Estimate and Validation Cefixime and Azithromycin Simultaneoslyy in Tablt Dosage Froms by RP-HPLC Method

ESTIMATE AND VALIDATION CEFIXIME AND AZITHROMYCIN
SIMULTANEOSLYY IN TABLT DOSAGE FROMS BY RP-HPLC

METHOD
Thesis Submitted to
Jawaharlal Nehru Technological University, Kakinada
In partialfulfilmentt of the requirement for the award of the degree of
MASTER OF PHARMACY
In
PHARMACEUTICAL ANALYSIS AND QUALITY ASSURANCE
By
REDDYBATHULA LEELA
M.Pharm
[Reg. No. 14CD1S0423]
Under The Guidance of
INSTITUITIONAL GUIDE: INDUSTRIAL GUIDE:

Mr. K.Naga Prashanth, M.Pharm Mr.
D.AnandKumar,M.pharm,
Asst. Prof, NIPS, Narasaraopet. Quest solutions pharma,
Kukatpally, HYD.
Department of Pharmaceutical Analysis

and Quality Assurance
NARASARAOPETA
INSTITUTE OF PHARMACEUTICAL
SCIENCES
Kotappakonda Road, Yallamanda(P), Narasaraopet (M)
Guntur (Dt.) Pin: 522601
2016
NARASARAOPETA INSTITUTE OF PHARMACEUTICAL SCIENCES
NARASARAOPET
CERTIFICATE
TO ESTIMATE CEFIXIME AND AZITHROMYCIN SIMULTANEOUSLY IN TABLET
DOSAGE FORMS BY RP-HPLC METHOD
This is to certify that the thesis entitiled . To Estimate Cefixime And

Azithromycin Simultaneously In Tablet Dosage Forms By RP-HPLC
Method is the bonafide research work carried out by Mis. Reddybathula
Leela of II year M. Pharmacy bearing Reg No. 14CD1S0423 in partial
fulfilment of the requirement for the award of master of Pharmacy in
Pharmaceutical analysis and quality assurance as per the norms of the Jahwarlal
Nehru Technological University Kakinada for the academic year 2015-16.
Signature of the Guide Principal
Dr. J.N. Suresh Kumar
DEPARTMENT OF PHARMACEUTICAL ANALYSIS AND QUALITY ASSURANCE, NIPS Page

NARASARAOPETA INSTITUTE OF PHARMACEUTICAL SCIENCES
NARASARAOPET

DECLARATION BY THE CANDIDATE
I Reddybathula Leela of II Year M.Pharmacy bearing Regd. No
14CD1S0423 hereby declare that the research work entitled Estimate
Cefixime and Azithromycin Simultaneously In Tablet Dosage Forms By
RP-HPLC Method was carried out by me under the guidance of
Mr. P. Prasanth Kumar(Associate prof.) The work embodied in this thesis is
original and has not been submitted in part of in full for any degree or diploma
of this or other university.
ACKNOWLEDGEMENT
As I inscribe this acknowledgement, all the pleasant memories are flooding my mind.
Right from getting a chance to work under great guides to finishing my work in two years, it
has been dream tenure for me. As I remember the good and the tough times I had, I

remember the people who helped me in various ways in my endeavour and made my
experience a special one. I take this opportunity to thank all of them.
I fondly thank with deepest gratitude to my honourable guide K,Naga Prashanth Asst.
Professor, Narsaraopet Institute of Pharmaceutical sciences who with his deep knowledge,
keen interest of the subject, kind co-operation, untiring patience, constructive criticism,
constant help and encouragement has enabled me to complete my dissertation work. His
principles, discipline, simplicity caring attitude and provision of fearless work environment
will be cherished in all walks of my life.
I First of all, I would like to thank the Super power, the nature, for all that happens in life.
I am thankful to Principal Mr.J.N.Suresh kumar, who have been a constant source of

encouragement and treasure of valuable inspiring guidance I immensely thank my friends
and my class mates Harish, Sandhy and GML reddy for their joyful encouragement.
I would like to thank everyone who directly or indirectly helped me in my project work. Last
but not the least I pay my reverence to our institute, Narsaraopet Institute of pharmaceutical
Sciences,I am undeniably proud to be associated with this college.
Thank you everyone..!
Leela!!!
S.NO CONTENTS P.NO

1 ABBREVIATIONS
2 LIST OF TABLES
3 LIST OF FIGURES

4 INTRODUCTION 1
5 LITERATURE REVIEW 25
6 DRUG PROFILE 27
7 AIM AND PLAN OF WORK 31
8 MATERIALS AND METHODS 32
9 RESULTS AND DISCUSSIONS 39
10 SUMMARY AND CONCLUSION 78
11 BIBILOGRAPHY 79
ABBREVIATIONS
Symbols Description
g Microgram
L Microlitre
AR Analytical reagent

BDS Base deactivated silane

Conc. Concentration
fg fico gram
Fig. Figure
GC Gas chromatography
gm Gram
GPC Gel permeation chromatography
HETP Height equivalent to Theoretical Plates
HIC Hydrophobic interaction chromatography
HILIC Hydrophilic interaction chromatography
HPLC High-performance liquid chromatography
I.D Internal diameter
ICH International Conference on Harmonization
IEC Ion exchange chromatography
IP Ion-pair
LC Liquid chromatography
LC-MS Liquid chromatography-mass spectroscopy
LOD Limit of detection
LOQ Limit of quantification
Min Minutes
mL Millilitre
mm millimetre
mM Millimolar
MS Mass spectroscopy
NARP Non-aqueous reverse phase
ng Nano gram
NPC Normal phase chromatography
pg Pico gram
RPC Reversed phase chromatography
Rs Resolution
RSD Relative standard deviation
RT Retention time
SD Standard deviation
SEC Size-exclusion chromatography
TF Tailing factor
TP Theoretical plate

INTRODUCTION
Pharmaceutical Analysis plays a very vital role in the quality assurance and quality
control of bulk drugs and their formulations. Pharmaceutical analysis is a specialized branch
of analytical chemistry which involves separating, identifying and determining the relative
amounts of components in a sample of matter. It is concerned with the chemical
characterization of matter both quantitative and qualitative.
In recent years, several analytical techniques have been evolved.
1. SPECTROPHOTOMETRIC METHODS
Spectrophotometer is generally preferred especially by small-scale industries as the cost of

the equipment is less and the maintenance problems are minimal. The method of analysis is
based on measuring the absorption of a monochromatic light by colourless compounds in the
near ultraviolet path of spectrum (200-380nm). The photometric methods of analysis are
based on the Bouger-Lambert-Beers law, which establishes the absorbance of a solution is
directly proportional to the concentration of the analytic. The fundamental principle of
operation of spectrophotometer covering UV region consists in that light of definite interval
of wavelength passes through a cell with solvent and falls on to the photoelectric cell that
transforms the radiant energy into electrical energy measured by a galvanometer.
The important applications are
Identification of many types of organic, inorganic molecules and ions.

Quantitative determination of many biological, organic and inorganic species.
Monitoring and identification of chromatographic of effluents.
2. HPLC METHOD DEVELOPMENT

The term Chromatography covers those processes aimed at the separation of the
various species of a mixture on the basis of their distribution characteristics between a
stationary and a mobile phase.
2.1.MODES OF CHROMATOGRAPHY
Modes of chromatography are defined essentially according to the nature of the
interactions between the solute and the stationary phase, which may arise from hydrogen
bonding, Vander walls forces, electrostatic forces or hydrophobic forces or basing on the size
of the particles (e.g. Size exclusion chromatography).
Different modes of chromatography are as follows:

Normal Phase Chromatography

Reversed Phase Chromatography
Reversed Phase ion pair Chromatography
Ion-Exchange Chromatography
Size Exclusion Chromatography
REVERSED PHASE CHROMATOGRAPHY

The objective was to make less polar or non polar so that polar solvents can be used to
separate water-soluble polar compounds. Since the ionic nature of the chemically modified
silica is now reversed i.e. it is non-polar or the nature of the phase is reversed. The
chromatographic separation carried out with such silica is referred to as reversed- phase
chromatography.
A large number of chemically bonded stationary phases based on silica are available
commercially. Silica based stationary phases are still most popular in reversed phase
chromatography however other adsorbents based on polymer (styrene-divinyl benzene
copolymer) are slowly gaining ground.
Simple compounds are better retained by the reversed phase surface, the less water-
soluble (i.e. the more non-polar) they are. The retention decreases in the following order:
aliphatic > induced dipoles (i.e. CCl4) > permanent dipoles (e.g.CHCl3) > weak Lewis bases
(ethers, aldehydes, ketones) > strong Lewis bases (amines) > weak Lewis acids (alcohols,
phenols) > strong Lewis acids (carboxylic acids). Also the retention increases as the number
of carbon atoms increases.
As a general rule the retention increases with increasing contact area between sample
molecule and stationary phase i.e. with increasing number of water molecules, which are
released during the adsorption of a compound. Branched chain compounds are eluted more
rapidly than their corresponding normal isomers.
Chemically bonded octadecyl silane (ODS) an alkaline with 18 carbon atoms it is the
most popular stationary phase used in pharmaceutical industry. Since most pharmaceutical
compounds are polar and water soluble, the majority of HPLC methods used for quality
assurance, decomposition studies, quantitative analysis of both bulk drugs and their
formulations use ODS HPLC columns. The solvent strength in reversed phase
chromatography is reversed from that of adsorption chromatography (silica gel) as stated
earlier. Water interacts strongly with silanol groups, so that, adsorption of sample molecules
become highly restricted and they are rapidly eluted as a result.
Exactly opposite applies in reversed phase system water cannot wet the non-polar
(hydrophobic) alkyl groups such as C18 of ODS phase and therefore does not interact with the
bonded moiety. Hence water is the weakest solvent of all and gives slowest elution rate. The
elution time (retention time) in reversed phase chromatography increases with increasing
amount of water in the mobile phase.

ADSORPTION CHROMATOGRAPHY OR NORMAL PHASE

CHROMATOGRAPHY
In normal phase chromatography, the stationary phase is a polar adsorbent and the mobile
phase is generally a mixture of non-aqueous solvents.
The silica structure is saturated with silanol groups at the end. These OH groups are
statistically disturbed over the whole of the surface. The silanol groups represent the active
sites (very polar) in the stationary phase.
These situations arise when the molecule has one or several atoms with lone pair electron
or a double bond. The adsorption strengths and hence k values (elution series) increase in the
following order. Saturated hydrocarbon < olefins < aromatics < organic halogen compounds
< sulphides < ethers< esters < aldehydes and ketones < amines < sulphones < amides <
carboxylic acids. The strength of interactions depends not only on the functional groups in
the sample molecule but also on steric factors. If a molecule has several functional groups,
then the most polar one determines the reaction properties.
The aminopropyl and cyanopropyl phases provide opportunities for specific interactions
between the analyte and the stationary phases and thus offer additional options for the
optimizations of separations.
Resolution with water in weak mobile phase may be most conveniently achieved by drying
the solvents and then adding a constant concentration of water or some very polar modifier
such as acetic acid or trietylamine (TEA) to the mobile phase. The
addition of such polar modifiers serves to deactivate the more polar shape as well as the
reproducibility of the retention times.
Chromatographic methods can be classified most practically according to the stationary
and mobile phases, as shown in the table 1:
Table.2.1: Classification of Chromatographic Methods
Stationary phase Mobile phase Method

Adsorption column, thin-layer, ion
Solid Liquid exchange, High performance
liquid chromatography.
Partition, column, thin-layer,
Liquid
HPLC, paper chromatography.
Liquid
GasLiquid Chromatography
Gas
The modern form of column chromatography has been called high performance, high
pressure, and high-resolution and high-speed liquid chromatography.

High-Performance Liquid Chromatography (HPLC) is a special branch of column

chromatography in which the mobile phase is forced through the column at high speed. As a
result the analysis time is reduced by 1-2 orders of magnitude relative to classical column
chromatography and the use of much smaller particles of the adsorbent or support becomes
possible increasing the column efficiency substantially.
The essential equipment consists of an eluent, reservoir, a high-pressure pump, and
an injector for introducing the sample, a column containing the stationary phase, a detector
and recorder. The development of highly efficient micro particulate bonded phases has
increased the versatility of the technique and has greatly improved the analysis of multi
component mixtures.
The various components of a HPLC system are herewith described.
Solvent container Pump damping unit Injection port Column

Recorder Detector Effluent
2.2 SYSTEM COMPONENTS:

Solvent delivery system:
The mobile phase is pumped under pressure from one or several reservoirs and flows
through the column at a constant rate. With micro particulate packing, there is a high-pressure
drop across a chromatography column. Eluting power of the mobile phase is determined by
its overall polarity, the polarity of the stationary phase and the nature of the sample
components. For normal phase separations eluting power increases with increasing polarity of
the solvent but for reversed phase separations, eluting power decreases with increasing
solvent polarity. Optimum separating conditions can be achieved by making use of mixture of
two solvents. Some other properties of the solvents, which need to be considered for a
successful separation, are boiling point, viscosity, detector compatibility, flammability and
toxicity.
The most important component of HPLC in solvent delivery system is the pump,
because its performance directly effects the retention time, reproducibility and detector
sensitivity. Among the several solvent delivery systems (direct gas pressure, pneumatic
intensifier, reciprocating etc.) reciprocating pump with twin or triple pistons is widely used,
as this system gives less baseline noise, good flow rate reproducibility etc.
Solvent degassing system:
The constituents of the mobile phase should be degassed and filtered before use.
Several methods are employed to remove the dissolved gases in the mobile phase. They
include heating and stirring, vacuum degassing with an aspirator, filtration through 0.45
filters, vacuum degassing with an air-soluble membrane, helium purging ultra sonication or
purging or combination of these methods. HPLC systems are also provided an online
degassing system, which continuously removes the dissolved gases from the mobile phase.
Gradient elution devices: HPLC columns may be run isocratically, i.e., with constant eluent
or they may be run in the gradient elution mode in which the mobile phase composition

varies during run. Gradient elution is a means of overcoming the problem of dealing with a
complex mixture of solutes.
Sample introduction systems:
Two means for analyte introduction on the column are injection in to a flowing stream
and a stop flow injection. These techniques can be used with a syringe or an injection valve.
Automatic injector is a microprocessor-controlled version of the manual universal injector.
Usually, up to 100 samples can be loaded in to the auto injector tray. The system parameters
such as flow rates, gradient, run time, volume to be injected, etc. are chosen, stored in
memory and sequentially executed on consecutive injections.
Liquid chromatographic detectors: The function of the detector in HPLC is to

monitor the mobile phase as it emerges from the column. Generally, there are two types of
HPLC detectors, bulk property detectors and solute property detectors.
1. Bulk property detectors:
These detectors are based on differential measurement of a property, which is

common to both the sample and the mobile phase. Examples of such detectors are refractive
index, conductivity and dielectric constant detectors.
2. Solute property detectors:
Solute property detectors respond to a physical property of the solute, which is
not exhibited by the pure mobile phase. These detectors measure a property, which is specific
to the sample, either with or without the removal of the mobile phase prior to the detection.
Solute property detectors which do not require the removal of the mobile phase before
detection include spectrophotometric (UV or UV-Vis) detector, fluorescence detectors,
polarographic, electro-chemical and radio activity detectors, whilst the moving wire flame
ionization detector and electron capture detector both require removal of the mobile phase
before detection.
UV-Vis and fluorescent detectors are suitable for gradient elution, because many
solvents used in HPLC do not absorb to any significant extent.
3.Column and Column-packing materials:
The heart of the system is the column. In order to achieve high efficiency of
separation, the column material (micro-particles, 5-10 m size) packed in such a way that
highest numbers of theoretical plates are possible. Silica (SiO 2, H2O) is the most widely used
substance for the manufacture of packing materials. It consists of a network of siloxane
linkages (Si-O-Si) in a rigid three dimensional structure containing inter connecting pores.
Thus a wide range of commercial products is available with surface areas ranging from 100
to 800 m2/g. and particle sizes from 3 to 50 m.
The most popular material is octadecyl-silica (ODS-Silica), which contains C 18

chains, but materials with C2, C6, C8 and C22 chains are also available. During manufacture,
such materials may be reacted with a small mono functional silane (e.g. trimethyl chloro

silane) to reduce further the number of silanol groups remaining on the surface (end-capping).
The useful pH range for columns is 2 to 8, since siloxane linkages are cleaved below pH-2
while at pH values above eight silica may dissolve.
In HPLC, generally two types of columns are used, normal phase columns and
reversed phase columns. Using normal phase chromatography, particularly of non-polar and
moderately polar drugs can make excellent separation. It was originally believed that
separation of compounds in mixture takes place slowly by differential adsorption on a
stationary silica phase.
4 .Column-packing materials
The heart of the system is the column. In order to achieve high efficiency of separation, the
column material (micro-particles, 5-10 m size) packed in such a way that highest numbers
of theoretical plates are possible.
Silica (SiO 2 X H2O) is the most widely used substance for the manufacture of
packing materials. It consists of a network of siloxane linkages (Si-O-Si) in a rigid three
dimensional structure containing inter connecting pores. Thus a wide range of commercial
products is available with surface areas ranging from 100 to 800 m 2/g. and particle sizes from
3 to 50 m.
The silanol groups on the surface of silica give it a polar character, which is exploited in
adsorption chromatography using non-polar organic eluents. Silica can be drastically altered
by reaction with organo chloro silanes or organo alkoxy silanes giving Si-O-Si-R linkages
with the surface. The attachment of hydrocarbon change to silica produces a non-polar
surface suitable for reversed phase chromatography where mixtures of water and organic
solvents are used as eluents. The most popular material is octadecyl-silica (ODS-Silica),
which contains C18 chains, but materials with C2, C6, C8 and C22 chains are also available.
During manufacture, such materials may be reacted with a small mono functional silane (e.g.
trimethyl chloro silane) to reduce further the number of silanol groups remaining on the
surface (end-capping). There is a vast range of materials which have intermediate surface
polarities arising from the bonding to silica of other organic compounds which contain
groups such as phenyl, nitro, amino and hydroxyl. Strong ion exchangers are also available in
which sulphonic acid groups or quaternary ammonium groups are bonded to silica. The
useful pH range for columns is 2 to 8, since siloxane linkages are cleaved below pH-2 while
at pH values above eight, silica may dissolve.
In HPLC, generally two types of columns are used, normal phase columns and reversed phase
columns. Using normal phase chromatography, particularly of non-polar and moderately
polar drugs can make excellent separation. It was originally believed that separation of
compounds in mixture takes place slowly by differential adsorption on a stationary silica
phase. However, it now seems that partition plays an important role, with the compounds
interacting with the polar silanol groups on the silica or with bound water molecules.

While normal phase seems the passage of a relatively non-polar mobile phase
over a polar stationary phase, reversed phase chromatography is carried out using a polar
mobile phase such as methanol, acetonitrile, water, buffers etc., over a non-polar stationary
phase. Ranges of stationary phases (C18, C8, -NH2, -CN, -phenyl etc.) are available and very
selective separations can be achieved. The pH of the mobile phase can be adjusted to
suppress the ionization of the drug and thereby increase the retention on the column. For
highly ionized drugs ion-pair chromatography is used.
BONDED PHASES FOR HPLC AND THEIR ABBREVIATIONS
Phase Description
Silica
Classic normal phase material. Suitable for separating polar non-ionic
Si organic compounds.
TMS, SAS,
Tri methyl silane
C1 Reversed phase material. Unique selectivity for polar and

multifunctional compounds. Least retentive of all alkyl group bonded
phases for non-polar solvents.
RP-2, Dimethyl
C2 Reversed phase material, less retentive than C4, C8, or C18. More retentive than C1.
Propyl: Reversed phase material, used in hydrophobic interaction chromatography

C3 (HIC) of proteins and peptides.

Butyl: Reversed phase material, useful for ion-pairing chromatography offers less
retention than C8 and C18 phases for non-polar solutes. When bonded to 300 silica, it
is an ideal phase for analyzing large proteins and hydrophobic peptides.
C4
Hexyl: Reversed phase material, useful for ion-pairing chromatography. Less

C6 retentive than C8 and C18 phases.
MOS, RP-8, LC8, Octyl
C8 Reversed phase material, similar selectivity to C18 but less retentive. Wide
applicability (e.g. pharmaceuticals, nucleosides, steroids). When bonded to300
silica, it is an ideal phase for peptides, peptide mapping and small hydrophilic
proteins.
ODS, RP-18, LC18, Octadecyl

Classic reversed phase material is most retentive for non-polar solutes and is
excellent for ion-pairing chromatography. It is having wide applicability for the
C18 assay of nucleosides, nucleotides, steroids, pharmaceuticals, vitamins, fatty acids
and environmental compounds when bonded to 300 silica, this phase is perfect
for separating small hydrophilic peptides.

It is a reversed phase material and exhibits unique selectivity. It is useful for

analyzing aromatic compounds. When bonded to 300 silica, this phase is useful
for HIC.
C6H5
Phenyl
CPS, PCN, Cyano,
CN Cyanopropyl, Nitrile
It can be employed as either a reversed phase or normal phase material. It is
slightly polar and exhibits unique selectivity for polar compounds in both RP
and NP modes. It equilibrates very rapidly and suitable for gradient
separations. It has many pharmaceutical applications (e.g. tricyclic
antidepressants).
APS, Amino, Amino propyl silyl
NH2
Can be employed as reversed phase, normal phase or weak anion exchange material.
Reversed phase: useful for separating carbohydrates. Normal phase: alternative
selectivity to silica, not deactivated by small amounts of water. Ion Exchange: weak
anion exchanger when used with buffers separates anions and organic acids.
Nitro
NO2
Normal phase material. Separates aromatic compounds and compounds with double
bonds.
Diol, Glycerol
OH
Can be employed as either a reversed phase or normal phase material. Reversed
phase: Used for Gel Filtration Chromatography (GFC) of proteins and peptides.
Normal phase: Similar selectivity to silica not deactivated by small amounts of
water.

SAX SB,Quaternaryamine,Strong Base
Ion exchange material. Strong anion exchangers (basic) are useful for separating
nucleosides, nucleotides and organic acids.
SAX SB,Quaternaryamine,Strong Base
Ion exchange material. Strong anion exchangers (basic) are useful for separating
nucleosides, nucleotides and organic acids.
SA, Sulfonic acid, Strong Acid

SCX
Ion exchange material. Strong cation exchangers (acidic) are useful for separating
organic bases.
PEI, DEAE, Polyethyleneimine,

WAX
Diethylaminoethyl, Weak Base
Ion exchange material. Weak anion exchangers (basic) are most useful for analyzing
acidic proteins and peptides.
CM, Carboxymethyl,
WCX
Weak Acid
Ion exchange material. Weak cation exchangers (acidic) are most useful for
analyzing basic proteins and peptides.
5.Derivatization:
In HPLC derivatization is used to enhance the sensitivity and selectivity of
detection when available detectors are not satisfactory for the underivatized compounds. Both
ultra violet absorbing and fluorescence derivatives have been widely used. Ultra violet
derivatization reagents include N-succinimidyl p-nitro phenyl acetate, phenyl hydrazine and
3, 5-dinitro benzyl chlorides, while fluorescent derivatives can be formed with reagents such
as dansyl chloride, 4-bromo methyl-7-methoxy-coumarin and fluorescamine. Derivative
formation can be carried out before the sample is injected on to the column or by online
chemical reactions between the column out let and the detector.

6. Gradient elution:
Gradient elution or solvent programming is the change of solvent composition
during a separation in which the solvent strength increases from the beginning to the end of
the separation. It is well suited to the analysis of samples of unknown complexity since good
resolution is automatically provided for a wide range of sample polarities. There are two
types of gradient systems: Low-pressure gradient mixtures and high- pressure gradient
mixtures. In the former the solvents are mixed at atmosphere pressure and then pumped to the
column, where as in the later, solvents are pumped in to a mixing chamber at high pressure
before going in to the column.
Performance calculations:
Calculating the following values (which can be include in a custom report) used
to access overall system performance.
Relative retention, Theoretical plates, Capacity factor
Resolution, Peak asymmetry, Plates per meter
The parameters used to calculate these system performance values for the separation of
two chromatographic components.
Relative retention (Selectivity):
= (t2 - ta) / (t1 - ta)
Theoretical plates:
n = 16 (t / W) 2
Capacity factor:
K' = (t2 / ta) - 1
Rsolution:
R = 2 (t2 - t1) / (W2 + W1)
Peak asymmetry:
T = W0.05 / 2f
Plates per meter:
N=n/L
HETP: L/n
Where, = Relative retention.
t2 = Retention time of the second peak measured from point of injection.

t1 = Retention time of the first peak measured from point of injection.

ta = Retention time of an inert peak not retained by the column, measured from point
of injection.
n = Theoretical plates.
t = Retention time of the component.
W = Width of the base of the component peak using tangent method.
K' = Capacity factor.
R = Resolution between a peak of interest (p2) and the peak preceding it (p1)
W2 = Width of the base of component peak 2.
W1 = Width of the base of component peak 1.
T = Peak asymmetry, or tailing factor.
W0.05 = Distance from the leading edge to the tailing edge of the peak,
Measured at a point 5 % of the peak height from the baseline.
f = Distance from the peak maximum to the leading edge of the peak.
L = length of column, in meters
N = Number of plates per meter
HPLC troubleshooting 3
Abnormal pressure
No pressure reading, no flow
Possible cause Solution
Power off Turn on power
Fuse blown Replace fuse
Controller setting or failure Verify proper settings, repair or replace controller
Broken piston Replace piston
Air trapped in pump head Degas mobile phase, bleed air from pump and prime pump
Insufficient mobile phase Replenish reservoir, replace inlet frit if it is blocked
Faulty check valves Replace check valves

Major leak Tighten or replace fittings
Now pressure reading, flow is normal

Faulty meter Replace meter
Faulty pressure transducer Replace transducer
High back pressure

Flow rate set too high Adjust settings
Blocked column frit Backflush column if it is permitted, replace frit according to the
manufacturer's instructions and warranty conditions or replace
column
Improper mobile phase, Use correct mobile phase, wash column
precipitated buffer
Improper column Use proper column
Injector blockage Clear blockage or replace injector
Column temperature too Raise temperature
low
Controller malfunction Repair or replace controller
Blocked guard column Remove/replace guard column
Blocked in-line filter Remove/replace in-line filter
You should find out, what caused high back pressure - column or system? We recommend
following procedure:
Remove column from the system and turn on pump. If high back pressure still appears, then
the blockage is in the system:
blocked or crimped tubing

dirty pump frit
or clogged injection valve
If the pressure is normal, there is a problem with the column:
clogged or damaged pre-column filter, guard column or frit
precipitation of sample or buffer in column
Low back pressure 9

Flow set too low Adjust flow rate
Leak in the system Locate and correct
Improper column Use proper column
Column temperature too high Lower temperature
Controller malfunction Repair or replace controller
Fluctuating pressure
See section High back pressure.
Pressure dropping to zero
See sections No pressure reading, no flow and No pressure reading, flow is normal
Pressure dropping, but not to zero
See section Low pressure
Pressure cycling
Possible case Solution

Air in pump Degas solvent and/or bleed air from the pump
Faulty check Replace check valve(s)

valve(s)
Pump seal failure Replace pump seal
Insufficient Degas solvent and/or change degassing methods (e.g. use vacuum
degassing degasser)
Leak in system Locate leak and correct it
Using gradient Pressure cycling is normal due to viscosity change
elution
Leaks
Leaks are usually stopped by tightening or replacing a fitting. Be aware, however, that
overtightened metal compression fittings can leak and plasrtic fingertight fittings can
wear out. If a fitting leak does not stop when the fitting is tightened a little, take the
fitting apart and inspect for damage (e.g. distored ferrule, or particles on the sealing
surface). If the fitting or ferrule is damaged, replace it with new one.
Leaky fittings

Loose fitting Tighten the fitting
Stripped fitting Replace the fitting
Overtightened fitting Loosen and retighten the fitting. If the fitting is damaged, replace it.
Overtightened fitting Loosen and retighten the fitting. If the fitting is damaged, replace it.
Dirty fitting Disassemble fitting and clean it. If the fitting is damaged, replace it.
Mismatched parts Use all parts from the same brand/type.
Leaks at the pump

Loose check valves Tighten check valve (do not overtighten) or replace check valve
Loose fittings Tighten fittings (do not overtighten)
Mixer seal failure Repair or replace
Pump seal failure Repair or replace the seal
Pulse damper failure Replace pulse damper
Proportionin valve Check diaphragms, replace if leaky and/or check for fitting

failure damage, replace

Purge valve Tighten valve or replace it if it is faulty
Injector leaks

Rotor seal failure Rebuild or replace rotor
Blocked loop Clean or replace loop
Loose injector port seal Adjust
Improper syringe needle diameter Use correct syringe
Waste line siphoning Keep waste line above surface vaste, with proper slope
Waste line blockage Replace waste line
Column leaks
Loose end fitting Tighten end fitting
Column packing in ferrule Disassemble, rinse ferrule, reassemble
Improper frit thickness Use proper frit(*)
*Note: When the particle size of stationary phase is 3 to 4 m, use frit 0.5 m. When particle
size of stationary phase is 5 to 20 m, use frit 2 m.
Detector leaks

Cell gasket failure Prevent excessive backpressure or replace gasket

Cracked cell window(s) Replace cell window(s)

Leaky fittings Tighten or replace fittings
Blocked waste line Replace waste line
Blocked flow cell Rebuild or replace flow cell
Problems with the chromatogram
Many issues in the LC system appear as changes in the chromatogram. Some of these can be
solved by changes in the instrument, however, other problems require modification of the
assay procedure. Setting the proper column type, pre-column or guard column, tubings,
detector cell and mobile phase are keys to good chromatography.
Peak tailing

Blocked frit Reverse flush column (if it is allowed) or replace frit (if it is
allowed) or replace column
Column void Fill void
Interfering peak Use longer column or change mobile phase and/or column
selectivity
Wrong mobile phase pH Adjust pH. For basic compounds, lower pH usually provides
more symmetric peaks
Sample reacting with Add ion pair reagent or volatile basic modifier or change
active sites column
Peak fronting


Low temperature Increase column temperature
Wrong sample Use mobile phase for injection solvent
solvent
Sample overload Decrease sample concentration
Bad column Reverse flush column (if it is allowed) or replace inlet frit (if it is
aalowed) or replace the column.
Split peaks

Contamination on Remove guard column and attempt analysis. Replace guard column
guard or analytical if necessary. If analytical column is obstructed, reverse and flush (if
column inlet it is allowed). If problem persists, column may be fouled with
strongly retained contaminants. Use appropriate restoration
procedure (see column care information). If problem persists, inlet
is probably plugged. Change frit or replace column.
Sample solvent Change solvent. whenever possible, inject samples in mobile phase.
incompatible with
mobile phase
Distortion of larger peaks
The peak distortion can be caused by sample overload. Reduce the sample size.
Distortion of early peaks

The distortion of early eluting peaks can be caused by wrong injection solvent. Reduce the
injection volume, or use weaker injection solvent.
Tailing, early peaks more than later ones
Extra-column Replumb the system (shorter, narrower tubing), use smaller volume
effects detector cell
Increased tailing as k' increases
Secondary retention Add triethylamine (basic samples) or add acetate (acidic

effects, reversed-phase samples) or add salt or buffer (ionic samples) or try a different
mode column.
Secondary retention Add triethylamine (basic compounds) or add acetic acid (acidic
effects, normal-phase compounds) or add water (poly-functional compounds). Only
mode for normal-phase methods which use water-miscible solvents.
You can also try a different LC method.
Secondary retention Add triethylamine (basic samples)
effects, ion-pair
chromatography
Acidic or basic peaks tail
Inadequate Use 50 to 100 mM buffer concentration, use buffer with pKa equal to
buffering pH of mobile phase

Extra peaks

Ghost peaks Impurities in the sample, reagents or material used. Change clean-up
procedure and/or check possible source of contamination (glassware,
vials, used reagents, solvents, etc...)
Late eluting peak Increase run time or gradient slope and/or increase flow rate
from previous
injection
METHOD OPTIMISATION: 12
Selection of stationary phase / column: Selection of the column is the first and the most
important step in method development.
Some of the important parameters considered while selecting chromatographic columns
are:
Length and diameter of the column.

Packing material.
Shape of the particles.
Size of the particles..
The column is selected depending on the nature of the solute and the information
about the analyte. Reversed phase mode of chromatography facilitates a wide range of
columns like dimethyl silane (C2), butylsilane (C4), octylsilane (C8), octadecylslane (C18),
base deactivated silane (C18) BDS phenyl, cyanopropyl (CN), nitro, amino etc. C 18 was
chosen for this study since it is most retentive one. The sample manipulation becomes easier
with this type of column
Generally longer columns provide better separation due to higher theoretical plate
numbers. Columns with 5-m particle size give the best compromise of efficiency,
reproducibility and reliability. In this case, the column selected had a particle size of 5 m
and a internal diameter of 4.6 mm
A useful and practical measurement of peak shape is peak asymmetry factor and peak
tailing factor. Peak asymmetry is measured at 10% of full peak height and peak tailing factor
at 5%. Reproducibility of retention times and capacity factor is important for developing a
rugged and repeatable method.
Selection of mobile phase: The primary objective in selection and optimization of mobile
phase is to achieve optimum separation of all the individual impurities and degradants from
each other and from analyte peak
In liquid chromatography, the solute retention is governed by the solute distribution
factor, which reflects the different interactions of the solute stationary phase, solute
mobile phase and the mobile phase stationary phase .For a given stationary phase, the
retention of the given solute depends directly upon the mobile phase, the nature and the
composition of which has to be judiciously selected in order to get appropriate and required
solute retention. The mobile has to be adapted in terms of elution strength (solute retention)
and solvent selectivity (solute separation) Solvent polarity is the key word in

chromatographic separations since a polar mobile phase will give rise to low solute retention
in normal phase and high solute retention in reverse phase LC. The selectivity will be
particularly altered if the buffer pH is close to the pKa of the analytes; the solvent strength is
a measure of its ability to pull analyte from the column. It is generally controlled by the
concentration of the solvent with the highest strength.
The following are the parameters, which shall be taken into consideration while
selecting and optimizing the mobile phase.
Buffer,
pH of the buffer
Mobile phase composition.
Selection of detector:
The detector was chosen depending upon some characteristic property of the analyte
like UV absorbance, fluorescence, conductance, oxidation, reduction etc. characteristics that
are to be fulfilled by a detector to be used in HPLC determination are,
High sensitivity, facilitating trace analysis
Negligible baseline noise. To facilitate lower detection
Low dead volume
Non destructive to sample
Inexpensive to purchase and operate
For the greatest sensitivity max should be used. Higher wavelengths give greater
selectivity.
3. METHOD VALIDATION 17
Method validation can be defined as (ICH) estabilishing documented evidence which
provides a high degree of assurance that specific activity will consistenty produce a desired
result or product meeting its predetermined specifications and quality characteristics.
Method validation is an integral part of the method development; it is the
process of demonstrating that analytical procedures are suitable for their intended use
and that they support the identity, quality, purity, an and drug products. Simply, method
validation is the process of proving that and potency of the drug substances analytical method
is acceptable for its intended purpose.
For chromatographic methods used in analytical applications there is more
consistency in validation practice with key analytical parameters
(a) Recovery (b) Response function (c) Sensitivity (d) Presicion (e) Accuracy (f) limits
of detection (g) Limit of quantitation (h) Ruggedness (i) Robustness (j) stability (k)
system suitability
(a)Recovery:
The absolute recovery of analytical method is measured as the response of a processed
spiked matrix standard expressed as a percentage of the response of pure standard which has

not been subjected to sample pre treatment and indicates whether the method provides a
response for the entire amount of analyte that is present in the sample.
Absolute recovery = Response of an analyte spike into matrix (processed)

-------- ------------------------------------------------------------ 100
Response of analyte of pure standard (unprocessed)
b) Sensitivity:
The method is said to be sensitive if small changes in concentration cause large
changes in response function. The sensitivity of an analytical method is determined from the
slope of the calibration line. The limits of quantification (LOQ) or working dynamic range of
bio analytical method are defined as the highest and lowest concentrations, which can
determined with acceptable accuracy. It is suggested that, this be set at 15% for both the
upper and lower limit of quantitation respectively. Any sample concentration that falls outside
the calibration range cannot be interpolated from the calibration line and extrapolation of the
calibration curve is discouraged. If the concentration is over range, the sample should be
diluted in drug-free matrix and re-assayed.
c) Precision:
The purpose of carrying out a determination is to obtain a valid estimate of a true
value. When one considers the criteria according to which an analytical procedure is selected,
precision and accuracy are usually the first time to come to mind. Precision and accuracy
together determine the error of an individual determination. They are among the most
important criteria for judging analytical procedures by their results.
Precision refers to the reproducibility of measurement within a set, that is, to the scatter of
dispersion of a set about its central value. The term set is defined as referring to a number
(n) of independent replicate measurements of some property. One of the most common
statistical terms employed is the standard deviation of a population of observation. Standard
deviation is the square root of the sum of squares of deviations of individual results for the
mean, divided by one less than the number of results in the set. The standard deviation S, is
given by
1 n
xi x
2
S= n 1 i1
Standard deviation has the same units as the property being measured.
The square of standard deviation is called variance (S 2). Relative standard deviation is
the standard deviation expressed as a fraction of the mean, i.e., S/x. It is sometimes multiplied

by 100 and expressed as a percent relative standard deviation. It becomes a more reliable
expression of precision.
% Relative standard deviation = S x 100 / x
d) Accuracy:
Accuracy normally refers to the difference between the mean x****, of the set of
results and the true or correct value for the quantity measured. According to IUPAC accuracy
relates to the difference between results (or mean) and the true value. For analytical methods,
there are two possible ways of determining the accuracy, absolute method and comparative
method.
Accuracy is best reported as percentage bias, which is calculated from the expression
(measured value - true value)
%Bias = X 100
true value
The accuracy of analytical method is then determined at each concentration by assessing the
agreement between the measured and nominal concentrations of the analytes in the spiked
drug free matrix sampler.
e) Limit of detection (LOD):

The limit of detection (LOD) of an analytical method may be defined as the
concentration, which gives rise to an instrument signal that is significantly different from the
blank. For spectroscopic techniques or other methods that rely upon a calibration curve for
quantitative measurements, the IUPAC approach employs the standard deviation of the
intercept (Sa), which may be related to LOD and the slope of the calibration curve, b, by
LOD = 3 Sa/ b
f) Limit of quantitation (LOQ)
The LOQ is the concentration that can be quantitate reliably with a specified level of
accuracy and precision. The LOQ represent the concentration of analyte that would yield a
signal-to-noise ratio of 10.
LOQ = 10 Sa/ b
Where, Sa- the estimate is the standard deviation of the peak area ratio of analyte to IS (5
injections) of the drugs. b -is slope of the corresponding calibration curve.
g) Ruggedness
Method Ruggedness is defined as the reproducibility of results when the method is
performed under actual use conditions. This includes different analysts, laboratories,
columns, instruments, source of reagents, chemicals, solvents etc. Method ruggedness may
not be known when a method is first developed, but insight is obtained during subsequent use
of that method.
h) Robustness

The concept of robustness of an analytical procedure has been defined by the ICH as a
measure of its capacity to remain unaffected by small but deliberate variations in method
parameters. The robustness of a method is the ability to remain unaffected by
small changes in parameters such as pH of the mobile phase, temperature, %organic solvent
strength and buffer concentration etc. to determine the robustness of the method experimental
conditions were purposely altered and chromatographic characters were evaluated.
i) System suitability
System suitability experiments can be defined as tests to ensure that the method can generate
results of acceptable accuracy and precision. The requirements for system suitability are
usually developed after method development and validation have been completed. (or) The
USP (2000) defines parameters that can be used to determine system suitability prior to
analysis.
The criteria selected will be based on the actual performance of the method as
determined during its validation. For example, if sample retention times form part of the
system suitability criteria, their variation (SD) during validation can be determined system
suitability might then require that retention times fall within a 3 SD range during routine
performance of the method.

LITERATURE REVIEW40
Satish A. Patel and Jinalben V Patel*:
A simple and sensitive reversed phase High Performance Liquid Chromatographic method
has been developed and validated for the simultaneous analysis of the Cefiximetrihydrate
(CEF) and Linezolid (LIN) in tablet dosage form. The separation was carried out using
mobile phase consisting of buffer and methanol with pH 2.5 in the ratio of 70:30, v/v. The
column used was ACE 5 C18, (150 mm x 4.6 mm i.d., 5 m) with flow rate 1.2 ml/min using
PDA detection at 250 nm. The method was linear over a concentration range of 23.33 40
g/ml and 70 120 g/ml for CEF and LIN, respectively. The retention time of CEF and LIN
were found to be 3.30 min and 8.07 min, respectively. Results of analysis were validated
statistically and by recovery studies. The mean recovery was 101.9 0.82 and 102.6 1.15
for CEF and LIN, respectively. The limit of detection (LOD) and the limit of quantification
(LOQ) for CEF and LIN were found to be 0.78 and 2.37 g/ml and 2.42 and 7.34 g/ml,
respectively. The results of the study showed that the proposed RPHPLC method is simple,
rapid, precise and accurate which is useful for the routine determination of CEF and LIN in
its pharmaceutical dosage form.
Vijaya krishna.C.Aradhya, Ranjitha L.R*, Satishkumar Shetty.A,

Manzoor Ahmed,Anil Kumar.S.M, Kuppast I.J:
The objective of the current study was to develop a simple, accurate, precise and rapid RP-
HPLC method with subsequently validate as per ICH guidelines for the determination of
Azithromycin (AZI) and Cefpodoxime Proxetil (CEF) using mobile phase [A mixture of

acetonitrile and Phosphate buffer- pH-7.0 in the ratio of 65:35 was considered to be the
optimal composition] as the solvent. The proposed method involves the measurement of
Retention time at selected analytical wavelength. 270.0 nm was selected as the analytical
wavelength. The retention time of AZI and CEF was found to be 7.318 and 3.521
respectively. The linearity of the proposed method was investigated in the range of 10-50
g/ml (r = 0.9999) for AZI and 8-40 g/ml (r = 0.9998) for CEF respectively. The method
was statistically validated for its linearity, accuracy and precision. Both inter-day and intra-
day variation was found to be showing less % RSD (Relative Standard Deviation) value
indicating high grade of precision of the method
saccurate reversed phase high-performance liquid chromatographic (RP-HPLC) method has

been developed andsubsequently validated for the simultaneous determination of
azithromycin and levofloxacin in pharmaceutical dosage form. TheHPLC analysis was
performed on the C18 column (250 mm x 4.6 mm id, 5 m particle size) in isocratic mode, at
30C temperatureusing a water consisting of methanol: potassium dihydrogen phosphate
buffer (60:40, v/v) at a flow rate of 1 mL/min. The detectionwas carried out at 279.6 nm for
azithromycin and levofloxacin. The retention time for azithromycin and levofloxacin were
found tobe 5.08 min, and 2.90 min, respectively. The method was validated for precision,
recovery, robustness, specificity, and detection andquantification limits, in accordance with
ICH Q2R1 guidelines. Linearity was observed in the concentration range from 500-
1500g/mL (r2=0.99) for azithromycin and levofloxacin (r2=0.99), the limit of detection and
quantification of AZT were 2.68 g/mL and8.94 g/mL, respectively. While for LFC it was
2.42 mg/mL and 8.08 mg/mL, respectively. The % recovery was found to be within 97-103%
for Azithromycin and 98-102% for Levofloxacin. The % RSD below 2.0 showed the high
precision of proposed method.

DRUG PROFILE
CEFIXIME8, 9
structure :
IUPAC : 6R,7R)-7-{[2-(2-Amino-1,3-thiazol-4-yl)-2-
(carboxymethoxyimino)acetyl]amino}-3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-
2-carboxylic acid
Molecular formula : C16H15N5O7S2
Molecular weight : 453.452 g/mol
Description : Cefixime, an antibiotic, is a third-generation cephalosporin like ceftriaxone and
cefotaxime. Cefixime is highly stable in the presence of beta-lactamase enzymes. As a result,
many organisms resistant to penicillins and some cephalosporins due to the presence of beta-
lactamases, may be susceptible to cefixime. The antibacterial effect of cefixime results from
inhibition of mucopeptide synthesis in the bacterial cell wall.
Categories :
Anti-Bacterial Agents
Cephalosporins
Antibacterials for Systemic Use

Antiinfectives for Systemic Use

Third-Generation Cephalosporins
Mechanism of action : ike all beta-lactam antibiotics, cefixime binds to specific penicillin-
binding proteins (PBPs) located inside the bacterial cell wall, causing the inhibition of the
third and last stage of bacterial cell wall synthesis. Cell lysis is then mediated by bacterial cell
wall autolytic enzymes such as autolysins; it is possible that cefixime interferes with an
autolysin inhibitor.
Pharmcodynamics : Cefixime, an antibiotic, is a third-generation cephalosporin like
ceftriaxone and cefotaxime. Cefixime is highly stable in the presence of beta-lactamase
enzymes. As a result, many organisms resistant to penicillins and some cephalosporins due to
the presence of beta-lactamases, may be susceptible to cefixime. The antibacterial effect of
cefixime results from inhibition of mucopeptide synthesis in the bacterial cell wall.
Solubility : It is soluble in water and methanol.
Boiling point : ~405.2 C at 760 mmHg (Predicted)
Melting point : 234-236 C
USES : t is primarily used in the treatment of urinary tract infections, although it may be used
against any susceptible aerobic bacterial species It may also be used to treat and prevent
Pneumocystis jiroveci pneumonia. It is generally not recommended for the treatment of
anaerobic infections such as Clostridium difficile colitis (the leading cause of antibiotic-
induced diarrhea).Resistance to trimethoprim is increasing, but it is still a first line antibiotic
in many countries.
AZITHROMYCIN
Structure :
IUPAC name : (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-

3,5,6,8,10,12,14-heptamethyl-15-oxo- 11-{[3,4,6-trideoxy-3-(dimethylamino)--D-xylo-

hexopyranosyl]oxy}-1-oxa-6-azacyclopentadec-13-yl 2,6-dideoxy-3-C-methyl-3-O-methyl-
-L-ribo-hexopyranoside
Molecular formula : C38H72N2O12
Molecular weight : 748.984 gmol1
Categories :
Anti-Bacterial Agents
Macrolides
Antibiotics
Ophthalmologicals
Sensory Organs
Antibacterials for Systemic Use
Antiinfectives for Systemic Use
Antiinfectives
Macrolides, Lincosamides and Streptogramins
Cytochrome P-450 CYP1A2 Inhibitors
Cytochrome P-450 CYP1A2 Inducers
CYP2A6 Inhibitors
CYP2A6 Inhibitors (strong)
CYP2A6 Inhibitors (moderate)
CYP2A6 Inducers
CYP2A6 Inducers (strong)
CYP3A4 Inhibitors
Combined Inhibitors of CYP3A4 and P-glycoprotein
Description : Azithromycin is a semi-synthetic macrolide antibiotic of the azalide class.

Like other macrolide antibiotics, azithromycin inhibits bacterial protein synthesis by binding
to the 50S ribosomal subunit of the bacterial 70S ribosome. Binding inhibits peptidyl
transferase activity and interferes with amino acid translocation during the process of
translation. Its effects may be bacteriostatic or bactericidal depending of the organism and the
drug concentration. Its long half life, which enables once daily dosing and shorter
administration durations, is a property distinct from other macrolides.
Mechanism of action : Azithromycin binds to the 50S subunit of the 70S bacterial
ribosomes, and therefore inhibits RNA-dependent protein synthesis in bacterial cells.

Pharmcodynamics: Azithromycin, a semisynthetic antibiotic belonging to the macrolide

subgroup of azalides, is used to treat STDs due to chlamydia and gonorrhea, community-
acquired pneumonia, pelvic inflammatory disease, pediatric otitis media and pharyngitis, and
Mycobacterium avium complex (MAC) in patients with advanced HIV disease. Similar in
structure to erythromycin. azithromycin reaches higher intracellular concentrations than
erythromycin, increasing its efficacy and duration of action.
Solubility : Soluble in DMSO (100 mg/ml at 25 C), ethanol (100 mg/ml at 25 C), water
(<1 mg/ml at 25 C), DMF (16 mg/ml), and ethanol:PBS(1:1 pH 7.2) (~0.5 mg/ml).
Melting point : ~114 C
Boiling point : ~822.1 C at 760 mmHg (Predicted)
Uses : Prevention and treatment of acute bacterial exacerbations of chronic obstructive
pulmonary disease due to H. influenzae, M. catarrhalis, or S. pneumoniae. The benefits of
long-term prophylaxis must be weighed on a patient-by-patient basis against the risk of
cardiovascular and other adverse effects. Acute bacterial sinusitis due to H. influenzae, M.
catarrhalis, or S. pneumoniae. Other agents, such as amoxicillin/clavulanate are generally
preferred, however.. Community-acquired pneumonia' due to C. pneumoniae, H. influenzae,
M. pneumoniae, or S. pneumonia. Acute otitis media caused by H. influenzae, M. catarrhalis
oS. pneumoniae. Azithromycin is not, however, a first-line agent for this condition.
Amoxicillin or another beta lactam antibiotic is generally preferred. Pharyngitis or tonsillitis
caused by S. pyogenes as an alternative to first-line therapy in individuals who cannot use

first-line therapy .Uncomplicated skin and skin structure infections due to S. aureus, S.
pyogenes, or S. agalactiaeUrethritis and cervicitis due to C. trachomatis or N.
gonorrhoeaeGenital ulcer disease (chancroid) in men due to H. ducreyiIn combination with
ceftriaxone, azithromycin is part of the United States Centers for Disease Control-
recommended regimen for the treatment of gonorrhea. Azithromycin is active as
monotherapy in most cases, but the combination with ceftriaxone is recommended based on
the relatively low barrier to resistance development in gonococci.
AIM AND SCOPE OF PRESENT WORK

1. To Estimate Cefixime and Azithromycin simultaneously in tablet dosage forms by RP-
HPLC method.
2. To validate the method according to ICH guidelines.
PLAN OF WORK
The experimental work has been planned as follows:
Review of the literature for Cefixime and Azithromycinregarding their physical and chemical
properties, various analytical methods that were conducted for Cefixime and
Azithromycinforms the basis for development of new analytical RP-HPLC method
Cefiximeand Ketorolaccombination.
DEVELOPMENT OF THE METHOD BY RP- HPLC

1. Selection of the solvent to be used as diluents and mobile phase:
Choosing the suitable solvent in which the drug is soluble and stable.
They must be easily available, economical and of the HPLC grade
2. Selection of Mobile phase:

For the mobile phase, the first variable to be decided is whether an organic or aqueous eluent
should be used. With the RP-HPLC analysis, either an aqueous eluent or a very polar organic
solvent such as methanol or acetonitrile should be fixed. If the K values are too large with an
aqueous solvent, organic solvent should be tried. If the K value are too low with organic
solvent the separation should be attempted using a mixture of two solvents with various
properties.
K-capacity factor is a measurement of the degree where the peak of the

interest is located with respect to void volume, i.e. Elution time of non-
retained components. Generally the value of K is > 2.
If a buffer is used, the pH as well as ionic strength of the buffer can be tried
3. In order to select the wavelength to carry out the analysis, critical examination of the
Ultraviolet absorbance spectra of the drug should be done.
4. A perfect study of the structure of drug and its physicochemical properties; to select the
chromatographic parameters.
5. Selection of method for quantitative chromatographic analysis. Determination of working
concentration range.
6. Validation of the developed method by following ICH guidelines.
MATERIALS AND METHODS

Instruments:
HPLC Waters Model NO.2690/5 series Compact System Consisting of
Inertsil-C18 ODS column.
Electronic balance (SARTORIOUS)
Sonicator( FAST CLEAN)
Chemicals:
Methanol HPLC Grade.
Buffer(KH2PO4)Hplc Grade.
Raw Material:
Cefixime and AzithromycinWorking Standards.
5.1 METHOD DEVELOPMENT FOR HPLC:
The objective of this experiment was to optimize the assay method for simultaneous
estimation of Cefixime and Azithromycinon the literature survey made. So here the trials
mentioned describes how the optimization was done.
Trial: 1
Mobile Phase: Degassed Acetonitrile: Methanal 65:45.
Preparation of Standard Solution:

Weigh down 10mgs of Cefiximeand Azithromycin drugs and dissolved in 10ml of

Mobile phase taken in two 10ml of volumetric flasks seperately and sonicated for 20 minutes
to get 1000ppms and 1 ml was taken from each solution into a 10ml volumetric flask and
diluted to 10 ml with mobile phase.
Chromatographic Conditions:
Flow rate : 1.0ml/min
Column : Inertsil - C18, ODS column
Detector wavelength :275nm
Column temp : Ambient
Injection volume : 20l
Run time : 10min
Retention time : 3.045min for Cefiximeand 4.933 for Azithromycin.
Observation: Two peaks are merged and not separated completely. The trial 1 chromatogram
result was shown in Fig:1.
Trail: 2
Mobile Phase: Degassed Acetonitrile and Water in the ratio of 90:10 V/V.

Weigh down 10mgs of Cefiximeand Azithromycin drugs and dissolved in 10ml of Mobile
phase taken in two 10ml of volumetric flasks seperately and sonicated for 20 minutes to get
1000ppms and 1 ml was taken from each solution and diluted to 10 ml with mobile phase.
Flow rate : 1ml/min
Column : Inertsil -C18, BDS co
lumn
Detector wavelength :275nm
Run time : 10min
Retention time : 2.793 min for Cefiximeand 3.188 min for
Azithromycin.
Observation: The two peaks are separated completely but peak shapes are not good. The
trial 2 chromatogram result was shown in Fig:2.
Trail: 3
Mobile Phase: Degassed Acetonitrile and Methanol in the ratio of 75:25 V/V.

Weigh down 10mgs of Cefiximeand Azithromycin drugs and dissolved in 10ml of

Mobile phase taken in two 10ml of volumetric flasks separately and sonicated for 20 minutes
to get 1000ppms and 1 ml was taken from each solution and diluted to 10 ml with mobile
phase.
Flow rate : 1.0ml/min
Column : Inertsil - C18, BDS column
Detector wavelength : 275nm
Run time : 10min
Retention time : 2.844 min for Cefiximeand 3.538 min for
Azithromycin.
Observation: peaks are seperated completely,But get extra peak.. The trial 3chromatogram
result was shown in Fig:3
5.2 OPTIMIZED METHOD
Mobile Phase: Degassed Methanol and Potassium Dihydrogen Phosphate Buffer in the ratio
of 85:15 V/V.
Preparation of stock solution:
Reference solution: The solution was prepared by dissolving 20.0 mg of accurately weighed
Cefixime and 25.0 mg Azithromycin in Mobile phase, in two 100.0 mL volumetric flasks
separately and sonicate for 20min. From the above solutions take 10.0 mL from each solution
into a 50.0 mL volumetric flask and then makeup with mobile phase and sonicate for 10min.
Preparation of working standard solution:
The stock solutions equivalent to 20ppm to 80ppm with respect to both drugs were prepared
in combination of Cefixime and Azithromycin above, sonicated and filtered through 0.45
membrane.
Optimized chromatographic conditions:
Parameters Method
Stationary phase (column)

Inertsil -ODS C18(250 x 4.6 mm, 5 )

Mobile Phase Methanol : Buffer (85:15)
Flow rate (ml/min) 1.0 ml/min
Run time (minutes) 10 min
Column temperature (C) Ambient
Volume of injection loop (l) 20
Detection wavelength (nm) 275nm
Drug RT (min) 2.186min for Cefiximeand 3.968 for

Azithromycin.
METHOD VALIDATION
5.3.1SYSTEM SUITABILITY:
A Standard solution was prepared by using Cefiximeand Azithromycin working
standards as per test method and was injected Five times into the HPLC system.
The system suitability parameters were evaluated from standard chromatograms by
calculating the % RSD from five replicate injections for Cefixime and Azithromycin ,
retention times and peak areas.
ACCEPTANCE CRITERIA:
1. The % RSD for the retention times of principal peak from 5 replicate injections of each
Standard solution should be not more than 2.0 %
2. The % RSD for the peak area responses of principal peak from 5 replicate injections of
each standard Solution should be not more than 2.0%.
3. The number of theoretical plates (N) for the Cefiximeand Azithromycin peaks is NLT 3000.
4. The Tailing factor (T) for the Cefiximeand Azithromycin
peaks is NMT 2.0
OBSERVATION:
The %RSD for retention times and peak areas were found to be within the limit.
Refer table: 1 As shown in fig 6 10.

5.3.2 SPECIFICITY:
Cefiximeand Azithromycin :
Solutions of standard and sample were prepared as per the test method are injected into
chromatographic system.
ACCEPTENCE CRITERIA:
Chromatograms of standard and sample should be identical with near Retention time.
OBSERVATION:
The chromatograms of Standard and Sample were same identical with same retention
time. As shown in fig: 12 and fig: 13
5.3.3 PRECISION:
5.3.3.1Repeatability:
a System precision: Standard solution prepared as per test method and injected five
times.
b Method precision: Prepared six sample preparations individually using single as per
test method and injected each solution.
ACCEPTANCE CRITERIA: The % relative standard deviation of individual Cefiximeand
Azithromycin , from the six units should be not more than 2.0%.
The individual assays of Cefiximeand Azithromycin should be not less than 98% and
not more than 102.0%.
OBSERVATION:
Test results are showing that the test method is precise. Refer tables 2 and 3 for
system precision and for method precision.
5.3.3.2Intermediate precision (analyst to analyst variability):
A study was conducted by two analysts as per test method
ACCEPTENCE CRITERIA:
The individual assays of Cefiximeand Azithromycin should be not less than 98% and not
more than 102% and %RSD of assays should be NMT2.0% by both analysts.
OBSERVATION:
Individual %assays and %RSD of Assay are within limit and passes the intermediate
precision, Refer table: 4
5.3.4 ACCURACY (RECOVERY):
A study of Accuracy was conducted. Drug Assay was performed in triplicate as per
test method with equivalent amount of Cefiximeand Azithromycin into each volumetric flask
for each spike level to get the concentration of Cefiximeand Azithromycin equivalent to
50%, 100%, and 150% of the labeled amount as per the test method. The average % recovery
of Cefiximeand Azithromycin were calculated.
The mean % recovery of the Cefiximeand Azithromycin at each spike level should be
not less than 98.0% and not more than 102.0% for both the drugs separately.

OBSERVATION:
Amount found
%Recovery = ---------------------- 100
Amount added
The recovery results indicating that the test method has an acceptable level of
accuracy. Refer table: 5
5.3.5LINEARITY OF TEST METHOD:

A Series of solutions are prepared using Cefiximeand Azithromycin working standards at
concentration levels from 20ppm to 80 ppm of target concentration .Measure the peak area
response of solution at Level 1 and Level 6 six times and Level 2 to Level 5 two times.
Correlation Coefficient should be not less than 0.9990.
% of y- Intercept should be 2.0.
% of RSD for level 1 and Level 6 should be not more than 2.0%.
OBSERVATION:
The linear fit of the system was illustrated graphically. The results are presented in table6.
5.3.6 RUGGEDNESS OF TEST METHOD:

a) System to system variability:
System to system variability study was conducted on different HPLC systems, under
similar conditions at different times. Six samples were prepared and each was analyzed as per
test method.
Comparison of both the results obtained on two different HPLC systems, shows that the
assay test method are rugged for System to system variability.
The % relative standard deviation of Cefiximeand Azithromycin from the six sample
preparations should be not more than 2.0%
The % assay of Cefiximeand Azithromycin should be between 98.0%-102.0%.
OBSERVATION:
The % RSD was found within the limit. Ref tables: 3 &7.
b) column to column variability:
Column to column variability study was conducted by using different columns. Six
samples were prepared and each was analysed as per test method

The %RSD of Cefiximeand Azithromycin tablets should be NMT2.0%. The %assay of
Cefiximeand Azithromycin should be between 98.0% and 102.0% for individual drugs.
OBSERVATION:
The results obtained by comparing with both two types were within limit. Refer tables: 3
&9
5.3.7 ROBUSTNESS:
a) Effect of variation of flow rate:
A study was conducted to determine the effect of variation in flow rate. Standard
solution prepared as per the test method was injected into the HPLC system using flow rates,
1.0ml/min and 1.2ml/min. The system suitability parameters were evaluated and found to be
within the limits for 1.0ml/min and 1.2ml/min flow.
Cefiximeand Azithromycin and was resolved from all other peaks and the retention times
were comparable with those obtained for mobile phase having flow rates 1.0ml/min.
The Tailing Factor of Cefiximeand Azithromycin standards should be NMT 2.0 for
Variation in Flow.
OBSERVATION:
The tailing factor for Cefiximeand Azithromycin was found to be within the limits. As
shown in table 10.
b) Effect of variation of temperature:

A study was conducted to determine the effect of variation in temperature. Standard
solution prepared as per the test method was injected into the HPLC system at 20C
temperature. The system suitability parameters were evaluated and found to be within the
limits for a temperature change of 20c.
Similarly sample solution was chromatographed at 25C temperature. Cefiximeand
Azithromycin were resolved from all other peaks and the retention times were comparable
with those
The Tailing Factor of Cefiximeand Azithromycin standard and sample solutions should
be NMT 2.0 for Variation in temperature.
OBSERVATION:
The tailing factor for Cefiximeand Azithromycin
x is found to be within the limits. As shown in table 11.

5.3.8 LIMIT OF DETECTION AND QUANTITATION (LOD and LOQ):

From the linearity data calculate the limit of detection and quantitation, using the following
formula.
LOD= 3.3
S
= standard deviation of the response
S = slope of the calibration curve of the analyte.
LOQ = 10
S
= standard deviation of the response
S = slope of the calibration curve of the analyte.
RESULTS AND DISCUSSION
6.1 Method development:
Fig1: Chromatogram of Trial 1
0.14
3.045
0.12
0.10
0.08
AU
4.933
0.06
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
Minutes
Inference :
Two peaks are seperated, second peak is splited.

S.NO Name of the peak Retention

time(min)
1 Cefixime 3.045
2 Azithromycin 4.933
Fig 2: Chromatogram of Trial 2:
0.025
2.79 3
0.020
3.18 8
0.015
AU
0.010
0.005
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

Minutes
Inference: Peaks shapes are not good.
time(min)
1 Cefixime 2.793
Fig 3: Chromatogram of Trial3

0.040
3.538
0.035
0.030
2.844
0.025
AU
0.020
0.015
0.010
0.005
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

Minutes
Inference : peaks are seperated but peak shape is not good.

time(min)
1 Cefixime 2.844
6.2 OPTIMIZED METHOD
Fig 4: Chromatogram of standard

0.080
Cefixime - 2.186
0.070
0.060
Azithromycin - 3.963
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: Got chromatogram at RTs of 2.186min to Cefiximeand 3.963min to
Azithromycin

time(min)
1 Cefixime 2.186
Fig5:Chromatogram of sample
0.080
Cefixime - 2.183
0.070
0.060
0.050
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: Got same chromatogram with same RT values as of standard.

time(min)
1 Cefixime 2.183

2 3.960
Azithromycin
6.3 VALIDATION DATA
6.3.1 SYSTEM SUITABILITY:
TABLE- 1(a): Data of System Suitability for Cefixime
Injection RT Peak Area USP Plate count USP Tailing
1 2.183 2975976 21895.54212 1.16974
2 2.178 2978251 21654.25418 1.18741
3 2.179 2976324 21784.89743 1.13279
4 2.179 2973269 21587.75412 1.12547
5 2.177 2970542 21876.21874 1.15476
Mean 2.1792 2974872 21759.7333 1.154034
SD 0.00228 3002.635 ------- -------
% RSD 0.104642 0.100933 ------- -------
TABLE-1(b): Data of System Suitability for Azithromycin
Injection RT Peak Area USP Plate count USP Tailing
1 3.960 429156 8457.254 1.24875
2 3.955 429821 8408.784 1.23654

3 3.957 429058 8427.547 1.29875
4 3.956 429519 8479.874 1.26321
5 3.954 429458 8476.874 1.29876
Mean 3.9564 429402.4 8450.0666 1.269202
SD 0.002302 304.6462 ------- -------
% RSD 0.058189 0.070947 ------- -------
Fig: 6-10 Chromatograms of system suitability (standards 1-5)

0.080
Cefixime - 2.183
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: System suitability Chromatogram for standard 1
0.080
Cefixime - 2.178
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

0.080
Cefixime - 2.179
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: System suitability Chromatogram for standard - 3
0.080
Cefixime - 2.179
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

Inference: System suitability Chromatogram for standard - 4
0.080
Cefixime - 2.177
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
6.3.2: SPECIFICITY:
Fig 11: Chromatogram of Blank
0.0015
0.0010
0.0005
0.0000
-0.0005
AU
-0.0010
-0.0015
-0.0020
-0.0025
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

Fig 12: Chromatogram of std
0.080
Cefixime - 2.177
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: Got a peak for sample at an Rt of 2.177min for Cefiximeand 3.954min for
Azithromycin
6.3.2: PRECISION:
6.3.2.1Repeatability:
(a) System precision:TABLE-2(i):
Data of Repeatability (System precision) for Cefixime
Peak Areas of
Injection
Cefixime %Assay
Concentration
1 2978965 100.51
40ppm
2 2970867 100.24
3 2973742 100.34
4 2978761 100.51

5 2978642 100.50
Mean 2976195 100.42

Statistical
SD 3696.277 0.123895
Analysis
% RSD 0.124195 0.123377
TABLE-2(ii): Data of Repeatability (System precision) for Azithromycin
Peak Areas of
Injection
Azithromycin %Assay
1 429853 100.34
2 429741 100.31
Concentration
40ppm
3 429784 100.32
4 429403 100.23
5 429746 100.31
Mean 429705.4 100.302

Statistical
SD 174.8751 0.04207
Analysis
% RSD 0.040697 0.041945

Fig13-14 Chromatograms of system precision
0.080
Cefixime - 2.186
0.070
0.060
0.050 Azithromycin - 3.963
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: Chromatogram for system precision (standard - 1)

C e fix im e - 2 .1 8 3
0.080
0.070
A z ith ro m y c in - 3 .9 6 0
0.060
0.050
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

0.080
Cefix im e - 2.183
0.070
0.060
A z ithrom y c in - 3.960
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.178
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

0.080
Cefixime - 2.179
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
(b)Method precision:
TABLE-3(i): Data of Repeatability (Method precision) for Cefixime
Peak Areas of
Injection
Cefixime %Assay
1 2970873 100.24
Concentration 2 2978461 100.50
40ppm 3 2978462 100.50
4 2978462 100.50
5 2976542 100.43
6 2978642 100.50

Mean 2976907 100.445

Statistical 3059.528 0.104259
Analysis SD
% RSD 0.0102775 0.103797
TABLE-3(ii): Data of Repeatability (Method precision) for Azithromycin
Peak Areas of
Injection %Assay
Azithromycin
1 429827 100.33
Concentration 2 429391 100.23

40ppm
3 429085 100.56
4 429786 100.32
5 429375 100.23
6 429274 100.20
Mean 429456.3 100.3117

Statistical
SD 292.606 0.132577
Analysis
% RSD 0.068134 0.132165
Fig 15-17: Chromatograms of Repeatability
Inference: Chromatogram for Repeatability (standard - 1)

Cefix im e - 2.179
0.080
0.070
0.060
A z ithrom y c in - 3.957
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.179
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

0.080
Cefixime - 2.176
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

0.080
Cefixime - 2.177
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.175
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
6.3.2.2 Intermediate precision:
For Analyst 1 ref: Table3.
Table4:
(i) Data of Intermediate precision (Analyst 2) forCefixime

Peak Areas of
Injection
Cefixime %Assay
1 2970478 100.23
Concentrati
2 2978492 100.50
on
3 2970874 100.24
40ppm
4 2977892 100.48
5 2970845 100.24
6 2978632 100.50
Mean 2974536 100.365

Statistical 4175.907 0.140819
Analysis SD
% RSD 0.140389 0.140307
(ii)Data of Intermediate precision (Analyst 2) for Azithromycin
Peak Areas of
Injection
Azithromycin %Assay
1 429874 100.34
Concentrati
2 429654 100.29
on
3 429658 100.29
40ppm
4 429631 100.29
5 429874 100.34
6 429631 100.34
Mean 429720.3 100.315

Statistical 119.5603 0.027386
Analysis SD
% RSD 0.027823 0.0273

Fig18-20: Chromatograms of Intermediate precision
0.080
Cefixime - 2.174
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: Chromatogram for Intermediate Precision

0.080
Cefixime - 2.173
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: Chromatogram for Intermediate Precision

Cefixime - 2.173
0.080
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
6.3.4 ACCURACY (RECOVERY
TABLE-5:

(i)Data of Accuracy forCefixime
Concentration Amount Amount

Area % Statistical Analysis
% of spiked added found
Recovery of % Recovery
level (ppm) (ppm)
50% 1486721
20 19.89 99.47 MEAN 99.5433
Injection 1
50%
20 1489764 19.94 99.68
Injection 2
50%
20 1486795 19.90 99.48 %RSD 0.119006
Injection 3
100 % 100.463
40 2978864 40.20 100.51 MEAN
Injection 1 3
100 %
40 2976794 40.18 100.44
Injection 2
100% 0.04022
40 2976874 40.18 100.44 %RSD
Injection 3 8
150%
60 4465274 60.44 100.73 MEAN 100.783
Injection 1
150%
60 4467892 60.47 100.79
Injection 2
150% 0.04994
60 4469874 60.50 100.83 %RSD
Injection 3 1
(ii) Data of Accuracy for Azithromycin
Concentratio
Amount Amount
n Area % Statistical Analysis
added found
% of spiked Recovery of % Recovery
(ppm) (ppm)
level
50% 214836
20 19.92 99.62 MEAN 99.593
Injection 1

50% 214975
20 19.94 99.68
Injection 2
50% 214558 0.10305
20 19.90 99.48 %RSD
Injection 3 1
100 % 429754
40 40.13 98.92 MEAN 99.66
Injection 1
100 % 429634
40 40.11 99.75
Injection 2
100% 429754 0.70174
40 40.13 100.31 %RSD
Injection 3 3
150% 644876 100.346
60 60.35 99.96 MEAN
Injection 1 7
150% 644968
60 60.36 100.59
Injection 2
150% 644308 0.00734
60 60.29 100.49 %RSD
Injection 3 4
Fig21 -22Chromatograms for accuracy (50%)

0.040
Cefixime - 2.191
0.035
0.030
0.025
AU
0.020
0.015
0.010
0.005
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: Chromatogram for standard 1

0.040
Cefixime - 2.190
0.035
0.030
0.025
AU
0.020
0.015
0.010
0.005
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Fig 23-24: Chromatograms for accuracy (100%)

Cefixime - 2.173
0.080
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

Cefixime - 2.173
0.080
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Fig 25-26: chromatograms For Accuracy (150%)
0.12
Cefixime - 2.171
0.10
0.08
0.06
AU
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

0.12
Cefixime - 2.170
0.10
0.08
AU
0.06
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
6.3.5 LINEARITY:
TABLE6:
(i) Data of Linearity (Cefixime)
Concentrati Average Statistical Analysis

on (ppm) Area
0 0 Slope 73469
20 1488271 y-Intercept 25088
30 2232407 Correlation Coefficient 0.999
40 2976541
50 3720677
60 4464812
70 5208948
80 5823083
Fig: 41(a) Linearity Plot (Concentration Vs Response) ofCefixime

7000000
6000000
f(x) = 73468.67x + 25087.91
5000000 R = 1
4000000
3000000 Linear ()
2000000
1000000
0
0 10 20 30 40 50 60 70 80 90
(2)Data of Linearity (Azithromycin)
Concentrati Average Statistical Analysis

on (ppm) Area
0 0 Slope 10638
20 214938 y-Intercept 2894
30 322407 Correlation Coefficient 0.999
40 429875
50 537344
60 644813
70 752282
80 844751
Fig: 41(b) Linearity Plot (Concentration Vs Response) of Azithromycin

900000
800000 f(x) = 10637.86x + 2894.82

R = 1
700000
600000
500000
400000 Linear ()
300000
200000
100000
0
0 10 20 30 40 50 60 70 80 90

Fig:27-28Chromatograms for 20 ppm:
0.040
Cefixime - 2.199
0.035
0.030
0.025
AU
0.020
0.015
0.010
0.005
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: Chromatogram for 20 ppm standard 1
Fig: 29-30 chromatograms for30ppm, 40 ppm

0.060
Cefixime - 2.189
0.050
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

0.080
Cefixime - 2.186
0.070
0.060
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Fig31:32 Chromatograms for 50 ppm, 60 ppm

Cefixime - 2.171
0.10
0.08
0.06
AU
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.12
Cefixime - 2.171
0.10
0.06
AU
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Fig31:32 Chromatograms for 70 ppm, 80 ppm

0.12
Cefixime - 2.170
0.10
0.08
AU
0.06
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.16
Cefixime - 2.170
0.14
0.12
0.10
0.08
AU
0.06
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

6.3.6 Ruggedness:
a) System to System variability:
For system 1 Refer: Table3
TABLE: 7
(i)Data of system to system variability (Cefixime)
System-2
Assay % of
S.NO: Peak area Cefixime
1
2975974 100.41
2 2978542 100.50
3 2978792 100.51
4 2970875 100.23
5 2978453 100.50
6 2971542 100.26
Mean 2975696.33 100.4017
%RSD 0.121967 0.126522
(ii) Data of system to system variability (Azithromycin)

System-2
Assay % of
S.NO: Peak area Azithromycin
1 429871 100.34
2 429637 100.29
3 429654 100.34
4 429875 100.34
5 429875 100.34
6 428754 99.08
Mean 429611. 100.121
%RSD 0.101139 0.5100815
Chromatograms of system to system variability
Inference: Chromatogram of system to system variability std- 1
0.16
Cefixime - 2.170
0.14
0.12
0.10
0.08
AU
0.06
0.04
0.02
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

0.080
Cefixime - 2.183
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
0.080
Cefixime - 2.178
0.070
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

0.080
Cefixime - 2.179
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

0.080
Cefixime - 2.179
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
6.3.7 Robustness:
TABLE: 10(i) Data for Effect of variation in flow rate (Cefixime):
Flow Std Tailing Flow Std Tailing Flow Std Tailing

0.8 ml Area factor 1.0 ml Area factor 1.2 ml Area factor
2968201 1.09937 297843 1.12845 2984051 1.12187

2 1 1 5
2958708 1.10358 297684 1.11225 2986371 1.12225

7 8 7 4
2968754 1.111587 297846 1.12128 2983078 1.12435

2 7 7

2965882 1.11786 297089 1.12475 2987628 1.12389

1 4 2 5
2962082 1.11954 297845 1.12387 2986071 1.09915

7 2 4 7
Avg 2964725 1.11039 Avg 297661 1.12212 Avg 2985440 1.11830

1 7 4 8
SD 4267.42 0.00878 SD 3273.68 0.00608 SD 1841.23 0.01075

9 6 4 8 7
%RS 0.14394 0.79125 %RS 0.10998 0.54218 %RS 0.06167 0.96190

D 5 D 2 D 4 8
TABLE: 10(ii) Data for Effect of variation in flow rate (Azithromycin)
Flow Std Tailing Flow Std Tailing Flow Std Tailing

0.8 ml Area factor 1.0 ml Area factor 1.2 ml Area factor
428631 1.23891 429860 1.25165 430584 1.26227

5 8 6
428894 1.23063 429631 1.24543 430963 1.25158

7 5 1
428634 1.24085 429874 1.26246 430217 1.23787

8 4 5
428761 1.23899 429364 1.23701 430492 1.23982

5 8 4
428637 1.24107 429874 1.23901 430674 1.23825

3 0 7

Avg 429711. 1.23809 Avg 429720. 1.24711 Avg 430586 1.24596

4 6 6 7 3
SD 115.966 0.00429 SD 225.548 0.01032 SD 271.511 0.01072

8 8 5 6
%RS 0.02705 0.34647 %RS 0.05225 0.82817 %RS 0.06305 0.86083

D 7 D 4 2 D 6 5
Fig42-44Chromatograms of robustness
a) Effect of variation of flow rate (for 0.8 ml/min flow)
0.080
0.070
Cefixime - 2.397
0.060
0.050
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
b) Inference: Chromatogram for robustness standard - 1

0.080
Cefixime - 2.397
0.070
0.060
0.050
0.040
AU
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: Chromatogram for robustness standard - 2
Fig45-46: chromatograms for 1ml/min
0.080
Cefixime - 2.179
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Inference: Chromatogram for robustness standard 1

0.080
Cefixime - 2.177
0.070
0.060
0.050
AU
0.040
0.030
0.020
0.010
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Fig47-49: Chromatograms for 1.2ml/min

Cefixime - 1.980
0.20
0.15
AU
0.10
0.05
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes

Cefixime - 1.982
0.20
0.15
AU
0.10
0.05
0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
6.3.8LIMIT OF DETECTION AND LIMIT OF QUANTITATION (LOD and LOQ):
Cefixime:
From the linearity plot the LOD and LOQ are calculated:
LOD = 3.3
S
3.32124.413
= ---------------------- = 0.34
20193
LOQ = 10
S

102124.413
= -------------------- = 1.05 20193
Azithromycin;
LOD = 3.3
S
3.32431.578
= ---------------------- = 0.25
31282
LOQ = 10
S
102431.578
= ---------------------- = 0.77
31282

Summary and conclusion
The analytical method was developed by studying different parameters. First of all, maximum
absorbance was found to be at 237nm Cefixime for and 275nm for Azithromycin.Common
wavelength will be 275nm and the peaks purity was excellent. Injection volume was selected
to be 20l which gave a good peak area. The column used for study was Inertsil C 18, ODS
chosen good peak shape. Ambient temperature was found to be suitable for the nature of drug
solution. The flow rate was fixed at 1.0ml/min because of good peak area, satisfactory
retention time and good resolution. Different ratios of mobile phase were studied, mobile
phase with ratio of 55:45 Methanol : Buffer was fixed due to good symmetrical peaks and for
good resolution. So this mobile phase was used for the proposed study.
The present recovery was found to be 98.0-101.50 was linear and precise over the same
range. Both system and method precision was found to be accurate and well within range.
Detection limit was found to be 0.25 Cefixime and 0.34 for Azithromycin. Linearity study

was, correlation coefficient and curve fitting was found to be. The analytical method was
found linearity over the range of 20-80ppm of the target concentration for both the drugs. The
analytical passed both robustness and ruggedness tests. On both cases, relative standard
deviation was well satisfactory.
BIBILOGRAPHY
1. The Merck Index, 14th ed., Merck and Co. Inc., Whitehouse Station, NJ, 2006.
2. Suhagia BN, Shah SA, Rathod IS, Patle HM, Doshi KR, Determination of azithromycin in
pharmaceutical dosage forms by spectrophotometric method, Indian J Pharm Sci., 68, 2006,
242-245.
3. Nirogi R, Kandikere V, Shukla M, Mudigonda K, Maurya S, Boosi R, Yerramilli A,

Sensitive and selective liquid chromatographytandem mass spectrometry method for the
quantification of azithromycin in human plasma, Analytica Chimica Acta., 553, 2005, 1-8.
4. Khedr A, Sheha M, Quantitative thin-layer chromatographic method of analysis of

azithromycin in pure and capsule forms, J. Chromatogr. Sci., 41, 2003, 10-16.
5. R. Yanamandra, A. Chaudhary, S. Bandaru, Patro B, MURTHY YLN, RAMAIAH P,

SASTRY CSP, UPLC Method for simultaneous separation and estimation of secnidazole,
fluconazole and azithromycin in pharmaceutical dosage forms, E-journal of chemistry, 7,
2010, S363-S371.
6. Devia ML, Chandrasekharb KB, A validated stability-indicating RP-HPLC method for

levofloxacin in the presence of degradation products, its process related impurities and
identification of oxidative degradant, J. Pharmaceut. Biomed., 50, 2009, 710-717.

7. Kumar TM, Gurrala S, Rao VJ, Sambasiva Rao KRS, Development and validation of
HPLC-UV method for the estimation of levofloxacin in human plasma, International journal
of pharmacy and pharmaceutical sciences, 3, 2011, 247-250.
8. Patel P, Shah H, Patel K, Patel M, Development and validation of spectrophotometric

methods for simultaneous estimation of cefixime trihydrate and levofloxacin hemihydrate in
their combined tablet dosage forms, International journal of pharmaceutical research and bio
science, 1, 2012, 502-515
.
9. Kavar RC, Savaliya BM, Lakkad AJ, Soriya SV, Kapuriya KG, Faldu SD, Q-absorbance
ratio spectrophotometric method for the simultaneous estimation of cefpodoxime proxetil and
levofloxacin hemihydrate in their combined dosage forms, International bulletin of drug
research, 2, 2013, 22-30.
10. Dhanhukiya VR, Tiwari PS, Godavariya VD, Development and validation of RP-HPLC
method for simultaneous estimation of levofloxacin hemihydrates and cefixime trihydrate in
marketed formulation, International research journal of pharmacy, 4, 2013, 81-83.
11. El-Shanawany AA, El-Adl SM, Abdel-Aziz LM, Hashem HA, Sebaiy MM, Rapid RP-
HPLC Method for simultaneous estimation of levofloxacin hydrochloride, lomefloxacin
hydrochloride, gatifloxacin and sparfloxacin, Asian. J. Research. chem., 4, 2011, 1688-1694.
12. Chepurwar SB, Shirkhedkar AA, Bari SB, Fursule RA, Surana SJ, Validated HPTLC
method for simultaneous estimation of levofloxacin hemihydrate and ornidazole in
pharmaceutical dosage form, J. Chromatogr. Sci., 45, 2007, 531-536.
13. Raja MS, Shan SH, Perumala P, Moorthy MTS, RP-HPLC method development and
validation for the simultaneous estimation of azithromycin and ambroxol hydrochloride in
tablets, International journal of pharmtech research., 2, 2007, 36-39.
14. Agrawal OD, Shirkhedkar AA, Surana SJ, Simultaneous determination of levofloxacin
hemihydrates and ambroxol hydrochloride in tablets by thin-layer chromatography combined
with densitometry, J. Anal. Chem., 65, 2010, 418- 422.
15. ICH Harmonized Tripartite Guidelines, Text on Validation of Analytical Procedures,

Q2A, 1996.
16. Methods for the Measurement of Fluid Flow in Pipes, Part 1. Orifice Plates, Nozzles
and Venturi Tubes. British Standards Institute.
17. [Was: "fluidengineering.co.nr/Manometer.htm". At 1/2010 that took me to bad link.

Types of fluid Manometers]
18. Beckwith, Thomas G.; Roy D. Marangoni & John H. Lienhard V (1993). "Measurement
of Low Pressures". Mechanical Measurements (Fifth ed.). Reading, MA: Addison-Wesley.
pp. 591595. ISBN 0-201-56947-7.

19. Boyes, Walt (2008). Instrumentation Reference Book (Fourth ed.). Butterworth-
Heinemann. p. 1312.
20 A. Chambers, Basic Vacuum Technology, pp. 100-102, CRC Press, 1998 ISBN
0585254915.
21. John F. O'Hanlon, A User's Guide to Vacuum Technology, pp. 92-94, John Wiley &
Sons, 2005 ISBN 0471467154.
22. Robert M. Besanon, ed. (1990). "Vacuum Techniques". The Encyclopedia of

Physics (3rd ed.). Van Nostrand Reinhold, New York. pp. 12781284. ISBN 0-442-00522-9.
23. Arayne MS, Sultana N, Qureshi F, Ahmed Siddiqui F, Zeeshan Mirza A, et al.
(2009) Simultaneous determination of tranexamic acid and losartan potassium in dosage
formulations and human serum by RP-LC. Chromatographia 70: 789-795.
24. Huertas-Prez JF, Heger M, Dekker H, Krabbe H, Lankelma J et al. (2007) Simple, rapid,
and sensitive liquid chromatography-fluorescence method for the quantification of
tranexamic acid in blood. J chromatogr A 1157: 142-150.
25. Ansari TM, Raza A, Rehman A (2005) Spectrophotometric determination of tranexamic

acid in pharmaceutical bulk and dosage forms. Anal Sci 21: 1133- 1135.
26. Shiha Y, Wub KL, Sueb JW, Senthil Kumar A, Zen JM (2008) Determination of
tranexamic acid in cosmetic products by high-performance liquid chromatography coupled
with barrel plating nickel electrode. J Pharm Biomed Anal 48: 1446-1450.
27. Alarfaj NA, Altamimi SA , Almarshady LZ (2009) Spectrophotometric determination of

mefenamic acid in pharmaceutical preparations. Asian J Chem 21: 217-226.
28. Dahivelkar PP, Mahajan VK, Bari SB, Shirkhedkar AA, Fursule RA, et al.
(2007) Simultaneous derivative and multicomponent spectrophotometric determination of
drotaverine hydrochloride and mefenamic acid in tablets. Indian J Pharm Sci 69: 812-814.
29. Derle DV, Bela M, Kasliwal N (2008) In vitro and in vivo evaluation of mefenamic acid
and its complexes with - cyclodextrin and HP- - cyclodextrin. Asian J Pharm 2: 30-34.
30. Santini AO, Pezza HR, Pezza L (2007) Development of a potentiometric mefenamate ion
sensor for the determination of mefenamic acid in pharmaceuticals and human blood
serum. Sensors and Actuators B: Chemical 128: 117-123.
31. Chang Q, Yin OQ, Chow MS (2004) Liquid chromatographytandem mass spectrometry
method for the determination of tranexamic acid in human plasma. J Chromatogr B Analyt
Technol Biomed Life Sci 805: 275- 280.

32. Delyle SG, Abi E, Batisse A, Tremey B, Fischer M, et al. (2010) A validated assay for the
quantitative analysis of tranexamic acid in human serum by liquid chromatography coupled
with electrospray ionisation mass spectrometry. Clinicia Chimica Acta 411: 438-443.
33. Aroud KAE, Abushoffa AM, Abdellatef HE (2007) Spectrophotometric and

spectrofluorimetric methods for the determination of tranexamic acid in pharmaceutical
formulations. Chem Pharm Bull 55: 364-367.
34. Dusci LJ, Hackett LP (1978) Gas liquid chromatographic determination of mefenamic
acid in human serum. J Chromatogr 161: 340-342.
35. Rouini MR, Asadipour A, Ardakani YH, Aghdasi F (2004) Liquid chromatography
method for the determination of mefenamic acid in human serum. J Chromatogr B Analyt
Technol Biomed Life Sci 800 : 172-189.
36. Liu L, Song J (2006) Voltametric determination of mefenamic acid at lanthanum

hydroxide nanowires modified carbon paste electrodes. Anal Biochem 354: 22- 27.
37. Shaikha KA, Patil SD, Devkhileb AB (2008) Development and validation of a reversed-
phase HPLC method for simultaneous estimation of ambroxol hydrochloride and
azithromycin in tablet dosage form. J Pharm Biomed Anal 48: 1481-1484.
38. Ratinavel G, Umanath U, Valarmathy J, Samualjoshua L, Selvin Thanuja C, et al.

(2009) RP-HPLC method for the simultaneous estimation of rosiglitazone and gliclazide in
tablets. Eurasian J Anal Chem 6: 1188-1192.
39. Kumudhavalli MV, Jayakar B, Margret R, Kumar M, Saravanan C (2010) Method

development and validation of RP-HPLC method for simultaneous estimation of ibuprofen
and methocarbamol. Int J Chem Anal Science 1: 28-30.
40. Topagi KS, Jeswani RM, Sinha PK, Damle MC (2010) A validated normal phase HPLC
method for simultaneous determination of drotavarine hydrochloride and omeprazole in
pharmaceutical formulation. AJPCR 3: 20-24.
41. Saeed Arayne M, Sultana N, Qureshi F, Siddiqui FA, Bahaldur SS, et al.
(2009) Simultaneous Determination of Tranexamic Acid and Losartan Potassium in Dosage
Formulations and Human Serum by RP-LC. Chromatographia 70: 789- 795.
42. Siddiqui MR, Tariq A, Chaudary M, Dinesh Reddy K, Negi PS, et al. (2009)
Simultaneous estimation of pantaprazole and domperidone by spectrophotometry. Am J Appli
Sci 10: 1781-1787.
43. Liu H, Wang H, Sunderland VB (2005) An isocratic ion exchange HPLC method for the
simultaneous determination of flucloxacillin and amoxicillin in a pharmaceutical formulation
for injection. J Pharm Biomed Anal 37: 395-398.
44. Dhoka MV, Gawande VT, Joshi PP (2010) Simultaneous estimation of cefixime and
Trihydrate erdostenine in pharmaceutical dosage form by using reverse phase-high
performance liquid chromatography. Int J Chem Tech Res 2: 79-87.

45. Choudhary B, Goyal A, Khokra SL, Kaushik D (2009) Simultaneous estimation of

diclofenac sodium and rabeprazole by high performance liquid chromatography method in
combined dosage forms. IJPSDR 1: 43-45.
46. Joshi SJ, Karbharia PA, Bhoira SI, Bindub KS, Dasc C (2010) RP-HPLC method for
simultaneous estimation of bisoprolol fumarate and hydrochlorothiazide in tablet
formulation. J Pharm Biomed Anal 52: 362-371.
47. Hadad GM, Gindy AE, Mahmoud WMM (2007) Optimization and validation of an
HPLC-UV method for determination of tranexamic acid in a dosage form and in human
urine. Chromatographia 66: 311-317.

Estimate and Validation Cefixime and Azithromycin Simultaneoslyy in Tablt Dosage Froms by RP-HPLC Method

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ESTIMATE AND VALIDATION CEFIXIME AND AZITHROMYCIN

SIMULTANEOSLYY IN TABLT DOSAGE FROMS BY RP-HPLC

Jawaharlal Nehru Technological University, Kakinada

In partialfulfilmentt of the requirement for the award of the degree of

INSTITUITIONAL GUIDE: INDUSTRIAL GUIDE:

Department of Pharmaceutical Analysis

This is to certify that the thesis entitiled . To Estimate Cefixime And

Signature of the Guide Principal

Dr. J.N. Suresh Kumar

DEPARTMENT OF PHARMACEUTICAL ANALYSIS AND QUALITY ASSURANCE, NIPS Page

NARASARAOPETA INSTITUTE OF PHARMACEUTICAL SCIENCES

DEPARTMENT OF PHARMACEUTICAL ANALYSIS AND QUALITY ASSURANCE, NIPS Page

DECLARATION BY THE CANDIDATE

I Reddybathula Leela of II Year M.Pharmacy bearing Regd. No

14CD1S0423 hereby declare that the research work entitled Estimate

Cefixime and Azithromycin Simultaneously In Tablet Dosage Forms By

RP-HPLC Method was carried out by me under the guidance of

Mr. P. Prasanth Kumar(Associate prof.) The work embodied in this thesis is

of this or other university.

DEPARTMENT OF PHARMACEUTICAL ANALYSIS AND QUALITY ASSURANCE, NIPS Page

I am thankful to Principal Mr.J.N.Suresh kumar, who have been a constant source of

Thank you everyone..!

S.NO CONTENTS P.NO

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7 AIM AND PLAN OF WORK 31

8 MATERIALS AND METHODS 32

9 RESULTS AND DISCUSSIONS 39

10 SUMMARY AND CONCLUSION 78

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BDS Base deactivated silane

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Spectrophotometer is generally preferred especially by small-scale industries as the cost of

The important applications are

Identification of many types of organic, inorganic molecules and ions.

2. HPLC METHOD DEVELOPMENT

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Normal Phase Chromatography

REVERSED PHASE CHROMATOGRAPHY

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ADSORPTION CHROMATOGRAPHY OR NORMAL PHASE

Table.2.1: Classification of Chromatographic Methods

Stationary phase Mobile phase Method

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High-Performance Liquid Chromatography (HPLC) is a special branch of column

Solvent container Pump damping unit Injection port Column

2.2 SYSTEM COMPONENTS:

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Liquid chromatographic detectors: The function of the detector in HPLC is to

1. Bulk property detectors:

These detectors are based on differential measurement of a property, which is

The most popular material is octadecyl-silica (ODS-Silica), which contains C 18

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DEPARTMENT OF PHARMACEUTICAL ANALYSIS AND QUALITY ASSURANCE, NIPS Page

BONDED PHASES FOR HPLC AND THEIR ABBREVIATIONS

C1 Reversed phase material. Unique selectivity for polar and

Propyl: Reversed phase material, used in hydrophobic interaction chromatography

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Hexyl: Reversed phase material, useful for ion-pairing chromatography. Less

MOS, RP-8, LC8, Octyl

ODS, RP-18, LC18, Octadecyl

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It is a reversed phase material and exhibits unique selectivity. It is useful for

CPS, PCN, Cyano,