of the ldentification Yields the reactions characteristic of potassium
resulting solution at 350 nm, does not exceed O. 023 ( 0401 ) , salts and phosphates ( 0301 ) .
using water as the blank solution. Alkalinity Dissolve l. O g in 20 ml of water, pH 8. 5-9. 6
Water Not more than O. 2 % ( 0832 method 1 ( 1 ) ) . <0631 >.
Related substances Dilute an accurately weighed quantity of Clarity and colour of solution Dissolve l. O g in 10 ml of
the substance being examined with the interna! standard water, the solution is clear and colourless ( 0901 and 0902).
 Dipotassium Hydrogen Phosphate Trihydrate
Chloride Carry out the limit test far chlorides ( 0801 ) ,
using l. 5 g. Any opalescence produced is not more
pronounced than that of a reference solution using 6. O ml of
sodium chloride standard solution (0. 004%).
Sulfate Dissolved 2. O g in about 40 ml of water, adjust to
pH< 6 with hydrochloric acid, Carry out the limit test far
sulfates ( 0802 ) . Any opalescence produced is not more
pronounced than that of a reference solution using 3. 4 ml of
potassium sulfate standard solution (O. 017 % ) .
Carbonate Dissolve 2. O g in 10 ml of water, boil and cool,
add 2 ml of hydrochloric acid, no effervescence is produced.
Condensed phosphate Dissolve 2. O g in a 100 ml volumetric
flask, dilute to volume with water and mix well. Transfer
5. O ml of the solution to a Nessler cylinder, add l. O ml of
dilute acetic acid, 5. O ml of the acetic acid-sodium acetate
solution ( mix 17 ml of 1 mol/L sodium hydroxide solution
and 40 ml of dilute acetic acid, dilute with water to 100 mD,
and sufficient water to produce 15 ml, add 2 ml of barium
chloride TS and mix well. Allow to stand at 25 ± 2ºC far
15 minutes, no opalescence is produced.
Water-insoluble substances Dissolve 10. O g in 100 ml of hot
water, filter through a tared No. 4 sintered glass crucible,
previously dried to constant weight, and wash the residue
with 200 ml of hot water in 10 portions. The residue, when
dry at 105ºC for 2 hours, weighs not more than 2 mg (0. 02%).
Reducing substances Dissolve 5. O g in freshly boiled and
cooled water, and dilute to 50 ml with the water. Transfer
5. O ml of the solution. add 5 ml of dilute sulfuric acid and
O. 25 ml of potassium permanganate (O. 02 mol/U VS and
heat on a water bath far 5 minutes. The solution remains a
purplish red colour.
Loss on drying When dried to constant weight at 130ºC,
loses not more than 2. O% of its weight ( 0831 ) .
Iron Dissolve l. O g in 20 ml of water, add 1 ml of
hydrochloric acid solution 0-2) and 2mlof10% sulfosalicylic
acid solution, mix well, add 5 ml of ammonia TS and mix well.
Any colour produced is not more intense than that of a reference
solution prepared in the same manner, using l. O ml of iron
standard solution ( 0807) (0. 001%).
Sodium Dissolve l. 00 g with water in a 100 ml volumetric
flask, dilute to volume with water and mix well as the test
stock solution. Accurately measure 5 ml of the above
solution to a 100 ml volumetric flask, dilute to volume with
water and mix well, as the test solution. Dissolve O. 5084 g
of sodium chloride, previously dried to the constant weight at
100-105ºC far 3 hours and accurately weighed, in a 100 ml
volumetric flask, dilute to volume with water, mix well, as
the standard solution I . Accurately measure 2. 5 ml of the
standard solution I to a 100 ml volumetric flask, dilute to
volume with water and mix well, as the standard solution 11
(50 µg/ml, Na+); accurately measure 5 ml of the test stock
solution and 1 ml of the standard solution 11 to a 100 ml
volumetric flask, dilute to volume with water and mix well,
as the standard solution. Carry out the method of atomic
absorption spectrophotometry ( 0406 method 2) , use flame
atomizer to measure the absorbance of the standard solution
and the test solution at 589 nm, the absorbance of the
standard solution as a , and the absorbance of the test
solution as b. b is less than (a-b): not more than O. 1%.
Heavy metals Dissolve 2. O gin 15 ml of water, and adjust to
pH 4 with hydrochloric acid, add 2 ml of acetate BS (pH 3. 5)
and sufficient water to produce 25 ml. Carry out the limit test
for heavy metals ( 0821 method 1): not more than O. 001%.
Arsenic Dissolve l. O g in 23 ml of water, add 5 ml of
hydrochloric acid. Carry out the limit test far arsenic ( 0822
method 1 ) : not more than O. 0002 % .
Baterial endotoxin ( For lnjection) Carry out the test far
baterial endotoxin ( 1143): not more than l. 1 EU per mg of
dipotassium hydrogen phosphate.
Sterility (For sterile product without terminal sterilization
process) Complies with the test far sterility (1101).
Assay Dissolve about 3 g, accurately weighted, in 50 ml of
freshly boiled and cooled water. Carry out the method far
potentiometric titration ( 0701 ) , titrate with sulfuric acid
(O. 5 mol/U VS. Each ml of sulfuric acid (O. 5 mol/U VS
is equivalent to 17 4. 2 mg of Kz HP04.
Category Pharmaceutical excipients, pH regulator and buffer,
etc.
Storage Preserve in tightly closed containers, stored in a dry
place.
Dipotassium Hydrogen Phosphate
Trihydrate
KzHP04•3H20 228. 22
[16788-57-1]
Dipotassium hydrogen phosphate trihydrate contains not less
than 99. O% of K2 HP04 , calculated on the dried basis.
Description Colourless or white crystal or masses;
hygroscopic.
Very soluble in water; practically insoluble in ethanol.
ldentification Yields the reactions characteristic of potassium
salts and phosphates ( 0301).
Alkalinity Dissolve l. O g in 20 ml of water, pH 8. 9-9. 4
<0631 >.
Clarity and colour of solution Dissolve l. O g in 10 ml of
water, the solution is clear and colourless ( 0901 and 0902).
Chloride Carry out the limit test far chlorides ( 0801 ) ,
using 2. 5 g. Any opalescence produced is not more
pronounced than that of a reference solution using 5. O ml of
sodium chloride standard solution (0. 002%).
Sulfate Dissolved 2. O g in about 40 ml of water, adjust to
pH<6 with hydrochloric acid, Carry out the limit test far
sulfates ( 0802 ) . Any opalescence produced is not more
pronounced than that of a reference solution using 2. O ml of
potassium sulfate standard solution (0. 01%).
Carbonate Dissolve 2. O gin 10 ml of water, boil and allow
to cool, add 2 ml of hydrochloric acid, no effervescence is
produced.
Condensed phosphate Dissolve 2. O g in water in a 100 ml
volumetric flask, dilute to volume and mix well. Transfer
5. O ml of the solution to a Nessler cylinder, add l. O ml of
dilute acetic acid, 5. O ml of the acetic acid-sodium acetate
solution ( mix 17 ml of 1 mol/L sodium hydroxide solution
and 40 ml of dilute acetic acid, dilute with water to 100 mD,
and sufficient water to produce 15 ml, add 2 ml of barium
chloride TS and mix well. Allow to stand at 25 ± 2ºC far
15 minutes, no opalescence is produced.
Water-insoluble substances Dissolve 10. O gin 100 ml of hot
water, filter with a tared No. 4 sintered glass crucible,
previously dried to constant weight, and wash the residue

with 200 ml of hot water in 10 portions. The residue, when
dry at 105ºC for 2 hours, weighs not more than 1 mg
(0. 01%).
Reducing substances Dissolve 5. O g in freshly boiled and
cooled water, and dilute to 50 ml with the water. Transfer
5. O ml of the solution, add 5 ml of dilute sulfuric acid and
O. 25 ml of potassium permanganate (O. 02 mol/L) VS and
heat on a water bath for 5 minutes. The solution remains a
purplish red colour.
Loss on drying When dried to constant weight at 180ºC,
loses not less than 22. O% and not more than 26. O% of its
weight <0831 ) .
Iron Dissolve l. O g in 20 ml of water, add 1 ml of
hydrochloric acid solution ( 1 - 2 ) and 2 ml of 10 %
sulfosalicylic acid solution, mix well, add 5 ml of ammonia
TS and mix well. Any colour produced is not more intense
than that of a reference solution prepared in the same
manner, using l. O ml of iron standard solution < 0807)
(O. 001%).
Sodium Dissolve l. 00 gin a 100 ml volumetric flask, dilute
to volume with water and mix well as the test stock solution.
Accurately measure 5 ml of the above solution to a 100 ml
volumetric flask, dilute to volume with water and mix well,
as the test solution. Dissolve O. 5084 g of sodium chloride,
previously dried to the constant weight at 100-105ºC for 3
hours and accurately weighed, in a 100 ml volumetric flask,
dilute to volume with water, mix well, as the standard
solution I . Accurately measure 2. 5 ml of the standard
solution I to a 100 ml volumetric flask, dilute to volume
with water and mix well, as the standard solutionll ( 50 µg/ ml,
Na+). Accurately measure 5 ml of the test stock solution and
1 ml of the standard solution II to a 100 ml volumetric flask,
dilute to volume with water and mix well, as the standard
solution. Carry out the method of atomic absorption
spectrophotometry <0406 method 2) , use flame atomizer to
measure the absorbance of the standard solution and the test
solution at 589 nm, absorbance of the standard solution as a,
and the absorbance of the test solution as b. b is less than
(a -b): caculated as Na not more than O. 1%.
Heavy metals Dissolve 2. O g in 15 ml of water, and adjust
to pH 4 with hydrochloric acid, add 2 ml of acetate BS
(pH 3. 5) and sufficient water to produce 25 ml. Carry out
the limit test for heavy metals <0821 method 1): not more
than O. 001%.
Arsenic Dissolve 2. O g in 23 ml of water, add 5 ml of
hydrochloric acid. Carry out the limit test for arsenic <0822
method 1): not more than O. 0001%.
Baterial endotoxin ( For Injection) Carry out the test for
baterial endotoxin ( 1143): not more than l. 1 EU per mg of
dipotassium hydrogen phosphate trihydrate.
Sterility (For sterile product without terminal sterilization
process) Complies with the test for sterility <1101 ).
Assay Dissolve about 3. 8 g, accurately weighted, in 50 ml
of freshly boiled and cooled water. Carry out the method for
potentiometric titration < 0701 ) , titrate with sulfuric acid
(O. 5 mol/L) VS. Each ml of sulfuric acid (O. 5 mol/L) VS
is equivalent to 17 4. 2 mg of Kz HP04.
Category Pharmaceutical excipients, pH regulator and
buffer, etc.
Storage Preserve in tightly closed containers, stored in a dry
place.
, ..... ,,.:·· .;·»
¡·.·. .· .· .· . .· :·. · .· .·
Disodium Edetate ·:5lli ';:'<'••••
Disodium Edetate
C10 H14 Nz Na2 Os• 2H2 O 372. 24
[6831-92-6]
Disodium Edetate is disodium ethylenediamine tetraacetate
dihydrate. It contains not less than 99. O% and not more
than 101. O% of C10 Hl4 Nz Na2 Üs• 2H2 O.
Description A white or almost white crystalline powder;
odourless; soluble in water; practically insoluble in methanol, in
ethanol and in chloroform.
Identification (1) Dissolve 2 gin 25 ml of water, add 6 ml
of 3. 3 % lead nitrate solution, shake and add 3 ml of
potassium iodide TS. No yellow precipitate is formed. Add 3
ml of ammonium oxalate TS. No precipitate is formed.
(2) When dried under reduced pressure at 50ºC for 4 hours.
The infrared absorption spectrum <0402) is concordant with
the reference spectrum CIR Album No. 978).
(3) Yields the reactions characteristic of sodium salt
(0301).
Chelating capacity test Dissolve an accurately weighed
quantity of the substance being examined in water to produce
a solution containing about O. 01 mol/L, and use as the test
solution. Place O. 10 g of calcium carbonate, dried for 2
hours at 200ºC, accurately weighed, in a 100 ml volumetric
flask, add 10 ml of water and O. 4 ml of 6 mol/L of
hydrochloric acid solution to dissolve, and adjust to pH 7. O
with ammonia TS, add water to volume, shake well and
use as solution ( 1) (O. O1 mol/L). In a 1 OO ml volumetric
flask, dissolve O. 250 g of cupric sulphate CCuSÜ4• 5H2 0),
accurately weighed, with water, and dilute to volume, shake
well and use as solution (2) (O. 01 mol/U. Toan accurately
measured 5 ml of test solution add 3 drop of ammonia TS and
2. 5 ml of 4 % ammonium oxalate solution, add 5. O ml of
solution (1) with shaking constantly, the solution is clear, if
opalescence after shaking for 1 minute, add O. 2 ml of the
test solution, shaking for 1 minute, the solutions are clear.
To accurately measured 5 ml of the test solution add O. 5 ml
of ammonia TS and O. 5 ml of 1O% potassium ferrocyanide
solution, add 4. 8 ml of solution (2) with shaking
constantly, the solution is light blue, and not any red.
Acidity Dissolve O. 50 g in 10 ml of water. pH 4. 0-5. O <0631 ).
Clarity and colour of solution Dissolve O. 50 g in 10 ml of
water. The solution is clear and colourless. <0901 and 0902).
Chlorides Dissolve l. O g in 25 ml of water, add 10 ml of
dilute nitric acid TS and shake well, allow to stand for 10 minutes,
after the precipitation completely, filter, rinse the filter with
small volume of water in portions, combine the washings and
filtra te. Carry out the limit test for Chlorides <0801 ) . Any
opalescence produced is not more pronounced than that of a
reference solution prepared by using 4. O ml of sodium
chloride standard solution (0. 004%).
lron Place O. 50 g in a 50 ml Nessler cylinder, add a volume
of water to dissolve, add 2 ml of 20 % citric acid solution and
 Disodium Hydrogen Phosphate Anhydrous
O. 5 g of calcium chloride, shake to dissolve, add O. 1 ml of
thioglycollic acid and shake, alkaline to litmus TP by the
addition of ammonia TS, and dilute to 50 ml with water,
allow to stand for 5 minutes. Carry out the limit test for Iron
( 0807). Any opalescene produced is not more pronounced
than that of a reference solution prepared by using l. O ml of
iron standard solution (0. 002%).
Heavy metals To l. O g add l. O ml of sulfuric acid TS, heat
until charring completely, and then ignite at 500-600ºC until
incinerated completely. Carry out the limit test for heavy
metals ( 0821 method 2) : Not more than O. 001 % .
Assay Dissolve about O. 4 g, accurately weighed, in about
40 ml of water, add 10 ml of ammonia-ammonium chloride
BS (pH 10. 0), titrate with zinc (0. 05 mol/L) VS, add a
small quantity of Eriochrome Black T indicator towards the
end point, titrate continually until the colour of solution
changes from blue to purplish-red. Each ml of zinc
CO. 05 mol/U VS is equivalent to 18. 61 mg of C10 H14 N2 Na2 Os•
2H20
Category Pharmaceutical excipients, chelating agent.
Storage Preserve in tightly closed containers, stored in a dry
place.
Disodium Hydrogen Phosphate
Anhydrous
Na2 HP04 142. O
[7558-79-4]
Disodium Hydrogen Phosphate contains not less than 99. O%
of Na2 HP04, calculated on the dried basis.
Description A white or almost white powder; hygroscopic;
Freely soluble in water; insoluble in ethanol.
ldentification Yields the reaction characteristic of sodium
salts and phosphates ( 0301).
Alkalinity Dissolve l. O g in 20 ml of water, pH 9. 0-9. 4
<0631 >.
Clarity and colour of solution Dissolve l. O g in 10 ml of
water, shake thoroughly, the solution is clear and colourless
( 0901 and 0902).
Chloride Carry out the limit test for chlorides ( 0801 ) ,
using 5. O g. Any opalescence produced is not more
pronounced than that of a reference solution using 5. O ml of
sodium chloride standard solution (0. 001 %).
Sulfate Carry out the limit test for sulfates ( 0802) , using
2. O g. Any opalescence produced is not more pronounced
than that of a reference solution using 2. O ml of potassium
sulfate standard solution (0. 01%).
Carbonate Dissolve 2. O gin 10 ml of water, boil and allow
to cool, add 2 ml of hydrochloric acid. No effervescence is
produced.
Water-insoluble substances Dissolve 20. O g with 100 ml of
hot water, filter with a tared No. 4 sintered glass crucible,
previously dried to constant weight at 105ºC, and wash the
residue with 200 ml of hot water in 10 portions. The
residue, when dry at 105ºC for 2 hours, weights not more
than 10 mg (0. 05%).
Reducing substances Dissolve 5. O g in freshly boiled and
cooled water, and dilute to 50 ml with the same solvent. To
5. O ml of the solution add 5 ml of dilute sulfuric acid and
O. 25 ml of potassium permanganate (O. 02 mol/L) VS, heat
on a water bath for 5 minutes. The solution remams a
purplish red colour.
Sodium dihydrogen phosphate Calculate the content of
sodium dihydrogen phosphate using the results under the
Assay as follows, not more than 2. 5 % .
N2-N3X100%
N3-N1
Loss on drying When dried to constant weight at 130ºC,
losses not more than l. O% of its weight ( 0831 ) .
Iron Dissolve O. 50 g in 20 ml of water, add 1 ml of
hydrochloric acid solution e 1 - 2) and 2 ml of 10%
sulfosalicylic acid solution, mix well, add 5 ml of ammonia
TS and mix well. Any colour produced is not more intense
than that of a reference solution prepared in the same
manner, using l. O ml of iron standard solution ( 0807)
(0. 002%).
Heavy metals Dissolve 2. O g in 15 ml of water, and adjust
to pH 4 with a quantity of hydrochloric acid, add 2 ml of
acetate BS (pH 3. 5) and a quantity of water to produce 25
ml. Carry out the limit test for heavy metals ( 0821 method
1): not more than O. 001%.
Arsenic Dissolve l. O g in 23 ml of water, add 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822
method 1 ) : not more than O. 0002 % .
Assay Dissolve about 2. 5 g, accurately weighed, in 25 ml
of freshly boiled and cooled water, add accurately 25 ml of
hydrochloric acid C1 mol/L) VS. Carry out the method for
potentiometric titration ( 0701 ) , titrate with sodium
hydroxide (1 mol/L) VS, record the volume used CN1 ) of
e
sodium hydroxide 1 mol/L) vs at the 1st point of inflexion
and N2 at the 2nc1 point of inflexion. Perform a blank determination
( N ) and make any necessary correction. Each ml of
hydrochloric acid (1 mol/L) VS is equivalent to 142. O mg of
Na2HP04.
Category Pharmaceutic excipients, pH regulator and buffer
agent.
Storage Preserve in tightly closed containers.
Disodium Hydrogen Phosphate
Dodecahydrate
Na2HP0 4•12H20 358.14
[10039-32-4]
Disodium Hydrogen Phosphate Dodecahydrate contains not
less than 98. O% of Na2 HP0 4 , calculated on dried basis.
Description Colourless or white crystals or masses;
odourless; efflorescent in dry air.
Freely soluble in water, practically insoluble in ethanol.
Identification The aqueous solution yields the reactions
characteristic of sodium salts and phosphates <0301 ) .
Akalinity Dissolve l. O g in 20 ml of water, pH 9. 0-9. 4
<0631>.
Clarity and colour of solution Dissol ve 1. O g in 1O ml of
water by shaking thoroughly. The solution is clear and
colourless.
Chlorides Carry out the limit test for chlorides ( 0801 ) ,
using 5. O g. Any opalescence produced is not more pronounced

than of a reference solution using 5. O mi of sodium chloride
standard solution (0. 001 %).
Sulfates Carry out the limit test for sulfates ( 0802) , using
2. O g. Any opalescence produced is not more pronounced
than of a reference solution using 2. O mi of sodium sulfate
standard solution (O. 01%).
Carbonate. Dissolve 2. O g in 10 mi of water, boil, allow to
cool, and add 2 ml of hydrochloric acid: no effervescence is
produced.
Water-insoluble substances Dissolve 20. O gin 100 ml of hot
water, filter through a filtering crucible which was tared at
105ºC, wash the insoluble residue with 200 ml hot water for
10 times, and dry at 105ºC for 2 hours, the weight of the
insoluble residue is not more than 10 mg (O. 05%).
Reducing substances Dissolve 5. O g in freshly boiled and
cooled water, and dilute to 50 ml with the same solvent. To
5. O ml of the solution add 5 ml of dilute sulfuric acid and
O. 25 ml of potassium permanganate (O. 02 mol/L) VS,
heat on a water bath for 5 minutes. The solution remains a
purplish red colour.
Monosodium phosphate Use the volume of N 1 , N 2 and N3
( ml) obtained in the assay, calculate by the following formula,
not more than 2. 5%.
Nz -N3 XlOO%
N3-N1
Loss on drying When dried to constant weight at 130ºC,
loses not less than 55. O% and not more than 64. O% of its
weight ( 0831).
Iron Dissolve O. 50 g in 20 ml of water, add 1 ml of
hydrochloric acid solution ( 1 - 2 ) and 2 ml of 10 %
sulfosalicylic acid solution, mix well, add 5 ml of ammonia
TS and mix well. Any colour produced is not more intense
than that of a reference solution in the same manner, using
l. O ml of iron standard solution ( 0807) (0. 002%).
Heavy metals Dissolve 2. O g in 15 ml of water, adjust to
pH 4 with a quantity of hydrochloric acid. Add 2 ml of
aceta te BS ( pH 3. 5) and a quantity of water to produce
25 ml. Carry out the limit test for heavy metals ( 0821
method 1): not more than O. 0001%.
Arsenic Dissolve l. O g in 23 ml of water, add 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822
method 1 ) : not more than O. 0002 % .
Assay Dissolve about 4. O g, accurately weighed, in 25 ml
of freshly boiled and cooled water. Accurately add 25 ml of
hydrochloric acid (1 mol/L) VS. Carry out the method for
potentiometric titration ( 0701 ) , titrate with sodium
hydroxide (1 mol/L) VS. Record the volume required at the
pt inflexion point CN 1 mD. Continue the ti tration to the 2ºd
inflexion point, as total volume of sodium hydroxide (1 mol/L)
VS required ( N 2 ml). Calculate the content with N 1 ,
perform a blalnk determintation ( N 3 ) and make any
necessary correction. F.ach ml of hydrochloric acid (1 mol/L) VS
is equivalent to 142. O mg of Na2 HP0 4 •
Category Parrnaceutical Excipients, buffering agent, alkalizing
agent.
Storage Preserve in tightly closed containers.
Egg Yolk Lecithin
Egg Yolk Lecithin
[93685-90-6]
Egg Yolk Lecithin is a mixture of phospholipids obtained
from the yolk of hens' eggs or egg yolkpowder by extracting
and refining with the appropriate solvent. It contains l. 75 %
to l. 95 % of nitrogen and 3. 5 % to 4. 1 % of phosphorus, not
less than 68% of phosphatidylcholine, not more than 20% of
phosphatidylethanolamine and not less than 80 % of the total
of phosphatidylcholine and phosphatidylethanolamine, calculated
on the anhydrous.
Description A creamy white or pale yellow powder or waxy
solid, odour, slightly greasy to the touch ( touching creamy).
Soluble in ethanol, in ether, in chloroform and in petroleum
ether ( 40-60ºC) , practically insoluble in acetone and in water.
Acid value Not more than 20. O ( 0713 >.
Saponification value 195-212 ( 0713 >.
lodine value 60-73 (0713).
Peroxide value Accurately weigh 2. O g into a 250 ml iodine
flask. Carry out the limit test for peroxide value ( 0713). Not
more than 3. O.
ldentification (1) Place O. 1 g in a crucible, add 3 g of a
mixture of sodium carbinate and potassium carbonate
(2 : 1), mix well, slightly heat, the vapor evolved change a
mositened red litmus paper to blue.
(2) Take about 100 mg of the residue obtained from the
identification (1), ignite gently to carbonise until carbide is
completely disappeared. Allow to cool and add 30 ml of
water to dissolve the residue with slightly heat, filtrate to a
test tube. Add sulf uric acid dropwise until no bubble is
produced, then add 4 drops of sulfuric acid and a small
quantity of potassium molybdate, heat, the solution become
yellowish-green.
(3) The retention time of principal peaks in the substanc-
ebeing examined in the chromatogram obtained in the Assay
of phosphatidyl choline and phosphatidyl ethanolmine are
identical with that the principal peaks in the chromatogram of
the reference solution covrespondingly.
Free fatty acids Reference solution Place O. 512 g of
palmitic acid in a 50 ml volumetric flask, add n-heptane to
dissolve and dilute to volume with the same solvent, shake
well. Transfer accurately measured 2 ml to a 50 ml
volumetric flask, dilute to volume with n-heptane and shake
well.
Test solution Place 1 g, accurately weighed, in a 25 ml
volumetric flask, add isopropyl alcohol to dissolve and dilute
to volume with the same solvent, shake well.
Procedure Into two identical 20 ml test-tubes with stopper,
introduce respectively 1 ml of test solution and reference
solution. To each test-tube add 5. O ml of a mixture of
isopropyl alcohol-heptane-0. 5 mol/L sulfuric acid ( 40 : 10 : 1) ,
shake for 1 minute and allow to stand for 10 minutes. To the
test solution add 3 ml of each of n-heptane and water, and
then introduce 2 ml of n-heptane and 4 ml of water into the
reference solution, stopper and overturn the two test-tubes
repeatedly for 1O times, stand for not less than 15 minutes
until the separation of the layers is obtained in each test-
tube. Transfer accurately measured 3 ml of the upper layer
to a 10 ml centrifuge tube, add 1 ml of nile blue IS (dissolve
 :~:·· ~
,,Y5Jtt'. Egg Yolk Lecithin
O. 04 g of nile blue in 200 ml of water, add 100 ml of n-
heptane, shake and then discard the upper layer of n-
heptane, repeat the extracting procedure for 4 times. To 20
ml of the lower aqueous phase add 180 ml of anhydrous
ethanol and mix well. Transfer the resulting solution to an
amber flask and store at the room temperature, use the
solution within 1 month). Under the nitrogen gas, titrate
with sodium hydroxide (O. 01 mol/L) VS until the solution
produce a slightly purple colour. The volume of sodium
hydroxide (0. 01 mol/L) VS consumed by test solution is not
more than that consumed by reference solution ( not more
than 1%).
Triglyceride, cholesterol and palmitic acid Carry out the
method for thin-layer chromatography <0502). Using silica
gel G as the coating substance and hexane -ehter -glacial
acetic acid (70 : 30 : 1) as the mobile phase.
Test solution Dissolve a quantity of the substance in a
mixture of hexane-isopropyl alcohol-water ( 40 : 50 : 8) to
produce a solution containing 20 mg per ml.
Reference solution Into 3 identical volumetric flask,
introduce respectively a quantity of triglyceride CRS,
cholesterol CRS and palmitic acid CRS, add the same
mixture solvent to produce three ref erence solutions
containing O. 6 mg per ml of triglyceride, O. 6 mg per ml of
cholesterol and O. 2 mg per ml of palmitic acid respectively.
Procedure Apply to the plate 5 µl of each of the test
solution, triglyceride reference solution and cholesterol
reference solution, and 1 µl of palmitic acid reference
solution. Take the plate to a tank with a filter paper wetted
by mobile phase in the internal surface. After developing and
removal of the plate, dry it and spray with 10% (W/V)
copper sulfate dilute phosphoric acid (8%, W/V) solution,
dry again in a current of warm air and heat at l 70ºC for 10
minutes. Any spot, apart from the principal spot, in the
chromatogram obtained with test solution is not more intense
than the principal spot obtained with reference solution
(Triglyceride: not more than 3%; cholesterol: not more
than 2%; palmitic acid: not more than O. 2%).
Residual solvents Carry out the method for residual solvents
(0861 ). Using the column ( HP-PLOT/Q, O. 53 mm X
30 mX 40 µm). Maintain the column temperature at 160ºC
for 8 minutes, raise the temperature to 190ºC by 5ºC per
minute, and maintain for 6 minutes. The temperature of
injection is 250ºC . The temperature of detector is 260ºC,
split ratio is 20 : 1, flowing at a rate of about 2 ml per
minute. Equilibrium the head-space vial 80ºC for 45 minutes.
Injection volume is 1 ml. Inject the headspace of the
reference solution into the column, the resolution factor
between each pair of the adjacent peaks complies with the
requirements.
Test solution Place O. 2 g, accurately weighed, in a 20 ml
head-space vial, add accurately measured 2 ml of water to
dissolve and close the vial.
Reference solution Dissolve an accurately weighed quantity
of ethanol, acetone, diethyl ether, petroleum ether and
hexane in water to produce a mixture solution containing 200
µg, 200 µg, 200 µg, 50 µg, 27 µg per ml of the solvents.
Procedure Inject equal volume of the headspace of the test
solution and the reference solution onto the column, and
record the chromatogram. Calculate the content with respect to
the peak area in the chromatogram by the externa! standard
method. The each content of ethanol, acetone and diethyl ether
is not more than O. 2 %, the content of petroleum ether is not
more than O. 05 %, the content of hexane is not more than
O. 02 %, the total residual solvents are not more than O. 5 %.
Water Not more than 3 % <0832 method 1 ( 1 ) ) .
Heavy metals Ignite 2. O g gently to carbonise, add 2 rnl of
nitric acid, heat cautiously to dryness, add 2 rnl of sulfuric acid
and heat until completely carbonised. Ignite at 500-600ºC until
the incineration is completed, allow to cool. Carry out the limit
test for heavy metals < 0812 method 2), not more than
o. 0005%.
Arsenic Place l. O g of the substance in a kjeldahl flask, add
5 ml of sulfuric acid, heat gently to be carbonised ( add not
more than 10 ml of sulfuric acid if necessary). Add gradually
a few drops of concentrated hydrogen peroxide solution,
allow the reaction to stop and heat again. Add concentrated
hydrogen peroxide solution until the solution is colourless,
cool and add 10 ml of water, evaporate until hydrogen
perooxide fumes are no longer evolved. Add 5 ml of
hydrochloride acid and a quantity of water, carry out the
limit test for arsenic < 0822 method 1 ) , not more than
o. 0002%.
Microbial limit Comply with the requirements for
microbiological limit ( 1105 and 1106), the total aerobic
bacteria count is not more than 1000 cfu per g and the total
combined yeast/mold count is not more than 100 cfu per g of
the substance being examined, Escherichia coli should not be
detected per g, and salmonella species should not be detected
per 10 g.
Assay Nitrogen To about O. 1 g, accurately weighed,
carry out the determination of nitrogen < 0704).
Phosphorus Carry out the method for ultravilot-visible
spectrophotometry <0401 ) .
Reference solution Place about O. 13 g of potassium
dihydrogen phosphate previously dried to constant weight at
105ºC, accurately weighed, in a 100 ml volumetric flask,
add water to dissolve with water and dilute to volume with
the same solvent. Transfer accurately measured 10 ml of the
solution into a 100 ml volumetric flask, dilute to volume with
water, shake well (containing 30 µg per ml of phosphorus).
Test solution Place about O. 1 g, accurately weighed, in a
crucible, add 2 ml of chloroform to dissolve, add 2 g of zinc
oxide, evaporate to dryness; heat gently to make the
substance carbonised. lgnite at 600ºC for 1 hour, allow to
cool, add 10 ml of hydrochloric acid solution 0-2), boíl for
5 minutes to dissolve the residue, transfer completely to a
100 ml volumetric flask, dilute to volume with water, shake
well.
Procedure Into five identical 25 ml volumetric flasks,
introduce O ml, 2 ml, 4 ml, 6 ml and 10 ml of the reference
solution respectively, to each of flasks add 10 ml of water,
1 ml of ammonium molybdate-sulfuric acid solution (dissolve
5 g of ammonium molybdate in 100 ml of O. 5 mol/L sulfuric
acid) , 1 ml of hydroquine-sulfuric acid solution ( dissolve
O. 5 g of hydroquine in 100 ml of O. 25 mol/L of sulfuric acid
solution, freshly prepared befare use) and then 3 ml of 50 %
of sodium aceta te solution respectively, dilute to volume with
water, shake well and allow to stand for 5 minutes. Measure
the absorbance of each of the resulting solutions at 720 nm,
using the solution in the first flask as the blank, prepare a
calibration curve with the absorbances of each concentration
on the ordinate and the corresponding concentration on the
abscissa. Transfer accurately measured 4 ml of the test
solution to a 25 ml volumetric flask, repeat the operation
beginning at the words "add 10 ml of water". Calculate the

content of phosphorus by the regression equation.
Phosphatidyl choline and Phosphatidyl ethanolmine Carry
out the method for high performance liquid chromatography
(0512).
System suitability test Using a column packed with silica
gel (Alltima sillica 250 nmX 4. 6 mm, 5 µm) anda mixture
of methanol-water-glacial acetic acid-triethylamine (85 : 15 :
O. 45 : O. 05) as the mobile phase A, and a mixture of
hexane-isopropanol-mobile phase A ( 20 : 48 : 32) as the
mobile phase B. The flow rate is l. O ml per minute and elute
linear gradient as the below table. Using evaporation light
scattering detector. ( Reference standard: the drift tube
temperature is 72ºC, the carrier gas flow rate is 2 ml per
minute.)
Time The mobile The mobile
(minutes) phase A (%) phase B C%)
o 10 90
20 30 70
35 95 5 Acid value
36 10 90
41 10 90
Dissolve a quantity of phosphatidylethanolamine CRS,
phosphatidylinositol CRS, lysophosphatidyl ethanolamine
CRS, yolk phosphatidylcholine CRS, sphiugomyelim CRS,
lysophosphatidylcholine CRS in chloroform-methanol ( 2 : 1)
to produce a mixture solution containing 50, 100, 100,
200, 200, 200 µg per ml, respectively. lnject 20 µl of the
mixture solution onto the colurnn and record the chromatogram.
The resolution factor between each pair of the adjacent peaks in
the chromatogram complies Yvith thc requirements. The
number of the theoretical plates calculated for the peaks due
to phosphatidylethanolamine and yolk phosphatidylcholine is
not less than 1500.
Procedure Dissolve an accurately weighed quantity of
phosphatidylethanolamine CRS and yolk phosphatidylcholine
CRS, in chloroform-methanol (2 : 1) to produce 6 pieces of
solution containing different concentrations of phosphatidylcholine
and phosphatidylethanolamine as the reference solutions.
The reference solutions should cover a range of 60 % to
140 % concentration of the test solution. Inject 20 µl of each
of the series reference solutions onto the column and record
the chromatograms, prepare calibration curves with the
logarithm of peak area as the ordinate and the logarithm of
amount of above substances as the abscissa. Place 15 mg,
accurately weighed, in a 50 ml volumetric flask, add a
volume of the mixture of chloroform-methanol ( 2 : 1 ) to
dissolve, and dilute to the quantity with the same solvent,
shake well and use as the test solution. lnject 20 µl of the
test solution onto the column and record the chromatogram.
Calculate the contents of phosphatidylethanolamine and
phosphatidylcholine by the regression equation.
Category Pharmaceutical expients, emulsifer and solubilizer,
etc.
Storage Preserve in tightly closed containers, protected
from light and stored ata temperature below -18ºC.
Egg Yolk Lecithin (For Injection)
Egg Yolk Lecithin {For Injection}
[93685-90-6]
Egg Yolk Lecithin is a mixture ofphospholipids obtained
from the yolk of hens' eggs or egg yolk powder by extracting
and refining with the appropriate solven t. It contains l. 75 %
to l. 95% of nitrogen and 3. 5% to 4. 1% of phosphorus, not
less than 68% of phosphatidylcholine, not more than 20% of
phosphatidylethanolamine and not more than 80 % of the
total of phosphatidylcholine and phosphatidylethanolamine,
calculated on the anhydrous basis.
Description A creamy white or pale yellow powder or waxy
so lid, odour, slightly greasy to the touch ( touching creamy).
Soluble in ethanol, in ether, in chloroform or in petroleum
ether ( boiling range of 40-60ºC), practically insoluble in
acetone and in water.
Not more than 20. O ( 0713).
Saponification value 19 5-212 <O713 ) .
lodine value 60-73 (0713).
Peroxide value Accurately weigh 2. O g into a 250 ml iodine
flask. Carry out the limit test for peroxide value ( 0713), not
more than 3. O.
Identification (1) Place O. 1 g in a crucible, add 3 g of a
mixture of sodium carbonate and potassium carbonate
(2 : 1), mix well, slightly heat, the vapor evolved change a
moistened red litmus paper to blue.
( 2 ) Take about 100 mg of the residue obtained from
theidentification ( 1) , ignite gently to carbon ise until carbide is
completely disappeared. Allow to cool and add 30 ml of water to
dissolve the residue with slightly heat, filtrate to a test tube.
Drop sulfuric acid TS until no bubble is produced, then add 4
drops of sulfuric acid TS and a small quantity of potassium
molybdate, heat, the solution becomes yellowish-green.
(3) In the assay of phosphatidylcholine and phosphatidyle-
thanolamine, the retention time of the principal peak in the
chromatogram obtained with the test solution is identical
with that of the principal peak in the chromatogram obtained
with the reference solution.
Free fatty acids Reference solution Place O. 512 g of
palmitic acid in a 50 ml volumetric flask, add n-heptane to
dissolve and dilute to volume with the same solvent, shake
well. Transfer accurately 2 ml to a 50 ml volumetric flask,
dilute to the volume with n-heptane, shake well.
Test solution Place 1 g, accurately weighed, in a 25 ml
volumetric flask, add isopropyl alcohol to dissolve and dilute
to the volume with the same solvent, shake well.
Procedure lnto two identical 20 ml test-tubes with stopper,
introduce respectively 1 ml of test solution and reference
solution. To each test-tube add 5. O ml of a mixture of
isopropyl alcohol-heptane-0. 5 mol/L sulfuric acid (40 : 10 : 1),
shake for 1 min and allow to stand for 10 minutes. To the
test solution add 3 ml of each of n-heptane and water, and
then introduce 2 ml of n-heptane and 4 ml of water into
reference solution, stopper and overturn the two test-tubes
repeatedly for 10 times, stand for no less than 15 minutes
until the separation of the layers is obtained in each test-
tube. Transfer accurately measured 3 ml of the upper phase
to a 10 ml centrifuge tube, add 1 ml of nile blue IS (dissolve
O. 04 g of nile blue in 200 ml of water, add 100 ml of n-
 Egg Yolk Lecithin (For Injection)
heptane, shake and then discard the upper layer of n-
heptane, repeat the extracting procedure for 4 times. To 20
ml of the lower aqueous phase add 180 ml of absolute ethanol
and mix well. Transfer the resulting solution to an amber
flask and store at the room temperature, use the solution
within 1 month. ). Under the nitrogen gas, titrate with
sodium hydroxide (O. 01 mol/L) VS until the solution
produce a slightly purple colour. The volume of sodium
hydroxide (0. 01 mol/L) VS consumed by test solution is not
more than that consumed by reference solution ( not more
than 1 %).
Triglyceride, cholesterol and palmitic acid Carry out the
method for thin-layer chromatography ( 0502). Using silica
gel G as the coating substance and hexane -ehter -glacial
acetic acid (70 : 30 : 1) as the mobile phase.
Test solution Dissolve a quantity of the substance in a
mixture of hexane-isopropyl alcohol-water ( 40 : 50 : 8) to
produce a solution containing 20 mg per ml.
Reference solution Into 3 identical volumetric flask, introduce
respectively a quantity of triglyceride CRS, cholesterol CRS and
palmitic acid CRS, add the same mixture solvent to produce three
reference solutions containing O. 6 mg per ml of triglyceride, O. 2
mg per ml of cholesterol and O. 2 mg per ml of palmitic acid
respectively.
Procedure Apply to the plate 5 µl of each of the test
solution, triglyceride reference solution and cholesterol
reference solution, and 1 µl of palmitic acid reference
solution. Take the plate to a tank with a filter paper wetted
by mobile phase in the internal surface. After developing and
removal of the plate, dry it and spray with the 10% (W/V)
copper sulfate dilute phosphoric acid (8%, W /V) solution,
dry again in a current of warm air and heat at l 70ºC for 10
minutes. Any spot, apart from the principal spot, in the
chromatogram obtained with test solution is not more intense
than the principal spot obtained with reference solution
(Triglyceride: not more than 3%, cholesterol: not more
than 2%, palmitic acid: not more than O. 2%).
Related substances Using the chromatographic conditions as
described under Assay.
Test solution Place 125 mg, accurately weighed, in a 25 ml
volumetric flask, add a volume of a mixture of chloroform-
methanol ( 2 : 1) to dissolve and dilute to volume with the
same solvent.
Reference solution Dissolve a quantity of phosphatidy--linositol
CRS, lysophosphatidylethanolamine CRS, phosphosphingolipid
CRS, and lysophosphatidylcholine CRS in a mixture of
chloroform-methanol ( 2 : 1 ) to produce a mixture solution
containing phosphatidylinositol 10 µg, 20 µg, 40 µg, 60 µg,
100 µg per ml, lysophosphatidylethano-lamine 50 µg, 100 µg,
200 µg, 300 µg, 400 µg per ml, phosphosphingolipid 50 µg,
100 µg, 200 µg, 300 µg, 400 µg per ml, and lysophospha-
tidylcholine 10 µg, 20 µg, 60 µg, 100 µg, 200 µg per ml,
respectively.
Procedure Inject separately 20 µl of the reference solution
onto the column and record the chromatograms. Calculate
the regression equation in which the logarithm of peak area as
the ordinate and the logarithm of amount of above substances
as the abscissa. Inject 20 µl of the test solution on to the
column and record the chromatogram, calculate the content
of phosphatidylinositol, lysophosphatidylethanolamine, phosp
hosphingolipid, and lysophosphatidylcholine from the regression
equation. Not more than 5. 0% of phosphatidy-linositol (PI)
is detected, not more than 1% of lysophosphatidylethanolamine
(LPE) is detected; not more than 3. 0% of phosphosphingo-lipid
( SPM) is detected; and the quality of lysophosphatidyl-
choline ( LPC) is not more than 3. 5 %, the total quality of
lysophosphatidyle-thanolamine (LPE) and lysophosphatidylcholine
(LPC) is not more than 4. O%, the quality of the related
substances mentioned above is not more than 8. O%.
Residual solvents Carry out the method for residual solvents
(0861 ). Using the column ( HP-PLOT/Q, O. 53 mm X
30 mX 40 µm). Maintain the column temperature at 160ºC
for 8 minutes, raise the temperature to 190ºC by 5ºC per
minute, and maintain for 6 minutes. The temperature of
injection is 250ºC . The temperature of detector is 260ºC,
split ratio is 20 : 1, flowing at a rate of about 2 ml per
minute. Equilibrium the head-space vial 80ºC for 45 minutes.
Injection volume is 1 ml. Inject the headspace of the
reference solution onto the column, the resolution factor
between each pair of the adjacent peaks complies with the
requirements.
Test solution Place O. 2 g, accurately weighed, in a 20 ml
head-space vial, add accurately measured 2 ml of water to
dissolve and close the vial.
Reference solution Dissolve an accurately weighed
quantities of ethanol, acetone, diethyl ether, petroleum ether
and hexane, in water to produce a mixture solution
containing about 200 µg, 200 µg, 200 µg, 50 µg, 27 µg per
ml of the solvents.
Procedure Inject equal volume of the headspace of the test
solution and the reference solution onto the column, and
record the chromatograms. Calculate the content with
respect to the peak area in the chromatogram by the external
standard method. The each content of ethanol, acetone and
diethyl ether is not more than O. 2 %, the content of petroleum
ether is not more than O. 05 %, the content of hexane is not more
than O. 02%, the total residual solvents are not more than
0.5%.
Water Not more than 3 % <0832 method 1 ( 1 ) ) .
Protein To l. O g, add 10 ml of hexane, slightly heat to
dissolve, the solution is clear. Given presence of insolubles,
centrifuge at 3000 rpm for 5 minutes, discard the
supernatant, add 5 ml of hexane into the residue, stir to
dissolve, repeat 2 times, remove the hexane by decompression
drying from the residual, add 1 ml of water, vibrate to
dissolve, add 4 ml of biuret test solution (to l. 5 g of copper
sulfate and 6. O g of potassium sodium tartrate, add 500 ml
of water to dissolve, stir and add 300 ml of 10 % sodium
hydroxide solution, dilute with water to 1000 mD, stand for
30 minutes, the solution is not blue purple or purple red.
Heavy metals Ignite 2. O g gently to carbonise, add 2 ml of
nitric acid, heat cautiously to dryness, add 2 ml of sulfuric
acid and heat until completely carbonised. Ignite at 500-
600ºC until the incineration is completed, allow to cool.
Carry out the limit test for heavy metals ( 0821 method 2),
not more than O. 0005 %.
Arsenic Place l. O g of the substance in akjeldahl flask, add
5 ml sulfuric acid, heat gently to be carbonised, ( add not
more than 10 ml of sulfuric acid if necessary). Add gradually
a few drops of concentrated hydrogen peroxide solution,
allow the reaction to stop and heat again. Add concentrated
hydrogen peroxide solution until the solution is colourless,
cool and add 10 ml of water, evaporate until hydrogen
pero xi de fumes are no longer evol ved. Add 5 ml of
hydrochloride acid anda volume of water, carry out the limit
test for arsenic ( 0822 method 1 ) , not more than O. 0002 %.

Bacterial endotoxins Dissove a quantity of the substance
being examined in anhydrous ethanol, and dilute with water
( far bacteria! endotoxins test ) to the experiment
concentration ( the concentration of ethanol is less than
20 %) . Carry out the test far Bacteria! endotoxins < 1143 ,
turbidity method) : not more than 2. O EU per g.
Microbial limit Comply with the requirements far
microbiological limit ( 1105 and 1106) , the total aerobic bacteria
count is not more than 1000 cfu per g and the total combined
yeast/mold count is not more than 100 cfu per g of the substance
being examined, Escherichia coli should not be detected per g,
and salmonella species should not be detected per 1O g.
Sterile ( applicable to sterile preparations without sterilization
process) Carry out the test far the sterile ( 1101 ) , it
complies the requirements.
Assay Nitrogen To about O. 1 g, accurately weighed, carry
out the determination of nitrogen ( 0704).
Phosphorus Carry out the method far ultraviolet-visible
spectrophotometry ( 0401 ) .
Reference solution Place about O. 13 g of potassium
dihydrogen phosphate previously dried to constant weight at
105ºC, accurately weighed, in a 100 ml volumetric flask,
add water to dissolve and dilute to volume with the same
solvent. Transfer accurately measured 10 ml of the solution
into a 100 ml volumetric flask, dilute with water to volume,
shake well and use as the reference solution ( containing
30 µg per ml of phosphorus).
Test solution Place about O. 1 g, accurately weighed, in a
crucible, add 2 ml chlorofarm to dissolve, add 2 g of zinc
oxide, evaporate to dryness; heat gently to make the
substance carbonised. Ignite at 600ºC far 1 hour, allow to
cool, add 10 ml of hydrochloric acid solution 0-2), boil far
5 minutes to dissolve the residue, transíer completely to a
100 ml volumetric flask, dilute to volume with water, shake
well and use as the test solution.
Procedure Into five identical 25 ml volumetric flasks,
introduce O ml, 2 ml, 4 ml, 6 ml, and 10 ml of the test solution
respectively, to each of flasks add 10 ml of water, 1 ml of
ammonium molybdate-sulfuric acid solution ( dissolve 5 g of
ammonium molybdate in 100 ml of O. 5 mol/L sulfuric acid) , 1
ml of hydroquinol-sulfuric acid solution ( dissolve O. 5 g of
hydroquinol in 100 ml of O. 25 mol/L of sulfuric acid solution,
freshly prepared befare use) and then 3 ml of 50 % of sodium
acetate respectively, dilute to volume with water, shake well and
allow to stand far 5 minutes. Measure the absorbance of each of
the resulting solutions at 720 nm, using the solution in the first
flask as the blank, calculate the regression equation by using the
absorbance as the orainate and the concentration of series of
calibration curve solutions as the abscissa. Transfer accurately
measured 4 ml of the test solution to a 25 ml volumetric flask,
repeat the operation beginning at the words "add 10 ml of
water... " Calculate the percentage content of phosphorus by
the regression equation.
Phosphatidyl choline and Phosphatidyl ethanolmine Carry
out the method far high performance liquid chromatography
(0512).
System suitability test Using a column packed with silica
gel (Alltima Sillica, 250 nm X 4. 6 mm X 5 µm) anda
mixture of methanol-water-glacial acetic acid-triethylamine
(85 : 15 : O. 45 : O. 05) as the mobile phase A, and a
mixture of hexane-isopropanol-mobile phase A (20 : 48 : 32)
as the mobile phase B. The flow rate is l. O ml per minute
and elute linear gradient as the below table. Using evaporation
Enterosoluble Vacant Gelatin Capsules
light scattering detector.
Time The mobile The mobile
(minutes) phase A (%) phase B (%)
o 10 90
20 30 70
35 95 5
36 10 90
41 10 90
Dissolve a quantity of phosphatidylethanolamine CRS,
phosphatidylinositol CRS, lysophosphatidyl ethanolamine
CRS, yolk phosphatidylcholine CRS, sphingomyelin CRS,
lysophosphatidylcholine CRS with chloroform-methanol (2 : 1)
to produce a mixture solution containing 50, 100, 100, 200,
200, 200 µg per ml, respectively. Inject 20 µl of the mixture
solution onto the column and record the chromatogram. The
resolution factor between each pair of the adjacent peaks in the
chromatogram complies with the requirements. The number of
the theoretical platescalculated for the peaks due to yolk
phosphatidylethanolamine and phosphatidylethanolamine is
not less than 1500.
Procedure Dissolve an accurately weighed quantity of
phosphatidylethanolamine CRS and yolk phosphatidyl-choline
CRS in chloroform-methanol ( 2 : 1) to produce 6 pieces of
reference solution containing phosphatidyle-thanolamine and
phosphatidylcholine at different concenration, ranging from
60% to 140% of that concentration of the test solutions.
Inject 20 µl of each of the series reference solutions onto the
column and record the chromatograms, plot standard curves
\Vith the logarithm of peak area as the ordinate and the
logarithm of amount of above substances as the abscissa.
Place 15 mg of the substance being examined, accurately
weighed, in a 50 ml volumetric flask, add a volume of the
mixture of chlorofarm-methanol ( 2 : 1) to dissolve, and
dilute to the volume with the same solvent, shake well and
use as the test solution. Inject 20 µl of the test solution onto the
column and record the chromatogram. Calculate the percentage
contents ofphosphatidylethanolamine and phosphatidylcholine by
the regression equation.
Category Pharmaceutical excipients, emulsifier and solubilizer,
liposome materials.
Storage Preserve in tightly closed containers, protected
from light and stored at a temperature below- l8°C.
Enterosoluble Vacant Gelatin Capsules
Enterosoluble Vacant Gelatin Capsules are made of gelatin
for capsules, with expicients and proper enterosoluble
material, and divided into enterosoluble capsules and colonic
soluble capsules.
Description Cylindrical, hard, elastic empty capsules,
which consist of cap and body pieces. Capsules should have a
clean, smooth and uniformly coloured surface, odourless,
well trimmed and shaped without defarmation. It could be
transparent ( no opacifying agent in both pieces ) , half
transparent ( opacifying agent in either piece), and opaque
(opacifying agent in both pieces).
 _536 Ethanol
ldentification ( 1) Dissolve O. 25 g in 50 ml of water by
heating, cool and shake thoroughly. To 5 ml of the solution
add a few drops of a mixture of potassium dichromate TS and
dilute hydrochloric acid ( 4 : 1 ) , an orange flocculent
precipitate is formed.
(2) To 1 ml of the solution obtained in Identification ( 1),
add 50 ml of water, mix well and add a few drops of tannic
acid TS. An opalescence is produced.
(3) Place O. 3 g in a test tube, add a quantity of soda lime.
The gas generated turns the moistened red litmus TP blue.
Tests Disintegration For normal enterosoluhle capsules Fill
6 capsules with tale, carry out the test for disintegration of
enterosoluble capsules ( 0921 ) . Ali capsules should comply
with the requirements.
For colonic soluble capsules Fill 6 capsules with tale,
carry out the test for disintegration of colonic soluble
capsules ( 0921 ). Ali capsules should comply with the
req uirem en ts.
Compacto~, Sulfite, Parabens, chlorohydrin, ethylene oxide,
~ on drying, Residual on ignition, Chromium, Heavy metals
and Microbial limit Comply with the corresponding
requirements described under Vacant Gelatin Capsules.
Category Pharmaceutical excipents, for delayed release
capsules preparation.
Storage Preserve in tightly closed containers, stored at a
temperature of 10-25ºC anda relative humidity of 35%-65%.
Ethanol
See Ethanol in monograph of volume II .
Category Pharmaceutical excipients, solvent.
Storage Preserve in tightly closed containers, protected from
light.
Ethyl Acetate
C4 Hs 02 88. 11
[141-78-6]
Ethyl Acetate contains not less than 99. 5 % (g/ g) of C4 Hs 02 ,
calculated on the anhydrous basis.
Description A colourless, clear volatile, and flammable liquid;
fruit odour.
Soluble in water; Miscible with dichloromethenme, ethanol,
ether and acetone.
Relative density O. 898-0. 902 ( 0601 ) .
Refractive index l. 370-1. 373 ( 0622).
ldentification ( 1 ) When burned, a yellow flame and an
acetous odour are produced.
(2) The infrared absorption spectrum ( 0402) is concordant
wíth that of ethyl acetate CRS.
Clarity and colour of solution Dilute l. O ml in 15 ml of
water, the solution is clear and colourless ( 0901 and 0902).
Acidity Mix 2. O ml with 10 ml of neutralized ethanol, add 2
drops of phenolphthalein IS and titrate with sodium hydroxide
(O. 1 mol/L) VS until the colour changes to pink. Not more
than O. 10 ml of sodium hydroxide (O. 1 mol/L) VS is required.
Readily carbonizable substances Transfer 2. O ml to a 25 ml
colourimetric tube with stopper, add 10 ml of sulfuric acid
carefully, mix well, allow to stand for 15 minutes. No
colour is produced.
Non-volatile matter Transfer 20. O ml toan evaporating dish
previously dried to constant weight, evaporate on a water
bath to dryness and dry at 105ºC for 1 hour. The residue is
not more than O. 6 mg.
Water Not more than O. 1 % ( 0832 method 1 ( 2) )
Related suhstances Carry out the method for Gas
chromatography ( 0521), using a capillary column coated 6%
cyanopropylphenyl-94 % dimethylpolysiloxane ( or stationary
phase with similar polarity), maintaining the temperature of
the injection port at 260ºC and that of the detector at 280ºC;
maintain the column temperature at 90ºC for 5 minutes, then
increased at a rate of 28ºC per minute to 240ºC for
2 minutes. Prepare the mixture of chloroform, ethyl acetate,
isobutyl aceta te and n-butyl aceta te ( 3 : 1 : 1 : 1) as the
solution for the system suitability test. Inject 1 µl into the
column and record the chromatograms. The tailing factor of
ethyl aceta te is not more than l. 5, and the resolutions
between any two peaks meet the requirements.
Procedure Inject 1 µl of the substance being examined into
the column and record the chromatogram. Calculate the
contents by the peak area normalization method. The sum of
the areas of all peaks other than the principal peak is not
more than O. 2 % times of the principal peak.
Assay Weigh accurately about l. 5 gin a 250 ml conical flask,
add accurately 50 ml of sodium hydroxide (O. 5 mol/L) VS,
heat under reflex for 1 hour. Allow to cool, add 1 drop of
phenolphthalein IS, ti trate with hydrochloric acid (O. 5 mol/L)
VS. Perform a blank determination and make any necessary
correction. Each ml of sodium hydroxide (O. 5 mol/L) VS is
equivalent to 44. 05 mg of C4 Hs 02.
Category Pharmaceutical excipients, solvents.
Storage Preserve in well closed containers.
Ethyl Acrylate and Methyl Methacry-
late Copolymer Dispersion
Ethyl Acrylate and Methyl Methacrylate Copolymer Dispersion is
a 30 % aqueous dispersion of a copolymer of ethyl acrylate and
methyl methacrylate having an average molecular weight of
about 800 000. lt includes l. 5% nonoxynol 100 as emulsifier.
Description Milky white liquid with low viscosity and weak
special smell.
It is miscible with any proportion of water, and shows milky
white. When it is mixed with acetone, ethanol or isopropanol
(1 : 5), the precipitate would appear at the beginning, then
disappear after adding the excess solvent, and the solution is
transparent or slightly turbid viscous. When it is mixed with
1 mol/L sodium hydroxide solution ( 1 : 2) , the dispersion
would not be dissolved and still be milky white.
Relative Density l. 037-1. 047 (0601 ).
Viscosity Carry out the method for determination of
viscosity ( 0633 ) , using rotational viscometer and No. O
 Ethyl Oleate 5$1
rotator with the rotation speed of 30 rpm, the dynamic and a amount of water to 28 ml. Carry out the limit test far
viscosity at 20ºC is not more than 50 mPa•s. arsenic ( 0822 method 1), not more than O. 0002%.
ldentification (1) Pour it on the glass plate, a transparent Microbial limit Comply with the requirements far
film appears after evaporating. microbiological limit ( 1105 and 1106) , the total aerobic bacteria
(2) Transfer O. 1 ml into a evaporating dish, evaporate in a count is not more than 1000 cfu per g and the total combined
water bath, then dissolve the residues with a few drops of yeast/ mold count is not more than 100 cfu per g of the substance
acetone, drop the dissolved residues on a potassium bromide being examined, Escherichia coli should not be detected .
tablet, dry under infrared light. Carry out the method far
Category Pharmaceutical excipients, release coating material,
infrared spectrophotometry ( 0402 ) . The infrared absorption
matrix sustained release tablet adhesive and retardant, etc.
spectrum is concordant with the attached reference spectrum.
Storage Preserve in a tightly closed containers. Stored at 5-25ºC
pH 5. 5-8. 6 (0631 ).
Annex The infrared reference spectrum.
Coagulum content Take 100. O g, filter it through No. 7
sieve previoushly dried at 105ºC far 5 hours. Wash the lOOr-----...,--
95
residues with water until the eluate is clear, dry at 105ºC far 90
85
5 hours, not more than 1 %. 80
,,.-.._ 75
Monomer Carry out the method far high performance liquid "$- 70
"-" 65
chromatography ( 0512). E 60
~ 55
Using a column packed with octadecylsilyl silica gel and a § 50
·¡¡; 45
mixture of acetonitrile and water ( 15 : 85) as the mobile -~ 40
phase; the detection wavelength is 205 nm. The resolution ~ 35
§ 30
factor between peaks of ethyl acrylate and methyl l:J 25
20
methacrylate complies with the requirements. 15
10
The preparation of reference solution Dissolve a quantity 5
of ethyl acrylate CRS and methyl methacrylate CRS with 4000 3500 3000 2500 2000 1500 1000
tetrahydrofuran to produce a solution containing 2 µg per ml Wavenumber ( cm- 1 )
ethyl acrylate CRS and methyl methacrylate CRS respectively, as
the reference stock solution. Accurately measure 10 ml of the
reference stock solution, add 5 mi of sodium perchlorate
solution [ take 3. 5 g of sodium perchlorate ( NaC104 •Hz O) ,
dissolve and dilute to 100 ml with water J dropwise Ethyl Oleate
accurately, mix well, transfer 5 ml accurately into 10 ml
volumetric flask, dilute to the volume with water, mix well,
use as the reference solution.
The preparation of test solution Transfer 1 g accurately,
into 50 ml volumetric flask, dissolve and dilute to the volume
with tetrahydrofuran, mix well, transfer 10 ml accurately, add 5
ml of sodium perchlorate solution accurately accompanied by
stirring, remove the precipitate by centrifuge, transfer 5 ml of 310. 51
C20H3sÜ2
subsequent filtrate accurately into 10 ml volumetric flask, dilute [111-62-6]
to the volume with water, mix well and use as the test solution. Ethyl Oleate is a mixture cons1stmg of the ethyl esters of
fatty acids, contains mainly oleic acid.
Procedure lnject 20 µl of the reference solution and test
solution respectively onto the column and record the Description A colourless to pale yellow clear liquid.
chromatograms. Calculate the content by the externa! Practically insoluble in water, miscible with ethanol, with
standard method. The total content of ethyl acrylate and methylene chloride and with petroleum ether (40ºC to 60ºC).
methyl methacrylate is not more than O. 01%. Relative density O. 866-0. 874 <0601 ).
Loss on drying Weigh accurately 1 g, evaporate in a water bath, Refractive index l. 443-1. 450 at 25 ºC ( 0622).
dry at llOºC far 3 hours, loses 68. 5%-71. 5% of its weight.
Acid value Not more than O. 5 ( 0713).
Residue on ignition Weigh accurately l. O g, transfer into
Saponification value 177-188 <O713 ) .
the crucible previously dried to constant weight, dry
moderately in a water bath, ignite to the constant weight. lodine value 75-90 < 0713).
The remained residue is not more than O. 4 %. Peroxide value Not more than 10. O (0713).
Heavy metals Carry out the limit test far heavy metals Oleic acid Weigh l. O g of the substance being examined
( 0822 method 2) , using the residue obtained in the test far into a 25 ml round-bottomed flask with a ground-glass neck
Residue on ignition. Not more than O. 001%. fitted with a reflux condenser and a gas port into the flask.
Arsenic Transfer 1 g into a conical flask, add 5 ml of Add 10 ml of anhydrous methanol and O. 2 ml of a 6%
sulfuric acid, heat under a low temperature until the solution of potassium hydroxide in methanol. Attach the
incineration is completed, add hydrogen peroxide solution by reflux condenser, pass nitrogen through the mixture ata rate
drops (if a large amount of faam appears, stop heating and of about 50 ml per minute, shake and heat to boiling. When
rotate the conical flask to avoid the unreacted substance the solution is clear ( usually after about 10 minutes),
coagulates in the bottom of flask) until the solution becomes continue heating far a further 5 minutes. Cool the flask under
colourless, allow to cool, add 10 ml of water, then heat until running water and transfer the contents to a separating
the white smoke appears, cool, add 5 ml of hydrochloric acid funnel. Rinse the flask with 5 ml of heptane and transfer the
 538 Ethylcellulose
rinsings to the separating funnel and shake. Add 10 ml of
20 % sodium chloride solution and shake vigorously. Allow
to separate and transfer the organic layer to a vial containing
anhydrous sodium sulphate. Allow to stand, then filter, take
the successive filtrate as the test solution. Carry out the
method far gas chromatography < 0521 >, using a column
packed with bonded polyethyleneglycol as the stationary
phase, the initial temperature of column is maintained at
160ºC, then raise the temperature to 230ºC by 3ºC per
minute, the temperature of injection port is 250ºC, the
temperature of detector is 250ºC, a flame ionization detector
is used. Inject 1 µl of the test solution into the column and
record the chromatograms. Calculate the content of oleic acid
(based on methyl oleate) with respect to the area obtained in
the chromatogram by the area normalization method. It is
not less than 60. O%. Disregard the peak of the solvent and
any peak with an area less than O. 05 % of the total area.
Water Not more than l. O% <0832 method 1 ( 1 ) >.
Residue on ignition Not more than O. 1 % <0841 >.
Category Pharmaceutical excipients, plasticiser and ointment
base.
Storage Preserve in well closed containers, protected from
light.
Ethylcellulose
[9004-57-3]
Ethylcellulose is an ethyl ether cellulose. It contains not less
than 44. O% and not more than 51. O% of ethoxy groups
(-0C2 H 5 ) , calculated on the dried basis.
Description White or almost white granules or powder;
odourless; tasteless.
Freely soluble in toluene and in ether; insoluble in water.
Identification (1) Dissolve 5 g in 100 ml of a mixture of
ethanol-toluene (1 : 4), shake, a clear and slightly yellow
solution is produced. Pour a quantity of the solution on a
glass plate and evaporate the water. A thin and elastic film is
farmed. The film can be flammable.
(2) The Infrared absorption spectrum <0402 > is concordant
with that of ethylcellulose CRS.
Viscosity Tranfer 2. 5 g, accurately weighed, calculated on
the dried basis, into a conical flask with stopper. Add
accurately 50 ml of a mixture of ethanol-toluene (1 : 4) with
shaking until the substance being examined is completely
dissolved. Allow to stand far 8 to 10 hours. Adjust the
temperature of the solution to 25 ± O. 1 ºC . Determine the
kinetic viscosity ( 0633 method 1 , select capillary with
suitable inside diameter to get an appropriate dropping time
greater than 200 seconds). The viscosity is not less than
90. O% and not more than 110. O% of the value stated which
nominal viscosity not less than 1 O mPa • s; the viscosity is not
less than 80. O% and not more than 120. O% of the value
stated which nominal viscosity of 6-1 O mPa • s; the viscosity is
not less than 75. O% and not more than 140. O% of the value
stated which nominal viscosity not more than 6 mPa •s.
Acidity or alkalinity To O. 50 g add 25. O ml of water and
shake far 15 minutes to dissolve. Filter through a sintered-
glass filter No. 3. To 10. O ml of the filtrate add O. 1 ml of
phenolphthalein IS and O. 50 ml of sodium hydroxide
(0. 01 mol/L) VS, the solution is pink. To 10. O ml of the
filtrate add O. 1 ml of methyl red IS and O. 50 ml of
hydrochloric acid (0. 01 mol/L) VS, the solution is red.
Chloride To O. 25 g add 40 ml of water, heat to boíl and
allow to cool, add water to 50 ml and mix well. Filter and
discard the first 10 ml of the filtrate. Carry out the limit test
far chloride ( 0801 >,
using 10. O ml of the successive filtrate.
Any opalescence produced is not more pronounced than that
of a reference solution using 5. O ml of sodium chloride
standard solution <O. 1 % >.
Acetaldehyde Take 3. O g into a 250 ml conical flask with
stopper, add 10 ml of water, insert the stopper, stir
mechanically far 1 hour. Allow to stand far 24 hours, filter
and dilute to 100 ml with water, mix well. Transfer 5. O ml,
to a 25 ml volumetric flask, add 5. O ml of a O. 05 %
methylbenzothiazolone hydrazone hydrochloride solution and
heat in a water bath at 60ºC far 5 minutes. Add 2 ml of ferric
chloride-sulfaminic acid solution ( dissolve 1 g of ferric
chloride and sulfaminic acid respectively in 100 ml of water),
heat again in a water bath at 60ºC far 5 minutes. Cool and
dilute to volume with water, use the solution as test
solution. The colour of the test solution is not more intense
than that of a reference solution prepared at the same time
and in the same manner using 5. O ml of acetaldehyde
standard solution (Dilute l. O g of acetaldehyde, accurately
weighed, with isopropanol to 100 ml, mix well, transfer 5. O
ml in a 500 ml volumetric flask, dilute to volume with
water, mix well, transfer 3. O ml in a 100 ml volumetric
flask, dilute to volume with water, mix well. Prepared befar
using) (0. 01%).
Loss on drying When dried at 105ºC far 2 hours, loses not
more than 3. O% of its weight ( 0831 >.
Residue on ignition Not more than O. 4% <0841 >, using
l. o g.
Heavy metals Carry out the limit test far heavy metals
<0821 method 2 >, using the residue obtained in the test far
Residue on ignition: not more than O. 002%.
Arsenic Mix O. 67 g with l. O g of calcium hydroxide, add
water and stir well. After dryness, heat gently until it is
thoroughly charred, and then ignite at 500-600ºC until the
incineration is completed. Dissolve the cooled residue in a
mixture of 8 ml of hydrochloric acid and 23 ml of water.
Carry out the limit test far arsenic <0822 method 1 >: not
more than O. 0003 % .
&&ly Ethoxy group Carry out the method far determination
of methoxy, ethoxy and hydroxypropoxy ( 0712 >.
In the
method 2 ( volumetric method), weigh accurately a quantity
of substance being examined equivalent to about 10 mg of
ethoxy groups, adjust the temperature of oil bath to 150-
160ºC, and extend the heating time to 1-2 hours, other
operations proceed in the same manner. Each ml of sodium
thiosulfate (0. 1 mol/L) VS is equivalent to O. 7510 mg of
ethoxy group.
Category Pharmaceutical excipients, coating and release
retardan t.
Storage Preserve in well closed containers and stored in a
dry place.
Labeling Sta te the viscosity with mPa • s and Pa •s.

Ethylcellulose Aqueous Dispersion
Ethylcellulose aqueous dispersion contains not less than
90. O% and not more than 110. O% of the labeled amount of
ethylcellulose. It contains suitable amounts of Cetyl alcohol
and Sodium lauryl sulfate, which assist in the formation and
stabilization of the dispersion. It may contain suitable defoaming
agent and preservative.
Description A creamy white dispersion.
ldentification ( 1) Transfer a quantity of the substance
being examined to a clear glass plate, distribute evenly, and
dry in an oven at about 60ºC. A continuous transparent or
translucent film is formed.
(2) To 1 ml of the substance being examined, add 9 ml of
water and 25 ml methylene solution (place O. 7 ml of sulfuric
acid and 5 g of anhydrous sodium sulfate in a beaker, slowly
add 90 ml of water and O. 3% methylene solution to 100 ml,
mix well) , mix well, add 15 ml of chloroform and shake
vigorously. Allow to stand, the under layer is blue.
(3) Dissolve O. 2 g of the film prepared in Identification (1)
in 20 ml of chloroform, use this solution as the test solution.
Accurately weigh a quantity of Cetyl alcohol CRS and dilute
with chloroform to make a solution containing about O. 1 g
per ml, use the solution as the reference solution. Carry out
the method for gas chromatography ( 0521), using a column
with stationary phase: dimethylpolysiloxane. The temperature of
injection is 250ºC. The temperature of detector is 250ºC; maintain
the column temperature at 50ºC for 5 minutes, then increased
at a rate of 20ºC per minute to 220ºC, maintain for 2
minutes. Inject 1 µl of the test solution and the reference
solution respectively into the column and record the
chromatograms. The retention time principal peak of cetyl
alcohol in the chromatogram of the test solution is identical
with that of the principal peak of cetyl alcohol CRS in the
chromatogram of the reference solution.
( 4) The infrared spectrom of the film prepared in
identification (1) exhibits maxima absorptions between 3600-
2600 am- 1 and 1500-800 cm- 1 which concordant with the
reference spectrum of ethylcellulose CRS <0402) .
Viscosity Determine viscosity at 25ºC ± O. 1 ºC < 0633
method 2), using a NDJ-79 rotating viscosimeter with a
suitable rotator. Record readings after 60, 90 and 120
seconds and the readings should between 10 % and 90 % of
full-scale. The average of the three readings is the viscosity.
The viscosity is not more than 150 mPa •s.
pH 4.0-7.0 (0631).
Dichloromethane [ Note-Perform the test only if it is
included in the manufacture. ]
Weigh accurately a quantity of the substance being examined,
dissolve and dilute with dimethyl sulphoxide to produce a
solution containing about 75 mg per ml, as the test solution.
Weigh accurately a quantity of dichloromethane, dissolve and
dilute with dimethyl sulphoxide to produce the reference
solution containing about 45 µg of dichloromethane per ml.
Accurately measure 5 ml of the test solution and the
reference solution respectively into the headspace vial and
seal. Carry out the method for residual solvents < 0861
method 2). Use flame ionization detector and a column with
stationary phase: 6 % cyanopropylphenyl-94 % dimethyl-
polysiloxane Cor stationary phase with similar polarity). The
temperature of injection port is 250ºC. The temperature of
Ethylcellulose Aqueous Dispersion Type B
detector is 250ºC; maintain the column temperature at 50ºC
for 4 minutes, then increased ata rate of 30ºC per minute to
200ºC, maintaining for 2 minutes. Equilibration temperature
of headspace vials is 80ºC and equilibration time is 20
minutes. Inject separately the gaseous sample in the test vial
and the standard vial into the column and record the
chromatograms. Calculate the content of methylene chloride
by external standard method. The result complies with the
requirements.
Loss on drying Add 5 ml of the substance being examined to
about 10 g of standard 20 to 30 mesh sand, previously dried
to constant weight at 60ºC, mix well and weigh accurately.
Dry at 60ºC to constant weight, loses not more than 71. O%
of its weight ( 0831 ).
Heavy metals Carry out the limit test for heavy metals
<0821 method 2), using l. O g: not more than O. 001 %.
Assay Carry out the method of determination of methoxy,
ethoxy and hydroxypropoxy ( 0712 ) . In the method 2
( volumetric method), weigh accurately a quantity of
substance being examined equivalent to about 10 mg of
ethoxy groups, adjust the temperature of oil bath to 150-
160ºC, and extent the heating time to 1-2 hours, other
operations proceed in the same manner. Each ml of sodium
thiosulfate (0. 1 mol/L) VS is equivalent to O. 751 O mg of
ethoxy group.
Category Pharmaceutical excipients, coating material
Storage Preserve in tight containers, protect from freezing.
Ethylcellulose Aqueous Dispersion 1Ype B
Ethylcellulose dispersion type B is a stabilized dispersion of
ethylcellulose in water. It contains not less than 90. O% and not
more than 110. 0% of the labeled amount of ethylcellulose. It may
contain suitable amounts of plasticizers, stabilizers and glidants.
~ption A creamy white dispersion with an odour
characteristic of amonia.
Identification ( 1) Transfer a quantity of the substance being
examined to a clear glass plate, distribute evenly, and dry in an
oven about 60ºC not less than 60 minutes. A continuous
transparent or translucent film is formed.
(2) The retention time of the principal peak of the test solution in
the chromatogram obtained in Assay is identical with that of the
principal peak of the reference solution.
(3) Use the film prepared in Identification ( 1) and potassium
bromide to prepare the plate < 0402 ) , the infrared absorption
spectrum in the 3600 to 2600 cm- 1 and1500 to 800 cm- 1 regions
exhibits maxima wave-numbers concordant with the reference
spectrum of ethylcellulose CRS.
Viscosity Determine viscosity at 25 ± O. 1 ºC < 0633
method 2), using a Brookfield DV-S rotating viscosimeter
with No. 2 rotator at the speed of 20 rpm. The viscosity is
400-1500 mPa•s.
pH 9. 5-11. 5 (0631 ).
Dibutyl Sebacate and Oleic Acid [ Note-Perform the test
only if they are included in the labeling. ]
Weigh accurately about 1 g of the substance being examined
in a 50 ml volumetric flask, add 25 ml of tetrahydrofuran and
mix by shaking for 15 minutes. Dilute with tetrahydrofuran
to volume and mix well, then filter and use the successive
fitrates as the test solution. Weigh accurately a quantity of
 Ethylcellulose Aqueous Dispersion Type B
dibutyl sebacate CRS and oleic acid CRS, dissolve and dilute
with tetrahydrofuran to produce a solution containing about
O. 74 mg of dibutyl sebacate and O. 48 mg of oleic acid per
ml, using the solution as reference solution. Carry out the
method for gas chromatography ( 0521 ) . Use flame
ionization detector anda column with stationary phase: TPA
modified PEG-20 M ( or stationary phase with similar
polarity). The temperature of injection port is 280ºC. The
temperature of detector is 280ºC ; maintain the column
temperature at 150ºC for 2 minutes, then increased at a rate
of lOºC per minute to 250ºC, maintain for 10 minutes. Inject
O. 5 µl of the reference solution into the column and record
the chromatogram. The resolution between the dibutyl
sebacate and oleic acid is not less than 2. O, and RSD of
continuous 6 injections is not more than 5. O%. Inject O. 5 µl
of the test solution and the reference solution respectively into the
column and record the chromatogram. Calculate the content by
extemal standard method. The results of dibutyl sebacate and
oleic acid comply with the requirement dueto the labeling. The
ratio of dibutyl sebacate to ethylcellulose is less than O. 25. The
ratio of oleic acid to ethylcellulose is less than O. 15.
n-Butanol [Note-Perform the test only if it is included in
the labeling. J
Weigh accurately about 2 g of the substance being examined
in a 25 ml volumetric flask, add 15 ml of methanol and mix
by shaking for 15 minutes. Dilute with methanol to volume
and mix well, using the solution as the test solution. Weigh
accurately a quantity of n-butanol, dissolve and dilute with
methanol to produce a solution containing about O. 16 mg of
n-butanoi per mi, using the solution as reference solution.
Carry out the method for residual solvents ( 0861 method 3).
Use flame ionization detector and a column with stationary
phase: PEG-20 M ( or stationary phase with similar
polarity). The temperature of injection port is 250ºC. The
temperature of detector is 250ºC; maintain the column
temperature at 45ºC for 5 minutes, then increased ata rate of
lOºC per minute to 220ºC, maintain for 10 minutes. Inject
O. 5 µl of the reference solution into the column and record
the chromatogram. The tailing factor of n-butanol is not
more than 2. O. Inject O. 5 µl of the test solution and the
reference solution respectively into the column and record the
chromatograms. Calculate the content by externa! standard
method. The content of n-butanol is not more than O. 2%.
Glycerin [Note-Perform the test only if it is included in the
labeling.]
Weigh accurately about 2 g of the substance being examined in a
25 ml volumetric flask, add 15 ml of methanol and mix by
shaking for 15 minutes. Dilute with methanol to volurne and mix
well, using the solution as the test solution. Weigh accurately a
quantity of glycerin, dissolve and dilute with methanol to produce
a solution containing about O. 48 mg of glycerin per ml, using the
solution as reference solution. Carry out the method for residual
solvents ( 0861 method 3). Use flame ionization detector and a
column with stationary phase: 6% cyanopropylphenyl-94%
dimethylpolysiloxane ( or stationary phase with similar polarity).
The temperature of injection port is 280ºC. The temperature of
detector is 280ºC ; maintain the column temperature at 120ºC for
2 minutes, then increased at a rate of lOºC per minute to 240ºC,
maintain for 10 minutes. Inject 1 µl of the standard solution into
the column and record the chromatogram. The tailing factor of
glycerin is not more than 2. 5. Inject 1 µ1 of the test solution and
the reference solution respectively into the column and record the
chromatograms. Calculate the content by external standard
method. The content of glycerin is not more than O. 6%.
Dichloromethane [ Note-Perform the test only if it is
included in the manufacture.]
Weigh accurately about l. 9 g of the substance being
examined in a 25 ml volume flask, dissolve and dilute with
dimethyl sulphoxide to volume, mix well, as the test
solution. Weigh accurately a quantity of Dichloromethane,
dissolve and dilute with dimethyl sulphoxide to produce a
solution containing about 45 µg of methylene chloride per
ml, as the reference standard. Accurately measure 5 ml of
the test solution and the standard solution respectively into
the headspace vial and seal. Carry out the method for
residual solvents ( 0861 method 2 ) . Use flame ionization
detector and a column with stationary phase: 6%
cyanopropylphenyl-94 % dimethylpolysiloxane ( or stationary
phase with similar polarity). The temperature of injection
port is 250ºC. The temperature of detector is 250ºC;
maintain the column temperature at 50ºC for 4 minutes, then
increased ata rate of 30ºC per minute to 200ºC, maintain for
2 minutes. Equilibration temperature of headspace vials is
80ºC and equilibration time is 20 minutes. The injection
volume is l. O ml. Inject separately the gaseous sample in the
test vial and the standard vial into the column and record the
chromatogram. Calculate the content of methylene chloride
by externa! standard method. The result should comply with
the requirement.
Medium Chain Triglycerides [Note-Perform the test only if
it is included in the labeling.]
Use the test solution under Assay as the test solution. Weigh
a quantity of glycerin tricaprylate, dissolve and dilute with
tetrahydrofuran to produce a solution containing O. 6 mg of
glycerin tricaprylate per ml, using the solution as the
reference solution. Carry out the method under Assay. Inject
separately 20 µl of the test solution and the reference solution
into the column and record the chromatogram. Calculate the
content by externa! standard method. The results of medium
chain triglycerides comply with the requirement due to the
labeling . The ratio of medium chain triglycerides to
ethylcellulose is less than O. 25.
Total Solids When dried to constant weight at 105ºC, the
residue is 23. O%-26. O% of its weight, using 1 g.
Residue on ignition [ Note-Perform this test only if it
contains inorganic nonvolatile material in the labeling.]
Not more than l. 95 % ( 0841 ) .
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2 ) , using l. O g ( or using the residue
obtained in the test for Residue on ignition if this test is
carried out): not more than O. 001%.
Assay Carry out the method of high performance liquid
chromatography ( 0512), using a refractive index detector at
a temperature of 45ºC and a column packed with poly
( styrene-divinylbenzene ). Using tetrahydrofuran as the
mobile phase, the flow rate is O. 5 ml per minute. The
column temperature is 45ºC. Weigh a quantity of
ethylcellulose CRS, glycerin tricaprylate CRS and oleic acid
CRS, dissolve and dilute with tetrahydrofuran to produce a
solution containing about 3. 75 mg of ethylcellulose, O. 6 mg
of glycerin tricaprylate and O. 4 mg of oleic acid per ml, as
the solution for system suitability test. Inject 10 µl into the
column and record the chromatogram. The resolution factor
between peaks of glycerin tricaprylate and ethylcellulose is
not less than 2. O. The resolution factor between peaks of
glycerin tricaprylate and oleic acid is not less than l. 2. The
tailing factor of ethylcellulose is not more than 2. O.
Procedure Weigh accurately about 1 g of the substance
being examined in a 50 ml volumetric flask, add 30 ml of

tetrahydrofuran and mix well by shaking far 15 minutes.
Dilute with tetrahydrofuran to volume and mix well, then
filter and use the successive fitrates as the test solution.
Weigh accurately a quantity of ethylcellulose CRS, dissolve
and dilute with tetrahydrofuran to produce a solution
containing about 3. 75 mg of ethylcellulose per ml, as the
reference solution. Inject 10 µl of the test solution and the
reference solution separately into the column and record the
chromatogram. Calculate the content by externa! standard
method.
Category Pharmaceutical excipients, coating material and
release retardant.
Storage Preserve in tight containers at a temperature below
25ºC. Protect from freezing.
Labeling The labeling states the names of all the ingredients.
Ethylparaben
o Loss on drying When dried in vacuum in a desiccators
~O~CH3
HO AJ
CiH10Ü3 166.18
[120-47-8]
Ethylparaben is ethyl 4-hydroxybenzoate. It is prepared by
the esterification of ethanol and p-hydroxybenzoic acid. It
contains not less than 98. O% and not more than 102. O% of
Ci H10 Ü3, calculated on the dried basis.
Description A whi te crystalline powdero
Freely soluble in methanol, in ethanol and in ether; slightly
soluble in glycerol; practically insoluble in water.
Melting point 115-118 ºC ( 0612 )
Identification ( 1) The retention time of principal peak of
the test solution obtained in Assay is identical with that of
principal peak of ethylparaben CRS in the chromatogram.
(2) Weigh a quantity of the substance being examined to
produce a solution of 5 µg per ml in ethanol, the light
absorption of the solution exhibits maximum at 259 nm
( 0401 ).
(3) The infrared absorption spectrumis concordant with the
reference spectrum CIR Album No. 850).
Acidity To 2 ml of the solution prepared under clarity and
colour of solution add 2 ml of ethanol and 5 ml of water, mix
well, add 2 drops of bromoeresol green IS, and titrate with
sodium hydroxide (0. 1 mol/U VS: not more than O. 1 ml of
sodium hydroxide (O. 1 mol/U VS is required to change the
colour of the indicator to blue.
Clarity and colour of solution Dissolve 1. O g of the
substance being examined in 10 ml of ethanol, the solution is
clear and colourless ( 0901 and 0902); any colour produced
is not more intense than that of reference solution Y1 or YG1
( 0901 method 1 ) .
Related substances Dissolve a quantity, accurately weighed,
in the mobile phase to produce the solution of 1 mg per ml as
test solution. Accurately transfer 1 ml, dilute with the mobile
phase to volume in a 100 ml volumetric flask and mix well as
reference solution. Carry out the method as described under
Assay. Inject 20 µl of the reference solution into the column
and adjust the attenuation so that the principal peak height in
Ethylparaben
the chromatogram is about 25 % of full scale of the chart,
then inject separately 20 µl of each of the test solution and
reference solution into the column, record the chromatogram
far 4 times the retention time of the principal peak. The area
of any impurity peaks in the chromatogram is not more than
O. 4 times the area of the principal peak in the chromatogram
obtained with the reference solution (0. 4%), the sum of the
areas of all impurity peaks is not more than O. 8 times area of
the principal peak in the chromatogram obtained with the
reference solution (O. 8 % ) .
Chloride Heat 2. O g of the substance being examined with
50 ml of water at 80ºC water bath far 5 minutes, cool and
filter. Carry out the limit test far chlorides ( 0801), using 5
ml of the filtrate. Any opalescence produced is not more
pronounced than that of a reference solution using 7. O ml of
sodium chloride standard solution (O. 035 % ) .
Sulfate Carry out the limit test far sulfates ( 0802), using
25 ml of the filtrate obtained in the test far chloride. Any
opalescence produced is not more pronounced than that of a
reference solution using 2. 4 ml of potassiumsulfate standard
solution (0. 024%).
containing silica gel to constant weight, loses not more than
O. 5 % of its weight ( 0831 ) .
Residue on ignition
l. o g.
Not more than O. 1 % ( 0841 ) , using
Heavy metals Carry out the limit test far heavy metals
( 0821 method 2), using the residue obtained in the test far
residue on ignition: not more than O. 002%.
Arsenic Mix l. O g of the substance being examined with
l. O g of calcium hydroxide, add a small volume of water and
mix well. Dry, heat gently until it is thoroughly charred,
then ignite at 500-600°C until the incineration is complete,
cool, add 5 ml of hydrochloric acid and 23 ml of water.
Carry out the limit test far arsenic ( 0822 method 1 ) : not
more than O. 0002%.
Assay Carry out the method for high performance liquid
chromatography ( 0512 ) , using a column packed with
octadecylsilane bonded silica gel and a mixture of methanol-
1 % glacial acetic acid ( 60 : 40) as the mobile phase.
Detection wavelength is 254 nm. Dissolve a quantity of
methylparaben and ethylparaben in mobile phase to produce a
mixed solution containing 10 µg per ml. Inject 20 µl of the
resulting solution onto the column. The resolution factor
between peaks of methylparaben and ethylparaben complies
with the requirement.
Procedure Dissolve a quantity, accurately weighed, in the
mobile phase to produce the test solution of O. 1 mg
ethylparaben per ml. Inject 20 µl of the resulting solution,
accurately measured, into the column and record the peak
areas corresponding obtained in the chromatogram.
Accurately weigh ethylparaben CRS and repeat the
operation. Calculate the contents of ethylparaben with
respect to the peak area obtained in the chromatogram by the
externa! standard method.
Category pharmaceutical excipients, presenvative.
Storage Preserve in well closed containers.
 Eugenol
Eugenol
OH
~ absolute ethanol to produce a solution containing about 40 mg
H2C~OCH3
C10 H12 02 164. 20
Eugenol is 2-methoxy-4- ( prop-2-enyl) -phenol. It is
obtained by distillation from clove oil, clove leaf oil or other
aromatic oil containing eugenol. It contains not less than
98. O% and not more than 102. O% of C10 H12 02.
Description A clear, colourless or pale yellow liquid;
odour, aromatic, resembling that of clove; taste, pungent;
gradually deteriorated on exposure to air or long storage.
Soluble in ethanol, in chloroform and in ether, very slightly
soluble in water.
Relative density l. 060-1. 068 at 25ºC ( 0601 hydrostatic
method).
Boiling range Not less than 90. o% Cml/mD of the liguid is
to be distilled within 252-255ºC ( 0611).
Refractive index l. 538-1. 542 <0622).
ldentification ( 1 ) Dissolve about O. 05 ml in 2 ml of
ethanol, add O. 1 ml of f erric chloride TS and mix well. A
dark green coiour is produced which changes to yeiiowish-
green gradually after standing.
(2) Dissolve a quantity in ethanol to produce a solution
containing 2 mg per ml as the test solution. Dissolve a
quantity of eugenol CRS in ethanol to produce a solution
containing 2 mg per ml as the reference solution. Carry out
the method for thin-layer chromatography ( 0502), using
silica gel GF254 as the coating substance and a mixture of
ethyl acetate-toluene ( 10 : 90) as the mobile phase. Apply
separately to the plate 5 µl each of the test solution and
reference solution, after developing and removal of the plate,
dry it in air and examine under ultra-violet light ( 254 nm).
The position and colour of the principal spot in the
chromatogram obtained with the test solution corresponds to
the principal spot obtained with the reference solution. Spray
with anisaldehyde TS, heat at 105ºC for 10 minutes. The
position and colour of the principal spot in the chromatogram
obtained with the test solution corresponds to the principal
spot obtained with the reference solution.
( 3) The retention time of the principal peak of the test
solution in the chromatogram obtained in the Assay is
identical with that of the principal peak in the chromatogram
of the reference solution.
( 4 ) The infrared absorption spectrum ( film method) is
concordant with the reference spectrum CIR Album No. 19).
(2) or (3) may be used alternatively.
Hydrocarbons To 1 ml in a 50 ml stoppered cylinder, add
5 ml of 8. 5% sodium hydroxide solution and 30 ml of water,
mix well, the solution is clear and yellow.
Water-soluble phenol To 1 ml add 20 ml of hot water, mix
well and allow to cool, filter through moistened filter paper.
Add 1 drop of ferric chloride TS to the filtrate. A blue or
purple colour should not be produced except a transient
greyish-green colour.
Dimeric and oligomeric compounds Dissolve O. 150 g in
absolute ethanol and dilute to 100 ml. The absorbance of the
solution at 330 nm is not more than O. 25 ( 0401 ) .
Related substances Dissolve about 2 g in absolute ethanol in
a 10 ml volumetric flask, dilute with absolute ethanol to
volume and mix well as the test solution. Transfer accurately
1 ml into a 100 ml volumetric flask, dilute with absolute
ethanol to volume and mix well as the reference solution.
Dissolve a quantity of eugenol CRS and vanillin CRS in
and 10 mg respectively as the system suitability solution.
Carry out the method for gas chromatography ( 0521), using
a capillary column packed with (5% phenyl) -methylsilicone
Cor similar polarity) as the stationary phase. Maintain the
column temperature at 80ºC for 2 minutes, then raise the
temperature to 280ºC by 8ºC per minute, maintain for 20
minutes. The temperature of the injection port is 250ºC and
that of detector is 280ºC. The spilt ratio is 40 : l. Inject 1 µl
of the system suitability solution into the column, the
relative retention time of vanillin is about l. 1 to eugenol, the
resolution factor between the peaks of eugenol and vanillin
complies with the related requirements. Inject 1 µl of the
reference solution into the column, adjust the attenuation so
that the principal peak height in the chromatogram is 20 % of
the full scale of the chart. Inject accurately 1 µl each of the
test solution and the reference solution in to the column and
record the chromatograms. The area of any impurity peak is
not more than O. 5 times of the area of the principal peak in
the chromatogram obtained with the reference solution
(0. 5%). The sum of the areas of all impurity peaks other
than the principal peak is not more than 2 times of the area of
the principal peak in the chromatogram obtained with the
reference solution ( 2. O%). Disregard any peak with an area
less than O. 05 times of the area of the principal peak in the
chromatogram obtained with the reference solution.
Residue on ignition Not more than O. 1 % ( 0841 ) , using
l. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), using the residue obtained in the test for
Residue on ignition: not more than O. 002%.
AasSay Carry out the method for gas chromatography
( 0521 ) , using a capillary column packed with FFAP
(nitroterephthalic acid modified polyethylene glycoD as the
stationary phase (30 mXO. 32 mm, O. 25 µm). Maintain the
column temperature at 80ºC for 1 minute, then raise the
temperature to 200ºC by 5ºC per minute, maintain for 15
minutes. The temperature of injection port is 230ºC and that
of detector is 250ºC. The spilt ratio is 10 : l. The resolution
factor between the peaks of eugenol and methyl salicylate is
more than 2. 5.
Internal standard solution Dissolve a quantity of methyl
salicylate in absolute ethanol to produce a solution of 1 mg
per ml.
Procedure Dissolve about 50 mg of the substance being
examined, accurately weighed, in internal standard solution
in a 50 ml volumetric flask and dilute to volume, mix well.
Inject accurately O. 5 µl into the column and record the
chromatogram. Repeat the operation, using 50 mg of eugenol
CRS instead of the substance being examined. Calculate the
content of C10 H12 0 2 with respect to the peak area obtained in the
chromatogram by the interna! standard method.
Category Pharmaceutical excipients, taste masking agent,
etc.
Storage Preserve in tightly closed containers, protected
from light and stored in a cool place.

Fructose*
See the same monograph in Volume Il.
Category Pharmaceutical excipients, tas te masking agent
and filling agent, etc.
Fumaric Acid
o (O. 5 mol/L) VS is equivalent to 29. 02 mg of C4 H4 0 4 •
HO-.. ~Jl
lf ~ 'OH
o
C4 H4 04 116. 07
[110-17-8]
Fumaric acid is E-2-Butenedioic acid. It contains not less
than 99. O% of C4H4 04 , calculated on the anhydrous basis.
Description White or almost white, granules or crystalline
powder; odourless.
Soluble in ethanol; slightly soluble in water and in ether;
practically insoluble in dichloromethane.
ldentification (1) Dissolve about O. 5 g in 10 ml of water,
heat to boiling, add a few drops of bromine TS, the colour is
faded.
(2) Dissolve an accurately weight quantity of the substance
being examined and fumaric acid CRS in mobile phase
described under Related substances to produce a solution of
about 10 µg per ml as test solution and reference solution.
Using the chromatographic conditions described under
Related substances, inject 10 µl of test solution and reference
solution separately into the column. Record the chromatogram,
the retention time of the principal peak in the chromatogram
obtained with the test solution is identical with that of the
principal peak dueto fumaric acid CRS in the chromatogram
obtained with the reference solution.
(3) The infrared absorption spectrum is concordant with that
of fumaric acid CRS ( 0402).
Maleic acid and other related sumtances Dissolve an accurately
weighted quantity of the substance being examined in mobile
phase to produce a solution of about 1 mg per ml as test
solution. Dissolve an accurately weighted quantity of fumaric
acid CRS and maleic acid CRS in mobile phase to produce a
mixed solution containing about 1 µg of fumaric acid CRS and
maleic acid CRS separately per ml as reference solution.
Carry out the method for high performance liquid
chromatography ( 0512 ) , using a column packed with
octadecylsilane bonded silica gel and a mixture of water
( adjust pH to 3. O with phosphoric acid) -acetonitrile
(85 : 15) as the mobile phase. The detection wavelength is
210 nm. Inject 10 µl of reference solution into the column
and record the chromatogram. The resolution factor between
the peaks of fumaric acid and maleic acid is not less than
2. 5. Inject 10 µl each of the test solution and the reference
solution into the column and record the chromatograms for 3
times the retention time of the peak of fumaric acid. The
content of maleic acid calculated with respect to the peak area
obtained in the chromatogram of test solution by the extemal
standard method is not more than O. 1 % . The area of the
,... '
.···'·~:;,
. t=A···º.••.·.••..•..
Gallic Acid
other individual impurity in the chromatogram obtained with
the test solution is not greater than O. 1 % calculated with
respect to the area of peak of fumaric acid obtained in the
chromatogram of the reference solution. The sum of contents
of impurity is not more than O. 2 % .
Water Not more than O. 5 % ( 0832 method 1 ( 1 ) ) .
Residue on ignition Not more than O. 1 % ( 0841 ) , using l. O g.
Heavy metals Carry out the limit test for heavy metals
( 0841 method 2) , using the residue obtained in the test for
Residue on ignition: not more than O. 001%.
Assay Dissolve about l. O g, accurately weighted, in 50 ml
of methanol by heating gently on a water bath and cool. Add
a few drops of phenolphthalein IS, titrate with sodium
hydroxide (O. 5 mol/L) VS. Each ml of sodium hydroxide
Category Pharmaceutical excipients, pH regulator and
effervescent agent.
Storage Preserve in well closed containers.
Gallic Acid
HO
HO-h--'r° ,H20
HO
y bH
C, H6 Os• H2 O 188. 13
[5995-86-8]
Gallic acid is 3, 4, 5-trihydroxybenzoic acid monohydrate. lt
contains not less than 98. O% and not more than 102. O% of
C, H 6Os, calculated on the dried basis.
Description White or slightly yellow crystals or crystalline
powder, odorless.
Freely soluble in hot water, in methanol, in ethanol and in
acetone. Slightly soluble in water and in ether. Practically
insoluble in benzene, in chloroform and in petroleum ether.
ldentification (1) Dissolve O. 10 g in 100 ml of water, add
a drop of ferric chloride TS to 2 ml of the above solution; a
blue-black colour is produced.
(2) The retention time of the principal peak of the test
solution in the chromatogram obtained in the Assayis
identical with that of the principal peak in the chromatogram
of the standard solution.
(3) The infrared absorption spectrum is concordant with the
spectrum of gallic acid CRS ( 0402).
Acidity Dissolve 0.10 gin 100 ml of water, pH 3. 0-3. 8 (0631 ).
Clarity and colour of solution Dissolve l. O g in 20 ml of
dehydrate ethanol, the solution is clear and colourless ( 0901
and 0902 ) , any colour produced is not more intense than
that of reference solution Y3 ( 0901 method 1 ) .
Water solubility Dissolve l. O g with 20 ml of water (higher
than 80ºC) and observe immediately. The solution is clear.
Tannins Dissolve l. O g with 20 ml hot water. Put it in the
refrigerator ( 0-5ºC), cool untill gallic acid crystallization
precipitate is formed. Filter, immediately. To the filtrate add 5-
6 drops of 1 % gelatin-sodium chloride solution [Dissolve O. 5 g
of gelatin and 5 g of sodium chloride in 50 ml water (no higher
than 60ºC) , freshly prepared befare use]. The solution is clear.
 Gelatin For Capsules
Chloride Dissolve 2. O g with 90 ml hot water in 100 ml
volumetric flask. Allow it to cool to the room temperature,
dilute with water to volume, mix well. Put it in the
refrigerator C0-5ºC), cool untill gallic acid crystallization
precipitate is formed and filter, immediately. Carry out the
limit test for chlorides <0801), using 25 mi of the successive
filtrate. Any opalescence produced is not more intense than
that of a reference using 5. O mi of sodium chloride standard
solution (O. 01%).
Sulfate Measure accurately 25. O mi of successive filtrate
obtained from Chloride in a 50 mi Nessler cylinder. Add
O. 3 mi of hydrochloric acid solution (2-3), 3 mi of ethanol
and 1 mi of 10% of barium chloride solution. Mix well and
allow to stand for 10 minutes. Carry out the limit test for
sulfates < 0802 ) . Any opalescence produced is not more
intense than that of a reference using 2. 5 ml of potassium
sulfate standard solution eo. 005 % ).
Loss on drying When dried to constant weight at 105ºC,
loses not more than 10. O% of its weight <0831 ) .
Residue on ignition Not more than O. 1 % <0841 ) , using
2. O g and ignited to constant weight at 550-600ºC.
Iron Place 2. O g in a crucible, heat gently until thoroughly
charred, allow to cool to room temperature and moisten the
residue with O. 5 mi of sulfuric acid, heat at low temperature
and evaporate until the vapor of sulfuric acid is completely
expelled. lgnite at 550-600ºC until the in cineration is
complete, and cool to room temperature. Add 5 mi of
hydrochloric acid and 10 ml of water, heat on a water-bath,
cooi and íiiter. Wash the crucible with a smali quantity of
water, combine the filtrate and washing to a 50 mi nessler
tube, dilute water to 35 ml and add 50 mg of ammonium
persulfate. Carry out the limit test for iron < 0807) , any
colour produced is not more intense than that of a reference
using 2. O mi of iron standard solution (0. 001%).
Heavy metals Carry out the limit test for heavy metals
<0821 method 2) , using the residue obtained in the test for
Residue on ignition: not more than O. 001%.
Assay Carry out the method of high performance liquid
chromatography < 0512 ) . Using a column packed with
octadecylsilane bonded silica gel, a mixture of O. 05 %
phosphate solution- methanol (93 : 7) as the mobile phase.
Detection wavelength is 271 nm and the number of
theoretical plates calculated for the gallic acid peak is not less
than 4000.
Procedure Dissolve about O. 1 g of substance being
examined, accurately weighed, in a 100 ml volumetric flask,
dilute with the mobile phase to volume, mix well. Dissolve a
quantity with the mobile phase to produce a solution
containing about 20 µg per ml. Filter through a membrane
with a pore size of O. 45 µm. Discard 5 mi of the initial
filtrate, use the successive filtrate as the test solution. Inject
20 µl of the resulting solution into column and record the
chromatogram. Dissolve a quantity of gallic acid CRS,
accurately weighed, in the mobile phase, and dilute with the
mobile phase to produce a reference solution containing 20 µg
per ml, repeat operations, using the reference solution
iustead of the test solution, calculate the content of gallic
acid with respect to the peak areas obtained in the
chromatogram by external standard method.
Category Pharmaceutical excipients, chelating agent and
antioxidants.
Store Preserve in tightly closed containers and stored in a
dry place.
Gelatin For Capsules
Gelatin is obtained by the partial acid hydrolysis, partial
alkaline hydrolysis or enzymatic hydrolysis of collagen in
skin, bone, tendon and ligament of animals. It may also be a
mixture of above three diff erent types.
Description Pale yellow to yellow, transparent or tran- ·
slucent faintly lustrous thin sheets or granules; odourless and
tasteless. It swells and softens when immersed in water, and
absorbs water 5 to 10 times of its own weight.
Freely soluble in hot water; soluble in acetic acid or a hot
mixture of glycerol and water; insoluble in ethanol.
Identification C1) Dissolve O. 5 g in 50 ml of water by
heating, cool and shake thoroughly. To 5 mi of the solution
add a few drops of a mixture of potassium dichromate TS and
dilute hydrochloric acid e4 : 1 ) ' an orange flocculent
precipitate is formed.
(2) To 1 mi of the solution obtained in ldentification (1) add
100 mi of water, mix well and add a few drops of tannic acid
TS. An opalescence is produced.
(3) Heat with a quantity of soda lime, an ammoniacal odour
is perceptible.
Gel strength Conly for the hard capsules) Perform the test
in duplicate. Place 7. 50 g of the substance to be examined in
each gelbottle. Add water to make a 6. 67 % gel solution;
place a cover each bottle and allow to stand for 1-4 hours.
Heat in a water bath at 65ºC ± 2ºC for 15 minutes. While
heating, gently stir to ensure that the gel solution is
uniform. Allow to cool at room temperature for 15 minutes
and transfer the bottles to a thermostatically controlled water
bath at lOºC ±O. 1ºC, and close the bottles with a rubber
stopper and allow to stand for 17 ± 1 hours. Remove the
bottles from the water bath and quickly wipe the water from
the exterior of the bottles. Place the bottles on the platform
of the apparatus and start the measurements. Report the
result as the average of the two measurements. Gel strength
should be not less than 180 Bloom g.
Acidity or alkalinity Shake to dissolve l. O g in 100 ml of
hot water, shake thoroughly, cool to 35ºC, pH 4. 0-7. 2
<0631>.
Light transmission Weight 2. O g, dissolve and dilute with
water of 50-60ºC to produce a 6. 67 % solution, cool to 45ºC.
Measure the transmission at 450 nm and 620 nm <0401 ) .
The light transmission should be not less than 50 % and 70 %
respectively.
Conductivity Weight l. O g, dissolve and dilute with water
of no more than 60ºC to produce a l. O% solution as test
solution. Take 100 ml of water as blank solution. Transfer
the test solution and blank solution to a thermostatically
controlled water bath at 30ºC ± 1ºC for 1 hour. Using
platinum electrode as test electrode, wash platinum electrode
with water for three times, measure the conductivity of blank
solution with conductivity meter. The conductivity of blank
solution should be not more than 5. O µS/ cm. Wash the
platinum electrode with test solution far three times,
measure the conductivity of test solution, the conductivity of
test solution should be not more than O. 5 mS/ cm.
Sulfite Ccalculated as S0i) Place 10. O g in a long neck
round bottomed flask in 150 mi of water and stand for
1 hour. Heat to dissolve in a water bath of 60ºC, add 5 mi of

phosphoric acid and 1 g of sodium bicarbonate, and connect
the flask with a condenser immediately. ( Excessive foaming
can be alleviated by adding a few drops of a suitable
antifoaming agent, such as silicone oiD. Distill and collect 50
ml of the distillate under the surface of 15 ml of O. 05 mol/L
iodine solution. Dilute the solution to 100 ml with water.
Mix well and transfer 50 ml to evaporate on a water bath,
adding water timely and continue to evaporate until the liquid
is nearly colourless. Dilute the solution to 40 ml with water,
carry out the limit test far sulfates ( 0802). Any opalescence
produced is not more pronounced than that of a reference
solution using 7. 5 ml of potassium sulfate standard solution
(0. 01%).
Peroxide Place 10 g in a 250 ml flask with a stopper, add
140 mi of water, allow to stand far 2 hours, heat to dissolve
in a water-bath at 50ºC , cool immediately. Add 6 ml of
sulfuric acid solution ( 1- 5), O. 2 g of potassium iodine,
2 ml of 1% starch solution and 1 ml of O. 5% ammonium
molybdic acid solution, stopper closely and mix well, allow
to stand in the dark far 10 minutes: no blue colour is
produced.
Loss on drying When dried at 105ºC far 15 hours, loses not
more than 15. O% of its weight ( 0831 ) .
Residual on ignition Not more than 2. O% <0841 ) , using
l. o g.
Chromium Place O. 5 g in a PTFE vessel, add 5-10 mi of
nitric acid, shake thoroughly, allow to predigest far 2 hours
at lOOºC. Stopper and allow to digest in a microwave
digestion system. After digestion has been completed,
evaporate the solution on the electric heating plate until
reddish-brown fumes are no longer evolved and near to
dryness by gently heating. Transfer to a 50 mi PTFE
volumetric flask with 2% nitric acid, mix well, and dilute to
volume as test solution. Perform a blank test omitting the
substance being examined. Use the solution obtained as
blank solution. Transfer accurately a quantity of chromium
standard solution, dilute with 2 % nitric acid solution to
produce a solution of l. O µg per ml as chromium standard
stock solution. Befare determination, transfer accurately a
quantity of chromium standard stock solution, dilute with
2 % nitric acid solution to produce a successive solutions of 0-
80 ng per ml as chromium reference solutions. Carry out the
method far atomic absorbance spectrophotometry ( 0406
method 1) or inductively coupled plasma mass spectrometry
( 0412 method 1 ) , measure the absorbances of reference
solutions and test solution. Calculate the content of
chromium: not more than O. 0002%.
Heavy metal Carry out the limit test far heavy metals
( 0821 method 2) , using the residue obtained in the test far
Residual on ignition: not more than O. 003%.
Arsenic Mix 2. O g with O. 5 g of starch, l. O g of calcium
hydroxide anda small quantity of water, stir well and dry.
Heat gently until well charred, ignite at 500-600ºC until
incineration is complete, allow to cool, add 8 ml of
hydrochloric acid and 20 ml of water to dissolve the residue.
Carry out the limit test far arsenic ( 0822 method 1 ) : not
more than O. 0001%.
Microbial limit Comply with the requirements far
microbiological limit ( 1105 and 1106 ) , the total aerobic
bacteria count is not more than 1000 cfu per g and the total
combined yeast/mold count is not more than 100 cfu per g of
the substance being examined, Escherichia coli should not be
detected per g, and salmonella species should not be detected
per 10 g.
Glacial Acetic Acid 548······.
Category Pharmaceutical excipients, far hard capsules
preparation.
Storage Preserve in tightly closed containers, stored ata dry
place.
Glacial Acetic Acid
o
H3C)lOH
Cz H4 Üz
60. 05
[64-19-7]
Glacial Acetic Acid contains not less than 99. o% (g/g) of
C2H4Üz.
Description A clear colorless liquid or colourless crystalline
mass; odour, strong.
Miscible with water, ethanol, glycerin, and most of the
volatile oils and fatty oils.
CongeaJing point Not lower than 14. 8°C (0613).
ldentification ( 1) Dilute 1 ml with 1 ml of water,
neutralize with sodium hydroxide TS, add ferric chloride TS,
a deep red colour is produced. Boil, a reddish-brown
precipitate is formed which dissolves in hydrochloride acid to
forro a yellow solution.
(2) Heat a small quantity in a mixture of sulfuric acid anda
small amount of ethanol, an odour of ethyl acetate is
produced.
Chloride Dilute 10 ml with 20 ml of water. Carry out the
limit test far chlorides ( 0801 ) . Any opalescence produced is
not more pronounced than that of a reference soiution using
4. O ml of sodium chloride standard solution (0. 0004%).
Sulfate To 20 ml, add 1 ml of 1% anhydrous sodium
carbonate solution, evaporate to dryness on a water bath.
Carry out the limit test far sulfates ( 0802). Any opalescence
produced is not more pronounced than that of a reference
solution using l. O ml of potassium sulfate standard solution
(O. 0005%).
Formic acid and readily oxidizable substances Dilute 5 ml
with 10 ml of water. To 5 ml, add 2. 5 ml of potassium
dichromate (0. 016 67 mol/U VS and 6 ml of sulfate acid,
allow to stand far 1 minute, add 20 mi of water, cool to
15ºC, add 1 mi of potassium iodide TS; a deep yellow or
brown colour is produced.
Acetaldehyde Dilute l. 8 mi, accurately weighed, with
water to 10 mi, mix well, transfer 2. 5 mi into a headspace
vial, add 2. 5 ml of 3. 2 mol/L sodium hydroxide solution,
immediately seal, shake well and use as the test solution.
Dilute a quantity of acetaldehyde CRS, accurately weighed,
with l. 6 mol/L sodium acetate solution to produce a solution
containing O. 05 mg per ml, accurately measure 5 ml in a
headspace vial, seal and use as the reference solution. Carry
out the method far residual solvent ( 0861 method 2), using
a capillary column packed with polyethyleneglycol polysiloxane as
the stationary phase; maintain the column temperature at
35ºC far 5 minutes, then raise the temperature to 120ºC by
30ºC per minute, maintain far 2 minutes. The temperature
of injection port is 200ºC; the temperature of detection is
250ºC; the equilibrium temperature of the headspace vial is
80ºC and maintain far 30 minutes. Inject each of the gases
from headspace vials of the test solution and the reference
 546 Glycerol
solution onto the column. Calculate the content of
acetaldehyde with respect to the area obtained in the
chromatogram by the externa! standard method: not more
than O. 01%.
Potes.sium permanganate reducing substances Dilute 2 ml
with 10 ml of water, add O. 10 ml of potassium permanganate
(O. 02 mol/L) VS, mix well, and allow to stand for
30 minutes; the pink colour <loes not disappear completely.
Non-volatile substances Measure 20 ml in an evaporating
dish previously dried to constant weight at 105ºC, evaporate
to dryness on a water bath, and dry to constant weight at
l05ºC. The residue is not more than 1 mg.
Water Not more than O. 20 % ( 0832 method 1 ) .
Iron Evaporate 2 ml to dryness on a water bath, add 15 ml
of water and heat gently to dissolve, add sufficient water to
produce 25 ml. Carry out the limit test for iron ( 0807). Any
opalescence produced is not more pronounced than that of a
reference solution using l. O ml of iron standard solution
(O. 0005%).
Heavy metals Evaporate 10 ml to dryness on a water bath,
add 2 ml of acetate BS (pH3. 5) and 15 ml of water, heat
gently to dissolve, add water to produce 25 ml. Carry out the
limit test for heavy metals ( 0821 method 1): not more than
o. 0002%.
Assay Accurately measure about 2 ml in a conical flask with
stopper, add 40 ml of freshly boiled and cooled water and 3
drops of phenolphthalein IS, titrate with sodium hydroxide
( 1 mol/L) VS. Each mi oí sodium hydroxide ( 1 moiíL) VS
is equivalent to 60. 05 mg of C2 H 4 0 2.
Category pharmaceutical excipients, acidifying agent and
sol ven t.
Storage Preserve in tightly closed containers.
Glycerol
C3 Hs 03 92. 09
[56-81-5]
Glycerol is 1, 2, 3-propanetriol. It contains not less than
95. O% of C3 Hs 03 , calculated on the anhydrous basis.
Description A clear, colourless syrupy liquid; tas te, sweet;
hygroscopic; the aqueous solution 0-10) exhibits a neutral
reaction.
Miscible with water and with ethanol; slightly soluble m
acetone; insoluble in chloroform and in ether.
Relative density Not less than l. 257 at 25ºC ( 0601 ) .
Refractive index l. 4 70-1. 475 <0622 >.
ldentiflcation The infrared absorption spectrum is concordant
with the reference spectrum CIR Album No. 77).
Acidity or alkalinity Dilute 25. O g with 50 ml of water, add
O. 5 ml of phenolphthalein IS, the solution is colourless. Add
O. 2 ml of O. 1 mol/L sodium hydroxide solution, a pink
colour is produced.
Colour Measure 50 ml to a Nessler cylinder; any colour
produced is not more intense than that of a reference solution
prepared by diluting o. 2 ml of potassium dichromate es with
water to 50 ml.
Chloride Carry out the limit test for chlorides ( 0801 ) ,
using 5. O g. Any opalescence produced is not more
pronounced than that of a reference solution using 7. 5 ml of
sodium chloride standard solution (O. 001 5%).
Sulfate Carry out the limit test for sulfates ( 0802) , using
10 g. Any opalescence produced is not more pronounced than
that of a reference solution using 2. O ml of potassium sulfate
standard solution (O. 002%).
Aldehydes and reducing substances Weigh 1 g to a 50 ml
volumetric flask, dilute with 25 ml of water, add 2 ml of 10%
methylbenzo-2-thiazolonehydrazone hydrochloride (adjust pH to
4. O with O. 02 mol/L sodium hydroxide solution. Freshly
prepared. ) , and allow to stand for 30 minutes. Add 5 ml of
freshly prepared O. 5% ferric chloride solution, mix well,
allow to stand for 5 minutes, dilute with methanol to the
volume, mix well as the test solution. The light absorption
of the test solution at 655 nm ( 0401 ) is not more than that
of 2. O ml of the reference solution [5. O µg of formaldehyde
(CH2 0) per ml] prepared in the same manner.
Sugars To 5. O g add 5 ml of water, mix well, add 1 ml of
dilute sulfuric acid, heat on a water bath for 5 minutes, add
3 ml of 2 mol/L carbonate-free sodium hydroxide solution,
add dropwise 1 ml of copper sulfate TS and mix well. The
solution is clear and blue. Continue to heat on a water bath
for 5 minutes. The solution remains blue and no precipitate
is formed.
Fatty acids and esters Mix 40 g with 40 mi of freshly boiled
and cooled water, add accurately 10 ml of sodium hydroxide
(0. 1 mol/L) VS, mix well and boil for 5 minutes. Allow to
cool, add a few drops of phenolphthalein IS, titrate with
hydrochloric acid ( O. 1 mol/L) VS until the pink colour
disappears. Perform a blank determination and make any
necessary correction. Not more than 4. O ml of sodium
hydroxide (O. 1 mol/L) VS is required.
Readily carbonizable substances To 4. O g add dropwise 5 ml
of sulfuric acid with shaking at a temperature not exceeding
20ºC, allow to stand for 1 hour, any colour produced is not
more intense than that of an equal volume of a reference
solution prepared by mixing O. 2 ml of standard cobaltous
chloride es, l. 6 ml of standard potassium dichromate es
and 8. 2 ml of water.
Related substances Weigh accurately about 10 g into a 25 ml
volumetric flask, add accurately 5 ml of the internal standard
solution ( dissolve a quantity of n-hexanol in methanol to
produce a solution of about O. 5 mg per mD, dilute with
methanol to volume as the test solution. Dissolve a quantity
of diethylene glycol, ethylene glycol and 1, 2-propylene
glycol, accurately weighed, in methanol to produce a solution
of about O. 5 mg each of diethylene glycol, ethylene glycol
and 1, 2-propylene glycol per ml. Transfer accurately 5 ml
into a 25 ml volumetric flask, add accurately 5 ml of the
internal standard solution, dilute with methanol to volume as
the reference solution. Dissolve a quantity of diethylene
glycol, ethylene glycol, 1, 2-propylene glycol, n-hexanol
and glycerol, accurately weighed, in methanol to produce a
solution of 400 mg of glycerol per ml and O. 1 mg of
diethylene glycol, ethylene glycol and 1, 2-propylene glycol,
n-hexanol per ml respectively as the solution for system
suitability. Carry out the method for gas chromatography
( 0521 ) , using a capillary column packed with 6 %
cyanopropylphenyl-94 % dimethylpolysiloxane ( the capillary
column with the similar polarity coating can be substituted
each other); maintain the column temperature at lOOºC for 4

minutes, raise the temperature to 120ºC by 50ºC per minute,
maintain for 10 minutes, then raise the temperature to 220ºC
by 50ºC per minute, maintain for 20 minutes. The
temperature of injection port is 200ºC; the temperature of
detection is 250ºC. Inject 1 µl of the solution for system
suitability onto the column, and record the chromatogram for
twice the retention time of the principal peak. The resolution
factor between the peaks complies with the requirements.
The relative standard deviation (RSD) for replicate injections
of the reference solution is not more than 5 % . Inject
separately 1 µl, accurately measured, each of the test
solution and the reference solution onto the column and
record the chromatograms. Calculate the contents of
diethylene glycol and ethylene glycol with respect to the area
in the chromatogram obtained with the test solution by the
internal standard method; the content of diethylene glycol
and ethylene glycol is not more than O. 025 % , respectively,
and the content of 1, 2-propylene glycol is not more than
O. 1%. Calculate the content of any impurity by the peak area
normalization method other than the peak of n-hexanol: not
more than O. 1 % . The total impurity ( including diethylene
glycol, ethylene glycol and 1, 2-propylene glycol) is not
more than l. O%.
Water Not more than 2. O% ( 0832 method 1 ( 1 ) ) .
Residue on ignition Heat 20. O g to kindling, allow to burn
without further heating, cool. Carry out the test for residue
on ignition ( 0841): not more than 2 mg.
Anunonium To 4. O g add 5 ml of 10% potassium hydroxide
solution, mix well and stand for 5 minutes at 60ºC; no odour
of ammonia is produced.
Iron Carry out the limit test for iron ( 0807 ) , using
10. O g. Any colour produced is not more intense than that of
a reference solution using 2. O ml of standard iron solution
(0. 0002%).
Heavy metals Carry out the limit test for heavy metals
( 0821), using 5. O g: not more than O. 0002%.
Arsenic Mix 6. 65 g with 23 ml of water and 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822
method 1 ) : not more than O. 00003 % .
Assay To about O. 1 g, accurately weighed, add 45 ml of
water, mix well, add accurately 25 ml of 2. 14% sodium
periodate solution, mix well and allow to stand in the dark
for 15 minutes. Add 10 ml of 50 % ethylene glycol solution,
mix well and allow to stand in the dark for 20 minutes. Add
O. 5 ml of phenolphthalein IS, titrate with sodium hydroxide
(O. 1 mol/L) VS a red colour resists for 30 seconds. Perform
a blank determination and make any necessary correction.
Each ml of sodium hydroxide (O. 1 mol/L) VS is equivalent
to 9. 21 mg of C3HsÜ3.
Category Pharmaceutical excipients, solvent and suspending
agents.
Storage Preserve in tightly closed containers, stored in a dry
place.
Notes Glycerol can forma complex with boric acid, which
decomposes, producing toxic acrolein, when overheated. It
may explode when grinded with strong oxidizers. It becomes
black on exposure to light or in contact with alkaline bismuth
nitrate or oxidizers.
Glycerol (Far Injection) 547
Glycerol ( For lnjection)
C3 Hs 03 92. 09
[56-81-5]
Glycerol is 1, 2, 3-propanetriol. It contains not less than
98. O% of C3 H 8 03 , calculated on the anhydrous basis.
Description A clear, colourless syrupy liquid; taste, sweet;
hygroscopic; the aqueous solution 0-10) exhibits a neutral
reaction.
Miscible with water and with ethanol; slightly soluble m
acetone; insoluble in chloroform and in ether.
Relative density Not less than l. 257 at 25ºC ( 0601).
Refractive index l. 470-1. 4 75 <0622).
ldentification The infrared absorption spectrum is
concordant with the reference spectrum (IR Album No. 77).
Acidity or alkalinity Dilute 25. O g with 50 mi of water, add
O. 5 mi of phenolphthalein IS, the solution is colourless. Add
O. 2 mi of O. 1 mol/L sodium hydroxide solution, a pink
colour is produced.
Colour Measure 50 ml to a Nessler cylinder; any colour
produced is not more intense than that of a reference solution
prepared by diluting o. 2 mi of potassium dichromate es with
water to 50 ml.
Chloride Carry out the limit test for chlorides ( 0801 ) ,
using 5. O g. Any opalescence produced is not more
pronounced than that of a reference solution using 3. O ml of
sodium chloride standard solution (O. 0006 % ) .
Sulfate Carry out the limit test for sulfates ( 0802), using
1O g. Any opalescence produced is not more pronounced than
that of a reference solution using 2. O mi of potassium sulfate
standard solution (O. 002%).
Aldehydes and reducing substances Weigh 1 g to a 50 ml
volumetric flask, dilute with 25 ml of water, add 2 ml of
10% methylbenzo-2-thiazolonehydrazone hydrochloride ( adjust
pH to 4. O with O. 02 mol/L sodium hydroxide solution.
Freshly prepared.), and allow to stand for 30 minutes. Add
5 mi of freshly prepared O. 5 % ferric chloride solution, mix
well, allow to stand for 5 minutes, dilute with methanol to
the volume, mix well. The light absorption of the test
solution at 655 nm ( 0401 ) is not more than that of 2. O mi
of the reference solution [ 5. O µg of formaldehyde ( CH2 O)
per mi] prepared in the same manner.
Sugars To 5. O g add 5 mi of water, mix well, add 1 mi of
dilute sulfuric acid, heat on a water bath for 5 minutes, add
3 mi of 2 mol/L carbonate-free sodium hydroxide solution,
add dropwise 1 mi of freshly prepared copper sulfate TS and
mix well. The solution is clear and blue. Continue to heat on
a water bath for 5 minutes. The solution remains blue and no
precipitate is formed.
Fatty acids and esters Mix 40 g with 40 ml of freshly boiled
and cooled water, add accurately 10 mi of sodium hydroxide
(O. 1 mol/L) VS, mix well and boil for 5 minutes. Allow to
cool, add a few drops of phenolphthalein IS, titrate with
hydrochloric acid ( O. 1 mol/L) VS until the pink colour
disappears. Perform a blank determination and make any
 Glyceryl Behenate
necessary correction. Not more than 2. O ml of sodium
hydroxide (0. 1 mol/L) VS is required.
Readily carbonizable substances To 6. 3 g add dropwise 5 ml
of sulfuric acid with shaking at a temperature not exceeding
20ºC, allow to stand for 1 hour, any colour produced is not
more intense than that of an equal volume of a reference
solution prepared by mixing O. 2 ml of standard cobaltous
chloride es, l. 6 ml of standard potassium dichromate es
and 8. 2 ml of water.
Related substances Weigh accurately about 10 g into a 25 ml
volumetric flask, add accurately 5 ml of the internal standard
solution ( dissolve a quantity of n-hexanol in methanol to
produce a solution of about O. 5 mg per ml) , dilute with
methanol to volume as the test solution. Dissolve a quantity
of diethylene glycol, ethylene glycol and 1, 2-propylene
glycol, accurately weighed, in methanol to produce a solution
of about O. 5 mg each of diethylene glycol, ethylene glycol
and 1, 2-propylene glycol per ml. Transfer accurately 5 ml
into a 25 ml volumetric flask, add accurately 5 ml of the
internal standard solution, dilute with methanol to volume as
the reference solution. Dissolve a quantity of diethylene
glycol, ethylene glycol, 1, 2-propylene glycol, n-hexanol
and glycerol, accurately weighed, in methanol to produce a
solution of 400 mg of glycerol per ml and O. 1 mg of
diethylene glycol, ethylene glycol and 1 , 2-propylene glycol,
n-hexanol per ml respectively as the solution for system
suitability. Carry out the method for gas chromatography
( 0521 ) , using a capillary column packed with 6 %
cyanopropylphenyl-94% dimethylpolysiloxane ( the capillary
column with the similar polarity coating can be substituted
each other); maintain the column temperature at lOOºC for 4
minutes, raise the temperature to 120ºC by 50ºC per minute,
maintain for 10 minutes, then raise the temperature to 220ºC
by 50ºC per minute, maintain for 20 minutes. The
6
temperature of injection port is 200 C; the temperature of
detection is 250ºC . lnject 1 µl of the solution for system
suitability onto the column, and record the chromatogram for
twice the retention time of the principal peak. The resolution
factor between the peaks complies with the requirements.
The relative standard deviation CRSD) for replicate injections
of the reference solution is not more than 5 % . Inject
separately 1 µl, accurately measured, each of the test
solution and the reference solution onto the column and
record the chromatogram. Calculate the contents of
diethylene glycol and ethylene glycol with respect to the area
in the chromatogram obtained with the test solution by the
internal standard method; the content of diethylene glycol
and ethylene glycol is not more than O. 025 % , respectively,
and the content of 1, 2-propylene glycol is not more than
O. 1 % . Calcula te the content of any impurity by the peak area
normalization method other than the peak of n-hexanol: not
more than O. 1 % . The total impurity ( including diethylene
glycol, ethylene glycol and 1 , 2-propylene glycol) is not
more than O. 5%.
Water Not more than 2. O% ( 0832 method 1 ( 1 ) ) .
Residue on ignition Heat 20. O g to kindling, allow to bum
without further heating, cool. Carry out the test for residue
on ignition ( 0841): not more than 2 mg.
Ammonium To 4. O g add 5 ml of 10% potassium hydroxide
solution, mix well and stand for 5 minutes at 60ºC; no odour
of ammonia is produced.
Iron Carry out the limit test for iron ( 0807), using
20. O g. Any colour produced is not more intense than that of
a reference solution using l. O ml of standard iron solution
(0. 00005%).
Heavy metals Carry out the limit test for heavy metals
( 0821), using 5. O g: not more than O. 0002%.
Arsenic Mix 6. 65 g with 23 ml of water and 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822
method 1 ) : not more than O. 00003 % .
Microbial limit Comply with the requirements for
microbiological limit ( 1105 and 1106 ) , the total aerobic
bacteria count is not more than 1000 cfu per g and the total
combined yeast/ mold count is not more than 100 cfu per g of
the substance being examined, Escherichia coli should not be
detected.
Bacterial endotoxins Carry out the test for bacterial
endotoxins ( 1143 ) : not more than 1O EU per g.
Sterility ( intended use for the sterile preperations without
terminal sterilization) Complies with the test for sterility
(1101).
Assay To about O. 1 g, accurately weighed, add 45 ml of
water, mix well, add accurately 25 ml of 2. 14% sodium
periodate solution, mix well and allow to stand in the dark
for 15 minutes. Add 10 ml of 50 % ethylene glycol solution,
mix well and allow to stand in the dark for 20 minutes. Add
O. 5 ml of phenolphthalein IS, titrate with sodium hydroxide
(0. 1 mol/L) VS until a red colour resists for 30 seconds.
Perform a blank determination and make any necessary
correction. Each ml of sodium hydroxide (O. 1 mol/U VS is
equivalent to 9. 21 mg of C3 Ha 03.
Category Pharmaceutical excipients, solvent and suspending
agent.
Storage Preserve in tightly closed containers, stored in a dry
place.
Notes Glycerol can form a complex with boric acid, which
decomposes, producing toxic acrolein, when overheated. It
may explode when grinded with strong oxidizers. It becomes
black on exposure to light or in contact with alkaline bismuth
nitrate or oxidizers.
Glyceryl Behenate
Glyceryl Behenate is a mixture of glycerides, mainly glycerol
monobehenate, glycerol dibehenate, and glycerol tribehenate
obtained by esterification of glycerol with behenic acid.
Description A white or almost white powder or hard waxy
mass, faint odor.
Soluble in chloroform; practically insoluble in water and in
ethanol.
Melting point 65-77°C ( 0612).
Acid value Not more than 4 (0713).
Iodine value Not more than 3 ( 0713 >, using 3. O g.
Saponification value 145-165 ( 0713).
Peroxide value Not more than 6 ( 0713).
Identification ( 1) Dissolve a quantity of the substance
being examined in chloroform to produce a solution
containing about 60 mg per ml, and use as the test solution.
Dissolve a quantity of glyceryl behenate CRS in chloroform to
produce a solution containing 60 mg per ml and use as the
reference solution. Carry out the method for thin-layer
chromatography ( 0502), using silica gel G as the coating

substance and a mixture of chloroform-acetone ( 96 : 4) as
the mobile phase. Apply separately to the same plate
(previously preconditioned by migration of ether, after the
development and removal from the chamber, allow to dry
and then immerse in a 2. 5 % boric acid solution in ethanol for
1 minute, remove the plate, dry it and heat at llOºC for 30
minutes) 10 µl of the test solution and the reference
solution. Spray with a O. 02% dichlorofluorescein solution in
ethanol and examine under ultraviolet light (254 nm). The
colour and posit1ou of the principal spots in the
chromatogram obtained with the test solution correspoud to
the principal spot obtained with reference solution.
(2) In the composition of fatty acids, the retention time of
the principle peak in the chromatogram obtained with the test
solution is identical with that of the principie peak due to
methyl behenate in the chromatogram obtained with the
reference solution.
Free glycerol Using the combined aqueous extracts obtained
as directed in the test for concent of monoacylglycerols.
Carry out the method as described under content of
monoacylglycerols, beginning with "add 50. O ml of periodic
acid solution", operate the same manner. Prepare a blank by
75 ml of water. Each ml sodium thiosulfate (0. 1 mol/L) VS
is equivalent to 2. 3 mg of glycerol CC3 Hs 03 ) . Not more
than l. 0%.
Water Not more than l. O% <0832 method 1 ( 1) ) , using
pyridine as the solvent.
Residue on ignition Not more than O. 1% <0841), using
l. o g.
Nickel Dilute a volume of nickel standard solution with
O. 5 % dilute nitric acid solution to produce a solution
containing O. 001 µg per ml, as the reference solution. To
about O. 5 g, accurately weighed, add 10 ml of nitric acid Cor
other suitable reagent) to digest. Transfer the digested
solution to a 25 ml volumetric flask, wash the digestiong
container with two 2 ml portions of water, combines the
washing to the flask. Add 1 ml each of 1 % magnesium
nitra te solution and 10 % ammonium dihydrogen phosphate
solution, dilute with water to volume and shake well, as the
test solution. Prepare a blank solution using the same
manner. Carry out the method for atomic absorption
spectrophotometry ( 0406) , measure the absorbance of each
of the resulting solutiongs at 232 nm, not more than
o. 0001%.
Heavy metals Carry out the limit test for heavy metals
<0821 method 2) using the residue obtained in the test for
residue on ignition, not more than O. 001%.
Arsenic To l. O g add l. O g of calcium hydroxide, mix and
stir well with a small volume of water. Allow it to dry,
ignite gently to carbonize and then ignite at 500-600ºC until
free from carbon, cool and dissolve the residue in a mixture
of 5 ml of hydrochloric acid and 23 ml of water. Carry out
the limit test for arsenic <0822 method 1 ) : not more than
o. 0002%.
Compostion of fatty acids Place O. 1 g in a 50 ml conical
flask, add 2 ml of O. 5 mol/L sodium hydroxide solution in
methanol. Heat the mixture in a water bath at 65ºC under a
reflux condenser for 30 minutes. Cool, add 2 ml of 15%
trifluoride boron solution in methanol through the condenser
and reflux in a water bath at 65ºC for 30 minutes. Cool and
add 4 ml of heptane through the condenser and reflux for 5
minutes. Allow to cool and add 10 ml of saturated sodium
chloride solution, shake and allow to stand until separation of
the layers is obtained, collect the upper layer and wash with
Glyceryl Behenate 1 ¡
3 portions, each of 2 ml of water and dry over anhydrous
sodium sulfate, as the test solution. Carry out the method
for gas chromatography <0521 ) , using the column packed
with polyethylene glycol as the stationary phase. Maintain
the column temperature at 230ºC for 11 minutes, raise the
temperature to 250ºC by 5ºC per minute, maintain for 10
minutes. The temperature of injection port is 260ºC and the
temperature of detector is 270ºC. Dissolve methyl palmitate,
methyl stearate, methyl arachidate, methyl behenate,
methyl erucate and methyl tetracosanonate CRS in n-hexane
to produce a mixture solution containing O. 1 mg per ml
respectively. The number of the theoretical plates calculated for
the methyl behenate peak is not less than 10 000, the
resolution factor between each pair of adjacent peaks
complies with the requirements. Inject 1 µl of the test
solution into the column and record the chromatogram. The
substances are eluted in the following sequence, palmitic
acid, stearic acid, arachidic acid, behenic acid, erucic acid,
lignoceric acid. Calculate the content of fatty acid with
respect to the peak area obtained in the chromatogram by
normalization method. Palmitic acid: not more than 3. 0%;
Stearic acid: not more than 5. 0%; Arachidic acid: not more
than 10. 0%; Behenic acid: not more than 83. 0%; Erucic
acid: not more than 3. 0%; Lignoceric acid: not more than
3.0%.
Content of monoacylglycerols Place about 1 g, accurately
weighed, in a 100 ml conical flask, add 25 ml of
chloroformw to dissolve. Transfer the solution completely to
a separating funnel, wash the flask with 25 ml of
chloroformW and then 25 ml of water, combine the washings
to the same separating funnel, shake vigorously for 1
minute, and allow to stand until the separation of the layers
is obtained ( Add 1 ml to 2 ml of glacial acetic acid to break
any emulsion formed ). Transfer the aqueous layer in a
500 ml glass-stoppered conical fíask, and extract the
chloroform layer with two 25 ml portions of water. Retain
the combined aqueous extracts for the test of free glycerol.
Transfer the chloroform layer to a 500 ml glass-stoppered
conical flask, add accurately 50 ml of periodic acid solution
(Dissolve 5. 4 g of periodic acid in 100 ml of water, add 1900
ml of glacial acetic acid. Store in a dark place) , allow to· stand
for 60 minutes with occasionally shaking; add 20 ml of
potassium iodide TS and allow to stand for 5 minutes, add
100 ml of water and titrate with sodium thiosulfate (O. 1
mol/L) VS until the colour of the mixture solution changes
from brown to pal e yellow; add 2 ml of starch IS and
continue to titrate until the blue colour disappeared. Perform
a blank determination using a mixture of 50 ml of chloroform
and 10 ml of water, and make any necessary correction. Each
ml sodium thiosulfate titration (0. 1 mol/U VS is equivalent
to 20. 73 mg of monoacylglycerols. It contains not less than
of 12. O% and not more than 18. O% of monoacylglycerols.
Category Pharmaceutical excipients, lubricant and release
retardant.
Storage Preserve in well closed containers.
Q)Chloroform for the test of Content of monoacylglycerols
To each of three 500 ml flasks add accurately 50 ml of periodic
acid solution respectively. Add 50 ml of accurately chloroform
and 10 ml of water to two of the flasks, and add 50 ml of water
to the third flask. To each flask add 20 ml of potassium iodide
TS, shake well, and proceed as directed under coutent of
monoacylglyerols, beginning at the words "allow to stand for 5
minutes". The difference between the volumes of sodium
thiosulfate (O. 1 mol/L) VS required in the titrations with
and without the chloroform is not greater than O. 5 ml.
 .·I Glycine
Glycine
See the same monograph in Volume II .
Category Pharmaceutical excipients, solubilizer and ant-
ioxidant synergist.
Hard Fat
Hard Fat is a mixture of monoglycerides, diglycerides and
triglycerides of saturated fatty acids of Cs-Cis.
Description A white or almost white, waxy salid; odour,
characteristic of oil; greasy to the touch.
Freely soluble in chloroform and in ether; soluble in
petroleum ether ( 60-90ºC); practically insoluble in water
and in ethanol.
Melting point 33-35ºC ( type 34); 35-37°C ( type 36); 37-
39ºC ( type 38); 39-41 ºC ( type 40) ( 0612 method 2).
Acid value Not more than l. O <O713 >.
Saponification value laberlled saponification value is 215-260
<0713), the saponification value is not less than 95 % and
not more than 105 % of the labelled saponification value.
Hydroxyl value Not more than 60 <0713 >.
lodine value Not more than 2. O <0713 >.
Peroxide value Not more than 3 <0713).
ldentification Dissolve l. O g of the substance being
examined in 10 ml of chloroform as the test solution. Carry
out the method for thin-layer chromatography < 0502 ) ,
using silica gel G as the coating substance and a mixture of
chloroform-acetone ( 20 : O. 5) as the mobile phase. Apply
to the plate 5 µl of the test solution, after developing over a
path of 12 cm and removal of the plate, dry it, expose the
plate to iodine vapour and examine, the chromatogram
obtained with the test solution shows at least four spots.
Alkaline impurities To 2. O g, add l. 5 ml of ethanol and
3. O ml of ether, heat in a water bath at 40ºC to dissolve, add
O. 05 ml of bromophenol blue IS, titrate with hydrochloric
acid (0. 01 mol/L) VS until the colour changes to yellow, and
not more than O. 15 ml of hydrochloric acid (O. 01 mol/L) VS
is required.
Ash Place about 2 g, accurately weighed, in a crucible
previously ignited to constant weight, ignite slowly (be
careful to avoid combustion) until completely carbonized.
lncinerate completely to constant weight at 500-600ºC. The
residual ash is not more than O. 05 % of its weight.
Heavy metals To 1 g add 20 ml of saturated sodium chloride
solution, heat on a water bath to dissolve, then cool in the
ice bath, filter and transfer the filtrate to a 50 ml Nessler
cylinder, add 2 ml of dilute acetic acid anda quantity of water
to produce 25 ml. Carry out the limit test for heavy metals
<0821 method 1): not more than O. 001%.
Category pharmaceutical excipients, suppository base and
release retardant.
Storage Preserve in well closed containers, stored in a cool
place.
Histidine
See the same monograph in Volume II.
Category Pharmaceutical excipients, solubilizer and cry-
oprotectant.
Hydrochloric Acid
HCl 36. 46
[7647-01-0]
Hydrochloric Acid contains not less than 36. 0% and not
more than 38. 0% (g/g) of HCl.
Description A clear, colourless, fuming liquid; odour,
pungent; it is strongly acidic.
Identification ( 1 ) Yields the reactions characteristic of
chlorides <0301 ) .
(2) Dip a glass rod moistened with ammonia TS, toach the
surface of the substace being examined, while fumes air
produced.
(3) Dilute 1 ml to 100 ml with water, turns the blue litmus
paper to red.
Free chlorine or bromine Dilute 5. 4 g to 20 ml with water.
Cool, add O. 2 ml of zinc starch-iodide IS, a blue colour is not
produced within 10 minutes.
Bromide or iodide Dilute 3 ml to 10 ml with water. Allow to
cool, add 1 ml of chloroform and 1 drop of O. 002 mol/L
potassium permanganate solution, shake, the chloroform
layer is colourless.
Sulfate Mix 25 g with 2 drops of sodium carbonate TS,
evaporate to dryness on a waterbath, dissolve the residue in
20 ml of water. Carry out the limit test for sulfates <0802) ,
any opalescence produced is not more pronou~ced than that
of a reference solution using l. 25 ml of potassium sulfate
standard solution (O. 0005 % ) .
Sulfite Mix 1 g of potassium iodide, O. 15 ml of O. 005 mol/L
iodine solution and l. 5 ml of starch IS with 50 ml of freshly
boiled and cooled water, shake. The blue colour does not
disappear on the addition of a mixture of 5 ml of the
substance being examined and 50 ml of freshly boiled and
cooled water.
Residue on ignition Add 2 drops of sulfuric acid to 100 g,
evaporate to dryness, carry out the test for residue on
ignition <0841): not more than 2 mg (0. 002%).
lron Evaporate 30 g on a water bath to dryness, add 25 ml
of water to the residue, carry out the limit test for iron
<0807), any colour produced is not more intense than that of
a reference solution using 3. O ml of iron standard solution
(0. 0001%).
Heavy metals Evaporate 10 g on a water bath to dryness,
add 2 ml of aceta te BS ( pH 3. 5) and dilute to 25 ml with
water. Carry out the limit test for heavy metal < 0821
method 1 ) : not more than O. 0002 % .
Arsenic Dilute 2. O g in 22 ml of water, add 5 ml of
hydrochloric acid, complies with the limit test for arsenic
( 0822 method 1) (0. 0001 %).

As.say Transfer about 3 ml to a tared glass-stoppered flask
containing 20 ml of water and weigh accurately. Add 25 ml
of water and 2 drops of methyl red IS, titrate with sodium
hydroxide ( 1 mol/L) VS. Each ml of sodium hydroxide
( 1 mol/L) VS is equivalent to 36. 46 mg of HCL
Category Pharmaceutical excipients, pH regulator.
Storage Preserve in tightly closed containers.
Hydrogenated Castor Oil
C3Hs CC1sH3sÜ3)3 939.50
[8001-78-3]
Hydrogenated Castor Oil is obtained by hydrogenation from
Virgin Castor Oil. It consists mainly of the triglyceride of
12-hydroxystearic acid.
Description Almost white to pale yellow powder, masses or
flakes.
Slightly soluble in methylene chloride, very slightly soluble
in ethanol, practically insoluble in water and in petroleum
ether.
Melting point 85-88ºC <0612 >.
Acid value Not more than 4. O (0713).
Hydroxyl value 150-165 (0713).
lodine value Not more than 5. O ( 0713).
Saponification value 176-182 <0713 >.
Alkaline impurities To l. O g add l. 5 ml of ethanol and 3 ml
of toluene to dissolve with gentle heating, add 1 drop of
O. 04 % bromophenol blue solution in ethanol. Ti trate the
warm solution with hydrochloric acid (O. 01 mol/L) VS.
Not more than O. 2 ml of hydrochloric acid (0. 01 mol/L) VS
is required to change the colour of the indicator to yellow.
Nickel Carry out the method far atomic absorption
spectrometry ( 0406 method 1 ) .
Reference solution Dilute a volume of the nickel standard
solution with O. 5 % dilute nitric acid soluiton to produce a
series solutions containing O, O. 005, O. 025, O. 050 and
O. 075 mg per ml respectively, and use as the series reference
solutions.
Test solution Place about O. 5 gin digestion vessel to digest
with 10 ml of nitric acid. Transfer the digested mixture to a
25 ml volumetric flask, add 1 ml each of O. 04 mol/L
magnesium nitrate solution and O. 87 mol/L ammonium
dihydrogen phosphate solution, dilute to 25 ml with water,
shake well and use as the test solution.
Blank solution Prepare the blank solution using the same
manner as described far the test solution without the
substance being examined.
Procedure Measure the absorbance of the each of the
resulting solutions at 232. O nm. The content of nickel is not
more than O. 0005%.
Heavy metals Not more than O. 001 % ( 0821 method 2),
using l. O g.
Arsenic Transfer l. O g to a 150 ml conical flask, add 5 ml
of sulfuric acid, heat until the substance is completely
carbonized. Add dropwise strong hydrogen peroxide solution
(if too much bubbles occur, stop heating and revolve the
conical flask to protect the materials unreacted from being
Hydrogenated Soybean Oil
sintering in the bottom) until the solution is colourless.
Allow to cool, cautiously add 10 ml of water. Evaporate the
solution until the dense smoke of sulfur trioxide appeared.
Allow it to cool and slowly add water to 28 ml. It complies
with limit test far arsenic <0822 method 1): not more than
o. 0002%.
Composition of fatty acids Carry out the method far gas
chromatography ( 0521 ) , using the column packed with
bonding macrogol as the stationary phase. Maintain the
column temperature at 230ºC far 11 minutes, raise the
temperature of the column at arate of 5ºC/min from 230ºC
to 250ºC and maintain the column temperature at 250ºC far
10 minutes.
Test solution Place O. 1 gin a 50 ml conical flask, add 2 ml
of O. 5 mol/L sodium hydroxide solution in methanol. Reflux
the mixture in a water bath at 65ºC far 30 minutes. Add 2. O
ml of 15 % boron trifluoride solution in methanol through the
condenser and reflux in a water bath at 65ºC far 30 minutes.
Allow to cool and add 4 ml of heptane through the condenser
and reflux far 5 minutes. Cool and add 10 ml of saturated
sodium chloride solution. Shake well and allow to stand until
the separation of the layers is obtained, to 2 ml of the upper
layer wash with 3 portions, each of 2 ml of water, dry the
upper layer over anhydrous sodium sulfate.
Reference solution Dissolve a quantity of methyl palmitate
CRS, methyl stearate CRS, methyl arachidate CRS, methyl
12-oxostearate CRS and methyl 12-hydroxystearate CRS in
n-hexane to produce a mixture solution containing O. 1 mg per ml
respectively.
Procedure Inject 1 µl of the reference solution onto the
column and record the chromatogram, the number of the
theoretical plates calculated far the 12-hydroxystearate peak
is not less than 10 000. The resolution factor between each
pair of the adjacent peaks complies with the requirements.
Inject 1 µl of the test solution onto the column and record the
chromatogram. Calculate the content of fatty acid with
respect to the peak area obtained in the chromatogram by
normaligation method: palmitic acid: not more than 2. 0%;
stearic acid: 7. O%. to 14. O% ; arachidic acid: not more than
l. 0%; 12-oxostearic acid: not more than 5. 0%; 12-
hydroxystearic acid: 78. 0% to 91. 0%; any other fatty acid:
not more than 3. 0%.
Category Pharmaceutical excipients, emulsifier and ointment
base.
Storage Store in well-closed containers, protected from
light.
Hydrogenated Soybean Oil
[8016-70-4]
Hydrogenated Soybean Oil is the product prepared by
refining, bleaching, hydrogenation and deodorization of oil
obtained from seeds of Glycine soya bentham.
Desription A white to pale yellow mass or powder which
melts to a clear, pale yellow liquid when heated.
Freely soluble in dichloromethane and in toluene, practically
insoluble in water and in ethanol.
Melting range 66ºC to 72ºC <0612 method 2).
Acid value Place 10. O g, accurately weighed, in a 250 ml
conical flask, add 50 ml of a mixture of ethanol and toluene
 ·····.:$$2:······ •I Hydroxyethyl Cellulose
(1 : 1) [add O. 5 ml of phenolphthalein IS before use, make
to neutral by adding sodium hydroxidevs (O. 1 mol/L) VS]
and heat to dissolve completely. Titrate the hot resulting
solution with sodium hydroxide (O. 1 mol/L) VS immedi-
ately to produce a pink colour resists far 30 seconds. The
acid value is not more than O. 5 ( 0713).
Peroxide value Not more than 5. O <0713).
ldentification In the test far Fatty acid composition, the
retention time of the principal peaks due to methyl palmitate
and methyl stearate in the chromatogram obtained with the
test solution are identical with those of two principal peaks in
the chromatogram obtained with the reference solution.
Unsaponifiable matter Place 5. O g, accurately weighed, in a
250 ml conical flask, add 50 ml of potassium hydroxide
solution in ethanol ( dissolve 12 g of potassium hydroxide in
10 ml of water, dilute to 100 ml with ethanol), heat under
reflux far an hour, cool to a temperature below 25ºC, and
transfer to a separating funnel. Wash the flask with two
portions, each of 50 ml of water, and combine the washings
to the separating funnel. Extract with three portions, each of
100 ml of ether, combine the extracts and wash with three
potions, each of 40 ml of water and discard the
washings. Wash the ether extract successively with three
40 ml portions of 3 % sodium hydroxide solution and three 40
ml portions of water, then wash with a 40 ml portian of
water repeatly until the lasting washing is not reddened by
addition of two drops of phenolphthalein IS. Transfer the
ether extract to an evaporating dish, previously dried to
constant weight, wash the separating funnel with 1O ml of
ether, transfer the washing to the evaporating dish and
evaporate to dryness on a water bath at a temperature of
50ºC, dissolve the residue with 6 ml of acetone. Expel the
acetone in current air. Dry at 105ºC until the variation
between two consecutive weights is less than 1 mg, the
content of the unsaponifiable matter is not more than l. 0%.
Dissolve the residue with 20 ml of neutralized ethanol, add a
few drops of phenolphthalein IS and titrate with ethanolic
sodium hydroxide VS (O. 1 mol/L) CD until a pink colour is
produced and last for 30 seconds. The test is invalid if more
than O. 2 ml of ethanolic sodium hydroxide VS (O. 1 mol/L)
is consumed.
CD Ethanolic sodium hydroxide (O. 1 mol/L) VS
Preparation To 2 ml of 50 % sodium hydroxide solution
add 250 ml of ethanol ( allow to stand over night if the
solution is opalescence, and using the supernatant for
standardization).
Standardization To about O. 2 g of benzoic acid, accurately
weighed, add 10 ml of ethanol and 2 ml of water to dissolve,
add two drops of phenolphthalein IS and titrate with ethanolic
sodium hydroxide (O. 1 mol/L) VS until a persistent pink
colour is produced. Each ml of ethanolic sodium hydroxide
(0. 1 mol/L) VS is equivalent to 12. 21 mg of benzoic acid.
Alkaline impurities Dissolve 2. O g of hydrogenated soybean
oil in a mixture of l. 5 ml of ethanol and 3 ml of toluene with
gently heating. Add O. 05 ml of O. 04 % bromophenol blue
solution in ethanol, and ti trate with hydrochloric acid (O. 01
mol/L) VS until the colour turns to yellow. Not more than
O. 4 ml of hydrochloric acid (O. O1 mol/L) VS is required.
Water Not more than O. 3 % ( 0832 method 1 ( 2) ) , using
l. o g.
Nickel Dilute a volume of nickel standard solution, with
water to produce a solution containing O. 1 µg per ml and use
as the reference solution. Introduce 5. O g, accurately
weighed, in a crucible, cautiously heat until it is thoroughly
charred. lgnite at 600ºC until white ash is obtained. Cool,
dissolve the residue in 4 ml of dilute hydrochloric acid and
transfer to a 25 ml volumetric flask, add O. 3 ml of nitric acid
and dilute to volume with water, shake well and use as the
test solution. Place accurately measured O ml, l. O ml,
2. O ml and 3. O ml of the reference solution respectively in
faur 10 ml volumetric flasks, add accurately measured 2 ml
of the test solution respectively, dilute to volume with water
and shake well. Carry out the method for atomic absorption
spectophotometry ( 0406 method 2 ) , measure the
absorbance of each of the above solutions at 232. O nm. The
content of the nickel is not more than O. 0001%.
Fatty acid compositions Place O. 1 g in a 50 ml round-
bottomed flask, add 4 ml of O. 5 mol/L methanolic sodium
hydroxide solution, and heat under reflux on a water bath for
10 minutes, allow to cool and add 5 ml of 14% boron
trifluoride methanol solution, and heat under reflux for 2
minutes, cool. Add 5 ml of n-heptane and heat under reflux
far 1 minute. Cool, add 10 ml of saturated sodium chloride
solution, shake well and allow to stand. Take the supernatant
and dry over a small quantity of anhydrous sodium sulfate,
as the test solution. Carry out the method for gas
chromatography ( 0521 ) , using a column packed with 100 %
poly cyanopropyl siloxane as the stationary phase, maintain
the column temperature at 120ºC for 3 minutes, then raise at
a rate of lOºC per minute to 180ºC, maintain for 5. 5
minutes, then raise at a rate of 15ºC per minute to 215ºC,
and maintain far 3 minutes. The temperature of the injection
port is maintained at 250ºC and that of detection is at 280ºC.
Dissolve methyl myristate CRS, methyl palmitate CRS,
methyl stearate CRS, methyl oleate CRS, methyl linoleate
CRS, methyl linolenate CRS, methyl hebenate CRS, and
methyl arachidate CRS in n-heptane to produce a mixture
containing O. 5 mg per ml respectively. lnject O. 2 µl of the
mixture solution onto the column and record the
chromatograms, the number of the theoretical plates
calculated for the methyl palmitate peak is not less than
20 000. Inject O. 2 µl of the test solution onto the column and
calculate the peak areas by area normalization method.
Saturated fatty acids of carbon chain length less than 14: not
more than O. 1 % ; myristic acid: not more than O. 5 % ;
palmitic acid: 9. 0%-16. o%; stearic acid: 79. 0%-89. o%;
oleic acid: not more than 4. O% ; linoleic acid: not more than
l. 0%; linolenic acid: not more than O. 2%; arachidic acid:
not more than l. O% ; behenic acid: not more than l. O%.
Category Pharmaceutical excipients, lubricant and release
retardan t.
Storage Preserve in tightly closed containers, protected
from light and stored in a cool and dark place.
Hydroxyethyl Cellulose
[9004-62-0]
Hydroxyethyl Cellulose is a nonionic water soluble cellulose
ether, obtained from etherification of alkalinic cellulose and
ethylene oxide ( or 2-chloroethanoD.
Description White or greyish white or pale yellow white
powder or granules.
Swells in hot or cold water and produce a colloidal solution,
practically insoluble in acetone, in ethanol and in meth-
ylbenzene.

Viscosity Determine the viscosity of 1 % aqueous solution of
the substance being examined, using a rotating viscometer
with No. 2 rotator at the speed of 12 rpm at 25ºC ±O. 1 ºC
( 0633 method 3). Allow to follow the labeled concentration
and condition of solution to determine, the viscosity is not
less than 50 % and not more than 150 % of the labeled
viscosity.
Identification (1) Dissolve 1 gin 100 ml of water, stir till
completely dissolved to get a colloidal solution, the solution
remains clear when heated to 60ºC.
(2) Transfer 1 ml of the solution prepared in Identification
(1) on a glass plate, allow to evaporate water, a film is
formed.
(3) Transfer 10 ml of the solution prepared in Identification
(1), add O. 3 ml of diluted acetic acid and 2. 5 ml of 10%
tannic acid solution, yellowish white floccule is disappeared
when add diluted ammonia solution.
( 4) Dilute 1 ml of O. 05 % aqueous solution with 1 ml of 5 %
phenol aqueous solution, add 5 ml of sulfuric acid, shake,
cool. The colour of the solution changes to orange.
Acidity or alkalinity Dissolve 1 g with 100 ml of water, pH
6. 0-8. 5 <0631) .
Chlorides Dissolve O. 5 g with 100 ml of water. Carry out
the determination < 0801 ) , using l. O ml. Any colour
produced is not more intense than that of a reference solution
using 5. O ml of sodium chloride standard solution (l. 0%).
Nitrate Test solution Weigh accurately O. 5 g to a 100 ml
volumetric flask, dissolve and dilute to volume with buffer
solution and mix well.
Standard stock solution W eigh accurately O. 2038 g of
potassium nitrate to a 250 ml volumetric flask, dissolve and
dilute to volume with buffer solution etransfer 135 g of
potassium dihydrogen phosphate to a 1000 ml voltL.~etric
flask, dissolve in appropriate amount of water, add 50 ml of 1
mol/L sulphuric acid, diluted to volume with water, and
mix. Transfer 80 ml of this solution, add water to 2000 ml,
and mix) , and mix to produce potassium nitrate standard
stock solution Cthe concentration of N03 is about O. 5 mg per
mD.
Reference solution 1 Capply to samples with viscosity not
more than 1000 mPa • s) Transfer accurately 10 ml, 20 ml
and 40 ml of standard stock solution to 100 ml volumetric
flask respectively, dilute to volume with buffer solution and
mix well.
Reference solution 2 (apply to samples with viscosity more
than 1000 mPa • s) Transfer accurately 1 ml, 2 ml and
4 ml of standard stock solution to 100 ml volumetric flask
respectively, dilute to volume with buffer solution and mix
well.
Procedure Carry out the method for potentiometric
titration <0701 ) , using ni trate indicator electrode and silver-
silver chloride reference electrode. Determine electric
potential value of referece solution 1 or 2. Prepare standard
curre of E-lgc by linear regression between electric potential
CmV) and logarithm of coucentration of ni trate Clgc).
Determine electric potential value of test solution, calculated
on dried basis. Not more than 3. O% Cfor labeling viscosity
not more than 1000 mPa • s) or not more than O. 2% ( for
labeling viscosity more than 1000 mPa • s) .
Glyoxal Dissolve l. O g with 10. O ml of anhydrous ethanol
in a test tube with stopper. Stopper the tube and stir with
magnetic stirrer for 30 minutes, centrifuge. To 2. O ml of the
supernatant, add 5. O ml of a O. 4% solution of methylbenzo--
Hydroxyethyl Cellulose
2-thiazolonehydrazone hydrochloride in an 80 % solution of
glacial acetic acid, shake well. Allow to stand 2 hours, the
colour of solution is not more intense than that of a glyoxal
standard solution using 2. o ml etake appropriate amount of
calibrated glyoxal solution, diluted with dehydrated alcohol
to get reference solution with Ci Hz Oz of 20 µg per mD
instead of supernatant (0. 002%).
Calibration of glyoxal Weigh accurately l. O g of glyoxal
solution e 40 % ) ' add 20 ml of 7 % hydroxylamine
hydrochloride solution and 50 ml of water, shake well, stand
for 30 minutes, add 1 ml of methyl red mixed indicator 0%
methyl red-O. 02 % methylene blue in ethonal solution) ,
titration with sodium hydroxide (1 mol/L) VS until solution
become green from red. Perform a blank determination, each
ml of sodium hydroxide (1 mol/L) VS is equivalent to 29. 02
mg of glyoxal CCi Hz Oz ).
Ethylene oxide W eigh accurately 1 g of the substance being
examined into a headspace vial and add l. O ml of super
purified water, seal and mix well as test solution.
Transfer 300 µl of ethylene oxide Cequivalent to O. 25 g of
ethylene oxide) in a 100 ml volumetric flask that contains
50 ml of processed polyethylene glycol 400 ewhich was rotary
evaporated for 6 hours at 60ºC and l. 5-2. 5 kPa to remove
volatile ingredients), weigh, dilute to volume with the same
solvents and mix well as standard stock solution.
Dissolve 1 g of cooled ethylene oxide standard stock solution
in a 50 ml volumetric flask that contains 40. O g of processed
polyethylene glycol 400, dilute to volume with the same
solvents, and mix well. Transfer 10 g of this solution in a 50
ml volumetric flask that contains 30 ml of water, dilute to
volume with water and mix well to produce solution with 10
µg per ml of ethylene oxide as reference solution e1).
Weigh accurately 1 g of the substance being examined into a
headspace vial and add O. 1 ml of reference solution ( 1) and
O. 9 ml of water, seal, mix well as reference solution (2).
Transfer accurately O. 1 ml of reference solution C1) into a
vial, add O. 1 ml of freshly prepared O. 001% acetaldehyde
solution, and mix well as the system suitability test solution.
Carry out the method for gas chromatography ( 0521 ) .
Using polydimethylsiloxane as the stationary phase, maintain
initial temperature at 50ºC for 5 minutes, raise temperature
to 180ºC by rate of 5ºC per minute, then raise temperature to
230ºC by rate of 30ºC per minute, maintain 5 minutes.
Injection port temperature is 150ºC, and the detector
temperature is 250ºC, Maintain head space tube at 70ºC for
45 minutes. Inject system suitability test solution into the
chromatograph, adjust detection sensitivity to make sure the
peak height of ethylene oxide and acetaldehyde is about 15 %
of full height, and the resolution of ethylene oxide and
acetaldehyde is not less than 2. O. Inject separately test
solution and reference solution, repeat at least 3 times. The
relative standard deviation of ethylene oxide area is not more
than 15 % , calculate ethylene oxide content by standard
addition method, the limit is O. 0001%.
Calibration of ethylene oxide reference stock solution.
Transfer accurately 10 ml of 50 % magnesium chloride
supension in anhydrous ethanol, add accurately 20 ml of
hydrochloric acid ethonal CO. 1 mol/L) VS, shake well,
stand overnight. Then add accurately 5 g of ethylene oxide
standard stock solution, stand for 30 minutes. Carry out the
method for potentionmetric titrition < 0701 ) , titrate with
potassium hydroxide ethonal CO. 1 mol/U VS. Perform a blank
determination, each ml of potassium hydroxide ethonal eo. 1
mol/L) VS is equivalent to 4. 404 mg of ethylene oxide.
Ethylene glycol and diethylene glycol Weigh accurately O. 1 g
 554 Hydroxypropyl Betadex
into a 25 ml volumetric flask, transfer accurately 1 ml of
internal standard solution ( Transfer O. 2 g of 1, 3-butylene
glycol CRS into a 100 ml volumetric flask, add anhydrous
ethanol to dissolve and dilute to volume, shake welD, add
anhydrous ethanol to swell and dilute to volume, shake well
and filter, use subsequent filtra te as test solution.
Weigh accurately O. 2 g of ethylene glycol and diethylene
glycol respectively into a 100 ml volumetric flask, add
anhydrous ethanol to dissolve and dilute to volume, shake
well, then accurately transfer 5 ml into a 100 ml volumetric
flask, add 5 ml of internal standard solution, add anhydrous
ethanol to volume, shake well, transfer 2 ml into a 50 ml
volumetric flask, add anhydrous ethanol to volume, mix well
as reference solution.
Carry out the method for gas chromatography ( 0521 ) .
Using a mixture of phenyl group-polydimethylsiloxane (50 :
50) as the stationary phase, maintain initial temperature at
60ºC for 5 minutes, raise temperature to lOOºC by rate of
lOºC per minute, then raise temperature to l 70ºC by rate of
4 ºC per minute, raise temperature to 270ºC by rate of lOºC
per minute, maintain 2 minutes. Carrier gas is high purity
nitrogen. Injection port temperature is 270ºC, and the
detector temperature is 290ºC. Calculate ethylene glycol and
diethylene glycol content by internal standard method, not
more than O. 01 % respectively.
Loss on drying When dried at 105ºC for 3 hours, loses not
more than 10. O% of its weight <0831 ) , using l. 000 g.
Residue on ignition Not more than 5. O% <0841 ) , using
l. o g.
Heavy metals Carry out the limit test for heavy metals
<0821 method 2) , using the residue obtained in the test for
Residue on ignition, not more than O. 002%.
Category Pharmaceutical excipients, thickening agent
coating material, stabilizer adhesive and suspending agent,
etc.
Storage Preserve in tight container.
Labling Stated the viscosity with mPa • s or Pa •s.
Hydroxypropyl Betadex
[128446-35-5 J
Hydroxypropyl betadex is ,B-cyclodextrin 2-hydroxypropyl
ether. It contains is not less than 19. 6% and not more than
26. 3% of hydroxypropoxy (-OCH2CHOHCH3), calcul-
ated on the anhydrous basis.
Description A white or almost white, amorphous or
crystalline powder, odourless; tas te, slightly sweet; very
hygroscopic.
Very soluble in water; freely soluble in methanol and in
ethanol; practically insoluble in acetone and in chloroform.
ldentification Transfer O. 5 ml of the 5 % aqueous solution
into a 10 ml test tube, add 2 drops of 10 % solution of
a-naphthol in ethanol and shake well. Add slowly 1 ml of
sulfuric acid along the inner wall of the test tube, and a
purple ring develops at the interface of the two solutions.
Acidity or alkalinity Dissolve l. O g in 40 ml of water, pH
5.0-7.5 (0631>.
Clarity and colour of solution Dissolve 2. 5 g in 25 ml of
water, solution is clear and colourless <0901 and 0902) .
Chlorides Carry out the limit test for chlorides <0801 ) ,
using O. 1 g. Any opalescence produced is not more
pronounced than that of a reference solution prepared by
using 5. O ml of sodium chloride standard solution (0. 05%).
Betadex Carry out the method for high performance liquid
chromatography ( 0512), using a column packed with amino
bonded silica gel, a mixture of acetonitrile-water (65 : 35) as
the mobile phase, and diff erential refractometers as a
detector. Maintain the column temperature at 35ºC .
Transfer about l. O g of the substance being examined,
accurately weighed, to a 10 ml volumetric flask, add a
volume of water to dissolve and dilute to volume with water,
shake well and using the resulting solution as the test
solution. Transfer about 50 mg of betadex CRS, accurately
weighed, to a 100 ml volumetric flask, dissolve and dilute to
volume with water, shake well and using the resulting
solution as the reference standard solution. Inject 20 µl of
each of the reference standard solution and the test solution
into the column respectively, and record the chromatograms.
Calculate the content of hydroxypropyl betadex with respect
to the peak area obtained in the chromatogram by the
external standard method, not more than O. 5 % .
1, 2-Propylene glycol Carry out the method for deter-
mination of residual solvents < 0861 ) , using a capillary
column coated with 100 % dimethyl polysiloxane. Maintain
the column temperature at 70ºC, then raise the temperature
to lOOºC at a rate of lOºC per minute, and again raise the
temperature to 220ºC ata rate of 40ºC per minute. Maintain
for 4 minutes. The injector temperature is 250ºC, and that
of the detector is 280ºC. The number of the theoretical plates
calculated for the 1, 2-propyleneglycol peak is not less than
3000. Transfer about 1 g of the substance being examined,
accurately weighed, into a 10 ml volumetric flask, add a
volume of methanol to dissolve and dilute to volume with the
same solvent, shake well and using the resulting solution as
the test solution; Transfer about 50 mg of 1, 2-propylene
glycol, accurately weighed, into a 100 ml volumetric flask,
dilute to volume with methanol, shake well and using the
resulting solution as the reference solution. Inject equal
volume of the reference solution and test solution, into the
column respectively, and record the chromatograms.
Calculate the content of propylene glycol with respect to the
peak area obtained in the chromatogram by the external standard
method, not more than O. 5 % .
Water Not more than 6. O% ( 0832 method 1 ( 1 ) ) .
Residue on ignition Not more than O. 2 % <0841 ) , using
l. o g.
Heavy metals Carry out the limit test for heavy metals
<0821 method 2), using the residue obtained in the test far
residue on ignition: not more than O. 001%.
Assay Hydroxypropoxy Carry out the method for the
determination of methoxyl, ethoxy and hydroxypropoxy <0712) ,
using about O. 1 g of the substance being examined, accurately
weighed. Calculate the content of-OCH2 CHOHCH3.
Category Pharmaceutical excipients, packing agent and
stabilizer.
Storage Preserve in tightly closed containers and protected
from light.

Hydroxypropyl Cellulose
OH
Fº~
OR
o~
H
OR
n/2
[9004-64-2]
Hyprolose cellulose is partly substituted of 2-hydroxypropyl
ether cellulose. It contains not less than 53. 4 % and not more
than 77. 5 % of hydroxypropoxy group C-OCH2 CHOHCH3 ) ,
calculated on dried basis.
Description A white or almost white granule or powder.
Hygroscopic after drying.
Disperses and swells in water or in ethanol to produce a
colloidal solution; practically insoluble in ether.
Identification ( 1 ) weigh l. O g and add 100 ml of hot
water. Stir to farm slurry liquid. Cool in ice bath to produce
a viscous liquid; transfer 2 ml of the liquid to a test tube and
add 1 ml of O. 035 % anthrone solution in sulfuric acid slowly
along the tube inner wall, stand far 5 minutes, a bluish-
green ring is produced at the interface of the two layers.
(2) Place a volurne of the viscous liquid obtained m
Identification ( 1) on a glass plate. Allow the water to
evaporate anda film is farmed.
(3) Transfer 10 ml of viscous liquid obtained in Identification
test (1), add 1 g sodium hydroxide, and shake well. To O. 1
ml add sulfuric acid (9-+ 10) 9 ml, shake. Heat over water
bath far 3 minutes and cool in ice bath immediately. After
cooling, add O. 6 ml of ninhydrin solution ( dissolve O. 2 g
ninhydrin with 10 ml water, prepare the solution immediately
befare use) and shake well. Stand at room temperature and
the red colour is produced immediately. It turns to purple
after standing far 100 minutes.
( 4) Place a volume of the viscous liquid obtained in
Identification test ( 1), heat in water bath while stirring
and produce turbid or a fluocculent precipitate when the
temperature is over 40ºC. Allowed to cool, the solution is
clear again.
Viscosity Dissolve a quantity of substance being examined,
calculated on the dried basis, in hot water of 90ºC to produce
a 2. o% (g/g) solution, or 5. o% (g/g) ( if the labeled
viscosity below 150 mPa • s) solution. Stir thoroughly far
about 10 minutes until the granules are dispersed and
moistened completely ( all granules dissolved completely on
inner wall of the flask). Cool in ice bath while stirring,
remove any air bubbles and adjust the weight with cold
water. Determine the viscosity ( 0633 method 3 ) with
suitable single column type rotational viscometer (Brookfield
type LV Model, or equivalent), at 20ºC ± O. 1 ºC, the
visco si ty is not less than 7 5 % and not more than 140 % of
labeled.
Acidity or alkalinity Dissolve l. O g ( calculated on dried
basis) in 50 ml water ( 90ºC) while stirring. Allow to cool
Hydroxypropyl Cellulose 555:
and dilute to 100 ml with water. Stir until dissolves
completely and determine the pH ( 0631 ) , pH 5. 0-8. 5.
Chloride Dissolve O. 10 g in 50 ml of hot water while
stirring, mix well. Cool in ice bath and transfer to 100 ml
volumetric flask. Dilute to volume with water and shake
well. Carry out the limit test far chlorides ( 0801 ) using
10 ml of above solution. Any colour produced is not more
intense than that of a reference solution using 5. O ml of
standard sodium chloride solution (O. 5 % ) .
Residual solvents Isopropyl alcohol and toluene Weigh
accurately 50 mg to a headspace bottle, add 2 ml dimethyl
sulfaxide accurately to dissolve the substance. Seal it as test
sample solution; weigh a quantity of isopropyl alcohol and
toluene accurately and dilute with dimethyl sulfaxide to
produce a mixed solution with O. 125 mg of isopropyl alcohol
and O. 022 mg of toluene per ml. Transfer accurately 2 ml to
the headspace bottle and seal as the reference solution. Carry
out the test far residual solvents ( 0861 method 2). Using a
capillary chromatographic column with 6% cyanopropy-
lphenyl-94 % dimethyl siloxane as the stationary phase ( or
other stationary phase with similar polarity); initial tem-
perature is 50ºC, maitain far 2 minutes. raise the tem-
perature to 220ºC with arate of 15ºC per minute and maitain
far 1 minute; the temperature of detector is 250ºC; the
temperature of injection is 220ºC. The balance temperature
of headspace bottle is 70ºC, the balance time is 30 minutes.
The resolutions among different peaks of components far the
reference solution meet the requirement. Inject the test
solution and reference solution separately and record the
chromatogram. Calculate the peak area by externa! standard
method. It should comply with the requirement.
Loss on drying When dried at 105ºC to constant weight,
loses not more than 7. O% ( 0831 ) .
Silicon dioxide Transfer l. O g in platinum crucible and
ignite at lOOOºC to constant weight. Moisten the residue with
water and add 10 ml of hydrofluoric acid. Dry on water bath.
Allow to cool. Add 10 ml of hydrofluoric acid and O. 5 ml of
sulfuric acid. Dry on water bath. Then heat over an electric
stove to evaporate the acid vapour. Ignite at lOOOºC to
constant weight. Allow to cool and weight accurately. The
difference value compared with Residue on ignition is the
weight of silicon dioxide. Not more than O. 6 % , calculated
on dried basis.
Residue on ignition Not more than l. O% <0841 ) , using
l. o g.
Heavy metals Carry out the limit test far heavy metals
( 0821 method 2), using the residue obtained in the test far
Residue on ignition: not more than O. 002%.
Arsenic Mix l. O g with l. O g calcium hydroxide well. Add
a volume of water, stir well. After drying, heat gently until
completely charred, and then ignite at 500-600ºC until the
incineration is completed. Allow to cool, add 8 ml of
hydrochloric acid and 23 ml of water to dissolve the residue.
Carry out the limit test far arsenic ( 0822 method 1 ) : not
more than O. 0002%.
Assay Hydroxypropoxy groups Carry out the method far
determination of methoxy, ethoxyl and hydroxypropoxy
( 0712 ) . Far method 2 ( volumetric method), weigh
accurately O. 1 g of the substance.
Category Pharmaceutical excipients, disintegranting agent
and filler, etc.
Storage Stored in well closed container in dry place.
 Hypromellose
Labeling Stated the visicosity with mPa •s.
Hypromellose
[9004-65-3]
Hypromellose is 2-hydroxypropyl ether methyl cellulose, a
semi-synthetic fiber, produced by two methods: (1) Treated
lint or wood pulp fibers with sodium hydroxide, and then has
a reacts with chloromethane and propylene oxide, refined,
crushed; (2) Treated suitable levels of methyl cellulose with
sodium hydroxide, and propylene oxide at high temperature
and pressure to reflect the desired level, purified. Molecular
weight range from 10 000 to 1 500 000.
Hypromellose was divided into 1828, 2208, 2906 and 2910
types based on the content of methoxy and hydroxypropoxyl.
The content of methoxy and hydroxypropoxyl was calculated
on the dried basis and meet requirements of the following
table.
Replace type methoxy hydroxypropoxyl
1828 16. 5%-20. o% 23. o %-32. o%
2208 19. 0%-24. 0% 4. 0%-12. 0%
2906 27. 0%-30. 0% 4. 0%-7. 5%
2910 27. o%-3o. o% 7. 0%-12. o%
Description A white or almost white fibrous powder or
granular powder, odorless.
Practically insoluble in dehydrated ethanol, in acetone and in
ether. Disperses and swells in cold water to produce a clear
or slightly opalescent colloidal liquid.
Identification ( 1) Dissolve 1 g with 100 ml of hot water
(80-90ºC) while stirring, cool in an ice bath to give a viscous
liquid. Transfer 2 ml of the liquid to a test tube and add 1 ml
of O. 035 % anthrone solution in sulfuric acid slowly along the
inner wall of the tube, stand for 5 minutes, a bluish-green
ring is produced at the interface of the two layers.
(2) Place a volurne of the viscous liquid obtained m
Identification ( 1) on a glass plate and allow the water to
evaporate, an elastic film is formed.
(3) Dissolve O. 5 g with 50 ml of boiling water while stirring
to form insoluble slurry, cool to lOºC with continued stirring
and become clearly and slight precipitation, add 50 ml water,
stirring and heating at the speed of 2-5ºC per minute to form
muddy, and the temperature is not less than 50ºC.
Viscosity For viscosity labeled less than 600 rnPa • s, follow
method 1, the viscosity is not less than 80 % and not more
than 120 % of labeled. For viscosity labeled more than
600 mPa • s, follow method 2 , the viscosi ty is not less than
75% and not more than 140% of labeled. To a quantity of
the substance being exarnined, calculated on the dried basis,
add hot water of 90ºC to produce a 2. 0% (g/g) solution.
Stir thoroughly for about 10 minutes until the granules are
dispersed and moistened completely, cool in an ice bath and
stirring, remove any air bubbles of solution and adjust the
weight of the solution with cool water if necessary. Remove
all bubbles as test solution.
Method 1 : Measure the kinematic viscosity ( v) of solution at
20ºC ±O. 1 ºC, using a appropriate diameter of ubbelohde
viscosimeter by elute time is not less than 200 seconds, and
testing the density ( p) of solution at the same condition,
calcula te the dynamic viscosity as ( r¡) = pv.
Method 2: Carry out the test for viscosity with a suitable
single colurnn type rotational viscometer ( BrookfieldtypeLV
Model, or equivalent), at 20ºC ± O. 1 ºC, reference the
following table parameters ( 0633 method 3) , rotating for 2
minutes and read digital, stand for 2 minutes and duplica te
test for 2 times, adoption mean.
Labeled viscosity (mPa•s) Type of rotor Speed (r/min)
600-1400 3 60
1400-3500 3 12
3500-9500 4 60
9500-99 500 4 6
>99 500- 4 3
Acidity or alkalinity Dissolve l. O g in 50 ml hot water of
90ºC, calculated on the dried basis, and stir for dissolving,
after cooled add water to 100 ml, stirring until fully dissolve,
pH 5. 0-8. O <0631 ) .
Water-insoluble substances Transfer l. O g to a beaker, add
100 ml of hot water (80-90ºC) to swell for about 15 minutes.
Cool in an ice bath, add 300 ml of water (add more water for
high viscosity sample to make sure that the solution is
passed), stir well and filter with No. 1 sintered glass
crucible, previously dried at 105ºC to constant weight, wash
the beaker vlith v;ater and combine thc vvashings to thc
crucible, filter. Dry the residue to constant weight at 105ºC.
The weight of the residue is not more than 5 mg (O. 5%).
Loss on drying When dried at 105ºC for 2 hours, losses not
more than 5. O% ( 0831 ) .
Residue on ignition Not more than l. 5 % ( 0841 ) , using
l. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2) , using the residue obtained in the test for
Residue on ignition, not more than O. 002%.
Arsenic Mix l. O g with l. O g of calcium hydroxide, add
water and mix well. After dryness, heat gently until it is
thoroughly charred, ignite at 600ºC until the incineration is
completed. Dissolve the cooled residue in a mixture of 5 ml
of hydrochloric acid and 23 ml of water. Carry out the limit
test for arsenic ( 0822 method 1 ) , not more than O. 0002 % .
Assay Methoxy group Carry out the method for deter-
mination of methoxy, ethoxy and hydroxypropoxy ( 0712 ).
If carry out method 2 ( volumetric method) , weigh accurately
the substance being examined and proceed as described.
Calculate the content of -OCH 3 from the following exp-
ress1on.
Content of -CH30 C%)=A-CBX31/75XO. 93)
Where A is the assay value of methoxy group ( % ) ;
B is the assay value of hydroxypropoxy group ( % ) .
Hydroxypropoxy group Carry out the method for deter-
mination of methoxy, ethoxy and hydroxypropoxy ( 0712 ).
If carry out method 2 ( volumetric method) , weigh accurately
about O. 1 g and proceed as described.
Category Pharmaceutical excipients, release retardants and
coa ting material , etc.
Storage Preserve in well closed containers.
Labeling Stated the replace type and viscosity in mPa •s.

Hypromellose Acetate Succinate
[71138-97-1]
Hypromellose Acetate Succinate is a mixture of acetic acid
and succinic acid esters of hydroxypropyl methylcellulose. It
contains not less than 12. O% and not more than 28. O% of
methoxy groups, not less than 4. 0% and not more than
23. 0% of 2-hydroxypropoxy groups, not less than 2. 0% and
not more than 16. O% of acetyl groups, and not less than
4. O% and not more than 28. O% of succinoyl groups,
calculated on the dried basis.
Description A white or pale yellow powder or granules,
odorless, tasteless.
Practically insoluble in ethanol and in water, soluble in
methanol and in acetone, disperse and swells in cold water
and produce a clear or slightly opalescent colloidal liquid.
Viscosity To 2. 00 g of the substance being examined
(previously dried), add sodium hydroxide solution to 100 g,
and constant shaking for 30 minutes. The kinetic viscosity at
20ºC ± O. 1 ºC is not less than 80 % and not more than 120 %
of labeled ( 0633 method 2).
Acetic and succinic acids Place 102 mg of substance being
examined, accurately weighed into a conical flask. Transfer
4. O ml of phosphate solution (adjust O. 02 mol/L potassium
dihydrogen phosphate solution to a pH of 7. 5 with 1 mol/L
sodium hydroxide) to the flask and constant stirring for 2
hours. Add 4. O ml of the phosphoric acid solution ( transfer
l. O ml of l. 25 mol/L phosphoric acid into a 50 ml
volumetric flask, and dilute to volume with water) . Shake,
centrifuge, using the supematant as test solution. Weigh
accurately about 130 mg of succinic acid into a 100 ml
volumetric flask. Add a quantity of water, shake the
contents until the succinic acid is fully dissolved, and dilute
to volume with water as succinic acid stock solution. Add
approximately 20 ml of water to a 100 ml volumetric flask,
weigh. Add 2. O ml of the glacial acetic acid to the flask, and
record the weight of the acid added. Dilute to volume with
water. Transfer accurately 6. O ml of the solution into a
100 ml volumetric flask, and dilute to volume with water as
acetic acid stock solution. Transfer separately 4. O ml of the
acetic acid stock solution and succinic acid stock solution into
the same 25 ml volumetric flask and dilute to volume with
mobile phase as standard solution. Carry out the method for
high performance liquid chromatography ( 0512), using a
column packed with bonded silica gel and O. 02 mol/L
potassium dihydrogen phosphate solution (adjust O. 02 mol/L
potassium dihydrogen phosphate solution to a pH of 2. 8 with
6 mol/L phosphoric acid solution) as the mobile phase, flow
rate is l. O ml per minute, detection wavelength is 215 nm.
Inject 10 µl of standard solution, the number of theoretical
plates of the column is not less than 8000, calculated with
reference to the peak of succinic acid peak. lnject separately
10 µl each of test solution and standard solution into the
column, and measure the peak areas corresponding to acetic
and succinic acids. Calculate the percentage of free acetic acid
and succinic acid using the following expression.
Percentage of acetic acid=O. 0768 (WA/W) CruA/rSA),
Where: WA is the weight of glacial acetic acid, in mg, used
to prepare the acetic acid stock solution; W is the weight of
hypromellose acetate succinate, in mg, used to prepare the
test solution; and ruA and rsA are the peak area for acetic acid
,,
Hypromellose Acetate Succinate 551'
1
obtained from the test solution and the standard solution,
respectively.
Percentage of free succinic acid=l. 28 CWs/ Wus) (rus/ rss)
Where: Ws is the weight of succinic acid, in mg, used to
prepare the succinic acid stock solution; W us is the weight of
Hypromellose acetate succinate, in mg, used to prepare the
test solution; rus and rss are the peak responses for succinic
acid obtained from the test solution and the standard
solution, respectively.
The sum of free acetic acid and free succinic acid found is not
more than l. O%.
Loss on drying When dried to constant weight at 105ºC,
loses not more than 5. O% of its weight ( 0831 ) .
Residue on ignition Not more than O. 2% ( 0841 ) , using
1 g.
Heavy metals Carry out the limit test for heavy metals
( 0821), using the residue obtained in the test for residue on
ignition, not more than O. 001%.
Arsenic Mix l. O g of the substance being examined with
l. O g of calcium hydroxide, add water and mix well. Dry,
heat gently until it is thoroughly charred, then ignite at 500-
600ºC until the incineration is completed. Cool, add 5 ml of
hydrochloric acid and 23 ml of water. Carry out the limit test
for arsenic ( 0822 method 1), not more than O. 0002%.
Assay Content of acetyl and succinoyl groups carry out the
method for high performance liquid chromatography
( 0512), chromatography conditions and chromatographic
system as directed in the test for acetic and succinic acids.
Weigh accurately about 12. 4 mg of the substance being
examined into a conical flask. Add 4. O ml of l. O mol/L
sodium hydroxide to the flask, and stir the solution for 4
hours. Then, add 4. O ml of l. 25 mol/L phosphoric acid to
the same vial to adjust the pH of the solution to 3 or less,
and pass through a O. 22 µm filter. Use the filtrate as test
solution. Inject separately 10 µl of standard solution in the
test for Acetic and succinic acids and test solution into the
column, record the chromatograms, and measure the peak
areas. Calculate the percentage of acetic acid A by the
formula.
Percentage of acetic acid A =O. 0768(WAI Wu)(ruAI rSA)
Where: WA is the weight of acetic acid, in mg, used to
prepare the acetic acid stock solution; W u is the weight of
substance being examined, in mg, used to prepare the test
solution; and ruA and rSA are the peak responses for acetic
acid obtained from the test solution and the standard
solution, respectively.
Calculate the percentage of acetyl groups by the formula.
Percentage of acetyl groups=O. 717 (A -Arree), Where A1ree
is the percentage of free acetic acid, as determined in the test
for acetic and succinic acids; and A is as defined above.
Calculate the percentage of succinic acid by the formula.
Percentage of succinic acid=l. 28 (Ws/ Wu) (rus/ rss),
Where: W s is the weight of succinic acid, in mg, used to
prepare the Succinic acid stock solution; Wu is as defined
above; and rus and rss are the peak responses for succinic
acid obtained from the test solution and the standard
solution, respectively.
Calculate the percentage of succinoyl groups by the formula.
Percentage of succinoyl groups =O. 856 CS- Srree), Where:
S is the percentage of succinic acid, and S1ree is the
percentage of free succinic acid, as determined in the test for
acetic and succinic acids.
Note: Wash in turns the column -with a mixture of water-
acetonitrile ( 1 : 1 ) and -with methanol for 60 minutes, and
store in methanol.
 Hypromellose Phthalate
Content of methoxy and 2-hydroxypropoxy groups
Methoxy Carry out the method for determination of methoxy
( 0712). If carry out method 2 ( volumetric method), weigh
accurately the substance being examined and proceed as
described. Calculate the content of methoxy using the
following expression. Content of methoxy =A - (B X 31/
75XO. 93)
Where A is the assay value of methoxy group ( % ) ;
B is the assay value of hydroxypropoxy group ( % ) .
Hydroxypropoxy group Carry out the method as
described ( 0712 ) . Use the substance and proceed as
described.
Category Pharmaceutical excipients, coating material.
Storage Preserve in well tightly closed containers.
Labling Stated the viscosity with mPa • s or Pa •s.
Hypromellose Phthalate
[9050-31-1]
Hypromellose phthalate is a monophtha late ester of
hypromellose and phthalate. It contains not less than 21. O%
and not more than 35. O% of phthalyl groups, calculated on
the dried basis.
Description A white or almost white powder or granule,
odorless and tasteless.
Practically insoluble in water and anhydrous alcohol, slightly
soluble in acetone and in toluene, soluble in a mixture of
aceto ne and methanol ( 1 : 1) and a mixture of methanol and
methylene chloride ( 1 : 1).
Viscosity Dissolve 10 g of hypromellose phthalate,
previously dried at 105ºC for 1 hour, add 90 g of a mixture of
methanol and methylene chloride ( 1 : 1) ( g/ g). Carry out
the determination of viscosity at 20ºC ±O. 1 ºC ( 0633 method
2) , the viscosity is not less than 80 % and not more than
120 % of the labeled viscosity.
ldentification The infrared absorption spectrum is con-
cordant with the reference spectrum ( 0402).
Chlorides Dissolve O. 1 g in 40 ml of O. 2 mol/L sodium
hydroxide solution, add 1 drop of phenolphthalein IS and add
diluted nitric acid with stirring until the red colour
disappears. Add 5 ml of diluted nitric acid, heat to boil, and
a colloidal precipitate is produced. Cool and filter. Wash the
precipitate with a small amount of distilled water for several
times. Combine the filtrate and mix well, transfer to a 50 ml
nessler cylinder as the test solution. Carry out the limit test
for chlorides ( 0801), the colour is not more intense than a
reference solution by using 7. O ml of sodium chloride
standard solution (O. 07%).
Free phthalicacid Weigh accurately O. 2 g of substance to be
examined into a 100 ml volumetric flask, add about 50 ml of
acetonitrile and dissolve partially by ultrasonication. Then
add 10 ml of water, and ultrasonic to dissolve completely.
Dilute to the volume with acetonitrile and mix well, as the
test solution. Weigh accurately 10 mg of phthalic acid CRS
and dissolve with acetonitrile in a 50 ml volumetric flask,
dilute to the volume and shake well. Transfer accurately 5 ml
of solution prepared above into a 50 ml volumetric flask, add
5 ml of water, dilute to the volume with acetonitrile and
shake well, as the reference solution. Carry out the method
for high performance liquid chromatography ( 0512), using
a column packed with octadecylsilanebonded silica gel and a
mixture of acetonitrile-0. 1 % trifluoroaceticacid ( 1 : 9) as
the mobile phase, flow rate is 2. O ml per minute; detection
wavelength is 235 nm. Inject 10 µl of reference solution into
column for 6 times, the relative standard deviation (RSD) of
the peak areas is not more than l. 0%. Inject separately 10 µl
of each of test solution and reference solution into the
column, record the chromatograms. When a peak has the
identical retention time with that of phthalic acid in the
chromatogram obtained with solution, its peak area is not
more than l. O%, calculated by the externa! standard
method.
Water Not more than 5. O% ( 0832 method 1 ( 1 ) ) .
Residue on ignition Not more than O. 2% ( 0841), using l. O g.
Heavy metals Carry out the limit test for heavy metals using
the residue obtained from residue on ignition ( 0821 method
2): not more than O. 001 %.
Arsenic Mix l. O g of the substance being examined with
l. O g calcium hydroxide, add water and mix well. After
dryness, heat gently until it is thoroughly charred, and ignite
at 600ºC until the incineration is completed. Cool and
dissolve the residue in a mixture of 5 ml of hydrochloride acid
and 23 ml of water. Carry out the limit test for arsenic
( 0822 method 1 ) , not more than O. 0002 % .
Assay Weight accurately l. O g, add 50 ml of a mixture of
ethanol, acetone and water (2 : 2 : 1). Add 2 drops of
phenolphthalein IS and ti trate with sodium hydroxide (O. 1
mol/L) \TS. Perform a blank determination and make any
necessary correction. Calculate the content of phthaloyl
groups by the following equation:
O. 01XVXFX149. 1
Content of phthaloyl groups ( % )
Cl-a)W
-2 X 149.1 X S
166. 1
Where 149. 1: molecular weight of phthaloyl groups;
166. 1: molecular weight of phthalic acid;
W: weight of substance being examined, g;
F: concentration correction factor of the sodium
hydroxide (O. 1 mol/L) VS;
V: volume of sodium hydroxide VS consumed, ml;
a: content of water in substance being examined
(%);
S : content of free phthalic acid in substance being
examined.
Category Pharmaceutical excipients, coating material.
Storage Preserve in tightly closed containers.
Labling Stated the viscosity with mPa • s or Pa •s.
Isopropyl Alcohol
C3HsO 60.10
[67-63-0]
Isopropyl alcohol is 2-propanol
Description A colourless and clear liquid.
Miscible with water, with methanol, with ethanol and with
ether.

Relative density O. 785-0. 788 <0601 method 2).
Refractive index l. 376-1. 379 <0622).
ldentification ( 1) Transfer 1 ml, add 2 ml of iodine TS
and 2 ml of sodium hydroxide TS, shake, a light yellow
precipitate and a characteristic odour of iodofarm are
produced.
(2) Mix 5 ml and 20 ml of potassium dichromate TS, add
5 ml of sulfuric acid carefully. A gas is evolved by gentle
heating on a water bath, which turns the filter paper,
previously wetted with a solution of salicylaldehyde in
ethanol ( 1 : 10) and with 30 % hydroxide solution, to
reddish brown.
(3) The infrared absorption spectrum <0402) is concordant
with the reference spectrum of isopropyl alcohol CRS.
Acidity Mix 50. O ml with 100 ml of water. Add 2 drops of
phenolphthalein IS and titrate with sodium hydroxide
(0. 01 mol/L) VS until a pink colour persists 30 seconds.
Not more than l. 4 ml of sodium hydroxide (O. 01 mol/U
VS is req uired.
Clarity and colour of solution Mix l. O ml with 20 ml of
water. Allow to stand far 5 minutes. The solution is clear
and colourless <0901 and 0902).
Ahsorbance The absorbance of the substance being examined is
not more than O. 30, O. 10, O. 03, O. 02 and O. 01 at 230 nm,
250 nm, 270 nm, 290 nm and 310 nm, respectively ( 0401 ).
Water insoluble substance Mix 2. O ml with 8 ml of water
and shake. Allow to stand far 5 minutes, the solution
maintains clear.
Nonvolatile matter Transfer 50. O ml to an evaporating dish
previously dried to constant weight at 105ºC on a water bath
to dryness, and dry at 105ºC far 1 hour: the residue is not
more than LO mg [O. 002% (g/m])],
Readily oxidizable substances Transfer 10. O ml to a
colourimetric tube with stopper, adjust the temperature to
15ºC, add O. 50 ml of potassium permanganate (O. 02 mol/U
VS, stopper and shake, allow to stand at 15ºC far 15 min,
the pink colour of the solution <loes not disappear
completely.
Readily carbonizable substances Transfer 5 ml of sulfuric
acid to a dry colourimetric tube, cool to lOºC, and add
dropwise 5. O ml of the substance being examined with
shaking ( the temperature <loes not exceed 20ºC). Any colour
produced is not more intense than that of the reference
solution Y1 ( 0901 ) .
Carbonyl compound Place O. 50 ml in a colourimetric tube
with stopper, add 1 ml of 2, 4-dinitrophenylhydrazine
solution (To 50 mg of 2, 4-dinitrophenylhydrazine, add 2 ml
of hydrochloric acid, and dilute to 50 ml with the solution
AO), stopper the cylinder and shake, allow to stand far 30
minutes. Add 8 ml of pyridine, 2 ml of water and 2 ml of
potassium hydroxide-methanol solution (to 15 ml of 33%
potassium hydroxide solution, add 50 ml of solution A,),
mix well, allow to stand far 30 minutes, and dilute to 25 ml
with solution A, mix well, the dark red colour produce is not
more intense than that of O. 50 ml of the reference solution
(co• ) obtained in the same way.
Water Not more than O. 2 % <0832 method 1 ( 1 ) ) .
Benzene and related substances Carry out the method far gas
chromatography < 0521 ) , using a capillary column coated
6 % cyanopropylphenyl -94 % dimethylpolysiloxane ( or
stationary phase with similar polarity), maintaining the
temperature of the injection port at 280ºC and the detector at
Kaolin
280ºC; maintain the column temperature at 40ºC far 12
minutes, then increased at a rate of lOºC per minute to 240ºC
far 10 minutes. The resolution between the peak of propano!
and the peak of 2-butanol is not less than 10.
Procedure Using the substance being examined as test
solution (a). Accurately measure 1 ml of 2-butanol in a 50 ml
volumetric flask, dilute with the substance being examined to
volume and shake well, accurately measure 5 ml in a 100 ml
volumetric flask, dilute with the substance being examined to
volume and shake well, as test solution ( b). Accurately
measure O. 5 ml of 2-butanol and O. 5 ml of propano! in a
50 ml volumetric flask, dilute with the substance being
examined to volume and shake well, accurately measure 5 ml
in a 50 ml volumetric flask, dilute with the substance being
examined to volume and shake well, as reference solution
(a). Accurately measure 100 µl of benzene in a 100 ml
volumetric flask, dilute with the substance being examined to
volume and shake well, accurately measure O. 2 ml in a
100 ml volumetric flask, dilute with the substance to be
examined to volume and shake well, as reference solution
(b). Inject respectively 1 µl of test solution (a), test solution
(b), reference solution (a) and reference solution (b) into
the column and record the chromatograms. The peak of
benzene in test solution (a) is not more than half of the area
of the corresponding peak in the chromatogram obtained with
reference solution ( b) (O. 0002%). The total peak of
impurities apart from 2-butanol in test solution (b) is not
more than 3 times the area of the peak due to 2-butanol in the
chromatogram obtained with reference solution (a) (0. 3%).
Category Pharmaceutical excipients, solvents.
Storage Preserve in tightly closed containers, protected
from light.
OSolution A: To 2000 ml of methanol, add 10 g of 2, 4-
dinitrophenylhydrazine and O. 5 ml of hydrochloric acid,
reflux 2 hours on a water bath, discard 50 ml of the initial
distillate and collect the distillate in a brown flask.
8Reference solution CCO): Weight accurately 10. 43 g of
acetone (equivalent to 5. 000 g of CO) to a 100 ml volumetric
flask, dilute with the solution A to volume and mix well.
Measure accurately 2. O ml to a 100 ml volumetric flask,
dilute with the solution A and mix well. Measure accurately
2. O ml to a 50 ml volumetric flask, dilute with the solution A
and mix well.
Kaolin
[68515-07-1]
Kaolin is a natural hydrated aluminum silicate, freed from
most of its impurities by elutriation with water, treated with
dilute mineral acid, washed with water repeatedly and dried.
Description A fine, almost white powder. When moistened
with water, it has an odour of clay and darkens in colour.
Practically insoluble in water, in dilute mineral acids, and in
sodium hydroxide solutions.
Identification Place 1 g in a porcelain evaporating dish, add
10 ml of water and 5 ml of sulfuric acid, heat until white
fumes of sulfur trioxide appear. Cool, add slowly 20 ml of
water, boil far 2-3 minutes and filter; a gray residue remains
on the filter. The filtrate yields the reactions characteristic of
aluminum salts (0301 ).
Chloride Boil O. 20 g with 25 ml of water and 1 drop of
 580 Lactase
nitric acid for 5 minutes and filter. Carry out the limit test
for chlorides < 0801 ) , using the filtrate. Any opalescence
produced is not more intense than that of a reference using
6. O ml of sodium chloride standard solution (O. 03%).
Sulfate Boil O. 3 g with 40 ml of water and 2 ml of dilute
hydrochloric acid for 5 minutes, cool and filter. Carry out
the limit test for sulfates <0802 ) , using the filtrate. Any
opalescence produced is not more intense than that of a
reference using 3. O ml of potassium sulfate standard solution
(0. 1%).
Acidsoluble substances Boil l. O g with 50 ml of hydrochloric
acid solution 08-1000) for 5 minutes, filter, evaporate the
filtrate to dryness and ignite to constant weight; the residue
weighs not more than 1O mg.
Loss on ignition When ignited to constant weight, loses not
more than 15. 0% of its weight.
Sand particles Place 2 gin a beaker, add 50 ml of water and
stir well, pour to a moistened sieve No. 7. Wash the beaker
several times with water until the substance being examined
is completely transferred to the sieve. Wash the sieve with
water to gather the residue. Stroke the sieve with fingers; no
feeling of gritty particles of sand.
Iron Boil O. 42 g with 25 ml of each of dilute hydrochloric
acid water for 2 minutes, cool and filter. Add water to the
filtrate to produce 100 ml and mix well. To 20 ml, add
50 mg of ammonium persulfate, dilute with water to 35 ml.
Carry out the limit test for iron < 0807 ) . Any colour
produced is not more intense than that of a reference using
5. O ml of iron standard solution (O. 06%).
Heavy metals Boil 4. O g with 4 ml of acetate BS CpH 3. 5)
and 46 ml of water, cool, filter, add water to the filtrate to
produce 50 ml and mix well. Carry out the limit test for
heavy metals <0821 method 1), using 25 ml: not more than
o. 001%.
Arsenic To l. O g, add 5 ml of hydrochloric acid and 23 ml
of water. Carry out the limit test for arsenic <0822 method
1), complies with the requirements (0. 0002%).
Category Pharmaceutical excipients, absorbent agent and
suspending agent.
Storage Preserve in well closed containers.
Laetose
C12H22011•H20 360. 31
[5989-81-1]
Lactase 1s 4-0-jJ-D-Galactopyranosyl-D-glucopyranose
monohydrate. It contains not less than 98. O% and not more
that 102. O% of C12 H22 011 , calculated on the anhydrous
base.
Description White crystalline granules or a crystalline power;
odorless; taste, slightly sweet.
Freely soluble in water; insoluble in ethanol, in chloroform
and in ether.
Specific optical rotation +52. Oºto +52. 6°, in a solution of
O. 1 g of lactase and O. 02 ml of ammonia TS per ml in water
<0621 >.
ldentification (1) To O. 2 g add 5 ml of sodium hydroxide
TS and heat gently, the colour of the solution becomes
yellow, then reddish-brown and a red precipitate of cuprous
oxide is produced in addition of a few drops of copper sulfate
TS.
(2) The retention time of principal peak of the test solution
obtained in Assay is identical with that of principal peak of
the reference solution in the chromatogram.
(3) The infrared absorption spectrum is concordant with the
reference spectrum CIR Album No. 256) (0402).
Acidity An aqueous solution of 50 mg per ml, pH 4. 0-7. O
<0631 >.
Clarity and colour of solution Dissolve l. O g in 10 ml of
boiling water, the resulting solution is clear and colourless
<0901 and 0902), any colour produced is not more intense
than that of reference solution Y2 <0901 method 1 ) .
Related substances Dissolve a quantity, accurately weighed,
in water to produce a test solution of 100 mg per ml,
measure accurately 1 ml to 100 ml volumetric flask, dilute
with water to volume, mix well, as the reference solution.
Carry out the method as described under Assay, record the
chromatograms for two times of the retention time of the
principal peak. The sum of the areas of all impurity peaks
other than the solvent peak and the principal peak in the
chromatogram obtained with the test solution is not greater
than O. 5 times area of the principal peak in the chrom-
atogram obtained with the reference solution (O. 5%).
Absorbance of impurity Dissolve an accurately weighed
quantity, in warm water to produce a solution containing 100 mg
per ml. Carry out the ultraviolet-visible spectrophotometry
method <0401 ) . The absorbance of the solution at 400 nm is
not more than O. 04. Dilute 1 ml, accurately measured of the
solution to 10 ml with water, the absorbance of the solution
at 210-220 nm < 0401 ) is not more than O. 25; the
absorbance of the solution at 270-300 nm is not more than
o. 07.
Protein Dissolve 5. O g in 25 ml of hot water, cool. Add
O. 5 ml of mercuric nitrate TS; no flocculent precipitate is
produced within 5 minutes.
Loss on drying When dried in vacuum to constant at 80ºC,
loses not more than l. O% of its weight <0831 ) .
Water 4. 5 %-5. 5 % , using a mixture of 1 volume of
formamide and 2 volumes of methanol as the solvent <0832
method 1 (1) ).
Residue on ignition Not more than O. 1 % <0841 ) , using
l. o g.
Heavy rnetals Dissolve 3. O g in 20 ml of warm water, add
2 ml of acetate BS (pH 3. 5) and dilute with water to 25 ml.
Carry out the limit test for heavy metals <0821 method 1):
not more than O. 0005%.
Arsenic Dissolve the residue obtained in the test for residue
on ignition in 5 ml of hydrochloric acid and 23 ml of water.
Carry out the limit test for arsenic <0822 method 1 ) ; not
more than O. 0002%.
Microbial lirnit Comply with the requirements for
microbiological limit < 1105 and 1106), the total aerobic

bacteria count is not more than 1000 cf u per g and the total
combined yeast/mold count is not more than 100 cfu per g of
the substance being examined, Escherichia coli should not be
detected.
Assay Carry out the method of high performance liquid
chromatography ( 0512), using a column packed with amino
bonded silica gel, a mixture of acetonitrile-water (70 : 30) as
the mobile phase and using a refractive index detector. The
column temperature is 45ºC and the detector temperature is
40°C. Dissolve an accurately weighed quantity of lactase CRS
and sucrose CRS in water to produce a solution containing
each of 1 mg per ml. Inject 10 µl into the column. The
resolution factor between peak of lactase and sucrose
complies with the requirements. The number of the
theoretical plates of the column is not less than 5000,
calculated with reference to the peak of lactase.
Procedure Dissolve an accurately weighed quantity in water
to produce a test solution of about 1 mg per ml. Inject 10 µl
the resulting solution into column and record the
chromatogram. Repeat the operations using lactase CRS
instead of the substance being examined, calculate the content of
C12 H 22 0 11 with respect to the peak areas obtained in the
chromatogram by external standard method.
Category
agent.
Pharrnaceutical excipients, filler and taste masking
Storage Preserve in tightly closed containers.
Lanolin
[ 8006-54-0 J
Lanolin is a refined waxy substances obtaíned from wooi.
Description A pale-yellow or browish-yellow waxy substance.
Emplastic and creamy; odour, slight and characteristic.
Freely soluble in chloroform and in ether, soluble in hot
ethanol, very slightly soluble in ethanol, insoluble in water
but mix with about twice its weight of water without
difficulty.
Melting range 36-42ºC ( 0612 method 2).
Acid value Not more than l. 5 ( O713 >.
Saponification value 92-106 ( 0713) Cheat under reflux for
2 hours).
lodine value 18-35 ( 0713) (allow to stand in the dark for 4
hours).
ldentification Dissolve O. 5 g in 5 mi of chloroform, add
1 mi of acetic anhydride and 2 drops of sulfuric acid; a deep
green colour is produced.
Acidity or alkalinity Melt 10 g with 50 mi of water on a
water bath, stir continuously, cool, remove the separated
fat, the solution is clear. To 10 ml, add 1 drop of
phenophthalein IS, no red colour is produced. To another 10
mi, add 1 drop of methyl red IS, no red colour is produced.
Chloride To O. 2 gin a conical flask, add 27 ml of ethanol,
heat under reflux for several minutes, cool, add O. 5 mi of
nitric acid and filter. To the filtrate add 5 drops of ethanolic
silver nitrate solution Cl-50). Any opalescence produced is
not more than pronounced than that of a reference solution
[to 20 ml of ethanol, add 7. O ml of sodium chloride standard
solution, O. 5 ml of nitric acid and 5 drops of ethanolic silver
nitrate solution O- 50) ] (O. 035%).
Laurocapram
Readily oxidizable substances To 10 mi of the solution
obtained in test for Acidity or alkalinity, add 1 drop of
potassium permanganate (O. 02 mol/L) VS, the red colour
<loes not completely disappear within 5 minutes.
Ethanol-insoluble substances Boíl O. 5 g with 40 mi of dehy-
drated ethanol, the solution is clear or very slightly turbid.
Loss on drying When dried with frequent stirring to
constant weight at lü5ºC, loses not more than O. 5% of its
weight ( 0831 ) .
Residue on ignition Not more than O. 15% (0841 ).
Category Pharmaceutical excipients, ointment base and
emulsifier, etc.
Storage Preserve in tightly closed containers, stored in a
cool place.
Laurocapram
a ~CH3
C1s H3s NO 281. 48
[59227-89-3]
Laurocapram is 1-dodecylhexahydro-2H-azepin-2-one. It
contains not less than 97. 0% and not more than 102. 0% of
C1sH3sNO.
Description A clear colourless viscous liquid; almost
odourless; tasteless.
Very soluble in dehydrated ethanol, in ethyl acetate, in
ether, and in cyclohexane; insoluble in water.
Relative density O. 906-0. 926 <0601 ) .
Refractive index l. 470-1. 4 73 ( 0622).
Viscosity Kinematic viscosity 32-34 mm 2/s, at 25ºC ( 0633
method 1), using a capillary tube: l. 2 mm± O. 05 mm in
internal diameter.
ldentification (1) To 2 ml add 2 ml of methanol, 1 ml of
1 mol/L hydroxylamine hydrochloride solution Cprepare the
solution immediately befare use), and 1 granule of potassium
hydroxide, heat on a water bath, allow to cool, and add 1
drop of ferric chloride TS, shake well. Heat on a water bath
again, a brownish-violet colour is produced.
(2) The infrared absorption spectrum is concordant with the
reference spectrum (IR Album No. 48).
Acidity or alkalinity To 5 ml of the substance being
examined add 5 ml of neutral ethanol, heat gently to dissolve
allow to cool, the litmus TP moisted by the solution exhibits
a neutral reaction.
Hexaiactam and related substances Transfer O. 5 g to a 10 ml
volumetric flask, dissolve with methanol and dilute to
volume, mix well, as test solution. Dissolve an appropriate
quantity of hexaiactam, accurately weighed, in methanol to
produce a test solution of O. 05 mg per ml as reference
solution. Carry out the method of residual solvent ( 0861
method 2) , using a capillary column packed with dimethyl
polysiloxane with 100 % eor similar polarity) as stationary
liquid capillary maintain the column temperature at lüüºC for
1 minute, raise the temperature of the column temperature at
 Lauroyl Macrogolglycerides ( 6)
a rate of 15ºC per minute from 240ºC and maintain the
temperature until 2 times the retention time of the principal
peak; The detector temperature is 300ºC; lnjection port
temperature is 250ºC. lnject 1 µl reference solution into the
column, adjust the detection sensitivity until the peak height
of the principal peak in the chromatogram is approximately
25 % of full scale of the chart. Accurately inject 1 µl of test
solution and reference solution into the column respectively,
record chromatograms. The area of impurity of which
retention time is identical with the retention time of
hexaiactam is not more than the area of the principal peak of
reference solution in the chromatogram obtained with the
reference solution (O. 1 %) . Calcula te the contents of other
impurities with respect to the peak areas obtained in the
chromatogram by the normalization procedure method, any
impurity peak is not more than l. 5 %, and the sum of all
impurity peaks is not more than 3. O%.
Bromide Place l. O g of the substance being examined in
10 ml of water, shake thoroughly, add 3 drops of
hydrochloride acid and 1 ml of chloroform, then add 3 drops
of 2 % chloroamine T solution ( prepare the solution
immediately before use) dropwise with shaking. Any colour
produced in the chloroform layer is not more intense than that
of a reference solution using l. O ml of potassium bromide
standard solution ( dissolve accurately weighed O. 1489 g of
potassium bromide previously dried to constant weight at
105ºC in water to 100 ml, shake welD prepared in the same
manner (0. 1%).
Residue on ignition Not more than O. 1% ( 0841); using
2. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2) , using the residue obtained in the test for
Residue on ignition: not more than O. 001%.
Assay Carry out the method for gas chromatography
( 0521 ) , using a capillary column packed with dimethyl
polysiloxane with 100 % ( or similar polarity) as stationary
liquid capillary maintain the column temperature at lOOºC for
1 minute, raise the temperature of the column temperature at
arate of 15ºC per minute from 240ºC, maintain 45 minutes;
The detector temperature is 300ºC; Injection port
temperature is 250ºC. The number of the theoretical plates
calculated for the Laurocapram peak is not less than 1000,
and the resolution factor between the peaks due to
Laurocapram and the internal substance complies with the
requirements.
Determination of correction factor Dissolve an accurately
weighed quantity of tetracosane in n-hexane to produce a
solution containing 2 mg per ml as the internal solution.
Place accurately weighed about 20 mg of laurocapram CRS in
a 10 ml volumetric flask, add the internal solution to dissolve
and dilute to volume with the same solvent and shake well.
lnject 1 µl into the column and calculate the correction
factor.
Procedure Place accurately weighed about 20 mg of the
substance being examined in a 10 ml volumetric flask, add
the internal solution to dissolve and dilute to volume with the
same solvent and shake well. Inject each 1 µl onto the column,
and calculate the content of laurocapram CCl8 H 35 NO).
Category Pharmaceutical excipients, penetration enhancer.
Storage Preserve in tightly closed containers, protected
from light.
Lauroyl Macrogolglycerides ( 6)
Lauroyl Macrogolglycerides ( 6) are mixtures of monoesters,
diesters and triesters of glycerol and monoesters and diesters
of macrogol 300, obtained by partial alcoholysis of saturated
oils using macrogol, or by esterification of glycerol and
macrogol 300 with fatty acids, or by mixing glycerol esters
and condensates of ethylene oxide with fatty acids.
Description A pale yellow waxy solid.
Freely soluble in dichloromethane, practically insoluble but
dispersible in water.
Acid value Not more than 2 ( 0713).
Saponification value 190-204 ( 0713).
Hydroxyl value 65-85 ( 0713).
lodine value Not more than 2 ( O713).
Peroxide value Not more than 6 ( 0713).
ldentification ( 1) Dissolve a quantity of the substance of
being examined and Lauroyl Macrogolglycerides ( 6 ) CRS
separately in dichloromethane to produce solutions each of 50
mg per ml. Carry out the method for thin-layer
chromatography ( 0502 ) , using silica gel G as the coating
substance and a mixture of ether-n-hexane ( 7 : 3) as the
mobile phase. Apply separately to the plate 10 µl each of
above two solutions, after developing and removal of the
plate, dry it in air and visualize in iodine vapor. The
chromatograms obtained with the test solution and the
reference solution both at least show 5 clearly separated
spots. The position and colour of the principal spots in the
chromatogram obtained with the test solution correspond to
the principal spots obtained with the reference solution.
(2) The infrared absorption spectrum is concordant with the
reference spectrum of lauroyl macrogolglycerides ( 6) CRS
( 0402).
Alkaline impurities Transfer 5. O g to a test tube and add a
mixture of O. 3 ml of water, 10 ml of ethanol and two drops
of O. 4 g/L neutralised solution of bromophenol blue in
ethanol. Shake and allow to stand. Not more than l. O ml of
hydrochloric acid (0. 01 mol/L) VS is required to change the
colour of the upper layer to yellow.
Free glycerol Dissolve l. 2 g in 25 ml of dichloromethane.
Heat if necessary. After cooling, add 100 ml of water. Shake
and add 25 ml of sodium periodate solution in acetic acid
( transfer O. 446 g of sodium periodate to a 100 ml volumetric
flask, dissolve with 2. 5 ml of 25 % sulfuric acid solution,
dilute with glacial acetic acid to volume). Shake and allow to
stand for 30 minutes. Add 40 ml of a 75 g/L solution of
potassium iodide. Allow to stand for 1 minute. Add 1 ml of
starch solution. Titrate with sodium thiosulfate (0. 1 mol/L)
VS. Carry out a blank titration. Each ml of sodium
thiosulfate (O. 1 mol/L) VS is equivalent to 2. 3 mg of
glycerol. Not more than 3. 0%.
Ethylene oxide and dioxane Transfer 1 g, accurately
weighed, to a headspace vial, add l. O ml of N, N-
dimethylacetamide and O. 2 ml of water. Close tightly and
mix well as the test solution. Transfer 99. 75 g of
polyethylene glycol 400 ( using a rotary evaporator remove
any volatile components by applying a temperature of 60ºC
and a pressure of l. 5-2. 5 kPa for 6 hours) , accurately
weighed, to a 100 ml vial Cor other suitable container), close

tightly. Using a glass syringe, previously cooled to - lOºC,
transfer 300 /ll of ethylene oxide ( equivalent to O. 25 g) ,
accurately weighed, to the above vial, mix well as the
reference stock solution of ethylene oxide ( freshly prepare or
calibrate before use). Transfer accurately 1 g of the cold
reference stock solution, accurately weighed, to a vial
containing 49 g of cold polyethylene glycol 400, clase tightly
and mix well. Transfer 10 g, accurately weighed, to a 50 ml
volumetric flask containing 30 ml of water, dilute with water
to volume as ethylene oxide reference solution ( 10 /lg per
mD. Disslove a quantity of dioxane, accurately weighed, in
water to produce a solution of O. 5 mg per ml as dioxane
reference solution. Transfer l. O g of the substance being
examined, accurately weighed, to a headspace vial,
accurately add l. O ml of N, N-dimethylacetamide, O. 1 ml
of ethylene oxide reference solution and O. 1 ml of dioxane
reference solution, clase tightly and mix well as the reference
solution. Transfer O. 1 ml of ethylene oxide reference
solution to a headspace vial, add O. 1 ml of freshly prepared
O. 001 % acetaldehyde solution and O. 1 ml of dioxane
reference solution, close tightly and mix well as the system
suitability solution. Carry out the method for gas
chromatography ( 0521 ) , using a column packed with
dimethylpolysiloxane as the stationary phase. Maintain the
column temperature at 50ºC for 5 minutes, raise the
temperature to 180ºC by 5ºC per minute, and then raise the
temperature to 230ºC by 30ºC per minute, maintain for 5
minutes (can be adjusted depending on the circumstance).
The temperature of injection port is 150ºC and the
temperature of detector is 250ºC using a flame ionization
detector. Headspace equilibrium temperature is 70ºC,
equilibrium time is 45 minutes. Inject the system suitability
solution into the column, adjust the attenuation so that the
peak heights of ethylene oxide and acetaldehyde are 15 % of
the full scale of the chart. The resolution factor betvv,.een the
peaks of acetaldehyde and ethylene oxide is not less than 2. O.
The peak of dioxane is detected with a signal-to-noise ratio of
at least 5. Headspace equilibrium temperature is 90ºC,
equilibrium time is 45 minutes. lnject separately the test
solution and the reference solution into the column, repeat
the injection at least three times. The relative standard
deviation of the peak area of ethylene oxide is not more than
15 % , the relative standard deviation of dioxane peak area is
not more than 10% . Calculated by standard addition
method, the contents of ethylene oxide and dioxane are not
more than O. 0001 % and O. 001 % respectively.
Calibration of the reference stock solution of ethylene
oxide To 10 ml of 50 % magnesium chloride suspension in
absolute ethanol, add accurately 20 ml of ethanolic
hydrochloric acid (O. 1 mol/L) VS, mix well and allow to
equilibrate overnight. Transfer 5 g of the reference stock
solution of ethylene oxide, accurately weighed, to the above
solution, mix well and allow to stand for 30 minutes. Carry
out the method for potentiometric titration ( 0701 ) , titrate
with ethanolic potassium hydroxide ( O. 1 mol/L) VS.
Perform a blank determination using polyethylene glycol 400
and make any necessary correction. Each ml of alcoholic
potassium hydroxide (O. 1 mol/U VS is equivalent to 4. 404
mg of ethylene oxide.
Ethylene glycol, diethylene glycol and triethylene glycol
Transfer 4 g of the substance being examined and O. 004 g of
1, 3-butanediol, accurately weighed, to a 100 ml volumetric
flask, dissolve in ethanol and dilute with the same solvent to
volume as the test solution. Transfer O. 0025 g of glycol,
O. 004 g of diethylene glycol, O. 004 g of triethylene and
O. 004 g of 1, 3-butanediol, accurately weighed, to a 100 ml
Lauroyl Macrogolglycerides ( 6)
volumetric flask, dissolve in ethanol and dilute with the same
solvent to volume as the reference solution. Carry out the
method for gas chromatography ( 0521), using a capillary
column packed with 50 % phenyl-50 % methylpolysilicone as
the stationary phase (film thickness is l. O /lm). Maintain the
column temperature at 60ºC for 5 minutes, raise the
temperature to l 70ºC by 5ºC per minute, and then raise the
temperature to 280ºC by 15ºC per minute, maintain for 50
minutes (can be adjusted depending on the circumstances).
The temperature of injection port is 270ºC and the
temperature of detector is 290ºC using flame ionization
detector. Use the reference solution as the system suitability
solution, the carrier gas is nitrogen, the flow rate is 4. O ml
per minute, the split ratio is 2 : 1, the injection volume is
l. O /ll. The resolution factor between the peaks of glycol,
diethylene glycol, triethylene glycol and the interna! standard
1, 3-butanediol is not less than 2. O. The tailing factors for
all peaks comply with the requirements. The relative
standard deviation of peak area ratio of glycol, diethylene
glycol and triethylene glycol to the interna! standard 1, 3-
butanediol is not more than 5. O%. Calculated by the interna!
standard method, the contents of glycol, diethylene glycol and
triethylene glycol are all not more than O. 1 % .
Water Not more than l. O% ( 0832 method 1 ( 1 ) ) , using
methanol- dichloromethane (3 : 7) as solvent.
Residue on ignition Not more than O. 1 % ( 0841 ) , using
l. o g.
Heavy metals Carry out the limit test for heavy metals
<0821 method 2) , using the residue obtained in the test for
Residue on ignition: not more than O. 001%.
Composition of fatty acids Transfer about l. O g to a 25 ml
round-bottomed flask with two necks, add 10 ml of
anhydrous methanol and O. 2 ml of 60 g/L methanolic
potassium hydroxide solution, shake to dissolve, pass
through nitrogen (50 ml per minute), heat to boiling. When
the solution becomes transparent ( about 10 minutes),
continue heating for 5 minutes, cool the flask with water,
and transfer to a separator. Wash the flask with 5 ml of n-
heptane, transfer the solution to the separator and mix well.
Add 10 ml of 200 g/L sodium chloride solution, mix well and
allow to separate. Transfer the organic layer to a vial
containing anhydrous sodium sulfate and dry, take the
filtrate as the test solution. Dissolve a quantity of the
following fatty acid methyl esters CRS in n-heptane,
accurately weighed, to produce the mixture solution ( 1)
containing l. O mg of methyl caprylate, l. O mg of methyl
decanoate, 3. O mg of methyl laurate, l. 5 mg of methyl
myristate, l. 5 mg of methyl hexadecanoate, and 2. O mg of
methyl stearate per ml. Transfer l. O ml to a 10 ml
volumetric flask, dilute with n-heptane to volume, mix well
as the mixture solution ( 2). Carry out the method for gas
chromatography ( 0521 ) , using a capillary column packed
with polyethylene glycol as the stationary phase. Maintain
the column temperature at 170ºC, raise the temperature to
230ºC by 2ºC per minute, maintain for 10 minutes. The
temperature of injection port is 250ºC and the temperature of
detector is 250ºC. Inject separately 1 /ll each of the mixture
solution (1) and the mixture solution (2) into the column,
and record the chromatogram. The resolution factor between
the peaks of the methyl ester peaks in the mixture solution
( 1) is not less than l. 8. The number of theoretical plates of
the column is not less than 30 000, calculated with the
reference to the peak of methyl caprylate. The minimum
peak height of methyl ester in the mixture solution ( 2) is
detected with a signal-to-noise ratio of at least 5. Inject 1 /ll
 Lauroyl Macrogolglycerides ( 8)
of the test solution into the column, calculate the peak areas
by area normalization method. Caprylic acid: not more than
15. o%; Capric acid: not more than 12. o%; Lauric acid:
30. 0%-50. 0%; Myristic acid: 5. 0%-25. o%; Palmitic acid:
4. 0%-25. 0%; Stearic acid: 5. 0%-35. 0%.
Category Pharmaceutical excipients, solubilizer
emulsifier.
Storage Preserve in tightly closed and nitrogen filled
containers, stored in a cool and dry place.
Lauroyl Macrogolglycerides ( 8)
Lauroyl Macrogolglycerides (8) are mixtures of monoesters,
diesters and triesters of glycerol and monoesters and diesters
of macrogol 400, obtained by partial alcoholysis of saturated
oils using macrogol, or by esterification of glycerol and
macrogol 400 with fatty acids, or by mixing glycerol esters
and condensates of ethylene oxide with fatty acids.
Description A pale yellow waxy solid.
Freely soluble in dichloromethane, practically insoluble but
dispersible in water.
Acid value Not more than 2 (0713).
Saponification value 170-190 (0713).
Hydroxyl value 60-80 ( O713 ) .
lodine value Not more than 2 ( 0713).
Peroxide value Not more than 6 (0713).
ldentification C1) Dissolve a quantity of the substance
being examined and Lauroyl Macrogolglycerides C8) CRS
separately in dichloromethane to produce solutions each of 50
mg per ml. Carry out the method for thin-layer
chromatography ( 0502) , using silica gel G as the coating
substance and a mixture of ether-n-hexane e7 : 3) as the
mobile phase. Apply separately to the plate 10 µl each of
above two solutions, after developing and removal of the
plate, dry it in air and visualize in iodine vapor. The
chromatograms obtained with the test solution and the
reference solution both at least show 5 clearly separated
spots. The position and colour of the principal spots in the
chromatogram obtained with the test solution correspond to
the principal spots obtained with the reference solution.
(2) The infrared absorption spectrum is concordant with the
reference spectrum of lauroyl macrogolglycerides C8) CRS
( 0402 ).
Alkaline impurities Transfer 5. O g to a test tube and add a
mixture of O. 3 ml of water, 10 ml of ethanol and two drops
of O. 4 g/L neutralised solution of bromophenol blue in
ethanol. Shake and allow to stand. Not more than 1. O ml of
hydrochloric acid (0. 01 mol/L) VS is required to change the
colour of the upper layer to yellow.
Free glycerol Dissolve l. 2 g in 25 ml of dichloromethane.
Heat if necessary. After cooling, add 100 ml of water. Shake
and add 25 ml of sodium periodate solution in acetic acid
etransfer o. 446 g of sodium periodate to a 100 ml volumetric
flask, dissolve with 2. 5 ml of 25 % sulfuric acid solution,
dilute with glacial acetic acid to volume). Shake and allow to
stand for 30 minutes. Add 40 ml of a 75 g/L solution of
potassium iodide. Allow to stand for 1 minute. Add 1 ml of
starch solution. Titrate with sodium thiosulfate (O. 1 mol/L)
VS. Carry out a blank titration. Each ml of sodium
thiosulfate CO. 1 mol/L) VS is equivalent to 2. 3 mg of
glycerol. Not more than 3. O%.
Ethylene oxide and dioxane Transfer 1 g, accurately
weighed, to a headspace vial, add l. O ml of N, N-dimethy-
lacetamide and O. 2 ml of water. Clase tightly and mix well
and as the test solution. Transfer 99. 75 g of polyethylene glycol
400 ( using a rotary evaporator remove any volatile
components by applying a temperature of 60ºC and a pressure
of l. 5-2. 5 kPa for 6 hours), accurately weighed, to a 100 ml
vial Cor other suitable container), clase tightly. Using a
glass syringe, previously cooled to -lOºC, transfer 300 µl of
ethylene oxide eequivalent to o. 25 g)' accurately weighed,
to the above vial, mix well as the reference stock solutíon of
ethylene oxide efreshly prepare or calíbrate before use).
Transfer accurately 1 g of the cold reference stock solutíon,
accurately weighed, to a vial containing 49 g of cool
polyethylene glycol 400, clase tíghtly and mix well. Transfer
10 g, accurately weighed, to a 50 ml volumetric flask
containing 30 ml of water, dilute with water to volume as
ethylene oxide reference solution (10 µg per mD. Disslove a
quantity of dioxane, accurately weighed, in water to produce
a solution of O. 5 mg per ml as dioxane reference solution.
Transfer l. O g of the substance being examined, accurately
weighed, to a headspace vial, accurately add l. O ml of N,
N-dimethylacetamíde, O. 1 ml of ethylene oxide reference
solution and O. 1 ml of dioxane reference solution, close
tightly and mix well as the reference solutíon. Transfer
O. 1 ml of ethylene oxide reference solution to a headspace
vial, add O. 1 ml of freshly prepared O. 001% acetaldehyde
solution and O. 1 ml of dioxane reference solution, clase
tightly and míx well as the system suitability solution. Carry
out the method for gas chromatography ( 0521 ) , using a
column packed with dimethylpolysiloxane as the statíonary
phase. Maintain the column temperature at 50ºC for 5
minutes, raise the temperature to 180ºC by 5ºC per minute,
and then raise the temperature to 230ºC by 30ºC per minute,
maintain for 5 minutes ecan be adjusted depending on the
circumstance). The temperature of injection port is 150ºC
and the temperature of detector is 250ºC using a flame
ionization detector. Headspace equilibrium temperature is
70ºC, equilibrium time is 45 minutes. Inject the system
suitability solution into the column, adjust the attenuation so
that the peak heights of ethylene oxide and acetaldehyde are
15 % of the full scale of the chart. The resolution factor
between the peaks of acetaldehyde and ethylene oxide is not
less than 2. O. The peak of dioxane is detected with a signal-
to-noise ratio of at least 5. Headspace equilibrium
temperature is 90ºC, equilibrium time is 45 minutes. lnject
separately the test solution and the reference solution in to the
column, repeat the injection at least three times. The relative
standard deviation of the peak area of ethylene oxide is not
more than 15%, the relative standard deviation of dioxane
peak area is not more than 10 %. Calculated by standard
addition method, the contents of ethylene oxide and dioxane
are not more than O. 0001 % and O. 001 % respectively.
Calibration of the reference stock solution of ethylene
oxide To 10 ml of 50% magnesium chloride suspension in
absolute ethanol, add accurately 20 ml of ethanolic
e
hydrochloric acid o. 1 mol/L) vs, mix well and allow to
equilibrate overnight. Transfer 5 g of the reference stock
solution of ethylene oxide, accurately weighed, to the above
solutíon, mix well and allow to stand for 30 minutes. Carry
out the method for potentíometric titration ( 0701 ) , titrate
with ethanolic potassíum hydroxide ( O. 1 mol/L) VS.
Perform a blank determination using polyethylene glycol 400
and make any necessary correctíon. Each ml of alcoholic

potassium hydroxide (O. 1 mol/U VS is equivalent to 4. 404
mg of ethylene oxide.
Ethylene glycol, diethylene glycol and triethylene glycol
Transfer 4 g of the substance being examined and O. 004 g of
1, 3-butanediol, accurately weighed, to a 100 ml volumetric
flask, dissolve in ethanol and dilute with the same solvent to
volume as the test solution. Transfer O. 0025 g of glycol,
O. 004 g of diethylene glycol, O. 004 g of triethylene and
O. 004 g of 1, 3-butanediol, accurately weighed, to a 100 ml
volumetric flask, dissolve in ethanol and dilute with the same
solvent to volume as the reference solution. Carry out the
method for gas chromatography ( 0521 ) , using a capillary
column packed with 50 % phenyl-50 % methylpolysilicone as
the stationary phase (film thickness is l. O µm). Maintain the
column temperature at 60ºC for 5 minutes, raise the
temperature to l 70ºC by 5ºC per minute, and then raise the
temperature to 280ºC by 15ºC per minute, maintain for 50
minutes (can be adjusted depending on the circumstances).
The temperature of injection port is 270ºC and the
temperature of detector is 290ºC using flame ionization
detector. Use the reference solution as the system suitability
solution, the carrier gas is nitrogen, the flow rate is 4. O ml
per minute, the split ratio is 2 : 1, the injection volume is
l. O µl. The resolution factor between the peaks of glycol,
diethylene glycol, triethylene glycol and the internal standard
1, 3-butanediol is not less than 2. O. The tailing factors for
all peaks comply with the requirements. The relative
standard deviation of peak area ratio of glycol, diethylene
glycol and triethylene glycol to the interna! standard 1, 3-
butanediol is not more than 5. 0%. Calculated by the internal
standard method, the contents of glycol, diethylene glycol
and triethylene glycol are all not more than O. 1 % .
Water Not more than l. O% ( 0832 method 1 ( 1 ) ) , using
methanol- dichloromethane (3 : 7) as solvent.
Residue on ignition Not more than O. 1% ( 0841 ) , using
l. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), using the residue obtained in the test for
Residue on ignition: not more than O. 001%.
Composition of fatty acids Transfer about l. O g to a 25 ml
round-bottomed flask with two necks, add 10 ml of
anhydrous methanol and O. 2 ml of 60 g/L methanolic
potassium hydroxide solution, shake to dissolve, pass
through nitrogen (50 ml per minute), heat to boiling. When
the solution becomes transparent ( about 10 minutes),
continue heating for 5 minutes, cool the flask with water,
and transfer to a separator. Wash the flask with 5 ml of n-
heptane, transfer the solution to the separator and mix well.
Add 10 ml of 200 g/L sodium chloride solution, mix well and
allow to separate. Transfer the organic layer to a vial
containing anhydrous sodium sulfate and dry, take the
filtrate as the test solution . Dissolve a quantity of the
following fatty acid methyl esters CRS in n-heptane,
accurately weighed, to produce the mixture solution ( 1)
containing l. O mg of methyl caprylate, l. O mg of methyl
decanoate, 3. O mg of methyl laurate, l. 5 mg of methyl
myristate, l. 5 mg of methyl hexadecanoate, and 2. O mg of
methyl stearate per ml. Transfer l. O ml to a 10 ml
volumetric flask, dilute with n-heptane to volume, mix well
as the mixture solution ( 2). Carry out the method for gas
chromatography ( 0521 ) , using a capillary column packed
with polyethylene glycol as the stationary phase. Maintain
the column temperature at l 70ºC, raise the temperature to
230ºC by 2ºC per minute, maintain for 10 minutes. The
temperature of injection port is 250ºC and the temperature of
Lauroyl Macrogolglycerides ( 12) 565
detector is 250ºC. Inject separately 1 µl each of the mixture
solution (1) and the mixture solution (2) into the column,
and record the chromatograms. The resolution factor
between the peaks of the methyl ester peaks in the mixture
solution ( 1) is not less than l. 8. The number of theoretical
plates of the column is not less than 30 000, calculated with
the reference to the peak of methyl caprylate. The mínimum
peak height of methyl ester in the mixture solution ( 2) is
detected with a signal-to-noise ratio of at least 5. Inject 1 µl
of the test solution into the column, calculate the peak areas
by area normalization method. Caprylic acid: not more than
15. 0%; Capric acid: not more than 12. 0%; Lauric acid:
30. 0%-50. o%; Myristic acid: 5. 0%-25. 0%; Palmitic acid:
4. 0%-25. 0%; Stearic acid: 5. 0%-35. 0%.
Category Pharmaceutical excipients, solubilizer and emulsifier.
Storage Preserve in tightly closed and nitrogen filled
containers, stored in a cool and dry place.
Lauroyl Macrogolglycerides ( 12)
Lauroyl Macrogolglycerides ( 12 ) are mixtures of
monoesters, diesters and triesters of glycerol and monoesters
and diesters of macrogol 600, obtained by partial alcoholysis
of saturated oils using macrogol, or by esterification of
glycerol and macrogol 600 with fatty acids, or by mixing
glycerol esters and condensates of ethylene oxide with fatty
acids.
Description A pale yellow waxy solid.
Freely soluble in dichloromethane practically insoluble but
dispersible in water.
Acid value Not more than 2 ( 0713).
Saponification value 150-170 ( 0713).
Hydroxyl value 50-70 ( 0713 ).
lodine value Not more than 2 (0713).
Peroxide value Not more than 6 (0713).
ldentification ( 1) Dissolve a quantity of the substance
being examined and Lauroyl Macrogolglycerides ( 12) CRS
separately in dichloromethane to produce solutions each of 50
mg per ml. Carry out the method for thin-layer chromat-
ography ( 0502) , using silica gel G as the coating substance
anda mixture of ether-n-hexane (7 : 3) as the mobile phase.
Apply separately to the plate 10 µl each of above two
solutions, after developing and removal of the plate, dry it in
air and visualize in iodine vapor. The chromatograms
obtained with the test solution and the reference solution
both at least show 5 clearly separated spots. The position
and colour of the principal spots in the chromatogram
obtained with the test solution correspond to the principal
spots obtained with the reference solution.
(2) The infrared absorption spectrum is concordant with the
reference spectrum of lauroyl macrogolglycerides (12) CRS
<0402).
Alkaline impurities Transfer 5. O g to a test tube and add a
mixture of O. 3 ml of water, 10 mi of ethanol and two drops
of O. 4 g/L neutralised solution of bromophenol blue in
ethanol. Shake and allow to stand. Not more than l. O ml of
hydrochloric acid (0. 01 mol/U VS is required to change the
colour of the upper layer to yellow.
Free glycerol Dissolve l. 2 g in 25 ml of dichloromethane.
 Lauroyl Macrogolglycerides ( 12)
Heat if necessary. After cooling, add 100 ml of water. Shake
and add 25 ml of sodium periodate solution in acetic acid
( transfer O. 446 g of sodium periodate to a 100 ml volumetric
flask, dissolve with 2. 5 ml of 25 % sulfuric acid solution,
dilute with glacial acetic acid to volume). Shake and allow to
stand for 30 minutes. Add 40 ml of a 75 g/L solution of
potassium iodide. Allow to stand for 1 minute. Add 1 ml of
starch solution. Ti trate with sodium thiosulfate (O. 1 mol/L)
VS. Carry out a blank titration. Each ml of sodium
thiosulfate (O. 1 mol/L) VS is equivalent to 2. 3 mg of
glycerol. Not more than 3. 0%.
Ethylene oxide and dioxane Transfer 1 g, accurately weighed,
to a headspace vial, add l. O ml of N , N-dimethylacetamide
and O. 2 ml of water. Clase tightly and mix well as the test
solution. Transfer 99. 75 g of polyethylene glycol 400 ( using
a rotary evaporator remove any volatile components by
applying a temperature of 60ºC anda pressure of l. 5-.2. 5 kPa
for 6 hours), accurately weighed, to a 100 ml vial Cor other
suitable container), clase tightly. Using a glass syringe,
previously cooled to - lOºC, transfer 300 µl of ethylene
oxide ( equivalent to O. 25 g), accurately weighed, to the
above vial, mix well as the reference stock solution of
ethylene oxide ( freshly prepare or calibra te befare use).
Transfer accurately 1 g of the cold reference stock solution,
accurately weighed, to a vial containing 49 g of cold
polyethylene glycol 400, clase tightly and mix well. Transfer
10 g, accurately weighed, to a 50 ml volumetric flask
containing 30 ml of water, dilute with water to volume as
ethylene oxide reference solution (10 µg per mD. Disslove a
quantity of dioxane; accurately v:eighed, in Vlater to produce
a solution of O. 5 mg per ml as dioxane reference solution.
Transfer l. O g of the substance being examined, accurately
weighed, to a headspace vial, accurately add l. O ml of N,
N-dimethylacetamide, O. 1 ml of ethylene oxide reference
solution and O. 1 ml of dioxane reference solution, clase
tightly and mix well as the reference solution. Transfer
O. 1 ml of ethylene oxide reference solution to a headspace
vial, add O. 1 ml of freshly prepared O. 001% acetaldehyde
solution and O. 1 ml of dioxane reference solution, clase
tightly and mix well as the system suitability solution. Carry
out the method for gas chromatography ( 0521 ) , using a
column packed with dimethylpolysiloxane as the stationary
phase. Maintain the column temperature at 50ºC for 5
minutes, raise the temperature to 180ºC by 5ºC per minute,
and then raise the temperature to 230ºC by 30ºC per minute,
maintain for 5 minutes (can be adjusted depending on the
circumstance). The temperature of injection port is 150ºC
and the temperature of detector is 250ºC using a flame
ionization detector. Headspace equilibrium temperature is
70ºC , equilibrium time is 45 minutes. Inject the system
suitability solution into the column, adjust the attenuation so
that the peak heights of ethylene oxide and acetaldehyde are
15 % of the full scale of the chart. The resolution factor
between the peaks of acetaldehyde and ethylene oxide is not
less than 2. O. The peak of dioxane is detected with a signal-
to-noise ratio of at least 5. Headspace equilibrium
temperature is 90ºC, equilibrium time is 45 minutes. Inject
separately the test solution and the reference solution into the
column, repeat the injection at least three times. The relative
standard deviation of the peak area of ethylene oxide is not
more than 15 % , the relative standard deviation of dioxane
peak area is not more than 10 % . Calculated by standard
addition method, the contents of ethylene oxide and dioxane
are not more than O. 0001 % and O. 001 % respectively.
Calibration of the reference stock solution of ethylene oxide
To 10 ml of 50% magnesium chloride suspension in absolute
ethanol, add accurately 20 ml of ethanolic hydrochloric acid
(O. 1 mol/L) VS, mix well and allow to equilibrate
overnight. Transfer 5 g of the reference stock solution of
ethylene oxide, accurately weighed, to the above solution,
mix well and allow to stand far 30 minutes. Carry out the
method for potentiometric titration ( 0701 ) , titrate with
ethanolic potassium hydroxide (O. 1 mol/L) VS. Perform a
blank determination using polyethylene glycol 400 and make
any necessary correction. Each ml of alcoholic potassium
hydroxide ( O. 1 mol/L) VS is equivalent to 4. 404 mg of
ethylene oxide.
Ethylene glycol, diethylene glycol and triethylene glycol
Transfer 4 g of the substance being examined and O. 004 g of
1, 3-butanediol, accurately weighed, to a 100 ml volumetric
flask, dissolve in ethanol and dilute with the same solvent to
volume as the test solution. Transfer O. 0025 g of glycol,
O. 004 g of diethylene glycol, O. 004 g of triethylene and
O. 004 g of 1, 3-butanediol, accurately weighed, to a 100 ml
volumetric flask, dissolve in ethanol and dilute with the same
solvent to volume as the reference solution. Carry out the
method for gas chromatography ( 0521 ) , using a capillary
column packed with 50 % phenyl-50 % methylpolysilicone as
the stationary phase (film thickness is l. O µm). Maintain the
column temperature at 60ºC for 5 minutes, raise the
temperature to 170ºC by 5ºC per minute, and then raise the
temperature to 280ºC by 15ºC per minute, maintain for 50
minutes (can be adjusted depending on the circumstances).
The temperature of injection port is 270ºC and the
temperature of detector is 290ºC using flame ionization
detector. Use the reference solution as the system suitabílity
solution, the carrier gas is nitrogen, the flow rate is 4. O ml
per minute, the split ratio is 2 : 1, the injection volume is
l. O µl. The resolution factor between the peaks of glycol,
diethylene glycol, triethylene glycol and the interna! standard
1, 3-butanediol is not less than 2. O. The tailing factors for
all peaks comply with the requirements. The relative
standard deviation of peak area ratio of glycol, diethylene
glycol and triethylene glycol to the interna! standard 1, 3-
butanediol is not more than 5. O%. Calculated by the interna!
standard method, the contents of glycol, diethylene glycol
and triethylene glycol are all not more than O. 1 % .
Water Not more than l. O% ( 0832 method 1 (1 ) ) , using
methanol-dichloromethane (3 : 7) as solvent.
Residue on ignition Not more than O. 1% ( 0841), using
l. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2) , using the residue obtained in the test for
Residue on ignition: not more than O. 001%.
Composition of fatty acids Transfer about l. O g to a 25 ml
round-bottomed flask with two necks, add 10 ml of anhy-
drous methanol and O. 2 ml of 60 g/L methanolic potassium
hydroxide solution, shake to dissolve, pass through nitrogen
(50 ml per minute), heat to boiling. When the solution
becomes transparent (about 10 minutes), continue heating
far 5 minutes, cool the flask with water, and transfer to a
separator. Wash the flask with 5 ml of n-heptane, transfer
the solution to the separator and mix well. Add 10 ml of 200
g/L sodium chloride solution, mix well and allow to
separate. Transfer the organic layer to a vial containing
anhydrous sodium sulfate and dry, take the filtrate as the test
solution. Dissolve a quantity of the following fatty acid
methyl esters CRS in n-heptane, accurately weighed, to
produce the mixture solution ( 1 ) containing l. O mg of
methyl caprylate, l. O mg of methyl decanoate, 3. O mg of
methyl laurate, l. 5 mg of methyl myristate, l. 5 mg of

methyl hexadecanoate, and 2. O mg of methyl stearate per
ml. Transfer l. O ml to a 10 ml volumetric flask, dilute with
n-heptane to volume, mix well as the mixture solution (2).
Carry out the method for gas chromatography < 0521 ) ,
using a capillary column packed with polyethylene glycol as
the stationary phase. Maintain the column temperature at
l 70ºC , then raise the temperature to 230ºC by 2ºC per
minute, maintain for 10 minutes . The temperature of
injection port is 250ºC and the temperature of detector is
250ºC. Inject separately 1 µl each of the mixture solution (1)
and the mixture solution (2) into the column, and record the
chromatogram. The resolution factor between the peaks of
the methyl ester peaks in the mixture solution (1) is not less
than l. 8. The number of theoretical plates of the column is
not less than 30 000, calculated with the reference to the
peak of methyl caprylate. The minimum peak height of
methyl ester in the mixture solution ( 2) is detected with a
signal-to-noise ratio of at least 5. Inject 1 µl of the test
solution into the column, calculate the peak areas by area
normalization method. Caprylic acid: not more than 15. 0%;
Capric acid: not more than 12. 0%; Lauric acid: 30. 0%-
50. 0%; Myristic acid: 5. 0%-25. o%; Palmitic acid : 4. 0%-
25. 0%; Stearic acid: 5. 0%-35. 0%.
Category Pharmaceutical excipients, solubilizer and emulsifier.
Storage Preserve in tightly closed and nitrogen filled
containers, stored in a cool and dry place.
Lauroyl Macrogolglycerides ( 32)
Lauroyl l\.1acrogolglycerides ( 32) are mixtures of monoesters,
diesters and triesters of glycerol and monoesters and diesters
of macrogol 1500, obtained by partial alcoholysis of saturated
oils using macrogol, or by esterification of glycerol and
macrogol 1500 with fatty acids, or by mixing glycerol esters
and condensates of ethylene oxide with fatty acids.
Description A pale yellow waxy solid.
Freely soluble in dichloromethane, practically insoluble but
dispersible in water.
Acid value Not more than 2 (0713).
Saponification value 79-93 <0713).
Hydroxyl value 36-56 <0713).
Iodine value Not more than 2 (0713).
Peroxide value Not more than 6 <0713).
ldentification ( 1) Dissolve a quantity of the substance
being examined and Lauroyl Macrogolglycerides ( 32) CRS
separately in dichloromethane to produce solutions each of
50 mg per ml. Carry out the method for thin-layer chro-
matography < 0502 ) , using silica gel G as the coating
substance and a mixture of ether-n-hexane ( 7 : 3) as the
mobile phase. Apply separately to the plate 10 µl each of
above two solutions, after developing and removal of the
plate, dry it in air and visualize in iodine vapor. The
chromatograms obtained with the test solution and the
reference solution both at least show 5 clearly separated
spots. The position and colour of the principal spots in the
chromatogram obtained with the test solution correspond to
the principal spots obtained with the reference solution.
(2) The infrared absorption spectrum is concordant with the
reference spectrum of lauroyl macrogolglycerides ( 32) CRS
<0402 ).
Lauroyl Macrogolglycerides ( 32)
Alkaline impurities Transfer 5. O g to a test tube and add a
mixture of O. 3 ml of water, 10 ml of ethanol and two drops
of O. 4 g/L neutralised solution of bromophenol blue in
ethanol. Shake and allow to stand. Not more than l. O ml of
hydrochloric acid (0. 01 mol/L) VS is required to change the
colour of the upper layer to yellow.
Free glycerol Dissolve l. 2 g in 25 ml of methylene chloride.
Heat if necessary. After cooling, add 100 ml of water. Shake
and add 25 ml of sodium periodate solution in acetic acid
( transfer O. 446 g of sodium periodate to a 100 ml volumetric
flask, dissolve with 2. 5 ml of 25 % sulfuric acid solution,
dilute with glacial acetic acid to volume). Shake and allow to
stand for 30 minutes. Add 40 ml of a 75 g/L solution of
potassium iodide. Allow to stand for 1 minute. Add 1 ml of
starch solution. Titrate with sodium thiosulfate (O. 1 mol/L)
VS. Carry out a blank titration. Each ml of sodium
thiosulfate (O. 1 mol/L) VS is equivalent to 2. 3 mg of
glycerol. Not more than 3. O%.
Ethylene oxide and dioxane Transfer 1 g, accurately weighed,
to a headspace vial, add l. O ml of N, N-dimethylacetamide
and O. 2 ml of water. Close tightly and mix well as the test
solution. Transfer 99. 75 g of polyethylene glycol 400 ( using
a rotary evaporator remove any volatile components by
applying a temperature of 60ºC and a pressure of l. 5-2. 5 kPa
for 6 hours), accurately weighed, to a 100 ml vial (or other
suitable container), close tightly. Using a glass syringe,
previously cooled to - lOºC, transfer 300 µl of ethylene
oxide ( equivalent to O. 25 g), accurately weighed, to the
above vial, mix well as the reference stock solution of
ethylene oxide ( freshly prepare or calibra te befare use).
Transfer accurately 1 g of the cold reference stock solution,
accurately weighed, to a vial containing 49 g of cold
polyethylene glycol 400, close tightly and mix well. Transfer
10 g, accurately weighed, to a 50 ml volumetric flask
containing 30 ml of water, dilute with water to volume as
ethylene oxide reference solution (10 µg per mD. Disslove a
quantity of dioxane, accurately weighed, in water to produce
a solution of O. 5 mg per ml as dioxane reference solution.
Transfer l. O g of the substance being examined, accurately
weighed, to a headspace vial, accurately add l. O ml of N,
N-dimethylacetamide, O. 1 ml of ethylene oxide reference
solution and O. 1 ml of dioxane reference solution, close
tightly and mix well as the reference solution. Transfer
O. 1 ml of ethylene oxide reference solution to a headspace
vial, add O. 1 ml of freshly prepared O. 001 % acetaldehyde
solution and O. 1 ml of dioxane reference solution, close
tightly and mix well as the system suitability solution. Carry
out the method for gas chromatography <0521 ) , using a
column packed with dimethylpolysiloxane as the stationary
phase. Maintain the column temperature at 50ºC for 5
minutes, raise the temperature to 180ºC by 5ºC per minute,
and then raise the temperature to 230ºC by 30ºC per minute,
maintain for 5 minutes ( can be adj usted depending on the
circumstance). The temperature of injection port is 150ºC
and the temperature of detector is 250ºC using a flame
ionization detector. Headspace equilibrium temperature is
70ºC, equilibrium time is 45 minutes. Inject the system
suitability solution into the column, adjust the attenuation so
that the peak heights of ethylene oxide and acetaldehyde are
15 % of the full scale of the chart. The resolution factor
between the peaks of acetaldehyde and ethylene oxide is not
less than 2. O. The peak of dioxane is detected with a signal-
to-noise ratio of at least 5. Headspace equilibrium
temperature is 90ºC, equilibrium time is 45 minutes. Inject
separately the test solution and the reference solution into the
column, repeat the injection at least three times. The relative
 568 Leucine
standard deviation of the peak area of ethylene oxide is not
more than 15 % , the relative standard deviation of dioxane
peak area is not more than 10 % . Calculated by standard
addition method, the contents of ethylene oxide and dioxane
are not more than O. 0001% and O. 001% respectively.
Calibration of the reference stock solution of ethylene oxide
To 10 ml of 50 % magnesium chloride suspension in absolute
ethanol, add accurately 20 ml of ethanolic hydrochloric acid
(O. 1 mol/L) VS, mix well and allow to equilibrate
overnight. Transfer 5 g of the reference stock solution of
ethylene oxide, accurately weighed, to the above solution,
mix well and allow to stand for 30 minutes. Carry out the
method for potentiometric titration ( 0701 >, titrate with
ethanolic potassium hydroxide (O. 1 mol/L) VS. Perform a
blank determination using polyethylene glycol 400 and make
any necessary correction. Each ml of alcoholic potassium
hydroxide (O. 1 mol/U VS is equivalent to 4. 404 mg of
ethylene oxide.
Ethylene glycol, diethylene glycol and triethylene glycol
Transfer 4 g of the substance being examined and O. 004 g of
1, 3-butanediol, accurately weighed, to a 100 ml volumetric
flask, dissolve in ethanol and dilute with the same solvent to
volume as the test solution. Transfer O. 0025 g of glycol,
O. 004 g of diethylene glycol, O. 004 g of triethylene and
O. 004 g of 1, 3-butanediol, accurately weighed, to a 100 ml
volumetric flask, dissolve in ethanol and dilute with the same
solvent to volume as the reference solution. Carry out the
method for gas chromatography ( 0521 >, using a capillary
column packed with 50 % phenyl-50 % methylpolysilicone as
the stationary phase (film thickness is l. O µm). Maintain the
column temperature at 60ºC for 5 minutes, raise the
temperature to l 70ºC by 5ºC per minute, and then raise the
temperature to 280ºC by 15ºC per minute, maintain for 50
minutes (can be adjusted depending on the circumstances).
The temperature of injection port is 270ºC and the
temperature of detector is 290ºC using flame ionization
detector. Use the reference solution as the system suitability
solution, the carrier gas is nitrogen, the flow rate is 4. O ml
per minute, the split ratio is 2 : 1, the injection volume is
l. O µl. The resolution factor between the peaks of glycol,
diethylene glycol, triethylene glycol and the internal standard
1, 3-butanediol is not less than 2. O. The tailing factors for
all peaks comply with the related requirements. The relative
standard deviation of peak area ratio of glycol, diethylene
glycol and triethylene glycol to the internal standard 1, 3-
butanediol is not more than 5. 0%. Calculated by the internal
standard method, the contents of glycol, diethylene glycol
and triethylene glycol are all not more than O. 1 % .
Water Not more than l. O% ( 0832 method 1 ( 1 ) >, using
methanol-dichloromethane (3 : 7) as solvent.
Residue on ignition Not more than O. 1 % ( 0841 >, using
l. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2 >, using the residue obtained in the test for
Residue on ignition: not more than O. 001%.
Composition of fatty acids Transfer about l. O g to a 25 ml
round-bottomed flask with two necks, add 10 ml of anhydrous
methanol and O. 2 ml of 60 g/L methanolic potassium
hydroxide solution, shake to dissolve, pass through nitrogen
( 50 ml per minute) , heat to boiling. When the solution
becomes transparent ( about 10 minutes), continue heating
for 5 minutes, cool the flask with water, and transfer to a
separator. Wash the flask with 5 ml of n-heptane, transfer
the solution to the separator and mix well. Add 10 ml of 200
g/L sodium chloride solution, mix well and allow to
separate. Transfer the organic layer to a vial contammg
anhydrous sodium sulfate and dry, take the filtrate as the
test solution. Dissolve a quantity of the following fatty acid
methyl esters CRS in n-heptane, accurately weighed, to
produce the mixture solution ( 1 ) containing l. O mg of
methyl caprylate, l. O mg of methyl decanoate, 3. O mg of
methyl laurate, l. 5 mg of methyl myristate, l. 5 mg of
methyl hexadecanoate, and 2. O mg of methyl stearate per
ml. Transfer l. O ml to a 10 ml volumetric flask, dilute with
n-heptane to volume, mix well as the mixture solution (2).
Carry out the method for gas chromatography ( 0521 >,
using a capillary column packed with polyethylene glycol as
the stationary phase. Maintain the column temperature at
170ºC, raise the temperature to 230ºC by 2ºC per minute,
maintain for 10 minutes. The temperature of injection port is
250ºC and the temperature of detector is 250ºC . Inject
separately 1 µl each of the mixture solution ( 1 ) and the
mixture solution ( 2) into the column, and record the
chromatogram. The resolution factor between the peaks of
the methyl ester peaks in the mixture solution (1) is not less
than l. 8. The number of theoretical plates of the column is
not less than 30 000, calculated with the reference to the
peak of methyl caprylate. The minimum peak height of
methyl ester in the mixture solution ( 2) is detected with a
signal-to-noise ratio of at least 5. Inject 1 µl of the test
solution into the column, calculate the peak areas by area
normalization method. Caprylic acid: not more than 15. 0%;
Capric acid: not more than 12. 0%; Lauric acid: 30. 0%-
50. 0%; Myristic acid: 5. 0%-25. o%; Palmitic acid: 4. 0%-
25. o%; Stearic acid: 5. 0%-35. 0%.
Category Pharmaceutical excipients, solubilizer and emulsifier.
Storage Preserve in tightly closed and nitrogen filled
containers, stored in a cool and dry place.
Leucine
See the same monograph in Volume 1I.
Category Pharmaceutical excipients, antioxidant and solubilizer.
Light Liquid Paraffin
[8012-95-1]
Light Liquid Paraffin is a purified mixture of liquid saturated
hydrocarbons obtained from petroleum.
Description Colourless, transparent, oily liquid; odourless,
tasteless; free from fluorescence in daylight.
Miscible with chloroform, with ether, and with most fatty
oil except the castor oil, slightly soluble in ethanol,
practically insoluble in water.
Relative density O. 830-0. 860 ( 0601 >.
Viscosity Kinematic viscosity ( 0633 method 1 ) at 40ºC
( using a capillary tube: l. O mm ± O. 05 mm in internal
diameter) is not less than 12 mm2 /s.
ldentification ( 1) Heat about 5 ml of the substance being
examinder in a crucible and burns with a bright flame, and
the odour of paraffin is perceptible.
(2) To about O. 5 g of the substance being examined in a
dried test-tune add an equal quantity of sulfur, shake and

heat, the odour of hydrogen sulfide is perceptible.
Acidity or alkalinity To 10 ml, add 10 ml of boiling water
and a drop of phenolphthalein IS, shake vigorously, the
solution is colourless. Not more than O. 20 ml of sodium
hydroxide (O. 02 mol/L) VS is required to change the colour
of the solution to pink.
Sulfide To 4. Oml of the substance being examined, add 2
drops of saturated solution of lead oxide in sodium hydroxide
solution 0 -5) and 2 ml of ethanol, shake well, heat and
shake in a water bath at 70ºC for 10 minutes, allow to cool.
No brownish-black colour is produced.
Polycylic aromatic hydrocarbons Accurately measured 25 ml
of the substance being examined into a separating funnel, and
25 ml of n-hexane, mix well and add an accurately measured
5 ml of dimethyl sulphoxied, shake vigorously for 2 minutes
and allow to stand until two clear layers are formed.
Transfer the dimethyl sulphoxide layer into a second
separating funnel, add 2 ml of n-hexane and shake, allow to
stand until two clear layers are formed ( centrifuge if
necessary). Separate the dimethyl sulphoxide layer as the test
solution and measure the absorbance between 260 nm and 350
nm ( 0401), the maximum absorbance is not greater than O. 10,
using the dimethyl sulphoxide layer obtained by vigorously
shaking 5. O ml of dimthyl sulphoxide with 25 ml of n-hexane in
a 50 ml separating funnel for 2 minutes as the blank solution.
Solid paraffins Dry a quantity of the substance being
examined at 105ºC for 2 hours and cool in a desiccators over
sulfuric acid. Place the dried substance in a 50 ml Nessler
cylinder to 50 ml, stopper the tube and cool at OºC for
4 hours: any opalescence produced is not more pronounce
than that of the reference solution (to O. 15 ml of O. 01 mol/L
hydrochloric acid solution add 6 ml of dilute nitric acid and
l. O ml of silver nitrate TS, dilute to 50 ml with water, allow
to stand in the dark for 5 minutes).
Readily carbonizable substances Transfer 5 ml of the
substance being examined to a colour matching tube about
160 mm long and 25 mm in interna! diameter, add 5 ml of
sulfuric acid [94. 5%-95. 9% (g/g) ], heat in a water bath
for 30 seconds and remove the tube from the bath rapidly.
Stopper the tube and shake vigorously in longitudinal
direction 3 times, the amplitude is more than 12 cm, the
time is not more than 3 seconds. Place the tube again in the
water bath and shake as above at intervals of 30 seconds. At
the end of 10 minutes of heating, remove the tube from the
water bath for readily carbonizable substances ( 0842 ) , the
paraffin layer is colourless; any colour produced in the
sulfuric acid layer is not more intense than that of a reference
solution (a mixture of l. 5 ml of potassium bichromate colour
standard solution, l. 3 ml of cobaltous chloride colour
standard solution, O. 5 ml of copper sulfate colous standard
solution, l. 7 ml of water and 5 ml of the substance being
examined).
Heavy metals Place l. O g of the substance being examined
in a crucible, ignite slowly until completely carbonized.
Incinerate completely at 450-550ºC. Allow to cool, add 2 ml
of hydrochloric acid, and evaporate to dryness on a water
bath. Carry out the limit test for heavy metals ( 0821
method 2): not more than O. 001%.
Arsenic Place l. O g of the substance being examined in a
crucible, add 10 ml of 2% magnesium nitrate solution in
ethanol, ignite until carbonized ( if any uncarbonized
obserced, add a small volume of nitric acid and ignite once
more until the incineration is complete), allow to cool, add
5 ml of hydrochloric acid, heat on water bath to dissolve,
Light Magnesium Oxide 569
add 23 ml of water. Carry out the limit test for arsenic
( 0822 method 1 ) : not more than O. 0002 % .
Category Pharmaceutical excipients, lubricant and ointment
base, etc.
Storage Preserve in tightly closed containers.
Light Magnesium Oxide
MgO 40. 03
[1309-48-4]
Light Magnesium Oxide contains not less than 96. 5 % of
MgO, calculated with reference to substance ignited to
constant weight.
Description A white or almost white powder, odourless;
tasteless; gradually absorbs carbon dioxide from the air.
Practically insoluble in water or in ethanol; soluble in diluted
acids.
Identification Its solution in dilute hydrochloric acid yields
the reactions characteristic of magnesium salts ( 0301 ) .
Apparent volume Place 15. O g into a graduate cylinder, the
apparent volume is not less than 100 ml without vibration.
Alkalinity Boil l. O gin 50 ml of water for 5 minutes. Filter
while hot, wash the residue with a volume of water.
Combine the washings with the filtrate, add a few drops of
methyl red IS and 2. O ml sulfuric acid (O. 05 mol/L) VS,
yellow colour changes to red.
Color of the solution Dissolve l. O g in 15 ml of acetic acid
and 5 ml of water, boil for 2 minutes and cool, add water to
20 ml, fi!ter if necessary; the solution is colourless. Any
colour produced is not more intense than that of reference
solution YG2 ( 0901 method 1 ) .
Chloride Dilute l. O ml of the solution prepared in the test
for calcium oxide with water to 25 ml. Carry out the limit
test for chlorides ( 0801 ) . Any opalescence produced is not
more pronounced than that of a ref erence prepared by using
5. O ml of sodium chloride standard solution (O. 1%).
Sulfate Dilute 2. O ml of the solution prepared in the test for
calcium oxide with water to 20 ml. Carry out the limit test
for sulfates ( 0802). Any opalescence produced is not more
pronounced than that of a reference prepared by using 3. O ml
of potassium sulfate standard solution (0. 3%).
Carbonates Boil O. 10 g with 5 ml of water, allow to cool
and add 5 ml of acetic acid, no effervescence occurs.
Acid insoluble substances Dissolve 2. O g in 25 ml of
hydrochloric acid on a water bath and add 100 ml of water.
Filter through a sintered-glass crucible (No. 4) previously
dried to constant weight at 105ºC. Wash the residue with
water until the washing shows no reaction of chlorides. Dry
the residue to constant weight at 105ºC. The residue is not
more than 2. O mg (O. 10%).
Soluble substances Boil l. O g with 100 ml of water for 5
minutes, filter while hot, wash the residue with small quantity
of water, combine the filtrate an the washings, evaporate to
dryness in a evaporating dish previously dried to constant weight
at 105ºC on a water bath, and dry to constant weight at 105ºC.
The residue is not more than 2. O%.
Loss on ignition When ignited to constant weight, loses not
more than 5. O% , using O. 50 g.
 Liquid Paraffin
Calciwn oxide Weigh accurately 5. O g substance being
examined, freshly ignited and cooled, dissovle in 30 ml of
water and 70 ml of acetic acid, boil for 2 minutes. Cool and
filter, wash the residue with dilute acetic acid, combine the
filtrate and washings into a 100 ml volumetric flask, add
dilute acetic acid to volume. Mix well, as the test solution.
Transfer accurately 10 ml of the test solution, add 300 ml of
water, 10 ml of triethanolamine solution (3-+-10) and 10 ml
of 45 % potassium hydroxide solution. Allow to stand for 5
minutes, add O. 1 g of calcium purpurin indicator mixture,
titrate with disodium edetate (O. 01 mol/L) VS until the
colour changes from purple to blue. Perform a blank
determination and make any necessary correction. Each ml of
disodium edetate (0. 01 mol/L) VS is equivalent to O. 5608
mg of CaO: not more than O. 50%.
Iron Dissolve 50 mg with 2 ml of dilute hydrochloric acid
and 23 ml of water. Carry out the limit test for iron ( 0807).
Any colour produced is not more intense than that of a
reference solution prepared by using 2. 5 ml of iron standard
solution (0. 05%).
Manganese To l. O g add 20 ml of water, 5 ml of nitric
acid, 5 ml of sulphuric acid and 1 ml of phosphoric acid, boil
for 2 minutes and cool. Add 2. O g of potassium periodate,
boil for 5 minutes and cool. Transfer to a 50 ml Nessler
cylinder, dilute to volume with non-reducing water (add 3 ml
of nitric acid and 5 g of potassium periodate to 1000 ml of
water, boil for 2 minutes and cool) , mix well. Any colour
produced is not more intense than that of a reference solution
prepared by using O. 30 ml of manganese standard solution
(Dissolve O. 275 g of anhydrous manganese sulfate previously
ignited to constant weight at 400-500ºC in a 1000 ml
volumetric flask, dilute to volume with water and mix well.
Each ml of manganese standard solution is equivalent to O. 10
mg of Mn) (0. 003%).
Heavy rnetals Dissolve by heating O. 50 g with 10 ml of
dilute hydrochloric acid and 5 ml of water, boil for 1 minute,
allow to cool and filter. To the filtrate add 1 drop of
phenolphthalein IS and add dropwise ammonia TS until the
colour of the solution becomes pink. Add 2 ml of acetate BS
(pH 3. 5) and sufficient water to produce 25 ml, add O. 5 g
of ascorbic acid and shake to dissolve. Carry out the limit
test for heavy metals ( 0821 method 1), compare the colour
after 5 minutes: not more than O. 002 % .
Arsenic Dissolve O. 50 g with 5 ml of hydrochloric acid TS
and 23 ml of water. Carry out the limit test for arsenic
( 0822 method 1 ) : not more than O. 0004 % .
Assay Dissolve O. 4 g, accurately weighed, in 25 ml of
sulfuric acid (O. 5 mol/L) VS, accurately measured, add 1
drop of methyl orange IS. Ti trate with sodium hydroxide ( 1
mol/L) VS. Perform a blank determination and make any
necessary correction. The quantity of sulfuric acid consumed
by MgO is calculated by subtracting the quantity consumed
by CaO from the total amount of sulfuric acid consumed.
Each ml of sulfuric acid (O. 5 mol/L) VS is equivalent to
20. 15 mg of MgO or 28. 04 mg of CaO.
Category Pharmaceutical excipients, filling agent and
alkalizing agent.
Storage Preserve in tightly closed containers.
Liquid Paraffin
[8012-95-1]
Liquid Paraffin is a purified mixture of liquid saturated
hydrocarbons obtained from petroleum.
Description Colourless, transparent, oily liquid; odourless,
tasteless; free from fluorescence in da ylight.
Miscible with chloroform and with ether, slightly soluble in
ethanol, insoluble in water.
Relative density O. 845-0. 890 ( 0601 ) .
Viscosity The kinematic viscosity ( 0633 method 1 ) at
40ºC (using a capillary tube: l. O mm±O. 05 mm in interna!
diameter) is not less than 36 mm2 /s.
ldentification ( 1) Heat about 5 ml in a crucible and burns
with a bright flame, and the odour of paraffin is perceptible.
(2) To O. 5 g in a dried test-tube add an equal quantity of
sulfur, shake and heat to melt, the odour of hydrogen sulfide
is perceptible.
Acidity or alkalinity To 15 ml add 30 ml of boiling water,
shake vigorously for 1 minute. Cool, separate the aqueous
layer and filter. To 10 ml of the filtrate, add 2 drops of
phenolphthalein IS, the solution is colourless. Not more than
l. O ml of sodium hydroxide (O. 02 mol/L) VS is required to
change the colour of the solution to pink.
Sulfide To 4. O ml add 2 drops of saturated solution of lead
oxide in sodium hydroxide solution Cl-+-5) and 2 ml of ethanol,
mix well, heat and shake in a water bath at 70ºC for 10 minutes,
allow to cool. No brownish-black colour is produced.
Polycyclic arornatic hydrocarbons Transfer accurately 25 ml
to a 250 ml separator, add 25 ml of n-hexane, mix well and
add accurately 5 ml of dimethyl sulphoxide. Shake vigorously
for 2 minutes and allow to stand until two clear layers are
formed. Transfer the dimethyl sulphoxide layer to another
50 ml separator, add 2 ml of n-hexane and shake, allow to
stand until two clear layers are formed ( centrifuge if
necessary). Separate the dimethyl sulphoxide layer as the test
solution. Transfer 25 ml of n-hexane to a 50 ml separator,
add accurately 5 ml of dimethyl sulphoxide. Shake vigorously
far 2 minutes and allow to stand until two clear layers are
formed. Separate the dimethyl sulphoxide layer as the blank
solution. Measure the absorbance in the range of 260-350 nm
( 0401), the maximum absorbance of the test solution is not
greater than O. 10.
Solid paraffins Dry a quantity of the substance being
examined at 105ºC for 2 hours and cool in a desiccators over
sulfuric acid. Transfer the dried substance in a 50 ml Nessler
cylinder to 50 ml, stopper the tube and cool at OºC for 4
hours, any opalescence produced is not more pronounced
than that of the reference solution (to O. 15 ml of O. 01 mol/L
hydrochloric acid solution add 6 ml of dilute nitric acid and
l. O ml of silver nitrate TS, dilute to 50 ml with water, allow
to stand in the dark for 5 minutes).
Readily carbonizable substances Transfer 5 ml to a stoppered
tube about 160 mm long and 25 mm in internal diameter, add
5 ml of sulfuric acid ( 94. 5 %-95. 5 % ( g/ g) ) , heat in a
water bath for 30 seconds and remove the tube from the bath
rapidly. lnsert the stopper and shake vigorously in
longitudinal direction of the tube three times. Make sure that
the amplitude is above 12 cm and the shaking time is no more

than 3 seconds. Then place the tube in the water bath,
remove it from the bath at intervals of 30 seconds and shake
as described above. At the end of 10 minutes of heating,
remove the tube from the water bath and allow to stand until
two clear layers are formed. Carry out the limit test for
readily carbonizable substances ( 0842) , the paraffin layer is
colourless; any colour produced in the sulfuric acid layer is
not more intense than that of a reference solution (a mixture
of l. 5 ml of potassium bichromate colour standard solution,
l. 3 ml of cobaltous chloride colour standard solution, O. 5 ml
of copper sulfate colour standard solution, l. 7 ml of water
and 5 ml of the substance being examined).
Heavy metals Place l. O g in a crucible, ignite slowly until
completely carbonized. lncinerate completely at 450-550ºC.
Allow to cool, add 2 ml of hydrochloric acid, and evaporate
to dryness on a water bath. Carry out the limit test for heavy
metals ( 0821 method 2): not more than O. 001%.
Arsenic Place l. O g in a crucible, add 10 ml of 2%
magnesium nitrate solution in ethanol, ignite until carbonized
(if any uncarbonized observed, add a small volume of nitric
acid and ignite once more until the incineration is complete),
allow to cool, add 5 ml of hydrochloric acid, heat on a water
bath to dissolve, add 23 ml of water. Carry out the limit test
for arsenic <0822 method 1 ) : not more than O. 0002 % .
Category Pharmaceutical excipients, lubricant and ointment
base.
Storage Preserve in tightly closed containers.
Low-substituted Hydroxypropyl Cellulose
OH
O~
H OR
OR
n/2
[9004-64-2]
Low-substituted hydroxypropyl cellulose is a low-substituted
2-hydroxypropyl ether of cellulose. Cellulose is treaded by
caustic solution, etherification with propylene oxide (PO) ,
then through neutralization, recrystallization, washing,
drying, grinding and screening. Hydroxypropoxy groups
(-OCH2CHOHCH3) is not less than 5. 0% and not more
than 16. o%' calculated on the dried basis.
Description A white or almost white powder; odorless;
tasteless. Practically insoluble in ethanol, in acetone and in ether.
ldentification (1) To 40 mg, into a test tube, add 2 ml of
water, shake to a suspension, add slowly 1 ml of a O. 035%
solution of anthrone in sulfuric acid. A blue to greenish-blue
colour ring is produced at the interface of the two lays.
(2) To O. 1 g, add 10 ml of water and shake, add 1 g of
sodium hydroxide, shake until it becomes homogeneous as
the test solution. To O. 1 ml of test solution, add 9 ml of
sulfuric acid solution (9-10), shake well. Heat in a water
bath for 3 minutes and cool in an ice bath at once. Add
Macrogol 300 (For lnjection) 1 ;:
O. 6 ml of ninhydrin solution ( freshly prepared immediately
before using by dissolving O. 2 g of ninhydrin in 10 ml of
water) , shake and allow to stand. The red colour is produced
immediately and turns to violet within 100 minutes.
(3) To 5 ml of the test solution in Identification (2), add 10
ml of acetone-methanol ( 4 : 1) and shake. A white
flocculent precipitate is formed.
Acidity or alkalinity To O. 10 g, add 10 ml of water, shake
to a suspension. pH 5. 0-7. 5 ( 0631) .
Chloride To O. 10 g, add 30 ml of hot water, heat in a
water bath for 10 minutes, filter the supernatant while hot.
Wash the residue with hot water for 4 times, each of 15 ml,
combine the washings and the filtrate into a 100 ml
volumetric flask, cool, dilute to volume with water and
shake. using 10 ml, carry out the test for chlorides ( 0801 ).
Any colour produced is not more intense than that of a
reference solution using 2. O ml of sodium chloride standard
solution (O. 20%).
Los.son drying When dried at 105ºC to constant weight,
loses not more than 8. O% of its weight ( 0831 ) .
Residue on ignition Not more than l. O% ( 0841 >, using
l. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2) , using the residue obtained in the test for
Residue on ignition: not more than O. 002%.
Iron lgnite l. O g as described under Residue on ignition.
Dissolve the residue with 5 ml of dilute hydrochloride acid by
heat in a water bath, add water to 25 ml and mix well. Carry
out the limit test for iron ( 0807 ) , using 5 ml of the
solution. The colour produced is not more intense than that
of a standard using 2. O ml of iron standard solution
(0. 010%).
Arsenic Mix l. O g with l. O g of calcium hydroxide, add a
few quantity of water, stir well and dry. Ignite gently to
carbonize and then at 500-600ºC until free from carbon.
Allow to cool and dissolve the residue with a mixture of 8 ml
of hydrochloric acid and 23 ml of water. Carry out the limit
test for arsenic ( 0822 method 1), not more than O. 0002%.
Assay Hydroxypropyloxy groups Carry out the method
for determination of methoxy, ethoxyl and hydroxypropoxy
( 0712 ) . For method 2 ( volumetric method), weighed
accurately O. 1 g, calcula te the content of hydroxpropoxy
e-oc3 H6 om.
Category Pharmaceutical excipients, disintegrant and filler,
etc.
Storage Preserve in well closed containers.
Macrogol 300 ( For Injection)
[25322-68-3]
Macrogol 300 is a mixture of polymer of ethylene oxide and
water, represented by the general molecular formula HO
(CH2CH2Ü)n H, where n is the average number of ethylene
oxide groups.
Description A colourless and clear viscous liquid, slight
odour.
Freely soluble in water, in ethanol and in ethylene glycol,
insoluble in ether.
Relative density 1.120-1.130, at 20ºC (0601 >.
 572 Macrogol 300 (For Injection)
Viscosity Kinematic viscosity is 59-73 mm 2 /s, at 25ºC
<0633 method 1 >, using a capillary tube of l. 2 mm m
interna! diameter.
Identification ( 1) To O. 05 g, add 5 ml of dilute
hydrochloric acid and 1 ml of barium chloride, shake and
filter. To the filtra te add 1 ml of 10 % phosphomolybdic acid
solution, a yellow-green precipitate is formed.
(2) Place O. 1 g in a test tube, add O. 1 g each of potassium
thiocyanate and cobaltous nitrate, mix and add 5 ml of
dichloromethane, the solution exhibits a blue colour.
Average molecular weight Place about l. 2 g, accurately
weighed, in a dry 250 ml conical flask with stopper, add
25 ml, accurately measured, of phthalic anhydride solution
in pyridine (dissolve 14 g of phthalic anhydride in 100 ml of
anhydrous pyridine and allow to stand over night). Mix well
and heat for 30-60 minutes in a boiling water bath. Allow to
cool, add 50 ml of sodium hydroxide (0. 5 mol/L) VS,
accurately measured, and a solution of phenolphthalein in
pyridine ( 1 - 100) as indicator and titrate with sodium
hydroxide (O. 5 mol/L) VS until a pink colour is produced.
Perform a blank determination and make any necessary
correction. Multiply the weight ( g) of substance being
examined by 4000 and divide by the volume ( ml) of sodium
hydroxide (0. 5 mol/L) VS consumed, the average molecular
weight is 285-315.
Acidity Dissolve l. O g in 20 ml of water, pH 4. 5-7. 5
<0631>.
Clarity and colour of solution Dissolve 5. O g in 50 ml of
water, the solution is clear and colourless. Any opalescence
produced is not more pronounced than that of reference
suspension 2 <0902 method 1 >. Any colour produced is not
more intense than that of reference solution Y 2 < 0901
method 1 >.
Ethylene glycol, diglycol and triglycol W eigh 400 mg each of
ethylene glycol, diglycol and triglycol into a 100 ml
volumetric flask, dilute to volume with anhydrous ethanol,
mix well as the reference stock solution. Weigh 400 mg of 1,
3- butanol as internal standard into a 100 ml volumetric
flask, dilute to volume with anhydrous ethanol, mix well as
the interna! reference stock solution. Measure 1 ml of each
above solutions into a 100 ml volumetric flask, dilute to
volume with anhydrous ethanol, mix well as the reference
solution. Weigh 4. O g of the substance being examined into a
100 ml volumetric flask, add 1 ml of the interna! reference
stock solution, dilute to volume with anhydrous ethanol, mix
well as the test solution. Carry out the method for gas
chromatography < 0521 >, using a column packed with
phenyl-polydimethylsiloxane ( 50 : 50 ) as the stationary
phase, maintain the column temperature at 60ºC for 5
minutes, raise the temperature to l 70ºC by 2ºC per minute
and maintain for 5 minutes, then raise to 280ºC by 15ºC per
minute and maintain for 50 minutes. The temperature of
injection port is 270ºC. The temperature of the detector is
290ºC. Carrier gas is high purity nitrogen, the fuel gas is
hydrogen, the auxiliary gas is compressed air, the velocity of
carrier gas is 4. O ml per minute. Calculate the content by the
internal standard method, the each content of ethylene
glycol, diglycol and triglycol is not more than O. 1%.
Ethylene oxide and dioxin Accurately weigh 1 g in a head-
space vial, add l. O ml of purified water, accurate1y
measured, seal the vial and mix well as the test solution.
Take 300 µl of ethylene oxide ( equivalent to about O. 25 g)
into a 100 ml volumetric flask containing 50 ml of treated
polyethylene glycol 400 ( using a rotary evaporator remove
any volatile components at a temperature of 60ºC and
pressure of l. 5 to 2. 5 kPa for 6 hours), dilute to volume
with the same solvent, mix well as ethylene oxide reference
stock solution. Accurately weigh 1 g of cold ethylene oxide
stock solution, into a 50 ml volumetric flask containing 40 ml
of treated polyethylene glycol 400, dilute to volume with the
same solvent. Accurately weigh 10 g into a 50 ml volumetric
flask containing 30 ml of water, dilute to volume with water.
Accurately measure 10 ml into a 50 ml volumetric flask,
dilute to volume with water, mix well as ethylene oxide
reference solution. Dilute a quantity of dioxan, accurately
weighed, with water to produce a solution of O. 1 mg per ml,
as dioxan reference solution. Accurately weigh 1 g of the
substance being examined, in a headspace vial, add
accurately O. 5 ml of ethylene oxide reference solution and
O. 5 ml of dioxan reference solution, seal and mix well as the
reference solution. Measure O. 5 ml of ethylene oxide
reference solution in a headspace vial, add O. 1 ml of O. 001 %
acetaldehyde solution with freshly prepared and O. 1 ml of
dioxan solution, seal and mix well as the system suitability
test solution. Carry out the method for gas chromatography
<0521 >, using a column packed with polydimethylsiloxane as
the stationary phase. Maintain the column temperature at
35ºC for 5 minutes, raise the temperature to 180ºC by 5ºC
per minute, then raise to 230ºC by 30ºC per minute and
maintain for 5 minutes ( adjust if necessary ). The
temperature of the injection port is 150ºC. The temperature
of the detector is 250ºC . Use the head-space injection
method. The equilibration temperature is 70ºC and maintain
for 4S minutes. Inject the gaseous sample of the system
suitability test solution onto the column and record the
chromatogram. Adjust the detection sensitivity so that the
peak heights of ethylene oxide and acetaldehyde in the
chromatogram are about 15 percent of the full scale of the
chart. The resolution factor between the peaks corresponding
to acetaldehyde and ethylene oxide is at least 2. O. The
signal-to-noise ratio of the peak of dioxan is not less than 5.
Inject separately the gaseous sample each of the test solution
and reference solution onto the column. Repeat the procedure
at least three times. The relative standard deviation of the
three areas of peak of ethylene oxide is not greater than 15
percent and the relative standard deviation of the three areas
of peak of dioxan is not greater than 10 percent. Calculate
the content according to the peak area obtained in the
chromatogram by the standard addition method, ethylene
oxide is not more than O. 0001 % and dioxan is not more than
o. 001%.
Standardization of ethylene oxide reference stock solution
Measure 10 ml of 50 % suspension of magnesium chloride in
absolute ethanol, accurately add 20 ml of ethanolic
hydrochloric acid (O. 1 mol/L) VS, allow to stand overnight.
Weigh 5 g of the ethylene oxide ref erence stock solution into
the same flask and stand for 30 minutes. Carry out the method
for potentiometric titration <0701 >, titrate with ethanolic
potassium hydroxide (O. 1 mol/L) VS. Perform a blank
determination and make any necessary correction. Each ml of
ethanolic potassium hydroxide (0. 1 mol/L) VS is equivalent
to 4. 404 mg of ethylene oxide.
Fonnaldehyde To 1 g of the substance being examined,
accurately weighed, add O. 25 ml of O. 6 % sodium
chromophate solution, cool in an ice water and add 5 ml of
sulphuric acid, mix well. Allow to stand for 15 minutes and
transfer slowly to a 25 ml volumetric flask containing 10 ml
of water, allow to cool and dilute to volume with water, and
mix well as the test solution. Dilute O. 27 g of formaldehyde
solution ( equivalent to O. 1 g of formaldehyde), accurately

weighed, to 100 ml volumetric flask with water. Dilute 1ml
of this solution to 100 ml with water. Take accurately 1 ml,
proceeded with the same procedure beginning at the words
"add O. 25 ml of O. 6% sodium chromophate TS", and use
the resulting solution as the reference solution. Determine
the absorbance at 567 nm < 0401 ) , perform a blank
determination, and make any necessary correction. The
absorbance obtained with the test solution is not greater than
that of the reference solution (0. 001%).
Water Not more than l. O% <0832 method 1 ( 1 ) ) , using
2. o g.
Reducing substances Transfer l. O g of the substance being
examined into a neutral cylinder which is colourless and
transparent with 12 mm of outer diameter, dissolve in 1 ml
of 1 % resorcinol solution ( heat if necessary) , and add 2 ml
of hydrochloric acid. Transfer reference solution into another
cylinder, allow to stand for 5 minutes, view clown vertically
the cylinders against a white background, and the colour of
the test solution is not more intense than that of reference
solution OR2.
Residue on ignition Not more than O. 1% <0841 ).
Heavy metals Dissolve 4. O g in 5 ml of hydrochloric acid
solution (9-1000) and a quantity of water, adjust to pH
3. 0-4. O with dilute acetic acid or ammonia TS, then dilute
with water to 25 ml. Carry out the limit test for heavy
metals ( 0821 method 1): not more than O. 0005%.
Arsenic Transfer O. 67 g into a kjeldahl flask, add 5 ml of
sulfuric acid, digest gently at a temperature not exceeding
120ºC, add sulfuric acid if necessary, keep the total quantity
not more than 10 ml. Add cautiously concentrate hydrogen
peroxide solution in dropwise until the reaction goes to
completion. Continue to heat the reaction mixture, and add
conccntratc hydrogen peroxide solution in dropvlÍse until the
solution becomes colourless. Allow to cool, add 10 ml of
water, evaporate until the fume of excess hydrogen peroxide
evolves completely. Add 5 ml of hydrochloric acid and a
quantity of water to the resulting solution. Carry out the
limit test for arsemc ( 0822 method 1 ) , not more than
o. 0003%.
Bacterial endotoxin Carry out the test for bacteria!
endotoxin < 1143): not more than O. 012 EU per mg of
macrogol 300.
Sterility ( intended for the sterile preparations without
terminal sterilization) Carry out the method for sterility
test <1101), it complies with the requirement.
Category Pharmaceutical excipients, solvent and plasticizer.
Storage Preserve in tightly closed containers.
Macrogol 400
Macrogol 400 is a mixture of polymer of ethylene oxide and
water with the molecular formula represented by HO
( CH 2CH 2O ) n H, where n is the average number of
ethyleneoxy groups.
Description A colourless or almost colourless viscous
liquid, odour, slightly characteristic.
Freely soluble in water and in ethanol, insoluble in ether.
Congealing point 4-SºC <0613 ).
Relative density 1.110-1.140 (0601 ).
Macrogol 400 573
Viscosity kinematic viscosity is 37-45 mm2/s, at 40ºC
<0633 method 1 ) , using a capillary tube of l. 2 mm in
internal diameter.
ldentification ( 1) To O. 05 g add 5 ml of dilute hydrochloric
acid and 1 ml of barium chloride TS, shake and filter, to the
filtra te add 1 ml of 10 % phosphomolybdic acid solution, a
yellow-green precipitate is formed.
(2) Place O. 1 g in a test tube, add O. 1 g each of potassium
thiocyanate and cobaltous nitrate, mix and add 5 ml of
dichloromethane, the solution exhibits a blue colour.
Average molecular weight Place about l. 2 g, accurately
weighed, in a dry 250 ml conical flask with stopper, add
25 ml, accurately measured, of phthalic anhydride solution
in pyridine (dissolve 14 g of phthalic anhydride in 100 ml of
anhydrous pyridine and allow it to stand overnight). Mix well
and heat for 30-60 minutes in a boiling water bath. Allow it
to cool, add 50 ml of sodium hydroxide (0. 5 mol/L) VS,
accurately measured, and a solution of phenolphthalein in
pyridine Cl-100) as indicator and titrate with sodium hydr-
oxide ( O. 5 mol/L) VS until a pink colour is produced.
Perform a blank determination and make any necessary
correction. Multiply the weight ( g) of substance being
examined by 4000 and divide by the volume ( ml) of sodium
hydroxide (0. 5 mol/L) VS consumed, the average molecular
weight is 380-420.
Acidity Dissolve l. O g in 20 ml water, pH 4. 0-7. O
(0631 ).
Oarity and colour of solution Dissolve 5. O g in 50 m1 of water,
the solution is clear and colourless. Any opalescence
produced is not more pronounced than that of reference
suspension 2 ( 0902 ) . Any colour produced is not more
intense than that of reference solution Y2 ( 0901 method 1 ) .
Ethylene glycol, diglycol and triglycol \Veigh 400 mg each of
ethylene glycol, diglycol and triglycol into a 100 ml
volumetric flask, dilute to volume with anhydrous ethanol,
mix well as the stock reference solution. Weigh 400 mg
internal standard 1, 3-butanol into a 100 ml volumetric
flask, dilute to volume with anhydrous ethanol, mix well as
the stock internal ref eren ce solution. Measure each of
solution 1 ml into a 100 ml volumetric flask, dilute to volume
with anhydrous ethanol, mix well as the reference solution.
Weigh 4. O g into a 100 ml volumetric flask, add 1 ml of the
stock internal reference solution, dilute to volume with
anhydrous ethanol, mix well as the test solution. Carry out
the method for gas chromatography ( 0521), using a column
packed with phe-nyl-polydimethylsiloxane ( 50 : 50) as the
stationary phase, maintain the column temperature at 60ºC
for 5 minutes, raise the temperature to l 70ºC by 2ºC per
minute and maintain for 5 minutes, then to 280ºC by 15ºC
per minute and maintain for 50 minutes. The temperature of
injection port is 270ºC. The temperature of the detector is
290ºC. Carrier gas is high purity nitrogen, the fuel gas is
hydrogen, the auxiliary gas is compressed air, the velocity of
carrier gas is 4. O ml per minute. Calculate the content by the
internal standard method, the each content of ethylene glycol,
diglycol and triglycol is not more than O. 1 % .
Ethylene oxide and dioxan Weigh accurately 1 g in a
headspace vial, add 1 ml of purified water, accurately
measured, seal the vial and mix well as the test solution.
Take 300 µl ethylene oxide (equivalent to about O. 25 g) into
a 100 ml volumetric flask containing 50 ml of treated
polyethylene glycol 400 ( using a rotary evaporator remove
any volatile components at a temperature of 60ºC and
pressure of l. 5 to 2. 5 kPa for 6 hours) , dilute to volume
 Macrogol 400 (For Injection)
with the same solvent, mix well as ethylene oxide stock
reference solution. Weigh accurately 1 g of cold ethylene
oxide stock solution, into a 50 ml volumetric flask containing
40 ml of treated polyethylene glycol 400, dilute to volume
with the same solvent. Weigh accurately 10 g into a 50 ml
volumetric flask containing 30 ml of water, dilute to volume
with water. Transfer accurately 10 ml into a 50 ml
volumetric flask, dilute to volume with water, mix well as
ethylene oxide reference solution. Dilute a quantity of
dioxan, accurately weighed, with water to produce a solution
of O. 1 mg per ml, as dioxan reference solution. Weigh
accurately 1 g of the substance to be examined, in a headspace
vial, add accurately, O. 5 ml of ethylene oxide reference
solution and O. 5 ml of dioxan reference solution, and seal the
vial, mix well as the reference solution. Measure O. 5 ml of
ethylene oxide reference solution in a headspace vial, and add
O. 1 ml of freshly prepared O. 001 % acetaldehyde solution and
O. 1 ml of dioxan solution, seal the vial and mix well as the
system suitability solution. Carry out the method for gas
chromatography < 0521 ) , using a column packed with
polydimethylsiloxane as the stationary phase, maitain the
column temperature at 35ºC for 5 minutes, raise the
temperature to 180ºC by 5ºC per minute, then to 230ºC by
30ºC per minute and maintain for 5 minutes ( adjust if
necessary). The temperature of the injection port is 150ºC.
The temperature of the detector is 250ºC. Use the head-
space injection method. The equilibration temperature is
70ºC and maintain for 45 minutes. Proceed with the system
suitability solution under the above operating conditions.
Adjust the dection sensitivity so that the peak heights of
ethylene oxide and acetaldehyde in the chromatogram are
about 15 % of the full scale of the chart. The resolution
factor between the peaks corresponding to acetaldehyde and
ethylene oxide is at least 2. O, the signal-to-noise ratio of the
peak of dioxan is not less than 5. Inject separately the gaseous
sample each in the test vial and reference vial onto the
column. Repeat the procedure at least three times. The
relative standard deviation of the three areas of peak of
ethylene oxide is not greater than 15 % and the relative
standard deviation of the three areas of peak of dioxan is not
greater than 10 % . Calculate the content according to the
peak area obtained in the chromatogram by the standard
addition method, ethylene oxide is not more than O. 0001 %
and dioxan is not more than O. 001%.
Formaldehyde To 1 g of the substance being examined,
accurately weighed, add O. 25 ml of O. 6 % sodium
chromophate solution, cool in an iced water and add 5 ml of
sulphuric acid, mix well. Allow to stand for 15 minutes and
transfer slowly to a 25 ml volumetric flask containing 10 ml
of water, allow to cool and dilute to volume with water, mix
well, as the test solution. Dilute O. 81 g of formaldehyde,
accurately weighed, to 100 ml with water. Dilute 1 ml of this
solution to 100 ml volumetric flask with water. Take
accurately 1 ml, proceeded with the same procedure
beginning at the words "add O. 25 ml of O. 6% sodium
chromophate TS", use the resulting solution as the reference
solution. Determine the absorbance at 567 nm < 0401 ) ,
perform a blank determination and make any necessary
correction. The absorbance obtained with the test solution is
not greater than that of the reference solution (O. 003 %) .
Water Not more than l. O% <0832 method 1 ( 1 ) ) , using
o g.
2.
Residue on ignition Not more than O. 1 % <0841 ) .
Heavy metals Dissolve 4. O g in 5 ml of hydrochloric acid
solution (9-1000) and a quantity of water, adjust to pH
3. 0-4. O with dilute acetic acid or ammonia TS, then dilute to
25 ml with water. Carry out the limit test for heavy metals
<0821 method 1 ) : not more than O. 0005 % .
Arsenic Transfer O. 67 g into a kjeldahl flask, add 5 ml of
sulfuric acid, digest gently at a temperature not exceeding
120ºC, add sulfuric acid if necessary, keep the total quantity
not more than 10 ml. Add cautiously, in dropwise, of
concentrate hydrogen peroxide solution until the reaction goes
to completion. Continue to heat the reaction mixture and
add, in dropwise, concentrate hydrogen peroxide solution
until the solution becomes colourless. Allow it to cool, add
10 ml of water, evaporate until the fume of excess hydrogen
peroxide evolves completely. Add 5 ml of hydrochloric acid
and a quantity of water to the resulting solution. Carry out
the limit test for arsenic <0822 method 1), not more than
o. 0003%.
Category Pharmaceutical excipients, solvent and plasticizer,
etc.
Storage Preserve in tightly closed containers.
Macrogol 400 ( For Injection)
[25322-68-3]
Macrogol 400 is a mixture of polymer of ethylene oxide and
water, represented by the general molecular formula HO
(CH 2 CH 2 0)n H, Nhere n is the average number of ethylene
1
oxide groups.
Description A colourless or almost colourless viscous
liquid, odour, slightly characteristic.
Freely soluble in water and in ethanol, insoluble in ether.
Relative density l. 110-1. 140 <0601 ) .
Viscosity Kinematic viscosity is 37-45 mm 2 / s, at 40ºC
<0633 method 1 ) , using a capillary tube of l. 2 mm or the
suitable size in internal diameter.
Identification ( 1 ) To O. 05 g, add 5 ml of dilute
hydrochloric acid and 1 ml of barium chloride TS, shake, and
fil ter. To the filtrate add 1 ml of 10 % phosphomolybdic acid
solution, a yellow-green precipitate is formed.
(2) Place O. 1 gin a test tube, add O. 1 g each of potassium
thiocyanate and cobaltous nitrate, mix and add 5 ml of
dichloromethane, the solution exhibits a blue colour.
Average molecular weight Place about l. 2 g, accurately
weighed, in a dry 250 ml conical flask with stopper, add
25 ml, accurately measured, of phthalic anhydride solution
in pyridine (dissolve 14 g of phthalic anhydride in 100 ml of
anhydrous pyridine and allow it to stand over night). Mix
well and heat for 30-60 minutes in a boiling water bath.
Allow it to cool, add 50 ml of sodium hydroxide (O. 5 mol/L)
VS, accurately measured, and a solution of phenolphthalein
in pyridine ( 1-100) as indicator and titrate with sodium
hydroxide (O. 5 mol/L) VS until a pink colour is produced.
Perform a blank determination and make any necessary
correction. Multiply the weight ( g) of substance being
examined by 4000 and divide by the volume (ml) of sodium
hydroxide (0. 5 mol/L) VS consumed, the average molecular
weight is 380-420.
Acidity Dissolve l. O g in 20 ml of water, pH 4. 0-7. O
<0631 >.
Clarity and colour of solution Dissolve S. O g in 50 ml of
water, the solution is clear and colourless. Any opalescence

produced is not more pronounced than that of reference
suspension 2 ( 0902 >. Any colour produced is not more
intense than that of reference solution Y 2 ( 0901 method 1 >.
Ethylene glycol, diglycol and triglycol W eigh 400 mg each of
ethylene glycol, diglycol and triglycol into a 100 ml
volumetric flask, dilute to volume with anhydrous ethanol,
mix well as the reference stock solution. Weigh 400 mg of 1,
3- butanol as interna! reference into a 100 ml volumetric
flask, dilute to volume with anhydrous ethanol, mix well as
the interna! reference stock solution. Measure 1 ml of each
above solutions into a 100 ml volumetric flask, dilute to
volume with anhydrous ethanol, mix well as the reference
solution. Weigh 4. O g of the substance being examined into a
100 ml volumetric flask, add 1 ml of the interna! reference
stock solution, dilute to volume with dehydrated ethanol,
mix well as the test solution. Carry out the method far gas
chromatography ( 0521 >, using a column packed with
phenyl-polydimethylsiloxane (50% : 50%) as the stationary
phase, maintain the column temperature at 60ºC far 5
minutes, raise the temperature to l 70ºC by 2ºC per minute
and maintain far 5 minutes, then raise to 280ºC by 15ºC per
minute and maintain far 50 minutes. The temperature of
injection port is 270ºC. The temperature of the detector is
290ºC. Carrier gas is high purity nitrogen, the fuel gas is
hydrogen, the auxiliary gas is compressed air, the velocity of
carrier gas is 4. O ml per min. Calculate the content by the
interna! standard method, the each content of ethylene
glycol, diglycol and triglycol is not more than O. 1 %.
Ethylene oxide and dioxin Accurately weigh 1 g in a head-
space vial, add 1 ml of purified water, accurately measured,
seal the vial and mix well as the test solution. Take 300 µl of
ethylene oxide ( equivalent to about O. 25 g) into a 100 ml
volumetric flask containing 50 ml of treated polyethylene
glycol 400 ( using a rotary evaporator to remove any volatile
components at a temperature of 60ºC and pressure of l. 5 to
2. 5 kPa far 6 hours) , dilute to volume with the same
solvent, mix well as ethylene oxide reference stock solution.
Accurately weigh 1 g of cold ethylene oxide stock solution,
into a 50 ml volumetric flask containing 40 ml of treated
polyethylene glycol 400, dilute to volume with the same
solvent. Accurately weigh 10 g into a 50 ml volumetric flask
containing 30 ml of water, dilute to volume with water.
Accurately measure 10 ml into a 50 ml volumetric flask,
dilute to volume with water, mix well as ethylene oxide
reference solution. Dilute a quantity of dioxan, accurately
weighed, in water to produce a solution of O. 1 mg per ml, as
dioxan reference solution. Accurately weigh 1 g of the
substance being examined, in a head-space vial, add
accurately O. 5 ml of ethylene oxide reference solution and
O. 5 ml of dioxan reference solution, seal and mix well as the
system suitability test solution. Measure O. 5 ml of ethylene
oxide reference solution in a head-space vial, add O. 1 ml of
O. 001 % acetaldehyde solution with freshly prepared and
O. 1 ml of dioxan solution, seal and mix well as the system
suitability test solution. Carry out the method far gas
chromatography ( 0521 >, using a column packed with
polydimethylsiloxane as the stationary phase, maintain the
column temperature at 35ºC far 5 minutes, raise the
temperature to 180ºC by 5ºC per minute, then raise to 230ºC
by 30ºC per minute and maintain far 5 minutes ( adjust if
necessary). The temperature of the injection port is 150ºC.
The temperature of the detector is 250ºC. Use the head-space
injection method. The equilibration temperature is 70ºC and
maintain far 45 minutes. Inject the gaseous sample of the
system suitability test solution onto the column and record
the chromatogram. Adjust the attenuation so that the peak
Macrogol 400 (Far lnjection) 515
heights due to ethylene oxide and acetaldehyde in the
chromatogram are about 15 percent of the full scale of the
chart. The resolution factor between the peaks corresponding
to acetaldehyde and ethylene oxide is at least 2. O; the signal-
to-noise ratio of the peak of dioxan is not less than 5. Inject
separately the gaseous samples of the test solution and
reference solution onto the column. Repeat the procedure at
least three times. The relative standard deviation of the 3
areas of peak of ethylene oxide is not greater than 15 % and
the relative standard deviation of the 3 areas of peak of
dioxan is not greater than 10 %. Calcula te the content with
respect to the peak area obtained in the chromatogram by the
standard addition method, ethylene oxide is not more than
O. 0001% and dioxan is not more than O. 001%.
Standardization of ethylene oxide reference stock sol-
ution Measure 10 ml of 50% suspension of magnesium
chloride in absolute ethanol, accurately add 20 ml of
ethanolic hydrochloric acid (O. 1 mol/L) VS, allow to stand
overnight. W eigh 5 g of the ethylene oxide ref erence stock
solution into the same flask and stand far 30 minutes. Carry
out the method far potentiometric titration ( 0701 >, titrate
with ethanolic potassium hydroxide ( O. 1 mol/L) VS.
Perfarm a blank determination and make any necessary
correction. Each ml of ethanolic potassium hydroxide (O. 1
mol/L) VS is equivalent to 4. 404 mg of ethylene oxide.
Formaldehyde To 1 g of the substance being examined,
accurately weighed, add O. 25 ml of O. 6% sodium chromophate
test solution, cool in an iced water and add 5 ml of sulphuric
acid, mix well. Allow to stand far 15 minutes and transfer
slowly to a 25 ml volumetric flask containing 10 ml of water,
allow to cool and dilute to volume with water, and mix well
as the test solution. Dilute O. 27 g of farmaldehyde solution
(equivalent to O. 1 g of formaldehyde), accurately weighed,
to 100 ml with water. Dilute l ml of this solution to 100 ml
with water. Measure accurately 1 ml, proceeded with the
same procedure beginning at the words "add O. 25 ml of
O. 6 % sodium chromophate test solution", and use the
resulting solution as the reference solution. Determine the
absorbance at 567 nm ( 0401 >,perfarm a blank
determination and make any necessary correction. The
absorbance obtained with the test solution is not greater than
that of the reference solution (0. 001%).
Water Not more than l. O% ( 0832 method 1 ( 1 ) >, using
2. o g.
Reducing substances Transfer l. O g of the substance being
examined into a neutral cylinder which is colourless and
transparent with 12 mm of outer diameter, dissolve in 1 ml
of 1% resorcinol solution (heat if necessary), and add 2 ml
of hydrochloric acid. Transfer reference solution into another
cylinder, allow to stand far 5 minutes, view clown vertically
the cylinders against a white background, and the colour of
the test solution is not more intense than that of reference
solution OR2.
Residue on ignition Not more than O. 1% ( 0841 >.
Heavy metals Dissolve 4. O g in 5 ml of hydrochloric acid
solution (9-1000) and a quantity of water, adjust to pH
3. 0-4. O with dilute acetic acid or ammonia test solution,
then dilute with water to 25 ml. Carry out the limit test far
heavy metals (0821 method 1 >, not more than O. 0005%.
Arsenic Transfer O. 67 g into a kjeldahl flask, add 5 ml of
sulfuric acid, digest gently at a temperature not exceeding
120ºC, add sulfuric acid if necessary, keep the total quantity
not more than 10 ml. Add cautiously concentrate hydrogen
peroxide solution in dropwise until the reaction goes to
 Macrogol 600
completion. Continue to heat the reaction mixture, and add
concentrate hydrogen peroxide solution in dropwise until the
solution becomes colourless. Allow to cool, add 10 ml of
water, evaporate until the fume of excess hydrogen peroxide
evolves completely. Add 5 ml of hydrochloric acid anda quantity
of water to the resulting solution. Carry out the limit test far
arsenic <0822 method 1 ) , not more than O. 0003 % .
Bacterial endotoxin Carry out the test far bacteria!
endotoxin < 1143): not more than O. 012 EU per mg of
macrogol 400.
Sterility ( intended far the sterile preparations without
terminal sterilization) Carry out the method far sterility test
<1101), it complies with the requirement.
Category Pharmaceutical excipients, solvent and plasticizer,
etc.
Storage Preserve in tightly closed containers.
Macrogol 600
Macrogol 600 is a mixture of polymer of ethylene oxide and
water with the molecular formula represented by HO
( CH2 CH2 O ) n H, where n is the average number of
ethyleneoxy groups.
Description Colourless or almost colourless viscous liquid,
or translucent waxy soft substance; odour~ slight ;md
characteris tic.
Freely soluble in water and in ethanol, insoluble in ether.
Congealing point 15-25 ºC <0613 ) .
Relative density l. 115-1. 145 <0601).
Viscosity kinematic viscosity is 56-62 mm2/s, at 40ºC
<0633 method 1 ) , using a capillary tube of 1. 2 mm in
interna! diameter.
Identification ( 1 ) To O. 05 g add 5 ml of dilute
hydrochloric acid and 1 ml of barium chloride TS, shake and
filter, to the filtrate add 1 ml of 10% phosphomolybdic acid
solution, a yellow-green precipitate is formed.
(2) Place O. 1 g in a test tube, add O. 1 g each of potassium
thiocyanate and cobaltous nitrate, mix and add 5 ml of
dichloromethane, the solution exhibits a blue colour.
Average molecular weight Place about l. 2 g, accurately
weighed, in a dry 250 ml conical flask with stopper, add
25 ml, accurately measured, of phthalic anhydride solution
in pyridine (dissolve 14 g of phthalic anhydride in 100 ml of
anhydrous pyridine and allow it to stand overnight). Mix well
and heat far 30-60 minutes in a boiling water bath. Allow it
to cool after removal, add 50 ml of sodium hydroxide
(0. 5 mol/L) VS, accurately measured, and a solution of
phenolphthalein in pyridine Cl-100) as indicator and titrate
with sodium hydroxide (O. 5 mol/L) VS until a pink colour is
produced. Perform a blank determination and make any
necessary correction. Multiply the weight (g) of substance
being examined by 4000 and divide by the volume ( mD of
sodium hydroxide (O. 5 mol/L) VS consumed, the average
molecular weight is 570-630.
Acidity Dissolve l. O gin 20 ml water, pH 4. 0-7. O (0631 ).
Clarity and colour of solution Dissolve 5. O g in 50 ml of
water, the solution is clear and colourless. Any opalescence
produced is not more pronounced than that of reference
suspension 2 < 0902 ) . Any colour produced is not more
intense than that of reference solution Y2 <0901 method 1 ) .
Ethylene glycol, diglycol and triglycol W eigh 400 mg each of
ethylene glycol, diglycol and triglycol in to a 100 ml
volumetric flask, dilute to volume with dehydrated ethanol,
mix well as the stock reference solution. Weigh 400 mg
internal standard 1, 3-butanol into a 100 ml volumetric
flask, dilute to volume with anhyrous ethanol, mix well as
the stock internal reference solution. Measure each of
solution 1 ml into a 100 ml volumetric flask, dilute to volume
with anhyrous ethanol, mix well as the reference solution.
Weigh 4. O g into a 100 ml volumetric flask, add 1 ml of the
stock internal reference solution, dilute to volume with
anhyrous ethanol, mix well as the test solution. Carry out
the method far gas chromatography <0521), using a column
packed with phenyl-polydimethylsiloxane ( 50 : 50) as the
stationary phase, maintain the column temperature at 60ºC
far 5 minutes, raise the temperature to l 70ºC by 2ºC per
minute and maintain far 5 minutes, then to 280ºC by 15ºC
per minute and maintain far 50 minutes. The temperature of
injection part is 270ºC. The temperature of the detector is
290ºC. Carrier gas is high purity nitrogen, the fuel gas is
hydrogen, the auxiliary gas is compressed air, the velocity of
carrier gas is 4. O ml per minute. Calculate the content by the
internal standard method, the each content of ethylene glycol,
diglycol and triglycol is not more than O. 1 % .
Ethylene oxide and dioxan Weigh accurately 1 g in a headspace
vial, add 1 ml of purified water, accurately measured, seal
the vial and mix well as the test solution. Take 300 pl
ethylene oxide ( equivalent to about O. 25 g) into a 100 ml
volumetric flask containing 50 ml of treated polyethylene
glycol 400 ( using a rotary evaporator remove any volatile
components ata temperature of 60ºC and pressure of l. 5 to
2. 5 kPa far 6 hours) , dilute to volume with the same
solvent, mix well as ethylene oxide stock reference solution.
Weigh accurately 1 g of cold ethylene oxide stock solution,
into a 50 ml volumetric flask containing 40 ml of treated
polyethylene glycol 400, dilute to volume with the same
solvent. Weigh accurately 10 g into a 50 ml volumetric flask
containing 30 ml of water, dilute to volume with water.
Transfer accurately 10 ml into a 50 ml volumetric flask,
dilute to volume with water, mix well as ethylene oxide
reference solution. Dilute a quantity of dioxan, accurately
weighed, with water to produce a solution of O. 1 mg per ml,
as dioxan reference solution. Weigh accurately 1 g of the
substance to be examined, in a headspace vial, add accurately
O. 5 ml of ethylene oxide reference solution and O. 5 ml of
dioxan reference solution, and seal the vial, mix well as the
reference solution. Measure O. 5 ml of ethylene oxide
reference solution in a headspace vial, and add O. 1 ml of
freshly prepared O. 001 % acetaldehyde solution and O. 1 ml of
dioxan solution, seal the vial and mix well as the system
suitability solution. Carry out the method far gas chrom-
atography <0521 ) , using a column packed with polydime-
thylsiloxane as the stationary phase; maitain the column
temperature at 35ºC far 5 minutes, raise the temperature to
180ºC by 5ºC per minute, then to 230ºC by 30ºC per minute
and maintain far 5 minutes ( adjust if necessary ). The
temperature of the injection port is 150ºC. The temperature
of the detector is 250ºC . Use the head-space injection
method. The equilibration temperature is 70ºC and maintain
far 45 minutes. Proceed with the system suitability solution
under the above operating conditions. Adjust the dection
sensitivity so that the peak heights of ethylene oxide and
acetaldehyde in the chromatogram are about 15 % of the full
scale of the chart. The resolution factor between the peaks
corresponding to acetaldehyde and ethylene oxide is at least

2. O, the signal-to-noise ratio of the peak of dioxan is not less
than 5. Inject separately the gaseous sample each in the test
vial and reference vial onto the column. Repeat the procedure
at least three times. The relative standard deviation of the
three areas of peak of ethylene oxide is not greater than 15 %
and the relative standard deviation of the three areas of peak
of dioxan is not greater than 10%. Calculate the content
according to the peak area obtained in the chromatogram by
the standard addition method, ethylene oxide is not more
than O. 0001% and dioxan is not more than O. 001%.
Formaldehyde To 1 g of the substance being examined,
accurately weighed, add O. 25 ml of O. 6 % sodium
chromophate, cool in an ice water and add 5 ml of sulphuric
acid, mix well. Allow to stand for 15 minutes and transfer
slowly to a 25 ml volumetric flask containing 10 ml of water,
allow to cool and dilute to volume with water, mix well, as
the test solution. Dilute O. 81 g of formaldehyde, accurately
weighed, to 100 ml with water. Dilute 1 ml of this solution
to 100 ml with water. Take accurately 1 ml proceeded with
the same procedure, beginning at the words "add O. 25 ml of
O. 6% sodium chromophate TS", and use the resulting
solution as the reference solution. Determine the absorbance
at 567 nm <0401 ) , adjusted by a blank solution proceeded
with the same procedure. The absorbance obtained with the
test solution is not greater than that of the reference solution
(O. 003%).
Water Not more than l. O% <0832 method 1 ( 1 ) ) , using
2. o g.
Residue on ignition Not more than O. 1 % <0841 ) .
Heavy metals Dissolve 4. O g in 5 ml of hydrochloric acid
solution (9-1000) and a quantity of water, adjust to pH
3. 0-4. O with dilute acetic acid or ammonia test solution,
then dilute to 25 ml with water. Carry out the limit test for
heavy metals <0821 method 1): not more than O. 0005%.
Arsenic Transfer O. 67 g into a kjeldahl flask, add 5 ml of
sulfuric acid, digest gently at a temperature not exceeding
120ºC, add sulfuric acid if necessary, keep the total quantity
not more than 10 ml. Add cautiously, in dropwise, of
concentrate hydrogen peroxide solution until the reaction goes
to completion. Continue to heat the reaction mixture and
add, in dropwise, concentrate hydrogen peroxide solution
until the solution becomes colourless. Allow it to cool, add
10 ml of water, evaporate until the fume of excess hydrogen
peroxide evolves completely. Add 5 ml of hydrochloric acid
and a quantity of water to the resulting solution. Carry out
the limit test for arsenic <0822 method 1 ) , not more than
o. 0003%.
Category Pharmaceutical excipients, solvent and plasticizer,
etc.
Storage Preserve in tightly closed containers.
Macrogol 1000
Macrogol 1000 is a mixture of polymer of ethylene oxide and
water with the molecular formula represented by HO
( CH2 CH2 O ) n H, where n is the average number of
ethyleneoxy groups.
Description Colourless or almost colourless viscous liquid or
translucent waxy soft substance, odour, slightly characteristic.
Freely soluble in water and in ethanol, insoluble in ether.
Macrogol 1000 ·577
Congealing point 33-38ºC <0613).
Viscosity Dissolve 25. O g in a 50 ml volumetric flask in
water, dilute to volume with water and mix well, the kinetic
viscosityat 40ºC is 8.5-11.0 mm2/s, (0633 method ]),
using a capillary tube of O. 8 mm in internal diameter.
ldentification ( 1 ) To O. 05 g add 5 ml of dilute
hydrochloric acid and 1 ml of barium chloride TS, shake and
filter, to the filtrate add 1 ml of 10% phosphomolybdic acid
solution, a yellow-green precipitate is formed.
(2) Place O. 1 gin a test tube, add O. 1 g each of potassium
thiocyanate and cobaltous nitrate, mix and add 5 ml of
dichloromethane, the solution exhibits a blue colour.
Average molecular weight Place about 3. O g, accurately
weighed, in a dry 250 ml conical flask with stopper, add
25 ml, accurately measured, of phthalic anhydride solution
in pyridine (dissolve 14 g of phthalic anhydride in 100 ml of
anhydrous pyridine and allow it to stand overnight). Mix well
and heat for 30-60 minutes in a boiling water bath. Allow it
to cool, add 50 ml of sodium hydroxide (0. 5 mol/L) VS,
accurately measured, and a solution of phenolphthalein in
pyridine ( 1 - 100) as indicator and titrate with sodium
hydroxide (O. 5 mol/L) VS until a pink colour is produced.
Perform a blank determination and make any necessary
correction. Multiply the weight ( g) of substance being
examined by 4000 and divide by the volume ( ml) of sodium
hydroxide (O. 5 mol/L) VS consumed, the average molecular
weight is 900-1100.
Acidity Dissolve l. O g in 20 ml water, pH 4. 0-7. O
(0631 ).
Clarity and colour of solution Dissolve 5. O g in 50 ml of
water, the solution is clear and colourless. Any opalescence
produced is not more pronounced than that of reference
suspension 2 < 0902 ) . Any colour produced is not more
intense than that of reference solution Y2 <0901 method 1 ) .
Ethylene glycol, diglycol and triglycol W eigh 400 mg each of
ethylene glycol, diglycol and triglycol into a 100 ml
volumetric flask, dilute to volume with dehydrated ethanol,
mix well as the stock reference solution. Weigh 400 mg
internal standard 1, 3-butanol into a 100 ml volumetric
flask, dilute to volume with anhydrous ethanol, mix well as
the stock interna} reference solution. Measure each of
solution 1 ml into a 100 ml volumetric flask, dilute to volume
with anhydrous ethanol, mix well as the reference solution.
Weigh 4. O g into a 100 ml volumetric flask, add 1 ml of the
stock internal reference solution, dilute to volume with
anhydrous ethanol, mix well as the test solution. Carry out
the method for gas chromatography <0521 ) , using a column
packed with phenyl-polydimethylsiloxane ( 50 : 50) as the
stationary phase, maintain the column temperature at 60ºC
for 5 minutes, raise the temperature to l 70ºC by 2ºC per
minute and maintain for 5 minutes, then to 280ºC by 15ºC
per minute and maintain for 50 minutes. The temperature of
injection port is 270ºC. The temperature of the detector is
290ºC. Carrier gas is high purity nitrogen, the fuel gas is
hydrogen, the auxiliary gas is compressed air, the velocity of
carrier gas is 4. O ml per minute. Calculate the content by the
internal standard method, the each content of ethylene glycol,
diglycol and triglycol is not more than O. 1 % .
Ethylene oxide and dioxan Weigh accurately 1 g in a headspace
vial, add 1 ml of purified water, accurately measured, seal
the vial and mix well as the test solution. Take 300 µl
ethylene oxide ( equivalent to about O. 25 g) into a 100 ml
volumetric flask containing 50 ml of treated polyethylene
glycol 400 ( using a rotary evaporator remove any volatile
 578 Macrogol 1500
components at a temperature of 60ºC and pressure of l. 5 to
2. 5 kPa for 6 hours), dilute to volume with the same
solvent, mix well as ethylene oxide stock reference solution.
Weigh accurately 1 g of cold ethylene oxide stock solution,
into a 50 ml volumetric flask containing 40 ml of treated
polyethylene glycol 400, dilute to volume with the same
solvent. Weigh accurately 10 g into a 50 ml volumetric flask
containing 30 ml of water, dilute to volume with water.
Transfer accurately 10 ml into a 50 ml volumetric flask,
dilute to volume with water, mix well as ethylene oxide
reference solution. Dilute a quantity of dioxan, accurately
weighed, with water to produce a solution of O. 1 mg per ml,
as dioxan reference solution. Weigh accurately 1 g of the
substance to be examined, in a headspace vial, add
accurately, O. 5 ml of ethylene oxide reference solution and
O. 5 ml of dioxan reference solution, and seal the vial, mix
well as the reference solution. Measure O. 5 ml of ethylene
oxide reference solution in a headspace vial, and add O. 1 ml
of freshly prepared O. 001 % acetaldehyde solution and O. 1 ml
of dioxan solution, seal the vial and mix well as the system
suitability solution. Carry out the method for gas chrom-
atography ( 0521 ) , using a column packed with poly-
dimethylsiloxane as the stationary phase, maitain the column
temperature at 35ºC for 5 minutes, raise the temperature to
180ºC by 5ºC per minute, then to 230ºC by 30ºC per minute
and maintain for 5 minutes ( adjust if necessary ). The
temperature of the injection port is 150ºC. The temperature
of the detector is 250ºC . Use the head-space injection
method. The equilibration temperature is 70ºC and maintain
for 45 minutes. Procccd vvith thc systcm suitability solution
under the above operating conditions. Adjust the dection
sensitirity so that the peak heights of ethylene oxide and
acetaldehyde in the chromatogram are about 15 % of the full
scale of the chart. The resolution factor between the peaks
corresponding to acetaldehyde and ethylene oxide is at least
2. O, the signal-to--noise ratio of the peak of dioxan is not less
than 5. Inject separately the gaseous sample each in the test
vial and reference vial onto the column. Repeat the procedure
at least three times. The relative standard deviation of the
three areas of peak of ethylene oxide is not greater than 15 %
and the relative standard deviation of the three areas of peak
of dioxan is not greater than 10 % . Calcula te the content
according to the peak area obtained in the chromatogram by
the standard addition method, ethylene oxide is not more
than O. 0001% and dioxan is not more than O. 001%.
Fonnaldehyde To 1 g of the substance being examined,
accurately weighed, add O. 25 ml of O. 6% sodium chromo--
phate, cool in an iced water and add 5 ml of sulphuric acid,
mix well. Allow to stand for 15 minutes and transfer slowly
to a 25 ml volumetric flask containing 10 ml of water, allow
to cool and dilute to volume with water, mix well, as the test
solution. Dilute O. 81 g of formaldehyde, accurately
weighed+ to 100 ml volumetric flask with water. Dilute 1 ml
of this solution to 100 ml with water. Take accurately 1 ml,
proceeded with the same procedure beginning at the words
"add O. 25 ml of O. 6 % sodium chromophate ", use the
resulting solution as the reference solution. Determine the
absorbance at 567 nm ( 0401 ) , perform a blank determin-
ation, and make any necessary correction. The absorbance
obtained with the test solution is not greater than that of the
reference solution (0. 003%).
Water Not more than l. O% ( 0832 method 1 ( 1 ) ) , using
2. o g.
Residue on ignition Not more than O. 1 % ( 0841 ) .
Heavy metals Dissolve 4. O g in 5 ml of hydrochloric acid
solution (9-1000) and a quantity of water, adjust to pH
3. 0-4. O with dilute acetic acid or ammonia TS, then dilute
with water to 25 ml. Carry out the limit test for heavy
metals ( 0821 method 1): not more than O. 0005%.
Category Pharmaceutical excipients, ointment bases and
lubricant, etc.
Storage Preserve in tightly closed containers.
Macrogol 1500
Macrogol 1500 is a mixture of polymer of ethylene oxide and
water with the molecular formula represented by HO
(CH2CH2Ü)n H, where n is the average number of ethy-
leneoxy groups.
Description White waxy solids, plates or granular powder,
odour, slightly characteristic.
Freely soluble in water and in ethanol, insoluble in ether.
Congealing point 41-46ºC ( 0613).
Viscosity Dissolve 25. O g in a 50 ml volumetric flask in
water, dilute to volume with water and mix well, the kinetic
viscosity at 40ºC is 3. 0-4. O mm2/ s ( 0633 method 1 ) , using
a capillary tube of O. 8 mm in internal diameter.
ldentification ( 1 ) To O. 05 g add 5 ml of dilute
hydrochloric acid and 1 ml of barium chloride TS, shake and
filter; to the filtrate a<l<l 1 ml of 10% phosphornolybdic acid
solution, a yellow-green precipitate is formed.
(2) Place O. 1 gin a test tube, add O. 1 g each of potassium
thiocyanate and cobaltous nitrate, mix and add 5 ml of
dichloromethane, the solution exhibits a blue colour.
Average molecular weight Place about 4. 5 g, accurately
weighed, in a dry 250 ml conical flask with stopper, add
25 ml, accurately measured, of phthalic anhydride solution
in pyridine (dissolve 14 g of phthalic anhydride in 100 ml of
anhydrous pyridine and allow it to stand overnight). Mix well
and heat for 30-60 minutes in a boiling water bath. Allow it
to cool, add 50 ml of sodium hydroxide (0. 5 mol/L) VS,
accurately measured, and a solution of phenolphthalein in
pyridine ( 1 - 100) as indicator and titrate with sodium
hydroxide (O. 5 mol/U VS until a pink colour is produced.
Perform a blank determination and make any necessary
correction. Multiply the weight ( g) of substance being
examined by 4000 and divide by the volume ( mD of sodium
hydroxide (0. 5 mol/U VS consumed, the average molecular
weight is 1350-1650.
Acidity Dissolve l. O g in 20 ml water, pH 4. 0-7. O ( 0631 ) .
Clarity and colour of solution Dissolve 5. O g in 50 ml of
water, the solution is clear and colourless. Any opalescence
produced is not more pronounced than that of reference
suspension 2 ( 0902 method 1 ) . Any colour produced is not
more intense than that of reference solution Y2 ( 0901
method 1 ).
Ethylene glycol, diglycol and triglycol W eigh 400 mg each of
ethylene glycol, diglycol and triglycol in to a 100 ml
volumetric flask, dilute to volume with anhydrous ethanol,
mix well as the stock reference solution. Weigh 400 mg
internal standard 1, 3-butanol into a 100 ml volumetric
flask, dilute to volume with anhydrous ethanol, mix well as
the stock internal standard solution. Measure each of
solution 1 ml into a 100 ml volumetric flask, dilute to volume
with anhydrous ethanol, mix well as the reference solution.

Weigh 4. O g into a 100 ml volumetric flask, add 1 ml of the
stock internal reference solution, dilute to volume with
anhydrous ethanol, mix well as the test solution. Carry out
the method for gas chromatography ( 0521 ) , using a column
packed with phenyl-polydimethylsiloxane ( 50 : 50) as the
stationary phase, maintain the column temperature at 60ºC
for 5 minutes, raise the temperature to l 70ºC by 2ºC per
minute and maintain for 5 minutes, then to 280ºC by 15ºC
per minute and maintain for 50 minutes. The temperature of
injection port is 270ºC. The temperature of the detector is
290ºC. Carrier gas is high purity nitrogen, the fuel gas is
hydrogen, the auxiliary gas is compressed air, the velocity of
carrier gas is 4. O ml per minute. Calculate the content by the
internal standard method, the each content of ethylene glycol,
diglycol and triglycol is not more than O. 1 %.
Ethylene oxide and dioxan Weigh accurately 1 g in a head-
space vial, add 1 ml of purified water, accurately measured,
seal the vial and mix well as the test solution ( the test vial).
Take 300 111 ethylene oxide (equivalent to about O. 25 g) into
a 100 ml volumetric flask containing 50 ml of treated
polyethylene glycol 400 ( using a rotary evaporator remove
any volatile components at a temperature of 60ºC and
pressure of l. 5 to 2. 5 kPa for 6 hours), dilute to volume
with the same solvent, mix well as ethylene oxide stock
reference solution. Weigh accurately 1 g of cold ethylene
oxide stock solution, into a 50 ml volumetric flask containing
40 ml of treated polyethylene glycol 400, dilute to volue with
the same solvent. Weigh accurately 10 g into a 50 ml
volumetric flask containing 30 ml of water, dilute to volume
with water. Transfer accurately 10 ml into a 50 ml volu-
metric flask, dilute to volume with water, mix well as
ethylene oxide reference solution. Dilute a quantity of
dioxan, accurately weighed, with water to produce a solution
of O. 1 mg per ml, as dioxan reference solution. Weigh
accurately 1 g of the substance to be examined, in a
headspace vial, add accurately, O. 5 ml of ethylene oxide
reference solution and O. 5 ml of dioxan reference solution,
and seal the vial, mix well as the reference solution. Measure
O. 5 ml of ethylene oxide reference solution in a headspace
vial, and add O. 1 ml of freshly prepared O. 001 % acetal-
dehyde solution and O. 1 ml of dioxan solution, seal the vial
and mix well as the system suitability solution ( the reference
vial). Carry out the method for gas chromatography
( 0521 ) , using a column packed with polydimethylsiloxane as
the stationary phase, maitain the column temperature at
35ºC for 5 minutes, raise the temperature to 180ºC by 5ºC
per minute, then to 230ºC by 30ºC per minute and maintain
for 5 minutes (adjust if necessary). The temperature of the
injection port is 150ºC . The temperature of the detector is
250ºC. Use the head-space injection method. The equilibration
temperature is 70ºC and maintain for 45 minutes. Proceed
with the system suitability solution under the above operating
conditions Adjust the dection sensitivity so that the peak
heights of ethylene oxide and acetaldehyde in the chrom-
a togram are about 15 % of the full scale of the chart. The
resolution factor between the peaks corresponding to
acetaldehyde and ethylene oxide is at least 2. O, the signal-to-
noise ratio of the peak of dioxan is not less than 5. Inject
separately the gaseous sample each in the test vial and
reference vial onto the column. Repeat the procedure at least
three times. The relative standard deviation of the three
areas of peak of ethylene oxide is not greater than 15 % and
the relative standard deviation of the three areas of peak of
dioxan is not greater than 10 % . Calcula te the content
according to the peak area obtained in the chromatogram by
the standard addition method, ethylene oxide is not more
Macrogol 4000 579
than O. 0001% and dioxan is not more than O. 001%.
Fonnaldehyde To 1 g of the substance being examined,
accurately weighed, add O. 25 ml of O. 6% sodium chrom-
ophate, cool in an iced water and add 5 ml of sulphuric acid,
mix well. Allow to stand for 15 minutes and transfer slowly
to a 25 ml volumetric flask containing 10 ml of water, allow
to cool and dilute to volume with water, mix well, as the test
solution. Dilute O. 81 g of formaldehyde, accurately weighed, to
100 ml with water Dilute 1 ml of this solution to 100 ml
volumetric flask with water. Measure accurately 1 ml,
procecded with the same procedure beginning at the words
"add O. 25 ml of O. 6% sodium chromophate TS", use the
resulting solution as the reference solution. Determine the
absorbance at 567 nm ( 0401 ) , Perform a blank deter-
mination, and make any necessary correction. The absor-
bance obtained with the test solution is not greater than that
of the reference solution (O. 003 % ) .
Water Not more than l. O% ( 0832 method 1 ( 1 ) ) , using
2. o g.
Residue on ignition Not more than O. 1 % ( 0841 ) .
Heavy metals Dissolve 4. O g in 5 ml of hydrochloric acid
solution (9-1000) and a quantity of water, adjust to pH
3. 0-4. O with dilute acetic acid or ammonia TS, then dilute
with water to 25 ml. Carry out the limit test for heavy
metals ( 0821 method 1): not more than O. 0005%.
Category Pharmaceutical excipients, ointment bases and
lubricant, etc.
Storage Preserve in tightly closed containers.
Macrogol 4000
Macrogol 4000 is a mixture of polymer of ethylene oxide and
water with the molecular formula represented by HO
( CH2 CH2 O ) n H, where n is the average number of
ethyleneoxy groups.
Description White waxy solids plates or granular powder;
odour, slightly characteristic.
Freely soluble in water and in ethanol, insoluble in ether.
Congealing point 50-54 ºC <0613).
Viscosity Dissolve 25. O g in a 50 ml volumetric flask in
water, dilute to volume with water and mix well, the kinetic
viscosity at 40ºC is 5. 5-9. O mm2/ s ( 0633 method 1 ) , using
a capillary tube of O. 8 mm in interna! diameter.
Identification ( 1 ) Dissolve O. 05 g in 5 ml of dilute
hydrochloric acid and 1 ml of barium chloride TS, shake, and
filter. To the filtra te add 1 ml of 10 % phosphomolybdic acid
solution, a yellow-green precipitate is formed.
(2) Place O. 1 g in a test tube, add O. 1 g each of potassium
thiocyanate and cobaltous nitrate, mix and add 5 ml of
dichloromethane, the solution exhibits a blue colour.
Average molecular weight Weigh accurately about 12 g,
transfer to a dry 250 ml conical flask with stopper, add
25 ml, accurately measured, of phthalic anhydride solution
in pyridine (dissolve 14 g of phthalic anhydride in 100 ml of
anhydrous pyridine and allow it to stand overnight). Mix well
and heat for 30-60 minutes in a boiling water bath. Allow it
to cool, add 50 ml of sodium hydroxide (O. 5 mol/L) VS,
accurately measured, and a solution of phenolphthalein in
pyridine ( 1 - 100) as indicator and titrate with sodium
 .
iJOfi''
•.. r.··.
·.·.·'1J.
·. ' 1·•.1· .•.
Macrogol 6000
hydroxide (O. 5 mol/L) VS until a pink colour is produced.
Perform a blank determination and make any necessary
correction. Multiply the weight ( g) of substance being
examined by 4000 and divide by the volume ( mD of sodium
hydroxide (O. 5 mol/L) VS consumed, the average molecular
weight is 3400-4200.
Acidity Dissolve l. O g in 20 ml water, pH 4. 0-7. O ( 0631 ) .
Oarity and colour of solution Dissolve 5. O gin 50 ml of water,
the solution is clear and colourless. Any opalescence
produced is not more pronounced than that of reference
suspension 2 ( 0902 ) . Any colour produced is not more
intense than that of reference solution Y 2 ( 0901 method 1 ) .
Ethylene glycol, diglycol and triglycol Weigh 400 mg each of
ethylene glycol, diglycol and triglycol into a 100 ml
volumetric flask, dilute to volume with anhydrous ethanol,
mix well as the stock reference solution. Weigh 400 mg
internal standard 1, 3-butanol into a 100 ml volumetric
flask, dilute to volume with anhydrous ethanol, mix well as
the stock internal reference solution. Measure each of
solution 1 ml into a 100 ml volumetric flask, dilute to volume
with anhydrous ethanol, mix well as the reference solution.
Weigh 4. O g into a 100 ml volumetric flask, add 1 ml of the
stock internal reference solution, dilute to volume with
anhydrous ethanol, mix well as the test solution. Carry out
the method for gas chromatography ( 0521 ) , using a column
packed with phenyl-polydimethylsiloxane ( 50 : 50) as the
stationary phase, maintain the column temperature at 60ºC
for 5 minutes, raise the temperature to l 70ºC by 2ºC per
minute and maintain for 5 minutes; then to 280ºC by 15ºC
per minute and maintain for 50 minutes. The temperature of
injection port is 270ºC. The temperature of the detector is
290ºC. Carrier gas is high purity nitrogen, the fuel gas is
hydrogen, the auxiliary gas is compressed air, the velocity of
carrier gas is 4. O ml per minute. Calculate the content by the
internal standard method, the each content of ethylene
glycol, diglycol and triglycol is not more than O. 1 %.
Ethylene oxide and dioxin W eigh accurately 1 g in a head-
space vial, add 1 ml of purified water, accurately measured,
seal the vial and mix well as the test solution. Take 300 µl
ethylene oxide ( equivalent to about O. 25 g) into a 100 ml
volumetric flask containing 50 ml of treated polyethylene
glycol 400 ( using a rotary evaporator remove any volatile
components at a temperature of 60ºC and pressure of l. 5 to
2. 5 kPa for 6 hours) , dilute to volume with the same
solvent, mix well as ethylene oxide stock reference solution.
Weigh accurately 1 g of cold ethylene oxide stock solution,
into a 50 ml volumetric flask containing 40 ml of treated
polyethylene glycol 400, dilute to volue with the same
solvent. Weigh accurately 10 g into a 50 ml volumetric flask
containing 30 ml of water, dilute to volume with water.
Transfer accurately 10 ml into a 50 ml volumetric flask,
dilute to volume with water, mix well as ethylene oxide
reference solution. Dilute a quantity of dioxan, accurately
weighed, with water to produce a solution of O. 1 mg per ml,
as dioxan reference solution. Weigh accurately 1 g of the
substance to be examined, in a headspace vial, add accurately
O. 5 ml of ethylene oxide reference solution and O. 5 ml of
dioxan reference solution, and seal the vial, mix well as the
reference solution. Take O. 5 ml of ethylene oxide reference
solution in a headspace vial, and add O. 1 ml of freshly
prepared O. 001 % acetaldehyde solution and O. 1 ml of dioxan
solution, seal the vial and mix well as the system suitability
solution. Carry out the method for gas chromatography
( 0521 ) , using a column packed with polydimethylsiloxane as
the stationary phase; maitain the column temperature at
35ºC for 5 minutes, raise the temperature to 180ºC by 5ºC
per minute, then to 230ºC by 30ºC per minute and maintain
for 5 minutes (adjust if necessary). The temperature of the
injection port is 150ºC . The temperature of the detector is
250ºC. Use the head-space injection method. The equilibration
temperature is 70ºC and maintain for 45 minutes. Proceed
with the system suitability solution under the above operating
conditions. Adjust the detection sensitivity so that the peak
heights of ethylene oxide and acetaldehyde in the
chromatogram are about 15% of the full scale of the chart.
The resolution factor between the peaks corresponding to
acetaldehyde and ethylene oxide is at least 2. O; the signal-to-
noise ratio of the peak of dioxan is not less than 5. Inject
separately the gaseous sample each in the test vial and
reference vial onto the column. Repeat the procedure at least
three times. The relative standard deviation of the three
areas of peak of ethylene oxide is not greater than 15 % and
the relative standard deviation of the three areas of peak of
dioxan is not greater than 10 % . Calcula te the content
according to the peak area obtained in the chromatogram by
the standard addition method, ethylene oxide is not more
than O. 0001% and dioxan is not more than O. 001%.
Fonnaldehyde To 1 g of the substance being examined, accurately
weighed, add O. 25 ml of O. 6% sodium chromophate test
solution, cool in an iced water and add 5 ml of sulphuric
acid, mix well. Allow to stand for 15 minutes and transfer
slowly to a 25 ml volumetric flask containing 10 ml of water,
allow to cool and dilute to volume with water, mix well, as
the test solution. Dilute O. 81 g of formaldehyde solution,
accurately weighed, to 100 ml with water. Dilute 1 ml of
this solution to 100 ml with water. Take accurately 1 ml,
proceeded with the same procedure beginning at the words
"add O. 25 ml of O. 6 % sodium chromophate test solution",
and use the resulting solution as the reference solution.
Determine the absorbance at 567 nm ( 0401 ) , adjusted by a
blank solution proceeded with the same procedure. The
absorbance obtained with the test solution is not greater than
that of the reference solution (O. 003 %) .
Water Not more than l. O% ( 0832 method 1 ( 1 ) ) , using
2. o g.
Residue on ignition Not more than O. 1 % <0841 ) .
Heavy metals Dissolve 4. O g in 5 ml of hydrochloric acid
solution (9-1000) and a quantity of water, adjust to pH
3. 0-4. O with dilute acetic acid or ammonia test solution,
then dilute with water to 25 ml. Carry out the limit test for
heavy metals ( 0821 method 1 ) : not more than O. 0005 % .
Category Pharmaceutical excipients, ointment bases and
lubricant, etc.
Storage Preserve in tightly closed containers.
Macrogol 6000
Macrogol 6000 is a mixture of polymer of ethylene oxide and
water with the molecular formula represented by
HO CCH2CH2Ü)n H, where n is the average number of
ethyleneoxy groups.
Description White waxy solids, plates or granular powder,
odour, slightly characteristic.
Freely soluble in water and in ethanol, insoluble in ether.
Congealing point 53-58ºC <0613 >.

Viscosity Dissolve 25. O g in a 50 ml volumetric flask in
water, dilute to volume with water and mix well, the kinetic
viscosity at 40ºC is 10. 5-16. 5 mm 2 / s ( 0633 method 1 ) ,
using a capillary tube of 1. O mm in internal diameter.
Identification C 1 ) To O. 05 g add 5 ml of dilute
hydrochloric acid and 1 ml of barium chloride TS, shake and
filter, to the filtrate add 1 ml of 10% phosphomolybdic acid
solution, a yellow-green precipitate is formed.
(2) Place O. 1 g in a test tube, add O. 1 g each of potassium
thiocyanate and cobaltous nitrate, mix and add 5 ml of
dichloromethane, the solution exhibits a blue colour.
Average molecular weight Place about 12. 5 g, accurately
weighed, in a dry 250 ml conical flask with stopper, add 25
ml, accurately measured, of phthalic anhydride solution in
pyridine edissolve 14 g of phthalic anhydride in 100 ml of
anhydrous pyridine and allow it to stand overnight). Mix well
and heat for 30-60 minutes in a boiling water bath. Allow it
to cool, add 50 ml of sodium hydroxide (0. 5 mol/L) VS,
accurately measured, and a solution of phenolphthalein in
pyridine e1 - 100) as indicator and titrate with sodium
e
hydroxide o. 5 mol/L) vs until a pink colour is produced.
Perform a blank determination and make any necessary
correction. Multiply the weight Cg) of substance being
examined by 4000 and divide by the volume CmD of sodium
hydroxide (O. 5 mol/L) VS consumed, the average molecular
weight is 5400-7800.
Acidity Dissolve 1. O gin 20 ml water, pH 4. 0-7. O (0631 ).
Clarity and colour of solution Dissolve 5. O g in 50 ml of
water, the solution is clear and colourless. Any opalescence
produced is not more pronounced than that of reference
suspension 2 ( 0902 ) . Any colour produced is not more
intense than that of reference solution Y2 <0901 method 1 ) .
Ethylene glycol, diglycol and triglycol \Veigh 400 mg each of
ethylene glycol, diglycol and triglycol into a 100 ml
volumetric flask, dilute to volume with dehydrated ethanol,
mix well as the stock reference solution. Weigh 400 mg
internal standard 1, 3-butanol into a 100 ml volumetric
flask, dilute to volume with anhydrous ethanol, mix well as
the stock internal reference solution. Measure each of
solution 1 ml into a 100 ml volumetric flask, dilute to volume
with anhydrous ethanol, mix well as the reference solution.
Weigh 4. O g into a 100 ml volumetric flask, add 1 ml of the
stock internal reference solution, dilute to volume with
anhydrous ethanol, mix well as the test solution. Carry out
the method for gas chromatography ( 0521 ) , using a column
packed with phenyl-polydimethylsiloxane e50 : 50 ) as the
stationary phase, maintain the column temperature at 60ºC
for 5 minutes, raise the temperature to 170ºC by 2ºC per
minute and maintain for 5 minutes, then to 280ºC by 15ºC
per minute and maintain for 50 minutes. The temperature of
injection port is 270ºC. The temperature of the detector is
290ºC. Carrier gas is high purity nitrogen, the fuel gas is
hydrogen, the auxiliary gas is compressed air, the velocity of
carrier gas is 4. O ml per minute. Calculate the content by the
internal standard method, the each content of ethylene
glycol, diglycol and triglycol is not more than O. 1%.
Ethylene oxide and dioxan Weigh accurately 1 g in a
headspace vial, add 1 ml of purified water, accurately
measured, seal the vial and mix well as the test solution.
Take 300 µl ethylene oxide (equivalent to about O. 25 g) into
a 100 ml volumetric flask containing 50 ml of treated
polyethylene glycol 400 eusing a rotary evaporator remove
any volatile components at a temperature of 60ºC and
pressure of 1. 5 to 2. 5 kPa for 6 hours), dilute to volume
Macrogol 6000
with the same solvent, mix well as ethylene oxide stock
reference solution. Weigh accurately 1 g of cold ethylene
oxide stock solution, into a 50 ml volumetric flask containing
40 ml of treated polyethylene glycol 400, dilute to volue with
the same solvent. Weigh accurately 10 g into a 50 ml
volumetric flask containing 30 ml of water, dilute to volume
with water. Transfer accurately 10 ml into a 50 ml volu-
metric flask, dilute to volume with water, mix well as
ethylene oxide reference solution. Dilute a quantity of
dioxan, accurately weighed, with water to produce a solution
of O. 1 mg per ml, as dioxan reference solution. Weigh
accurately 1 g of the substance to be examined, in a
headspace vial, add accurately O. 5 ml of ethylene oxide
reference solution and O. 5 ml of dioxan reference solution,
and seal the vial, mix well as the reference solution. Measure
O. 5 ml of ethylene oxide ref erence solution in a headspace
vial, and add O. 1 ml of freshly prepared O. 001 % acetal-
dehyde solution and O. 1 ml of dioxan solution, seal the vial
and mix well as the system suitability solution. Carry out the
method for gas chromatography ( 0521 ) , using a column
packed with polydimethylsiloxane as the stationary phase;
maitain the column temperature at 35ºC for 5 minutes, raise
the temperature to 180ºC by 5ºC per minute, then to 230ºC
by 30ºC per minute and maintain for 5 minutes Cadjust if
necessary). The temperature of the injection port is 150ºC.
The temperature of the detector is 250ºC. Use the head-
space injection method. The equilibration temperature is
70ºC and maintain for 45 minutes. Proceed with the system
suitability solution under the above operating conditions.
Adjust the dection sensitility so that the peak heights of
ethylene oxide and acetaldehyde in the chromatogram are
about 15 % of the full scale of the chart. The resolution
factor between the peaks corresponding to acetaldehyde and
ethylene oxide is at least 2. O, the signal-to-noise ratio of the
peak of dioxan is not less than 5. Inject separately the
gaseous sample each in the test vial and reference vial onto
the column. Repeat the procedure at least three times. The
relative standard deviation of the three areas of peak of
ethylene oxide is not greater than 15 % and the relative
standard deviation of the three areas of peak of dioxan is not
greater than 10 % . Calcula te the content according to the
peak area obtained in the chromatogram by the standard
addition method, ethylene oxide is not more than O. 0001 %
and dioxan is not more than O. 001%.
Fonnaldehyde To 1 g of the substance being examined,
accurately weighed, add O. 25 ml of O. 6% sodium
chromophate, cool in an ice water and add 5 ml of sulphuric
acid, mix well. Allow to stand for 15 minutes and transfer
slowly to a 25 ml volumetric flask containing 10 ml of water,
allow to cool and dilute to volume with water, mix well, as
the test solution. Dilute O. 81 g of formaldehyde, accurately
weighed, to 100 ml with water. Dilute 1 ml of this solution
to 100 ml with water. Take accurately 1 ml proceeded with
the same procedure, beginning at the words "add O. 25 ml of
O. 6 % sodium chromophate test solution", and use the
resulting solution as the reference solution. Determine the
absorbance at 567 nm ( 0401 ) , adjusted by a blank solution
proceeded with the same procedure. The absorbance obtained
with the test solution is not greater than that of the reference
solution eo. 003 % ) .
Water Not more than 1. O% <0832 method 1 ( 1 ) ) , using
2. o g.
Residue on ignition Not more than O. 1 % <0841 ) .
Heavy metals Dissolve 4. O g in 5 ml of hydrochloric acid
solution (9-1000) and a quantity of water, adjust to pH
 582 Magnesium Chloride
3. 0-4. O with dilute acetic acid or ammonia TS, then dilute
with water to 25 ml. Carry out the limit test for heavy
metals ( 0821 method 1): not more than O. 0005%.
Category Pharmaceutical excipients, ointment bases and
lubricant, etc.
Storage Preserve in tightly closed containers, stored in a dry
place.
Magnesium Chloride
MgClz •6H20 203. 30
[7791-18-6]
Magnesium Chloride contains not less than 98. O% and not
more that 101. 0% of magnesium chloride (MgClz•6H20).
Description A colourless transparent crystal or crystalline
powder; odorless; taste bitter; freely deliquescent.
Freely soluble in water and in ethanol.
ldentification The aqueous solution yields the reactions
characteristic of magnesium salts and chlorides ( 0301 ) .
Acidity Dissolve 1 gin 20 ml of water, pH 4. 5-7. O ( 0631 ).
Clarity and colour of solution Dissolve 2. 5 g in 25 ml of
water, the solution is clear and colourless ( 0901 and 0902).
Bromide Dissolve 2. O g in water in a 100 ml volumetric
flask, dilute with water to volume, mix well. Transfer 5 ml,
accurately measured, to a 10 mi colourimetric tube, add
2. O ml of phenol red mixed solution [ Dissolve 25 mg of
ammonium sulphate in 235 ml of water, add 105 ml of 2
mol/L sodium hydroxide solution and 135 ml of 2 mol/L
acetic acid, mix well, add 25 ml of phenol red solution
(Dissolve 33 mg of phenol red in l. 5 ml of 2 mol/L sodium
hydroxide solution, dilute to 100 ml with water, mix well.),
mix well. If necessary, adj ust the pH of the mixture to
4. 7], and add l. O ml of O. 01% chloramines T solution
(freshly prepared), and mix immediately. After exactly 2
minutes, add O. 15 ml of O. 1 mol/L sodium thiosulphate
solution, mix, dilute to 10 ml with water, mix well, as the
test solution; transfer 5. O ml of potassium bromide standard
solution ( Accurately weigh 30 mg of potassium bromide,
previously dried to constant weight at 105ºC, add water to
produce 100 ml, mix well. Transfer 5 ml, accurately
measured, to a 100 ml volumetric flask, dilute with water to
volume, mix well. Each ml is equivalent to 10 µg of Br) to a
10 ml colourmetric tube, as the reference solution prepared
in the same manne. Carry out the method for ultraviolet-
visible absorption spectrophotometry ( 0401), using water as
the blank solution, measure the absorbance of the solution at
590 nm. The absorbance of the test solution is not greater
than that of the reference solution (O. 05 % ) .
Sulfates Carry out the limit test for sulfates ( 0802) , using
2. O g. Any opalescence produced is not more pronounced
than that of a reference solution using 2. O ml of potassium
sulfate standard solution (O. 01%).
Water 51. 0%-55. 0% <0832 method 1 (1) ).
Aluminum ( If intended for the manufacture of
haemodialysis solutions, it complies with the test for
aluminium)
Dissolve 4. O g in 100 ml of water and add 10 ml of pH6. O
acetate BS as the test solution; Prepare a mixture of 2. O ml
of standard aluminum solution (measure accurately a volume
of standard aluminum solution, dilute with water to produce
a solution of 2 µg of Al per mD, 98 ml of water, and 10 ml
of pH6. O acetate BS as the standard solution. Prepare a
mixture of 10 ml of pH6. O acetate BS and 100 ml of water as
the blank solution. Place above three solutions in a separator
respectively and shake with 2 quantities, each of 20 ml, and
then with one 10 ml quantity of a O. 5 % of 8-hydroxy-
quinoline in chloroform, combine the chloroform extracts in
a 50-ml volumetric flask. Dilute the combined extracts with
chloroform to volume, and mix well. Carry out the method
for fluorometry ( 0405 ) , determine the fluorescence
intensities of solutions at an excitation wavelength of 392 nm
and an emission wavelength of 518 nm. The fluorescence
intensity of the test solution is not greater than that of the
standard solution (0. 0001 %).
Barium Dissolve 1gin10 ml of water, add 1 ml of 1 mol/L
sulfuric acid solution; no opalescence is produced within
2 hours.
Calcium Dissolve O. 10 g in 15 ml of water, add 1 ml of
2 mol/L acetic acid solution and 1 ml of ammonium oxalate
TS, mix well and allow to stand for 15 minutes. Any
opalescence produced is not more pronounced than that of a
reference solution using 10 ml of calcium standard solution
(place 2. 5 g of calcium carbonate, accurately weighed,
previously dried to constant weight at 105-llOºC, in a
1000 ml volumetric flask, dissolve with 12 ml of 6 mol/L
acetic solution, dilute with water to the volume, mix well,
transfer 10 ml of the solution, accurately measure, into
another 1000 ml volumetric flask befare use, dilute with
water to the volume and mix well, each ml is equivalent to 10 µg
of Ca) (O. 1%).
Potassium Dissolve 5 g in 5 ml of water, add O. 2 ml of
sodium bitartrate TS; no opalescence is produced within 5
minutes.
Iron Carry out the limit test for iron ( 0807) , using 2. O g.
Any opalescence produced is not greater than that of a
reference solution using 2. O ml of iron standard solution
(O. 001%).
Heavy metals Dissolve l. O g in 20 ml of water, add 2 ml of
dilute acetic acid and sufficient water to 25 ml. Carry out the
limit test for heavy metals ( 0821 method 1 ) : not more than
o. 001%.
Arsenic Dissolve l. O g in 23 ml of water and 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822
method 1): not more than O. 0002%.
Assay Dissolve about O. 3 g, accurately weighed, in 50 ml
of water, add 10 ml of ammonia-ammonium chloride BS
(pH 10. O) and a few amount of eriochrome black T
indicator, ti trate with disodium edetate (O. 05 mol/L) VS
until the colour changes from purple red to pure blue.
Each ml of disodium edetate (O. 05 mol/L) VS is equivalent
to 10. 17 mg of MgClz•6H20.
Category Pharmaceutical excipients, osmotic pressure
regulator, local analgesic and buffer agent.
Storage Preserve in tightly closed containers.
Magnesium Oxide
MgO 40. 30
[1309-48-4]
Magnesium Oxide contains not less than 96. 5% of MgO,

calculated with reference to substance ignited to constant
weight.
Description A white powder, odourless; tasteless; gra-
dually absorbs carbon dioxide in the air.
Practically insoluble in water and in ethanol; soluble in dilute
acids.
ldentification Its solution in dilute hydrochloric acid yields
the reactions characteristic of magnesium salts ( 0301 ) .
Apparent volume Transfer 15. O g to a cylinder without
shaking, the volume is not more than 60 mi.
Alkalinity Boil l. O g with 50 mi of water for 5 minutes.
Filter while hot, wash the residue with a volume of water.
Combine the washings with the filtrate, add a few drops of
methyl red IS and 2. O ml of sulfuric acid (O. 05 mol/L) VS;
a yellow colour changes to red.
Colour of solution Dissolve l. O g with 15 ml of acetic acid
and 5 ml of water, boil for 2 minutes and cool, add water to
20 ml, filter if necessary, the solution is colourless; any
colour produced is not more intense than that of reference
solution YG2 ( 0901 method 1 ) .
Chlorides Dilute l. O ml of the solution prepared for the test
for calcium oxide with water to 25 ml. Carry out the limit
test for chlorides ( 0801 ) , any opalescence produced is not
more pronouncedthan that of a reference using 5. O ml of
sodium chloridestandard solution (O. 1 % ) .
Sulfates Dilute 2. O ml of the solution in the test for calcium
oxide, with water to 20 mi. Carry out the limit test for
sulfates ( 0802 ) . Any opalescence produced is not more
pronounced than that of a reference by using 3. O ml of
potassium sulfate standard solution (O. 3 % ) .
Carbonate Boil O. 10 g with 5 ml of water, allow to cool and
add S m l of acetic acid, no effervescence occurs.
Acid insoluble substances Dissolve 2. O g in 25 ml of
hydrochloric acid on a water bath and add 100 ml of water.
Filter through a sintered-glass crucible ( No. 4) previously
dried to constant weight at 105ºC. Wash the residue water
until the washing shows no reaction of chlorides. Dry the
residue to constant weight at 105ºC. The residue is not more
than 2. O mg (O. 10%).
Soluble substances Boil l. O g with 100 ml of water for 5
minutes, filter while hot, wash the residue with small
quantity of water, combine the filtrate and the washing,
evaporate to dryness in a evaporating dish previously dried to
constant weight at 105ºC on a water bath, and dry to
constant weight at 105ºC . The residue is not more than
2.0%.
Residue on ignition When ignited to constant weight, loses
not more than 5. 0% of its weight, using O. 50 g.
Calcium oxide Weigh accurately 5. O g after freshly ignited
and cooled, dissolve in 30 ml of water and 70 mi of acetic
acid, boil for 2 minutes, cool and filter, wash the residue
with dilute acetic acid, combine the filtrate and washings in
to 100 ml volumetric flask, add dilute acetic acid to volume,
mix well, as the test solution. Transfer accurately 10 ml of
the test solution, add 300 ml of water, 10 ml of
triethanolamine solution ( 3 - 10 ) and 10 ml of 45 %
potassium hydr-oxide solution. Allow to stand for 5
minutes, add O. 1 g of calcium purpurin indicator, titrate
with disodium edetate (O. 01 mol/L) VS until the colour
changes from purple to blue. Perform a blank determination
and make any necessary correction. Each ml of disodium
edetate (O. 01 mol/L) VS is equivalent to O. 5608 mg of
,., ... , ... ·......, ..•... ···''
Magnesium Stearate :518:·:···•.:••·:·::.
CaO: not more than O. 50 % .
Iron Dissolve 50 mg in 2 ml of dilute hydrochloric acid and
23 ml of water. Carry out the limit test for iron ( 0807). Any
colour produced is not more intense than that of a reference
solution using 2. 5 ml of iron standard solution (O. 05 % ) .
Manganese To l. O g add 20 ml of water, 5 ml of nitricacid,
5 ml of sulfuric acid and 1 ml of phosphoric acid, boil for 2
minutes and cool. Add 2. O g of potassium periodate, boil for
5 minutes and cool. Transfer the solution to a 50 ml Nessler
cylinder, dilute to volume with non-reducing water (add 3 ml
of nitric acid and 5 g of potassium periodate to 1000 ml of
water, boil for 2 minutes and cooD, mix well. Any colour
produced is not more intense than that of a reference solution
prepared by using O. 30 ml of manganese standard solution
(dissolve O. 275 g of anhydrous manganese sulfate previously
ignited to constant weight at 400-500ºC in a 1000 ml
volumetric flask, with water dilute to volume and mix well.
Each ml of manganese solution is equivalent to O. 10 mg of
Mn) (0. 003%).
Heavy metals Dissolve by heating O. 50 g with 10 ml of
dilute hydrochloric acid and 5 ml of water, boil for 1 minute,
allow to cool and filter. To the filtrate add 1 drop of
phenolphthalein IS and add dropwise ammonia TS until the
solution becomes pink. Add 2 ml of acetate BS (pH3. 5) and
sufficient water to produce 25 ml, add O. 5 g of ascorbicacid
and shake to dissolve. Carry out the limit test for heavy
metals ( 0821 method 1 ) , compare the colour after 5
minutes: not more than O. 002 % .
Arsenic Dissolve O. 50 g in 5 ml of hydrochloric acid and
23 ml of water. Carry out the limit test for arsenic ( 0822
method 1 ) : not more than O. 0004 % .
Assay Dissolve O. 4 g, accurately weighed, in 25 ml of
sulfuric acid (O. 5 mol/L) VS, accurately measured, add ldrop
of methyl orange IS. Titrate with sodium hydroxide ( 1 mol/L)
VS. Perform a blank determination and make any necessary
correction. The quantity of sulfuric acid consumed by MgO
is calculated by subtracting the quantity consumed by CaO
from the total amount of sulfuric acid consumed. Each ml of
sulfuric acid (O. 5 mol/L) VS is equivalent to 20. 15 mg of
MgO or 28. 04 mg of CaO.
Category Pharmaceutical excipients, filler and pH regulator.
Storage Preserve in well closed containers.
Magnesium Stearate
[557-04-0]
Magnesium Stearate is prepared by stearic acid and
magnesium. It is a mixture mainly consist of magnesium
stearate ( C36H1 o MgQ4) and magnesium palmitate
( C32 H6 2 Mg0 4) . It contains not less than 4. O% and not
more than 5. O% of Mg, calculated on the dried basis.
Description Fine, light, white powder, free from
grittiness; odour faint; unctuous to the touch with skin.
Insoluble in water, in ethanol and in ether.
ldentification (1) Place 5. O g in a round-bottom flask,
add 50 ml of peroxide-free ether, 20 ml of dilute nitric acid
and 20 ml of water, reflux until dissolved completely. Allow
to cool, transfer the contents of the flask to a separating
funnel. Shake and allow to stand until separation of the
layers is obtained, transfer the aqueous layer to another
 Magnesium Stearate
separating funnel. Extract the ether layer with two portions,
each of 4 ml of water, combine the extracts with the above
aqueous layer. Wash the aqueous extract with 15 ml of
peroxide-free ether, transf er the aqueous extract to a 50 ml
volumetric flask, dilute with water to volume, shake well
and use as the test solution. The solution yields the reactions
characteristic of magnesium salts ( 0301 ) .
(2) The retention time of principal peaks in the substance
being examined in the chromatogram obtained in the relative
contents of stearic acid and palmitic acid are identical with
that the principal peaks of stearic acid CRS and palmitic in
the chromatogram of the reference solution correspondingly.
Acidity or alkalinity Boil l. O g with 20 ml of freshly boiled
and cooled water on a water bath for 1 minute with constant
shaking, cool and filter. Add O. 05 ml of bromothymol blue
IS to 10. O ml the successive filtrate: not more than O. 05 ml
of hydrochloric acid (O. 1 mol/L) VS or sodium hydroxide
(O. 1 mol/L) VS is required to change the colour of the
solution.
Chlorides Carry out the limit test for chlorides ( 0801 ) ,
using l. O ml of the test solution obtained in Identification
test ( 1). Any opalescence produced is not more pronounced
than that of a reference solution prepared by using 10. O ml of
sodium chloride standard solution (O. 1O%).
Sulfates Carry out the limit test for sulfates ( 0802) , using
l. O ml of the test solution obtained in identification test ( 1).
Any opalescence produced is not more pronounced than that
of a reference solution prepared by using 6. O ml of potassium
sulfate standard solution (Oº 6 %) .
~ on drying When dried to constant weight at 80ºC ,
loses not more than 5. 0% of its weight ( 0831 >.
Iron lgnite O. 50 g and boil the residue with 5 ml of dilute
hydrochloric acid and 10 ml of water. Allow to cool and
filter. To the filtrate add 50 mg of ammonium persulfate,
dilute with water to 35 ml. Carry out the limit test for iron
( 0807). Any colour produced is not more intense than that
of a reference solution prepared by using 5. O ml of iron
standard solution (0. 01%).
Cadmium Place O. 05 g of the substance to be examined in
duplicate, accurately weighted, in a polytetrafluoroethylene
digestion bomb respectively. Add 2 ml of nitric acid in one,
after being digested, transfer the contents quantitatively to
100 ml of volumetric flask and dilute with water to volume,
shake well and use as test solution; Add O. 5 ml of cadmium
standard solution ( dilute cadmium single-standard solution
with water into a solution of O. 3 µg/ml of cadmium)
accurately in another, operate followed by the same method
and use as reference solution. Carry out Atomic absorption
spectrometry ( 0406 method 2 ) and determine the
absorbance respectively at wavelength 228. 8 nm, calculate:
not more than O. 0003 % .
Nickel Place O. 05 g of the substance to be examined in
duplicate, accurately weighted, in a polytetrafluoroethylene
digestion bomb respectively. Add 2 ml of nitric acid in one,
after being digested, transfer the contents quantitatively to
10 ml of volumetric flask and dilute with water to volume,
shake well and use as test solution; Add O. 5 ml of nickel
standard solution (dilute nickel single-standard solution with
water into a solution of O. 5 µg/ml of nickel) accurately in
another, operate followed by the same method and use as
reference solution. Carry out Atornic absorption spectrometry
( 0406 method 2) and determine the absorbance respectively
at wavelength 232. O nm, calculate: not more than O. 0005%.
Heavy metals lgnite gently 2. O g in a crucible until
thoroughly charred, cool, moisten the residue with O. 5-
1. O ml of sulfuric acid, ignite at a low temperature until
sulfurous acid fumes are no longer evolved, add O. 5 ml of
nitric acid, evaporate to dryness, heat until nitrous oxide
fumes are no longer evolved. Cool, incinerate completely at
500-600ºC, cool, add 2 ml of hydrochloric acid, evaporate to
dryness on a water bath, add 15 ml of water and 2 ml of
dilute acetic acid, heat to dissolve. Cool, add 2 ml of acetate
BS (pH 3. 5) and sufficient water to 25 ml, carry out the
limit test for heavy metals ( 0821 method 2) : not more than
o. 0015%.
Relative contents of stearic acid and palmitic acid Place O. 1 g
of the substance being examined, accurately weighed, in a
conical flask. Add 5 ml of methanol solution of boron
trifluoride [Dissolve a quantity of boron trifluoride mono-
hydrate or boron trifluoride dihydrate, equivalent to 14 g of
boron trifluoride in methanol to 100 ml, mix well], reflux
for 10 minutes to dissolve. Add 4 ml of n-heptane through
the condenser, and reflux for 10 minutes. Cool, add 20 ml of
saturated sodium chloride solution, shake and allow to stand
until the separation of the layers is obtained. Pass the n-
heptane layer through O. 1 g of anhydrous sodium sulfate
(previously washed with n-heptane) into a beaker, and use
as the test solution. Carry out the method for gas chromato-
graphy ( 0521 ) , using a capillary column coated with
polyethylene glycol ( PEG-20M), maintaining the column
temperature at 70ºC for 2 minutes, then raise the tem-
perature to 240ºC at a rate of 5ºC per minute, maintain at
24üºC for 5 minutes. The temperature of injection port is
220ºC, the temperature of the detection is 260ºC. To an
accurately wighed quantity of methyl palmitate CRS and
methyl stearate CRS add n-heptane to produce a mixture
solution containing methyl palmitate 15 mg per ml and
methyl stearate 10 mg per ml respectively. Inject 1 µl onto
the column, the resolution factor between peaks due to
methyl palmitate and methyl stearate is greater than 3. O.
Transfer accurately measured 1 ml of the test solution to a
100 ml volumetric flask, dilute with n-heptane to volume,
shake well. Inject 1 µl onto the column, adjust the sensitivity
of the detector so that the methyl palmitate peak and methyl
stearate peak can be detected. Repeat the operation, using
1 µl of the test solution, record the chromatogram. Calculate
the percentage of stearic acid in the fatty acid as follows:
Stearic acid% = ; X 100%
Where A is the area of the peak of methyl stearate;
B is the sum of the areas of all the peaks of fatty
acid ester.
Calculate the percentage of palmitic acid in the total fatty acid
with the same method. The relative content of stearic acid is
not less than 40 %; the sum of the relative contents of stearic
acid and palmitic acid is not less than 90 %.
Microbial limit Comply with the requirements for
microbiological limit ( 1105 and 1106), the total aerobic
bacteria count is not more than 1000 cfu per g and the total
combined yeast/mold count is not more than 100 cfu per g of
the substance being examined, Escherichia coli should not be
detected.
Assay To O. 2 g of the substance being examined, accurately
weighed, add 50 ml of a mixture of n-butanol and anhydrous
ethanol ( 1 : 1), 5 ml of concentrated ammonia solution,
3 ml of ammonia-ammonium chloride BS ( pH 10. O) , add
accurately measured 25 ml of disodium edetate (O. 05 mol/L)
VS anda small quantity of eriochrome black T indicator, mix

well, heat on a water bath at 40-50ºC until the solution is
clear. Titrate with zinc (O. 05 mol/L) VS until the colour
changes from blue to violet. Perform a blank determination,
and make any necessary correction. Each ml of disodium
edetate (0. 05 mol/L) VS is equivalent to l. 215 mg of Mg.
Category Pharmaceutical excipients, lubricant.
Storage Preserve in well closed containers.
Magnesium Trisilicate
[14987-04-3]
Magnesium Trisilicate is a hydrate magnesium silicate
( Mg2 Si3 0 8 • nH 20) with varying components. lt contains not
less than 20. O% of MgO and not less than 45. O% of Si02.
The ratio of Si0 2 to MgO is 2. 1-2. 3.
Description A white or almost white powder, odourless,
tasteless, slightly hygroscopic.
Insoluble in water and in ethanol.
ldentification ( 1) Prepare a bead by fusing a few crystals
of sodium ammonia phosphate on a platinum loop in the
nonluminous flame. Place the hot, transparent bead in
contact with the substance being examined and fose again,
silica floats about in the bead andan opaque bead with a web-
like structure is produced upon cooling.
(2) Mix about O. 5 g with 10 ml of dilute hydrochloric acid,
filter and neutralize the filtrate with ammonia TS; the filtrate
yields reactions characteristic of magnesium salts ( 2 )
<0301>.
Particle size and particle distribution (use for the glidant)
Carry out the determination of particle size and particle
distribution < 0982 method 3 ) , using laser diff raction
analyzer, water as dispersion media, determined by wet
method. The particle size over 250 µm is not more than 6 %
of the substance being examined.
Acid-consuming capacity Weigh accurately about O. 30 g into
a glass-stoppered conical flask. Add accurately 50 ml of
hydrochloric acid ( O. 1 mol/L) VS and 50 ml of water.
Warm the flask in a water bath at 37°C for 2 hours, shake
the mixture occasionally but leave it undisturbed during the
last 15 minutes, cool. To 50 ml of the supematant liquid add
1 drop of methyl orange IS and titrate the excess acid with
sodium hydroxide ( O. 1 mol/L) VS. 1 g of magnesium
trisilicate, calculated on the ignited basis, consumes not less
than 140 ml and not more than 170 ml of hydrochloric acid
(0. 1 mol/L) VS.
Free alkali Boíl 4. O g with 60 ml of water for 15 minutes
and filter with 2-3 layers of filter paper. Wash the residue
with water in portions, combine the washings and filtrate,
dilute to 100 ml with water and mix well. Add 2 drops of
phenolphthalein IS to 25 ml of the solution; any pink colour
is produced, not more than l. O ml of hydrochloric acid
(0. 1 mol/L) VS is required to discharge it.
Chlorides Mix l. O g with 4 ml of nitric acid and 4 ml of
water, boíl and shake. Add 20 ml of water, mix well, cool
and filter, wash the residue with water, combine the filtrate
and the washings into 50 ml volumetric flask, and dilute with
water to volume, mix well, use as the test solution. Carry
out the limit test for chlorides <0801 ) using 5. O ml of the
test solution. Any opalescence produced is not more
pronounced than that of a reference solution using 5. O ml of
Magnesium Trisilicate
sodium chloride standard solution (O. 05 %) .
Sulfates Transfer accurately 5 ml of the test solution in the
test for Chlorides, add 30 ml of water. Carry out the limit
test for sulfates <0802 ) . Any opalescence produced is not
more pronounced than that of a reference solution using
5. O ml of potassium sulfate standard solution (0. 5%).
Soluble Salts Evaporate 25 ml of the filtrate obtained in the
test for Free alkali to dryness and ignite to constant weight.
The weight of the residue is not more than 15 mg.
Los.s on ignition Weigh accurately about O. 5 g, ignite to
constant weight at 700-SOOºC, loses not more than 30. O% of
its weight.
Heavy metals Boíl 3. O g with 5 ml of hydrochloric acid and
40 ml of water for 20 minutes, cool, add 2 drops of
phenolphthalein IS and ammonia concentrated TS until a pink
colour is produced. Add 1 ml of O. 1 mol/L hydrochloric acid
solution to make it slightly acid. Filter and wash the residue
with small amount of water in portions, combine the
washings and filtrate. Add ammonia TS dropwise until a
pink colour is produced, add 8 ml of O. 1 mol/L hydrochloric
acid solution and sufficient quantity of water to produce 75
ml, mix well. Carry out the limit test for heavy metals
<0821 method 1), using 25 ml: not more than O. 002%.
Mercury Take two portions, each of l. O g into 25 ml of
volumetric flask, dissolve the one portion in 6 ml of
hydrochloric acid and dilute with water to volume, mix well
and filter, wash the residue with hydrochloric acid solution
(6-25), combine the filtrate and the washings into 50 ml
volumetric flask. Add O. 5 ml of 5 % potassium perman-
ganate solution, mix well, add 5 % hydroxylamine
hydrochloride dropwise until the purple colour disappear, and
dilute with hydrochloric acid solution ( 6- 25) to volume,
mix weii as test soiution. To another portion add accurateiy
5 ml of standard mercury solution ( measure accurately a
volume of mercury single-element standard solution dilute
with water to produce a solution of O. 1 µg Hg per mD ,
operated in the same manner to produce reference solution.
Carry out the method for atomic absorption spectro-
photometry ( 0406 method 2) and measure the absorbance of
the two solutions at 253. 6 nm. Not more than O. 00005%.
Arsenic Transfer accurately 20 ml of the test solution in the
test for chlorides, add 5 ml of hydrochloric acid and 3 ml of
water, carry out the limit test for arsenic <0822 method 1):
not more than O. 0005%.
~y Magnesium Oxide To about l. 5 g accurately
weighed, add 50 ml of sulfuric acid (O. 5 mol/L) VS,
accurately measured, heat on a water bath for 15 minutes
and allow to cool. Add 1 drop of methyl orange IS, titrate
with sodium hydroxide ( 1 mol/L) VS. Each ml of sulfuric
acid (O. 5 mol/L) VS is equivalent to 20. 15 mg of MgO.
Silicon Dioxide To about O. 4 g, accurately weighed, in a
porcelain dish add a mixture of 3 ml of sulfuric acid and 5 ml
of nitric acid, heat gently until the reaction is complete.
Evaporate on a sand-bath to dryness, allow to cool. Add
10 ml of dilute sulfuric acid and 100 ml of water, boíl to
dissolve the magnesium salt. Filter the supernatant liquid
through an ashless filter paper, wash the res id ue for three
times with hot water and filter the washings. Finally transfer
the residue to the filter paper, wash with hot water.
Transfer the filter paper and its contents to a platinum
crucible, dry, incinerate, and ignite for further 30 minutes,
cool and weigh accurately. Moisten the residue with water,
add 3 ml of hydrofluoric acid and 3 drops of sulfuric acid.
 Maize Starch
Evaporate to dryness, ignite for 5 minutes, cool, and weigh
accurately: the loss in weight represents the weight of Si0 2
in the substance being examined.
Category Pharmaceutical excipients, glidant, antiadherent,
suspending agent, adsorbants and filter-aid.
Storage Preserve in tightly closed containers.
Maize Starch
Maize Starch is polysaccharide granules obtained from the
caryopsis of maize (Zea mays L.)
Description A white or almost white powder.
Insoluble in water or in ethanol.
ldentification ( 1) .Boil about l. O g with 15 ml of water,
cool. A whitish translucent gelationous substance is produced.
(2) Add a drop of iodine TS to 1 ml of the gelationous
substance from Identification ( 1). A dark blue or dark violet
colour is produced which gradually disappears on heating.
(3) Examine under microscopy with glycerine-acetic acid TS
( 2001 ) Simple grains, polygonal or ovoid, 5-30 µm in
diameter. Central hilum, rounded or star-shaped. Straitions
indistinct. Under polarised microscope: with polarized cross,
intersecting at the hilum.
Acidity Dissolve 4. O g in 20 ml of water, shake for 5
minutes to mix, centrifuge, pH 5. 0-8. O ( 0631 ) .
Foreign matter Examine under the Identification ( 3) : no
starch grains of any other origin are present.
Sulfur dioxide Not more than O. 004 % ( 2331 ) .
Oxidizing substance Transfer 4. O g of the substance being
examined to a conical flask with stopper, add 50. O ml of
water, stopper and shake for 5 minutes, transfer to a
centrifuger tube with stopper, centrifuge until the solution is
clear. Transfer 30. O ml of the supernatant toan iodine flask,
add 1 ml of glacial acetic acid and l. O g of potassium iodide,
stopper tightly, shake, allow to stand for 30 minutes in a
dark place. Add 1 ml of starch IS, and titrate with sodium
thiosulfate ( O. 002 mol/L ) VS until the blue colour
disappears. Perform a blank determination and make any
necessary correction. Not more than l. 4 ml of sodium
thiosulfate (O. 002 mol/L) VS is consumed (0. 002%).
Loss on drying When dried at 130ºC for 90 minutes, loses
not more than 14. O% of its weight ( 0831).
Ash Not more than O. 3 % ( 2302).
Heavy metals Carry out the limit test for heavy metals
( 0821), using l. O g: Not more than O. 002%.
Iron Mix l. O g with 4 ml of dilute hydrochloric acid and
16 ml of water, shake for 5 minutes, filter and wash the
residue with a volume of water. Combine the filtrate and
washings to a 50 ml Nessler cylinder, add 50 mg of
ammonium persulfate and dilute with water to 35 ml. Carry
out the limit test for iron ( 0807). Any colour produced is not
more intense than that of a reference solution by using l. O ml
of iron standard solution (0. 001%).
Microbial limit Comply with the requirements for
microbiological limit ( 1105 and 1106), the total aerobic
bacteria count is not more than 1000 cfu per g and the total
combined yeast/mold count is not more than 100 cfu per g of
the substance being examined, Escherichia coli should not be
detected.
Category Pharmaceutical excipients, filler and disintegzating
agent.
Storage Preserve in well closed containers.
Maleic Acid
C4 H4 Ü4 116. 07
[110-16-7]
Maleic acid is (Z) -butenedioic acid. It contains not less than
99. O% of Maleic acid CC H4 04 ) , calculated on the
anhydrous basis.
Description A white or almost white crystalline powder,
odour characteristic.
Freely soluble in water and in acetone.
Melting point 133. 0-137. OºC ( 0612).
Identification ( 1) Dissolve O. 1 g in 10 ml of water, mix
well as test solution. To O. 3 ml of test solution add 3 ml of
resorcinol solution in sulfuric acid Cl--+-300), heat on a water
bath for 15 minutes, the solution is colourless. To 3 ml of
test solution add 1 ml of bromine TS, heat on a water bath
for 15 minutes, the colour of bromine fades, cool. To O. 2 ml
of the solution add resorcinol solution in sulfuric acid ( 1-
300), heat on a water bath for 15 minutes. A violet-pink
colour is produced.
(2) Dissolve separately the substance being examined and
maleic acid CRS in the mobile phase as described under
Related substances to produce two solutions of 10 µg per ml
as the test solution and the reference solution. Using the
chromatographic conditions described under Related sub-
stances. lnject 10 µl of test solution and reference solution
separately. The retention time of principal peak of the test
solution examined in the chromatogram is identical with that
of reference solution.
(3) The infrared absorption spectrum is concordant with the
spectrum of maleic acid CRS ( 0402).
Acidity To O. 5 g add 10 ml of water, shake to dissolve.
The pH is not more than 2. O ( 0631 ) .
Clarity and colour of solution Dissolve l. O g in 10 ml of
water, the solution is clear and colourless; any colour
produced is not more intense than that of ref erence solution
Y1 ( 0901 method 1 ) .
Fumaric acid and other related substances Dissolve a
quantity, accurately weighed, in the mobile phase to produce
the solution of 1 mg per ml as the test solution. Dissolve a
quantity of maleic acid CRS and fumaric acid CRS in mobile
phase to produce a ref erence solution of about 1 µg and 5 µg
as the ref erence solution. Carry out the method for high
performance liquid chromatography ( 0512), using a column
packed with octadecylsilance bonded silica gel. Mobile phase
is a mixture of water ( previously adjusted to pH 3. O with
phosphoric acid) -acetonitrile ( 85 15 ). Detection
wavelength is 210 nm. Inject 10 µl of reference solution, the
resolution factor between peaks of fumaric acid and maleic
acid is more than 2. 5. Inject 10 µl of test solution and
reference solution separately, record the chromatogram for
twice the retention time of the peak of maleic acid. The
content of fumaric calculated with respect to the peak area

obtained in the chromatogram by the external standard
method is not more than O. 5 %, the content of any other
impurity calculated with respect to the peak area of maleic
acid obtained with reference solution is not more than O. 1 %,
the sum of the areas of all impurity peaks is not more than
l. 0%.
Water Not more than 2. O% ( 0832 method 1 ( 1 ) ) .
Residue on ignition Not more than O. 1 % <0841 >, using
l. o g.
lron To l. O g add 25 ml of water, carry out the limit test
for iron ( 0807>. Any colour produced is not more intense
than that of a reference solution using O. 5 ml of iron standard
solution (O. 0005%).
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), using the residue obtained in the test for
Residue on ignition: not more than O. 001%.
Assay Dissolve about l. O g, accurately weighed, in 100 ml
of water, add a few drops of phenolphthalein IS, titrate
with sodium hydroxide ( 1 mol/L) VS. Each ml of sodium
hydroxide ( 1 mol/L) VS is equivalent to 58. 04 mg of
C4H4Q4.
Category Pharmaceutical excipients, pH regulator and
effervescent agent.
Storage Preserve in well closed containers.
DL-Malic Acid
C H6 05 134. 09
[617-48-1]
DL-Malic Acid is (RS) - ( ±) -hydroxybutanedioic acid. It
contains not less than 99. O% of C H 6 0 5 , calculated on the
anhydrous basis.
Description A white crystalline powder; odourless; tasteless.
Freely soluble in water and in ethanol; slightly soluble m
acetone.
Meltingpoint 128-132ºC (0612>.
ldentification (1) Dissolve O. 5 gin 10 ml of water, adjust
pH to neutral with concentrate ammonia solution, add 1 ml
of 1 % sulfanilic acid solution and heat on a boiling water bath
for 5 minutes, add 5 ml of 20 % sodium nitrite solution and
heat on water bath for 3 minutes, add 5 ml of 4% sodium
hydroxide solution, and a red colour is produced immediately.
(2) The infrared absorption spectrum is concordant with that
of DL-malic acid CRS <0402 >.
Clarity and Colour of solution Dissolve 10. O g in 100 ml of
water, the solution is clear and colourless ( 0901 and 0902).
Any opalescence produced is not more pronounced than that
of ref erence suspension 1 ( 0902 method 1 ) .
Optical rotation - O. 10º to +O. 10º, determined on a
solution of O. 2 g per ml in water <0621 ) .
Readily oxidizable substances Dissolve O. 10 g in 25 ml of
water and 25 ml of sulfuric acid solution ( 1 - 20) , in a
100 ml beaker, mix well, allow to cool in a water bath at
20± 1 ºC, add 5 ml of O. 02 mol/L potassium permanganate
DL-Malic Acid
· · ·. •. . ··.·•·.·e,
,, .•.....
··;··.~::·:_~>,·:::~·
.,..... ,. '
solution, allow to stand for 3 minutes, the colour of the
solution <loes not disappear completely.
Chloride Carry out the limit test for chlorides ( 0801 ) ,
using l. O g. Any opalescence produced is not more
pronounced than that of a reference solution prepared by
using 5. O ml of sodium chloride standard solution (0. 005%).
Sulfate Carry out the limit test for sulfates ( 0802), using
l. O g. Any opalescence produced is not more pronounced
than that of a reference solution prepared by using 3. O ml of
potassium sulfate standard solution (O. 03 %) .
Water-insoluble substances Dissolve 25. O g in 100 ml of
water, filter through a sintered-glass crucible (No. 4)
previously dried to constant weight at lOOºC. Wash the filter
with hot water for several times, and dry to constant weight
at lOOºC. The residue is not more than 0.1% of its weight.
Related substances Dissolve an accurately weighed quantity
in mobile phase to produce a test solution containing about
1 mg per ml. Dissolve an accurately weighed quantity of
fumaric acid CRS and maleic acid CRS in mobile phase to
produce a mixture solution containing about 1Oµg of fumaric
acid per ml and O. 5 µg of maleic acid per ml respectively, and
use as the reference solution. Carry out the method for high
performance liquid chromatography ( 0512), using a column
packed with octylsilicane bonded silica gel and a mixture of
O. 1 % phosphoric acid-methanol ( 90 : 10) as the mobile
phase. Detection wavelength is 214 nm. Dissolve a quantity
of fumaric acid CRS, maleic acid CRS and DL-malic acid CRS
in mobile phase to produce a mixture solution containing
about 10 µg of fumaric acid, 4 µg of maleic acid and 1 mg of
DL-malic acid per ml respectively and inject 10 µl into the
column. The components are eluted in the following
sequence: DL-malic acid, maleic acid and fumaric acid. The
theoretical plates calculated for DL-malic acid peak is not less
than 2000; the resolution between the each pair of adjacent
peaks due to fumaric acid, maleic acid and DL-malic acid
comply with the requirements. Inject 10 µl of the reference
solution into the column, adjust the sensitivity of the
detector so that the principal peak height in the chrom-
atogram is about 1O% of the full scale of the recorder. Inject
separately 10 µl of the test solution and the reference solution
into the column, and record the chromatogram for 3 times
the retention time of the principal peak. Calculate the content
of fumaric acid and maleic acid with respect to the peak area
by the external standard method, the content of fumaric acid
and maleic acid is not more than l. O% and O. 05 %
respectively. The area of the other individual impurity peak
in the chromatogram obtained with the test solution is not
greater than 2 times the area of maleic acid in the
chromatogram obtained with the reference solution (O. 1%).
The sum of ar.ea of all other impurity peaks in the chrornatogram
obtained with the test solution is not greater than 10 times of
the area of maleic acid in the chromatogram obtained with the
reference solution (O. 5 %) .
Water Not more than 2. O% ( 0832 method 1 ( 1 ) ) .
Residue on ignition Not more than O. 1 % <0841 >, using
l. o g.
Calcium Dissolve l. O gin 10 ml of water, add 20 ml of 5%
sodium acetate solution, shake well, to 15 ml of the solution
add 1 ml of 2 mol/L acetic acid solution, shake well and use
as the test solution. Transfer 10. O ml of calcium standard
solution (Place 2. 50 g of calcium carbonate, accurately
weighed, in a 1000 ml volumetric flask, add 12 ml of 5 mol/L
acetic acid solution and a volume of water to dissolve and
dilute with water to volume, shake well and use as calcium
 L-Malic Acid
stock solution. Transfer accurately 1 ml of the calcium stock
solution to a 100 ml volumetric flask immediately before use,
add water to volume and shake well. Each ml is equivalent to
10 µg of Ca) , add 1 ml of 2 mol/L acetic acid solution and
5 ml of water, shake well and use as the reference solution.
Transfer O. 2 ml of ethanolic calcium standard solution
(Transfer accurately 10 ml of the calcium stock solution to a
100 ml volumetric flask immediately before use, add ethanol
to volume and shake well. Each ml is equivalent to O. 1 mg of
Ca. ) to a Nessler tube, add 1 ml of 4 % ammonium oxalate
solution, after 1 minute, add the test solution, shake well
and allow to stand for 15 minutes. Any opalescence produced
is not more pronounced than that of a reference prepared in
the same manner (O. 02%).
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), using the residue obtained in the test for
residue on ignition: not more than O. 002%.
Arsenic Dissolve l. O g in 23 ml of water, and 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822
method 1 ) : not more than O. 0002 %.
Assay Place about l. O g of the substance being examined,
accurately weighed, in a 250 ml volumetric flask, add water
to dissolve and dilute to volume, shake well. Transfer
accurately 25 ml in a conical flask, add 2 drops of phen-
olphthalein IS and titrate with sodium hydroxide (O. 1 mol/L)
VS until a reddish colour persists for 30 seconds. Each ml of
sodium hydroxide (O. 1 mol/L) VS is equivalent to 6. 704 mg
of C4H60s.
Category Pharmaceutical excipients, acidifying agent and
antioxidant.
Storage Preserve in tightly closed containers, protected
from light.
L-Malic Acid
/'....,,/C0 2H
H02C .. _/"-,
HO H number of theoretical plates calculated for L-malic acid peak
C4 H6 Os 134. 09
[97-67-6]
L-Malic acid is L-hydroxybutanedioic acid, it is prepared by
enzyme engineering technology or fermentation reaction and
followed separation and purification process. It contains not
less than 99. o% of C4 H6 Os , calculated on the anhydrous
basis.
Description A white crystal or a crystalline powder,
odourless, tasteless.
Freely soluble in water and in ethanol, slightly soluble in
aceto ne.
Specific optical rotation - l. 6° to - 2. 6°, in a solution
containing 85 mg per ml in water ( 0621 ) .
ldentification (1) Dissolve O. 5 gin 10 ml of water, adjust
pH to neutral with concentrate ammonia solution, add 1 ml
of 1 % sulfanilic acid solution and heat on a boiling water bath
for 5 minutes, add 5 mi of 20% sodium nitrite solution and
heat on a water bath for 3 minutes, add 5 ml of 4% sodium
hydroxide solution, and a red colour is produced imm-
ediately.
(2) The infrared absorption spectrum is concordant with that
of L-Malic acid CRS ( 0402).
Clarity and Colour of solution Dissolve 10. O g in 100 ml of
water, the solution is clear and colourless ( 0901 and 0902).
Any opalescence produced is not more pronounced than that
of reference suspension 1 ( 0902 method 1 ) .
Readily oxidizable substances Dissolve O. 10 g in 25 mi of
water and 25 ml of sulfuric acid solution ( 1 - 20) , in a
100 ml beaker, allow to cool in a water bath at 20±lºC, add
5 ml of O. 02 mol/L potassium permanganate solution, allow
to stand for 3 minutes, the colour of the solution <loes not
disappear completely.
Chloride Carry out the limit test for chlorides ( 0801 ) ,
using l. O g. Any opalescence produced is not more pron-
ounced than that of a reference solution prepared by using
5. O ml of sodium chloride standard solution (O. 005%).
Sulfate Carry out the limit test for sulfates ( 0802), using
l. O g. Any opalescence produced is not more pronounced
than that of a reference solution prepared by using 3. O ml of
potassium sulfate standard solution (O. 03 %) .
Water-insoluble substances Dissolve 25. O g in 100 ml of
water, filter through a sintered-glass crucible (No. 4)
previously dried to constant weight at lOOºC. Wash the filter
with hot water for several times, and dry to constant weight
at lOOºC. The residue is not more than O. 1% of its weight.
Related substances Dissolve an accurately weighed quantity
in mobile phase to produce a test solution containing about
1 mg per ml. Dissolve an accurately weighed quantity of
fumaric acid CRS and maleic acid CRS in mobile phase to
produce a mixture solution containing about 10 µg of fumaric
acid per ml and O. 5 µg of maleic acid per ml respectively,
and use as the reference solution. Carry out the method for
high performance liquid chromatography ( 0512), using a
column packed with octylsilicane bonded silica gel and a
mixture of O. 1 % phosphoric acid-methanol ( 90 : 10) as the
mobile phase. Detection wavelength is 214 nm. Dissolve a
quantity of fumaric acid CRS, maleic acid CRS and L-malic
acid CRS in mobile phase to produce a mixture solution
containing about 10 µg of fumaric acid, 4 µg of maleic acid
and 1 mg of L-malic acid per mi respectively and inject 10 µl
onto the column. The components are eluted in the following
sequence: L-malic acid, maleic acid and fumaric acid. The
is not less than 2000; the resolution factors between the each
pair of adjacent peaks due to fumaric acid, maleic acid and
L-malic acid comply with the requirements. Inject 10 µl of
the reference solution onto the column, adjust the sensitivity
of the detector so that the principal peak height in the
chromatogram is about 1O% of the full scale of the recorder.
Inject separately 10 µl of the test solution and the reference
solution onto the column, and record the chromatogram for 3
times the retention time of the principal peak. Calculate the
content of fumaric acid and maleic acid with respect to the
peak area by the external standard method, the content of
fumaric acid and maleic acid is not more than l. O% and
O. 05 % respectively. The area of the other individual
impurity peak in the chromatogram obtained with the test
solution is not greater than 2 times the area of the principal
peak due to maleic acid in the chromatogram obtained with
the reference solution (O. 1 %) . The sum of areas of all other
impurity peaks in the chromatogram obtained with the test
solution is not greater than 10 times of the area of the
principal peak due to maleic acid in the chromatogram
obtained with the reference solution (0. 5%).
Water Not more than 2. O% ( 0832 method 1 ) .
Residue on ignition Not more than O. 1 % ( 0841 ) , usmg

l. o g.
Calcium Dissolve l. O gin 10 ml of water, add 20 ml of 5%
sodium acetate solution, shake well, to 15 ml of the solution
add 1 ml of 2 mol/L acetic acid solution, shake well and use
as the test solution. Transfer 10. O ml of calcium standard
solution CPlace 2. 50 g of calcium carbonate, accurately
weighed, in a 1000 ml volumetric flask, add 12 ml of 5 mol/L
acetic acid solution and a volume of water to dissolve and
dilute with water to volume, shake well and use as calcium
stock solution. Transfer 1 ml of the stock solution, accur-
ately measured, to a 100 ml volumetric flask immediately
befare use, add water to volume and shake well. Each ml is
equivalent to 10 µg of Ca), add 1 ml of 2 mol/L acetic acid
solution and 5 ml of water, shake well and use as the
reference solution. Transfer O. 2 ml of ethanolic calcium
standard solution etransfer 10 ml of the calcium stock
solution, accurately measured, to a 100 ml volumetric flask
immediately befare use, add ethanol to volume and shake
well. Each ml is equivalent to O. 1 mg of Ca) to a Nessler
tube, add 1 ml of 4% ammonium oxalate solution, after 1
minute, add the test solution, shake well and allow to stand
far 15 minutes. Any opalescence produced is not more
pronounced than that of a reference prepared in the same
manner (O. 02%).
Heavy metals Carry out the limit test far heavy metals
<0821 method 2) , using the residue obtained in the test far
Residue on ignition: not more than O. 002%.
Arsenic Dissolve l. O g in 23 ml of water and 5 ml of
hydrochloric acid. Carry out the limit test far arsenic <0821
method 1): not more than O. 0002%.
Assay Place about l. O g of the substance being examined,
accurately weighed, in a 250 ml volumetric flask, add water
to dissolve and dilute to volume, shake well. Transfer
accurately measured 25 ml in a conical flask, add 2 drops of
phenolphthalein IS and titrate with sodium hydroxide CO. 1 mol/L)
VS until a reddish colour persists far 30 seconds. Each ml of
e
sodium hydroxide o. 1 mol/L) vs is equivalent to 6. 704 mg
of C4H605.
Category Pharmaceutical excipients, acidifying agent and
antioxidant.
Storage Preserve in tightly closed containers, protected
from light and stored in a cool place.
Maltodextrin
ok l
~
OHO O H
HO O
OH
H titration. Operate in the same manner as pre-titration, and
y conduct a parallel test.
CC6 H10 Ü5 )n• H2 O
[9050-36-6]
Maltodextrin is prepared by the hydrolysis of edible starch
with suitable enzymes or acids then purified.
Description A white or almost white powder or granule;
slightly odor; tasteless or slightly sweet; hygroscopic.
Freely soluble in water, practically insoluble in absolute
ethanol.
Maltodextrin
Identification Dissolve about 1 gin 10 ml of water, add the
warm alkaline cupric tartrate TS slowly, a red precipitate is
produced.
Acidity Dissolve 2. O g in 10 ml of water, pH 4. 5-6. 5
<0631).
Water-insoluble matters Dissolve 5. O g, calculated on the
dried basis, to a beaker, in 50 ml of water (35-40ºC). Filter
while still warm through a No. 3 sintered glass crucible
previous dried to constant weight at 105ºC, wash the beaker
with 50 ml of water (35-40ºC) far several times, filter the
washings. Dry the residue to constant weight at 105ºC, the
weight of the residue is not more than l. O%.
Protein Transfer about 10 g, accurately weighed, to a
500 ml Kjeldahl flask, add 10 g of anhydrous potassium
sulfate and O. 5 g of copper sulfate, add 50 ml of sulfuric acid
slowly, carry out the method far determination of nitrogen
<0704 method 1 ) , multiply by 6. 25: not more than O. 1 %.
Sulfur dioxide Dissolve 5 g, accurately weighed, with
100 ml of water in a 250 ml iodine flask, add 5 ml of
hydrochloric acid and 1 ml of starch IS. Titrate with iodine
(0. 01 mol/L) VS immediately, until the colour turns from
pale yellow to light blue to purplish red, perform a blank
determination and make any necessary correction. Each ml of
iodine (0. 01 mol/L) VS is equivalent to O. 6406 mg of S02:
not more than O. 004 %.
~ on drying When dried to constant weight at 105ºC,
loses not more than 6. O% of its weight <0831 ) .
Residue on ignition Not more than O. 5% <0841), using
2. o g.
Heavy metals Carry out the limit test far heavy metals
<0821), using the residue obtained in Residue on ignition:
not more than O. 0005 % .
Arsenic Mix 2. O g with l. O g of calcium hydroxide, add a
volume of water, stir well and dry. Heat gently until
completely charred, and then ignited at 500-600ºC until the
incineration complete, allow to cool. Add 8 ml of
hydrochloric acid and 23 ml of water. Carry out the limit test
far arsenic <0822 method 1 ) : not more than O. 0001 % .
Dextrose equivalent Dissolve O. 5 g of the anhydrous glucose
CRS, accurately weighed, with water in a 250 ml volumetric
flask, dilute with water to volume and shake well and use as
glucose reference solution. Far pre-titration, to 10 ml of
alkaline copper tartrate TS in a conical flask, accurately
measured, add 20 ml of water, add 3 glass beads, add 24 ml
of glucose reference solution by a 50 ml burette, mix well,
place the solution on an electric furnace far heating to
boiling, maintain the solution at boiling, add 2 drops of 1 %
methylene blue solution, continue to titrate with glucose
reference solution until the blue colour disappears ( the all
titration process is completed in 3 minutes) ; far formal
titration, pre-add glucose reference solution O. 5 ml less than
the volume of glucose ref erence solution added far pre-
Dissolve a quantity (see the accompanying table), accurately
weighed, in hot water, cool, dilute to 250 ml, shake well
and use as the test solution. For pre-titration, to 10 ml of
alkaline copper tartrate solution, accurately measured, in a
conical flask, add 20 ml of water, add 3 glass beads, add a
volume of the test solution from the 50 ml burette, operate in
the same manner as glucose reference solution. For formal
titration, pre-add test solution O. 5 ml less than the volume of
test solution added far pre-titration. Operate in the same
 Maltol

manner as pre-titration, and conduct a parallel test.
Calculate the dextrose equivaleut value using the following
equation. Not more than 20, calculated on the dried basis.
X= CiVi X 100
C2V2
Where X is DE value [ sample dextrose equivalent value
( percentage of reducing sugar in the sample in the
dry matter) ] , % ;
C1 is concentration of glucose reference solution,
mg/ml;
V1 is total volume of glucose reference solution
consumed, ml;
C 2 is concentration of the test solution ( calculated
on anhydrous substance) , mg/ ml;
V2 is total volume of test solution consumed, ml;
Microbial limit Comply with the test far microbial limit
( 1105, 1106), the total aerobic bacteria count is not more
than 1000 cfu per gram and total combanced yeast/mold
count is not more than 100 cfu, and Escherichia coli should
be absent.
Category Pharmaceutical excipients, coating agent, diluent,
binder and thickener.
Storage Preserve in fightly closed containers, stored in a dry
place.
Table Reference sampling weight in the Dextrose equivalent
DE value 1 6 9 12
mercury and copper ( 2321 ) , not more than O. 001 % .
Heavy Metals Carry out the limit test far heavy metals
( 0821 method 2), using the residue obtained under the test
far residue on ignition: not more than O. 002%.
Arsenic Ignite O. 67 g of the substance being examined to
completely carbonized, at the temperature of 450-500ºC,
carry out the limit test for arsenic ( 0822 Method 1 ) , not
more than O. 0003 % .
Assay Place 50 mg of the substance being examined,
accurately weighed, in a 250 ml volumetric flask, diluted
with O. 1 mol/L hydrochloric acid solution to the volume,
shake well, and accurately measure 5 ml of the resulting
solution into a 100 ml volumetric flask, diluted with O. 1
mol/L hydrochloric acid solution to the volume, shake well.
Carry out the method far altraviolet-visible spectrophotometry
( 0401 ) , determine the absorbance at 27 4 nm, Repeating the
operation, using maltol CRS instead of the test solution.
Category Pharmaceutical excipients, taste-masking agent,
flavoring additives.
Storage Preserve in well closed containers, protected from
light.
Mal tose
15 19

:~OH ~HO.H
'>A 1A A C" ') (\
Sampling weight (g) t.v. f'\
v _¡_v. v f'\ C""
voV '"to v voV

OH O OH
The table abo ve can be ref erred to far the sampling weight of
samples with different DE values to prepare test solutions at OH H O OH H
certain concentrations. Pre-titration tests should be OH H
conducted first. H OH H OH

:~OH ~HOH OH O OH
Maltol OH H O OH H
OH H
H OH H OH

O OH C12 H22 Ou •Hz O 360. 31

ÓC O
1

CH3
[ 6363-53-7]
C12 H22 Ou 342. 30
[69-79-4]
C6 H6 03 126. 11 Maltose is 4-0-a-D-glucopyranosyl-,B-glucopyranose, mono-
[118-71-8] hydrated or anhydrous. It contains not less than 98. O% of
Maltol is 3-Hydroxy-2-methyl-4-pyrone. It contains not less C12 H22 Ün , calculated on the anhydrous basis.
than 99. O% of rnaltol CC6H6 0 3 ) , calculated on the
Description White crystals or a crystalline powder, taste
anhydrous basis.
sweetly.
Description a white crystalline power, caramel or cream Freely soluble in water, slightly soluble in methanol, very
odor. slightly soluble in ethanol, practically insoluble in diethyl
Soluble in ethanol or in propylene glycol, slightly soluble in ether.
water or in glycerin.
Specific optical rotation Dry at 80ºC for 4 hours, dissolve
Melting point 160-164 ºC <0612). about 10 g, accurately weighed, in O. 2 ml of ammonia TS in
ldentification ( 1 ) The light absorption of the solution a 100 ml volumetric flask, dilute to volume with water, and
obtained in the test far Assay, exhibits a maximum at mix well. The specific optical rotation of the solution is
217. O nm (0401 ). +126º to +131º (0621 ).
(2) The infrared absorption spectrum is concordant with the Identification (1) Dissolve O. 5 g in 5 ml of water, add
reference spectrum of maltol CRS ( 0402). 5 ml of ammonia TS, and heat on a water bath for 5
Water Not more the O. 5 % ( 0832 method 1 ) . minutes: an orange colour is produced.
(2) Add 2-3 drops of a solution of Maltose Cl-20) to 5 ml
Residue on ignition Not more than O. 2 % , using l. O g <0841 ) . of hot alkaline cupric tartrate TS: a red precipitate is
Lead Carry out the limit test far lead, cadmium, arsenic, formed.

(3) The retention time of principal peak of the test solution
in the chromatogram obtained in the Assay is identical with
that of the principal peak of the reference solution in the
chromatogram.
Acidity Dissolve l. O g in 10 ml of water, pH 4. 5-6. 5
<0631 >.
Chloride Carry out the limit test for chlorides ( 0801 ) ,
using O. 40 g. Any opalescence produced is not more
pronounced than that of a reference using 7. 2 ml of sodium
chloride standard solution (0. 018%).
Sulfate Carry out the limit test for Sulfates ( 0802) , using
l. O g. Any opalescence produced is not more pronounced
than that of a reference using 2. 4 ml of potassium sulfate
standard solution (O. 024%).
Dextrin, soluble starch and sulfite Dissolve l. O gin 10 ml of
water, and add 1 drop of iodine TS: a yellow colour is
produced. Then add 1 drop of starch TS: a blue colour is
produced.
Related substance Dissolve an accurately weighed quantity in
water to produce a solution of 50 mg per ml as the test
solution. Dilute 1 ml to 100 ml with water, and mix well as
the reference solution. Using the chromatographic conditions
in the Assay, inject 20 111 of the reference solution onto the
column. Adjust the attenuation so that the principal peak
height is 15 %-25 % of full scale of the chart. Inject 20 111
each of the test solution and the reference solution onto the
column and record the chromatograms for 2. 5 times the
retention time of the principal peak. The sum of the area of
all impurity peaks before the principal peak in the
chromatogram obtained with the test solution is not greater
than l. 5 times of the area of the principal peak in the
chromatogram obtained with the reference solution (l. 5%).
The sum of the area of all impurity peaks after the principal
peak in the chromatogram obtained with the test solution is
not greater than O. 5 times of the area of the principal peak in
the chromatogram obtained with the reference solution
(0. 5%).
Water Not more than l. 5 % for the anhydrous form; Not
less than 4. 5 % and not more than 65 % for the monohydrated
form ( 0832 method 1 ) .
Residue on ignition Not more than O. 05 % <0841 ) , using
l. o g.
Heavy metals Dissolve 5. O g in 23 ml of water, and add
2 ml of aceta te BS ( pH 3. 5). Carry out the limit test for
heavy metals ( 0821 method 1): not more than O. 0004%.
Arsenic Dissolve l. 5 g in 5 ml water, add 5 ml of dilute
sulfuric acid and 1 ml of bromine TS, heat on a water bath
for 5 minutes, then concentrate to 5 ml, cool and add slowly
5 ml of hydrochloric acid and 23 ml of water to dissolve the
residue. Carry out the limit test for arsenic ( 0822 ) : not
more than O. 00013%.
Microbial limit Comply with the requirements for micro-
biological limit ( 1105 and 1106), the total aerobic bacteria
count is not more than 1000 cfu per g and the total combined
yeast/mold count is not more than 100 cfu per g of the
substance being examined, Escherichia coli should not be
detected.
Assay Carry out the method for high performance liquid
chromatography ( 0512), using a column packed with amino
bonded silica anda mixture of acetonitrile-water (70 : 30) as
the mobile phase. Maintain the column temperature at 35ºC;
use a differential refractive index detector. Dissolve a
1-Menthol [ ••·• 'Bli
quantity of maltose CRS, glucose CRS and maltotriose CRS
in water to produce a solution containing each of 10 mg per
ml. lnject 20 111 onto the column, and record the chrom-
atogram. The resolution factors between the peaks of
maltose, glucose and maltotriose comply with the requirement.
Procedure Dissolve an accurately weighed quantity in water
to produce a solution containing 10 mg per ml. lnject 20 111
onto the column, and record the chromatogram. Repeat the
operation, using maltose CRS instead of the substance being
examined. Calcula te the content of C12 Hz2 Üu with respect to
the peak areas obtain in the chromatogram by the external
standard method.
Category Pharmaceutical excipients, filling agent, and
flavoring agent.
Storage Preserve in well closed containers.
l-Menthol
C10 H20 O 156. 27
[1490-04-6] or [89-78-1]
l-Menthol is l-1-methyl-4-isopropylcyclohexan-3-olobtained
by steam distilling, freezing, and recrystallizing from the
fresh stalks and leaves of Menthahaplocalyx Briq. It
contains not less than 95. O% and not more than 105. O% of
C10HzoO.
Description Colourless needle or prismatic crystals or a
white, crystalline powder, odour, aromatic, resembling that
of peppermint, taste, pungent, and then cool. A solution
inethanol exhibits a neutral reaction.
Very soluble in ethanol, in chloroform and in ether, very
slightly soluble in water.
Melting point 42-44 ºC <0612).
Specific optical rotation - 49º to - 50º, in a solution of
O. 1 g per ml in ethanol ( 0621 ) .
ldentification ( 1) Dissolve 1 g in 20 ml of sulfuric acid,
an orange-red colour is produced. After 24 hours, a
colourless oil layer without aromatic odour of menthol is
separated (distinction from thymol).
(2) Dissolve 50 mg in 1 ml of glacial acetic acid, add a cold
mixture of 6 drops of sulfuric acid and 1 drop of nitric acid.
Only a pale yellow colour is produced ( distinction from
thymoD.
Related substances Dissolve a quantity of the substance
being examined, accurately weighed, in absolute ethanol to
produce a solution containing 50 mg per ml as the test
solution. Dissolve a quantity of l-menthol CRS, accurately
weighed, in absolute ethanol to produce a solution containing
O. 5 mg per ml as the reference solution. Carry out the method
as described under Assay, the column temperature sets at
llOºC. Inject 1 111 of the reference solution into the column,
adjust the attenuation so that the principal peak height in the
chromatogram is 20-30 % of the full scale of the chart. Inject
separately 1 111 each of the test solution and the reference
solution into the column, record the chromatogram for twice
 \592'. CJ 1 Methacrylic Acid Copolymer I
the retention time of the principal peak. The sum of the areas
of all impurity peaks other than the principal peak is not
more than the area of the principal peak in the chromatogram
obtained with the reference solution (l. O%).
Non-volatile substances Transfer 2 g toan evaporating dish,
previously dried to constant weight, evaporate slowly on a
water bath and dry to constant weight at 105ºC. The residue
is not more than 1 mg.
Heavy metals and hannful element Carry out the method for
determination of Lead (Pb), Cadmium (Cd), Arsenic (As),
Mercury ( Hg) and Copper (Cu) ( 2321 ) : not more than
O. 0005% of Lead, not more than O. 00003% of Cadmium,
not more than O. 0002% of Arsenic, not more than
O. 00002 % of Mercury, not more than O. 002 % of Copper.
Assay Carry out the method for gas chromatography
( 0521), using a capillary column packed with cross linked
polyethylene glycol as the stationary phase. Maintain the
column temperature at 120ºC. The temperature of injection
port is 250ºC and the temperature of detector is 250ºC. The
split ratio is 10 : l. The number of the theoretical plates of
the column is not less than 10 000, calculated with the
reference to the peak of menthol.
Procedure Dissolve 10 mg of the substance being examined,
accurately weighed, in absolute ethanol in a 10 ml volumetric
flask, dilute to volume and mix well. Inject accurately 1 µl of
the resulting solution into the column and record the
chromatogram. Repeat the operation, using l-Menthol CRS
instead of the substance being examined. Calculate the
content of C10 H20 O with respect to the peak area obtained in
the chromatogram by the externa! standard method.
Category Pharmaceutical excipients, tas te masking agent
and aromatic, etc.
Storage Preserve in tightly closed containers, stored in a
cool place.
Methacrylic Acid Copolymer I cautiously 10 ml of water, heat again until the sulfur trioxide
Methacrylic acid copolymer I is a copolymer of methyl
methacrylate, ethyl acrylate and trimethyl ammonium
chloride ethyl methacrylate in a ratio of 60 : 30 : 10.
Description Almost white, translucent or transparent salid
with different shape and size.
Soluble in boilng water or in acetone, practically insoluble in
isopropanol.
Refractive index Dissolve l. 25 g in 10 ml of isopropanol-
acetone ( 6 : 4) , the refractive index is l. 380-1. 385 ( 0622).
Alkaline value Dissolve about l. O g, previously dried to
constant weight at llOºC (for about 5 hours) and accurately
weighed, in 25 ml of dichloromethane, add 50 ml of glacial
acetic acid and 5 ml of mercuric aceta te TS, mix well and add
3 drops of quinaldine red IS, titrate with perchloric acid
(O. 1 mol /L) VS until the colour of the solution changes
from red to colourless. Perform a blank determination and
make necessary correction. Each ml of perchloric acid
(0. 1 mol /L) VS is equivalent to 5. 61 mg of KOH. The
value is 23. 9-32. 3 mg/ g, calculated on the dried basis.
ldentification The infrared absorption spectrum <0402) is
concordant with the spectrum of Methacrylic acid copolymer
l CRS.
Viscosity Dissolve 6. O g in a 100 ml of 75 % ethanol
solution, the kinematic viscosity at 20ºC is not more than
O. 015 Pa•s, using a rotational type of viscometer with a
No. O rotator ( 0633 method 2) at the speed of 30 rpm.
Related substance Dissolve a quantity of the substance being
examined, accurately weighed, in methanol to produce a
solution of 1 mg per ml as the test solution. Carry out the
method for high performance liquid chromatography
( 0512), using a column packed with octadecylsilane bonded
silica gel and a mixture of methanol-phosphate BS [ dissolve
3. 55 g of disodium hydrogen phosphate and 3. 40 g of
potassium dihydrogen phosphate in 1000 ml of water, and
adjust pH to 2. O with phosphoric acid] (2 : 8) as the mobile
phase. The detector wavelength is 202 nm. Dissolve an
accurately weighed quantity of Methacrylate CRS, Ethyl
acrylate CRS and Methyl methacrylate CRS in methanol to
produce a reference solution of each 3 µg per ml. Inject
separately 20 µl each of the test solution and reference
solution into the column, and record the chromatograms.
The number of the theoretical plates calculated for the peak
of methacrylate is not less than 1000 and the resolution factor
between the peaks of ethyl acrylate and methyl methacrylate
complies with the related requirements. Calculate the content
of each monomer impurity separately with respect to the
peak area obtained in the chromatogram by the externa! standard
method. The sum of contents is not more than O. 3 % .
Loss on drying When dried at llOºC for 6 hours, loses not
more than 5. O% of its weight ( 0831).
Residue on ignition Not more than O. 3% ( 0821 ) , using
l. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2) , using the residue obtained in the test for
Residue on ignition; not more than O. 003%.
Arsenic Transfer l. O g in a 150 ml conical flask, add 5 ml
of sulfuric acid, heat until it is completely charred, add
concentrate hydrogen peroxide solution dropwise ( if a lot of
foam is evolved stop heating and rotate the conical flask to
prevent the unreacted substance conglomerating at the
bottom) until the solution is colourless. Allow to cool, add
is evolved, cool and add slowly 5 ml of hydrochloric acid and
a quantity of water to produce 28 ml. Carry out the limit test
for arsenic ( 0822 method 1 ) : not more than O. 0002 % .
Category Pharmaceutical excipients, coating material and
release retardant, etc.
Storage Preserve in tightly closed cintainers, stored in a
cool place.
Methacrylic Acid Copolymer Il
Methacrylic acid copolymer 11 is a copolymer of methyl
methacrylate, ethyl acrylate and trimethyl ammonium
Chloride ethyl methacrylate in a ratio of 65 : 30 : 5.
Description Almost white, translucent or transparent salid
with different shape and size.
Sparingly soluble in acetone, practically insoluble in boiling
water or in isopropanol.
Refractive index Dissolve l. 25 g in 10 ml of isopropanol-
acetone ( 6 : 4) , the refractive index is l. 380-1. 385 ( 0622).
Alkaline value Dissolve l. O g, previously dried to constant

weight at llOºC (far about 5 hours) and accurately weighed,
in 25 ml of dichloromethane, add 50 ml of glacial acetic acid
and 5 ml of mercuric acetate TS, mix well and add 3 drops of
quinaldine red IS, ti trate with perchloric acid (O. 1 mol/U
VS until the colour of the solution changes from red to
colourless. Perfarm a blank determination and make nec-
essary correction. Each ml of perchloric acid (O. 1 mol/L)
VS is equivalent to 5. 61 rng of KOH. The value is 12. 1-18. 3
rng/ g, calculated on the dried basis.
ldentification The infrared absorption spectrum ( 0402 ) is
concordant with the spectrum of Methacrylic acid copolymer
II CRS.
Viscosity Dissolve 6. O g in a 100 ml of 75% ethanol
solution, the kinematic viscosity at 20ºC is not more than
O. 015 Pa • s, using a rotational type of viscometer with a
No. O rotator ( 0633 method 2) , at the speed of 30 rpm.
Related substance Dissolve a quantity of the substance being
examined, accurately weighed, in methanol to produce a
solution of 1 mg per ml as the test solution. Carry out the
method for high performance liquid chromatography ( 0512),
using a column packed with octadecylsilane bonded silica gel
anda mixture of methanol-phosphate BS [dissolve 3. 55 g of
disodium hydrogen phosphate and 3. 40 g of potassium
dihydrogen phosphate in 1000 ml of water, and adjust pH to
2. O with phosphoric acid] (2 : 8) as the mobile phase. The
detector wavelength is 202 nm. Dissolve an accurately
weighed quantity of Methacrylate CRS, Ethyl acrylate CRS
and Methyl methacrylate CRS in methanol to produce a
reference solution of each 3 µg per ml. Inject separately 20 µl
each of the test solution and reference solution into the
column, and record the chromatograms. The number of the
theoretical plates calculated far the peak of methacrylate is
not less than 1000 and the resolution factor between the
µeaks of ethyl acrylate and methyl methacrylate complies
with the related requirements. Calculate the content of each
monomer impurity separately with respect to the peak area
obtained in the chromatogram by the extemal standard
method. The sum of contents is not more than O. 3 %.
Loss on drying When dried at llOºC far 6 hours, loses not
more than 5. O% of its weight ( 0831 ) .
Residue on ignition Not more than O. 3% ( 0841 ) , using
l. o g.
Heavy metals Carry out the limit test far heavy metals
( 0821 method 2) , using the residue obtained in the test for
Residue on ignition; not more than O. 003%.
Arsenic Transfer l. O g in a 150 ml conical flask, add 5 ml
of sulfuric acid, heat until it is completely charred, add
concentrate hydrogen peroxide solution dropwise ( if a lot of
faam is evolved stop heating and rotate the conical flask to
prevent the unreacted substance conglomerating at the
bottom) until the solution is colourless. Allow to cool, add
cautiously 10 ml of water, heat again until the sulfur trioxide
is evolved, cool and add slowly 5 ml of hydrochloric acid and
a quantity of water to produce 28 ml. Carry out the limit test
for arsenic ( 0822 method 1 ) : not more than O. 0002 % .
Category Pharmaceutical excipients, coating material and
release retardant, etc.
Storage Preserve in tightly closed cintainers, stored in a
cool place.
Methylcellulose 1 (:'; <' '$93
Methylcellulose
[9004-67-5]
Methylcellulose is a methylether cellulose. It contains not
less than 27. O% and not more than 32. O% of methoxy
groups (-0CH 3 ) , calculated on the dried basis.
Description A white or almost white fibrous powder or
granular; odorless; tasteless.
Disperse and swell in water to produce a clear or slightly
opalescent colloidal solution; insoluble m dehydrated
ethanol, in chloroform and in ether.
ldentification ( 1) To 1 g, add 100 ml of boiling water,
stir to mix welL Cool the suspension in an ice bath to
produce a clear or slightly opalescent solution. Place a
quantity of the solution in a test tube, add 2 ml of a solution
of O. 035 % anthrone in sulfuric acid slowly along the inner
wall of the tube and allow to stand. A bluish-green ring is
farmed at the interface of the two layers.
(2) Heat a quantity of the solution obtained in Identification
(1), a cloudy or flocculent precipitate is formed. The
solution becomes clear again when cooling.
(3) Place a quantity of the solution obtained in Identification
(1) on a glass plate and evaporate the water, a thin and
elastic film is formed
(4) To O. 1 ml of the solution obtained in Identification (1),
add 9 ml of sulfuric acid solution (9-10), shake, heat in a
boiling water bath far 3 minutes, and immediately cool in an
ice bath. Add O. 6 mi of O. 2 % ninhydrin solution, allow to
stand at 25ºC, the solution shows a red colour, and it <loes
not change to purple in 100 minutes.
(5) Add 50 mi of the solution obtained in Identification (1)
to 50 ml of water in a beaker. Insert a thermometer into the
solution. Stir the solution and begin heating, increasing the
temperature at a rate of 2-5ºC per minute. Determine the
temperature at which a turbidity increase begins to occur and
designate the temperature as the flocculation temperature,
the flocculation temperature is not lower than 50ºC.
Viscosity Weigh accurately a quantity of the substance being
examined equivalent to 4. O g of the dried substance. Add
196 g of water which is 90ºC and stir abundantly for 10
minutes. Continue the stirring in an ice bath for another 40
minutes. Adjust the solution mass if necessary to 200 g with
cold water. Adjust the temperature of the solution to 20 ±
O. 1 ºC, decompress or centrifuge the solution if necessary to
remove any air bubbles. Determine the kinetic viscosity
( 0633 method 1 ) , select a suitable viscosimeter with a
required intemal diameter of the capillary tube. The viscosity
is not less than 80 % and not more than 120 % of the value
stated on the label for samples with a nominal viscosity less
than 600 mPa • s; Weigh accurately a quantity of the
substance being examined equivalent to 10. O g of the dried
substance. Add 490 g of water which is 90ºC and stir
abundently for 10 minutes. Continue the stirring in an ice
bath for another 40 minutes. Adjust the solution mass if
necessary to 500 g with cold water. Adjust the temperature
of the solution to 20±0. 1 ºC. Determine the kinetic viscosity
( 0633 method 3 ) , using NDJ-1 rotating viscosimeter and
select the appropriate rotator and speed according to the
following table. The viscosity is not less than 75 % and not
more than 140% of the value stated on the label for samples
with a nominal viscosity not less than 600 mPa •s.
 594 Methylparaben

Norminal viscosity CRS in the chromatogram obtained in the Assay.

Rotor Revolution Calculation
(mPa • s) (2) Dissolve a quantity of the substance being examined in
number (r/min) multiplier
ethanol to produce a solution of 5 µg per ml, the light
600 to less than 1400 3 60 20 absorption of the solution exhibits a maximum at 258 nm
<0401>.
1400 to less than 3500 3 12 100 ( 3) The infrared absorption spectrum is concordant with the
reference spectrum (IR Album No. 853).
3500 to less than 9500 4 60 100
Acidity To 2 ml of the solution prepared under clarity and
not less than 9500 4 6 1000 colour of solution add 2 ml of ethanol and 5 ml of water, mix
well, add 2 drops of bromocresol green IS; not more than
Acidity or alkalinity Determine the pH of the solution O. 1 ml of sodium hydroxide (0. 1 mol/L) VS is required to
obtained in Viscosity, read the pH after the electrode has change the colour of the solution to blue.
been immersed in the solution far 5 ±O. 5 minutes, pH 5. 0-
8. o <0631>. Clarity and colour of solution Dissolve l. O g of the
substance being examined in 10 ml of ethanol, the solution is
Loss on drying When dried at 105ºC far 2 hours, loses not clear and colourless ( 0901 and 0902); any colour produced
more than 5. O% of its weight ( 0831 ) . is not more intense than that of reference solution Y 1 or YG1
Residue on ignition Not more than l. O% ( 0841 ) , using ( 0901 method 1 ) .
l. o g. Chloride Heat 2. O g of the substance being examined with
Heavy metals Carry out the limit test far heavy metals 50 ml of water at 80ºC far 5 minutes, cool, filter. Carry out
( 0821 method 2) , using the residue obtained in the test far the limit test far chlorides ( 0801 ) , using 5 ml of the
Residue on ignition: not more than O. 002% successive filtrate. Any opalescence produced is not more
pronounced than that of a reference solution using 7. O ml of
Arsenic Mix l. O g with l. O g of calcium hydroxide, add
sodium chloride standard solution (O. 035 % ) .
water and stir well. After dryness, heat gently until it is
thoroughly charred, and then ignite at 500-600ºC until the Sulfate Carry out the limit test far sulfates ( 0802) , using
incineration is completed. Dissolve the cooled residue in a 25 ml of the successive filtrate obtained in the test far
mixture of 8 ml of hydrochloric acid and 23 ml of water. chloride. Any opalescence produced is not more pronounced
Carry out the limit test far arsenic ( 0822 method 1 ) : not than that of a reference solution using 2. 4 ml of potassium
more than O. 0002 % . sulfate standard solution (0. 024%).
Assay Methoxy group To a quantity of the substance Related substances Dissolve a quantity of the substance
being examined, accurately weighed. Carry out the method being examined in the mobile phase to produce a solution of
far determination of methoxy, ethoxy and hydroxypropoxy 1 mg per ml as test solution; measure accurately 1 ml of the
( 0712). Calcula te the content of -OCH 3 • test solution, to a 100 ml volumetric flask, dilute to volume
with the mobile phase, mix well, as reference solution.
Category Pharmaceutical excipients, binder and suspending
Carry out the method as described under the Assay, inject
agent.
20 µl of the reference solution into the column and adjust the
Storage Preserve in well closed containers. attenuation so that the principal peak height in the
Labeling The labeling states the viscosity with the unit chromatogram is about 25 % of full scale of the chart, then
of mPa • s or Pa • s. inject separately 20 µl of the test solution and reference
solution into the column, record the chromatogram far 4
times the retention time of the principal peak. The area of
any impurity peaks in the chromatogram is not greater than
O. 4 times the area of the principal peak in the chromatogram
Methylparaben obtained with the reference solution (O. 4%), the sum of the
areas of all impurity peaks is not greater than O. 8 times area
of the principal peak in the chromatogram obtained with the

~OCH3 reference solution (0. 8%).

HO)l)
Cs Hs Ü3 152. 15
[99-76-3]
Methylparaben is methyl 4-hydroxybenzoate, esterified by
methanol and p-hydroxybenzoic acid. It contains not less
than 98.0% and not more than 102.0% of CsH 8 0 3 ,
calculated on the dried basis.
Description White or almost white crystals or a crystalline
powder.
Freely soluble in methanol, in ethanol and in ether; soluble
in hot water; slightly soluble in water.
Melting point 125-128ºC <0612).
ldentification ( 1) The retention time of the principie peak
of the substance being examined in the chromatogram is
identical with that of the principal peak of methylparaben
Methanol Dissolve a quantity, accurately weighed, in
N, N-dimethylfarmamide, shake well to produce the
solution of O. 1 g per ml as test solution; dissolve a quantity
of methanol, accurately weighed, in N, N-dimethy-
lformamide to produce the solution of O. 3 mg per ml as
reference solution. Carry out the method far residual solvent
( 0861 method 3 ) , using a capillary column packed with
100 % dimethyl polysiloxane as stationary phase; the
temperature increased from 40ºC to 80ºCat arate of 15ºC per
minute, and maintained far 5 minutes; then increased at a
rate of 6ºC per minute to 130ºC, and maintained for 1
minute; then increased at a rate of 40ºC per minute to
220ºC, and maintained far 3 minutes; the temperature of
injection port is 200ºC; the temperature of detector is 250ºC.
Inject 1 µl of the reference solution onto the column, the
resolution factor between each pair of adjacent peaks in the
chromatogram obtained complies with the requirement.
Inject 1 µl of the reference solution and the test solution

accurately measured into the column respectively, record the
chromatogram and calculate by the externa! standard
method, not more than O. 3%.
Loss on drying When dried in vacuum in a desiccators
containing silica gel to constant weight, loses not more than
O. 5 % of its weight <0831).
Residue on ignition Not more than O. 1 % <0841 ) , using
l. o g.
Heavy metals Carry out the limit test far heavy metals
<0821 method 2), using the residue obtained in the test far
residue on ignition; not more than O. 002 % .
Arsenic Mix l. O g of the substance being examined with
l. O g of calcium hydroxide, add a small volume of water and
mix well. Dry, heat gently until it is thoroughly charred,
then ignite at 500-600ºC until the incineration is complete,
cool, add 5 ml of hydrochloric acid and 23 ml of water. Carry
out the limit test far arsenic <0822 method 1 ) , not more
than O. 000 2%.
Assay Carry out the method far high performance liquid
chromatograph < 0512 ) , using a column packed with
octadecylsilane bonded silica gel and a mixture of methanol-
1 % glacial acid solution ( 60 : 40) as the mobile phase.
Detection wavelengnth is 254 nm. Dissolve a quantity of
methylparaben and ethylparaben in the mobile phase to
produce a mixture solution containing 10 µg each of the two
substances per ml. Inject 20 µl of the mixture solution into
the column and record the chromatogram. The resolution
factor between peaks of methylparaben and ethylparaben
complies with the requirements.
Procedure Dissolve a quantity, accurately weighed, in the
mobile phase to produce the test solution of O. 1 mg per ml.
lnject 20 µl of the resulting solution, accurately measured,
onto the column and record the peak areas corresponding
obtained in the chromatogram. Accurately weigh methyl-
paraben CRS and repeat the operation. Calculate the contents
of Cs H 8 0 3 with repect to the peak area obtained in the
chromatogram by the external standard method.
Category Pharmaceutical excipients, presercative.
Storage Preserve in well closed containers.
Microcrystalline Cellulose
OH
OH
H n/2
C6n H1on+Z Dsn+ 1
[9004-34-6]
Microcrystalline Cellulose is purified, partially depolymerized
cellulose prepared by treating alpha cellulose, obtained as a
pulp from fibrous plant material, with mineral acids.
Description A white or almost white powder or granular;
odourless; tasteless.
Practically insoluble in water, in ethanol, in ether; in dilute
sulfuric acid and in 5 % sodium hydroxide.
Microcrystalline Cellulose 515
ldentification ( 1) To 1O mg; add 2 ml zinc chloride iodine
TS. A blue colour is produced.
(2) Weigh accurately l. 3 g into an iodine flask. Accurately
add 25 ml of water and shake to disperse and humidify
microcrystalline cellulose. Inject nitrogen to exhaust the air
in the flask. Accurately add 25 ml of 1 mol/L cupriethy-
lenediamine hydcoxide solution on the premise of keeping
nitrogen injection. Remove the nitrogen pipe and sealing plug
and strongly shake to dissolve microcrystalline cellulose; use
the solution as the test solution. Transfer a volume of
solution into a 25±0. 1 ºC water bath. About 5 minutes
later, transfer it into an Ubbelohde viscometer (capillary ID
O. 7-1. O mm, selection of appropriate viscometer constant
K1). Measure the viscosity in the 25 ± O. 1 ºC water bath
<0633 method 2). Record the time t 1 when the test solution
flows through the two scales such as upper and lower scales
of the viscometer. Calculate the kinematic viscosity v1 of the
test solution as per the following formula.
v1=t1XK1
Measure accurately 25 ml of water and 25 ml of 1 mol/L
cupriethylenediamine hydroxide solution separately, mix to
uniformity, and use the solution as the blank solution. Take
an appropriate quantity of solution and place it into a
25ºC ±O. 1 ºC water bath. About 5 minutes later, transfer it into
an Ubbelohde viscometer (capillary ID O. 5-0. 6 mm, viscometer
constant Kz about O. 01). Measure the viscosity in the 25±0. 1ºC
water bath <0633 method 2). Record the time t 2 when the blank
solution flows through the two scales such as upper and lower
scales of the viscometer. Calculate the kinematic viscosity vz of
the blank solution using the following formula:
vz =tz XKz
Calculate the relative viscosity of microcrystalline cellulose
using the following formula:
1Jrel = Vl / Vz.
According to the calculated relative viscosity (lJrel), refer to
the attached table to obtain the value of [ 1J J c {product of
characteristic viscosity [1)] (ml/g) and concentration c (g/
100 ml) } . Calcula te the degree of polymerization ( P)
using the following formula, which is not more than 350.
P = 95 C1JJ c
m
Where m is the test product sample quantity (g), which is
calculated as dry product.
Acidity or alkalinity Take the supernatant liquid prepared
under the Conductivity; pH 5. 0-7. 5 < 0631 ) .
Chloride Dissolve O. 10 g in 35 ml of water; shake and
filtrate the solution; take the filtrate; carry out the limit test
far chloride <0801 ) . Any opalescence produced is not more
pronounced than that of a reference solution using 3. O ml
standard sodium chloride solution (O. 03%).
Water-soluble substance Dissolve 5. O g in 80 ml of water
and shake far 10 minutes, allowed to stand at room
temperature far 10-20 minutes. Filter through a microporous
membrane (pare size is not more than 2 µm) or a
quantitative analysis filter paper with the aid of vacuum.
Evaporate the filtvate on a water bath in a constant weight of
evaporating dish. Dry at 105ºC far 1 hour; the residue is not
more than O. 2%.
Ether-soluble substance Place 10. O g in a column about
20 mm in internal diameter and pass 50 ml of peroxide-free
ether through the volumn. Evaporate the elute in a constant
weight evaporating dish. Dry the residue at 105ºC to
constant weight. The residue is not more than O. 05 %.
Starch Dissolve O. 10 g with 5 ml of water and shake; add
O. 2 ml iodine TS. No blue colour is produced.
 •. ••. !B6 · :.1 Microcrystalline Cellulose

Conductivity Dissolve 5. O g with 40 ml of newly boiled cold
water ( room temperature) and shake for 20 minutes.
Centrifugation and take supernatant, measure the cond-
uctivity at 2ºC ±0. 1 ºC <0681 ) , measure the conductivity of
the water used to prepare the test solution too. The
difference: not more than 75 µS/ cm.
Los.son drying When dried at 105ºC for 3 hours, loses not
more than 7. O% of its weight <0831 ) , using l. O g.
Residue on ignition Not more than O. 1% <0841), using l. O g.
ArsenicMix l. O g with l. O g of calcium hydroxide and a
volume of water, stir well and dry. Heat gently until
completely charred; and then ignite it at 600ºC till the
incineration is complete, allow to cool, add 5 ml hydrochloric
acid and 23 ml water to dissolve the residue; carry out the
limit test for arsenic < 0822 method 1 ) : not more than
o. 0002%.
Category Pharmaceutical excipients, filler, disintegrant
agent, etc.

Heavy rnetals Carry out the limit test for heavy metals Storage Preserve in tightly closed containers.
<0821 method 2) , using the residue obtained in the test for Labeling State the type of product.
Residue on ignition: not more than O. 001%.
Table Conversion table of relative viscosity( 11 re1 )and product of characteristic viscosity and concentration( [11]C)
[r¡]C
r¡ rel
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 o. 08 o. 09
l. 1 0.098 o. 106 o. 115 o. 125 o. 134 o. 143 o. 152 o. 161 o. 170 o. 180
l. 2 o. 189 o. 198 0.207 0.216 0.225 0.233 0.242 0.250 o. 259 o. 268
l. 3 0.276 0.285 0.293 0.302 0.310 0.318 0.326 0.334 o. 342 o. 350
l. 4 0.358 0.367 0.375 0.383 0.391 0.399 0.407 0.414 o. 422 o. 430
l. 5 0.437 0.445 0.453 0.460 0.468 0.476 0.484 0.491 o. 499 o. 507
l. 6 o. 515 0.522 0.529 0.536 0.544 0.551 0.558 0.566 o. 573 o. 580
l. 7 o. 587 0.595 0.602 o. 608 0.615 0.622 0.629 0.636 o. 642 o. 649
l. 8 0.656 0.663 0.670 0.677 0.683 o. 690 0.697 0.704 o. 710 o. 717
l. 9 o. 723 0.730 o. 736 o. 743 o. 749 o. 756 o. 762 o. 769 o. 775 o. 782

2.0 o. 788 0.795 0.802 0.809 0.815 0.821 0.827 0.833 o. 840 o. 846
2. 1 0.852 0.858 0.864 0.870 0.876 0.882 0.888 0.894 o. 900 o. 906
2. 2 0.912 0.918 0.924 0.929 0.935 0.941 0.948 0.953 o. 959 o. 965
2.3 o. 971 0.976 0.983 0.988 0.994 l. 000 l. 006 l. 011 l. 017 l. 022
2.4 l. 028 l. 033 l. 039 1.044 l. 050 l. 056 l. 061 l. 067 l. 072 l. 078
2.5 l. 083 l. 089 l. 094 l. 100 l. 105 l. 111 l. 116 l. 121 l. 126 l. 131
2. 6 l. 137 l. 142 l. 147 l. 153 l. 158 l. 163 l. 169 l. 174 l. 179 l. 184
2. 7 l. 190 l. 195 l. 200 l. 205 l. 210 l. 215 l. 220 l. 225 l. 230 l. 235
2.8 l. 240 l. 245 l. 250 l. 255 l. 260 l. 265 l. 270 l. 275 l. 280 l. 285
2.9 l. 290 l. 295 l. 300 l. 305 l. 310 l. 314 l. 319 l. 324 l. 329 l. 333

3.0 l. 338 l. 343 l. 348 l. 352 l. 357 l. 362 l. 367 l. 371 l. 376 l. 381
3.1 l. 386 l. 390 l. 395 l. 400 l. 405 l. 409 l. 414 l. 418 l. 423 l. 427
3. 2 l. 432 l. 436 l. 441 l. 446 l. 450 l. 455 l. 459 l. 464 l. 468 l. 473
3. 3 l. 477 l. 482 l. 486 l. 491 l. 496 l. 500 l. 504 l. 508 l. 513 l. 517
3.4 l. 521 l. 525 l. 529 l. 533 l. 537 l. 542 l. 546 l. 550 l. 554 l. 558
3.5 l. 562 l. 566 l. 570 l. 575 l. 579 l. 583 l. 587 l. 591 l. 595 l. 600
3.6 l. 604 l. 608 l. 612 l. 617 l. 621 l. 625 l. 629 l. 633 l. 637 l. 642
3. 7 l. 646 l. 650 l. 654 l. 658 l. 662 l. 666 l. 671 l. 675 l. 679 l. 683
3. 8 l. 687 l. 691 l. 695 l. 700 l. 704 l. 708 l. 712 l. 715 l. 719 l. 723
3.9 l. 727 l. 731 l. 735 l. 739 l. 742 l. 746 l. 750 l. 754 l. 758 l. 762

4.0 l. 765 l. 769 l. 773 l. 777 l. 781 l. 785 l. 789 l. 792 l. 796 l. 800
4. 1 1.804 1.808 l. 811 l. 815 l. 819 l. 822 l. 826 l. 830 l. 833 l. 837
 Microcrys talline Cell ulose

continued
[r¡]C
1/ re!
o. 00 o. 01 o. 02 o. 03 o. 04 o. 05 o. 06 o. 07 o. 08 o. 09
4.2 1.841 1.845 1.848 1.852 1.856 1.859 1.863 1.867 1.870 1.874
4.3 l. 878 l. 882 l. 885 l. 889 l. 893 l. 896 l. 900 l. 904 l. 907 l. 911
4.4 1.914 1.918 1.921 1.925 1.929 1.932 1.936 1.939 1.943 1.946
4.5 1.950 1.954 1.957 1.961 1.964 1.968 1.971 1.975 1.979 1.982
4.6 1.986 l. 989 l. 993 l. 996 2.000 2.003 2.007 2.010 2.013 2.017
4. 7 2.020 2.023 2.027 2.030 2.033 2.037 2.040 2.043 2.047 2.050
4.8 2.053 2.057 2.060 2.063 2.067 2.070 2.073 2.077 2.080 2.083
4.9 2.087 2.090 2.093 2.097 2. 100 2. 103 2. 107 2. 110 2. 113 2. 116

5. o 2. 119 2. 122 2. 125 2. 129 2. 132 2. 135 2. 139 2. 142 2. 145 2. 148
5. 1 2. 151 2. 154 2. 158 2. 160 2. 164 2. 167 2. 170 2. 173 2. 176 2. 180
5.2 2. 183 2. 186 2. 190 2. 192 2. 195 2. 197 2.200 2.203 2.206 2.209
5. 3 2.212 2.215 2. 218 2.221 2. 224 2.227 2.230 2.233 2. 236 2.240
5.4 2.243 2.246 2.249 2.252 2.255 2.258 2.261 2.264 2.267 2.270
5.5 2.273 2. 276 2. 279 2. 282 2. 285 2. 288 2.291 2. 294 2.297 2. 300
5.6 2.303 2.306 2.309 2.312 2.315 2.318 2.320 2.324 2.326 2.329
5. 7 2. 332 2. 335 2. 338 2. 341 2. 344 2. 34 7 2. 350 2. 353 2. 355 2. 358
5.8 2.361 2.364 2. 367 2.370 2.373 2.376 2. 379 2. 382 2. 384 2. 387
5.9 2.390 2.393 2.396 2.400 2.403 2.405 2.408 2.411 2.414 2.417

6.0 2.419 2.422 2.425 2.428 2.431 2.433 2.436 2.439 2.442 2.444
6. 1 2.447 2.450 2.453 2.156 2.458 2.461 2.464 2.467 2.470 2.472
6. 2 2.475 2.478 2.481 2.483 2.486 2.489 2.492 2.494 2.497 2.500
6.3 2.503 2.505 2.508 2.511 2.513 2.516 2.518 2.521 2.524 2.526
6.4 2.529 2.532 2.534 2.537 2. 540 2.542 2.545 2.547 2. 550 2. 553
6.5 2.555 2.558 2.561 2.563 2.566 2.568 2.571 2.574 2.576 2.579
6. 6 2. 581 2. 584 2. 587 2.590 2. 592 2.595 2. 597 2. 600 2. 603 2. 605
6. 7 2.608 2. 610 2. 613 2.615 2. 618 2.620 2. 623 2.625 2. 627 2. 630
6.8 2.633 2.635 2.637 2.640 2.643 2.645 2.648 2.650 2.653 2.655
6. 9 2.658 2. 660 2.663 2. 665 2. 668 2.670 2. 673 2.675 2. 678 2. 680

7.0 2. 683 2. 685 2. 687 2.690 2. 693 2. 695 2. 698 2. 700 2. 702 2. 705
7. 1 2.707 2. 710 2.712 2.714 2.717 2. 719 2. 721 2. 724 2. 726 2. 729
7. 2 2. 731 2. 733 2. 736 2. 738 2. 740 2. 743 2. 745 2. 748 2. 750 2. 752
7. 3 2. 755 2. 757 2. 760 2. 762 2. 764 2. 767 2. 769 2. 771 2. 774 2. 776
7.4 2. 779 2. 781 2.783 2.786 2. 788 2.790 2. 793 2. 795 2. 798 2.800
7.5 2.802 2.805 2.807 2.809 2.812 2.814 2.816 2.819 2.821 2.823
7. 6 2.826 2.828 2.830 2.833 2.835 2.837 2.840 2.842 2.844 2.847
7. 7 2.849 2.851 2.854 2.856 2.858 2.860 2.863 2.865 2.868 2.870
7.8 2.873 2.875 2.877 2.879 2.881 2.884 2.887 2.889 2.891 2.893
7. 9 2.895 2.898 2.900 2.902 2. 905 2.907 2. 909 2. 911 2.913 2. 915

8.0 2.918 2.920 2.922 2.924 2.926 2.928 2.931 2.933 2.935 2.937
 Microcrystalline Wax

continued
[r¡]C
1J rel
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
8. 1 2.939 2.942 2.944 2. 946 2.948 2. 950 2.952 2.955 2.957 2.959
8.2 2.961 2.963 2. 966 2.968 2.970 2.972 2.974 2. 976 2. 979 2.981
8.3 2.983 2. 985 2.987 2.990 2.992 2.994 2.996 2.998 3.000 3.002
8.4 3.004 3.006 3.008 3.010 3.012 3.015 3.017 3.019 3.021 3.023
8.5 3.025 3.027 3.029 3.031 3.033 3.035 3.037 3.040 3.042 3.044
8. 6 3.046 3.048 3.050 3.052 3.054 3.056 3.058 3.060 3.062 3.064
8. 7 3.067 3.069 3.071 3.073 3.075 3. 077 3.079 3.081 3.083 3.085
8.8 3.087 3.089 3.092 3.094 3.096 3.098 3. 100 3.102 3. 104 3. 106
8.9 3. 108 3. 110 3.112 3. 114 3. 116 3. 118 3. 120 3. 122 3. 124 3. 126

9.0 3. 128 3. 130 3. 132 3. 134 3. 136 3. 138 3. 140 3. 142 3. 144 3. 146
9. 1 3. 148 3. 150 3. 152 3. 154 3. 156 3. 158 3. 160 3. 162 3. 164 3. 166
9. 2 3. 168 3. 170 3. 172 3. 174 3. 176 3. 178 3. 180 3.182 3. 184 3. 186
9. 3 3. 188 3. 190 3. 192 3. 194 3. 196 3. 198 3.200 3.202 3.204 3.206
9.4 3.208 3.210 3.212 3.214 3.215 3.217 3.219 3.221 3.223 3.225
9.5 3.227 3.229 3.231 3. 233 3.235 3.237 3.239 3.241 3.242 3.244
9.6 3.246 3.248 3.250 3.252 3.254 3.256 3.258 3.260 3.262 3. 264
9. 7 3.266 3.268 3.269 3.271 3.273 3.275 3.277 3.279 3.281 3.283
9. 8 3.285 3.287 3.289 3.291 3.293 3.295 3.297 3.298 3. 300 3. 302
9. 9 3.304 3.305 3.307 3.309 3. 311 3.313 3.316 3.318 3. 320 3.321

10 3.32 3. 34 3.36 3.37 3.39 3.41 3.43 3.45 3.46 3.48

11 3.50 3. 52 3.53 3.55 3.56 3.58 3.60 3.61 3.63 3.64
12 3.66 3.68 3.69 3. 71 3. 72 3. 74 3. 76 3. 77 3. 79 3.80
13 3.80 3.83 3.85 3.86 3.88 3.89 3.90 3.92 3.93 3. 95
14 3.96 3.97 3.99 4.00 4.02 4.03 4.04 4.06 4.07 4.09
15 4. 10 4.11 4. 13 4. 14 4. 15 4. 17 4. 18 4. 19 4.20 4.22
16 4.23 4.24 4.25 4. 27 4.28 4.29 4.30 4.31 4. 33 4.34
17 4.35 4.36 4.37 4.38 4.39 4.41 4.42 4.43 4.44 4.45
18 4.46 4.47 4.48 4.49 4.50 4.52 4.53 4.54 4.55 4.56
19 4.57 4.58 4.59 4.60 4.61 4.62 4.63 4.64 4. 65 4.66

sulfur, heat, hydrogen sulfide gas will be produced, import

Microcrystalline Wax
[63231-60-7]
Microcrystalline Wax is a mixture of straight-chain,
branched-chain, and cyclic hydrocarbons obtained from
petroleum.
Description White or almost white waxy solid, odourless.
Freely soluble in chloroform and in ether, slightly soluble in
absolute ethanol, insoluble in water.
Melting range 54-102ºC <0612 method 2).
Identification ( 1 ) Melt and bum a quantity of the
substance of being examined, a luminous flame with a smell
of oil flavor is formed.
(2) Mix about O. 5 g in a dry test tube with O. 5 g sublimed
the gas in 50 ml of lead acetate TS, the colour of the solution
gradually change from colourless to dark.
Acidity or alkalinity Place 35. O g in a 250 ml of separating
funnel, add 100 ml of boiled water, shake thorougly for
5 minutes, allow to stand, rinse with 50 ml of boiled water
for two times, combine the water layer, add 1 drop of
phenolohthalein IS, boiled, the solution is colourless; add
1 ml of methyl orange IS, no pink colour is produced.
Colour Melt about 10 g on water bath, any colour produced
by the melted liquid is not more intense than that of a
reference preparation (mix l. 2 ml of standard cobalt chloride
es, l. 8 ml of standard potassium dichromate es, and 2 ml
of water).
Organic acids Place 20. O g in 250 ml conical flask, add
100 ml of neutral dilute ethanol, heat under reflux for 10
minutes, add 1 ml of phenolphthalein IS; not more than

O. 4 ml of sodium hydroxide (O. 1 mol/L) VS is required to
change the colour of the solution to pink.
Fats and fatty oils or resins To 10. O g add 50 ml of 20 %
sodium hydroxide solution, and heat under reflux for 30
minutes. Cool, collect the water layer. To the water layer
add 200 ml of dilute sulfuric acid: neither oily matter nor
precipitate is produced.
Ash Ignite slowly about l. O g, accurately weighted, in a
crucible, previously ignited to constant weight, until the
substance being examined is completely carbonized, raise the
temperature gradually to 500-600ºC, incinerate completely to
constant weight, The residual ash is not more than O. 1%.
Heavy metals Ignite slowly 2. O g, in a crucible, until the
substance being examined is completely carbonized, raise the
temperature gradually to 450-550ºC, incinerate completely.
Cool, add 2 ml of hydrochloric acid, evaporate to dryness on
a water bath, add 2 ml of acetic acid and 15 ml of water.
Carry out the limit test for heavy metals <0821 method 2):
not more than O. 001%.
Category Pharmaceutical excipients, Coating, controlled
release carrier, etc.
Storage Preserve in tighely closed containers, protected
from light.
Nicotinamide
C6H6N20 122.13
[98-92-0]
Nicotinamide is 3-pyridinecarboxamide. It contains not less
than 99. 0% of C6H 6Nz0, calculated on the dried basis.
Description A white crystalline powder; odourless or
almost odourless; taste, bitter, slightly hygroscopic.
Freely soluble in water or in ethanol; soluble in glycerin.
Melting point 128-131 ºC <0612).
ldentification (1) Dissolve O. 1 g in 5 ml of water, add
5 ml of sodium hydroxide TS, heat gently, an ammonia gas
is evolved which turns moistened red litmus TP blue
(distinction from nicotinic acid); heat continuously until the
ammonia gas is removed, cool, add 1-2 drops of phenophthalein
IS, neutralize with dilute sulfuric acid, then add 2 rnl of cupric
sulfate TS; a light blue precipitate is produced.
(2) The light absorption of a solution of about 20 µg per ml
exhibits a maximum at 261 nm and a minimum at 245 nm;
the ratio of the absorption at 245 nm to that at 261 nm is
o. 63-0. 67 <0401).
(3) The infrared absorption spectrum <0402 > is concordant
with the reference spectrum CIR Album No. 421).
Acidity and alkalinity Dissolve l. O gin 10 ml of water, pH
5. 5-7. 5 <0631>.
Clarity and colour of solution Dissolvel. O g in 10 ml of
water, the solution is clear and colourless <0901 and 0902).
Readily carbonizablesubstances Carry out the limit test for
readily carbonizable substances < 0842), using O. 2 g; any
colour produced is not more intense than that of a reference
preparation by using 5 ml of mixture (mix l. O ml of standard
cobalt chloride es, 2. 5 ml of standard potassium dichromate
es, and 1 ml standard copper sulfate es, with water to
produce 50 ml).
Related substances Carry out the method for thin-layer
Nicotinic Acid 599
chromatography <0502 ) , using silica GF2s4 as the coating
substance and a mixture of chloroform-dehydrated ethanol-
water (48: 45 : 4) as the mobile phase. Apply separately to
the plate 5 µl each of five solutions in ethanol containing
(1) 40 mg, (2) O. 2 mg, (3) O. 1 mg of the substance being
examined per ml, (4) O. 2 mg of nicotonic acid CRS per ml,
and ( 5) a mix of O. 2 mg of nicotonic acid CRS per ml and
1 ml of the substance being examined per ml. After
developing and removal of the plate, dry it in air and examine
under ultra-violet light (254 nm). Two well separated clear
spots should be detected in the chromatogram obtained with
solution ( 5) , a spot is clearly visible in the chromatogram
obtained with solution (3). The spot due to nicotonic acid in
the chromatogram obtained with solution ( 1) is not more
intense than the principal spot obtained with solution ( 4) and
any other secondary spot is not more intense than the
principal spot obtained with solution (2) (O. 5 %).
Loss on drying When dried under reduced pressure over
phosphorous pentoxide for 18 hours, loses not more than
O. 5 % of its weight <0831 ) .
Residue on ignition Not more than O. 1 % <0841 ) .
Heavy metals Dissolve l. O g in 10 ml of water, add 6 ml of
hydrochloric acid solution ( 1 mol/L) and water to 25 ml.
Carry out the limit test for heavy metals ( 0821 method 1 >:
not more than O. 002%.
Arsenic Dissolve l. O g in 5 ml of hydrochloric acid and
23 ml of water. Carry out the limit test for arsenic ( 0822
method 1 ) : not more than O. 0002 % .
Assay Dissolve O. 1 g, accurately weighed, in 20 ml of
glacial acetic acid, add 5 ml of acetic anhydride and 1 drop of
crystal violet IS, titrate with perchloric acid (O. 1 mol/L)
VS, until a bluish green colour is produced. Perform a blank
determination and make any necessary correction. Each ml of
perchloric acid (0. 1 mol/L) VS is equivalent to 12. 21 mg of
C6H6Nz0.
Category Pharmaceutical excipients, cosolvent and stabilizer.
Storage Preserve in well closed containers, protected from
light.
Nicotinic Acid
o
d'OH
N
C6 Hs N02 123. 11
[59-67-6]
Nicotinic Acid is 3-pyridine carboxylic acid. It contains not
less than 99. O% of C6 Hs N0 2 , calculated on the dried basis.
Description A white crystal or crystalline powder;
odourless or with a slight odour; taste, feebly acid; aqueous
solution yields an acid reaction.
Soluble in boiling water or in boiling ethanol; sparingly
soluble in water: slightly soluble in ethanol; practically
insoluble in ether; freely soluble in sodium carbonate TS or
in sodium hydroxide TS.
ldentification (1) Dissolve 50 mg in 20 ml of water, add
O. 4% sodium hydroxide solution dropwise, until the solution
is neutral to litmus paper, add 3 ml of cupric sulfate TS; a
pale blue precipitate is produced slowly.
 600 Oleoyl Macrogolglycerides
(2) The light absorption of a solution of 20 µg per ml
exhibits a maximum at 262 nm and a minimum at 237 nm,
the ratio of the absorbance at 237 nm to that at 262 nm is
o. 35-0. 39 <0401 ) .
( 3) The infrared absorption spectrum <0402 ) is concordant
with the reference spectrum CIR Album No. 422).
Colour of Solution Carry out the test for the colour of
solution < 0901 ). Dissolve l. O g in 10 ml of sodium
hydroxide TS, the solution is not more intensely coloured
than an equal volume of a reference solution prepared by
mixing l. 5 ml of cobaltous chloride es, 17 ml of potassium
dichromate es, l. 5 ml of cupric sulfate es, and dilute with
water to 1000 ml.
3-cyanopyridine Carry out the method for thin-layer
chromatography <0502 ) , using silica GF2s4 as the coating
substance and a mixture of toluene-chloroform-acetic ether-
glacial acetic acid ( 7. 5 : 5 : 2 : O. 5) as the mobile phase.
Apply separately to the plate 40 µl of test solution of the
substance being examined in ethanol containing 10 mg per
ml, and 5 µl of reference solution of 3-cyanopyridine CRS in
ethanol containing O. 2 mg per ml. After developing and
removal of the plate, dry it in air and examine under ultra-
violet light ( 254 nm). The spot due to 3-cyanopyridine in
chromatogram obtained with the test solution is not more
intense than the principal spot obtained with the reference
solution (O. 25%).
Chloride Carry out the limit test for chlorides < 0801 ) ,
using O. 25 g. Any opalescence produced is not more
pronounced than that of a reference solution using 5. O ml of
sodium chloride standard solution (0. 02%).
Sulfate Carry out the limit test for sulfate <0802) , using
O. 50 g. Any opalescence produced is not more pronounced
than that of a reference solution using l. O ml of potassium
sulfate standard solution (0. 02%).
Loss on drying When dried in vacuum over phosphorous
pentoxide to constant weight, loses not more than O. 5% of
its weight <0831).
Residue on ignition Not more than O. 1 % < 0841 ) ; using
l. o g.
Heavy metals Carry out the limit test for heavy metals
<0821 method 2) : using the residue obtained in the test for
Residue on ignition: not more than O. 002%.
Arsenic Dissol ve 1. O g in 5 ml of hydrochloric acid and
23 ml of water. Carry out the limit test for arsenic <0822
method 1): not more than O. 0002%.
Assay Dissolve about O. 3 g, accurately weighed, in 50 ml
of freshly boiled and cooled water, add 3 drops of
phenophthalein IS, ti trate with sodium hydroxide (O. 1 mol/L)
VS. Each ml of sodium hydroxide ( O. 1 mol/L) VS is
equivalent to 12. 31 mg of C6 Hs N02.
Category Pharmaceutical excipients, cosolvent and carrier.
Storage Preserve in tightly closed containers.
Oleoyl Macrogolglycerides
Oleoyl Macrogolglycerides are mixtures of monoesters,
diesters and trimesters of glycerol and monoesters and
diesters of macrogol, obtained by partial alcoholysis of an
unsaturated oil and macrogols, or by esterification of glycerol
and macrogol with unsaturated fatty acids, or by m1xmg
glycerol esters and condensates of ethylene oxide with the
fatty acids of the unsaturated oil. It may contain free
macrogolspolyethylene glycol, with a mean relative
molecular mass between 300 and 400.
Description A light yellow oily liquid. Very soluble in
dichloromethane, practically insoluble but dispersible in water.
Relative density O. 925-0. 955 <0601 ) .
Refractive index l. 465-1. 4 75 <0622).
Viscosity Kinematic viscosity 30-45 mm 2/ s, at 40ºC <0633
method 1 ) , using a capillary tube: l. 2 mm in interna!
diameter or other suitable capillary tube interna! diameter.
Acid value Not more than 2 <0713).
Saponification value 150-170 (0713).
Hydroxyl value 45-65 <0713).
lodinevalue 75-95 (0713).
Peroxide value Not more than 12 <0713).
ldentification ( 1) Dilute a quantity with dichloromethane
to produce a solution containing 50 mg per ml as test
solution. Carry out the thin-layer chromatography ( 0502),
using silica gel G as the coating substance and a mixture of
ether-hexane (7 : 3) as the mobile phase. Apply to the plate
10 µl of above solution, after developing and removal of the
plate, dry it in air, put it in the iodine vapor until spots are
clear. The chromatogram of test solution shows at least 5
completely separate spots, which is due to triglycerides with
an Rf value oí about O. 9, and spots due to 1, 3-diglycerides
( R' f O. 7) , to 1, 2-diglycerides ( R' f O. 6) , to monoglycerides
CR'f O. 1) and to esters of macrogol CR'f 0).
(2) The infrared absorption spectrum <0402) is concordant
with the spectrum of oleoyl macrogolglycerides CRS.
Alkaline impurities To 5. O g of the substance being
examined, accurately weighed, into a tube. Add 30 ml of
ethanol and heat to dissolve. Carry out the method for
potentiometric titration <0701 ) , titrate with hydrochloride
(0. 01 mol/L) VS. The volume consumed by hydrochloride
(0. 01 mol/L) VS is V (ml) and the weight of sample is W
(g). Calculate the content of alkaline impurities by following
equation: Not more than O. 008%.
. . .. % 40 X 10 X VX 10-6 %
Alka lme impunt1es o= X 100 o
w
Free glycerol Dissolve l. 2 g of the substance being
examined, in 25 ml of dichloromethane, heat if necessary.
Cool and add 100 ml of water, shaking while adding 25 ml of
sodium periodate acetic acid solution ( dissolve O. 446 g of
sodium periodate acetic acid in a 100 ml volume flask, add
2. 5 ml of 25 % sulfuric acid, dilute to volume with glacial
acetic acid and mix well). Allow to stand for 30 minutes, add
40 ml of a 75 g/L solution of potassium iodide and allow to
stand for Each minute. Add 1 ml of starch solution IS.
Ti trate the iodine with sodium thiosulfate (O. 1 mol/L)
VS. Perform a blank determination and make any necessary
correction. Each ml of sodium thiosulfate (O. 1 mol/L) VS is
equivalent to 2. 3 mg of glycerol: not more than 3. O%.
Ethylene oxide and dioxane W eigh accurately 1 g of the
substance being examined in a headspace vial, add l. O ml of
N, N-dimethylacetamide and O. 2 ml of water, seal the vial
and mix well as the test solution. Measure 99. 75 g of
macrogol 400 (Using a rotary evaporator remove any volatile
components at a temperature of 60ºC and pressure of l. 5 to
2. 5 kPa for 6 hours) into a 100 ml of penicillin bottle ( or
other suitable container) , accurately measured and seal.

Puncture injection 300 µl of ethylene oxide ( equivalent to
about O. 25 g of ethylene oxide) by a glass syringe frozen to
-lOºC into the above penicillin bottle, accurately weighed,
as the ethylene oxide stock reference solution ( freshly
prepared or calibrated befare use). Weigh accurately 1 g of
cold ethylene oxide stock solution into a previously cooled
penicillin bottle containing 49 g of macrogol 400 described
above, seal and mix. Measure accurately 10 g into a 50 ml
volumetric flask containing 30 ml of water, dilute to volume
with water and mix well as ethylene oxide reference solution.
Dilute a quantity of dioxane, accurately weighed, with water
to produce a solution of O. 1 mg per ml, as dioxane reference
solution. Accurately weigh 1 g of the substance being
examined, in a headspace vial, add accurately l. O ml of N,
N-dimethylacetamide, O. 1 ml of ethylene oxide reference
solution and O. 1 ml dioxane reference solution, seal the vial,
mix well as the reference solution. Measure O. 1 ml of
ethylene oxide reference solution into a headspace vial, and
add O. 1 ml of freshly prepared O. 001 % acetaldehyde solution
and O. 1 ml of dioxane reference solution, seal the vial and
mix well as the system suitability solution. Carry out the
method for gas chromatograph ( 0521 ) , using a column
packed with polydimethysiloxane as the stationary phase;
maintain the column temperature at 50ºC for 5 minutes, raise
the temperature to 180ºC by 5ºC per minute, then to 230ºC
by 30ºC per minute and maintain for 5 minutes ( adjust if
necessary). The temperature of the injection is 150ºC. The
temperature of the flame ionization detector is 250ºC. Use
the headspace mJection method. The equilibration
temperature is 70ºC and maintain for 45 minutes. Inject the
gaseous sample in the system suitability vial onto the column
and record the chromatogram. Adjust the attenuation so that
the peak heights due to ethylene oxide and acetaldehyde in
the chromatogram are about 15 % of the full scale of the
chart. The resolution factor between the peaks corresponding
to acetaldehyde and ethylene oxide is at least 2. O, the signal-
to-noise ratio of the peak of dioxane is not less than 5. The
equilibration temperature is 90ºC and maintain for 45 minutes
Inject separately the gaseous sample each in the test vial and
the reference vial onto the column. Repeat the procedure at
least three times. The relative standard deviation of the 3
areas of peak of ethylene oxide is not greater than 15 % and
the relative standard deviation of the 3 areas of peak of
dioxane is not greater than 10%. Calculate the content with
respect to the peak area obtained in the chromatogram by the
standard addition method, ethylene oxide is not more than
O. 0001% and dioxane is not more than O. 001%.
Standardization of the ethylene oxide stock reference
solution Measure 10 ml of 50% suspension of magnesium
chloride in absolute ethanol, accurately add 20 ml of O. 1
mol/L ethanolic hydrochloric acid VS, allow to stand
overnight. Weigh accurately 5 g of the ethylene oxide stock
reference solution into the same flask and stand for 30
minutes. Carry out the method for potentiometric titration
( 0701 ) , titrate with ethanolic potassium hydroxide (O. 1
mol/L) VS. Perform a blank determination by using
macrogol 400 and make any necessary correction. Each ml of
ethanolic potassium hydroxide VS is equivalent to 4. 404 mg
of ethylene oxide.
Water Not more than l. O% , using methanol-dichloro-
methane ( 3 : 7) as the solvent ( 0832 method 1 ) .
Residue on ignition Not more than O. 1 % ( 0841 ) , using
l. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), using the residue obtained in the test for
Olive Oil ·60:1····
Residue on ignition: not more than O. 001%.
Composition of fatty acids Carry out the method for gas
chromatography ( 0521 ) . Dissolve l. O g of the substance
being examined in a 25 ml twin neck round bottom flask.
Add 10 ml of anhydrous methanol and O. 2 ml of 60 g/L
solution of potassium hydroxide in ethanol. Shake to dissolve
and pass nitrogen through the mixture at a rate of about
50 ml per minute, heat to boiling. When the solution is clear
(usually after about 10 minutes), continue heating for 5
minutes. Cool the flask under running water and transfer the
contents to a separating funnel. Rinse the flask with 5 ml of
n-heptane, transfer it to the separating funnel and shake.
Add 10 ml of 200 g/L sodium chloride solution and shake
thoroughly. Allow to separate and transfer the organic layer
to a vial containing anhydrous sodium sulfate. Filter and the
filtrate is as the test solution. Dissolve methyl palmitate
CRS, methyl stearate CRS, methyl oleate CRS, methyl
linolenate CRS, methyl lindenate CRS, methyl arachidate
CRS and methyl eicosenoate CRS in n-heptane to produce a
mixture reference solution (1) containing O. 8 mg per ml,
O. 6 mg per ml, 5. O mg per ml, 3. O mg per ml, O. 2 mg per
ml, O. 2 mg per ml and O. 2 mg per ml, respectively.
Transfer l. O ml and dilute to 10. O ml with n-heptane as the
mixture reference solution (2). Carry out the method for gas
chromatography <0521 ) , using a capillary column packed
with polyethylene glycol. The column temperature is l 70ºC.
Raise the temperature of the column to 230ºC by 2ºC per
minute and maintain for 10 minutes . Maintain the
temperature of injection at 250ºC, and that of detector at
250ºC. Inject 1 µl of the mixture reference solution (1) and
the mixture reference solution ( 2) onto the column and
record the chromatogram. The resolution factor between
each pair of adjacent peaks in the chromatogram obtained
with the reference solution ( 1) is not less than l. 8 and the
nurnber oí the theoretical plates calculated for methyl oleate
is not less than 30 000. The signal-to-noise ratio of the
minimum peak height of fatty acid methyl ester is not less
than 5 times in the mixture reference solution (2). Inject the
test solution 1 µl onto the column and calculate the content of
the fatty acid with respect to the peak area by area
normalization method. The palmitic acid is 4. 0% to 9. 0%,
stearic acid is not more than 6. O%, oleic acid is 58. O% to
80. O%, linoleic acid is 15. O% to 35. O%, linolenic acid is
not more than 2. O% , arachidic acid is not more than 2. O%
and eicosenoic acid is not more than 2. O%.
Category Pharmaceutical excipients, solubilizer and emulsifier.
Storage Tightly closed and contained at dry and cool place
with nitrogen.
Olive Oil
[8001-25-0]
Olive Oíl is the fatty oíl obtained by refining of the ripe
drupes of Olea euro paea L.
Description A clear, pale yellow transparent liquid,
odourless or nearly odourless.
Miscible with ether or with chloroform, very slightly soluble
in ethanol, practically insoluble in water.
Relative density O. 908-0. 915 < 0601 ) .
Acid value Not more than l. O (0713).
Saponification value 186-194 <0713).
 602 Olive Oil
lodinevalue 79-88 (0713).
Absorbance Place l. 00 g in a 100 ml volumetric flask, add
a volume of cyclohexane to dissolve and dilute to volume.
Carry out the method for ultraviolet-visible spectro-
photometry ( 0401), measure the absorbance of the resulting
solution at 270 nm, the absorbance is not more than l. 2.
Peroxide value In a 250 ml iodine flask, dissolve 10. O g in
30 ml of acetic acid-chloroform ( 60 : 40) with shaking.
Accurately add O. 5 ml of potassium iodide TS, stopper the
flask, shake thoroughly for 1 minute and add 30 ml of water.
Ti trate with sodium thiosulfate (O. 01 mol/L) VS. Add
O. 5 ml of starch IS towards the end point and continue the
titration until the blue colour disappears. Perform a blank
determination and make any necessary correction. Not more
than 10. O ml of sodium thiosulfate (O. 01 mol/L) VS is
consumed.
Unsaponifiable matter Accurately weigh 5. O g in a 250 ml
conical flask, add 50 ml of potassium hydroxide solution in
ethanol ( dissolve 12 g of potassium hydroxide in 10 ml of
water, dilute to 100 ml with ethanol and shake welD. Reflux
the mixture for 1 hour, cool to below 25ºC. Transfer the
solution to a separating funnel, wash the flask with two
portions, each of 50 ml, of water, combine the washings and
transfer to the separating funnel. Extract with three
portions, each of 100 ml, of ether, combine the ether layer
and wash with three portions, each of 40 ml, of water,
discard the aqueous layer. Wash the ether layer successively
with three portions, each of 40 ml, of 3% potassium
hydroxide solution and water, then wash the ether layer with
a 40-ml portion of water for several times until the last
washing is no longer appears red with two drops of
phenolphthalein IS. Transfer the ether extract to an
evaporating dish previously dried to constant weight, wash
the separeting funnel with 10 ml of ether, transfer the
washing to the evaporating dish, evaporate ether on a water
bath at 50ºC, dissolve the residue with 6 ml of acetone, expel
the acetone with current of air. Dry the residue at 105ºC until
the variation between two successive weighing is less than
1 mg, the content of the unsaponifiable matter is not more
than l. 5%.
Dissolve the residue in 20 ml of neutralized ethanol, add a
few drops of phenolphthalein IS and titrate with ethanolic
sodium hydroxide (O. 1 mol/L) VS until a pink colour
persists 30 seconds, the test is invalid if more than O. 2 ml
alcoholic sodium hydroxide (O. 1 mol/U VS is consumed,
and the test must be repeated. The total weight of residue
should not be taken as the weight of unsaponifiable matter.
Alkaline impurities In a test-tube add 10 ml of freshly
distilled acetone, O. 3 ml of water and 1 drop of O. 04%
bromophenol blue solution in ethanol. Neutralize the solution
with O. 01 mol/L hydrochloric acid or O. 01 mol/L sodium
hydroxide solution. Add 10 ml of the substance being
examined, shake and allow to stand. Not more than O. 1 ml
of O. 01 mol/L hydrochloric acid VS is required to change the
colour of the upper layer to yellow.
Cottonseed oil Place 5 ml in a test-tu be, add 5 ml of a
mixture of equal volumes of amyl alcohol and 1 % solution of
sulfur in carbon disulfide. Place the test-tube in a saturated
sodium chloride water bath, warm the mixture carefully,
when foam is produced ( carbon disulfide has been expelled) ,
heat for 15 minutes, no red colour appears.
Sesame oil To 10 ml add 10 ml of hydrochloric acid TS and
O. 1 ml of freshly prepared furfural solution in ethanol ( 1-
50), shake vigorously for 15 seconds: no pink to crimson
colour appears in the acid layer. If any colour appears in the
acid layer, add 10 ml of water, and again shake the mixture,
the colour disappears in the acid layer.
Water Not more than O. 5 % ( 0832 method I ( 1 ) ) ,
using a mixture of equal volumes of decanol and anhydrous
methanol as solvent.
Heavy metals Transfer 4. O g to a 50 ml porcelain eva-
porating dish, add 4 ml of sulfuric acid, mix well, heat
gently to expel the sulfuric fumes, add 2 ml of nitric acid and
5 drops of sulfuric acid TS, heat to expel oxide fumes and
ignite at 500-600ºC until the incineration is complete. Cool,
carry out the limit test for heavy metals ( 0821 method 2) :
not more than O. 001%.
Arsenic Mix l. O g with l. O g of calcium hydroxide, stir
well with a small volume of water. Allow it to dry, ignite
gently to carbonize and then ignite at 500-600ºC until the
incineration is complete, cool and dissolve the residue in a
mixture of 5 ml of hydrochloric acid and 23 ml of water.
Carry out the limit test for arsenic ( 0822 method 1 ) : not
more than O. 0002%.
Composition of fatty acids Carry out the method for gas
chromatography < 0521 ) , using the column packed with
polyethylene glycol as the stationary phase. Maintain the
injection temperature at 260ºC and the detector temperature
at 270ºC, maintain the column temperature at 230ºC for 11
minutes. Raise the temperature of the column by 5ºC per
minute from 230ºC to 250ºC and maintain the column
temperature at 250ºC for 10 minutes. The number of the
theoretical plates calculated for the methyl oleic acid peak is
not less than 10 000, the resolution factor between each pair
of adjacent peaks complies with the requirements.
Reference solution Dissolve methyl palmitic acid, methyl
palmitolic acid, methyl stearic acid, methyl oleic acid,
methyl linoleic acid, methyl linolenic acid, methyl arachidic
acid, methyl eicosenoic acid, methyl behenic acid, and
methyl lignoceric acid CRS in n-hexane to produce a mixture
solution containing O. 1 mg per ml respectively.
Test solution Place O. 1 g of the substance to be examined in
a 50 ml conical flask, add 2 ml of O. 5 mol/L sodium
hydroxide solution in methanol, Reflux the mixture in a
water bath at 65ºC under a reflux condenser for 30 minutes.
Allow to cool and add 2. O ml of 15 % boron trifluoride
solution in methanol through the condenser and reflux in a
water bath at 65 ºC for 30 minutes. Cool and add 4 ml of
heptane through the condenser continue to reflux for 5
minutes, allow to cool and add 10 ml of saturated sodium
chloride solution, shake well and allow to stand until
separation of the layers is obtained; collect the upper layer
and wash with 3 portions, each of 2 ml, of water and dry
with anhydrous sodium sulfate.
Procedure lnject 1 µl of the upper layer onto the column
and record the chromatogram. Calculate the content of fatty
acid with respect to the peak area obtained in the
chromatogram by normalization method. The content of
saturated fatty acids of chain length less than C16 is not more
than o. 1%' palmitic acid is 7. 5% to 20. o%' palmitolic acid
is not more than 3. 5 % , stearic acid is O. 5 % to 5. O%, oleic
acid iS 56. O% to 85. O%, linoleic acid is 3. 5 % to 20. O%,
linolenic acid is not more than l. 2 % , arachidic acid is not
more than O. 7 % , eicosenoic acid is not more than O. 4%,
behenic acid is not more than O. 2 % , and lignoceric acid is
not more than O. 2%.
Category Pharmaceutical excipients, solvent and dispersant,

etc.
Storage Preserved in tightly closed containers, protected
from light and stored in a dark and cool place.
Oxyquinoline Sulfate
(e9H1N0)2 •H2S04 •H20 406. 42
[134-31-6]
Oxyquinoline sulfate is 8-hydroxyquinoline sulfate monohy-
drate. It contains not less than 97. 0% and not more than
101.0% of (e9 H1NÜ)2•H2SÜ4, calculated on anhydrous
basis.
Description A yellow crystalline powder.
Very soluble in water; freely soluble in methanol; slightly
soluble in ethanol; practically insoluble in acetone or ether.
Identification ( 1 ) The infrared absorbance spectrum
(nujol mull method) is concordant with that of Oxyquinoline
Sulfate eRS < 0402 ) . If necessary, dissolve a quantity of
substance being examined and Oxyquinoline Sulfate eRS
respectively in water, filter; evaporate to dryness, allow the
residues to stand in desiccator for one night, then perform
the determination and compare the spectra.
(2) The aqueous solution ( 1 - 10) yields the reactions
characteristic of sulfates <0301 ) .
Water 4. O%-6. O% <0832 method 1 ( 1 ) ) .
Residue on ignition Not more than O. 3 % <0841 ) , using
1 g.
Heavy metals Not more than O. 002% <0821 method 2),
using the residue obtained in the test for Residue on ignition.
Assay Place about O. 1 g, accurately weighed, in a conical
flask with stopper, add 30 ml of glacial acetic acid to
dissolve. Add 25 ml of bromine (0. 05 mol/L) VS accurately
measured, 10 ml of potassium bromide solution (3-20) and
10 ml of hydrochloric acid, stopper immediately and shake
thoroughly, allow to stand in dark place for 15 minutes. Add
10 ml of potassium iodide solution ( 1-10) and 100 ml of
water quickly, stopper and shake. Rinse the wall of the flask
with water and shake. Titrate with sodium thiosulfate
(O. 1 mol/L) VS, add 3 ml of starch TS towards the end of
titration. Perform a blank determination and make any
necessary correction. Each ml of bromine (O. 05 mol/L) VS
is equivalent to 4. 855 mg of (C9H1NÜ)z•H2SÜ4.
Category Pharmaceutical excipients, preservative.
Storage Preserve in well closed containers.
Paraffin
[68476-81-3]
Paraffin is a mixture of solid hydrocarbons obtained from
petroleum or shale oil.
Description A colourless or white translucent mass, usually
Pectin 603
exhibit a crystalline structure; odourless; tasteless; slightly
greasy to the touch.
Soluble in chloroform or in ether; practically insoluble in
water or ethanol.
Melting range 50-65ºC (0612 method 2).
ldentification (1) When strongly heated, it burns with a
luminous flame to leave a carbonized residue.
(2) Heat about O. 5 g in a dry test tube with an equal
quantity of sulfur: the mixture is carbonized with the
evolution of hydrogen sulfide.
Acidity or alkalinity Melt about 5. O g, add an equal volume
of hot neutral ethanol and shake. Allow to separate, the
solution is neutral to litmus paper.
Readily carbonisable substances Transfer 4. O g to a test tube
with stopper, heat in a water bath at 65-70ºC. Add 5 ml of
95 % (g/ g) sulfuric acid and maintain at this temperature for
10 minutes, shake the mixture thoroughly for a few seconds
at intervals of every one minute. The paraffin remains
unchanged in colour, any colour produced in the acid layer is
not more intense than that of a reference solution ( mix O. 8
ml of standard cobaltous chloride es, o. 3 ml of standard
copper sulfate es, l. o ml of standard potassium dichromate
es and 2. 9 ml of water).
Sulfides To 4. O g, add 2 drops of the saturated solution of
lead oxide in sodium hydroxide solution 0-5) and 2 ml of
ethanol, shake well. Heat in a water bath at 70ºC for 10
minutes with shaking, cool. No brownish-black colour is
produced.
Polycyclic aromatic hydrocarbons Transfer O. 5 g, accurately
weighed, to a separating funnel, add 25 ml n-hexane to
dissolve, then add accurately 5 ml of dimethyl sulfoxide.
Shake vigorously for 2 minutes and allow to stand until two
clear layers are formed. Transfer the dimethyl sulfoxide layer
to another separator, add 2 ml of n-hexane and shake, allow
to stand until two clear layers are formed ( centrifuge if
necessary). Separate the dimethyl sulfoxide layer as the test
solution. Transfer 25 ml of n-hexane to a separating funnel,
add accurately 5 ml of dimethyl sulfoxide. Shake vigorously
for 2 minutes and allow to stand until two clear layers are
formed. Separate the dimethyl sulfoxide layer as a blank
solution. Measure the absorbance between 260 nm and 350
nm <0401), the maximum absorbance of the test solution is
not greater than O. 10.
Category Pharmaceutical excipients, ointment base, coating
material, etc.
Storage Preserve in well closed containers.
Pectin
Pectin is a purified carbohydrate product obtained from the
rind of citrus fruits or from apple pomace. It contains not
less than 6. 7 % of methoxy groups ( -OeH 3 ) and not less
than 74. o% of galacturonic acid ( e6 H10 01 ) , calculated on
the dried basis.
Description A white to light yellow granules or powder.
ldentification ( 1) Heat l. O g with 9 ml of water on a water
bath to dissolve, replacing water lost by evaporation: it
forms a stiff gel on cooling.
(2) To a solution (1 in 100) add an equal volume of ethanol:
a translucent, gelatinous precipitate is formed.
 604 Phosphoric Acid
(3) To 5 ml of a solution (1 in 100) add 1 ml of 2 mol/L
sodium hydroxide solution, and allow to stand at room
temperature for 15 minutes: a gel or semigel forms.
( 4) Acidify the gel or semigel from the preceding test with 3
mol/L hydrochloric acid solution, and shake: a certain
volume of colourless and gelatinous precipitate forms, which
upan boiling becomes white and flocculent.
Sugars and organic acids Place l. O g in a 500 ml flask,
moisten it with 3 to 5 ml of ethanol, rapidly pour in 100 ml
of water, shake to dissolve. To this solution add 100 ml of
ethanol containing O. 3 ml of hydrochloric acid solution, mix
well, and filter rapidly. Measure 25 ml of the filtrate into an
evaporating dish, previously dried to constant weight,
evaporate the liquid on a water bath and dry the residue in a
vacuum oven at 50ºC for 2 hours: the weight of the residue is
not more than 20 mg.
Loss on drying When dried at 105ºC for 3 hours, loses not
more than 10. O% of its weight <0831).
Heavy metals Weigh 2. O g, carry out the limit test for
heavy metals (0821 method 2): not more than O. 001%.
Arsenic Dissolve O. 5 g in 23 ml of water, add 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822
method 1 ) : not more than O. 0003 % .
Assay methoxy groups Transfer 5. O g, accurately
weighed, to a suitable beaker, and stir for 10 minutes with a
mixture of 60% ethanol-hydrochloric acid (20 : 1). Transfer
to a sintered-glass filter (30-60 ml crucible or Buchner type,
coarse), and wash with six 15 ml portions of the mixture
above, followed by 60 % ethanol until the filtra te is free from
chlorides. Finally wash with 20 ml of ethanol, dry for 1 hour
at 105ºC, cool, and weigh. Transfer exactly one-tenth of the
total net weight of the dried residue to a 250 ml conical flask,
and moisten with 2 ml of ethanol. Add 100 ml of freshly
boiled water, shake to dissolve completely. Add 5 drops of
phenolphthalein TS, titrate with sodium hydroxide (O. 5
mol/L) VS, and record the results as the initial titer (V1 ).
Add 20. O ml of sodium hydroxide (O. 5 mol/L) VS, insert
the stopper, shake vigorously, and allow to stand for 15
minutes. Add 20. O ml of hydrochloric acid (0. 5 mol/L) VS,
and shake until the pink colour disappears. Add phenol-
phthalein TS, and ti trate with sodium hydroxide (O. 5 mol/L)
VS until a faint pink colour appears: record this value as the
saponification titer (V2). Each ml of sodium hydroxide (0. 5
mol/L) VS used in the saponification titer is equivalent to
15. 52 mg of -OCH3.
galacturonic acid Each ml of sodium hydroxide (O. 5 mol/L)
VS used in the total titration ( the initial titer added to the
saponification titer) in the Assay for methoxy groups is
equivalent to 97. 07 mg of C6 H10 01.
Category Pharmaceutical excipients, thinkening agent, release
retardant, etc.
Storage Preserve in tightly closed containers.
Phosphoric Acid
H3PÜ4 98. 00
[7664-38-2]
Phosphoric acid contains not less than 85. O% and not more
than 90. O% (g/ g) of H3 P04.
Description A clear, colourless, sviscous liquid, corrosive,
miscible with water and with ethanol.
Relative density About l. 7 ( 0601 >.
ldentification Yields the reactions characteristic of phos-
pha tes ( 0301 >.
Clarity and color of the solution A solution of l. O gin 15 ml
of water is clear and colourless ( 0901 and 0902 >.
Substances precipitated with anunonia To 1. O g add 15 ml of
water and 12 ml of ammonia TS, no opalescence is produced.
Hypophosphorous acid and phosphorous acid To l. O g add
15 ml of water and 6 ml of silver nitrate TS, heat on a
waterbath for 5 minutes, no opalescence is produced.
Alkaline phosphate Add 6 ml of ether and 2 ml of ethanol to
1 ml of substance being examined, no opalescence is
produced.
Nitrate Dilute 2. 6 g with 3. 5 ml of water, successively add
O. 1 ml of indigo carmine TS and 5 ml of sulfuric acid, the
blue colour <loes not disappear within 1 minute.
Chloride Carry out the limit test for chloride ( 0801 >, using
2. O g. Any opalescence produced is not more pronounced than
that of a reference solution using 10. O ml of sodium chloride
standard solution (O. 005 % ) .
Sulfate Carry out the limit test for sulfate ( 0802 >, using
2. O g. Any opalescence produced is not more pronounced
than that of a reference solution using 2. O ml of potassium
sulfate standard solution (O. 01%).
Iron Dilute 2. O g with 30 ml of water, mix well, carry out
the iimit test for iron < 0807 >, using 3. O ml of the test
solution. Any colour produced is not more intense than that
of a reference solution using l. O ml of iron standard solution
(0. 005%).
Heavy metals To l. O g add l. 6 ml of ammonia TS and
dilute with water to 25 ml. Carry out the limit test for heavy
metals ( 0821 method 1 ) : not more than O. 001 % .
Arsenic Dissolve l. O g in 5 ml of hydrochloric acid and
22 ml of water. Carry out the limit test for arsenic ( 0822
method 1): not more than O. 0002%.
Assay Dilute l. O g, accurately weighed, with 120 ml of
water. Add O. 5 ml of thymolphthalein IS. Titrate with
sodium hydroxide ( 1 mol/L) VS. Each ml of sodium
hydroxide (1 mol/L) VS is equivalent to 49. 00 mg of
H3PQ4.
Category Pharmaceutical excipients, acidifying agent.
Storage Preserve in tightly closed containers.
Poloxamer 188
HCC2 H4 Ü)a (C3 H6 Ü)b CC2 H4 O) aOH
[9003-11-06]
Poloxamer 188 is a block copolymer of a-hydro-w-hydro-
xypoly ( oxyethylene ) ª poly ( oxypropylene ) b poly
(oxyethylene)a obtained by reaction of propylene oxide with
propylene glycerol form polyoxypropylene glycerol and then
addition of ethylene oxide to polymerize, where a is 75-85
and b is 25-30. The average content of oxyethylene unit is

79. 9 %-83. 7 %. The average molecular weight is 7680-9510.
Description White translucent waxy solids; odour, slightly
characteristic.
Freely soluble in water and in ethanol; soluble in dehydrated
ethanol and in ethyl acetate; practically insoluble in ether and
in petroleum ether.
Identification The infrared absorption spectrum is
concordant with the reference spectrum (IR Album No. 618).
Acidity or alkalinity Dissolve l. O g in 10 ml of water, pH
5.0-7.5 (063]).
Clarity and colour of the solution The solution under the test
of acidity or alkalinity is clear and colourless < 0901 and
0902).
Oxyethylene Dissolve O. 1-0. 2 g of the substance being
examined in 1 ml of deuterated water containing 1 % sodium
4, 4-dimethy-4-silapentane sulfonate, or use 1 ml of
deuterochloroform containing 1 % of tetramethysilane as the
solvent. Transfer the test sample solution into the NMR
tube. If the solvent is deuterochoroform, add 1 drop of
deuterated water and shake the tube. Sean the region from O
to 5 X 10- 6 , and calculate the percentage of oxyethylene
(EO) by the direct comparison method using the following
expression:
E0=3300a/ (33a+58)
Where a is (A2 / Al) -1
A 1 is the average area of the doublet appearing at
about l. 15 X 10- 6 , due to the methyl of the
oxypropylene;
A 2 is the average area of the composite band from
3. 2-3. 8 X 10- 6 , due to CH2 O groups of both
the oxypropylene and oxyethylene units and the
CHO groups of the oxyethylene units
EO is the percentage of oxyethylene in the entire
molecule, accounting for 79. 9 %-83. 7 %.
Unsaturation Weigh accurately about 15. O g of the pow-
dered poloxamer, add accurately 50 ml of mercuric acetate
solution, and mix on a magnetic stirrer until the poloxamer is
dissolved completely. Allow to stand for 30 minutes with
occasional shaking. Add 10 g of sodium bromide crystals,
and stir on a magnetic stirrer for 2 minutes, add 1 ml of
phenolphthalein IS immediately, titrate with methanolic
potassium hydroxide ( O. 1 mol/L) VS. Perform a blank
determination and determine the initial acidity [Dissolve 15. O
g of poloxamer 188 in 75 ml of neutral methanol (neutral to
phenolphthalein IS), titrate with methanolic potassium
hydroxide (O. 1 mol/L) VS until the solution is neutral to
phenolphthalein IS] to make any necessary correction. The
unsaturation is calculated, in mmol/g, with the following
equation: O. 018-0. 034 meq/ g.
Unsaturation= (V samrte-Vb1ank-V initia1) N /W
Where V samrte, V btank and V ini1ia1 are the volumes of
methanolic potassium hydroxide (O. 1 mol/L) VS
consumed for titrating the substance being
examined, the blank and the initial acidity,
respectively, ml;
N is the concentration of methanolic potassium
hydroxide (0. 1 mol/L) VS, mol/L;
W is weight of the substance being examined, g.
Average molecular weight Weigh accurately a quantity,
calculated by multiplying the molecular weight by O. 002 g,
of poloxamer, add accurately 25 ml of phthalic anhydride-
pyridine solution and a few glass beads. Heat under a reflux
condenser for 1 hour, allow to cool and wash the reflux
condenser wi th two portions of pyridine, each of 1O ml. Add
Poloxamer 188 605
10 ml of water, mix well, seal and allow to stand for 10
minutes. Add accurately 50 ml of O. 66 mol/L sodium
hydroxide solution and O. 5 ml of phenolphthalein- pyridine
solution (1-100), titrate with sodium hydroxide (O. 5 mol/L)
VS until the solution changes to a light pink colour that
persists for 15 seconds. Perform a blank determination and
make any necessary correction. Calculate the average
molecular weight for the substance being examined using the
following equation:
Average molecular weight= 2000W/ [ (B - S) N]
Where W is the weight of the substance being examined,
g;
B is the volume, of the sodium hydroxide (O. 5
mol/L) VS consumed for titrating blank, ml;
S is the volume, of the sodium hydroxide (O. 5
mol/L ) VS consumed for titrating the
substance being examined, ml;
N is the concentration of sodium hydroxide (O. 5
mol/L) VS, mol/L.
Ethylene oxide, propylene oxide and 1, 4-dioxane W eigh
accurately 1 g in a head-space vial, accurately add 5 ml of
dimethylfomamide, shake well, seal and use as the test
solution. Accurately measured a quantity of ethylene oxide,
propylene oxide and 1, 4-dioxane and dilute with dimethy-
lfomamide to produce a mixed solution containing O. 2 µg, 1
µg and 2 µg per ml respectively. Accurately measure 5 ml of
the solution in a head-space vial, seal and use as the reference
solution. Carry out the method for gas chromatography
<0521 ) . Use a capillary column (O. 25 mm X 30 m) coated
with a stationary phase of poly ( 6 % cyanopropyl phenyl)
(94% dimethyl) siloxane. The initial temperature of column
is 70ºC, then increase to 220ºC by 35ºC per minute, maintain
for 5 minutes. A flame ionization detector is used. The
temperature of injection is at 250ºC and the temperature of
detector is at 280ºC . The equilibrium temperature of the
head space vial is 80ºC and maintain for 30 minutes. Inject
the gas of the ref erence solution. The resolution factors
between the peaks of ethylene oxide, propylene oxide and 1,
4-dioxane comply with the related requirements. Inject the
gas from the head-space vial of test solution and ref erence
solution respectively and record the chromatograms. Cal-
culate the contents with respect to the peak area obtained in
the chromatogram by externa! standard method. The content
of ethylene oxide is not more than O. 0001 % , the content of
propylene oxide is not more than O. 0005 % and the content of
1, 4-dioxane is not more than O. 0005%.
Ethylene glycol and diethylene glycol Dissolve a quantity of
1, 3-butanediol, accurately weighed, in absolute ethanol to
obtain a solution containing O. O1 mg per ml as the internal
standard solution. Weigh accurately about O. 1 g of the
substance being examined into a 25 ml volumetric flask, add
accurately 1 ml of the internal standard solution, dilute to
volume with absolute ethanol and take the filtrate as the test
solution. Dissolve a quantity of ethylene glycol and
diethylene glycol, accurately weighed, in absolute ethanol to
produce a solution of about O. 01 mg of ethylene glycol and
diethylene glycol, respectively. Transfer accurately 1 ml into
a 25 ml flask, add accurately 1 ml of internal standard
solution and dilute to volume with absolute ethanol as the
reference solution. Carry out the method for gas chromato-
graphy ( 0521 ) , using a column packed with phenyl-
methylpolysiloxane ( 50 : 50 ) as stationary phase. The
temperature of injection is 270ºC . The temperature of
detector is 290ºC; maintain the temperature of the column at
60ºC for 5 minutes, then raise the temperature to lOOºC by
lOºC per minute, then raise the temperature to 170ºC by 4 ºC
 606 Poloxamer 407
per minute, then raise the temperature to 270ºC by lOºC per
minute and maintain for 2 minutes. Calculate the contents of
ethylene glycol and diethylene glycol with respect to the peak
area obtained in the chromatogram by the interna! standard
method. Ethylene glycol and diethylene glycol are not more
than O. 01%, respectively.
Propylene glycol Dissolve a quantity of the substance being
examined, accurately weighed, in absolute ethanol to obtain
a solution containing 1 mg per ml as the test solution.
Dissovle a quantity of propylene glycol, accurately weighed,
in absolute ethanol to obtain a solution containing O. 005 µg
per ml as the reference solution. Carry out the method for
gas chromatography ( 0521 ) , using a column packed with
polyethylene glycol 20 M as the stationary phase. The
temperature of injection is 230ºC . The temperature of
detector is 250ºC; maintain the temperature of the column at
130ºC for 1 minute, then raise the temperature to 240ºC by
lOºC per minute, and maintain for 1 minute. Inject separately
1 µl, accurately measured the test solution and the reference
solution. Calculate the contents of propylene glycol with
respect to the peak area obtained in the chromatogram by the
externa! standard method. Propylene glycol is not more than
o. 0005%.
Water Not more than l. O% ( 0832 method 1 ) .
Residue on ignition Not more than O. 4 % , using l. O g
<0841>.
Heavy metals Dissolve l. O gin 23 ml of water, add 2 ml of
aceta te BS CpH 3. 5). Carry out the limit test for heavy
metals <0821 methnd 2): not morf' th<rn O. 002%.
Arsenic To l. O g add 5 ml of hydrochloric acid and 23 ml of
water, shake to dissolve. Carry out the limit test for arsenic
( 0822 method 1 ) : not more than O. 000 2 % .
Bacterial endotoxin Capplicable to products for injection).
Carry out the test for bacteria! endotoxin ( 1143 ) : not more
than O. 012 EU per mg.
Sterility Capplicable to sterile products without sterilization
process) Complies with the test for sterility ( 1101).
Category Pharmaceutical excipients, solubilizer, emulsifier.
Storage Preserve in tightly closed containers and protect
from light.
Preparation of mercuric acetate solution Dissolve 50 g of
mercuric acetate in 900 ml of methanol containing O. 5 ml of
glacial acetic acid, dilute to 1000 ml with methanol and mix
well. Discard the solution if it changes to yellow. If it is
turbid, filter it. Discard it if it is still turbid or presents as
yellow. The solution should be freshly prepared, and
preserve in an amber glass bottle, stored in dark place.
Preparation and calibration of phthalic anhydride- pyridine
solution Preparation To 500 ml of pyridine containing less
than O. 1% of water Cor dissolve 30 g of phthalic anhydride in
500 ml of pyridine, distill and use the middle portian of the
distillate) add 72 g of phthalic anhydride, shake vigorously
or heat on a water bath at 40ºC to dissolve completely, allow
to stand overnight with protecting from light.
Standardization Mix 10 ml of the above resulting solution,
accurately measured, with 25 ml of pyridine and 50 ml of
water, allow to stand for 15 minutes, add O. 5 ml of
phenolphthalein-pyridine solution C1 - 100), titrate with
sodium hydroxide CO. 5 mol/L) VS: consumes between 37. 6 ml
to 40. O ml.
Poloxamer 407
H CC2 H4 Q) ª CC3 H6 Q) CC2 H4 Q) ª OH
b
[9003-11-06]
Poloxamer 407 is a block copolymer of a- hydrogen -w- hydr-
oxy poly Cethylene oxide)ª - poly Coxypropylene) b - poly
Cethylene oxide)ª , obtained by reaction of propylene oxide
with propylene glycerol to form polyoxypropylene glycerol
and then addition of ethylene oxide to polymerize. In the
copolymer, oxyethylene Ca) is 101, oxypropylene Cb) is 56,
and ethylene oxide CEO) content is 71. 5 %-74. 9 % . The
average molecular weight is 9840-14600.
Description A white to slightly yellow and semi-trans-
lucent, waxy salid; odour, slightly characteristic.
Freely soluble in water and in ethanol; soluble in absolute
ethanol and ethyl acetate; insoluble in diethyl ether and in
petroleum ether.
ldentification The infrared absorption spectrum is concor-
dant with the reference spectrum (IR Album 618).
Acidity or alkalinity Dissolve l. O g in 10 ml of water, pH
5. 0-7. 5 <0631>.
Clarity and colour of the solution The solution under Acidity
or alkalinity is clear and colourless ( 0901 and 0902). Any
colour produced is not more intense than that of reference
solution Y1 ( 0901 method 1 ).
Weight percent oxyethylene Transfer O. 5-1. O ml of 10 %-
20 % deuteration trichloromethane solution Cg/ml) cont-
aining 1% tetramethylsilane Cor deuterated water, use 1%
4, 4-dimethy-4-silapentane sulfonate as internal standard) to
the NMR tube, add 1 drop of deuterated water and shake.
Sean the region from OX 10- 6 to 5 X 10- 6 , and calcula te the
percentage of oxyethylene CEO) by the direct comparison
method using the following expression:
E0=3300a/ C33a+58)
Where is CA2/ A1) -1
is the average area of the doublet appearing at
about l. 15 X 10- 6 , due to the methyl of the
oxypropylene units;
A 2 is the average area of the composite band from
3. 2-3. 8X10- 6 , due to CH 2O groups of both
the oxypropylene and oxyethylene units and
the CHO groups of the oxyethylene units
EO is the proportion of oxyethylene in the entire
molecular, % .
Unsaturation Weigh accurately about 15. O g of the pow-
dered poloxamer, accurately add 50 ml of mercuric acetate
solution, and mix on a magnetic stirrer until the poloxamer is
dissolved completely. Allow to stand for 30 minutes with
occasional shaking. Add 10 g of sodium bromide crystals,
and stir on a magnetic stirrer for 2 minutes, add 1 ml of
phenolphthalein IS immediately, with methanolic potassium
hydroxide CO. 1 mol/L) VS. Perform a blank determination
and determine the initial acidity [ Dissolve 15. O g of
poloxamer 407 in 75 ml of neutral methanol Cneutral to
phenolphthalein IS), ti trate with methanolic potassium
hydroxide CO. 1 mol/L) VS until the solution is neutral to

phenolphthalein IS] to make any necessary correction. The
unsaturation is calculated, in mmol/ g, with the fallowing
equation and is O. 031- O. 065 meq/g.
Unsaturation = (V sampte - Vbtank -V ini1ia1) N /W
Where V sample, Vb1ank and V ini1ia1 are the volumes of
methanolic potassium hydroxide (O. 1 mol/L) VS
consumed far titrating the substance being
examined, the blank and the initial acidity,
respectively, ml;
N is the concentration of methanolic potassium
hydroxide (O. 1 mol/U VS, mol/L;
W is weight of the substance being examined, g.
Average molecular weight Weigh accurately a quantity,
calculated by multiplying the molecular weight by O. 002 g,
of poloxamer, add accurately 25 ml of phthalic anhydride -
pyridine solution and a few glass beads. Heat under a reflux
condenser far 1 hour, allow to cool and wash the reflux
condenser with two portions of pyridine, each of 10 ml. Add
10 ml of water, mix well, seal and allow to stand far 10
minutes. Add accurately 50 ml of O. 66 mol/L sodium
hydroxide solution and O. 5 ml of phenolphthalein- pyridine
solution 0-100) , ti trate with sodium hydroxide (O. 5 mol/
L) VS until the solution changes to a light pink colour that
persists far 15 seconds. Perform a blank determination and
make any necessary correction. Calculate the average
molecular weight of the substance being examined using the
fallowing equation:
Average molecular weight=2000W/ [ (B-S) N]
Where W is the weight of the substance being examined,
g;
B is the volume, of the sodium hydroxide (O. 5
mol/L) VS consumed far titrating blank, ml;
S is the volume, of the sodium hydroxide (O. 5
mol/L) VS consumed far titrating the sub-
stance being examined, ml;
N is the concentration of sodium hydroxide (O. 5
mol/L) VS, mol/L.
Ethylene oxide, propylene o~de and 1, 4-dioxane W eigh
accurately 1 g in a head-space vial, accurately add 5 ml of
dimethylfamamide, shake well, seal and use as the test
solution. Accurately measure a quantity of ethylene oxide,
propylene oxide and 1, 4-dioxane and dilute with dimeth-
ylfamamide to produce a mixed solution containing O. 2 µg, 1
µg and 2 µg per ml respectively. Accurately measure 5 ml of
the solution into a head-space vial, seal and use as the
reference solution. Carry out the method far gas chrom-
atography ( 0521 ). Use a capillary column (O. 25 mmX 30 m)
coated with a stationary phase of poly ( 6 % cyanopropyl
phenyD (97 % dimethyl) siloxane. The initial temperature of
the column is 70ºC, then increase to 220ºC by 35ºC per
minute, maintain far 5 minutes. A flame ionization dector is
used. The temperature of injection is at 250ºC and the
temperate of detector is at 280ºC . The equilibrium tem-
perature of the head space vial is 80ºC and maintain far 30
minutes. Inject the gas of the reference solution. The
resolution factors between the peaks of ethylene oxide,
propylene oxide and 1, 4-dioxane comply with the related
requirements. Calculate the contents with respect to the peak
area obtained in the chromatogram by externa! standard
method. The content of ethylene oxide is not more than
O. 0001 % , the content of propylene oxide is not more than
O. 0005 % and the content of 1, 4-dioxane is not more than
o. 0005%.
Ethylene glycol and diethylene glycol Dissolve a quantity of
1, 3-butanediol, accurately weighed, in absolute ethanol to
Poloxamer 407 607
obtain a solution containing O. 01 mg per ml as the interna!
standard solution. Weigh accurately about O. 1 g of the
substance being examined into a 25 ml volumetric flask, add
accurately 1 ml of the internal standard solution, dilute to
volume with absolute ethanol and take the filtrate as the test
solution. Dissolve a quantity of ethylene glycol and dieth-
ylene glycol, accurately weighed, in absolute ethanol to
produce a solution of about O. 01 mg per ml of ethylene glycol
and diethylene glycol, respectively. Transfer accurately 1 ml
to a 25 ml flask, add accurately 1 ml of interna! standard
solution and dilute with the absolute ethanol to the volume as
the reference solution. Carry out the method far gas
chromatography ( 0521 ) , using a column packed with
phenyl-methylpolysiloxane ( 50 : 50). A flame ionization
detector is used. The temperature of injection is 270ºC. The
temperature of detector is 290ºC; maintain the temperature
of the column at 60ºC far 5 minutes, then raise the
temperature to lOOºC by lOºC per minute, then raise the
temperature to l 70ºC by 4ºC per minute, then raise the
temperature to 270ºC by lOºC per minute and maintain far 2
minutes. Calculate the contents of ethylene glycol and
diethylene glycol with the respect to the area obtained in the
chromatogram by the interna! standard method. Ethylene
glycol and diethylene glycol are not more than O. 01 % ,
respectively.
Propylene glycol Dissolve a quantity of the substance being
examined, accurately weighed, in absolute ethanol to obtain
a solution containing 1 mg per ml as the test solution.
Dissolve a quantity of propylene glycol, accurately weighed,
in absolute ethanol to obtain a solution containing O. 005 µg
per ml as the reí erence solution. Carry out the method far
gas chromatography ( 0521 ) , using a column packed with
polyethylene glycol 20 M, a flame ionization detector is used.
The temperature of injection is 230ºC. The temperature of
detector is 250ºC; maintain the temperature of the column at
130°C far 1 minute, then raise the temperature to 240ºC by
lOºC per minute, and maintain far 1 minute. Inject
separately 1 µl, accurately measured the test solution and the
reference solution. Calculate the contents of propylene glycol
with respect to the peak area obtained in the chromatogram
by the externa! standard method. Propylene glycol is not
more than O. 0005 % .
Water Not more than l. O% ( 0832 method 1 ) .
Residue on ignition Not more than O. 4 % ( 0841 ) .
Heavy metals Dissolve l. O gin 23 ml of water and add 2 ml
of aceta te BS ( pH 3. 5). Carry out the limit test far heavy
metals ( 0821 method 2): not more than O. 002%.
Arsenic To l. O g add 5 ml of hydrochloric acid and 23 ml of
water and shake to dissolve. Carry out the limit test far
arsenic ( 0822 method 1 ) : not more than O. 0002 % .
Category Pharmaceutical excipients, solubilizer, emulsifier,
etc.
Storage Preserve in well closed containers and protected
from light.
Preparation of mercuric acetate solution Dissolve 50 g of
mercuric acetate in 900 ml of methanol containing O. 5 ml of
glacial acetic acid, dilute to 1000 ml with methanol and mix.
Discard the solution if it changes to yellow. If it is turbid,
filter it. Discard it if it is still turbid or presents as yellow.
The solution should be freshly prepared, and preserve in an
amber glass bottle, stored in dark place.
Preparation and calibration of phthalic anhydride- pyridine
solution Preparation To 500 ml of pyridine containing less
 Poly (lactide-co--glycolide) ( 5050) ( For Injection)
than O. 1 % of water (or dissolve 30 g of phthalic anhydride in
500 ml of pyridine, distill and use the middle portion of the
distillate) add 72 g of phthalíc anhydride, shake vigorously
or heat on a water bath at 40ºC to dissolve completely, allow
to stand overnight with protecting from light.
Standardization Mix 10 ml of the above solution,
accurately measured, with 25 ml of pyridine and 50 ml of
water, allow to stand for 15 minutes, add O. 5 ml of a
mixture of phenolphthalein- pyridine solution ( 1 - 100),
titrate with sodium hydroxide (0. 5 mol/L) VS: consumes
between 37. 6 ml and 40. O ml.
Poly {lactid~co-glycolide) {5050 ) {For
Injection)
CPLGA5050)
o o o o
11 11 11 11
H+o-1H--c-~1H-c-ifo-cH2-C-O-CH2-C*'O-H
CH3 CH3
H (C6 Ha Ü4 )n (C4 H4 04 )m OH
[26780-50-7]
PLGA 5050 is a ring opening copolymer of lactide and
glycolide cyclic dimer under the catalytical reaction of
nucleophilic initiator. The molar ratio of lactide and glycolide
is 50 : 50, the intrinsic viscosity meets the requirements in
the attached table, and the molecular vveight distribution
coefficient D (Mw/Mn) is not more than 2. 5.
Description White to slightly yellow powder or granules,
almost odorless.
Freely soluble in chloroform, dichloromethane, acetone and
dimethylfomamide; slightly soluble m ethyl acetate;
insoluble in water, ethanol and ether.
Intrinsic viscosity Weigh O. 5 g accurately, transfer into a
100 ml volumetric flask, add 70 ml of chloroform, sonicate
to dissolve, allow to cool to the room temperature, dilute to
the volume with chloroform, shake well. Carry out the
method at 25ºC for determination of viscosity ( 0633 method
2) , it complies with the requirements in the table below.
ldentification The infrared absorption spectrum of the
solution obtained from the test of intrinsic viscosity is
concordant with that of the reference solution ( 0402).
Acidity Take a quantity of the substance being examined,
grind, add a quantity water, sonicate for 10 minutes to
obtain a suspension of 2. O mg per ml, filter. The pH value
of successive filtra te is 5. 0-7. O ( 0631 ) .
Clarity of the solution Take O. 5 g, add 25 ml of dichl-
oromethane to dissolve. The solution is clear ( 0902).
Molecular weight distribution Take a quantity of the subs-
tance being examined, accurately weighed, dissolve and
dilute with furanidine to produce a solution of 3 mg per ml,
shake well, place overnight at the room temperature as the
test solution. To 5 portions of polystyrene molecular weight
reference substances ( the range of molecular weight covers
the molecular weight of the substance being examined) ,
dissolve with furanidine to produce a solution of 3 mg per ml
as the reference solution. Carry out the method for size-
exclusion chromatography ( 0514), using a column packed
with gel and the furanidine as the mobile phase, and
refractive index detector is used; the temperature of detector
is 35ºC. Inject 20 µl of acetonitrile into the column, record
the chromatogram. The number of theoretical plates of the
column is not less than 10 000, calculated with reference to
the peak of acetonitrile.
Inject 20 µl of the reference solution onto the column, record
the chromatogram, then calculate the regression equation
with GPC software. Inject 20 µl of the test solution and
determine with the same method. Calculate the weight-
average molecular weight, number-average molecular weight
and molecular weight distribution of the test solution with
GPC software. The weight-average molecular is 7000-
170 000, the polydispersity D ( Mw /Mn ) is not more than
2. 5.
Molar ratio of glycolide and lactide W eigh 1O to 20 mg of the
substances being examined, add O. 6 to O. 8 ml of trich-
loromethane-D (containing TMS) to dissolve. Carry out the
method for nuclear magnetic resonance spectrometry
( 0441 ) . Record the integral area of methyne proton ( 4. 4-5. O
ppm) of glycolide unit and methylene proton (5. 1-5. 5 ppm)
of lactide unit. Calculate the mole percentage of lactide and
glycolide, and the mole percentage is 45 %-55 % and 45 %-
55 % , respectively.
Glycolide and lactide Take a quantity of ethyl acetate, weighed
accurately, dissolve with dichloromethane to produce a
solution of O. 125 mg per ml as the interna! standard
solution; take about O. 1 g, weighed accurately, transfer to a
10 ml volumetric flask, add 2 ml of interna! standard
solution, dissolve and dilute to the volume with dichloro--
methane, mix well as the test solution; take a volume of
glycolide and lactide respectively; add accurately a quantity of
interna! standard solution, dissolve with dichloromethane to
produce a solution containing 50 µg of glycolide, 100 µg of
lactide and 25 µg of isobutyl acetate per ml, as the reference
solution. Carry out the method for gas chromatography
( 0521 ) , using a column packed with 5 % phenyl-methyl
polysiloxane ( or similar polarity) as the stationary phase, the
temperature of column is 135ºC, the temperature of injection
is 250ºC, the temperature of detector is 300ºC. Inject 3 µl of
the test solution and reference solution onto the column
respectively. Calculate with peak area by the interna!
standard method, the content of lactide is not more than
l. 5 % , and the content of glycolide is not more than O. 5 % .
Residual solvents Methanol, acetone, dichloromethane and
toluene Take about O. 1 g, weighed accurately, transfer into
a 10 ml volumetric flask, dissolve and dilute to the volume
with dimethylfomamide as the test solution. Take a quantity
of methanol, acetone, dichloromethane and toluene, weighed
accurately, dissolve and dilute with dimethylfomamide to
produce a mixture solution containing 30 µg of methanol,
50 µg of acetone, 6 µg of dichloromethane, and 8. 9 µg of
toluene per ml, as the reference solution. Carry out the limit
test for residual solvents ( 0861 method 3 ) . Using the
column packed with 6 % cyanopropyl phenyl-94 % methyl
polysiloxane ( or similar polarity) as the stationary phase;
maintain the column temperature at 40ºC for 8 minutes, raise
the temperature to 200ºC by lOºC per minute; the
temperature of injection port is 180ºC, the temperature of
detector is 250ºC . Inject 3 µl of the test solution and
reference solution onto the column respectively. Calculate the
content with the peak area by the externa! standard method.
The content of methanol is not more than O. 3 % , acetone is
not more than O. 5 % , dichloromethane is not more than
O. 05 % and toluene is not more than O. 05 % .
Water Not more than l. O% ( 0832 method 1 ) . using
chloroform as the solvent.
 Poly (lactide-co-glycolide) ( 7525) (For Injection)

Residue on ignition Not more than O. 2% , using l. O g ( 0841 ) .

Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), using the residues obtained from Residue
on ignition: not more than O. 001%. Poly ( lactide-co-glycolide) ( 7 525 ) ( For
Arsenic Take l. O g, add l. O g of calcium hydroxide, mix Injection)
well, add a quantity of water to stir well. After dryness,
heat gently until thoroughly charred, ignite at 500-600ºC CPLGA7525)
until complete incineration, allow to cool, add 5 ml of o o o o
hydrochloric acid and 23 ml of water. Carry out the limit test 11 11 11 11+
for arsenic ( 0822 method 1 ) : it complies with the H{o-TH-C-O-TH-c-ij-o-cH2-C-O-CH2-C mo~H
requirement eo. 0002 % ) . CH3 CH3
Tin Take O. 25 g into a PTFE digestion tank, add 6. O ml of H CCs Hs 04 )n (C4 H4 04 )m OH
nitric acid and 2. O ml of concentrated hydrogen peroxide [26780-50-7]
solution, cover the lid, tighten the coat, transfer into PLGA 7525 is a ring opening copolymer of lactide and
microwave digestion instrument for digestion. After the glycolide cyclic dimer under the catalytical reaction of
complete digestion, take out the inner digestion tank, heat nucleophilic initiator. The molar ratio of lactide and glycolide
on the electric hot plate slowly to expel the reddish brown is 75 : 25, the intrinsic viscosity meets the requirements in
gas, transfer the digestion solution into a 100 ml volumetric the attached table, and the molecular weight distribution
flask with ultra-pure water, and dilute to the volume with coefficient D CMw/Mn) is not more than 2. 5.
the ultra-pure water, mix well, as the test solution. Prepare
the blank solution by the same method. Carry out the Description White to slightly yellow powder or granules,
method for inductively coupled plasma atomic emission almost odorless.
spectrometry ( 0411 ) . The content of tin is not more than Freely soluble in chloroform, dichloromethane, acetone and
o. 015%. dimethylfomamide; slightly soluble in ethyl acetate;
insoluble in water, ethanol and ether.
Microbial limits Carry out the test for microbial limit
( 1105 and 1106), the number of total aerobic bacteria! is not Intrinsic viscosity Weigh O. 5 g accurately, transfer into a
more than 100 cfu per g, the number of coliform bacteria is 100 ml volumetric flask, add 70 ml of chloroform, sonicate
to dissolve, allow to cool to the room temperature, dilute to
not detected per g, salmonella is not detected per 1O g.
the volume with chloroform, mix well. Carry out the method
Bacteria endotoxin Carry out the limit test for bacteria! at 25ºC for determination of viscosity ( 0633 method 2), it
endotoxin ( 1143 ) : not more than O. 9 EU per 1 mg of complies with the requirements in the table below.
substance being examed.
ldentification The infrared absorption spectrum of the
Sterility Capplicable to sterile products without sterilization solution obtained from the test of intrinsic viscosity is
p~) Carry out the method for sterility test ( 1101). it concordant with that of the reference solution <0402).
complies with the requirement.
Acidity Take a quantity of the substance being examined,
Category Pharmaceutical excipients Cfor injection) , sust- grind, add a quantity of water, soniacte for 10 minutes, to
ained-release material. suspend and obtain a suspension of 2. O mg per ml, filter.
Storage Preserve in tightly closed containers, stored under The pH value of the successive filtra te is 5. 0-7. O ( 0631 ) .
the refrigerated or frozen condition C-20°C-8°C ). Maintain Clarity of the solution Take O. 5 g, add 25 ml of dichl-
the product' s temperature close to the room temperature oromethane to dissolve. The solution is clear ( 0902).
befare the seal is removed to decrease the degradation caused
Molecular weight distribution Take a quantity of the
by the condensation water.
substance being examined, accurately weighed, dissolve and
The limits of intrinsic viscosity dilute with furanidine to produce a solution of 3 mg per ml,
shake well, place overnight at the room temperature as the
The labeled Range of intrinsic test solution. To 5 portian of polystyrene molecular weight
viscosity eml/ g) viscosity eml/ g) reference substances ( the range of molecular weight covers
10 5-15 the molecular weight of the substance being examined) ,
dissolve with furanidine to produce a solution at 3 mg per ml
15 10-20 as the reference solution. Carry out the method for size-excl-
20 15-25 usion chromatography ( 0514), using a column packed with
gel and the furanidine as the mobile phase. Refractive index
25 20-30 detector is used; the temperature of detector is 35ºC. Inject
30 25-35 20 µl of acetonitrile onto the column, record the chrom-
atogram, the number of theoretical plates of the column is
35 30-40 not less than 10 000, calculated with reference to the peak of
40 35-45 acetoni trile.
Inject 20 µl of the reference solution into the column, record
50 45-55
the chromatogram, then calculate the regression equation
60 50-70 with GPC software. Inject 20 µl of the test solution and
determine with the same method. Calculate the weight-
70 60-80
average molecular weight, number-average molecular weight
80 70-90 and molecular weight distribution of the test solution with
GPC software. The weight-average molecular is 7000-
90 80-100
170 000, the poly dispersity D CMw/ Mn) is not more than
 Poly (lactide-co-glycolide) (7525) (For lnjection)

2. 5. requirement (O. 0002%).

Molar ratio of glycolide and lactide Weigh 1O to 20 mg of Tin Take O. 25 g into a PTFE digestion tank, add 6. O ml of
the substances being examined, add O. 6 to O. 8 ml of nitric acid and 2. O ml of concentrated hydrogen peroxide
trichloromethane -D ( containing TMS) to dissolve. Carry solution, cover the lid, tighten the coat, transfer into
out the method for nuclear magnetic resonance spectrometry microwave digestion instrument for digestion. After the
( 0441 ) . Record the integral area of methylene proton ( 4. 4- complete digestion, take out the inner digestion tank, heat
5. O ppm) of glycolide unit and methylene proton ( 5. 1-5. 5 on the electric hot plate slowly until the reddish brown gas
ppm) of lactide unit. Calculate the mole percentage of lactide evapora tes completely, transfer the digestion solution into a
and glycolide, and the mole percentage is 70 %-80 % and 100 ml volumetric flask with ultra-pure water, and dilute to
20 %-30 % , respectively. the volume with the ultra-pure water, mix well, as the test
Glycolide and lactide Take a quantity of ethyl acetate, weighed solution. Prepare the blank solution by the same method.
accurately, dissolve with dichloromethane to produce a solution Carry out the method for inductively coupled plasma atomic
of O. 125 mg per ml as the internal standard solution; take gmission spectronetry ( 0411 ) . The content of tin is not
about O. 1 g, weighed accurately, transfer to a 10 ml more than O. 015%.
volumetric flask, add 2 ml of internal standard solution, Microbial limits Comply with the test for microbial limit
dissolve and dilute to the volume with dichloromethane, mix ( 1105 and 1106) , the number of total aerobic bacterial is not
well as the test solution; take a quantity of glycolide and more than 100 cfu per g, coliform bacteria is less than 100
lactide respectively, add accurately a quantity of interna! CFU per g, salmonella is absent per 10 g.
standard solution, dissolve with dichloromethane to produce Bacteria endotoxin Carry out the limit test for bacterial
a solution containing 50 µg of glycolide, 100 µg of lactide and endotoxin ( 1143 ) : not more than O. 9 EU per 1 mg of
25 µg of isobutyl aceta te per ml, as the reference solution.
substance being examed.
Carry out the method for gas chromatography ( 0521 ) , using
a column packed with 5 % phenyl-methyl polysiloxane ( or Sterility ( intended use for the sterile preperations without
similar polarity) as the stationary phase, the temperature of terminal sterilization) Carry out the method for sterility test
column is 135ºC, the temperature of injection port is 250ºC, ( 1101), it complies with the requirement.
the temperature of detector is 300ºC. lnject 3 µl of the test Category Pharmaceutical excipients ( for injection ) ,
solution and reference solution onto the column respectively. sustained-release material.
Calculate with peak area by the internal standard method,
Storage Preserve in tightly closed containers, and stored
the content of lactide is not more than l. 5 % , and the content
under the refrigerated or frozen condition ( - 20ºC-8ºC).
of glycolide is not more than O. 5 %.
Maintain the product; s temperature close to the room
Residual solvents Methanol, acetone, dichloromethane and temperature befare the seal is removed to decrease the
toluene Take about O. 1 g, weighed accurately, transfer it degradation caused by the condensation water.
into a 10 ml volumetric flask, dissolve and dilute to the
The limits of intrinsic viscosity
volume with dimethylfomamide as the test solution. Take
quantities of methanol, acetone, dichloromethane and
toluene, weighed accurately, dissolve and dilute with dimeth- The labeled Range of intrinsic
ylfomamide to produce a mixture solution containing 30 µg of viscosity (ml/g) viscosity ( ml/ g)
methanol, 50 µg of acetone, 6 µg of dichloromethane,
8. 9 µg of toluene per ml, as the reference solution. Carry 10 5-15
out the limit test for residual solvents ( 0861 method 3).
Using the column packed with 6% cyanopropyl phenyl-94% 15 10-20
methyl polysiloxane ( or similar polarity) as the stationary
phase; maintain the column temperature at 40ºC for 8 20 15-25
minutes, raise the temperature to 200ºC by lOºC per minute;
the temperature of injection port is 180ºC, the temperature 25 20-30
of detector is 250ºC . lnject 3 /ll of the test solution and
reference solution onto the column respectively. Calculate the 30 25-35
cdntent with the peak area by the externa! standard method.
The content of methanol is not more than O. 3 % , acetone is 35 30-40
not more than O. 5 % , dichloromethane is not more than
O. 05 % and toluene is not more than O. 05 % . 40 35-45

Water Take a quantity of the substance beng examined, 50 45-55

using chloroform as the solvent: not more than l. 0% ( 0832
method 1 ). 60 50-70
Residue on ignition Not more than O. 2% ( 0841), using l. O g.
70 60-80
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), using the residues obtained from residue 80 70-90
on ignition: not more than O. 001%
Arsenic Take l. O g, add l. O g of calcium hydroxide, mix 90 80-100
well, add a quantity of water, and stir well. After dryness,
heat gently until thoroughly charred, ignite at 500-600ºC
until complete incineration, allow to cool, add 5 ml of
hydrochloric acid and 23 ml of water. Carry out the limit test
for arsenic ( 0822 method 1 ) : it complies with the
 Poly (lactide-co-glycolide) ( 8515) (For Injection)
Poly ( Iactide-co-glycolide ) ( 8515 )
( For lnjection)
(PLGA8515)
H (C6 Hs 04 )n (C4 H4 Ü4 )m OH
[26780-50-7]
PLGA 8515 is a ring opening copolymer of lactide and
glycolide cyclic dimer under the catalytical reaction of
nucleophilic initiator. The molar ratio of lactide and glycolide
is 85 : 15, the intrinsic viscosity meets the requirements in
the attached table, and the molecular weight distribution
coefficient D (Mw/Mn) is not more than 2. 5.
Description White to slightly yellow powder or granules,
almost odorless.
Freely soluble in chloroform, dichloromethane, acetone and
dimethylfomamide; slightly soluble m ethyl acetate;
insoluble in water, ethanol and ether.
lntrinsic viscosity Weigh O. 5 g accurately, transfer into a
100 ml volumetric flask, add 70 ml of chloroform, sonicate
to dissolve, allow to cool to the room temperature, dilute to
the volume with chloroform, shake well. Carry out the
method at 25ºC for determination of viscosity <0633 method
2) , it complies with the requirements in the table below.
ldentification The infrared absorption spectrum of the
solution obtained from the test of intrinsic viscosity is
concordant wíth that of the reference soiution ( 0402).
Acidity Take a quantity of the substance being examined,
grind, add a quantity of water, sonicate for 10 minutes, to
obtain a suspension of 2. O mg per ml, filter. The pH value
of the successive filtrate is 5. 0-7. O <0631).
Clarity of the solution Take O. 5 g, add 25 ml of dichloro-
methane to dissolve. The solution is clear <0902).
Molecular weight distribution Take a quantity of the
substance being examied, accurately weighed, dissolve and
dilute with furanidine to produce a solution of 3 mg per ml,
shake well, place overnight at the room temperature as the
test solution. To 5 portian of polystyrene molecular weight
reference substances ( the range of molecular weight covers
the molecular weight of the substance being examined) ,
dissolve it with furanidine to produce a solution of 3 mg
per ml as the reference solution. Carry out the method for
size-exclusion chromatography < 0514 ) , using a column
packed with gel and the furanidine as the mobile phase.
refractive index detector is used; the temperature of detector
is 35ºC. Inject 20 µl of acetonitrile onto the column, record
the chromatogram. The number of theoretical plates of the
column is not less than 10 000, calculated with reference to
the peak of acetonitrile.
Inject 20 µl of the reference solution onto the column, record
the chromatogram, then calculate the regression equation
with GPC software. Inject 20 µl of the test solution and
determine with the same method. Calculate the weight-average
molecular weight, number-average molecular weight and
molecular weight distribution of the test solution with GPC
software. The weight-average molecular is 7000-170 000, the
polydispersity D (Mw/Mn) is not more than 2. 5.
Molar ratio of glycolide and lactide W eigh 10 to 20 mg of
the substances being examined, add O. 6 to O. 8 ml of
trichloromethane -D ( containing TMS) to dissolve. Carry
out the method for nuclear magnetic resonance spectrometry
<0441 ) . Record the integral area of methylene proton ( 4. 4-
5. O ppm) of glycolide unit and methylene proton ( 5. 1-5. 5
ppm) of lactide unit. Calculate the mole percentage of lactide
and glycolide, and the mole percentage is 80 %-90 % and
10 %-20 % , respectively.
Glyoolide and lactide Take a quantity of ethyl acetate, weighed
accurately, dissolve with dichloromethane to produce a solution
of O. 125 mg per ml as the internal standard solution; take
about O. 1 g, weighed accurately, transfer to a 10 ml
volumetric flask, add 2 ml of internal standard solution,
dissolve and dilute to the volume with dichloromethane,
shake well as the test solution; take a quantity of glycolide
and lactide respectively, add accurately a quantity of internal
standard solution, dissolve with dichloromethane to produce
a solution containing 50 µg of glycolide, 100 µg of lactide and
25 µg of isobutyl acetate per ml, as the reference solution.
Carry out the method for gas chromatography <0521), using a
column packed with 5 % -phenyl methyl polysiloxane ( or similar
polarity) as the stationary phase, the temperature of column is
135ºC , the temperature of injection port is 250ºC, the
temperature of detector is 300ºC. Inject 3 µl of the test solution
and reference solution into the column respectively. Calculate
with peak area by the internal standard method, the content of
lactide is not more than l. 5 % , and the content of glycolide is
not more than O. 5 % .
Residual solvents Methanol , acetone , dichloromethane and
toluene Take about O. 1 g, weighed accurately, to a 10 ml
volumetric flask, dissolve and dilute to the volume with
dimethylfomamide as the test solution. Take a quantity of
methanol, acetone, dichloromethane and toluene, weighed
accurately, dissolve and dilute with dimethylfomamide to
produce a mixture solution containing 30 µg of methanol, 50
µg of acetone, 6 µg of dichloromethane, 8. 9 µg of toluene
per ml, as the reference solution. Carry out the limit test for
residual solvents < 0861 method 3 ) . Using the column
packed with 6 % cyanopropyl phenyl-94 % methyl poly-
siloxane ( or similar polarity) as the stationary phase;
maintain the column temperature at 40ºCfor 8 minutes, raise
the temperature to 200ºC by lOºC per minute; the
temperature of injection port is 180ºC, the temperature of
detector is 250ºC . Inject 3 µl of the test solution and
reference solution into the column respectively. Calculate the
content with the peak area by the external standard method.
The content of methanol is not more than O. 3 % , acetone is
not more than O. 5 % , dichloromethane is not more than
O. 05 % and toluene is not more than O. 05 % .
Water Take a quantity of the sunstance being examined,
using chloroform as the solvent: not more than l. O% <0832
method 1 ).
Residue on ignition Not more than O. 2% <0841), using l. O g.
Heavy metals Carry out the limit test for heavy metals
<0821 method 2) , using the residues obtained from residue
on ignition: not more than O. 001%.
Arsenic Take l. O g, add l. O g of calcium hydroxide, mix
well, add a quantity of water to stir well. After dryness,
heat gently until thoroughly charred, ignite at 500-600ºC
until complete incineration, allow to cool, add 5 ml of
hydrochloric acid and 23 ml of water. Carry out the limit test
for arsenic < 0822 method 1 ) : it complies with the
requirement (0. 0002%).
 Polyacrylic Resin II

Tin Take O. 25 g into a PTFE digestion tank, add 6. O ml of in ethanol.

nitric acid and 2. O ml of concentrated hydrogen peroxide
solution, cover the lid, tighten the coat, transfer into
microwave digestion instrument for digestion. After the
complete digestion, take out the inner digestion tank, heat
on the electric hot plate slowly until the reddish brown gas
evapora tes completely, transfer the digestion solution into a
100 ml volumetric flask with ultra-pure water, and dilute to
the volume with the ultra-pure water, mix well, as the test
solution. Prepare the blank solution by the same method.
Carry out the method for Inductively coupled plasma-atomic
emission spectrocutry ( 0411 ) . The content of tin is not
more than O. 015%.
Microbial limits Carry out the test for microbial limit
( 1105), the number of total aerobic bacteria! is not more
than 100 CFU per g, the number of coliform bacteria is less
than 100 CFU per g, salmonella is absent per 10 g.
Bacteria endotoxin Carry out the limit test for bacteria!
endotoxin ( 1143 >: not more than O. 9 EU per 1 mg of
substomce being examed.
Sterility ( intended use for the sterile preperations without
terminal sterilization) Carry out the method for sterility test
( 1101), it complies with the requirement.
Category Pharmaceutical excipients ( for injection ) ,
sustained-release material.
Storage Preserve in tightly closed containers, and stored
under the refrigerated or frozen condition ( - 20°C-8°C).
Maintain the product' s temperature clase to the room
temperature befare the seal is removed to decrease the
degradation caused by the water condensation water.
The limits of intrinsic viscosity
The labeled Range of intrinsic
viscosi ty ( ml/ g) viscosity (ml/g)
10 5-15
15 10-20
20 15-25
Soluble in warm ethanol within 1 hour (break the strip to
1 cm long and the powder <loes not need to tritura te) ,
insoluble in water.
Acid value Dissolve about O. 5 g, accurately weighed, in a
250 ml conical flask with 25 ml of 75 % ethanol (neutral to
phenolphthalein IS) by warming. Allow to cool, dropwise 15
ml of sodium hydroxide (O. 1 mol/L) VS accurately, add 5 g
of sodium chloride and 10 ml of water until a pink colour
persists for 30 seconds. The acid value is 300-330, calculated
on the dried basis ( 0713).
Identification The infrared absorption spectrum ( 0402 > is
concordant with the reference spectrum of Polyacrylic Resin
II CRS.
Viscosity To 6. O g, add 100 ml of ethanol, warm to
dissolve, the kinematic viscosity at 25ºC is not more than
50 mPa • s, using a rota tional visco meter ( 0633 method 2).
Acidity Dissolve 3. O gin 100 ml of 75% ethanol (pH about
7) by warming and allow to cool, pH 4. 0-6. O ( 0631 ).
Loss on drying Dry to constant weight at llOºC, loses not
more than 10. O% ( 0831).
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2) , using l. O g: not more than O. 003 % .
Arsenic Heat l. O g in a 150 ml conical flask with 5 ml of
sulfuric acid, heat until it is completely charred, add
concentrate hydrogen peroxide solution, dropwise (if a lot of
foam evolved, stop heating and rotate the conical flask to
prevent conglomeration of the unreactant on the bottom)
until the solution becomes colourless. Allow to cool, add
cautiously 10 ml of water, heat again, the sulfur trioxide is
evolved, cool and add slowly a quantity of water to 28 ml.
Carry out the limit test for arsenic ( 0822), it complies with
the requirement (0. 0002%).
Category Pharmaceutical excipients, coating material and
release retardant, etc.
Storage Preserve in tightly closed cintaíners, and stored in a
cool place.

25 20-30

30 25-35 Polyacrylic Resin ][

35 30-40
Polyacrylic Resin fil is a copolymer of methylacrylic acid and
40 35-45 methyl methylacrylate in a ratio of 35 : 65.

50 45-55 Description A white strip or powder, easy to conglomerate

in ethanol.
60 50-70 Soluble in warm ethanol within 1 hour (break the strip to
1 cm long and the powder <loes not need to tritura te) ,
70 60-80 insoluble in water.
80 70-90 Acid value Dissolve about O. 5 g, accurately weighed, in a
250 ml conical flask with 25 ml of 75 % ethanol (neutral to
90 80-100 phenolphthalein IS) by warming. Allow to cool, dropwise
15 ml of sodium hydroxide (0. 1 mol/U VS accurately, add
5 g of sodium chloride and coml of water until a pink colour
persists for 30 seconds. The acid value is 210-240, calculated
on the dried basis ( 0713).
Polyacrylic Resin ll Identification The infrared absorption spectrum ( 0402) is
concordant with the reference spectrum of Polyacrylic Resin
Polyacrylic Resin II is a copolymer of methylacrylic acid and fil CRS.
methyl methylacrylate in a ratio of 50 : 50.
Viscosity To 6. O g, add 100 ml of ethanol, warm to
Description A white strip or powder, easy to conglomerate dissolve, the kinematic viscosity at 25ºC is not more than

50 mPa • s, using a rotational viswmeter <0633 method 2).
Acidity Dissolve 3. O g in 100 ml of 75 % ethanol (pH about
7) by warming, stand to cool, pH 4. 0-6. O <0631 ) .
Los.s on drying Dry to constant weight at llOºC, loses not
more than 10. O% <0831 ) .
Heavy metals Carry out the limit test far heavy metals
<0821 method 2) , using l. O g: not more than O. 003 % .
Arsenic Transfer l. O g in a 150 ml conical flask, add 5 ml
of sulfuric acid, heat until it is completely charred, add
concentrate hydrogen peroxide solution, dropwise (if a lot of
foam is evolved stop heating and rotate the conical flask to
prevent conglomeration of the unreactant on the bottom)
until the solution becomes colourless. Allow to cool, add
cautiously 10 ml of water, heat again, the sulfur trioxide is
evolved, cool and add slowly a quantity of water to 28 ml.
Carry out the limit test far arsenic <0822) , it complies with
the requirement (0. 0002%).
Category Pharmaceutical excipients, coating material and
release retardant, etc.
Storage Preserve in tightly closed cintainers, stored in a
cool place.
Polyacrylic Resin N
Polyacrylic Resin N is a copolymer of dimethylaminaethyl
methylacrylate and methylacrylic esters.
Description Pale yellow granule or flake salid, odour,
characteristic.
Soluble in warm ethanol ( within 1 hour) ; sparingly soluble
in hydrochloric acid solution ( 9- 1000) ( within 1 hour) ,
insoluble in water.
Reltive density Dissolve 10. 25 g with isopropanal-acetone
(3 : 2) in a 100 ml volumetric flask and dilute to volume as
the test solution. The relative density of the test solution is
0.810-0.820 (0601).
Refractive index l. 380-1. 395 < 0622 ) , using the test
solution obtained in the test far Relative density.
Alkaline value Dissolve about O. 3 g, accurately weighed,
with 25 ml of neutral ethanol (reveals yellow to Bromophenol
blue IS), add 20 ml of hydrochloric acid (O. 1 mol/L) VS,
accurately measured, and several drops of bromophenol blue
IS, mix well. Titrate with sodium hydroxide (O. 1 mol/L)
VS until the solution becomes bluish-green, perform a blank
determination and make any necessary correction. Calculate
the alkaline value with following equation. The alkaline value
is 162. 0-198. O.
. l (B -A) X 5. 61
Alka lme va ue = W 30 minutes and maintain the temperature at 25 ±O. 1 ºC.
Where A is volume consumed of sodium hydroxid
(O. 1 mol /L) VS of the substance being examined;
B is volume consumed in blank determined;
W is the weight of the substance being examined.
ldentification Apply about 10 µl of the solution obtained in
the test far Viscosity to a blank potassium bromide clise of
13 mm in diameter, heat to evaporate the solvent. The IR
spectrum < 0402) is consistent with that of Polyacrylic Resin
N CRS treated in the same manner.
Viscosity Dissolve 12. 00 g in a 100 ml volumetric flask and
dilute to volume with ethanol, the kinematic viscosity at
Polyethylene Oxide 1 ¡~j,<
30ºC is 5-20 mPa • s, using a model NDJ-79 rotational
viscometer <0633 method 3 ) .
Colour of solution The absorbance at 420 nm is not more
than O. 20 <0401 ) , using the test solution obtained in the
test far Relative density.
Los.son drying Dry to constant weight at llOºC, loses not
more than 4. O% <0831 ) .
Residue on ignition Not more than O. 2% <0841 ) , using
l. o g.
Heavy metals Carry out the limit test far heavy metals
<0821 method 2) , using the residue obtained in the test far
Residue on ignition; not more than O. 001%.
Arsenic Add 1 O ml of sulfuric acid to 1. O g, heat until it
is completely charred, add hydrogen peroxide solution
dropwise until the solution is colourless, allow to cool.
Add 1 O ml of water, heat until the sulfur trioxide is
evolved. Cool and add a quantity of water to 28 ml. Carry
out the limit test far arsenic <0822), it complies with the
requirement (O. 0002 % ) .
Category Pharmaceutical excipients, coating material,
release retardant, etc.
Storage Preserve in tightly closed containers, and stored in
a cool place.
Polyethylene Oxide
n = 2000-20000
[25322-68-3]
Polyethylene oxide is a non-ionic homopolymer of epoxyethane
(polymerized ethylene) produced under high temperature and
high pressure in the presence of initiator and catalyst. The
molecular formula is HO ( CH2 CH2 O) n H, where n is
average number of oxyethylene group, n = 2000-20000.
Description White to almost-white gliding powder.
ldentification (1) The infrared absorption spectrum <0402)
is concordant with that of the reference spectrum of
polyethylene oxide CRS.
(2) Transfer 12 g of the substance being examined into a 800
ml of beaker, add 125 ml of dehydrated isopropanol to the
beaker. Stir the mixture at a rate to ensure that slurry is
formed. Add 588 ml of water and quickly stir far 1 minute
( avoid water splashing) and continue to stir slowly far
3 hours until no jelly can be seen. Put it in a Water bath far
Carry out the method far viscosity <0633 method 3 ) . The
viscosity is 10-180 cP.
Alkalinity pH 8. 0-10. O <0631 ) , using the solution obt-
ained in Identification (2).
Los.s on drying When dried over phosphrorouspentoxide to
constant weight at room temperature, loses not more than
l. o% <0831>.
Silicon dioxide Accurately weigh l. O g of the substance
being examined into a previously ignited platinum crucible.
Add 4 drops of sulfuric acid. Heat to remove the sulfuric acid
thoroughly. lgnite the crucible at 700ºC to constant weight.
 Polyethylene Oxide
Wet the residue carefully with 1 ml of water and add
20 drops hydrofluoric acid slowly. Evaporate slowly to
dryness, then ignite at 700ºC far 10 minutes, cool to room
temperature and weigh. Repeat the addition of hydrofluoric
acid, evaporation, and ignition to constant weight. Calculate
the percentage of silicon dioxide residue on ignition from the
difference between the net weights befare and after the
hydrofluoric acid treatment. The residue is not more than
3.0%.
Residue on ignition Accurately weigh l. O g of the substance
being examined into a previously ignited platinum crucible.
Add 4 drops of sulfuric acid. Heat to remove the sulfuric acid
thoroughly. lgnite the crucible at 700ºC to constant weight.
Wet the residue carefully with 1 ml of water and add
20 drops hydrofluoric acid slowly. Evaporate slowly to
dryness, and then ignite at 700ºC far 10 minutes, cool to
room temperature and weigh. Repeat the addition of
hydrofluoric acid, evaporation, and ignition, to constant
weight. Calculate the percentage of silicon dioxide residue on
ignition from the final net weight. The residue is not more
than 2. 0%.
Heavy metals Not more than O. 001% ( 0821 method 2),
using l. O g.
Alkaline earth metal W eigh accurately 1 g of the substance
being examined, add 100 ml of isopropanol, stir and add
600 ml of water, stir to dissolve with high speed. Add
2 5 ml of 3 O% triethanolamine solution and 2 5 ml of 1 O%
sodium hydroxide solution, accurately add 25 ml of disodium
edentate (O. 01 mol/L) VS and continue stirring far 15
minutes. Add about 1 g of hydroxynaphthol blue IS and
titrate with calcium nitrate O. 01 mol/L) VS until the colour
changes to fuchsia. Perfarm a blank determination and make
any necessary correction. Each ml of calcium nitrate
(O. 01 mol/L) VS is equivalent to O. 5608 mg of CaO. The
content of alkaline earth metal is not more than 1. O% ,
calculated on CaO.
Particle size Unless otherwise specified, comply with the
requirement far Determination of Particle Size and Particle
Size Distribution ( 0982 method 2 ) . 100 % of the total
sample mass is passing through the No. 1 sieve. More than
96% and less than 100% of the total sample mass is passing
through the No. 2 sieve.
Ethylene oxide and dioxin Accurately weigh l. O g of the
substance being examined in a 20 ml headspace vial, heat far
30 minutes at lOOºC. Cool to room temperature, as the test
solution. Accurately weigh 40 g of acetone into 1000 ml of
volumetric flask, add O. 05 g of ethylene oxide CRS and O. 5 g
of dioxin CRS, seal and mix, as the reference stock solution.
To l. O g of the substance being examined, accurately
weighed, in 4 portions, to a 20 ml headspace vials, add
accurately 2. O, 4. O, 6. O and 8. O µl of the reference stock
solution respectively, seal, heat at lOOºC far 30 minutes,
cool to room temperature as the reference solution. Add
O. 1 ml of O. 001 % acetaldehyde solution, freshly prepared,
to O. 5 ml of reference stock solution, seal, and shake as a
system suitability test solution ( 1 ). Transfer 6 µl of the
reference stock solution to a headspace vial, seal, and shake
as a system suitability test solution (2).
Carry out the method far gas chromatograph <0521), using
a column packed with divinylbenzene-ethylene glycol-
dimethylacrylate as the stationary phase ( G45 ) ; maintain the
column temperature at 70ºC far 5 minutes, raise the
temperature to 200ºC by lOºC per minute and maintain far
5 minutes. The temperature of the injection port is 200ºC.
The temperature of the detector is 250ºC. Use the headspace
injection method. The equilibration temperature is lOOºC and
maintain far 30 minutes. The volume of injection is l. O ml.
Inject the gaseous sample in the system suitability vial onto
the column and record the chromatogram. The resolution
factor between the peaks corresponding to acetaldehyde,
ethylene oxide, and dioxin is more than 2. O. The resolution
factor between acetone and ethylene oxide is not less than
2. O. Repeat the procedure at least 5 times. The relative
standard deviation of the 5 areas of peak of ethylene oxide is
not greater than 5 % .
lnject separately the gaseous sample each in the test vial and
the reference vial into the column, then record the peak
area of ethylene oxide and dioxin. Plot the responses of the
test solution and the reference solutions versus the content,
in µg, of ethylene oxide or dioxin. Draw the straight line
best fitting the five points, and calculate the correlation
coefficient far the line, which is not less than O. 99.
Extrapolate the line until it intercepts the content axis on the
negative side. From the intercept, determine the total
amount, in µg, of ethylene oxide or dioxin in the test
solution. Calculate the content with respect to the peak area
obtained in the chromatogram by the standard addition
method, ethylene oxide is not more than O. 0001 % and
dioxin is not more than O. OO1 % .
2, 6-Di-tert-butyl-4-methylphenol Dissolve a quantity of
n-heneicosane into acetone to obtain a solution containing
O. 6 mg per ml of n-heneicosane as the interna} reference
solution. Dissolve 20 mg of 2, 6-di-tert-butyl-4-methylphenol,
accurately weighed, to obtain a solution containing O. 2 mg per
ml of 2, 6-di-tert-butyl-4-methylphenol as the reference
stock solution. Transfer accurately 15 ml of the reference
stock solution and 5 ml of the interna} reference solution,
mix as the reference solution. Accurately weigh 3 g of the
substance being examined, to a 25 ml of volumetric flask.
Add 15 ml of acetone and 5 ml of the interna} reference
solution, shake far 30 minutes and centrifuge, take the
supernatant as the test solution. Separately inject 1 µl of the
test solution and the reference solution, carry out the method
far gas chromatograph ( 0521 ) , using a column packed with
5 % phenyl-95 % methylpolysiloxane as the stationary phase;
maintain the column temperature at 50ºC far 2 minutes,
raise the temperature to 300ºC by 15ºC per minute and
maintain far 10 minutes. The temperature of the injection is
275ºC. The temperature of the flame ionization detector
CFID) is 310ºC. The carrier gas is nitrogen and flow rate is
Z. 5 ml per minute. The resolution between the interna!
standard and 2, 6-di-tert-butyl-4-methylphenol complies
with the requirement. Calculate the content of 2, 6-di-tert-
butyl-4-methylphenol by external standard method: not more
than O. 1%.
Ethylene glycol and diethyleneglycol Dissolve 200 mg of
ethylene glycol CRS and 200 mg of diethylene glycol CRS
with absolute ethanol in a 100 ml volumetric flask, mix, as
the reference stock solution. Dissolve 200 mg of 1, 3-
butanediol CRS with absolute ethanol into a 100 ml
volumetric flask as the internal reference stock solution.
Transfer 2. 5 ml of the reference stock solution and 2. 5 ml of
the interna} reference stock solution to two 50 ml volumetric
flasks, as the reference solution ( 1) and the interna}
reference solution ( 1), respectively. Transfer accurately
each of l. O ml of the reference solution ( 1) and the internal
reference solution ( 1) to a 25 ml volumetric flask, dilute
with absolute ethanol to volume as the reference solution.
Weigh accurately about O. 1 g of the substance being
examined into a 25 ml volumetric flask, dilute with absolute
ethanol to volume mix well, filer, as the test solution. Carry

out the method far gas chromatograph ( 0521 ) , using a
column packed with 50 % phenyl-50 % methylpolysiloxane as
the stationary phase; maintain the column temperature at
60ºC far 5 minutes, raise the temperature to lOOºC by lOºC
per minute, then raise the temperature to l 70ºC by 4 ºC per
minute, then raise the temperature to 270ºC by lOºC per
minute and maintain far 2 minutes. The temperature of the
injection port is 270ºC . The temperature of the flame
ionization detector (FID) is 290ºC. The carrier gas is pure
nitrogen. Calculate the content of ethylene glycol and
diethylene glycol with respect to the area by the interna!
standard method, the content of ethylene glycol and
diethylene glycol is not more than O. 1 % respectively.
Category Pharmaceutical excipients, disintegrator,
dant, etc.
Storage Preserve in tightly closed containers and protect
from light.
Polyoxyl {35) Castor Oil
[61791-12-6]
Polyoxyl 35 Castor Oil contains mainly the tri-ricinoleate
ester of ethoxylated glycerol, with smaller amounts of
polyethylene glycol ricinoleate and the correspongding free
glycols. It results from the reaction of 1 mol of glycerol
ricinoleate with 35 mol of ethylene oxide.
Description A white, almost white or pale yellow paste or
viscous liquid; odour, faint and characteristic.
Very soluble in ethanol.
Relative density l. 05-1. 06 ( 0601 >.
Viscosity Kinematic viscosity 570-710 mm2 /s, at 25ºC
( 0633 method 1), using a capillary tube: 2 mm or suitable
sizein interna! diameter.
Acid value Not more than 2. O ( 0713), using 5 g.
Saponification value 65-70 ( 0713).
Hydroxyl value 65-78 (0713).
lodine value 25-35 ( 0713).
Peroxide value Not more than 5 ( 0713).
Identification ( 1) The infrared absorption spectrum (film
method) ( 0402) is concordant with the reference spectrum
of polyoxyl 35 castor oil CRS.
(2) To the aqueous solution ( 1 - 20) add bromine TS
dropwise, the colour of bromine TS disappears imme-
diately.
Acidity Dissolve l. O g in 10 ml of water, pH 5. 0-7. O
<0631 >.
Clarity and colour of solution Dissolve 5. O g in 50 ml of
water, the solution is clear and colourless ( 0901 and 0902).
Any opalescence produced is not more pronounced than that
of reference suspension 3 ( 0902 ) ; any colour produced is
not more intense than that of reference solution OY1 ( 0901
method 1 ).
Ethylene oxide and dioxane Transfer 1 g, accurately
weighed, to a 10 ml headspace vial, add accurately l. O ml of
purified water, clase tightly and mix well, equilibrate at
70ºC for 45 minutes as the test solution. Measure 300 µl of
ethylene oxide ( equivalent to about O. 25 g) into a 100 ml
volumetric flask containing 50 ml of treatedpolyethylene
Polyoxyl ( 35) Castor Oil 615
glycol 400 ( using a rotary evaporator removeany volatile
components by applying a temperature of 60ºC anda pressure
of l. 5 to 2. 5 kPa for 6 hours) , weigh the volumetric flask
befare and after adding ethylene oxide. Dilute to volume with
the same solvent, mix well as the reference stock solution of
ethylene oxide. Transfer accurately 1 g of the cold reference
stock solution of ethylene oxide to a 50 ml volumetric flask
containing 40 ml of treated polyethylene glycol 400, dilute to
volume with the same solvent. Weigh accurately 10 g to a 50
ml volumetric flask containing about 30 ml of water, dilute
to volume with water. Transfer accurately 10 ml to a 50 ml
volumetric flask, dilute to volume with water as ethylene
oxide reference solution. Dissolve a quantity of dioxane,
retar- accurately weighed, in water to produce a solution of O. 1 mg
per ml as dioxane reference solution. Transfer 1 g of the
substance being examined, accurately weighed, to a
headspace vial, add accurately O. 5 ml of ethylene oxide
reference solution and O. 5 ml of dioxane referencesolution,
clase tightly and mix well, equilibrate at 70ºC for 45 minutes
as the reference solution. Transfer O. 5 ml of ethyleneoxide
reference solution to a 10 ml headspace vial, add O. 1 ml of
freshly prepared O. 001 % acetaldehyde solution and O. 1 mlof
dioxane reference solution, clase tightly and mix well,
equilibrate at 70ºC for 45 minutes as the system suitability
solution. Carry out the method for gas chromatography
( 0521 ) , using a glass or quartzcapillary column packed with
dimethylpoly-siloxane as the stationary phase. Maintain the
column temperature at 50ºC for 5 minutes, raise the
temperature to 180ºC by 5ºC per minute, and then raise the
temperature to 230ºC by 30ºC per minute, maintain for 5
minutes (can be adjusted depending on the circumstance).
The temperature of injection port is 150ºC and the
temperature of detector is 250ºC using a flame ionization
detector. Headspace equilibrium temperature is 70ºC,
equilibrium time is 45 minutes. Inject l. O ml of the system
suitability solution the column. The linear velocity of carrier
gas is 20 cm/s, the spilt ratio is 1 : 20. Adjust the
attenuation so that the peak heights of ethylene oxide and
acetaldehyde are 15 % of the full scale of the chart. The
resolution factor between the peaks of acetaldehyde and
ethylene oxide is not less than 2. O, and the peak of dioxane is
detected with a signal-to-noise ratio of at least 5. Inject
separately l. O ml of the test solution and the reference
solution onto the column, repeat the injection at least three
times. The relative standard deviation of the 3 values
obtained for ethylene oxide is not more than 15 % , and the
relative standard deviation of the 3 values obtained for
dioxane is not more than 10% . Calculate by standard
addition method, the contents of ethylene oxide and dioxane
are not more than O. 0001 % and O. 001 % , respectively.
The content of ethylene oxide ( in parts per million) is
calculated from the expressions:
AtXC
Where is peak area of ethylene oxide m the
chromatogram obtained with the test
solution;
is peak area of ethylene oxide m the
chromatogram obtained with the reference
solution;
is mass of the substance being examined in
the test solution, g;
is mass of the substance being examined in
the reference solution, g;
e is the amount of ethylene oxide added to
the reference solution, µg.
 616 Polyoxyl ( 40) Stearate
The content ofdioxane (in parts per million) is calculated
from the expressions:
CDr XMt) - CDi XMr)
Where Di is peak area of dioxane in the chromatogram
obtained with the test solution;
Dr is peak area of dioxane in the chromatogram
obtained with the reference solution;
C is the amount of dioxane added to the ref erence
solution, µg.
Standardization of the reference stock solution of
ethylene oxide To 1 O ml of 5 O% magnesium chloride
suspension in absolute ethanol, add accurately 2 O ml of
ethanolichydrochloric acid O. 1 mol/L) VS, mix well and
allow to equilibrate overnight. Transfer 5 g of the reference
stück solution of ethylene oxide, accurately weighed, to the
above solution, mix well and allow to stand for 30 minutes.
Carry out the method for potentiometric titration ( 0701 >,
ti trate with ethanolic potassium ydroxide (O. 1 mol/U VS.
Perform a blank determination using polyethylene glycol 400
and make any necessary correction. Each ml of ethanolic
potassium hydroxide (O. 1 mol/U VS is equivalent to 4. 404 rng
of ethylene oxide.
Water Not more than O. 5 % <0832 method 1 ( 1 ) >.
Residual on ignition Not more than O. 2% <0841 >, using 1 g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2 >,
using the residue obtained in the test for
Residue on ignition: not more than O. 001%.
Arsenic Transfer L O g to a crucible, add 10 ml of
magnesium nitrate hexahydrate solution in 95 % ethanol
o-so)' heat gently to evaporate ethanol and then ignite.
To any residue of carbonization add a little nitric acid,
continue igniting. Cool and add 3 ml of hydrochloric acid,
heat in a water bath to dissolve the residue. Carry out the
limit test for arsenic < 0822 method 2 > : not more than
o. 0002%.
Sterility ( for sterile preparations without sterilizing filtration
procedure) Comply with the requirements for sterility test
<1101>.
Bacterial endotoxin ( for injection) Carry out the test for
bacteria! encotoxin <1143 >: not more than O. 012 EU per mg
of the substance being examined.
Category Pharmaceutical excipients, emulsifier solubilizer,
etc.
Storage Preserve in tightly closed containers, protected
from light.
Polyoxyl ( 40)
Stearate
[9004-99-3]
Poloxyl ( 40) Stearate is polyethylene glycol monostearate.
It is represented by the molecular formula C11 H 35 COO
CCH2CH2Ü)nH, where nis about 40.
Description White waxy solids, odourless.
Soluble in water and in ethanol, insoluble m ether and m
ethylene glycol.
Melting range 46-51 ºC <0612 >.
Acid value Not greater than 2 <0713 >.
Saponification value 25-35 <0713 >.
Hydroxyl value 22-38 <0713 >.
ldentification The infrared absorption spectrum ( 0402 > is
concordant with the reference spectrum of polyoxyl ( 40)
stearate CRS.
Alkalinity Dissolve 2. O g in 20 ml of ethanol, to 2 ml of the
solution add O. 05 ml of phenolsulfonphahalein IS, a red
colour is not produced.
Clarity and colour of solution Dissolve l. O g in 20 ml of
water, the solution is clear and colourless ( 0901 and 0902 >;
any colour produced is not more intense than that of reference
solution Y 6 <0901 >.
Free polyethylene glycols Accurately weigh about 6 g to a 500 ml
separator, add 50 ml of ethyl acetate, shake to dissolve,
extract the solution with two potions of 50 ml of 29 % sodium
chloride solution. Combine the aqueous layer, and extract with
50 ml of ethyl acetate. Separate the aqueous layer, extract
with two potions of 50 ml of chloroform, discard the agueous
layer combine the chloroform extracts and evaporate to dryness
over a water bath. Dissolve the residue in 15 ml of
chloroform, filter, wash the filter with small volume of
chloroform, combine the filtrate and washing, and evaporate
to dryness until odour of chloroform or ethyl acetate
disappear. Dry the residue in vacuum at 6üºC for 1 hour. Cool
and weigh: not less than 1 7 % and not more than 2 7 % of free
polyethylene glycols is found.
Water Not more than 3. O% ( 0832 method 1 ) .
Residue on ignition Not more than O. 3 % <0841 >.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 3 >,
using 2. O g: not more than O. 001 % .
Composition of fatty acids Dissolve O. 1 g of the substance
being examined in 2 ml of O. 5 mol/L sodium hydroxide
solution in methanol. Boil the solution under reflux in a
25 ml conical flask for 30 minutes. Add 2 ml of 14% boron
trifluoride solution in methanol through the condenser and
boil under reflux again for 30 minutes. Add 4 ml of n-
heptane through the condenser and continue boil under reflux
for 5 minutes, cool. Add 10 ml of a saturated solution of
sodium chloride. Shake for 15 seconds and add the saturated
solution to bottle neck. Shake thoroughly, allow to stand.
Transfer 2 ml of the upper layer, wash with 2 ml of water
for 3 times. Dry the upper layer with anhydrous sodium
sulphate. Carry out the method for gas chromatography
( 0521 >, using a capillary column coated with polyethylene
glycol as the stationary phase. The temperature of injection is
250ºC; the temperature of detector is 260"C. Maintain the
column temperature at 170ºC far 2 minutes, then raise the
temperature to 24üºC by lOºC per minute, maintain for
several minutes. Using nitrogen as carrier gas , flow rate is
2 ml per minute, split ratio is 10 : l. Inject 1 µl of the upper
liquid into the column. The elution arder is methyl
palmitate, methyl stearate, and the resolution factor between
the peaks of methyl palmitate and methyl stearate is not less
than 5. O. Record the chromatogram for 3 times the retention
time of methyl stearate peak. Calculate the contents of
stearic acid and palmitric acid by the normalization method.
The content of stearic acid is not less than 40. O%, the sum
of stearic acid and palmitric acid is not less than 90. O%.
Arsenic Carry out the limit test for arsenic ( 0822 method
2), using O. 67 g: not more than O. 0003%.
Category Pharmaceutical excipients, solubilizer, emusifier
and matrix.
Storage Preserve in tightly closed containers, stored in a dry

and cool place.
Polyoxyl Oleate
[9004-96-0]
Polyoxyl Oleate is a mixture of monesters and diesters of
oleic acid and polyethylene glycols. It may be obtained by
ethoxylation of loleic acid which from animal or vegetable
origin or by esterification of polyethylene glycols with oleic
acid. The molecular formula is C17 H33 COO CCH2 CH2 Ü)n
H and n is either 5-6 or 10.
Description A light yellow thick liquid.
Dispersible in water, soluble in ethanol and in isopropanol,
and arbitrarily miscible with fatty oils and paraffin.
Refractive index l. 464-1. 468 ( 0622).
Acid value Not more than 1 ( 0713 ).
Saponification value 105-120 (n is 5-6) or 65-85 (n is 10)
(0713).
Hydroxyl value 50-70 ( n Is 5-6) or 65-90 ( n is 10)
(0713).
lodine value 50-60 ( n is 5-6) or 27-34 ( n Is 10)
(0713).
Peroxide value Not more than 12 ( 0713).
Identification The infrared absorption spectrum ( 0402 ) is
concordant with the spectrum of polyoxyl oleate CRS.
Alkalinity To 2. O g of the substance being examined, add
20 ml of ethanol and mix. To 2 ml of the above solution add
O. 05 ml of phenol red IS. The solution is not red.
Ethylene oxide and dioxin Accurately weigh 1 g of the
substance being examined in a headspace vial, add l. O ml of
N, N-dimethylacetamide and O. 2 ml of water, seal the vial
and mix well as the test solution ( the test vial). Measure
99. 75 g of macrogol 400 (Remove any volatile components
by using a rotary evaporator at a temperature of 60ºC and
pressure of l. 5 to 2. 5 kPa for 6 hours) into a 100 ml of
penicillin bottle ( or other suitable container), accurately
measured and sealed Puncture inj ection 300 µl of ethylene
oxide ( equivalent to about O. 25 g) by a glass syringe frozen
to - lOºC into the above penicillin bottle, accurately
weighed, as the ethylene oxide stock reference solution
(freshly prepared or calibrated before use). Accurately weigh
1 g of cold ethylene oxide stock reference solution, into an
ampoule containing 49 g of cold macrogol 400 as above, seal
and mix. Accurately measure 10 g into a 50 ml volumetric
flask, dilute to volume with water, mix well as ethylene
oxide reference solution. Dilute a quantity of dioxin, accurately
weighed, with water to produce a solution of O. 5 mg per ml, as
dioxin reference solution. Accurately weigh 1 g of the
substance being examined, in a headspace vial, add
accurately l. O ml of N, N-dimethylacetamide, O. 1 ml of
ethylene oxide reference solution and O. 1 ml dioxin reference
solution, seal the vial, mix well as the reference solution.
Measure O. 1 ml of ethylene oxide reference solution in a
headspace vial, and add O. 1 ml of freshly prepared O. 001 %
acetaldehyde solution and O. 1 ml of dioxin reference
solution, seal the vial and mix well as the system suitability
solution. Carry out the method for gas chromatograph
( 0521 ) , using a column packed with polydimethysiloxane as
the stationary phase; maintain the column temperature at
50ºC for 5 minutes, raise the temperature to 180ºC by 5ºC
Polyoxyl Oleate 617
per minute, then to 230ºC by 30ºC per minute and maintain
for 5 minutes (adjust if necessary). The temperature of the
injection is 150°C. The temperature of the detector is 250ºC.
The equilibration temperature is 70ºC and maintain for 45
minutes. Inject the gas from headspace vial of the system
suitability into the column and record the chromatogram.
Adjust the attenuation so that the peak heights due to
ethylene oxide and acetaldehyde in the chromatogram are
about 15 % of the full scale of the chart. The resolution
factor between the peaks of acetaldehyde and ethylene oxide
is not less than 2. O, the signal-to-noise ratio is not less than
5 for the peak of dioxin. Inject separately the gas aaa each in
the test vial and the reference vial onto the column. Repeat
the procedure at least three times. The relative standard deviation
of the 3 areas of peak of ethylene oxide is not greater than
15 % and the rela ti ve standard deviation of the 3 areas of peak
of dioxin is not greater than 10%. Calculate the content with
respect to the peak area obtained in the chromatogram by the
standard addition method, ethylene oxide is not more than
O. 0001% and dioxin is not more than O. 001%.
Water Not more than 2. O% ( 0832 method 1 ( 1 ) ) , usmg
l. o g.
Residue on ignition Not more than O. 3% ( 0841 ) , using
l. o g.
Composition of fatty acids Dissolve l. O g of the substance to
be examined in a 25 ml twin neck round bottom flask. Add
10 ml of anhydrous methanol and O. 2 ml of 60 g/L
potassium hydroxide solution in methanol. Shake to dissolve
and pass nitrogen through the mixture at flow arate of about
50 ml per minute, heat to boiling. When the solution is clear
(usually about 10 minutes), continue heating for 5 minutes.
Cool the flask under running water and transfer the contents
to a separating funnel. Rinse the flask with 5 ml of n-
heµtane, transfer to the seµarating funnel and shake. Add
10 ml of 200 g/L of sodium chloride solution and shake
vigorously. Allow to separate and transf er the organic layer
to a vial containing anhydrous sodium sulfate. Allow to
stand, then filter. The filtrate is the test solution. Accurately
weigh and dissolve methyl myristate CRS, methyl palmitate
CRS, methyl palmitoleate CRS, methyl stearate CRS,
methyl oleate CRS, methyl linoleate CRS and methyl
linolenic CRS in n-heptane to produce a mixture reference
solution (1) containing O. 5 mg per ml, l. O mg per ml,
O. 5 mg per ml, O. 5 mg per ml, 6. O mg per ml, l. O mg per
ml and O. 5 mg per ml, respectively. Transfer l. O ml to a
10 ml volume flask, dilute to volumn with n-heptane and
mixwell, as the mixture reference solution (2). Carry out the
method for gas chromatography ( 0521 ) , using the column
packed with polyethylene glycol. The column temperature is
170ºC. Raise the temperature of the column to 230ºC by 2ºC
per minute and maintain for 10 minutes. Maintain the
temperature of injection at 250ºC, and that of detector at
250ºC. Inject 1 µl of the mixture reference solution (1) and
the mixture reference solution (2) into the column and record
the chromatogram. The resolution factor between each pair
of adjacent peaks in the chromatogram obtained with the
reference solution ( 1) is not less than l. 8 and the number of
the theoretical plates calculated for methyl oleate is not less
than 30 000. The signal-to-noise ratio of the minimum peak
height of fatty acid is not less than 5. Inject 1 µl of the test
solution into the column and calculate the content of the fatty
acid with respect to the peak area by area normalization
method. The myristic acid is not more than 5. 0%, palmitic
acid is not more than 16. O%, palmitolic acid is not more
than 8. O%, stearic acid is not more than 6. O%, oleic acid is
 618 Polysorbate 20
65. O% to 88. O%, linoleic acid is not more than 18. O%,
linolenic acid is not more than 4. O%, and other fatty acid is
not more than 4. O%.
Category Pharmaceutical excipients, solubilizer and emulsifier.
Storage Preserve in tightly closed containers, and stored in
a dry and cool place with nitrogen.
Polysorbate 20
[9005-64-5]
Polysorbate20 is polyoxyethylene (20) sorbitanmonooleate,
polymerized by sorbitan monolaurate and ethylene oxide.
Description A pale yellow to yellow, oily, viscous liquid,
odour, faint and characteristic.
Freely soluble in water, in ethanol, in methanol and in ethyl
acetate, slightly soluble in liquid paraffin.
Relative density l. 09-1. 12 <0601).
Viscosity Kinematic viscosity is 250-440 mm2 / s < 0633
method 1 ) determined at 25ºC, using a capillary tube of
2. O mm or suituable in inner diameter.
Acid Value Place 10 g, accurately weighed, in a 250 ml
conical flask, add 50 ml of ethanol ( neutralized to phen-
olphthalein IS) to dissolve. Boil under reflux far 10 minutes,
cool. and add 5 drops of phenolphthalein IS. Then be titrated
with sodium hydroxide (O. 1 mol/U VS. The acid value is
not mor than 2. O <0713).
Saponification Value 40-50 <0713 >.
Hydroxyl Value 96-108 <0713 >.
Peroxide Value Not more than 10 <0713 >.
Identification (1) To 5 ml of a solution (1 - 20) add 5 ml
of sodium hydroxide TS, boil far a few minutes and cool.
acidify with diluted hydrochloric acid TS. A creamy-white
turbidi ty is farmed.
(2) To 2 ml of a solution (1 - 20) add O. 5 ml of bromine
TS dropwise: the bromine is not decolorized.
(3) To 10 ml of a solution (1 - 20) add 5 ml ammonium
cobalt thiocyanate solution (dissolve 17. 4 g of ammonium
thiocyanate and 2. 8 g of cobalt nitrate in 100 ml of water),
shake well, Add 5 ml of chlorofarm, shake and allow to
stand, the colour of chlorofarm layer turns blue.
Acidity or alkalinity Dissolve O. 50 gin 10 ml of water, pH
4.0-7.5 (0631).
Colour Using 10 ml, not more intense than that of a
ref erence solution prepared by mixing 8. O ml of standard
potassium dichromate es, o. 8 ml of standard cobaltous
chloride es with sufficient water to produce 10 ml.
Ethylene glycol and diglycol Place about 4 g, accurately
weighed, in a 100 ml volumetric flask, add accurately 1 ml of
internal standard solution ( dilute a quantity of 1, 3-
butanediol with acetone to produce a solution of 4 mg per
mD, dilute to volume with acetone, mix well, as the test
solution. Place 40 mg of the Ethylene glycol CRS and
diglycol CRS, accurately weighed, into a 100 ml volumetric
flask, dilute to volume with acetone, mix well. Transfer
10 ml of the solution into a 100 ml volumetric flask, add
accurately 1 ml of internal standard solution, dilute to
volume with acetone, mix well as the reference solution.
Carry out the method far gas chromatography < 0521 ) ,
using a O. 53 mm X 30 m capillary column bonded with a
l. O µm layer of thickness of phase 50 % phenyl-50 % methyl-
polysiloxane; maintain the column temperature at 40ºC,
raise the temperature to 60ºC by lOºC per minute and
maintain far 5 minutes, raise the temperature to 170ºC by
lOºC per minute, then raise the temperature to 280ºC by
15ºC per minute and maintain far 1 hour ( adjust if
necessary). The temperature of the injection is 270ºC. The
temperature of the detector is 290ºC . Use the reference
solution as the system suitability test solution. The resolution
factor between each peak is not less than 2. O. Tailing factor
of each peak should comply with the requirement. Inject
separately 1 ul of the test solution and the reference solution
onto the column and record the chromatogram. Calculate the
content with respect to the peak area obtained in the
chromatogram by the internal standard method, the amount
of ethylene glycol and diglycol are both not more than
o. 01%.
Ethylene oxide and dioxan Accurately weigh 1 g in a
headspace vial, add accurately measured l. O ml of purified
water, seal the vial and mix well, as the test solution ( the
test vial). Measure a quantity of ethylene oxide standard
stock solution, equivalent to about O. 25 g , into a 100 ml
volumetric flask, add 50 ml of treated polyrthylene glycol
400 ( using a rotary evaporator remove any volatile
components at a temperature of 60ºC and pressure of l. 5-
2. 5 kPa far 6 hours) to dissolve and dilute well to produce a
solution of 1 ug per ml as ethylene oxide reference solution.
Accurately weigh a quantity of dioxan in water to produce a
solution of 10 µg per ml as the dioxan reference solution.
Accurately weigh 1 g of the substance being examined, in a
head-space vial, accurately add O. 5 ml of ethylene oxide
reference solution and O. 5 ml of dioxan reference solution,
and seal the vial, mix well as the reference solution ( the
ref erence vial ) . Measure O. 5 ml of the ethylene oxide
reference solution in a head-space vial, add O. 1 ml of freshly
prepared O. 001 % acetaldehyde solution and O. 5 ml of dioxan
reference solution, seal the vial and mix well as the system
suitability test solution ( the system suitability test vial).
Carry out the method far gas chromatography < 0521 ) ,
using a column packed with polydimethylsiloxane as the
stationary phase; maintain the column temperature at 35ºC
far 5 minutes, raise the temperature to 180ºC by 5ºC per
minute, raise the temperature to 230ºC by 30ºC per minute
and maintain far 5 min ( adjust if necessary ). The
temperature of the injection is 150ºC. The temperature of the
detector is 250ºC. Use the head-space injection method. The
equilibration temperature is 70ºC and maintain far 45
minutes. Inject the gaseous sample of the system suitability
test vial onto the column and record the chromatogram, flow
rate is 2. 5 ml per min, split ratio is 1 : 20. Adjust the
sensitivity of the detector so that the peak heights due to
ethylene oxide and acetaldehyde in the chromatogram are
about 15% of the full scale of the recorder. The resolution
factor between the peaks of acetaldehyde and ethylene oxide
is at least 2. O; the signal to noise ratio of the peak of dioxan
is not less than 5. Inject separately each of the gaseous
sample of the test vial and reference vial onto the column.
Repeat the procedure at least three times. The relative
standard deviation of the 3 areas of peak of ethylene oxide is
not more than 15 % and the relative standard deviation of the
3 areas of peak of dixan is not more than 1O%. Calcula te the
content with respect to the peak area obtained in the
chromatogram by the standard addition method, the amount
of ethylene oxide is not more than O. 0001 % and the amount
of dioxan is not more than O. 001 % .
Water Not more than 3. O% < 0832 method 1 ( 1 ) ) .

Residue on ignition Not more than O. 25% <0841), usmg
l. o g.
Heavy Metals Carry out the limit test far heavy metals
( 0821 method 2), using the residue obtained in the test far
Residue on ignition: not more than O. 001%.
Arsenic Place l. O g to a Kjeldahl flask and add 5 ml sulfuric
acid. Charring and keep the temperature below 120ºC ( add
sulfuric acid if necessary, but the total volume added should
not exceed 10 mD, add hydrogen peroxide solution (30%)
dropwise, allowing the reaction to subside and again heating
between drops, until solution becomes colorless. Allow it to
cool to room temperature and add 10 ml of water. Evaporate
to remove hydrogen peroxide, add 5 ml hydrochloric acid and
a quantity of water, carry out the limit test far asenic ( 0822 ,
method 1 ) : not more than O. 0002 % .
Composition of fatty acids Place O. 1 g to a 5 O ml conical
flask, add 2 ml of 2 % sodium hydroxide solution in
methanol, boil under reflux in a water bath at 65ºC far 30
minutes. Cool, add 2. O ml of 14% boron trifluoride
solution in methanol and boil under reflux again far 30
minutes and allow it to cool. Add 4 ml of n-heptane and boil
under reflux far 5 minutes, cool. Add 1 O ml of saturated
sodium chloride solution, shake, allow to stand. Wash the
upper layer with 4 ml of water far 3 times. Dry the upper
layer with anhydrous sodium sulphate, as the test solution.
Carry out the method far gas chromatography ( 0521 ) ,
using a O. 32 mm X 30 m capillary column bonded with a
O. 50 µm layer of thickness of phase 50 % phenyl-50 %
methylpolysiloxane. The temperature of injection port is
190ºC; the temperature of detector is 250ºC; raise the
column temperature from 90ºC to 160ºC by 20ºC per
minute, maintain far 1 minute, then raise the column
temperature to 220ºC by 2°C per minute, maintain far 20
minute, Dissolve and dilute a quantity of methylcaproate
CRS, methyl caprylate CRS, methyl caprate CRS, methyl
laurate CRS, methyl myristate CRS, methyl palmitate CRS,
methyl oleate CRS and methyl linoleate CRS in n-heptane to
produce a mix reference solution containing O. 1 mg per ml of
methyl hexanoate, methyl octanoate, methyl decanoate and
methyl laurate respectively and 1 mg per ml of methyl
myristate, methyl palmitate, methyl stearate, methyl
oleate and methyl linoleate respectively. lnject 1 ul of the
mix reference solution onto the column. The number of the
theoretical plates of the column is not less than 1 O OOO,
calculated with reference to the peak of methyl laurate. The
resolution factors between all adjacent peaks comply with
the requirements. Inject 1 µl of the test solution onto the
column, and record the chromatogram. Calculate the
content of each fatty acid by peak area normalization
method ( discard any peak below O. O5 % ) . Lauric acid is
40. 0-60. O%, myristic acid is 14. 0-25. O%, palmitic acid
is 7. 0-15. O% , hexanoic acid is not more than 1. O% ,
octanoic acid is not more than 1 O. O% , decanoic acid is not
more than 1 O. O% , s tearic a cid is not more than 7. O% ,
oleic acid is not more than 11. O% and linoleic acid is
3. 0%.
Category Pharmaceutical excipients, solubilizer, emulsifier,
etc.
Storage Preserve in tightly closed containers, and protected
from light.
Polysorbate 40 619
Polysorbate 40
[ 9005-66-7]
Polysorbate40 ispolyoxyethylene ( 20 ) sorbitanmonopal-
mitate, polymerized by sorbitanmonopalmitate and ethylene
oxide.
Description A creamy-white to yellow, viscous liquid or
frozen paste, odour, faint and characteristic.
Freely soluble in warm water, in ethanol, in methanol and in
ethyl acetate, slightly soluble in liquid paraffin.
Relative density l. 07-1. 10 ( 0601), determined at 25ºC.
Viscosity Kinematic viscosity 250-400 mm2 / s ( 0633 method
1), determined at 30ºC, using a capillary tube of 2. O mm or
suitable in internal diameter.
Acid Value Dissolve about 10 g with 50 ml of ethanol
( neutralize to phenolphthalein IS) in a 250 ml conical flask.
Boil under reflux far 10 minutes, allow to cool and add 5
drops of phenolphthalein IS. Then titrate with sodium
hydroxide (O. 1 mol/L) VS. The acid value is not more than
2.0 (0713).
Saponification Value 41-5 2 ( O713 ) .
Hydroxyl Value 89-105 ( 0713).
Peroxide Value Not more than 10 ( 0713).
ldentification (1) To 5 ml of a solution 0-20) add 5 ml
of sodium hydroxide TS. Boil far a few minutes, allow to
cool and acidify with dilute hydrochloric acid TS. A creamy-
white turbidity is farmed.
(2) To 2 ml of a solution Cl~2o) add O. 5 ml of bromine TS
dropwise: the bromine is not decolorized.
(3) To 6 ml add 4 ml of water, mix well; a gelatinous mass
is farmed.
(4) To 10 ml of a solution 0-20) add 5 ml of ammonium
cobalt thiocyanate solution ( dissolve 17. 4 g of ammonium
thiocyanate and 2. 8 g of cobalt nitrate in 100 ml of water),
shake well. Add 5 ml of chlorofarm, shake and allow to
stand, the colour of the chlorofarm layer turns blue .
Acidity or alkalinity Dissolve O. 50 gin 10 ml of water, pH
4. o - 7. 5 (0613).
Colour Using 10 ml, not more intense than that of an equal
volume of a reference solution prepared by mixing 8. O ml of
standard potassium dichromate es and o. 8 ml of standard
cobaltous chloride es with sufficient water to produce 10 ml.
Ethylene glycol and diglycol Place 4 g, accurately weighed,
in a 100 ml volumetric flask, add accurately 1 ml of internal
standard solution ( dilute a quantity of 1, 3-butanediol with
acetone to produce a solution of 4 mg per ml) , dilute to
volume with acetone, mix well, as the test solution. Place 40
mg of the Ethylene glycol CRS and diglycol CRS, accurately
weighed, into a 100 ml volumetric flask, dilute to volume
with acetone, mix well. Transfer 10 ml of the solution into a
100 ml volumetric flask, add accurately 1 ml of internal
standard solution, dilute to volume with acetone, mix well as
the reference solution. Carry out the method far gas
chromatography < 0521 ) , using a O. 53 mm X 30 m
capillary column bonded with a l. O µm layer of thickness of
phase 50 % phenyl-50 % methylpolysiloxane; maintain the
column temperature at 40ºC, raise the temperature to 60ºC
by lOºC per minute and maintain far 5 minutes, raise the
 620 Polysorbate 60

temperature to l 70ºC by lOºC per minute, then raise the ( 0821 method 2) , using the residue obtained in the test for
temperature to 280ºC by 15ºC per minute and maintain for 1 Residue on ignition: not more than O. 001%.
hour (adjust if necessary). The temperature of the injection Arsenic Place l. O g to a Kjeldahl flask and add 5 ml sulfuric
is 270ºC. The temperature of the detector is 290ºC. Use the acid. char gently under a temperature below 120ºC ( add
reference solution as the system suitability test solution. The sulfuric acid if necessary, but the total volume added should
resolution factor between all adjacent peaks not less than
not exceed 10 mD , add hydrogen peroxide solution (30%)
2. O. Tailing factor of each peak should comply with the
dropwise, allow the reaction to subside, heat again and add
requirement. Inject separately 1 ul of the test solution and
hydrogen peroxide solution, until solution becomes colorless.
the reference solution onto the column and record the
Allow it to cool to room temperature and add 10 ml of water.
chromatogram. Calculate the content with respect to the
Evaporate to remove hydrogen peroxide, add 5 ml
peak area obtained in the chromatogram by the internal
hydrochloric acid anda quantity of water, carry out the limit
standard method, the amount of ethylene glycol and diglycol
test for asenic ( 0822 method 1): not more than O. 0002%.
are both not more than O. 01%.
Composition of fatty acids Place O. 1 g to a 50 ml conical
Ethylene oxide and dioxan Accurately weigh 1 g in a
flask, add 2 ml of 2 % sodium hydroxide solution in
headspace vial, add accurately measured l. O ml of purified
methanol, boil under reflux in a water bath at 65ºC for 30
water, seal the vial and mix well, as the test solution ( the
minutes. Cool, add 2. O ml of 14% boron trifluoride solution
test vial). Measure 300 µl of ethylene oxide, equivalent to
in methanol and boil under reflux again for 30 minutes and
~bout O. 25 g , in to a 100 ml volumetric flask containing
allow it to cool. Add 4 ml of n-heptane and boil under reflux
~O ml of treated polyrthylene glycol 400 ( using a rotary
for 5 minutes, cool. Add 10 ml of saturated sodium chloride
evaporator remove any volatile components at a temperature
solution, shake, allow to stand. Wash the upper layer with
of 60ºC and pressure of l. 5-2. 5 kPa for 6 hours), dilute well
4 ml of water for 3 times. Dry the upper layer with anhydrous
to produce a solution of 1 ug per ml as ethylene oxide
sodium sulphate, as the test solution. Carry out the method
reference solution. Accurately weigh a quantity of dioxan in
for gas chromatography ( 0521 ) , using a O. 32 mm X 30 m
water to produce a solution of 10 µg per ml as the dioxan
capillary column bonded with a O. 50 µm layer of thickness of
reference solution. Accurately weigh 1 g of the substance to
phase PEG-20 M. The temperature of injection port is
be examined, in a head-space vial, accurately add O. 5 ml of
190ºC; The temperature of detector is 2SOºC . Raise the
ethylene oxide ref erence solution and O. 5 ml of dioxan
column temperature from 90ºC to 160ºC by 20ºC per minute,
reference solution, and seal the vial, mix well as the
maintain for 1 minute, then raise the column temperature to
reference solution ( the reference vial). Measure O. 5 ml of the
220ºC by 2°C per minute, maintain for 20 minute. Dissolve
ethylcnc oxide rcfcrcnce solution in a head-space vial, add
and dilute a quantity of methyl palmitate CRS in n-heptane to
O. 1 ml of freshly prepared O. 001 % acetaldehyde solution and
produce a mix solution containing 1 mg per ml as the
O. 5 ml of dioxan reference solution, seal the vial and mix
reference solution. Inject 1 µl of the solution onto the
well as the system suitability test solution ( the system
column. The number of the theoretical plates of the column
suitability test vial). Carry out the method for gas chroma-
is not less than 10 000, calculated with reference to the peak
tography ( 0521 ) , using a column packed with polydimeth-
of methyl palmitate. Inject 1 µl of the test solution onto the
ylsiloxane as the stationary phase; maintain the column
column, and record the chromatogram. Calculate the content
temperature at 35ºC for 5 minutes, raise the temperature to
of palmitic acid by peak area normalization method. The
180ºC by 5ºC per minute, raise the temperature to 230ºC by
30ºC per minute and maintain for 5 minutes ( adjust if amount of palmitic acid is not less than 92. 0%.
necessary). The temperature of the injection is 150ºC. The Category Pharmaceutical excipients, emulsifier, solubilizer,
temperature of the detector is 250ºC. Carry out the head- etc.
space injection method. The equilibration temperature is
Storage Preserve in tightly closed containers, and protected
70ºC and maintain for 45 minutes. Inject the gaseous sample
from light.
of the system suitability test vial onto the columu and record
the chromatogram, flow rate is 2. 5 ml per min, split ratio is
1 : 20. Adjust the sensitivity of the detector so that the peak
heights due to ethylene oxide and acetaldehyde in the
chromatogram are about 15 % of the full scale of the Polysorbate 60
recorder. The resolution factor between the peaks of
acetaldehyde and ethylene oxide is at least 2. O; the signal to [9005-67-8]
noise ratio of the peak of dioxan is not less than 5. Inject
Polysorbate 60 is polyoxyethylene ( 20 ) sorbitan mono-
separately each of the gaseous sample of the test vial and
stearate, polymerized by sorbitan monostearate and ethylene
reference vial onto the column. Repeat the procedure at least
oxide.
three times. The relative standard deviation ( RSD) of the
3 areas of peak of ethylene oxide is not more than 15 % and Description A creamy-white to yellow, viscous liquid or
the relative standard deviation CRSD) of the 3 areas of peak frozen paste, odour, faint and characteristic.
of dixan is not more than 10%. Calculate the content with Freely soluble in warm water, in ethanol, in methanol and in
respect to the peak area obtained in the chromatogram by the ethyl acetate, slightly soluble in liquid paraffin.
standard addition method, the amount of ethylene oxide is Relative deffiity l. 06-1. 09, at 2SºC ( 0601) .
not more than O. 0001% and the amount of dioxan is not
more than O. 001%. Viscosity Kinematic viscosity 30CH50 mm2 / s at 30ºC ( 0633
method 1 ) , using a capillary tube of 2. O mm or suitable in
Water Not more than 3. O% ( 0832 method 1 ( 1 ) ) . interna! diameter.
Residue on ignition Not more than O. 25 % ( 0841 ) , usmg Acid value Dissolve 10 g, accurately weighed, with 50 ml of
l. o g. ethanol ( neutralize to phenolphthalein IS) in a 250 ml
Heavy metals Carry out the limit test for heavy metals conical flask. Boil under reflux for 10 minutes, allow to cool

and add 5 drops of phenolphthalein IS, Then titrate with
sodium hydroxide (O. 1 mol/L) VS. The acid value is not
more than 2. O ( 0713 >.
Saponification value 45-55 <0713).
Hydroxyl value 81-96 ( 0713).
Peroxide value Not more than 10 <0713 >.
ldentification (1) To 5 ml of a solution 0-20) add 5 ml
of sodium hydroxide TS. Boíl far a few minutes, allow to
cool and acidify with dilute hydrochloric acid TS, A creamy-
white turbidity is produced.
(2) To 2 ml of a solution 0-20) add O. 5 ml of bromine TS
dropwise: the bromine is not decolorized.
(3) To 6 ml add 4 ml of water, shake well: a gelatinous
mass is farmed.
(4) To 10 ml of a solution 0-20) add 5 ml of ammonium
cobalt thiocyanate solution ( dissolve 17. 4 g of ammonium
thiocyanate and 2. 8 g of cobalt nitrate in 100 ml of water),
shake well. Add 5 ml of chlorofarm, shake and allow to
stand, the colour of the chlorofarm layer turns blue.
Acidity or alkalinity Dissolve O. 50 g in 10 ml of water, pH
4. 0-7. 5 (0631).
Colour Using 10 ml, not more intense than that of an equal
volume of a reference solution prepared by mixing 8. O ml of
standard potassium dichromate es and o. 8 ml of standard
cobaltous chloride es with sufficient water to produce 10 ml.
Ethylene glycol, diglycol Place 4 g, accurately weighed, in a
100 ml volumetric flask, add accurately 1 ml of internal
standard solution (dilute a quantity of 1, 3-butanediol with
acetone to produce a solution of 4 mg per mD , dilute to
volume with acetone, mix well, as the test solution. Place 40
mg of the Ethylene glycol CRS and diglycol CRS, accurately
weighed, into a 100 ml volumetric flask, dilute to volume
with acetone, mix well. Transfer 10 ml of the solution into a
100 ml volumetric flask, add accurately 1 ml of interna!
standard solution, dilute to volume with acetone, mix well as
the ref erence solution. Carry out the method far gas
chromatography ( 0521 >, using a O. 53 mm X 30 m
capillary column bonded with a l. O µm layer of thickness of
phase 50% phenyl-50% methylpolysiloxane; maintain the
column temperature at 40ºC, raise the temperature to 60ºC
by lOºC per minute and maintain far 5 minutes, raise the
temperature to 170ºC by lüºC per minute, then raise the
temperature to 280ºC by 15ºC per minute and maintain far 1
hour (adjust if necessary). The temperature of the injection
is 270ºC. The temperature of the detector is 290ºC. Use the
reference solution as the system suitability test solution, The
resolution factors between all adjacent peaks are not less than
2. O. Tailing factor of each peak complies with the
requirement. Inject separately 1 ul of the test solution and
the reference solution onto the column and record the
chromatogram. Calculate the content with respect to the
peak area obtained in the chromatogram by the interna!
standard method, the amount of ethylene glycol and diglycol
are both not more than O. 01 % .
Ethylene oxide and dioxan Accurately weigh 1 g in a
headspace vial, add accurately measured l. O ml of purified
water, seal the vial and mix well, as the test solution ( the
test vial). Measure 300 µl of ethylene oxide, equivalent to
about O. 25 g, into a 100 ml volumetric flask containing 50
ml of treated polyrthylene glycol 400 ( using a rotary
evaporator remove any volatile components at a temperature
of 60ºC and pressure of l. 5-2. 5 kPa far 6 hours), dilute well
to produce a solution of 1 µg per ml as ethylene oxide
Polysorbate 60 621
reference solution. Accurately weigh a quantity of dioxan in
water to produce a solution of 10 µg per ml as the dioxan
reference solution. Accurately weigh 1 g of the substance to
be examined, in a head-space vial, accurately add O. 5 ml of
ethylene oxide reference solution and O. 5 ml of dioxan
reference solution, and seal the vial, mix well as the
reference solution ( the reference vial). Measure O. 5 ml of the
ethylene oxide reference solution in a head-space vial, add
O. 1 ml of freshly prepared O. 001 % acetaldehyde solution and
O. 5 ml of dioxan reference solution, seal the vial and mix
well as the system suitability test solution ( the system
suitability test vial ) . Carry out the method far gas
chromatography ( 0521 ) , using a column packed with poly-
dimethylsiloxane as the stationary phase; maintain the
column temperature at 35ºC far 5 minutes, raise the tem-
perature to 180ºC by 5ºC per minute, raise the temperature
to 230ºC by 30ºC per minute and maintain far 5 min (adjust
if necessary). The temperature of the injection is 150ºC. The
temperature of the detector is 250ºC. Carry out the head-
space injection method. The equilibration temperature is
70ºC and maintain far 45 minutes. Inject the gaseous sample
of the system suitability test vial onto the columu and record
the chromatogram, flow rate is 2. 5 ml per min, split ratio is
1 : 20. Adjust the sensitivity of the detector so that the peak
heights due to ethylene oxide and acetaldehyde in the
chromatogram are about 15% of the full scale of the
recorder. The resolution factor between the peaks of
acetaldehyde and ethylene oxide is not less 2. O; the signal to
noise ratio of the peak of dioxan is not less than 5. Inject
separately each of the gaseous sample of the test vial and
reference vial onto the column. Repeat the procedure at least
three times. The relative standard deviation (RSD) of the 3
areas of peak of ethylene oxide is not more than 15 % and the
relative standard deviation CRSD) of the 3 areas of peak of
dixan is not more than 10 % . Calculate the content with
respect to the peak area obtained in the chromatogram by the
standard addition method, the amount of ethylene oxide is
not more than O. 0001 % and the amount of dioxan is not
more than O. 001 % .
Water Not more than 3. O% ( 0832 method 1 ( 1 ) >.
Residue on ignition Not more than O. 25 % ( 0841 ) , usmg
l. o g.
Heavy metals Carry out the limit test far heavy metals
( 0821 method 2), using the residue obtained in the test far
Residue on ignition: not more than O. 001%.
Arsenic Place l. O g to a Kjeldahl flask and add 5 ml sulfuric
acid. chargently under a temperature below 120ºC ( add
sulfuric acid if necessary, but the total volume added should
not exceed 1O mD , add hydrogen peroxide solution ( 30 % )
dropwise, allowing the reaction to subside and again heating
between drops, until solution becomes colorless. Allow it to
cool to room temperature and add 10 ml of water. Evaporate
to remove hydrogen peroxide, add 5 ml hydrochloric acid and
a quantity of water, carry out the limit test far asenic ( 0822
method 1 ) : not more than O. 0002 % .
Composition of fatty acids Place O. 1 g to a 50 ml conical
flask, add 2 ml of 2 % sodium hydroxide solution in
methanol, boil under reflux in a water bath at 65ºC far 30
minutes. Cool, add 2. O ml of 14% boron trifluoride solution
in methanol and boil under reflux again far 30 minutes and
allow it to cool. Add 4 ml of n-heptane and boil under reflux
far 5 minutes, cool. Add 10 ml of saturated sodium chloride
solution, shake, allow to stand. Wash the upper layer with 4
ml of water far 3 times. Dry the upper layer with anhydrous
sodium sulphate, as the test solution. Carry out the method
 622 Polysorbate 80
far gas chromatography <0521 ) , using a O. 32 mm X 30 m
capillary column bonded with a O. 50 µm layer of thickness of
phase PEG--20 M. The temperature of injection port is
190ºC; The temperature of detector is 250ºC . Raise the
column temperature from 90ºC to 160ºC by 20ºC per minute,
maintain far 1 minute, then raise the column temperature to
220ºC by 2°C per minute, maintain far 20 minute. Dissolve
and dilute a quantity of methyl stearate and methyl palmitate
CRS with n-heptane to produce a mix solution containing
1 mg per ml respectively as the reference solution. Inject 1 µl
of the solution onto the column. The number of the
theoretical plates of the column is not less than 10 000,
calculated with reference to the peak of methyl stearate, the
resolution factor between peaks due to methyl palmitate and
methyl stearate complies with the related requirements.
lnject 1 µl of the test solution onto the column, and record
the chromatogram. Calculate the content of palmitic acid by
peak area normalization method. The amount of stearic acid
is 40. O%-60. O% and the total of stearic acid and palmitic
acid is not less than 90. 0%.
Category Pharmaceutical excipients, emulsifier and solub-
ilizer.
Storage Preserve in tightly closed containers, protected
from light.
Polysorbate 80
[9005-65-6]
Polysorbate 80 is polyoxyethylene (20) sorbitan monooleate
polymerized by sorbitan monooleate and ethylene oxide.
Description A pal e yellow to orange-yellow, viscous liquid,
odour, faint and characteristic; taste, slightly bitter and
astringent with a warm feeling.
Freely soluble in water, in ethanol, in methanol and in ethyl
acetate, very slightly soluble in mineral oils.
Relative density l. 06-1. 09 <0601 ) .
Viscosity Kinematic viscosity is 350-550 mm 2 /s, at 25ºC
<0633 method 1), using a capillary tube of 2. 0-2. 5 mm in
interna! diameter.
Acid value Dissolve 10 g, accurately weighed, with 50 ml of
ethanol neutralize to phenolphthalein IS in a 250 ml conical
flask. Boil under reflux far 10 minutes, cool, add 5 drops of
phenolphthalein IS, titrate with sodium hydroxide (O. 1 mol/L)
VS. The acid value is not more than 2. O <0713).
Saponification value 45-65 < 0713).
Hydroxyl value 65-80 <0713).
lodine value 18-24 <0713).
Peroxide Value Not more than 10 <0713 ).
Identification (1) To 5 ml of a solution (1-+-20) add 5 ml
of sodium hydroxide TS. Boil far a few minutes, cool, and
acidify with dilute hydrochloric acid, a creamy-white
turbidity is produced.
(2) To a solution (1-+-20) add bromine TS dropwise; the
bromine is decolourized.
(3) To 6 ml add 4 ml of water, mix well, a gelatinous mass
is farmed.
(4) To 10 ml of a solution Cl-+-20) add 5 ml of ammonium
cobalt thiocyanate solution (dissolve 17. 4 g of ammonium
thiocyanate and 2. 8 g of cobalt nitrate in 100 ml of water),
mix well. Add 5 ml of chlorofarm, shake, allow to stand,
chlorofarm layer turns blue.
Acidity or alkalinity Dissolve O. 50 gin 10 ml of water, pH
5. 0-7. 5 <0631>.
Colour The colour of 10 ml of the substance being examined
is not more intense than that of an equal volume of a
reference solution prepared by mixing 8. O ml of potassium
dichromate es, o. 8 ml of cobaltous chloride es, and diluting
with water to 10 ml.
Ethylene glycol and diglycol Place 4 g, accurately weighed,
in a 100 ml volumetric flask, add accurately 1 ml of interna!
standard solution ( dilute a quantity of 1, 3-butanediol with
acetone to produce a solution of 4 mg per ml) , dilute to
volume with acetone, mix well, as the test solution. Place
40 mg of the ethylene glycol CRS and diglycol CRS,
accurately weighed, into a 100 ml volumetric flask, dilute to
volume with acetone, mix well. Transfer 10 ml of the
solution into a 100 ml volumetric flask, add accurately 1 ml
of internal standard solution, dilute to volume with acetone,
mix well as the reference solution. Carry out the method far
gas chromatography < 0521 ) , using a O. 53 mm X 30 m
capillary column bonded with a l. O µm layer of thickness of
phase 50 % phenyl-50 % methylpolysiloxane; maintain the
column temperature at 40ºC, raise the temperature to 60ºC
by lOºC per minute and maintain far 5 minutes, raise the
temperature to l 70ºC by lOºC per minute, then raise the
temperature to 280ºC by 15ºC per minute and maintain far 1
hour (adjust if necessary). The temperature of the injection
is 270ºC. The temperature of the detector is 290ºC. Use the
reference solution as the system suitability test solution, The
resolution factor between each peak is not less than 2. O.
Tailing factor of each peak complies with the requirement.
Inject separately 1 µl of the test solution and the reference
solution onto the column and record the chromatogram.
Calculate the content with respect to the peak area obtained
in the chromatogram by the internal standard method, the
amount of ethylene glycol and diglycol are both not more
than O. 01%.
Ethylene oxide and dioxan Accurately weigh 1 g in a heads-
pace vial, add accurately measured l. O ml of purified water,
seal the vial and mix well, as the test solution ( the test
vial). Measure a quantity of the ethylene oxide stock
reference solution into a volumetric flask, dissolve and dilute
to volume with the treated polyrthylene glycol 400 (Using a
rotary evaporator remove any volatile components at a
temperature of 60ºC and pressure of l. 5-2. 5 kPa far 6
hours) , mix well to produce a solution of 1 µg per ml as
ethylene oxide reference solution. Accurately weigh a
quantity of dioxan in water to produce a solution of 10 µg per
ml as the dioxan reference solution. Accurately weigh 1 g of
the substance to be examined, in a headspace vial, accurately
add O. 5 ml of ethylene oxide reference solution and O. 5 ml of
dioxan reference solution, and seal the vial, mix well as the
reference solution ( the reference vial). Accurately measure
O. 5 ml of the ethylene oxide reference solution in a headspace
vial, add O. 1 ml of freshly prepared O. 001% acetaldehyde
solution and O. 5 ml of dioxan reference solution, seal the vial
and mix well as the system suitability test solution ( the
system suitability test vial). Carry out the method far gas
chromatography < 0521 ) , using a column packed with
polydimethylsiloxane as the stationary phase; maintain the
column temperature at 35ºC far 5 minutes, raise the temper-
ature to 18üºC by 5ºC per minute, raise the temperature to
230ºC by 30ºC per minute and maintain far 5 min (adjust if
necessary). The temperature of the injection is 150ºC. The

temperature of the detector is 250ºC . Use the headspace
injection method. The equilibration temperature is 70ºC and
maintain for 45 minutes. Inject the gaseous sample of the
system suitability test vial onto the column and record the
chromatogram. Flow rate is 2. 5 ml per min, split ratio is
1 : 20. Adjust the sensitivity of the detector so that the peak
heights of ethylene oxide and acetaldehyde in the
chromatogram are about 15% of the full scale of the
recorder. The resolution factor between the peaks
corresponding to acetaldehyde and ethylene oxide is not less
than 2. O; the signal to noise ratio of the peak of dioxan is not
less than 5. lnject separately each of the gaseous sample of
the test vial and reference vial onto the column. Repeat the
procedure at least three times. The relative standard
deviation CRSD) of the 3 areas of peak of ethylene oxide is
not more than 15% and the relative standard deviation
CRSD) of the 3 areas of peak of dioxan is not more than
10 % . Calculate the content with respect to the peak area
obtained in the chromatogram by the standard addition
method, the amount of ethylene oxide is not more than
O. 0001 % and the amount of dioxan is not more than
o. 001%.
Freezing test Allow to stand for 24 hours m a glass
container at 5ºC ±2ºC, it is not frozen.
Water Not more than 3. O% ( 0832 method 1 ( 1 ) ) .
Residue on ignition Not more than O. 2% ( 0841 ) ; using
l. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2) , using the residue obtained in the test for
residue on ignition: not more than O. 001%.
Arsenic To l. O g in a kjeldahl flask, add 5 ml of sulfuric
acid, digest gently at a temperature below 120ºC ( add
sulfuric acid if necessary. keep the total volume not more
than 10 mD. Add cautiously, in dropwise, of concentrate
hydrogen peroxide solution until the reaction subsides.
Continue to heat the reaction mixture and add, in dropwise,
concentrate hydrogen peroxide solution until the solution
becomes colourless. Allow to cool, add 10 ml of water,
evaporate until the fume of excess hydrogen peroxide evolves
completely. Add 5 ml of hydrochloric acid and a quantity of
water to the resulting solution. Carry out the limit test for
arsenic ( 0822 method 1): not more than O. 0002%.
Composition of fatty acids Place O. 1 g to a 5 O ml conical
flask, add 2 ml of 2 % sodium hydroxide solution in
methanol, boil under reflux in a water bath at 65 ºC for 30
minutes. Cool, add 2. O ml of 14 % boron trifluoride
solution in methanol and boil under reflux again for 30
minutes and allow it to cool. Add 4 ml of n-heptane and
continue boil under reflux for 5 minutes, cool. Add 1 O ml of
saturated sodium chloride solution, shake, allow to stand.
W ash the upper layer with 4 ml of water for 3 times. Dry
the upper layer with anhydrous sodium sulphate, as the test
solution. Carry out the method for gas chromatography
( 0521), using a O. 32 mm X 30 m capillary column bonded
with a O. 50 µm layer of thickness of phase PEG-20 M. The
tempera ture of inj ection port is 19 OºC ; the tempera tu re of
detector is 2 5 OºC . Raise the col umn tempera ture from 9 OºC
to 160ºC by 20ºC per minute, maintain for 1 minute, then
raise the column temperature to 220ºC by 2°C per minute,
and maintain for 20 minute. Dissolve and dilute a quantity of
methyl myristate CRS, methyl palmitate CRS, methyl
palmitoleate CRS, methyl stearate CRS, methyl oleate CRS,
methyl linoleate CRS and methyl linolenate CRS in n-heptane
to produce a mix reference solution containing 1 mg per ml
Polysorbate 80 (For lnjection) 623
respectively. Inject 1 µl of the mix reference solution onto the
column. The number of the theoretical plates of the column
is not less than 10 000, calculated with reference to the peak
of methyl oleate. The resolution factors between all adjacent
peaks comply with the related requirements. Inject 1 µl of
the test solution onto the column, and record the chrom-
atogram. Calculate the content of each fatty acid by peak area
normalization method ( discard any peak below O. 05%).
Oleic acid is not less than 58. O%, myristic acid is not more
than 5. O% , palmitic acid is not more than 16. O% ,
palmitoleic acid is not more than 8. O%, stearic acid is not
more than 6. O% , linoleic acid is not more than 18. O% and
linolenic acid is not more than 4. O% .
Category Pharmaceutical excipients, solubilizer, emulsifier,
etc.
Storage Preserve in tightly closed containers, and protected
from light.
Polysorbate 80 ( For Inj ection)
[9005-65-6]
Polysorbate 80 for injection is polyoxyethylene ( 20 )
sorbitanmonooleate polymerized by sorbitan monooleate and
ethylene oxide from plant origin.
Description A colorless or light yellow viscous liquid,
odour, faint and characteristic, taste, slightly bitter and
astringent with warm feeling.
Freely soluble in water, in ethanol, in methanol and in ethyl
acetate, very slightly in mineral oils.
Relative density l. 06-1. 09 <0601 ) .
Viscosity Kinematic viscosity 350-550 mmz / s ( 0633 method
1), determined at 25ºC, using a capillary tube of 2. 0-
2. 5 mm in interna! diameter.
Acid value Place 10 g, accurately weighed, in a 250 ml
conical flask, add 50 ml of neutral ethanol ( neutralized to
phenolphthalein IS) to dissolve. Boil under reflux for 10
minutes, cool. Add 5 drops of phenolphthalein IS, titrate
with sodium hydroxide (O. 1 mol/L) VS. The acid value is
not more than l. O ( 0713).
Saponification value 45-55 (0713).
Hydroxyl value 65-80 <0713 >.
lodinevalue 18-24 (0713).
Peroxide value Not more than 3 ( O713 ) .
ldentification (1) To 5 ml of an aqueous solution (1 - 20)
add 5 ml sodium hydroxide TS, boil for a few minutes and
allow it to cool, acidify with diluted hydrochloric acid, a
creamy-white turbidity is produced.
(2) To 2 ml of an aqueous solution (1 - 20) add O. 5 ml of
bromine TS dropwise, the bromine is not decolorized.
(3) To 6 ml add 4 ml of water, mix well, a gelatinous mass
is formed.
(4) To 10 ml of a solution (1 - 20) add 5 ml ammonium
cobalt thiocyanate solution ( dissolve 17. 4 g of ammonium
thiocyanate and 2. 8 g cobalt nitrate in water to 100 mD, mix
well, add 5 ml of chloroform, shake and allow to stand, the
colour of chloroform layer turns blue.
Acidity or alkalinity Dissolve O. 50 gin 10 ml of water, pH
5.0-7.5 (0613).
Absorbance Place O. 1 g, accurately weighed, in a 25 ml
 Polysorbate 80 (Far lnjection)
volumetric flask, dissolve and dilute with a mixture solution
of acetonitrile-water (70 : 30) to volume, mix well. The
absorbance of the solution at 225 nm is not more than l. O;
the absorbance of the solution at 267 nm is not more than O. 10,
and not any maximum absorbance peak between 190-400 nm.
Colour Any colour produced is not more intense than that of
reference solution Y2 <0901 ) .
Ethylene glycol, diethylene glycol and triethylene glycol
Accurately weigh 4 g in a 100 ml volumetric flask, add
accurately O. 004 g of 1, 3-butanediol, dilute to volume with
acetone, mix well as the test solution. Accurately weigh
O. 0025 g of the ethylene glycol CRS, O. 004 g of the
diethylene glycol CRS and O. 004 g of the triethylene glycol
CRS into a 100 ml volumetric flask, add accurately O. 004 g
of 1, 3-butanediol, dilute to volume with acetone, mix well
as the reference solution. Carry out the method far gas
chromatography <0521 ) , using a O. 53 mm X 30 m capillary
column bonded with a l. O µm layer of thickness of phase
50 % phenyl-50 % methylpolysiloxane; maintain the column
temperature at 40ºC, raise the temperature to 60ºC by lOºC
per minute and maintain far 5 minutes, raise the temperature
to l 70ºC by lOºC per minute, then raise the temperature to
280ºC by 15ºC per minute and maintain far 1 hour (adjust if
necessary). The temperature of the injection port is 270ºC.
The temperature of the FID detector is 290ºC . Use the
reference solution as the system suitability test solution,
helium as carrier gas, flow rate is 5. O ml per minute, split
ratio is 2 : l. lnject 1 µl of the test solution onto the column.
The resolution factors between all adjacent peaks are not
ieast than 2. O. Taiiing factors compiy with the requirement.
The relative standard deviation ( RSD) far peak response
ratio of ethylene glycol to 1, 3-butanediol ( internal
standard) and that of diethylene glycol, or triethylene glycol
to 1, 3-butanediol ( internal standard) are not more than
5. O% . Calculate the percentage of ethylene glycol,
diethylene glycol, or triethylene glycol as fallows:
Result =(Ru/Rs) X CCsX Cu) X F X 100
Where Ru is the peak response ratio of the test substance
to the internal standard in the sample solution;
Rs is the peak response ratio of the reference
substance to the internal standard in the
reference solution;
Cs is the concentration of the reference substance
in the reference solution, µg/ml;
Cu is the concentration of the test substance in the
sample solution, (mg/mD;
F is the conversion factor, 103 mg/ g.
All the results of ethylene glycol, diethylene glycol and
triethylene glycol are not more than O. 01%.
Ethylene oxide and dioxan Accurately weigh 1 g in a head-
space vial, add accurately measured l. O ml of purified water,
seal the vial and mix well, as the test solution ( the test vial).
Allow to stand at 70ºC far 45 minutes. Measure 300 µl of
ethylene oxide, equivalent to about O. 25 g, into a 100 ml
volumetric flask containing 50 ml of treated polyethylene
glycol 400 ( using a rotary evaporator remove any volatile
components at a temperature of 60ºC and pressure of l. 5-
2. 5 kPa far 6 hours ) , dilute to volume with the same
solvent, mix well as the ethylene oxide stock reference
solution. Accurately weigh 1 g of the cold ethylene oxide
stock solution, into a 50 ml volumetric flask containing 40 mi
of cooled treated polyrthylene glycol 400, dilute to volume
with the same solvent. Accurately weigh 10 g into a 50 ml
volumetric flask containing 30 ml of water, dilute to volume
with water, mix well. Accurately measure 10 ml into a
50 mi volumetric flask, dilute to volume with water, mix
well as the ethylene oxide reference solution. Accurately
weigh a quantity of dioxan in water to produce a solution of
O. 1 mg per mi as the dioxan reference· solution. Accurately
weigh 1 g of the substance to be examined, in a head space
vial, accurately add O. 5 ml of ethylene oxide reference
solution and O. 5 ml of dioxan reference solution, and seal the
vial, mix well as the reference solution ( the reference vial ) .
Allow to stand at 70ºC far 45 minutes. Measure O. 5 ml of
the ethylene oxide reference solution in a headspace vial, add
O. 1 ml of freshly prepared O. 001 % acetaldehyde solution and
O. 1 ml of dioxan solution, seal the vial and mix well as the
system suitability test solution ( the system suitability test
vial). Allow to stand at 70ºC far 45 minutes. Carry out the
method far gas chromatography < 0521 ) , using a column
packed with polydimethylsiloxane as the stationary phase,
maintain the column temperature at 35ºC far 5 minutes, raise
the temperature to 180ºC by 5ºC per minute, then raise the
temperature to 230ºC by 30ºC per minute and maintain far
5 min (adjust if necessary). The temperature of the injection
is 150ºC. The temperature of the detector is 250ºC. Use the
head space injection method. The equilibration temperature
is 70ºC and maintain far 45 minutes, Inject the gaseous
sample of the system suitability test vial onto the column and
record the chromatogram. Adjust the sensitivity of the
detector so that the peak heights of ethylene oxide and
acetaldehyde in the chromatogram are about 15 % of the full
scale of the recorder. The resolution factor between the
peaks of acetaldehyde and ethylene oxide is not less than 2. O;
the signal-to-noise ratio of the peak of dioxan is not less than
5. Inject separately each of the gaseous sample of the test
vial and reference vial onto the column. Repeat the procedure
at least three times. The relative standard deviation CRSD)
of the 3 areas of peak of ethylene oxide is not more than 15 %
and the relative standard deviation (RSD) of the 3 areas of
peak of dixan is not more than 10 % . Calculate the content
with respect to the peak area obtained in the chromatogram
by the standard addition method, the amount of ethylene
oxide is not more than O. 0001 % and the amount of dioxan is
not more than O. 001%.
Standardization of the ethylene oxide stock reference
solution Measure 10 ml of 50% suspension of magnesium
chloride in absolute ethanol, accurately add 20 ml of
O. 1 mol/L ethanolic hydrochloric acid VS, allow to stand
overnight. Weigh 5 g of the ethylene oxide stock reference
solution into the same flask and stand far 30 minutes. Carry
out the method far potentiometric titration <0701 ) , titrate
with ethanolic potassium hydroxide ( O. 1 mol/L) VS.
Perform a blank determination and make any necessary
correction. Each mi of ethanolic potassium hydroxide VS is
equivalent to 4. 404 mg of ethylene oxide.
Freezing test Allow to stand far 24 hours in a glass
container at 5 ±2ºC, it is not frozen.
Water Not more than O. 5 % <0832 method 1 ( 1 ) ) .
Residue on ignition Not more than O. 1% <0841 ) , using
l. o g.
Heavy Metals Carry out the limit test far heavy metals
<0821 method 2) , using the residue obtained in the test far
residue on ignition: not more than O. 001 %.
Arsenic Place l. O g to a Kjeldahl flask and add 5 mi sulfuric
acid. chargently at temperature below 120ºC ( add sulfuric
acid if necessary, but the total volume added should not
exceed 10 ml), add hydrogen peroxide solution ( 30%)
dropwise, allow the reaction to subside and again heat

between drops, until solution becomes colorless. Allow it to
cool to room temperature and add 10 ml of water. Evaporate
to remove hydrogen peroxide, add 5 ml hydrochloric acid and
a quantity of water, carry out the limit test for asenic ( 0822
method 1): not more than O. 0002%.
Composition of fatty acids Place O. 1 g to a 50 ml conical
flask, add 2 ml of 2% sodium hydroxide solution in
methanol, boíl under reflux in a water bath for 30 minutes.
Cool, add 2. O ml of 14% boron trifluoride solution in
methanol and boil under reflux again for 30 minutes and
allow it to cool. Add 4 ml of n-heptane and contine boil
under reflux for 5 minutes, cool. Add 10 ml of saturated
sodium chloride solution, shake thoroughly, allow to stand.
Wash the upper layer with 4 ml of water for 3 times, using
as the test solution. Carry out the method for gas
chromatography ( 0521 ) , using a O. 25 mm X 100 m
capillary column bonded with a O. 20 µm layer of thickness of
phase 88 % cyanide propyl polysiloxane. The temperature of
injection port is 340ºC; The temperature of FID detector is
330ºC. Raise the column temperature from 90ºC to 160ºC by
20ºC per minute, maintain for 1 minute, then raise the
column temperature to 220ºC by 2ºC per minute, maintain
for 20 minute. Dissolve and dilute a quantity of methyl
myristate CRS, methyl palmitate CRS, methyl palmitoleate
CRS, methyl stearate CRS, methyl oleate CRS, methyl
linoleate CRS and methyl linolenate CRS in n-heptane to
produce a mix reference solution containing O. 1 mg per ml
respectively. Inject 1 µl of the mix reference solution onto
the column. The number of the theoretical plates of the
column is not less than 10 000, calculated with reference to
the peak of methyl oleate. The resolution factors between all
adjacent peaks comply with the related requirements. Inject
1 µl of the test solution onto the column, and record the
chromatogram. Calculate the content of each fatty acid by
peak area normaiization method ( discard any peak beiow
O. 05%). Oleic acid is not less than 98. 0%, myristic acid,
palmitic acid, palmitoleic acid, stearic acid, linoleic acid and
linolenic acid are not more than O. 5 % respectively.
Sterility Ontended use for the sterile prepacations without
terminal sterilization) Complies with the test for sterility
~ 1101).
Bacteria) endotoxin Carry out the test for bacteria!
endotoxin ( 1143): not more than O. 012 EU per mg of
polysorbate 80.
Category Pharmaceutical excipients, solubilizer, emulsifier,
etc.
Storage Preserve in tightly closed containers, protected
from light.
Polyvinyl Alcohol
[9002-89-5]
Polyvinyl Alcohol is obtained by alcoholysis of polyvinyl
acetate in methanol in the presence of alkali. lt isrepresented
by the formula ( CH2 CHOH )n ( CH2 CHOCOCH3 )m, in
+
which m n represents the average polymerization degree.
And the m/n value is 0-0. 35. The mean relative molecular
mass lies between 20 000 and 150 000.
Description Yellow to white powder or translucent granules,
odourless, tasteless.
Soluble in water, slightly soluble in ethanol, practically
Polyvinyl Alcohol ¡.:) '~/ .
insoluble in acetone.
Acid value Place 10. O gin 250 ml of water in a borosilicate
round-bottomed flask attached to a reflux condenser with
stirring in a water bath, heat for 30 minutes, cool to room
temperature with stirring. Measure accurately 50 ml of the
resulting solution, and carry out the limit test for fats and
fatty oils ( 0713): not more than 3. O.
ldentification Carry out the method for infrared absorption
spectrophotometry ( 0402 ) . The spectrum obtained shows
absorption maxima at 2940±10 cm- 1 and 2920±10 cm- 1 •
Viscosity Dissolve by heating in a water, in water to
produce solutions of 3. 8% g/g, 4. 0% g/g, 4. 2% g/g,
respectively. Allow to cool, place in a water bath at 20 ±
O. 1ºC to expel any entrapped air, and carry out the method
for viscosity ( 0633 method 3). Accurately weigh 1 g of
different concentrate solutions. dry to constant weight at
105 ºC. calculate the actual concentration of the solutions on
the dried basis.
Plot the viscosity against the concentration and determine the
standard curve best fitting the plotted points by linear
regression. Determine the kinetic viscosity of a 4. O%
solution at 20 ± O. 1ºC from the standard curve, 85. O%-
115. 0%.
Degree of hydrolysis Transfer 1 g of polyvinyl alcohol, to a
250 ml conical flask, fitted by means of a suitable glass joint
to a reflux condenser. Add 35 ml of dilute methanol ( 3 in
5) , and mix gently to assure complete wetting of the solid.
Add 3 drops of phenolphthalein TS, and add O. 2 mol/L
hydrochloric acid or O. 2 rnol/L sodium hydroxide, if
necessary, to neutralize. Add accurately 25 ml of sodium
hydroxide (O. 2 mol/L) VS, and reflux gently on a hot plate
for 1 hour. Wash the inner wall and bottorn of stopper of the
condenser with 10 ml of water, collect the washings in the
fiask, cooi, and titrate with hydrochioric acid (0. 2 mol/U
VS. Concomitantly perform a blank determination in the
same manner, using the same quantity of sodium hydroxide
(O. 2 mol/L) VS. Calculation of saponification value by the
formula:
5=(B-A) X56.11Xc/W
in which B and A are the volumes, in rnl, of h ydrochloric
acid (0. 2 mol/L) VS consumed in the titration of the blank
and the substance being examined, respectively; e is the
exact normality of the hydrochloric acid solution; W is the
weight, in g, of the portian of polyvinyl alcohol taken; and
56. 11 is the molecular weight of potassium hydroxide.
Calculate the degree of hydrolysis, expressed as percentage
of hydrolysis of polyvinyl acetate, by the formula:
{100-[7. 845/(100-0. 0755) ]} /100
in which 5 is the saponification value of the polyvinyl alcohol
taken: 85%-89%.
Acidity To 2 g add 50 ml of water, heat in a water bath
until dissolved, allow to cool, pH 4. 5-6. 5 ( 0641).
Clarity and color of solution Dissolve 10 g in a round-
bottomed flask, add 250 ml of water, heat on a water bath
for 30 minutes until dissolved. Allow to cool. Any opales-
cence produced is not more pronounced than that of reference
suspension 1 ( 0902 , method 1 ) . Any color produced is not
more intense than that of reference solution Y or YGl
( 0901 , method 1 ) .
Water-insoluble substances To about 6 g, accurately
weighed, add water to produce a 4. 0% (g/g) solution, heat
in a water bath to dissolve with fully stirring, filter through a
100-mesh sieve previously dried at 110ºC to constant weight
when the solution is hot, wash the residue with 25 ml of
 Potassium Bicarbonate
water twice, dry the residue at llOºC far 1 hour: not more
than O. 1%.
Residual solvents Methanol and methyl acetate Transfer O. 6 g
of acetone to a 1000 ml volumetric flask, dilute to volume
with water, and shake well, as the internal standard
solution. Transfer about O. 5 g of the substance being
examined, accurately weighed, to a 20 ml headspace vial,
add accurately measured 2. O ml of internal standard
solution, and shake well, stopper tightly, as the test
solution. Transfer separately O. 125 g of methanol and
methyl acetate, accurately weighed, to a 50 ml volumetric
flask, dilute to volume with the internal standard solution,
and shake well, accurately measure 2 ml to a 20 ml
headspace vial, stopper tightly, as the reference solution.
Carry out the method far determination of residual solvents
<0861, method 2>, using a DB-624 capillary column ( 6 %
cyanopropyl phenol-94% dimethyl polysiloxane, 30. O m X
O. 530 mm X 3. 00 µm). The temperature of injection port is
200ºC; the temperature of detector is 250ºC. Maintain the
temperature of column at 40ºC far 8 min, then raise the
temperature to 150ºC by lOºC per minute, maintain far 2
minutes. The equilibration temperature is 80ºC; the
equilibration time is 30 min. Inject the headspace gas of the
reference solution. Methanol, acetone and methyl acetate are
eluted successively. Resolution factors between all adjacent
peaks comply with related requirements. Inject separately the
headspace gas of the test solution and the reference solution,
and record the chromatograms. Calculate the contents of
methanol and methyl acetate with respect to the peak areas
by internai standard method: not more than l. O% íor each
of methanol and methyl acetate.
Loss on drying When dried to constant weight at 105ºC, the
loses is not more than 5. O% of its weight ( 0831 ) .
Residue on ignition Not more than O. 5 % ( 0841 ) , using
l. o g.
Heavy metals Carry out the limit test far heavy metals
( 0821 method 2), using the residue obtained in the test far
Residue on ignition: not more than O. 001%.
Arsenic Mix l. O g with l. O g of calcium hydroxide, add
water and stir well. After dryness, heat gently until it is
thoroughly charred and then ignite at 500-600ºC until the
incineration is completed. Dissolve the cooled residue in a
mixture of 5 ml of hydrochloric acid and 23 ml of water.
Carry out the limit test far arsenic ( 0822 method 1 ) : not
more than O. 0002%.
Category Pharmaceutical excipients, film-farming agent and
suspending agent, etc.
Storage Preserve in tightly closed containers.
Labling Stated the viscosity with mPa • s or Pa •s.
Potassium Bicarbonate
KHC0 3 100. 12
[298-14-6]
Potassium Bicarbonate is prepared by passing carbon dioxide
in saturated potassium carbonate solution, fallowed by
cooling and crystallization. It contains not less than 99. O%
of KHC03, calculated on the dried basis.
Description A white or almost white crystalline powder or
colorless crystals.
Freely soluble in water; practically soluble in ethanol.
ldentification Yields the reactions characteristic of potassium
salts (1) and bicarbonate ( 0301).
Alkalinity Dissolve 2. 5 g in 50 ml of water, pH: not more
than 8. 6 ( 0631 ) .
Clarity and colour of solution A solution of 5. O g in 100 ml
of water is clear and colourless ( 0901 and 0902).
Carbonates Triturate 3. O g with 25 ml of ethanol and 5 ml
of water, add 3 drops of phenolphthalein IS, and titrate with
barium chloride solution ( dissolve accurately weighed
12. 216 g of barium chloride in 300 ml of water in a 1000 ml
volumetric flask, and dilute to volume with ethanoD until the
suspension becomes colourless. Trituate far 2 minutes, any
pink colour produced requires barium chloride solution to
discharge; repeat the operation until the pink colour does not
appear. Each ml of barium chloride solutionis equivalent to
6. 911 mg of potassium carbonate: not more than 2. 5%.
Chloride Dissolve O. 33 g in 25 ml of water, add dropwise
nitric acid to make the solution slightly acidic, heat to expel
carbon dioxide completely on a water bath, and allow to
cool. Carry out the limit test far chlorides ( 0801 ) . Any
opalescence produced is not more pronounced than that of a
ref erence solution using 5. O ml of sodium chloride standard
solution (0. 015%).
Sulfate Dissolve l. O g in 40 ml of water, add dropwise
hydrochloric acid to make the solution slightly acidic, heat to
expel carbon dioxide completely on a water bath, and allow
to cooi. Carry out the limit test íor suHates ( 0802). Any
opalescence produced is not more pronounced than that of a
reference solution using l. 5 ml of potassium sulfate standard
solution (0. 015%).
Arnmonium Dissolve l. O gin 50 ml of water, add 2 ml of
alkaline mercuric potassium iodide TS, mix well, and allow
to stand far 15 minutes. Carry out the limit test far
ammonium ( 0808 ) . Any colour produced is not more
intense than that of a reference solution using 2. O ml of
ammonium chloride standard solution (O. 002%).
Loss on drying When dried with silica gel in a desiccator far
4 hours, using 4. O g, loses not more than O. 3% of its
weight ( 0831 ) .
Calcium Dissolve l. O g in 50 ml of freshly boiled and cooled
water, add 1 ml of ammonia TS and 2 ml of ammonium
oxalate TS, mix well, and allow to stand far 2 hours. Any
opalescence produced is not more pronounced than that of a
reference solution using l. O ml of standard calcium solution
(accurately weight O. 125 g of calcium carbonate to a 500 ml
volumetric flask, add a mixture of 5 ml of water and O. 5 ml
of hydrochloric acid, dilute with water to volume, and mix
well. Each ml is equivalent to O. 1 mg of Ca) (0. 01%).
Iron Dissolve l. O g in a quantity of water, add dilute
hydrochloric acid to make the solution slightly acidic, boil to
expel carbon dioxide, allow to cool, dilute with water to
produce 25 ml. Carry out the limit test far iron ( 0807). Any
colour produced is not more intense than that of a reference
solution using 2. O ml of iron standard solution (O. 002 % ) .
Sodium Dissolve O. 25 g in water in a 50 ml volumetric flask
and dilute to volume, shake well. Accurately measure 20 ml
in each of two 50 ml volumetric flasks, add separately 10 ml
of hydrochloric acid solution ( 1 in 2) : to one volumetric
flask, dilute with water to volume, mix well, transfer
accurately measured 10 ml into a 50 ml volumetric flask, and
dilute with water to volume as the test solution; to the other

volumetric flask, add 5 ml of sodium chloride standard
solution containing O. 1 mg of Na per ml, dilute with water
to volume, mix well, transfer accurately measured 10 ml
into a 50 ml volumetric flask, dilute with water to volume as
the reference solution. Carry out the method for atomic
absorption spectrophotometry ( 0406 method 2 ) , and
measure the absorbance of the two solutions at 589 nm: not more
than o. 50%.
Heavy metals To 2. O g add 12 ml of dilute hydrochloric acid
and 5 ml of water, boil for 5 minutes, and allow to cool.
Add 1 drop of phenolphthalein IS, add dropwise ammonia TS
until a pink colour is produced, allow to cool, add 2 ml of
aceta te BS ( pH 3. 5) and then sufficient water to produce
25 ml. Carry out the limit test for heavy metals ( 0821
method 1): not more than O. 001%.
Arsenic Dissolve l. O g in 23 ml of water, add 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822
method 1): not more than O. 0002%.
Assay Dissolve 2 g, accurately weighed, in 100 rnl of water,
add 10 drops of methyl red-bromocresol green IS, titrate with
hydrochloric acid (l. O mol/L) VS until the colour turns from
green to purplish-red. &il for 2 minutes, cool to room
temperature and continue the titration until the colour turns from
green to dark purple. Perform a blank determination and make
any necessary correction. Each rnl of hydrochloric acid
(l. O mol/L) VS is equivalent tolOO. 1 mg of KHC03 •
Category Pharmaceutical excipients, pH regulator, etc.
Storage Preserve in well closed containers.
Potassium Chloride
See the same monograph in Volume II .
Category Pharmaceutical excipients, osmotic pressure
regulator, etc.
Potassium Dihydrogen Phosphate
KH2 P04 136. 09
[7778-77-0 J
Potassium Dihydrogen Phosphate contains not less than
99. O% of KH2 P0 4 , calculated on the dried basis.
Description Colourless crystals or a white crystalline
powder or granule or mass; odourless.
Freely soluble in water, practically insoluble in ethanol.
Identification Yields the reactions characteristic of potas-
sium salts and phosphates ( 0301 ) .
Acidity An aqueous solution of 50 mg per ml, pH 4. 2-4. 5
<0631>.
Clarity and colour of solution Dissolve l. O g in 10 ml of
water, the solution is clear and colourless ( 0901 and 0902),
any opalescence produced is not more pronounced than that
of reference suspension 1 ( 0902).
Chloride Carry out the limit test for chloride <0801), using
5. O g. Any opalescence produced is not more pronounced
than that of a reference solution using 5. O ml of sodium
chloride standard solution (O. 001 %).
Potassium Hydroxide 621
Sulfate Carry out the limit test for sulfate ( 0802 ) , using
3. 3 g. Any opalescence produced is not more pronounced
than that of a reference solution using l. O ml of potassium
sulfate standard solution (O. 003%).
Carbonate Dissolve 2. O gin 10 ml of water, boil and allow
to cool, add 2 ml of hydrochloric acid, no effervescence is
produced.
Condensed phosphate Dissolve 2. O g in water in a 100 ml
volumetric flask, dilute to volume and mix well. Transfer
5. O ml of the solution to a Nessler cylinder, add l. O ml of
dilute acetic acid, 5. O ml of the acetic acid-sodium acetate
solution ( mix 17 ml of 1 mol/L sodium hydroxide solution
and 40 ml of dilute acetic acid, dilute with water to 100 mD,
and sufficient water to produce 15 ml, add 2 ml of barium
chloride TS and mix well. Allow to stand at 25 ± 2ºC for
15 minutes, no opalescence is produced.
Water-insoluble substances Dissolve 10. O gin 100 ml of hot
water, filter through a tared No. 4 sintered glass crucible,
previously dried to constant weight, and wash the residue
with 200 ml of hot water in 10 portions, dry at 105ºC for 2
hours: not more than 20 mg (O. 2 % ) .
Reducing substances Dissolve 5. O g in freshly boiled and
cooled water, and dilute to 50. O ml with the same solvent.
To 5. O ml of the solution add 5 ml of dilute sulfuric acid and
O. 25 ml of potassium permanganate (O. 02 mol/L) VS and
heat on a water bath for 5 minutes, the solution remains a
purplish red colour.
Loss on drying When dried to constant weight at 105ºC,
loses not more than O. 2 % of its weight ( 0831 ) .
Iron Dissolve l. O g in 20 ml of water, add 2 ml of 10%
sulfosalicylic acid solution, mix well, add 5 ml of ammonia
TS and mix well. Any colour produced is not more intense
than that uf a reference :solutiou µreµared iu the :same
manner, using l. O ml of iron standard solution ( 0807)
(0. 001%).
Heavy rnetals Carry out the limit test for heavy metals
( 0821 method 1), using 2. O g: not more than O. 001%.
Arsenic Dissolve l. O g in 23 ml of water, add 5 ml of
hydrochloric acid. Carry out the limit test for arsenic ( 0822
method 1 ) : not more than O. 0002 % .
Assay Dissolve about 2. 5 g, accurately weighted, in 100 ml
of freshly boiled and cooled water. Carry out the method for
potentiometric titration ( 0701 ) , titrate with sodium
hydroxide ( 1 mol/L) VS. Each ml of sodium hydroxide
(1 mol/L) VS is equivalent to 136. 1 mg of KH 2P0 4 •
Category Pharmaceutical excipients, pH regulater and
buffer, etc.
Storage Preserve in tightly closed containers.
Potassium Hydroxide
KOH 56.11
[1310-58-3]
Potassium Hydroxide is produced by the electrolysis of potassium
chloride. It contains not less than 85. O% of KOH.
Description White solid, pellets, sticks or flakes; hard,
brittle and showing a crystalline fracture; rapidly absorbs
carbon dioxide in the air.
Very soluble in water, freely soluble in ethanol.
 ·1128.:.·) .....
···.·.····.·.·.·········.·.·1 Potassium Nitrate
Identification 0) Dissolve 50 mg in 500 ml of water, the
solution is alkaline.
(2) The aqueous solution yields the reactions characteristic of
potassium salts ( 0301).
Clarity and colour of solution Dissolve 5 g in 50 ml of
freshly boiled and cooled water, the solution is clear and
colourless ( 0901 and 0902).
Chloride Take 5 ml of the test solution in the assay, drop
nitric acid to neutral, add water to 25 ml ( 0801 >. Any
opalescence produced is not more pronounced than that of a
reference solution using 2. O ml of sodiumchloride standard
solution (0. 01%).
Sulfate Dissolve 2. O g in a quantity of water, add
hydrochloric acid 0-2) to neutral, add water to 40 ml, and
then add 5 ml of hydrochloric acid C1 - 2) <0802 >. Any
opalescence produced is not more pronounced than that of a
reference solution using l. O ml of potassium sulfate standard
solution (0. 005%).
Carbonate Not more than 2. O%, calculated by the Kz C03
from Assay.
Phosphate Dissolve O. 5 g in a quantity of water, dropping
nitric acid to acidic and add water to 100 ml. Add 4 ml of
molybdate sulfuric acid TS and O. 1 ml of stannous chloride
solution TS, shake thoroughly and allow to stand for 10
minutes. Any opalescence produced is not more pronounced
than that of a reference solution using 2. O ml of phosphate
standard solution ( accurately weigh 143 mg of mono-
potassium phosphate in a 1000 ml volumetric flask, dilute to
volume with water and shake well. Befare use, transfer 5 ml
of the solution, accurately measured, to a 100 ml volumetric
flask, dilute to volume with water, and shake well. Each ml
of the solution is equivalent to 5 µg of P04 ) CO. 002 % ) .
Sodiurn Dissolve 2. O g in a quantity of water in a 250 ml
volumetric flask' neutralize with hydrochloric acid e1-2) '
dilute to volume with water and shake well. Accurately
transfer l. O ml to four 100 ml volumetric flasks respectively
and add O, O. 1, O. 2, O. 3 ml of sodium standard solution
Ceach ml is equivalent to 1 mg of Na). Add water to volume
and shake well. Carry out the method for atomic absorbance
spectrophotometry ( 0406 method 2 ) , measure the
absorbance at 589 nm. The absorbance of the test solution is
not greater than that of the standard solution O. 0%).
Aluminum Dissolve l. O g in a quantity of water and
neutralize with hydrochloric acid e1 - 2) ' dilute to 20 ml
with water. Add 2 ml of 30 % acetic acid solution and 2 ml of
10% ascorbic acid, shake well and add 20 ml of acetic acid-
ammonium aceta te buffer solution CpH 4. 5) and 3 ml of
ammonium aurin tricarboxylate solution eweigh o. 25 g of
ammonium aurin tricarboxylate and 5 g of gum arabic, add
250 ml of water to dissolve warmly, add 87 g of ammonium
acetate to dissolve. Add 50 ml of hydrochloric acid and dilute
to 500 ml with water), add water to dilute to 50 ml, shake
well and stand for 15 minutes. Any opalescence produced is
not more pronounced than that of a reference solution using
o. 5 ml of aluminum standard solution eaccurately weigh
l. 759 g of aluminum potassium sulfate in a 1000 ml
volumetric flask, add a quantity of water to dissolve, add
sulfuric acid solution 0-4) 10 ml and dilute to volume with
water and shake well. Each ml of the solution is equivalent to
O. 1 mg of AD (0. 005%).
Iron Dissolve l. O gin 10 ml of water and hydrochloric acid
0-2), adjust pH to 2, dilute with water to 25 ml. Carry
out the limit test for iron ( 0807) : any opalescence produced
is not more pronounced than that of a reference solution
using l. O ml of iron standard solution (0. 001%).
Heavy metals Dissolve l. O g in a quantity of water, add
2 ml of nitric acid, and evaporate to dry on water bath. Add
water to dissolve the residue and adjust pH to 4 with
O. 1 mol/L sodium hydroxide solution and add water to
20 ml. Add 2 ml of acetate BS (pH 3. 5) anda quantity of
water to produce 25 ml. Carry out the limit test for heavy
metals ( 0821 method 1): not more than O. 002%.
~y Dissolve about 10 g, accurately and rapidly weighed,
in freshly boiled and cooled water in a 250 ml volumetric
flask, allow to cool, dilute to volume with water and shake
well. Accurately transfer 50 ml to a 500 ml conical flask with
stopper, add 95 ml of freshly boiled and cooled water and
5 ml of 10% barium chloride solution, plugged, shake well.
Stand for 15 minutes, add 2 drops of phenolphthalein TS,
e
and titrate with hydrochloric acid 1 mol/L) vs until the
pink color disappears. Read the volume CV1) of hydrochloric
acid (1 mol/L) VS consumed; add 10 drops of methyl red-
bromcresol green TS, continue the titration from a green
color to a dark red color. &il the solution for 2 minutes and
cool, titrate to dark red. Read the volume CYz ) of
hydrochloric acid C1 mol/L) VS. Calcula te the assay of
KOH according to V 1 , and calculate the assay of Kz C0 3
according to the volume CV2 -Vi) consumed in the titration
with methyl red-bromcresol green TS. Each ml of
hydrochloric acid O mol/L) VS is equivalent to 56. 11 mg of
total alkali, calculated as KOH, or equivalent to 69. 10 mg of
KzC03.
Category Pharmaceutical excipients, pH regulator, etc.
Storage Preserve in tightly closed containers.
Potassium Nitrate
KN03 101. 10
[7757-79-1]
Potassium nitrate contains not less than 99. O% of KN0 3 ,
calculated on the dried basis.
Description A white or colorless transparent crystal.
Freely soluble in water and slightly soluble in ethanol.
Identiflcation Yields the reactions characteristic of potassium
salts and ni trates ( 0301).
Acidity or alkalinity Dissolve l. O g with 10. O ml of freshly
boiled cold water, add 1 drop of bromothymol blue TS; not
more than O. 5 mL of hydrochloric acid (0. 01 mol/L) VS or
sodium hydroxide (0. 01 mol/L) VS is required to change the
color of the solution.
Clarity and color of solution A solution of 1 g in 10 ml of
water is clear and colorless ( 0901 and 0902).
Chloride Carry out the limit test for chloride ( 0801 >, using
l. O g. Any opalescence produced is not more pronounced
than that of a reference solution by using 2. O ml of sodium
chloride standard solution (0. 002%).
Sulfate Carry out the limit test for sulfate ( 0801 ) , using
l. O g. Any opalescence produced is not more pronounced
than that of a reference solution by using l. 5 ml of
potassium sulphate standard solution (O. 015%).
Reducing substances Dissolve l. O g in 10 ml of water. Add
O. 5 ml of dilute sulfuric acid and 2 ml of zinc iodide starch

IS, a blue color is not produced within 2 minutes.
Loss on drying When dried to constant weight at l05ºC,
loses not more than O. 5 % of its weight <0831 ) .
Ammonium Carry out the limit test far ammonium
<0808 ) , using O. 4 g. Any color produced is not more
intense than that of a reference solution by using 2. O ml
ammionium chloride standard solution (O. 005 % ) .
Calcium Dissolve 2. O gin 15 ml of water. Use the solution
as the test solution. Transfer O. 2 ml of alcoholic standard
calcium solution ( weigh accurately 2. 50 g of calcium
carbonate into a 1000 ml volumetric flask, add 12 ml of
acetic acid, dissolve with a quantity of water to volume,
shake well, as the calcium stock solution. Immediately
befare use, accurately transfer 10 ml of calcium stock
solution into a 100 ml volumetric flask. Dilute with ethanol
to volume and shake well. Each ml is equivalent to O. 1 mg of
calcium.) into a Nessler tube. Add 1 ml of 4% ammonium
oxalate solution. After 1 minute, add the mixture of 1 ml of
dilute acetic acid and 15 ml of test solution. Shake well and
allow to stand far 15 minutes, as the test solution. Add
O. 2 ml of alcoholic standard calcium solution and 1 ml of 4 %
ammonium oxalate solution into another Nessler tube. After
1 minute, add 10. O ml of standard calcium solution
(immediately befare use, accurately transfer 1 ml of calcium
stock solution into a 100 ml volumetric flask. Dilute with
water to volume and shake well. Each ml is equivalent to
10 µ.g of calcium), 1 ml of dilute acetic acid and 5 ml of
water, shake well and allow to stand far 15 minutes, as the
reference solution. The colour of test solution is not more
intense than that of the reference solution (O. 005 % ) .
lron Carry out the limit test far iron <0807), using 2. O g.
Any color produced is not more intense than that of a
reference solution using 2. O ml iron standard solution
fA AA1 O/'\
\V. VV.l/OJ.
Sodium Weigh l. O g into a 100 ml volumetric flask. Dilute
with water to volume and shake well. Use the solution as the
sample solution. Take O. 509 g sodium chloride primary
standard (equivalent to O. 2 g of sodium) previously dried at
120ºC far 2 hours. Dilute with water into reference solutions
which contain O. 5 µ.g, l. O µ.g, l. 5 µ.g and 2. O µ.g of sodium
per ml, respectively. Carry out the method far atomic
absorption spectrophotometry <0406 method 1), measuring
the absorbance at 589. O nm and using water as the blank
solution. Calculate by standard curve method, not more than
0.10%.
Heavy metal Not more than O. 001% <0821 method 1) ,
using l. O g.
Assay Dissolve O. 2 g, accurately weight, with 20 ml of
water. Transfer the solution into a pretreated strong acidic
cation-exchange resin column. Wash the resin column with
water (at about 3 ml/min flow rate). Collect about 250 ml of
exchange liquid and washing. Add 1 ml of phenolphthalein IS
and ti trate with sodium hydroxide (O. 1 mol/L) VS to the
end point. Each mi of sodium hydroxide (0. 01 mol/L) VS is
equivalent to 1O. 11 mg of KNQ3.
The treatment method of cation-exchange resin: mix 15 g of
cation exchange resin in sodium salt state with a quantity of
water. Transfer the solution into the ion exchange resin
column. Add 30-40 ml of 2 mol/L hydrochloric acid solution
from the top. Open the tap. After hydrochloric acid soaks
the resin, clase the tap. Immerse overnight. Wash the resin
column with 300-500 ml of freshly boiled cold water and take
the last 100 ml of washing. Add 2-3 drops of phenolphthalein
IS and 1 drop of sodium hydroxide (O. 1 mol/L) VS. If a
Potassium Sorbate
pink color is produced, it can be used for test.
Category Pharmaceutical excipients, osmotic pressure regulator.
Storage Preserve in tightly closed containers.
Potassium Sorbate
H3C~OK
o
Cs H7 K02 150. 22
[509-00-01]
Potassium Sorbate is ( E, E ) -2 ,4-hexadienoic acid
potassium salt. Produced by the reaction of sorbic acid with
potassium carbonate or potassium hydroxide. It contains not
less than 99. O% of C; H 7K02 , calculated on the dried basis.
Description A white or almost white scaly or granular
crystalls or crystal powder.
Freely soluble in water; slightly soluble in ethanol.
ldentification (1) Dissolve about O. 1 g in 10 ml of water,
add 1 ml of acetone and adjust pH to acidic by adding dilute
hydrochloric acid, add 2 drops of bromine TS and shake, the
colour of bromine disappears.
(2) Transfer a quantity of the substance being examined,
dissolve in water to produce a solution of O. 2 mg per ml. To
a quantity of this solution, add O. 1 mol/L hydrochloric acid
solution to produce a solution of 2 µ.g per ml. The
absorbance of this solution exhibits a maximum at 264 nm
(0401>.
(3) The infrared absorption spectrum (0402) is concordant
with the reference spectrum (IR Album No. 688).
( 4 ) Its aqueous solution yields the flame reaction of
potassium salts <G301).
Acidity or alkalinity Dissolve l. O gin 20 ml of water, add 2
drops of phenolphthalein IS, if pale red colour is produced,
add O. 25 mi of hydrochloric acid (O. 1 mol/L) VS, the
colour disappears; if colourless, add O. 25 ml of sodium
hydroxide (0. 1 mol/L) VS, pale red colour is produced.
Clarity and colour of solution Dissolve O. 20 g in 5 ml of
water, the solution is clear and colourless <0901 and 0902);
any colour produced is not more intense than that of reference
solution Y3 <0901 method 1 ) .
Chloride Dissolve O. 40 g in 15 ml of water, add 10 mi of
dilute nitric acid with stirring, fil ter and wash the residue far
severa! times with 10 ml of water, combine the filtrate and
washed solution, dilute the solution to about 40 ml with
water. Carry out the limit test far chloride <0801 ) . Any
opalescence produced is not more pronounced than that of a
reference solution using 7. O ml sodium chloride standard
solution (O. 018%).
Sulfate Dissolve l. 05 g in 30 ml of water, add 2 ml of
dilute hydrochloric acid with stirring, filter and wash the
residue with 8 ml of water, combine the filtrate and washed
solution, dilute the solution to about 40 ml with water.
Carry out the limit test for sulfate <0802). Any opalescence
produced is not more pronounced than that of a reference
solution using 4. O ml potassium sulfate standard solution
(0. 038%).
Aldehydes Transfer l. O g of the substance being examined
to a 100 ml volumetric flask, dissolve with 50 ml of 2-
propanol and 30 ml of water, adjust to pH 4 with 1 mol/L
 630 Patato Starch
hydrochloric acid solution, dilute to volume with water and
shake well. Accurately transfer 10 ml of the solution to a
Nessler cylinder, add 1 ml of fuchsin-sulfurous acid TS (To
O. 2 g of basic fuchsin add 120 ml of hot water, cool and add
20 ml of 10 % crystal sodium sulfite solution and 2 ml of
hydrochloric acid, shake welD , shake well and allow to stand
for 30 minutes. Any colour in the solution is not more
intense than that of a formaldehyde standard solution
(Transfer a quantity of formaldehyde solution accurately,
dilute with water to produce a solution containing O. 1 mg of
formaldehyde per ml, to l. O ml of the solution add 5 ml of
2-propanol and 4 ml of water, shake well, prepare by the
same manner as the reference solution) (0. 1%).
Loss on drying When dried to constant weight at 105ºC,
loses not more than l. O% of its weight <0831 ) .
Heavy metals Weigh 2. O g of the substance to be examined
in a crucible, mix with O. 5 g of magnesium oxide.
Progressively heat and continue heating until an almost white
or greyish-white residue is obtained. Heat at 800ºC for 1
hour. Dissolve the residue with 10 ml of hydrochloric acid
solution Cl-2) in 2 times. Add phenolphthalein IS and then
dropwise ammonia solution until the solution is neutral.
Cool, add glacial acetic acid until the red colour disappears
and add O. 5 ml in excess, then add 2 ml of acetate BS (pH
3. 5). Transfer the solution to a Nessler cylinder, dilute to
25 ml with water as the test solution. Using 2. O ml of lead
standard solution instead of the substance to be examined,
mix with O. 5 g of magnesium oxide, prepare by the same
manner as the reference solution. Carry out the limit test for
heavy metals <0821 method 1): not more than O. 001%.
Arsenic W eigh O. 67 g of the substance to be examined, mix
with l. O g of calcium hydroxide. Add a little water, stirring
and drying. lgnite until carbonized, then heat at 500-600ºC
until incineration is complete. Cool and dissolve in 5 ml of
hydrochloric acid and 23 ml of water. Carry out the limit test
for arsenic <0822 method 1 ) : not more than O. 0003 % .
Assay Dissolve about O. 12 g, accurately weighed, in 24 ml
of glacial acetic acid and 1 ml of acetic anhydride. Add 1 drop
of crystal violet IS, titrate with perchloric acid (0. 1 mol/L)
VS until the colour changes to blue. Perform a blank
determination and make any necessary correction. Each ml of
perchloric acid (0. 1 mol/L) VS is equivalent to 15. 02 mg of
C6 H1K02.
Category Pharmaceutical excipients, preservative.
Storage Preserve in tightly closed containers.
Potato Starch
Potato Starch is polysaccharide granules obtained from the
tuber of patato (Solanum tuherosum L.)
Description A white or almost white powder.
Insoluble in water orin ethanol.
ldentification ( 1) Examine under a microscopy with
glycerine-acetic acid TS < 2001 ) : simple grains, either
irregularly shaped, ovoid or pear-shaped, usually 30-100 µm
in size but occasionally exceeding 100 µm, or rounded, 10-35
µm in size. There are occasional compound granules having
2-4 components. The ovoid and pear-shaped granules have an
eccentric hilum and the rounded granules acentric or slightly
eccentric hilum. All granules show clearly visible concentric
striations. Between orthogonally orientated polarising plates
or prisms, the granules show a distinct black cross
intersecting at the hilum.
(2) Boil about 1 g with 15 ml of water, cool. A whitish
translucent gelationous substance is produced.
(3) Add a drop of iodine TS to the gelationous substance
obtained from Identification (2) . A blue or dark blue colour
is produced which gradually disappears on heating.
Acidity or alkalinity Dissolve 5. O g in 25 ml of water, stir
for 1 minute to mix, stand for 15 minutes , pH 5. 0-8. O
(0631 ).
Foreign matter Examine under a microscopy with glycerine-
acetic acid TS <2001), no starch grains of any other origin
are present.
Sulfur dioxide Not more than O. 005 % <0331 ) .
Oxidizing substance Transfer 4. O g of the substance being
examined to a conical flask with stopper, add 50. O ml of
water, insert the stopper and shake for 5 minutes, transfer
to a centrifuger tube with stopper, centrifuge until the
solution is clear. Transfer 30. O ml of the supernatant to a
iodine flask, add 1 ml of glacial acetic acid and l. O g of
potassium iodide, stopper tightly, shake, allow to stand for
30 minutes in a dark place. Add 1 ml of starch IS, and titrate
with sodium thiosulfate (O. 002 mol/L) VS until the blue
colour disappears. Perform a blank determination and make
any necessary correction. Each ml of sodium thiosulfate
(O. 002 mol/L) VS is equivalent to 34 µg of oxidizing
substance ( calculated as H2 02 ) , not more than l. 4 ml of
sodium thiosulfate ( O. 002 mol/L ) VS is consumed
(0. 002%).
Loss on drying When dried at 130ºC for 90 minutes, loses
not more than 20. O% of its weight <0831 ) .
Ash Not more than O. 6 % <2302) .
lron Mix l. 50 g with 15. O ml of 2 mol/L hydrochloric acid
solution, shake for 5 minutes, filter. Transfer 10. O ml of
the filtrate to a 50 ml Nessler cylinder, add 50 mg of
ammonium persulfate and dilute with water to 35 ml. Carry
out the limit test for iron <0807). Any colour produced is not
more intense than that of a reference solution using l. O ml of
iron standard solution (O. 001%).
Microbial limit Comply with the requirements for microbial
limit <1105 and 1106), the total aerobic bacteria count is
not more than 1000 cfu per g and the total combined yeast/
mold count is not more than 100 cfu per g of the substance
being examined, Escherichia coli are absent.
Category Pharmaceutical excipients, diluent, adhesive,
etc.
Storage Preserve in well closed containers.
Povidone K30
CC6H9NO)n
[9003-39-8]
Povidone K30 is obtained from the polymerization of vinyl
pyrrolidone monomer, which is prepared by the reaction of

pyrrolidone with ethene under the pressure, in the presence
of catalyst. It consists of linear polymers of 1-vinyl-2-
pyrrolidone. Average molecular weight is 3. 8 X 104 ,
molecular formula is CC6 Hg NO )n • The "n" is average
polymer length.
Description A white or almost white powder; odourless or
slightly characteristic odour; tasteless; hygroscopic.
Soluble in water, in ethanol, in isopropanol and in
chloroform; insoluble in acetone and in ether.
Identification (1) To 2 ml of a solution 0-50) add 2 ml
of 1 mol/L hydrochloric acid and a few drops of potdassium
dichromate TS: an orange-yellow precipitate is formed.
(2) To 3 ml of a solution 0-50) add 15 mg cobalt nitrate
and 75 mg of ammunium thiocyanate, stir and acidify the
resulting solution by the dropwise addition of dilute
hydrochloric acid TS, a pale blue precipitate is formed.
(3) To 3 ml of a solution 0-50) add 1-2 drops of iodine
TS: a brown-red colour is produced. Stir, a brown-red
colour solution is produced.
K-value Place l. 00 g ( calculate on anhydrous basis),
accurately weighed, in a 100 ml volumetric flask, add a
volume of water to dissolve and dilute to volume. Place it in
a water bath at 25"C ±O. 05ºC far 1 hour, measure the
relative viscosity r¡r ( 0633 method 3) , calculate the K value
from the following formula, not less than 27. O and not more
than 32. O.
K l. 5Wlg1Jr-W+ ./300Wlg11r+CW+1. 5Wlg1Jr) 2
o. 15W+o. 003w2
Where W is the weight, in g, of the substance being
examined (calculate on anhydrous basis).
pH value Dissolve l. O g ( calculate on anhydrous basis) in
20 ml of water, pH 3. 0-7. O ( 0631 ).
Aldehyde Mix 20. O g (calcula te on anhydrous basis) with
180 ml of 4. 5 mol/L sulfuric acid solution in a distilling
flask, and heat under refulx far 45 minutes; allow to cool
and add 20 ml hydrooxylamine hydrochloride solution
(dissolve 6. 95 g hydrooxylamine hydrochloride in 100 ml of
water, adjust to pH3. 1 with ammonia TS), and then place
the distilling flask in an ice bath, insert conderser under the
liquid surface. Heat and distil, until about 120 ml of the
distillate is obtained, stop distillation, not more than 9. 1 ml
of sodium hydroxide (O. 1 mol/L) VS is required to ti trate
pH value to 3. 1, and perform a blank determination and
make any necessary correction.
N-vinyl pyrrolidone Dissolve 10. O g ( calculate on anhy-
drous basis ) in 80 ml of water, add 1 g sodium acetate and
add 10 ml of iodine (O. 05 mol/L) VS accurately, allow to
stand far 10 minutes, titrate with sodium thiosulfate
(0. 1 mol/L) VS until the colour disappears; add 2 ml of
starcch IS towards the end point, not more than 3. 6 ml of
sodium thiosulfate (O. 1 mol/U VS is required, perform a
blank determination and make any necessary correction.
Water Not more than 5. O% ( 0832).
Residue on ignition Not more than O. 1 % ( 0841 ) , using
l. o g.
Heavy metals Carry out the limit test far heavy metals
( 0821 method 2): not more than O. 001 %.
Nitrogen content Place about O. 1 g, accurately weighed, in
a Kjeldahl flask, add 10 g of potassium sulfate and O. 5 g of
copper sulfate, slowly add 20 ml of sulfuric acid TS along the
wall of flask, put a small funnel on the top of the Kjeldahl
flask, gently heat the flask by direct fire until a clear, green
Powdered Cellulose 1 <
solution is obtained. Continue the heating far 30 minutes,
allow to cool. Transfer completely to a 100 ml volumetric
flask, and dilute to the volume with water. Using accurately
measured 10 ml of the resulting solution, carry out the
method far determination of nitrogen ( 0704 method 2),
titrate with sulfuric acid (O. 005 mol/L) VS, perform a
blank determination and make any necessary correction. The
content of nitrogen should be not less than 11. 5 % and not
more than 12. 8%, calculated on the anhydrous basis.
Category Pharmaceutical excipients, binder, cosolvent,
etc.
Storage Preserve in tightly closed containers, stored in a dry
place and protected from light.
Powdered Cellulose
OH
H
C6 n H10n+2 Osn+ i
[9004-34-6]
Powdered Cellulose is purified, mechanically disintegrated a-
cellulose obtained from fibrous plant materials.
Description A white or almost white powder or granular
powder.
Practically insoluble in water, in acetone, in anhydrous
ethanol, in methylbenzene and in dilute hydrochloric acid.
Identification ( 1) Place 10 mg on a glass plate, dissolve
with 2 ml of zinc chloride solution ( completely dissolve 20 g
of zinc chloride and 6. 5 g of potassium iodide with 10. 5 ml
of water, add O. 5 g of iodine, shake far 15 minutes) and a
colour of bluish violet is produced.
(2) Weigh accurately O. 25 g into a conical flask with stopper.
Add accurately 25 ml of water and l. O mol/L cupriethy-
lenediamine solution, respectively, stopper tightly, shake to
completely dissolve. Transfer a volume of the solution to the
Ubbelohde viscometer ( the internal diameter of the capillary
column is O. 7-0. 8 mm), equilibrate in 25±0. 1 ºC water bath
far at least 5 minutes. Record the time t 1 (in seconds) when
the solution flows through the two scales up and clown of the
viscometer and calculate the kinematic viscosity (u 1 ) of the
solution. Mix a volume of l. O mol/L cupriethylenediamine
solution with equivalent amount of water. Repeat the
operations with Ubbelohde viscometer ( the internal diameter
of the capillary column is O. 5-0. 6 mm) and determine the
flow time t2 (in seconds), calculate the kinematic viscosity
Cu2) of the solvent ( 0633 method 2). Calculate the relative
viscosity ( r¡reI) of the substance being examined as the
following formula:
VI
1Jrel = -
U2
According to the calculated relative viscosity ( r¡re1) to find
the intrinsic viscosity [ r¡] C in the lntrinsic Viscosity Table
( see the attached table ) . Calcula te the degree of
polymerization (P) according to the following formula and is
 Powdered Cellulose

not less than 440.
95 [ 9J e dry at 105ºC for 30 minutes, the residue is not more than
p m [ (lOO-b) /100]
Where m is the sample amount of the substance being
examined (g);
b is the loses on drying of the substance being
examined, %.
Acidity or alkalinity Dissolve 10 g in 90 ml of water, stir
for 1 hour and stand, the pH of the supernatant is 5. 0-7. 5
<0631>.
Solubility Dissolve 50 mg in 10 ml of ammoniated tetrammine
copper (dissolve 6. 9 g of copper sulfate in 20 ml of water, stir
and drop concentrated ammonia solution until the produced
precipitate is dissolved completely. Cool to below 20ºC,
shake and add 6 ml of 10 mol/L sodium hydroxide solution,
filter through No. 3 sintered glass funnel, wash the
precipitate with water until the filtrate is clear, add 40 ml of
concentrated ammonia solution, stir to dissolve the precipitate
and conduct the suction filtration), shake well, dissolve
completely, leaving no residue.
Soluble substances in ether Place 10 g, accurately weighed,
into a chromatographic column with the internal diameter of
20 mm, elute with 50 ml of ether free of peroxide, the flow
rate is 20 drops per minute, evaporate the elution in
constant-weight evaporating dish previously dried at 105ºC,
15. O mg (0. 15%).
Soluble substances in water Weigh accurately 6 g of the
substance, add 90 ml of freshly boiled cold water, stir for
10 minutes, perform the vacuum filter, discard at least
10 ml of primary filtrate, measure 15 ml of the clear
successive filtrate and evaporate to dryness in an evaporating
dish previously dried at 105ºC to constant weight the residue
is not more than 15. O mg (l. 5%).
~ on drying When dried at 105ºC for 3 hours, loses not
more than 6. 5 % <0831 ) .
Residue on ignition Not more than O. 3 % calculated on dried
basis ( 0841), using l. O g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2) , using the residue obtained in the test for
Residue on ignition, not more than O. 001%.
Category Pharmaceutical excipients, adhesive, filler and
disintegrating agent.
Storage Preserve in well closed containers.
Attached table The relative viscosity ( T/rel) and the intrinsic
viscosity [r¡] e multiplied by concentration.

[11J e
l'jrel
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
l. 1 0.098 o. 106 o. 115 o. 125 o. 134 o. 143 o. 152 o. 161 o. 170 o. 180
l. 2 o. 189 o. 198 0.207 0.216 0.225 0.233 0.242 0.250 0.259 o. 268
l. 3 0.276 0.285 0.293 0.302 0.310 0.318 o. 326 0.334 0.342 0.350
l. 4 0.358 0.367 0.375 0.383 0.391 0.399 0.407 0.414 0.422 0.430
l. 5 0.437 0.445 0.453 0.460 0.468 0.476 0.484 0.491 0.499 0.507
l. 6 0.515 0.522 0.529 0.536 0.544 0.551 o. 558 0.566 0.573 0.580
l. 7 0.587 0.595 0.602 0.608 0.615 0.622 0.629 0.636 0.642 0.649
l. 8 0.656 0.663 0.670 o. 677 0.683 0.690 0.697 o. 704 o. 710 o. 717
l. 9 o. .723 o. 730 o. 736 o. 743 o. 749 o. 756 0.762 0.769 o. 775 o. 782

2.0 0.788 o. 795 0.802 0.809 0.815 0.821 0.827 0.833 0.840 0.846
2. 1 0.852 0.858 0.864 0.870 0.876 0.882 0.888 0.894 0.900 0.906
2. 2 0.912 0.918 0.924 0.929 o. 935 0.941 0.948 0.953 0.959 o. 965
2.3 0.971 0.976 0.983 0.988 o. 994 1.000 l. 006 l. 011 l. 017 l. 022
2.4 l. 028 l. 033 l. 039 l. 044 l. 050 l. 056 l. 061 l. 067 l. 072 l. 078
2.5 1.083 l. 089 l. 094 l. 100 l. 105 l. 111 l. 116 l. 121 l. 126 l. 131
2.6 l. 137 l. 142 l. 147 l. 153 l. 158 l. 163 l. 169 l. 174 l. 179 l. 184
2. 7 l. 190 l. 195 l. 200 l. 205 l. 210 l. 215 l. 220 l. 225 l. 230 l. 235
2.8 l. 240 l. 245 l. 250 l. 255 l. 260 l. 265 l. 270 l. 275 l. 280 l. 285
2.9 l. 290 l. 295 l. 300 l. 305 l. 310 l. 314 l. 319 l. 324 l. 329 l. 333

3.0 l. 338 l. 343 1.348 l. 352 l. 357 l. 362 l. 367 l. 371 l. 376 l. 381
3. 1 l. 386 l. 390 l. 395 1.400 l. 405 l. 409 l. 414 l. 418 l. 423 l. 427
3. 2 l. 432 l. 436 l. 441 l. 446 l. 450 l. 455 l. 459 l. 464 l. 468 l. 473
 Powdered Cellulose

continued

[11J e
lJrel
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
3. 3 1.477 1.482 1.486 1.491 1.496 1.500 1.504 1.508 1.513 1.517
3.4 1.521 1.525 1.529 1.533 1.537 1.542 1.546 1.550 1.554 1.558
3.5 1.562 1.566 1.570 1.575 1.579 1.583 1.587 1.591 1.595 1.600
3. 6 1.604 1.608 1.612 1.617 1.621 1.625 1.629 1.633 1.637 1.642
3. 7 1.646 1.650 1.654 1.658 l. 662 l. 666 l. 671 l. 675 l. 679 l. 683
3.8 1.687 1.691 1.695 l. 700 l. 704 1.708 l. 712 l. 715 1.719 l. 723
3.9 l. 727 1.731 1.735 1.739 1.742 l. 746 l. 750 l. 754 1.758 1.762

4.0 l. 765 1.769 l. 773 1.777 l. 781 1.785 l. 789 1.792 1.796 1.800
4. 1 1.804 1.808 1.811 1.815 1.819 1.822 1.826 1.830 1.833 1.837
4.2 1.841 1.845 1.848 1.852 1.856 1.859 1.863 1.867 1.870 1.874
4.3 1.878 1.882 1.885 1.889 1.893 1.896 1.900 1.904 1.907 1.911
4.4 1.914 1.918 1.921 1.925 1.929 1.932 1.936 1.939 1.943 1.946
4.5 1.950 1.954 1.957 1.961 1.964 1.968 1.971 1.975 1.979 1.982
4. 6 1.986 1.989 l. 993 1.996 2.000 2.003 2.007 2.010 2.013 2.017
4. 7 2.020 2.023 2.027 2.030 2.033 2.037 2.040 2.043 2.047 2.050
4.8 2.053 2.057 2.060 2.063 2.067 2.070 2.073 2.077 2.080 2.083
4. 9 2.087 2.090 2.093 2.097 2. 100 2. 103 2. 107 2.110 2. 113 2. 116

5.0 2. 119 2. 122 2. 125 2. 129 2. 132 2. 135 2. 139 2. 142 2. 145 2. 148
5. 1 2. 151 2. 154 2. 158 2. 160 2. 164 2. 167 2. 170 2. 173 2. 176 2. 180
5.2 2. 183 2. 186 2. 190 2. 192 2. 195 2. 197 2.200 2.203 2.206 2.209
5. 3 2.212 2.215 2.218 2.221 2.224 2.227 2.230 2.233 2.236 2.240
5.4 2.243 2.246 2.249 2.252 2.255 2.258 2.261 2.264 2.267 2.270
5. 5 2.273 2.276 2.279 2.282 2.285 2.288 2.291 2.294 2.297 2.300
5.6 2.303 2.306 2.309 2.312 2.315 2.318 2.320 2.324 2.326 2.329
5. 7 2.332 2.335 2.338 2.341 2.344 2.347 2.350 2.353 2.355 2.358
5. 8 2.361 2.364 2.367 2.370 2.373 2.376 2.379 2.382 2.384 2.387
5. 9 2.390 2.393 2.396 2.400 2.403 2.405 2.408 2.411 2.414 2.417

6.0 2.419 2.422 2.425 2.428 2.431 2.433 2.436 2.439 2.442 2.444
6. 1 2.447 2.450 2.453 2.456 2.458 2.461 2.464 2.467 2.470 2.472
6. 2 2.475 2.478 2.481 2.483 2.486 2.489 2.492 2.494 2.497 2.500
6.3 2.503 2.505 2.508 2.511 2.513 2.516 2.518 2.521 2.524 2.526
6. 4 2.529 2.532 2.534 2.537 2.540 2.542 2.545 2.547 2.550 2.553
6.5 2.555 2.558 2.561 2.563 2.566 2.568 2.571 2.574 2.576 2.579
6. 6 2.581 2.584 2.587 2.590 2.592 2.595 2.597 2.600 2.603 2.605
6. 7 2.608 2.610 2.613 2.615 2.618 2.620 2.623 2.625 2.627 2.630
6.8 2.633 2.635 2.637 2.640 2.643 2.645 2.648 2.650 2.653 2.655
6. 9 2.658 2.660 2.663 2.665 2.668 2.670 2.673 2.675 2.678 2.680
 Powdered Cellulose

continued

[11J e
1Jrel
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
7.0 2.683 2.685 2.687 2. 690 2.693 2.695 2.698 2.700 2. 702 2. 705
7. 1 2. 707 2. 710 2. 712 2. 714 2. 717 2. 719 2.721 2.724 2. 726 2.729
7.2 2.731 2. 733 2. 736 2. 738 2. 740 2. 743 2. 745 2.748 2.750 2. 752
7.3 2. 755 2. 757 2. 760 2. 762 2.764 2. 767 2.769 2. 771 2.774 2. 776
7.4 2. 779 2. 781 2. 783 2. 786 2. 788 2. 790 2. 793 2.795 2. 798 2.800
7. 5 2.802 2.805 2.807 2.809 2.812 2.814 2.816 2.819 2.821 2.823
7. 6 2.826 2.828 2.830 2.833 2.835 2.837 2.840 2.842 2.844 2.847
7. 7 2.849 2.851 2.854 2.856 2.858 2.860 2.863 2.865 2.868 2.870
7. 8 2.873 2.875 2.877 2.879 2.881 2.884 2.887 2.889 2.891 2.893
7. 9 2.895 2.898 2.900 2.902 2. 905 2.907 2.909 2. 911 2.913 2.915

8.0 2.918 2.920 2.922 2. 924 2.926 2.928 2.931 2.933 2.935 2. 937
8. 1 2.939 2.942 2.944 2.946 2.948 2.950 2.952 2.955 2.957 2.959
8.2 2.961 2.963 2.966 2. 968 2.970 2.972 2.974 2.976 2. 979 2.981
8.3 2.983 2.985 2.987 2. 990 2.992 2.994 2.996 2.998 3.000 3.002
8.4 3.004 3.006 3.008 3.010 3.012 3.015 3.017 3.019 3.021 3.023
8.5 3.025 3.027 3.029 3.031 3.033 3.035 3.037 3.040 3.042 3.044
o e
o.u
'l f"\Af'_'
0. V'±O
'l /"\AO
0. V'±O 3.050 3.052 3.054 3.056 3.058 3.060 3.062 3.064
8. 7 3.067 3.069 3. 071 3.073 3.075 3. 077 3.079 3.081 3.083 3.085
8.8 3.087 3.089 3.092 3.094 3.096 3.098 3.100 3.102 3.104 3. 106
8. 9 3. 108 3.110 3.112 3. 114 3. 116 3.118 3.120 3. 122 3. 124 3. 126

9.0 3. 128 3. 130 3. 132 3. 134 3. 136 3. 138 3. 140 3. 142 3. 144 3. 146
9. 1 3. 148 3. 150 3. 152 3. 154 3. 156 3. 158 3. 160 3. 162 3. 164 3. 166
9. 2 3. 168 3. 170 3. 172 3. 174 3. 176 3. 178 3. 180 3. 182 3. 184 3. 186
9. 3 3. 188 3. 190 3. 192 3. 194 3. 196 3. 198 3.200 3.202 3.204 3.206
9.4 3.208 3. 210 3.212 3.214 3.215 3.217 3.219 3.221 3.223 3.225
9. 5 3.227 3.229 3.231 3.233 3.235 3.237 3.239 3.241 3.242 3.244
9.6 3.246 3.248 3.250 3.252 3.254 3.256 3.258 3.260 3.262 3.264
9. 7 3.266 3.268 3.269 3. 271 3.273 3.275 3.277 3.279 3.281 3.283
9. 8 3.285 3.287 3.289 3. 291 3.293 3.295 3. 297 3. 298 3.300 3.302
9. 9 3.304 3.305 3.307 3.309 3. 311 3.313 3.316 3.318 3.320 3.321

10 3.320 3.340 3.360 3.370 3.390 3.410 3.430 3.450 3.460 3.480
11 3.500 3.520 3.530 3. 550 3.560 3.580 3.600 3.610 3.630 3.640
12 3.660 3.680 3.690 3. 710 3. 720 3.740 3. 760 3. 770 3. 790 3.800
13 3.800 3.830 3.850 3.860 3.880 3.890 3.900 3.920 3.930 3.950
14 3.960 3.970 3.990 4.000 4.020 4.030 4.040 4.060 4.070 4.090
15 4. 100 4.110 4.130 4. 140 4. 150 4. 170 4. 180 4. 190 4.200 4.220
16 4.230 4.240 4.250 4.270 4.280 4.290 4.300 4.310 4.330 4.340
17 4.350 4.360 4.370 4.380 4.390 4.410 4.420 4.430 4.440 4.450
18 4.460 4.470 4.480 4.490 4.500 4.520 4. 530 4.540 4. 550 4.560
19 4.570 4.580 4.590 4.600 4.610 4.620 4.630 4.640 4.650 4.660

Pregelatinized Hydroxypropyl Starch
Pregelatinized Hydroxypropyl Starch is obtained from hydr-
oxypropyl starch by mechanically processed to rupture all or
part of the granules and dried with or without heating. It
contains not less than 2. 5 % and not more than 8. 9 % of
hydroxypropyloxy group ( -OCH2CHOHCH3 ) , caculated
on the dried basis.
Descriptioo A white or almost white or pale yellow powder
or granules, or translucent strips or flakes; swells in water.
ldeotificatioo (1) Boíl about 1gwith50 ml of water, cool.
A transparent or translucent dilute thick liquid is produced.
(2) Place O. 5 g in 2 ml of water, mix well, add a drop of
iodine TS. A blue or bluish violet colour is produced.
( 3) The retention time of the principal peak in the
chromatogram of the test solution obtained in the assay is
identical with that of the principal peak in the chromatogram
of the reference solution.
Acidity or aJkalinity Place 3. O gin 100 ml of water, stir for
10 minutes to mix, pH 4. 5-8. O ( 0631 ) .
Iroo Mix O. 50 g with 4 ml of dilute hydrochloric acid and
16 ml of water, stir for 5 minutes, filter and wash the
residue with a quantity of water. Combine the filtrate and
washings, add 50 mg of ammonium persulfate and dilute
with water to 35 ml. Carry out the limit test for iron
( 0807). Any colour produced is not more intense than that of
a reference solution using l. O ml of iron standard solution
(O. 002%).
Sulfur dioxide Not more than O. 005 % ( 2331 ) .
Oxidizing substance Transfer 4. O g of the substance being
examined to a conical flask with stopper, add 50. O ml of
methanol and water ( 1 : 1), stopper tightly and shake for
5 minutes, transfer to a centrifuger tube with stopper,
centrifuge until the solution is clear. Transfer 30. O ml of the
supernatant toan iodine flask, add 1 ml of glacial acetic acid
and l. O g of potassium iodide, stopper tightly, shake, allow
to stand for 30 minutes in a dark place. Add 1 ml of starch
IS, and titrate with sodium thiosulfate (O. 002 mol/L) VS
until the blue colour disappears. Perform a blank
determination and make any necessary correction. Each ml of
sodium thiosulfate (0. 002 mol/L) VS is equivalent to 34 µg
of oxidizing substance (calculated as H2 02), not more than
l. 4 ml of sodium thiosulfate (O. 002 mol/L) VS is consumed
(O. 002%).
1, 2-Propyleoe glycol Transfer about 2. O g of the substance
being examined, accurately weighed, into a 100 ml
volumetric flask, add a volume of ethanol, sonicate for 10
minutes, cool and dilute to volume with ethanol, shake well.
Centrifuga! for 10 minutes ( 3000 r/min) and use the clear
supernatant liquid as test solution. Dissolve a quantity of 1,
2-Propylene glycol, accurately weighed, in ethanol to
produce a solution of 20 µg per ml as the reference solution.
Carry out the method for gas chromatography ( 0521), using
a quartz capillary column packed with 6 % cyanopropyl
phenyl-94%dimethyl polysiloxane (or with similar polarity)
as the stationary phase. Maintain the column temperature at
90ºC . The temperature of injector port is 250ºC, the
temperature of detector is 250ºC. Inject separately 1 µl of the
test solution and reference solution into the column, record
the chromatogram. The number of the theoretical plates
Pregelatinized Starch
calculated for the peak of 1, 2-propyleneglycol is not less than
10 000. The resolution factor between the peaks of 1, 2-
Propylene glycol and solvent complies with the related
requirements. Calculate the contents of 1, 2-propyleneglycol
with respect to the area obtained in the chromatogram by the
external standard method. Not more than O. 1 % .
Loss oo drying When dried at 130ºC for 90 minutes, loses
not more than 15. O% of its weight ( 0831 ) .
Residue oo lgnitioo Not more than O. 6 % ( 0841 ) , using
l. o g.
Heavy metaJs Carry out the test for heavy metals ( 0821
method 2 ) , using the residue obtained in Residue on
ignition: not more than O. 002%.
Arseoic Boíl and dissolve l. O g with 1 ml of hydrochloric
acid and 21 ml of water, add 4 ml of hydrochloric acid. Carry
out the limit test for arsenic ( 0822 method 1 ) : not more
than O. 0002%.
MicrobiaJ limit Comply with the requirements for microbial
limit ( 1105 and 1106 ) , the total aerobic bacteria count is
not more than 1000 cfu per g and the total combined yeast/
mold count is not more than 100 cfu per g of the substance
being examined, Escherichia coli are absent.
Assay hydroxypropyloxy groups Carry out method for
determination of methoxy, ethoxyl and hydroxypropoxy
( 0712 method 1 >. Calculate the content of hydroxypropyloxy
groups.
Category Phannaceutical excipients, filling agent and binder.
Storage Preserve in well closed containers.
Pregelatinized Siarch
Pregelatinized Starch is mechanically processed starch to
improve its flowability and compressibility.
Descriptioo A white or almost white powder.
ldeotificatioo (1) Stir and boíl about 1 g with 15 ml of
water, allow to cool. A whitish transparent or translucent
gelationous substance is formed.
(2) Mix about O. 1 g with 20 ml of water, add a few drops of
iodine TS, a bluish black, blue, violet or purplish red colour
is produced which gradually disappears on heating.
Acidity Shake 10. O g with 10 ml of neutral ethanol, add
100 ml of boiled and cooled water, stir for 5 minutes, pH
4. 5-7. o <0631).
Sulfur dioxide Not more than O. 004 % ( 2331 ) .
Oxidizing substance To 5. O g, add 20 ml of a mixture of
methanol and water (1 : 1), add 1 ml of 6 mol/L acetic acid
solution, stir until a homogenized suspension is obtained.
Centrifuge. Add O. 5 ml of a freshly prepared and saturated
potassium iodine solution, allow to stand for 5 minutes: No
distinct blue, brown, or purple colour is observed in the
supernatant liquid and the precipitate.
Loss oo drying When dried at 120ºC for 4 hours, loses not
more than 14. O% of its weight ( 0831 ) .
Ash Not more than O. 3 % ( 2302) , using 1. Og.
Heavy metaJs Carry out the limit test for heavy metals
( 0821 method 2) , using the residue obtained in the test for
Ash : Not more than O. 002%.
 Propionic Acid
lron Mix O. 50 g with 4 ml of dilute hydrochloric acid and
16 ml of water, shake for 5 minutes, filter and wash the
residue with small quantity of water. Combine the filtrate
and washings, add 50 mg of ammonium persulfate and dilute with
water to 35 ml. Carry out the limit test for iron <0807 ) . Any
colour produced is not more intense than that of a reference
solution using l. O ml of iron standard solution (O. 002%).
Microbial limit Comply with test for microbial limit <1105
and 1106) , the number of total acrobic bacterial count is not
more than 1000 cfu per g and the total combined yeasts/mold
is not more than 100 cfu per, Escherichia coli is absent.
Category Pharmaceutical excipients, filling agent, disintegrant,
etc.
Storage Preserve in well closed containers.
Propionic Acid
C3 H6 02 74. 08
[79-09-4]
lt contains not less than 99. 5 % of C3H6 02.
Description A colourless to light yellow oily liquid with
irritating and grease rancidity odour.
rvliscible v:ith v:ater, ethanol or ethcr.
Relative density O. 993-0. 997 <0601 >.
Distilling range 138. 5-142. 5ºC <0611 >.
Identification Mix 1 ml of propionic acid with 3 drops of
sulfuric acid and 1 ml of ethanol, heat, produce flavor of ester.
Non-volatile matter Place 20. O g of the substance being
examined in an evaporating dish, previously dried to constant
weight at 105ºC, evaporate to dryness and dry at 105ºC to
constant weight. The residue is not more than 2. O mg.
Aldehydes Transfer 10. O ml to an iodine flask with 50 ml
water and 10. 00 ml of l. 25 % sodium bisulfite solution,
insert the stopper tightly, and shake vigorously and allow to
stand for 30 minutes; Titrate with iodine (0. 05 mol/U VS
until the color change to brownish-yellow, perform a blank
determination. The difference between the volume of iodine
(O. 05 mol/L) VS consumed in the blank sample and the test
sample is not more than l. 75 ml.
Readily Oxidi7.able Substance Dissolve 15 g of sodium
hydroxide in 50 ml of water, cool, add 6 ml of bromine, stir
thoroughly to completely dissolve, and dilute with water to
2000 ml, mix well. Transfer accurately 25 ml of the upper
layer solution to iodine flask with 100 ml water, and add
10 ml of 20 % sodium acetate solution and 10. O ml of substance
being examined, mix well, allow to stand for 15 minutes.
Add 5 ml of 25 % potassium iodide solution and 10 ml of
hydrochloric acid. Titrate the solution with sodium thio-
sulfate (O. 1 mol/L) VS until the brown color disappears,
perform a blank determination. The difference between the
volume of sodium thiosulfate (O. 1 mol/L) VS consumed in
the blank sample and the test sample is not more than 2. 2 ml.
Water Not more than O. 15 % <0832 method 1 ) .
Heavy metals Dissolve the residue obtained in the test for Non-
volatile matter in 8 ml of O. 1 mol/L hydrochloric acid solution,
heat gently to dissolve, dilute with water to 100 ml, mix well.
Carry out the limit test for heavy metals <0821 method 1 ) , using
10 ml of the solution: not more than O. 001%.
Arsenic Place 5. O g in porcelain crucible, add 10 ml of 15 %
magnesium nitrate solution and 1 g of magnesium oxide
powder, mix well, immerse for 4 hours, and then evaporate
to dryness at low temperature or on a water bath. Heat
gently until completely charred, and then ignite at 550ºC
until the incineration is complete, cool to room temperature,
moisten the residue with a quantity of water, add 1 drop of
phenolphthale IS, and then titrate slowly with hydrochloric
acid 0-2) until the red color disappears, filter, transfer the
filtrate to 50 ml volumetric flask, then flush the porcelain
crucible 3 times with little amount of water, and combine the
washing in the above volumetric flask, and add water to
volume, mix well. Take 6. 67 ml of this solution, add
16. 3 ml of water and 5 ml of hydrochloric acid. Carry out
the limit test for arsenic <0822 method 1 ) : not more than
o. 0003%.
Assay Dissolve O. 8 g, accurately weighed, with 100 ml of
freshly boiled and cooled water, add 2 drops of phenolphthale
IS. Titrate with sodium hydroxide (O. 5 mol/L) VS until the
colour of pink appears and persists for not less than 30
seconds. Each ml of sodium hydroxide (O. 5 mol/L) VS is
equivalent to 37. 04 mg of C3 H6 02.
Category Pharmaceutical excipients, pH regulater, cosol-
vent, preservative, etc.
Storage Preserve in tightly closed containers and protected
from light.
Propylene Carbonate
C4 H6 03 102. 09
[108-32-7]
Propylene Carbonate is 4-methyl-1, 3-dioxolan-2-one.
It con tains not less than 9 9. O% of propylene carbonate
(C4 H6 Ü3 ).
Description A clear, colourless or light yellow transparent
liquid.
Relative density l. 203-1. 210 <0601 >.
Identification The infrared absorption spectrum is con-
cordant with that of propylene carbonate CRS <0402).
Acidity or alkalinity Dissolve 10 ml of the substance being
examined with O. 3 ml of saturated potassium chloride
solution, and dilute to 100 ml with water. pH value is 6. 0-
7. 5 (0631>.
Residue on Ignition Not more than O. 1 % <0841 ) , using
l. o g.
Assay Accurately weigh O. 6 g of the substance being
examined into a 250 ml iodine flask, add accurately 50 ml of
barium hydroxide solution [(Weigh 75 g of barium hydroxide
(Ba (OH ) 2 • 8H 2 O), dissolved with 1000 ml of carbon
dioxide-free cool water as barium hydroxide solution. Filter
the solution befare use)]. Flush it with nitrogen to expel the
air and carbon dioxide, and stopper tightly, moisten the
stopper with 3 drops of water, heat the flask on a water bath at
95-lOOºC for 15 minutes, add 6 drops of phenolphthalein, and

titrate while hot with hydrochloric acid (O. 5 mol/L) VS until
colorless remains. Perform a blank determination and make
any necessary correction, using the same Barium hydroxide
solution. Each ml of hydrochloric acid (O. 5 mol/L) VS is
equivalent to 25. 52 mg of propylene carbonate (C4 H6 03 ).
Category Pharmaceutical excipients, solvents, etc.
Storage Preserve in tightly closed containers.
Propylene Glycol
C3 Ha 02 76. 09
[57-55-6]
Propylene Glycol is 1, 2-propanediol. It contains not less
than 99. 5% of C3Ha02.
Description A viscous, clear, colorless liquid; odourless;
hygroscopic.
Miscible with water, with ethanol and with chloroform.
Relative density l. 035-1. 037 at 25ºC (0601 ).
Refractive index l. 431-1. 433 <0622).
Identification ( 1) The retention time of principal peak of
the test solution in the chromatogram obtained in the Assary
is identical with that of principal peak of propylene glycol
CRS reference solution.
(2) The infrared absorption spectrum <0402) is consistent
with the reference spectrum (IR Album No. 706).
Acidity Dissolve 10. O ml in 50 ml of freshly boiled and
cooled water and add 3 drops of bromothymol blue IS, titrate
with sodium hydroxide (O. 01 mol/L) VS until the color
changes to blue. Not more than O. 5 ml of sodium hydroxide
(O. 01 mol/L) VS is required.
Chloride Carry out the limit test for chloride < 0801 ) ,
using l. O ml. Any opalescence produced is not more
pronounced than that of a reference solution using 7. O ml of
sodium chloride standard solution (O. 007 % ) .
Sulfate Carry out the limit test for sulfate <0802) , using
5. O ml. Any opalescence produced is not more pronounced
than that of a reference solution using 3. O ml of potassium
sulfate standard solution (O. 006%).
Related substance Dissolve a quantity of the substance being
examined, accurately weighed, in dehydrated ethanol to
produce a solution contain O. 5 g per ml as the test solution.
Dissolve an accurately weighed quantity of diglycol CRS,
dipropylene glycol CRS, tripropytene glycol CRS and 1, 2-
epoxypropane in dehydrated ethanol to produce a solution of
5 µg of diglycol, 500 µg of dipropylene glycol, 150 µg of
tripropylene glycol and 5 µg of 1, 2-epoxypropane per ml as
the reference solution. Carry out the method for gas
chromatography <0521 ) . Using a capillary column packed
with polyethylene glycol 20 M as the stationary phase, the
injection port temperature is 230ºC, the FID detector
temperature is 250ºC, the initial temperature of the column
is 80ºC, maitain for 3 minutes and raise the temprature to
220ºC ata rate of 15ºC per minute, maintain for 4 minutes.
The resolution factor between the peaks complies with the
related requirements. Inject separately 1 µl each of the test
solutions and the reference solution in to the column. Record
Propylene Glycol (For lnjection)
the chromatograms, calculated with respect to the peak area
in the chromatogram by the extemal standard method. The
content of diglycol is not more than O. 001 % , the content of
dipropylene glycol is not more than O. 1 % , the content of
tripropylene glycol is not more than O. 03 % and the content
of 1,2-epoxypropane is not more than O. 001%.
Oxidizing substances To 5. O mi add l. 5 ml of potassium
iodide TS and 2 ml of dilute sulfuric acid, allow to stand in a
glass-stoppered iodine flask protected from light for 15
minutes and add 2 ml of starch IS, any blue color produced ·
requires not more than O. 2 ml of sodium thiosulfate (O. 005
mol/L) VS to disappear.
Reducing substances To 1 ml add 1 ml of ammoniate TS and
heat in a water-bath at 60ºC for 5 minutes. The solution is
not yellow. lmmediately add O. 15 ml of silver nitrate TS,
mix well and allow to stand for 5 minutes. The solution does
not change its appearance.
Water Not more than O. 2 % <0832 method 1 ( 1 ) ) .
Residue on ignition Weigh accurately about 50 g of propylene
glycol and heat to bum. Stop heating, and allow it to bum
without further application of heat in a place free from
drafts. lgnite to constant weight at 700-800ºC : the weight of
the residue does not exceed 2. 5 mg.
Heavy metals Transfer 4. O ml to 19 ml of water and 2 ml of
acetate BS (pH 3. 5) and mix well. Carry out the limit test for
heavy metals <0821 method 1 ) : not more than O. 0005 % .
Arsenic Mx l. O g with 23 ml of water and 5 ml of hydrochloric
acid Carry out the limit test for arsenic < 0822 ) : not more
than O. 0002%.
Assay Carry out the method for gas chromatography
<0521 ) , using a column packed with polyethylene glycol 20
M as the stationary phase, the initial temperature of the
column is 130ºC, maintain the temperature for 1 minute and
raise the temperature to 240ºC by lOºC per minute, maitain
for 1 minute, the temperature of the injection port is 230ºC
and that of the FID detector is 25üºC . The number of
theoretical plates of the column is not less than 10 000,
calculate with reference to the peak of propylene glycol.
Procedure Dissolve a quantity of the substance being
examined, accurately weighed, in dehydrated ethanol to
produce a solution containing 1 mg per ml. Inject 1 µl outo
the column and record the chromatogram. Repeat the
operation, using propylene glycol CRS instead of the
substance being examined, calculate the content of C3 Ha 0 2
with respect to the peak area obtained in the chromatogram
by the extemal standard method.
Category Pharmaceutical excipients, solvents and plastici-
zers, etc.
Storage Preserve in tightly closed containers and stored in a
dry place.
Propylene Glycol ( For Injection)
C3 Ha 02 76. 09
[57-55-6]
Propylene Glycol is 1 , 2-propanediol. It contains not less
 Propylene Glycol (For lnjection)
Description A viscous, clear, colourless liq uid; odourless;
hygroscopic.
Miscible with water, with ethanol and with chloroform.
Relative density l. 035-1. 037 at 25ºC <0601 ) .
ldentification ( 1) The retention time of principal peak of
the test solution in the chromatogram obtained in the Assay
is identical with that of principal peak of the reference
· solution propylene glycol CRS.
(2) The infrared absorption spectrum ( 0402) is concordant
with the reference spectrum CIR Album No. 706).
Acidity Dissolve 10. O ml in 50 ml of freshly boiled and
cooled water, add 3 drops bromothymol blue IS, titrate with
sodium hydroxide (0. 01 mol/L) VS until the colour changes
to blue. Not more than O. 5 ml of sodium hydroxide (O. 01
mol/L) VS is required.
Chloride Carry out the limit test for chloride ( 0801 ) ,
using l. O ml. Any opalescence produced is not more pro-
nounced than that of a reference solution using 7. O ml of
sodium chloride standard solution (O. 007 % ) .
Sulfate Carry out the limit test for sulfate ( 0802 ) , using
5. O ml. Any opalescence produced is not more pronounced
than that of a reference solution using 3. O ml of potassium
sulfate standard solution (O. 006 % ).
Related substances Dissolve a quantity of the substance
being examined, accurately weighed, in dehydrated ethanol
to produce a solution of O. 5 g per ml as the test solution.
Dissolve an accurately weighed quantity of diglycol CRS,
dipropylene glycol CRS, tripropytene glycol CRS and 1, 2-
epoxypropane CRS in dehydrated ethanol to produce a
mixture of 5 µg of diglycol, 500 µg of dipropylene glycol,
150 µg of tripropylene glycol and 5 µg of 1, 2-epoxypropane
per ml as the reference solution. Carry out the method for
gas chromatography ( 0521 ) , using a capillary column
packed with polyethylene glycol 20 M as the stationary
phase. The initial temperature of the column is 80ºC,
maintain for 3 minutes, raise the temperature to 220ºC by
15ºC per minute; and maintain for 4 minutes. The tempe-
rature of injection is 230ºC; the temperature of detection is
250ºC . The resolution factor between the peaks complies
with the related requirements. Inject accurately 1 µl each of
the test solution and the reference solution, separately, onto
the column and record the chromatogram. Calculate the
content with respect to the peak area in the chromatogram by
the extemal standard method. The content of diglycol is not
more than O. 001 % , the content of dipropylene glycol is not
more than O. 1 % , the content of tripropylene glycol is not
more than O. 03 % , the content of 1, 2-epoxypropane is not
more than O. 001%.
Ethylene glycol Weigh accurately 1 g to a 10 ml volumetric
flask, dilute with acetonitrile to volume and mix well as the
test solution. Weigh accurately O. 2 g of ethylene glycol CRS
to a 100 ml volumetric flask, dilute with acetonitrile to
volume, transfer 1 ml to a 100 ml volumetric flask, dilute
with acetonitrile to volume and mix well as the reference
solution. Carry out the method for gas chromatography
( 0521 ) , using a capillary column packed with 6%
cyanopropylphenyl-94 % dimethylpolysiloxane ( a capillary
column with the similar polarity can be substituted each
other), a flame ionization detector is used. The temperature
of injection is 230ºC; the split ratio is 30 : 1; the
temperature of detection is 250ºC. Maintain the temperature
of the column at 120ºC for 4 minutes, raise the temperature
to 140ºC by 8°C per minute, maintain it at 140ºC for 10
minutes, then raise the temperature to 220ºC by 8ºC per
minute, maintain the temperature at 220ºC for 5 minutes.
The number of theoretical plates calculated for the peak of
propylene glycol is not less than 10 000, the tailing factor is
less than 2. O, the resolution factor between the peaks is not
less than 2. O. lnject separately 1 µl each of the test solution
and the reference solution onto the column. Record the
chromatogram, calculated the content of ethylene glycol as
follows:
_ CAt XM,) %
content o f ethylene g1yco1 ( %o) - ClOOOA, XMt) X 100 o
Where At is peak area of ethylene glycol in the chromatogram
obtained with the test solution.
A, is peak area of ethylene glycol in the chroma-
togram obtained with the reference solution.
Mt is weight of the substance being examined in
the test solution (g).
M, is weight of ethylene glycol in the reference
solution (g).
The content of ethylene glycol is not more than O. 02%.
Oxidizing substances Measure 5. O ml to a glass-stoppered
iodine flask, add l. 5 ml of potassium iodide TS and 2 ml of
dilute sulfuric acid, allow to stand in the dark for 15
minutes, add 2 ml of starch IS, any blue color produced
requires not more than O. 2 ml of sodium thiosulfate
(O. 005 mol/L) VS to disappear.
Reducing substances To 1 ml add 1 ml of ammoniate TS,
and heat on a waterbath at 60ºC for 5 minutes; no yellow
colour is produced. lmmediately add O. 15 ml of silver nitrate
TS and mix well, allow to stand for 5 minutes. The solution
does not change its appearance.
Water Not more than O. 2 % ( 0832 method 1 ( 1 ) ) .
Residue on ignition Heat 50 g to buming and stop heating,
allow it to bum to dryness (if it does not bum, heat to expel
the steam completely). lgnite to constant weight at 700-
800ºC , the weight of the residue is not more than 2. 5 mg.
Heavy metals To 4. O ml add 19 ml of water and 2 ml of
acetate BS (pH 3. 5), mix well. Carry out the limit test for
heavy metals ( 0821 method 1 ) : not more than O. 0005 %.
Arsenic To l. O g add 23 ml of water and 5 ml of hydro-
chloric acid, shake well. Carry out the limit test for arsenic
( 0822) : not more than O. 0002 % .
Bacterial endotoxins Carry out the test for bacteria! endo-
toxins ( 1143): not more than O. 012 EU per mg.
Sterility ( Intended use for the sterile preparations without
terminal sterilization) Complies out the test for sterility
(1101 ). The result complies with the requirements.
Assay Carry out the method for gas chromatography ( 0521 ) ,
using a capillary column packed with polyethylene glycol 20
M as the stationary phase. The initial temperature of the
column is 130ºC, maintain the temperature for 1 minute,
raise the temperature to 240ºC by lOºC per minute, maintain
for 1 minute; the temperature of the injection is 230ºC; the
temperature of detection is 250ºC. The number of theoretical
plates calculated for propylene glycol is not less than 10 000.
Procedure Dissolve a quantity of substance being examined,
accurately weighed, in dehydrated ethanol to produce a
solution of 1 mg per ml. Inject accurately 1 µl onto the
column and record the chromatogram. Repeat the operation,
using propylene glycol CRS instead of the substance being
examined, calculate the content of C3 Hs 02 with respect to
the peak area obtained in the chromatogram by the extemal

standard method.
Category Pharmaceutical excipients, solvents, etc.
Storage Preserve in tightly closed containers and stored in a
dry place.
Propylparaben
C10 H12 03
180. 20
[94-13-3]
Propylparaben is propyl 4-hydroxybenzoate. It contains not
less than 98. O% and not more than 102. O% of C10 H12 03 ,
calculated on the dried basis.
Description White or almost white crystals or a crystalline
powder;
Freely soluble in methanol, in ethanol and in ether; slightly
soluble in hot water; practically insoluble in water.
Melting point 96-99ºC ( 0612).
ldentification ( 1) The retention time of the principie peak of
the substance being exarnined in the chromatogram is identical
with that of the principal peak of propylparaben CRS in the
chromatogram obtained in the Assay.
(2) Weigh a quantity of the substance being exarnined to produce
a solution of 5 µg per ml in ethanol. The light absorption of the
solution exhibits a maximum at 258 nm ( 0401 ) .
(3) The infrared absorption spectrum is concordant with the
reference spectrum CIR Album No. 852).
Acidity To 2 ml of the solution prepared under clarity and
colour of solution add 2 ml of ethanol and 5 ml of water, mix
well, add 2 drops of bromocresol green IS; not more than
O. 1 ml of sodium hydroxide (O. 1 mol/U VS is required to
change the colour of the solution to blue.
Oarity and rolour of solution Dissolve l. O g of the substance
being examined in 10 ml of ethanol, carry out the lirnit test for
clarity and colour ( 0901 and 0902), the solution is clear and
colourless; any colour produced is not more intense than that of
reference solution Y1 or YGi ( 0901 method 1 ) .
Chloride Heat 2. O g of the substance being examined with
50 ml of water at 80°C for 5 minutes, cool, filter. Carry out
the limit test for chloride ( 0801 ) , using 5. O ml of the
filtrate. Any opalescence produced is not more pronounced
than that of a reference solution using 7. O ml of sodium
chloride standard solution (O. 035 % ).
Sulfate Carry out the limit test for sulfate ( 0802 ) , using
25 ml of the successive filtrate obtained in the test for
chloride. Any opalescence produced is not more pronounced
than that of a reference solution using 2. 4 ml of potassium
sulfate standard solution (0. 024%).
Related substances Dissolve a quantity of the substance
being examined in the mobile phase to produce a solution of
1 mg per ml as test solution; measure accurately 1 ml of the
test solution, to a 100 ml volumetric flask, dilute to volume
with the mobile phase, mix well, as reference solution.
Carry out the method described under the Assay, inject 20 µl
of the reference solution onto the column and adjust the
Purple Ferric Oxide 639
attenuation so that the principal peak height in the
chromatogram is about 25 % of full scale of the chart, then
inject separately 20 µl of the test solution and reference
solution outo the column, record the chromatogram for 4
times the retention time of the principal peak. The area of
any impurity peaks in the chromatogram is not greater than
O. 4 times the area of the principal peak in the chromatogram
obtained with the reference solution (O. 4 % ) , the sum of the
areas of all impurity peaks is not greater than O. 8 times area
of the principal peak in the chromatogram obtained with the
reference solution (O. 8 % ).
Loss on drying When dried in vacuum in a desiccator
containing silica gel to constant weight, loses not more than
O. 5 % of its weight ( 0831).
Residue on ignition Not more than O. 1 % <0841 ) , using
l. o g.
Heavy metals Carry out the limit test for heavy metals
( 0821 method 2), using the residue obtained in the test for
residue on ignition: not more than O. 002%.
Arsenic Mix l. O g of the substance being examined with
l. O g of calcium hydroxide, add a small volume of water and
mix well. Dry, heat gently until it is thoroughly charred,
then ignite at 500-600ºC until the incineration is complete,
cool, add 5 ml of hydrochloric acid and 23 ml of water.
Carry out the limit test for arsenic ( 0822 method 1 ) : not
more than O. 0002 % .
Assay Carry out the method for high performance liquid
chromatograph ( 0512 ) , using a column packed with
octadecylsilane bonded silica gel and a mixture of methanol-
1 % glacial acid solution ( 60 : 40) as the mobile phase.
Detection wavelengnth is 254 nm. Dissolve a quantity of
methylparaben and ethylparaben in the mobile phase to
produce a mixture solution of 10 µg each of the two
substances per ml. Inject 20 µl of the mixture solution into
the column and record the chromatogram. The resolution
factor between peaks of methylparaben and ethylparaben
complies with the requirements.
Procedure Dissolve a quantity, accurately weighed, in the
mobile phase to produce the test solution of O. 1 mg per ml.
Inject 20 µl of the resulting solution, accurately measured,
onto the column and record the peak areas corresponding
obtained in the chromatogram. Accurately weigh Propylparaben
CRS and repeat the operation. Calculate the contents of C10 H 12
0 3 with respect to the peak area obtained in the chromatogram
by the extemal standard method.
Category Pharmaceutical excipients, preservative.
Storage Preserve in well close containers.
Purified Water*
See the same monograph in Volume II .
Category Pharmaceutical excipients, solution and diluent.
Storage Preserve in well dosed coutainers.
Purple Ferric Oxide
Purple Ferric Oxide is mixture of a fixed proportion of red
 640 Red F erric Oxide

ferric oxide and black ferric oxide. It contains not less than Category Pharmaceutical excipients, colourant and coating
98. O% of Fez 03 , calculated with reference to the substance material, etc.
ignited to constant weight. Storage Preserve in tightly closed containers.
Description A dark purple powder; odourless; tasteless.
Insoluble in water; freely soluble in boiling hydrochloric
a cid.
Identification Boil about O. 1 g with 5 ml of dilute hydro- Red Ferric Oxide
chloric acid, allow to cool, the solution yields the reactions
characteristic of f erric salts ( 0301 ) .
Fez 03 159. 69
Water soluble substances To 2. O g add 100 ml of water, [1309-37-1]
reflux on a water bath for 2 hours and filter. Wash the Red Ferric Oxide contains not less than 98. O% of Fez 0 3 ,
residue with a volume of water, evaporate the combined calculated with reference to substance ignited to constant
filtrate and washings to dryness in an evaporating dish weight.
previously dried to constant weight at 105ºC, dry at 105°C to
Description A dark red powder; odorless; tasteless.
constant weight: the residue not more than 10 mg (0. 5%).
Insoluble in water; freely soluble in boiling hydrochloric
Acid insoluble substances Dissolve 2. O g in 25 ml of hydro- acid.
chloric acid by heating on a water bath, add 100 ml of water,
Identification Dissolve about O. 1 g with 5 ml of dilute
filter through a sintered glass crucible (No. 4) previously dried to
hydrochloric acid, heat to boil, allow to cool, the solution
constant weight at 105ºC, wash the residue with hydrochloric
yields the reactions characteristic of ferric salts ( 0301 ) .
acid solution e1-100) until the washings become colourless'
then wash the residue with water until the washings give no Water soluble substances To 2. O g, add 100 ml of water,
reaction of chlorides, dry the residue to constant weight at reflux on a water bath for 2 hours and filter. Wash the
105ºC: the residue not more than 6 mg (O. 3%). residue with a small amount of water, evaporate the
combined filtrate and washings to dryness in an evaporating
Loss on ignition When ignited to constant weight at
dish previously dried to constant weight, dry the residue to
800ºC, loses not more than 4. O% of its weight ( 0831),
constant weight at 105ºC: not more than 10 mg (0. 5%).
using l. O g.
Acid insoluble substances To 2. O g, add 25 ml of
Barium To O. 2 g add 5 ml of hydrochloric acid, heat to
hydrochloric acid TS, heat on a water bath to dissolve, add
dissolve. Add 1 drop of hydrogen peroxide TS and 20 ml of
100 ml of vratcr and filtcr through a sintcrcd glass crucible
10 % sodium hydroxide solution, fil ter and wash the residue
(No. 4) previously dried to constant weight at 105ºC, wash
with 10 ml of water. Combine the filtrate and washings, add
the residue with hydrochloric acid solution Cl-100) until the
10 ml of sulfuric acid solution (2-10), no opalescence is
washings become colorless, then wash the residue with water
produced.
until the washings give no reaction of chlorides, dry the residue to
Lead Transfer 2. 5 g of the substance being examined to a constant weight at 105ºC: not more than 6 mg (O. 3%).
100 ml conical flask with stoppper, add 35 ml of O. 1 mol/L
Loss on ignition When ignited to constant weight at 800ºC,
hydrochloric acid solution, stir for 1 hour. Filter and wash
loses not more than 4. 0% of its weight, using l. O g ( 0831 ).
the residue with O. 1 mol/L hydrochloric acid solution,
combine the filtrate and washings in a 50 ml volumetric Barium To O. 2 g, add 5 ml of hydrochloric acid and heat to
flask, dilute with O. 1 mol/L hydrochloric acid solution to dissolve. Add 1 drop of hydrogen peroxide TS and 20 ml of
volume, shake well and use as the test solution. Carry out 10 % sodium hydroxide solution, fil ter and wash the residue
th