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3044 Enoxaparin / Official Monographs


Analysis: Proceed as directed in the Assay for Anti-Factor Xa
Activity, except to use Thrombin human solution instead of
Factor Xa solution and to use the Human antithrombin III
solution as described above.
Calculations: For each series, calculate the regression of the
absorbance against log concentrations of the Sample
solutions and of the Standard solutions, and calculate the
potency of the enoxaparin sodium in the Injection in IU of
Anti-Factor IIa activity/mL using statistical methods for
parallel-line assays. The four independent dilution estimates
are then combined to obtain the final weighted mean. Then
calculate the confidence limits.
Acceptance criteria: It has a potency of NL T 2000 and
NMT 3500 IU/mL.
FREE SULFATE CONTENT
Mobile phase: 3.0 mM sodium carbonate solution
System suitability solution: 3 g/mL of sulfate anion and 5
g/mL of oxalate anion
Standard sulfate stock solution: Prepare a solution of
sodium sulfate in Mobile phase in a suitable sulfate-free
container such that the concentration of sulfate is accurately
known at about 1 mg/mL. T ransfer 5 g of the solution to a
similar container, and add Mobile phase to obtain 25 g of
solution.
Standard solution A: 0.1 g/g of sulfate from Standard
sulfate stock solution in Mobile phase
Standard solution B: 0.5 g/g of sulfate from Standard
sulfate stock solution in Mobile phase
Standard solution C: 1 g/g of sulfate from Standard sulfate
stock solution in Mobile phase
Standard solution D: 2 g/g of sulfate from Standard
sulfate stock solution in Mobile phase
Standard solution E: 4 g/g of sulfate from Standard sulfate
stock solution in Mobile phase
Standard solution F: 5 g/g of sulfate from Standard sulfate
stock solution in Mobile phase
Sample solution: Transfer 200 mg of a 100-mg/mL
Injection to a suitable previously tared sulfate-free vial. Add
Mobile phase to obtain a total mass, M S, of about 20 g.
Chromatographic system
(See Chromatography 621, System suitability.)
Mode: Ion chromatography
Detector: Conductivity
Column
Guard: 4-mm 5-cm; packing L61
Analytical: 4-mm 25-cm; packing L61
[NOTEUse a micromembrane anion autosuppressor 2 or a
suitable chemical suppression system.]
Flow rate: 2.0 mL/min
Injection size: 25 L
System suitability
Sample: System suitability solution
Suitability requirements
Resolution: NLT 1 between the sulfate and oxalate peaks
Analysis
Samples: Standard solutions AF and Sample solution
Plot the standard cur ve of sulfate peak height as a function
of sulfate concentration (in g/g) in the Standard solutions
AF. From the sulfate peak height in the chromatogram
determine the concentration of sulfate, C, in g/g, in the
Sample solution using the standard cur ve. Calculate the
percentage of free sulfate content (w/w) in the portion of
Injection taken:
Result = [(C M S)/10m)] 100
MS
m

= total mass of the Sample solution (g)


= mass of Injection taken to prepare the Sample
solution (mg)
Acceptance criteria: The per centage of free sulfate is NMT
0.12%.
Available as Anion Self-Regenerating Suppressor (ASRS) from Dionex Inc, or
equivalent.
2

USP 35
STERILITY TESTS 71: Meets the requirements
PARTICULATE MATTER IN INJECTIONS 788: Meets the
requirements
OTHER REQUIREMENTS: It meets the requirements under
Injections 1.
ADDITIONAL REQUIREMENTS
PACKAGING AND STORAGE: Preserve in single-dose or multipledose containers in T ype I glass. Store between 20 and 25 ,
excursions permitted between 15 and 30 .
LABELING: Label it to indicate the amount (mg) of
Enoxaparin Sodium in the total volume of contents. The
label states also that the Enoxaparin Sodium starting material
is porcine derived.
USP REFERENCE STANDARDS 11
USP Benzyl Alcohol RS
USP Endotoxin RS
USP Enoxaparin Sodium RS
USP Enoxaparin Sodium Solution for Bioassays RS

Enrofloxacin

C19H22FN3O3 359.39
3-Quinolinecarboxylic acid, 1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-1,4-dihydro-4-oxo-;
1-Cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-1,4-dihydro-4oxo-3-quinolinecarboxylic acid
[93106-60-6].

Enrofloxacin contains not less than 98.5 percent and not more than 101.5 per cent of
C19H22FN3O3, calculated on the dried basis.
Packaging and storagePreserve in well-closed, tight, lightresistant containers.
LabelingLabel it to indicate that it is for veterinar y use only.
USP Reference standards 11
USP Enrofloxacin RS
USP Fluoroquinolonic Acid RS
USP N-Ethylpiperazine RS
USP Enrofloxacin Related Compound Mixture RS
Contains a mixture of USP Enrofloxacin RS, desfluoro-enrofloxacin, and USP Ciprofloxacin RS.
Desfluoro-enrofloxacin:1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-1,4-dihydro-4-oxo-3-quinoline-carboxylic acid.
Clarity of solution
Hydrazine sulfate solutionTransfer 1.0 g of hydrazine sulfate
to a 100-mL volumetric flask, dissolve in and dilute with water
to volume, and mix. Allow to stand for 4 to 6 hours.
Hexamethylenetetramine solutionTransfer 2.5 g of hexamethylenetetramine to a 100-mL volumetric flask, add 25.0
mL of water, insert a glass stopper, and mix to dissolve.
Primary opalescent suspension[NOTEThis suspension is stable for 2 months, provided it is stored in a glass container free
from surface defects. The suspension must not adhere to the
glass and must be well-mixed before use.] Transfer 25.0 mL of
the Hydrazine sulfate solution to the Hexamethylenetetramine solution in the 100-mL glass-stoppered flask. Mix, and allow to
stand for 24 hours.
Opalescence standard[NOTEThis suspension should not be
used beyond 24 hours after preparation.] Transfer 15.0 mL of
the Primary opalescent suspension to a 1000-mL volumetric flask,
dilute with water to volume, and mix.

Official from May 1, 2012


Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.

Accessed from 128.83.63.20 by nEwp0rt1 on Thu Nov 24 01:20:17 EST 2011

USP 35

Official Monographs / Enrofloxacin 3045

Reference suspensionTransfer 10.0 mL of the Opalescence


standard to a 100-mL volumetric flask, dilute with water to volume, and mix.
Test solutionTo 1.0 g of Enrofloxacin add about 0.25 g of
potassium hydroxide and 7 mL of water. Sonicate to dissolve,
and dilute with water to 10.0 mL.
ProcedureTransfer a sufficient portion of the Test solution,
the Reference suspension, and water to separate test tubes of
colorless, transparent, neutral glass with a flat base and an internal diameter of 15 to 25 mm to obtain a depth of 40 mm.
Compare the Test solution, the Reference suspension, and water
in diffused daylight 5 minutes after preparation of the Reference
suspension, viewing vertically against a black background (see
Visual Comparison under Spectrophotometry and Light-Scattering
851). [ NOTEThe diffusion of light must be such that the Reference suspension can be readily distinguished from water.] The
Test solution shows the same clarity as that of water or its opalescence is not more pronounced than that of the Reference
suspension.
Color of solution
Standard stock solutionCombine 9.6 mL of ferric chloride
CS, 0.2 mL of cobaltous chloride CS, and 0.2 mL of cupric
sulfate CS, and mix.
Standard solution[NOTEPrepare the Standard solution immediately before use.] Transfer 5.0 mL of the Standard stock
solution to a 100-mL volumetric flask, and dilute with dilute
hydrochloric acid (10 g per 1000 mL).
Test solutionTo 1.0 g of Enrofloxacin add about 0.25 g of
potassium hydroxide and 7 mL of water. Sonicate to dissolve,
and dilute with water to 10.0 mL.
ProcedureTransfer a sufficient portion of the Test solution,
the Standard solution, and water to separate test tubes of colorless, transparent, neutral glass with a flat base and an internal
diameter of 15 mm to 25 mm to obtain a depth of 40 mm.
Compare the Test solution, the Standard solution, and water in
diffused daylight, viewing vertically against a white background
(see Visual Comparison under Spectrophotometry and Light-Scattering 851). [ NOTEThe diffusion of light must be such that
the Reference suspension can be readily distinguished from
water.] The Test solution has the appearance of water or is not
more intensely colored than the Standard solution.
Identification
A: Infrared Absorption 197K.
B: Thin-Layer Chromatographic Identification Test 201
Test solution: Prepare a solution containing 10 mg of Enrofloxacin per mL of 0.05 M hydrochloric acid.
Application volume: 5 L.
Developing solvent solution: methylene chloride, methanol,
25% ammonia solution, and acetonitrile (2:2:1:0.5).
ProcedureProceed as directed in the chapter except apply
5-L portions of the Test solution and the Standard solution to
the starting line of the chromatographic plate. Place the plate
in an atmosphere of ammonia for approximately 15 minutes.
Develop the chromatogram in an unsaturated chamber with
the Developing solvent solution.
Loss on drying 731Dry a 2-g sample, accurately weighed,
5 to 7 hours under vacuum at 120 to constant weight: it loses
not more than 1.0% of its weight.
Residue on ignition 281: not more than 0.1%, based on a
test specimen of about 2 g.
Chloride 221Add 58 mL of water to 0.5 g of Enrofloxacin,
shake for 5 minutes, and pass through a chloride-free filter paper, add 2 mL of 2 M acetic acid, and mix. T ransfer 15.0 mL of
the filtrate to a 50-mL color-comparison tube (Test solution). To
a second matched 50-mL color-comparison tube transfer 10.0
mL of a Standard solution of sodium chloride having a concentration of 8.2 g per mL, corresponding to 5 g of chloride per
mL, add 5.0 mL of water, and mix. T o each tube add 1 mL of
2 N nitric acid, mix, add 1 mL of silver nitrate TS, and mix.
Allow the solutions to stand for 5 minutes, protected from light.
Examine the tubes vertically against a black background (see

Visual Comparison under Spectrophotometry and Light-Scattering


851). Any opalescence in the Test solution is not more intense
than that in the Standard solution (0.04%).
Sulfate 221Dissolve 0.5 g of Enrofloxacin in 5.0 mL of 2 N
acetic acid and 15.0 mL of water ( Test solution). To each of two
50-mL matched color-comparison tubes transfer 1.50 mL of a
Standard solution of potassium sulfate in 30% alcohol having a
concentration of 18.1 g per mL, equivalent to 10 g of sulfate
per mL. To each tube add, successively and with continuous
shaking, 1.0 mL of barium chloride solution (1 in 4), and allow
to stand for 1 minute. T o one of the tubes transfer 15.0 mL of
the Standard solution and 0.5 mL of 30% acetic acid, and mix.
To the second tube add 15.0 mL of the Test solution and 0.5
mL of 30% acetic acid, and mix. Allow solutions to stand for 5
minutes. Examine the tubes vertically against a black background (see Visual Comparison under Spectrophotometry and
Light-Scattering 851). Any opalescence in the Test solution is
not more intense than that in the Standard solution (0.04%).
Heavy metals, Method II 231: 0.002%.
Limit of N-ethylpiperazine
Internal standard solutionDissolve an accurately weighed
quantity of n-decane in chloroform, and dilute quantitatively,
and stepwise if necessar y, with chloroform to obtain a solution
having a known concentration of about 0.1 mg per mL.
Standard stock solutionDissolve an accurately weighed
quantity of USP N-Ethylpiperazine RS in chloroform, and dilute
quantitatively, and stepwise if necessar y, with chloroform to obtain a solution having a known concentration of about 9.0 mg
per mL.
Standard solutionTo 2.0 mL of the Internal standard solution
add 20 L of the Standard stock solution, and mix.
Test solutionTo 200 mg of Enrofloxacin, accurately
weighed, add 2.0 mL of the Internal standard solution, and mix.
Chromatographic system (see Chromatography 621)The
gas chromatograph is equipped with a flame-ionization detector
and a split injector system and contains a 0.32-mm 50-m
column with 100% liquid phase G1 with a film thickness of
about 5.0 m. The carrier gas is hydrogen (helium may be
used), flowing at a rate of about 2.9 mL per minute. The auxiliary gas is nitrogen flowing at a rate of about 30 mL per minute. The chromatograph is programmed as follows. Initially the
temperature of the column is equilibrated at 80 , then the temperature is increased at a rate of 10 per minute to 240 , and
maintained at 240 for 15 minutes. The split injector (25:1 split
ratio) temperature is maintained at 200 , and the detector is
maintained at 250 . Chromatograph the Standard solution, and
record the peak responses as directed for Procedure: the relative
retention times are about 0.90 for N-ethylpiperazine and 1.0 for
n-decane.
ProcedureInject a volume (about 1.0 L) of the Standard
solution and the Test solution into the chromatograph, record
the chromatogram, and measure the responses for the major
peaks. Calculate the per centage of the impurity in the portion
of Enrofloxacin taken by the formula:
100(CS / CU)(RU / RS)
in which CS is the concentration of the impurity, in mg per mL,
in the Standard solution; CU is the concentration, in mg per mL,
of Enrofloxacin in the Test solution; RU is the peak response ratio
of the impurity peak to the internal standard peak obtained
from the Test solution; and RS is the peak response ratio of the
impurity peak to the internal standard peak obtained from the
Standard solution. Not more than 0.1% of the impurity is found.
Related compounds
TEST 1 (FOR FLUOROQUINOLONIC ACID)
Adsorbent: 0.25-mm layer of chromatographic silica gel
mixture.
Test solutionPrepare as directed for the Test solution in
Identification test B.
DiluentTransfer 0.1 mL of 6 M ammonium to a 100-mL
volumetric flask, mix, and dilute with water to volume.

Official from May 1, 2012


Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.

Accessed from 128.83.63.20 by nEwp0rt1 on Thu Nov 24 01:20:17 EST 2011

3046 Enrofloxacin / Official Monographs


Standard stock solutionDissolve and mix an accurately
weighed quantity of USP Fluroquinolonic Acid RS with Diluent
to prepare a solution containing about 0.10 mg per mL, and
mix.
Standard solution 1 (0.1%)Transfer 1.0 mL of the Standard
stock solution to a 10-mL volumetric flask, dilute with water to
volume, and mix.
Standard solution 2 (0.2%)Transfer 2.0 mL of the Standard
stock solution to a 10-mL volumetric flask, dilute with water to
volume, and mix.
Application volume: 5 L.
Developing solvent system[NOTECarefully follow the mixing order stated below.] Shake butyl acetate, n-butanol, water,
and glacial acetic acid (50:9:15:25), and allow to settle. Use the
upper layer as the mobile phase and discard the lower layer.
ProcedureProceed as directed for Thin-Layer Chromatography under Chromatography 621. Apply separately the Test solution, Standard solution 1, and Standard solution 2 to the thinlayer plate, and chromatograph using the Developing solvent
system. Dry the developed chromatogram in the air under a
fume hood for 30 to 60 minutes, then view under short-wavelength UV light. Determine the quantity of fluoroquinolonic
acid by comparing the size and intensity of the spots from the
Test solution to the Standard solutions. The intensity of any spot
from the Test solution at about the same retardation factor, RF,
as that of the Standard solutions is not greater than the intensity
of the Standard solution 2 (0.2%) spot.
TEST 2 (FOR CIPROFLOXACIN, DES-FLUORO COMPOUND AND OTHER UNSPECIFIED IMPURITIES)
Phosphoric acid bufferPrepare 25 mM phosphoric acid, and
adjust with triethylamine to a pH of 3.0.
Mobile phasePrepare a solution containing Phosphoric acid
buffer and acetonitrile (87:13).
Control solutionDissolve about 5 mg of USP Enrofloxacin
Related Compound Mixture RS, accurately weighed, in Mobile
phase in a 5-mL volumetric flask, dilute with Mobile phase to
volume, and mix.
Test solution 1Dissolve about 50 mg of Enrofloxacin, accurately weighed, in Mobile phase in a 50-mL volumetric flask,
dilute with Mobile phase to volume, and mix.
Test solution 2Transfer 1.0 mL of Test solution 1 into a 50mL volumetric flask, dilute with Mobile phase to volume, and
mix. Transfer 1.0 mL of this solution into a 10-mL volumetric
flask, dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography 621)The
liquid chromatograph is equipped with a 278-nm detector and
a 4.6-mm 25-cm stainless steel column that contains 5- m
packing L1. The column temperature is maintained at 40 , and
the flow rate is about 1.5 mL per minute.
Chromatograph the Control solution, and record the peak responses as directed for Procedure: the relative retention times
are about 0.58 for the des-fluoro compound, 0.74 for ciprofloxacin, and 1.0 for enrofloxacin. The resolution, R, between the
des-fluoro compound and ciprofloxacin is not less than 1.5.
ProcedureInject a volume (about 25 L) of Test solution 1,
Test solution 2, and the Control solution into the chromatograph,
record the chromatogram, and measure the peak responses.
Identify the ciprofloxacin and the des-fluoro compound peaks in
Test solution 2 by comparing their retention times with those
from the Control solution. Calculate the per centage of each related compound in the portion of Enrofloxacin taken by the
formula:
100C(rI / rS)
in which C is the concentration of Enrofloxacin in Test solution 2
as a per centage of Test solution 1 (0.2%); rI is the individual
peak response of each related compound obtained from Test
solution 1; and rS is the individual peak area of enrofloxacin
obtained from Test solution 2: not more than 0.1% of des-fluoro
compound, not more than 0.3% of ciprofloxacin, not more

USP 35
than 0.1% of any unspecified impurity, and not more than
0.5% of total impurities are found.
AssayTransfer about 250 mg of Enrofloxacin, accurately
weighed, to a 125-mL flask, dissolve in 100 mL of anhydrous
acetic acid, and titrate with 0.1 M per chloric acid VS, determining the endpoint potentiometrically. Per form a blank determination, and make any necessar y correction. Each mL of 0.1 M
perchloric acid is equivalent to 35.94 mg of C 19H22FN3O3.

Ensulizole
.

C13H10N2O3S
1H-Benzimidazole-5-sulfonic acid-2-phenyl-;
2-Phenylbenzimidazole-5-sulfonic acid [27503-81-7].

274.30

DEFINITION
Ensulizole contains NLT 98.0% and NMT 102.0% of
C13H10N2O3S, calculated on the dried basis.
IDENTIFICATION
A. INFRARED ABSORPTION 197K
B. ULTRAVIOLET ABSORPTION 197U
Sample solution: 5 g/mL
[NOTETransfer 100 mg of Ensulizole to a 100-mL volumetric flask, dissolve in 4 mL of 1 N sodium hydroxide, and
dilute with water to volume. T ransfer 0.5 mL of the solution to a 100-mL volumetric flask, and dilute with water
to volume.]
Acceptance criteria: The absorptivities, calculated on the
dried basis, at the wavelength of maximum absorbance at
about 302 nm, do not differ by more than 3.0%.
ASSAY
PROCEDURE
Sample solution: 48 mg/mL of Ensulizole in 0.5 N sodium
hydroxide
Titrant: 0.5 N hydrochloric acid VS
Analysis: To the Sample solution, add phenolphthalein TS,
and titrate the excess with Titrant. Perform a blank determination, and make any necessar y corrections (see Titrimetry
541). Each mL of 0.5 N sodium hydroxide is equivalent to
137.15 mg of C 13H10N2O3S.
Calculate the per centage of C 13H10N2O3S in the portion of
Ensulizole taken:
Result = [(N (V B V A))/(W (1 LOD))] M r1 100
N
VB
VA

= actual normality of the Titrant


= volume of Titrant used for the blank (mL)
= volume of Titrant used for the Sample solution
(mL)
W
= weight of Ensulizole taken for the Sample solution
(mg)
LOD = percentage of loss on dr ying expressed as a
decimal fraction, as determined in the test for
Loss on Drying
= molecular weight of ensulizole (274.30)
Mr1
Acceptance criteria: 98.0%102.0% on the dried basis
SPECIFIC TESTS
LOSS ON DRYING 731: Dry a sample at 105 for 4 h: it loses
NMT 2.0% of its weight.
ADDITIONAL REQUIREMENTS
PACKAGING AND STORAGE: Preserve in tight containers in a
cool place.

Official from May 1, 2012


Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.