Kar Thick

Chapter1 Introduction
1. INTRODUCTION
Analytical chemistry is the science to analyze morphologies compositions and
quantities targets. These analytical results have played critical roles from the
understanding of basic science to a variety of practical applications such a biomedical
applications, environmental monitoring, quality control of industrial
manufacturing, and forensic science.
Analytical chemistry studies and uses instruments and methods used to

separate, identify, and quantify matter. In practice, separation, identification or
quantification may constitute the entire analysis or be combined with another
method. Separation isolates analytes.Qualitative analysis identifies analytes, while
quantitative analysis determines the numerical amount or concentration.
Analytical chemistry is concerned with much broader & more general aspects
of analysis ,while chemical analysis is confined to much narrower & ore specific
aspects of analysis.
Traditionally analytical chemistry has been split into two main types
qualitative and quantitative.
Qualitative inorganic analysis seeks to establish in the presence of a given

element or an inorganic compound in a sample.
Quantitative analysis seeks to establish the amount of a given element or

compound in a sample.
Analytical Techniques:
Some of the medicinal products are still being assayed by the time. A wide
diversity in the type of analytical techniques has been employed for estimation of
different components in formulation.
1. Titrimetric and Gravimetric methods

2. Colorimetric and UV Spectrometric methods
3. Paper Chromatography
4. Preparative Thin Layer Chromatography
Dept of Pharmaceutical Analysis 1 JKKMMRF’S COLLEGE OF PHARMACY

5. Column Chromatography
6. Ion-Exchange Chromatography
7. Flame photometry and Atomic Absorption Spectrometry
8. High Performance Liquid Chromatography
Variables in Quantitative Analysis:
 In homogeneity of the medication

 Sampling Errors
 Preparation of samples such as extraction
 Precision, accuracy and ruggedness of the method
 Random error including that of the operator
CHROMATOGRAPHY
Chromatographic methods are commonly used for the quantitative and
qualitative analysis of raw materials, drug substances, drug products and compounds
in biological fluids. The components monitored include chiral or achiral drug process
impurities, residual solvents, excipients such as preservatives, degradation products,
extractables and leachables from container and closure or manufacturing process,
pesticide in drug product from plant origin, and metabolites.
Methods validation should not be a one-time situation to fulfill Agency

filing requirements, but the methods should be validated and also designed by the
developer or user to ensure ruggedness or robustness. Methods should be reproducible
when used by other analysts, on other equivalent equipment, on other days or
locations, and throughout the life of the drug product. Data that are generated for
acceptance, release, stability, or pharmacokinetics will only be trustworthy if the
methods used to generate the data are reliable. The process of validation and method
design also should be early in the development cycle before important data are
generated. Validation should be on-going in the form of re-validation with method
changes.

I. Types of chromatography
A. High Performance Liquid Chromatography (HPLC)

HPLC chromatographic separation is based on interaction and differential
partition of the sample between the mobile phase and the stationary phase. The
commonly used chromatographic methods can be roughly divided into the following
groups,
1. Chiral
2. Ion-exchange
3. Ion-pair/affinity
4. Normal phase
5. Reversed phase
6. Size exclusion
1. Chiral Chromatography
Separation of the enantiomers can be achieved on chiral stationary phases by

formation of diastercomers via derivatizing agents or mobile phase additives on
achiral stationary phases. When used as an impurity test method, the sensitivity is
enhanced if the enantiomeric impurity elutes before the enantiomeric drug.
2. Ion-exchange Chromatography
Separation is based on the charge-bearing functional groups, anion exchange for
sample negative ion (X-), or cation exchange for sample positive ion (X+). Gradient
elution by pH is common.
3. Ion-pair/Affinity Chromatography
Separation is based on a chemical interaction specific to the target species. The
more popular reversed phase mode uses a buffer and an added counter-ion of opposite
charge to the sample with separation being influenced by pH, ionic strength,
temperature, concentration of and type of organic co-solvent(s). Affinity
chromatography, common for macromolecules, employs a ligand (biologically active
molecule bonded covalently to the solid matrix) which interacts with its homologous
antigen (analyte) as a reversible complex that can be eluted by changing buffer

conditions.
4. Normal Phase Chromatography
Normal phase chromatography is a chromatographic technique that uses
organic solvents for the mobile phase and a polar stationary phase. Here, the less
polar components elute faster than the more polar components.
5. Reversed Phase Chromatography

The test method most commonly used in reversed phase HPLC method. UV
detection is the most common detection technique. Reversed phase chromatography, a
bonded phase chromatographic technique, uses water as the base solvent. Separation
based on solvent strength and selectivity also may be affected by column temperature
and pH. In general, the more polar components elute faster than the less polar
components.
6. Size Exclusion Chromatography

Also known as gel permeation or filtration, separation is based on the
molecular size or hydrodynamic volume of the components. Molecules that are too
large for the pores of the porous packing material on the column elute first, small
molecules that enter the pores elute last, and the elution rates of the rest depend on
their relative sizes.
HPLC system:
 Small diameter (2-5 mm), reusable stainless steel columns;
 Column packings with very small (3,5 and 10 µm) particles;
 Relatively high inlet pressures and controlled flow of the mobile phase;
 Precise sample introduction without the need for large samples;
 Special continuous flow detectors capable of handling small flow rates and
detecting very small amounts;
 Automated standardized instruments;
 Rapid analysis:
 High resolution;
Initially, pressure was selected as the principal criterion of modern liquid
chromatography and thus the name was "high pressure liquid chromatography" or
HPLC. (www.labcompliance.com)

Stationary Phases (Adsorbents):

HPLC separation is based on the surface interactions, and depends on the
types ofthe adsorption sites (surface chemistry).
Main adsorbent parameters are:

 Particle size: 3 to 10 µm;
 Particle size distribution: as narrow as possible( within 10% of the mean)
 Pore size: 70 to 300 A0;

 Surface area: 50 to 250 mm;
 Bonding phase density (no.of adsorption sites per surface unit) 1-5nm.
The last parameter in the list represents an adsorbent surface chemistry

Depending on the type of the ligand attached to the surface, the adsorbent could
be normal phase (-OH,-NH2), or reversed-phase (C8, C18, Phenyl), and even anion
(NH4+), orcation (-COO-) exchangers.
Mobile phases (eluents):
In HPLC type, composition of the mobile phase (eluent) is one of the

variablesinfluencing the separation despite of the large variety of solvents used in
HPLC.
There are several common properties:

Purity, Detector compatibility, Solubility of the sampleLow viscosity,
Chemical inertness, Reasonable price
The following points should also be considered when choosing a mobile phase:
1. Drug is stable in the mobile phase.
2. Excessive salt concentrations should be avoided.
3. The mobile phase should have a pH 2.5 and pH 7.0.
4. Reduce cost and toxicity of the mobile phase
5. Minimize the absorbance of buffer.
6. Use volatile mobile phases when possible, to facilitate collection of products
and LC-MS analysis. Volatile mobile phases include ammonium acetate,
ammonium phosphate, formic acid, acetic acid, and trifluoroacetic acid, Some

caution is needed as these buffers absorb below 220 nm.
7. Ionizable compounds in some cases can present some problems when

analyzed byreverse phase chromatography.
Instrumentation
HPLC instrumentation includes:
 Pumps;
 Injectors;
 Columns;
 Detectors;
 Recorder or data system;
 Mobile phase reservoir;
The most common type of solvent reservoir is a glass bottle. Most of the
Manufacturers supply these bottles with the special caps.Teflon tubing and filters to
connect to pump inlet and to the purge gas (helium) used to remove dissolved air.
Helium purging and storage of the solvent under helium was found not to be sufficient
for degassing of aqueous solvents. It is useful to apply a vacuum for 5-10 min. and
then keepthe solvent under a helium atmosphere. (www.studyhplc.com)

Pumps
The components in a liquid chromatography system which is the solvent
delivery system. The two basic classifications are the constant-pressure and the
constant-flow pump.
The constant-pressure pump is used only for column packing. The constant-flow
pump is the most widely used in all common HPLC applications.
Standard HPLC pump requirements are:

Constant Flow Pumps
Constant-flow systems are generally of two basic types: reciprocating piston
and positive displacement (syringe) pumps. (www.hplc.chem.shu.edu)
Reciprocating piston pump can maintain a liquid flow for indefinitely long
time. In reciprocating piston pumps the pumping rate is controlled by piston retracts
or by the camrotating speed.
Syringe pump has to be refilled after it displaces the whole syringe volume.
For the micro-HPLC applications a syringe pump allows for the maintaining of a
constant flow atthe microliter per minute flow rate range.
Dual Piston Pumps

This pump provides a constant and almost pulse free flow. Both pump
chambers are driven by the same motor through a common eccentric cam; this
common drive allows one piston to pump while the other is refilling.
Check valves
These are present to control the flow rate of solvent & back pressure.
Pulse dampners
Most detectors, but in particular the refractive index detector, are sensitive to
flow "pulses". For both trace analysis and good quantization, it may be necessary to
eliminate the pulsations.
Coil damper
The most simple involves placing a large coil of narrow-bore tubing in the line
between the pump and the injector. As the pump strokes, the coil flexes, absorbing the
energy of the pulsations.

Problems in pumping liquids

The common problems are:
 Solvent degassing
 Corrosion, and
 Compressibility
Degassing
The gas may come out of the solution at the column exit or in the detector,
resulting in sharp spikes. Spikes are created by microscopic bubbles which change the
nature of the flowing stream making it heterogeneous. The drift may occur as these
microscopic bubbles gradually collect and combine in the detector cell. The main
problem is oxygen (from the air) dissolving in polar solvents, particularly water.
Corrosion
When attempting to convert a thin-layer or (glass) column chromatographic
method to HPLC, substitute another acid such as nitric, boric, or acetic for halogens.
lon- exchange systems should be monitored carefully for discolored eluent especially
when small particle packing that exhibit high back pressure (2500+ psi) are used,
since several researchers have observed highly accelerated corrosive reactions at these
pressures with some buffer systems.
Injectors
Sample introduction can be accomplished in various ways. The simplest
method is to use an injection valve. In more sophisticated LC systems, automatic
sampling devices are incorporated where sample introduction is done with the help of
auto samplers and microprocessors.
In liquid chromatography, liquid samples may be injected directly and solid

samples need only be dissolved in an appropriate solvent. The solvent need not be the
mobile phase, but frequently it is judiciously chosen to avoid detector interference,
column/component interference, loss in efficiency or all of these. It is always best to
remove particles from the sample by filtering, or centrifuging since continuous
injections of particulate material will eventually cause blockage of injection devices
or columns. (www.thermoscientific.com)

Automatic Injectors
With commercially available automatic sampling devices, LC can routinely
analyze large numbers of samples without operator intervention. Such equipment is
Popular for the analysis of routine samples (e.g., quality control of drugs )particularly
when coupled with automatic data-handling systems.
Columns
The Heart of the system is the column. The choice of common packing
material and mobile phases depends on the physical properties of the drug many
different reverse phase columns will provide excellent specificity for any particular
separation. It is therefore best to routinely attempt separations with a standard C 8 or
C18 column and determine if it provides good separations. If this column does not
provide good separation or the mobile phase is unsatisfactory alternate methods or
columns should be explored. Reverse phase columns differ by the carbon chain length
degree of end capping and percent carbon loading Diol, cyano and amino groups can
also be used for reverse phase chromatography
Typical HPLC columns are 5, 10, 15 and 25 cm in length and are filled with
small diameter (3,5 or 10µm) particles. The internal diameter of the columns is
usually 4.6 mm many laboratories use 4.6mm ID columns as a standard but it is
worth considering the use of 4mm ID columns as an alternative. These require only
75% of the solvent flow that a 4 mm column uses. This translates to a 25% solvent
saving over the life of the column and can be even more significant if a routine
method is developed for such a column this is considered the best compromise for
sample capacity, mobile phase consumption, speed and resolution. However if pure
substances are to be collected (preparative scale) then larger diameter columns may be
needed. Packing the column tubing with small diameter particles requires high skill
and specialized equipment. For this reason it is generally recommended that all but
the most experienced chromatographers purchase prepacked columns since it is
difficult to match the high performance of professionally packed LC columns without
a large investment in time andequipment.

Choice of the Column

The selection of the column in HPLC is somewhat similar to the selection of
columns in G.C, in the sense that, in the adsorption and partition modes, the
separation mechanism is based on inductive forces, dipole-dipole interactions and
hydrogen bond formation. In case of ion-exchange chromatography, the separation is
based on the differences in the charge, size of the ions generated by the sample
molecules and the nature of ionisable group on the stationary phase. In the case of
size-exclusion chromatography the selection of the column is based on the molecular
weight and size of the sample components.
Detectors
Current LC detectors have wide dynamic range normally allowing both
analyticaland preparative scale runs on the same instrument.
On-line detectors:
 Refractive index
 UV/Vis Fixed wavelength
 UV/Vis Variable wavelength
 UV/Vis Diode array
 Fluorescence
 Conductivity
 Mass spectrometric (LCMS)
 Evaporative light scattering
Off-line detectors
FTIR spiral disk monitor, requires sample transfer on the germanium disk and the
following scanning in FTIR instrument.
Ultraviolet Visible spectroscopic Detectors

According to the Lambert-Bear law absorbance of the radiation is proportional to
the compound concentration in the cell and the length of the cell. The electromagnetic
spectrum is traditionally divided into several regions:

Infrared (IR) 2500 - 50000 nm

Near infrared 800 - 2500 nm
Visible 400 - 800 nm
Ultraviolet (UV) 200 - 400 nm
The majority of organic compounds can be analyzed by UV/VIS detectors.
Almost 70% of published HPLC analyses were performed with UV/VIS detectors.
This fact, plus the relative ease of its operation, makes the UV detector the most
useful and themost widely used LC detector.
In UV detection, one expresses the detector range in absorbance units

(A).One absorbance unit correspond to the depreciation of the light intensity by 90%
the incident light.
A=log (I0 / I)
Fixed wavelength detectors.

Low-pressure mercury vapor lamp emits very intense light at 253.7 nm. By
filtering out all other emitted wavelengths, manufacturers have been able to utilize
this 254 nm line to provide stable, highly sensitive detectors capable of measuring sub
nanogram quantities of any components which contains aromatic ring. The 254 nm
was chosen since the most intense line of mercury lamp is 254 nm, and most of UV
absorbingcompounds have some absorbance at 254 nm.
Advantages:
 Cheapest
 Sensitive
Drawbacks:
 Not in production any more
 One fixed wavelength

Variable-wavelength detectors:
Depending on the sophistication of the detector, wavelength change is done
manually or programmed on a time basis into the memory of the system. There are
several wavelength detectors.
Refractive index Detector, Deflection detectors, Reflective detectors,

Fluorescence Detectors, Electrochemical Detectors, Electrolytic conductivity
Detectors, Evaporative light scattering Detectors and Diode-array detectors.
Diode array detectors
Diode array permits qualitative information to be obtained beyond simple

identification by retention time.
Advantages:
1. The best wavelength(s) can be selected for actual analysis.
2. Absorbance rationing at several wavelengths helps to decide whether
the peakrepresents a single compound or, is in fact, al composite peak.
3. The absorption spectrum helps to identify the compound.

Data systems.
The main goal in using electronic data systems is to increase analysis
accuracy and precision, while reducing operator attention in routine analysis, where
he automation (in terms of data management or process control is needed, a pre-
programmed computing integrator may be sufficient. For higher control levels, a more
intelligent device is necessary, such as a data station or minicomputer.
The advantages of intelligent processors in chromatographs:

Additional automation options become easier to implement;
Complex data analysis becomes more feasible;
Software safeguards can be designed to reduce accidental misuse of the system.
System suitability specifications and tests5
The accuracy and precision of HPLC data collected begin with a well behaved
chromatographic system. The system suitability specifications and tests are
parameters that provide assistance in achieving this purpose. It consists of following
factors.
Capacity factor (k´)

k´=(tR-tO)/tO
The capacity factor is a measure of where the peak of interest is located

withrespect to the void volume, i.e., elution time of the non retained components
Recommendations
The peak should be well resolved from other peaks and the void
volume.
Generally the value of k'is > 2.

Precision Injection repeatability (RSD) of < 1% for 'n'>5 is desirable.
Resolution (R)
RS is the measure of how well two peaks are separated for reliable
quantification: well-separated peaks are essential for quantification. This is very
useful parameter if potential interference peak(s) may be of concern. The closest
potential eluting peak to the analyte should be selected Rs is minimally influenced by
the ratio of the compounds being measured.
RS=2(tR2-tR1)/(W1+W2)

Recommendations
Resolution of > 2 is desirable between the peak of interest and closest
potential interfering peak (impurity, excipients, degradation product, internal
Standard.etc).
Precision/injection repeatability (RSD)

Injection precision expressed as RSD (relative standard deviation) indicates
the performance of the HPL chromatograph which includes the plumbing, column,
and environmental conditions, at the time the samples are analyzed. It should be noted
that sample preparation and manufacturing variations are not considered.
Tailing factor (T)

The accuracy of quantitation decreases with increase in peak tailing because of
the difficulties encountered by the integrator in determining where when the peak
ends and hence the calculation of the area under the peak. Integrator variables are
preset by the analyst for optimum calculation of the area for the peak of interest. If the
integrator is unable to determine exactly when an upslope or down slope
occurs,accuracy drops.
T=Wx/2f
Recommendations
T of< 1.5 is preferred.
Theoretical plate number (N)
Theoretical plate number is a measure of column efficiency, that is, how
manypeaks can be located per unit run-time of the chromatogram.
N=16 (tR/tW)2= L/H
N - Constant for each peak on a chromatogram with a fixed set of operating

conditions,H or HETP- Height equivalent of a theoretical plate.
L - Length of column
Parameters, which can affect N or H, include peak position, particle size in
column, flow-rate of mobile phase, column temperature, viscosity of mobile phase,
and molecular weight of the analyte.

Recommendations:
The theoretical plate number depends on elution time but in general should
be > 1500.
Relative retention (α)

α =k'l/k´2
Relative retention is a measure of the relative location of two peaks. This is

not anessential parameter as long as the resolution (Rs) is stated.
Retention volume (Rv)21

Retention Volume is the volume of carrier gas required to elute 50% of the
component from the column. It is the product of retention time and flow rate.
Retention volume = Retention time x flow rate (Ravi sankar S, 2006)
II Reference standards
A reference standard is a highly punted compound that is well
characterized.Chromatographic methods rely heavily on a reference standard to
provide accurate data. Therefore the quality and purity of the reference standard is
very important. Two types of reference standards, chemical and nuclidic, exist. With
the latter, the radiolabel purity should also be considered as well as the chemical
purity.
Samples and analytical data for methods Validation, the two categories of
chemical reference standards are as follows: USD/NF reference standard that does not
need characterization, and non-compendial standard that should be of the highest
purity that can be obtained by reasonable effort and should be thoroughly
characterized to assure its identity, strength, quality and purity.
Chromatographic test methods use either external or internal standards for

quantitation.
A. An external standard method-- is used when the standard is analyzed on a

separate chromatogram from the sample. Quantitation is based on a
comparison of the peak area height (HPLC or GC) or spot intensity (TLC) of
the sample to that of a reference standardof the analyte of interest.

B. With an internal standard method -- compound of known punity that does

not causeinterference in the analysis is added to the sample mixture.
Quantitation is based on the response ratio of compound of interest to the
internal standard vs the response ratio of a similar preparation of the reference
standard (HPLC or GC). This technique is rarely usedfor TLC methods.
The working concentration is the target concentration of the compound of

interest as described in the method. Keeping the concentrations of the sample and the
standard close to each other for the external standard method improves the
accuracy ofthe method.
VALIDATION
Validation is a key process for effective quality assurance. "Validation" is
established documented evidence, which provides a high degree of assurance that
specific process or equipment will consistently produce a product or result meeting its
predetermined specification and quality attributes.
USFDA defines validation as "establish the documented evidence which

provides a high degree of assurance that a specific process will consistently produce a
product of predetermined specifications and quantity attributes".
EUGMP define validation as "action of proving in accordance with the

principle of Good manufacturing practice (GMP), that any material, activity or system
actually lead toexpected result".
AUSTRALIAN GMP defines validation as "The action of proving that any

material, process, activity, procedure, system, equipment or mechanism used in
manufacture or control can and will be reliable and achieve the desire and intended
result."
1. The primary objective of validation is to form a basis for written procedure

for production and process control which are designed to assure that the drug
products have the identity, quality, and purity they purport or are represented
to possess.
2. Assurance of Quantity

3. Government Regulation
Importance of validation
1. As the quality of product cannot always be assured by routine quality control
because of testing of statistically insignificant number of sample, the
validation thus should
2. Provide adequacy and reliability of a system or product to meet the pre-

determined criteria or attributes providing high degree of confidence that the
same level of quality is consistently built into each of finished product from
batch to batch.
3. Retrospective Validation is useful for trend comparison of results complains to

cGMP to cGLP
4. For taking appropriate action in case of non-compliance.
Types of validation
The following are frequently required to be validated on a pharmaceutical
process:
1. Equipment, Environment. Materials, Methods, Controls, Process ,
Personnel's.Facilities and operating procedure. Based on these, the validation
program comprises.
2. Equipment Validation
3. Facility Validation including utilities
4. Process Validation
5. Cleaning Validation
6. Analytical Method Validation
Analytical method validation

Method validation is the process to confirm that the analytical procedure
employed for a specific test is suitable for its intended use. Analytical testing of a
pharmaceutical product is necessary to ensure its purity, stability, safety and efficacy.
Analytical method validation is an integral part of the quality control system.

Although a thorough validation cannot rule out all potential problems, the process of
method development and validation should address the most common ones
VALIDATION PARAMETERS
The validation of the assay procedure was carried out using the following parameters
SpecificityDefinition:
Specificity is the ability to asses unequivocally the analyte in the presence of
impurities, degradants, matrix etc (components) which may be expected to be present.
Lack of specificity of an individual analytical procedure may be compensated by
other supporting analytical procedures.
Determination:
The demonstration of specificity requires that the procedure is unaffected by
the presence of impurities or excipients. In practice this can be done by spiking the
drug substance or product with appropriate levels of impunities or excipients and
demonstrating that the assay result is unaffected by the presence of these extraneous
materials.
ICH Requirement:
The ICH documents state that when chromatographic procedures are used,
representative chromatograms should be presented to demonstrate the degree of
selectivity, and peaks should be appropriately labeled. Peak purity tests (e.g., using
diode array of mass spectrometry) may be useful to show that the analyte
chromatographic peak is not attributable to more than one component.
Accuracy
Definition:
The accuracy of an analytical procedure expresses the closeness of agreement
between the value which is accepted either as a conventional true value or an accepted
reference value and the value found.
Determination:
In case of assay of a drug in a formulated product, accuracy may be determined
by application of the analytical method to synthetic mixtures of the drug product
components to which the known amount of analyte have been added within the range

of the method If it is not possible to obtain all drug product components, it may be
acceptable either to add known quantities of the analyte to the drug product or to
compare results with those of a second, well characterized method, the accuracy of
which has been stated or defined Accuracy is the measure of how close the
experimental value is to the true value. Accuracy studies for drug substance and drug
product are recommended to be performed at the 80. 100 and 120% levels of label
claim as stated in the Guideline for Submitting Samples and Analytical Data for
Methods Validation. For the drug product, this is performed frequently by the addition
of known amounts of drug by weight or volume (dissolved in diluent) to the placebo
formulation working in the linear range of detection of the analyte. This would be a
true recovery for liquid formulations. For formulations such as tablet, suppository.
transdermal patch this could mean evaluating potential interaction of the active drug
with the excipients in the diluent. From a practical standpoint, it is difficult to
manufacture a single unit with known amount of active drug to evaluate recovery.
This test evaluates the specificity of the method in the presence of the excipients
under the chromatographic conditions used for the analysis of the drug product. It will
pick up recovery problems that could be encountered during the sample preparation
and the chromatographic procedures. However, it does not count the effect of the
manufacturing process. At each recommended level studied, replicate samples are
evaluated. The RSD of the replicates will provide the analysis variation or how
precise the test method is. The mean of the replicates, expressed as % label claim.
indicates how accurate the test method is.
ICH Requirement:
The ICH documents recommended that accuracy should be assessed using a
minimum of nine determinations over a minimum of three concentration levels.
covering the specified range (i.e., three concentrations and three replicates of each
concentration.
Precision :
Definition:
The precision of an analytical procedure expresses the closeness of agreement
between a series of measurements obtained from multiple sampling of the same
homogenous sample under the prescribed conditions. The precision of an analytical
procedure is usually expressed as the variance, standard deviation or coefficient of

variation of a series of measurements.(ICH Q2A 1996)
Precision can be categorized into two types as follows,

System precision:
A system precision is evaluated by measuring the peak response for six
replicate injection of the same standard solution prepared as per the proposed method.
The RSD is calculating it should not be more than 2%.
Method precision:
A method precision is evaluated by measuring the peak response for six
replicate injection of six different weight of sample solution prepared as per the
proposed method. The RSD is calculating it should not be more than 2%.
Determination:
The precision of an analytical method is determined by assaying a sufficient
number of aliquots of a homogenous sample to be able to calculate statistically valid
estimates of standard deviation or relative standard deviation.
ICH Requirement:
The ICH documents recommended that repeatability should be assessed using
a minimum of nine determinations covering the specified range for the procedure
(i.e., three concentrations and three replicates of each concentration or using a
minimum of sixdeterminations at 100% of the test concentration).
Linearity:
Definition:
The linearity of an analytical procedure is its ability (within a given range) to
obtain the test results which are directly proportional to the concentration amount of
analyte in the sample.
Determination:
Linearity of an analytical procedure is established, using a minimum of five
concentrations. It is established initially by visual examination of a plot of signals as a
function of analyte concentration of content. If there appears to be a linear
relationship, test results are established by appropriate statistical methods. (i.e. by
calculation of a regression line by the method of least squares).

ICH Requirement:
ICH recommends that, for the establishment of linearity, a minimum of five
concentrations normally be used. It is also recommended that the following minimum
specified ranges should be considered Assay of drug substance (or a finished product)
from 80% to 120% of the test concentration.
Limit of detection (LOD) :

Definition:
LOD is the lowest concentration of the substance that the method can detect but
not necessarily quantify. LOD simply indicates that the sample is below or above a
certain level.
Determination:
For non-instrumental methods, the detection limit is generally determined by
the analysis of samples with known concentrations of analyte and by establishing the
minimum level at which the analyte can be reliably detected.
ICH Requirement:
The ICH describes a common approach, which is to compare measured signal
from samples with known low concentrations of analyte with those of blank samples.
The minimum concentration at which the analyte can reliably be detected is
established.
Typically acceptable signal-to-noise ratios are 2:1 or 3:1.

Measurement is based on:
 Signal to noise ratio
 Visual evaluation (relevant chromatogram acceptable)
 The standard deviation of the response and the slope.
LOD= 3.3σ/S
Where
σ - The standard deviation of the response
S - The slope of the calibration curve (of analyte)

Limit of quantitation (LOQ)Definition:

LOQ is the lowest concentration of the substance that can be estimated
quantitatively with acceptable precision, accuracy and reliability by the proposed
method LOQ is determined by analysis of samples containing decreasing known
quantity of the substance and determining the lowest level at which acceptable level of
accuracy and precision is attained.
Determination:
For non-instrumental methods, the quantization limit is generally determined
by the analysis of samples with known concentrations of analyte and by establishing
the minimum level at which the analyte can be determined with acceptable accuracy
and precision
ICH Requirement:
The ICH describes a common approach, which is to compare measured signal
from samples with known low concentrations of analyte with those of blank samples.
The minimum concentration at which the analyte can reliably be quantified is
established. Typically acceptable signal-to-noise ratios are 10:1
LOQ=10σ/S
Where.
σ - The standard deviation of the response
S - The slope of the calibration curve (of the analyte)
Range Definition
The range of an analytical procedure is the interval between the upper and
lower concentration amounts) of analyte in the sample (including these
concentrations) for which it has been demonstrated that the analytical procedure has a
suitable level of precision accuracy and linearity.
Determination:
The range of the method is validated by verifying that the analytical method
provides acceptable precision, accuracy and linearity when applied to samples
containing analyte at the extremes of the range as well as within the range.

Robustness:
Definition:
The most of an analytical procedure is met of capacity remain unchanged by
small but deliberate variations in method parameters and provides an indication of
its reliability during normal usage.
Determination:
The robustness of method is determined by performing the way by
deliberately altering parameters (change in flow rate ±10%, change in mobile phase
ratio of ±2 change in Ph of mobile phase ±0.2. change in wave length detection
±5mn, change in temperature ± 1 to 5°) that the results are not influenced by the
changes in the above parameters.
Ruggedness:
Definition:
The ruggedness of an analytical method is the degree of reproducibility of
test results obtained by the analysis of the same samples under a variety of conditions,
such as different laboratories, different analysts, different instruments, different lots of
reagents, different elapsed assay times, different assay temperatures, different
days,etc.
Determination:
The Ruggedness of an analytical method is determined by the analysis of
aliquots from homogenous lots in different laboratories, by different analysts, using
operational and environmental condition that may differ but are still within the
specified parameters of the assay. The degree of reproducibility of the result is then
determined as a function of assay variables. This reproducibility may be compared to
the precision of assay under normal condition to obtain a measure of the ruggedness
of the analytical method.
Sample solution stability:

Solution stability of the drug substance or drug product after preparation
according to the test method should be evaluated according to the test method. Most
laboratories utilize auto samplers with overnight runs and the sample will be in
solution for hours in the laboratory environment before the test procedure is

completed. This is of concern especially for drugs that can undergo degradation by
hydrolysis, photolysis or adhesion toglassware.
Degradation Studies
Forced degradation or accelerated degradation is a process whereby the natural
degradation rate of a product or material increased by the application of additional
stress and demonstrate the stability indicating studies.
These studies also provide information about the degradation pathways and
degradation products that could form during storage. Forced degradation studies may
help facilitate pharmaceutical development as well in areas such as formulation
development, manufacturing, and packaging, in which knowledge of chemical
behaviour can be used improve a drug product
Forced degradation studies are conducting for the following reasons:
 Facilitate development and validation of stability indicating analytical

methods.
 Elucidate the structure of degradation products
 Provide understanding necessary to solve problems.
 Forecast possible formation of geotaxis compounds.
 Determination of the intrinsic stability of a drug substance molecule.
After getting satisfied set of analytical conditions, preliminary degradation
studies are conducted as follows,
Analyze forcibly degraded sample under elevated temperature, photo

degradation acid hydrolysis, base hydrolysis, water hydrolysis and oxidation. All
stress solutions should be analyzed by using photo diode array detector to ensure that
there is no-elution of peak.
Different stress conditions:

Thermal degradation
Forcibly degrade the sample at 600C temperature and below the melting point
of the sample for two days. Collect the sample on 1st day and 2nd day evaluate the
peak purity.

Photo degradation
Forcibly degrade the sample under UV light specific nm or 2 days. Collect the
sample on 1st and 2 nd day evaluate the peak purity.
Acid hydrolysis
Forcibly degrade the sample using 0.5 HCl at room temperature. Based on
compound dissolution, change the medium. Initially collect the sample after 1 hour.
Evaluate the purity. Based on that results, either it will be stopped or defined.
Base hydrolysis:
Forcibly degrade the sample using 0.5 N NaOH at room temperature. Based
on compound dissolution, change the medium. Initially collect the sample after 1
hour. Evaluate the purity. Based on that results either it will be stopped or defined.
Peroxide degradation
Forcibly degrade the sample using 3 % H2O2, at room temperature. Initially
collect the sample after 1 hour. Evaluate the purity, Based on the results, either it will
be stopped or continued or defined.
Degradation criteria
Try to get 5% to 15% degradation, or make a comment on the data. Identify
the degradation by co-injection in case of known impurities and compare spectra.
Identify the mass numbers for unknown components by using mass detector and try to
establish the structure, if possible and compare the synthetic process for justification,
after conducting these studies verify the chromatograms and observe any peaks
merging with respect to main peak and any critical parts. If any such situations were
arrived adjust the mobile phase compositions, column, etc.,feel good and conclude the
method parameters.
Range:-
Difference between the greatest and the smallest values of the varieties.
Range= largest value - smallest value.

Co-efficient of range:-
L-S
=
L+S
L =Largest value
S =Smallest value

Chapter 2 Literature Review
2.LITERATURE REVIEW
1. Awdhut Pimpale et al., Development and Validation for Simultaneous

Estimation of Rosuvastatin Calcium and Clopidogrel Bisulfate in
Pharmaceutical Dosage Form by Reverse Phase-High Performance Liquid
Chromatography(Int. Journal of Pharmacy and Biological Sciences-IJPBSTM
(2020))
The present work was focused on the development and validation of (RP-
HPLC) method which is simple, rapid, precise, accurate, sensitive, and economical for
the quantification of rosuvastatin calcium and clopidogrel bisulfate in bulk and tablet
formulation. The separation was attained on Princeton (C18) (250×4.6 mm, 5μ)
employing buffer which is mixture of water (pH 3.0, adjusted with ortho phosphoric
acid) and methanol in the ratio (20:80) v/v as mobile phase, at flow rate 1.0 ml/min
and detection was carried out at wavelength 240nm. The retention time under
optimized condition of rosuvastatin calcium and clopidogrel bisulfate was found to be
2.844 min and 4.388 min respectively. The linearity of the method was demonstrated
in the concentration range of 6-16 µg/ml and 45-120 µg/ml for rosuvastatin calcium
and clopidogrel bisulfate with a correlation coefficient (r2 ) of 0.9999 and 0.9996
respectively. The percentage relative standard deviation was ˂2% and percentage
recovery was found to be 100.12-101.37% and 99.72-101.09% for rosuvastatin
calcium and clopidogrel bisulfate respectively. Assay of marketed tablet formulations
was found to be 98.99% and 99.92% respectively. Conclusion: The developed RP-
HPLC method was found to be simple, specific, sensitive, rapid, linear, accurate,
precise and economical and could be used for regular quality control of rosuvastatin
calcium and clopidogrel bisulfate in bulk and tablet formulations.
2. Harshana Rothekar Development and Validation of RP-HPLC

Method For Simultaneous De-termination Of Rosuvastatin And
Clopidogrel In Tablet Dosage Form
The aimed of research the method development and validation by RP-HPLC
method of the Rosuvastatin calcium and Clopidogrel bisulphate. The method is a
simple, accurate, specific, precise, reproducible and sensitive. The ? max of ROSU
and CLOP was found to be 240nm. Coefficient correlation 0.999, Beer’s Law limit

50-150 µg/ml, from the four trial of different concentration of mobile phase was
selected Methanol:Water 80:20 v/v, pH 3.0 at 240nm, flow rate 1ml/min, sample inlet
20 µL, C 18 Prontosil, %RSD of ROSU 1.017 and CLOP 0.173, theoretical plates
ROSU 7797.53 and ROSU 8257.53, Retention time ROSU 3.483min and CLOP
4.983min, Tailing factor ROSU 1.1787 and CLOP 1.074, limits 2 NMT, Accuracy
ROSU 0.37 %RSD, Recovery 99.59% and CLOP 0.18 %RSD, recovery 100.41% was
show good efficacy and results. The methods indicate Future scope in analysis quality
control of the estimation of ROSU and CLOP for routine drug quality analysis
investigation.
3. Pradip Kumar Tiwari1 et al., Analytical Method Development and Validation

for the Simultaneous Estimation of Aspirin, Clopidogrel and Rosuvastatin in
Pharmaceutical Dosage Form( Journal of Drug Delivery & Therapeutics. 2019;)
A new, simple, novel, accurate, precise, reliable, rapid and linear (RP-HPLC)
method was developed and fully validated for simultaneous qualitative and
quantitative estimation of Rosuvastatin , Clopidogrel and Aspirin in bulk and
pharmaceutical dosage form as per ICH guidelines. In the present work, good
chromatographic separation was achieved by isocratic method using a Hypersil BDS
C18 column (250 mm ×4.6, 5 μm) and a mobile phase consisting of KH2Po4 buffer
pH-6.0: acetonitrile in the ratio 60:40, at a flow rate of 1 ml/min. The effluents
obtained were monitored at 242nm with the UV-visible detector. The calibration
curves obtained were linear (r2=0.999) over the concentration range of 7.5-22.5μg/ml
and 1- 3μg/ml for CLOP, ASP and ROS respectively. A run time of 7.0 minutes for
each sample made it possible to analyze more than 200 samples per day. The retention
time of ASP, CLOP and ROS was found to be 3.103 min, 4,277 min and 5.707 min
respectively. The high recovery values (99%-101%) indicate a satisfactory accuracy.
The low percent relative standard deviation (% RSD) values in the precision study
reveal that the method is precise therefore the method can be used for routine
monitoring of CLOP, ASP and ROS in industry in the assay of bulk drug and dosage
form.

4. Pooja pisal et al., Development and Validation of Stability Indicating

rp-hplc Method for Simultaneous Determination of Aspirin,
Rosuvastatin, Clopidogrel in bulk and Pharmaceutical Dosage form
(int j pharm pharm sci, jul 2018)
In the present work, good chromatographic separation was achieved by

isocratic method using a BISCOF HPLC C18 column (250 mm ×4.6, 5 µm) and a
mobile phase consisting of water at pH 2.51 with 0.1 % (v/v) orthophosphoric acid
(OPA): acetonitrile in the ratio 50:50, at a flow rate of 1 ml/min. The effluents
obtained were monitored at 237 nm with the UV-visible detector. The retention time
of aspirin, rosuvastatin, and clopidogrel was found to be 4.3 min, 7.6 min and 16.6
min respectively. For linearity seven-point calibration curves were obtained in a
concentration range from 1-7 µg/ml for aspirin, rosuvastatin and clopidogrel with
correlation coefficient 0.999, 0.9989, 0.9988 respectively. The high recovery values
(99%-101%) indicate a satisfactory accuracy. The low percent relative standard
deviation (% RSD) values in the precision study reveal that the method is precise. In
the present study stability indicating an RP-HPLC method for the combination was
tested by degrading the drugs together under various stress condition like acid, base
and neutral hydrolysis, oxidation, thermal and photolytic stress which is
recommended by ICH. The developed RP-HPLC method is simple, economic,
specific, accurate and precise for the simultaneous estimation of aspirin, rosuvastatin,
and clopidogrel in the combined capsule dosage form. The developed stability
indicating analytical method can be used to check the stability of the compounds and
was found suitable to determine % degradation of drugs in pharmaceutical dosage
form.
5. Vadthya rajesekar et al., RP-HPLC Method Development and Validation

for the Simultaneous Estimation of Rosuvastatin and Ezetimibe in Tablet
Dosage Form (IJPAR | Jan-Mar -2014)
A simple reversed-phase high-performance liquid chromatographic (RP-HPLC)
method has been developed andvalidated for simultaneous determination of
Rosuvastatin and Ezetimibe in pharmaceutical tablet dosage form. Chromatographic
analysis was performed on a Symmetry X-
at ambient temperature with a mixture of ortho phosphoric acid buffer and

Acetonitrile in the ratio 40:60 v/v as mobile phase, at a flow rate of 1.0 mL min-1 .
UV detection was performed at 237 nm.. The retention times of Rosuvastatin and
Ezetimibe were 2.490 and 3.173 min, respectively. The correlation coefficient of
Rosuvastatin and Ezetimibe was found to be 0.999. Calibration plots were linear over
the concentration ranges 10–50 μg mL-1 for Rosuvastatin and Ezetimibe, respectively.
The Limit of detection was 1.626 and 0.918µg mL-1 and the quantification limit was
4.927 µg mL-1 and 2.783µg mL-1 for Rosuvastatin and Ezetimibe, respectively. The
accuracy of the proposed method was determined by recovery studies and found to be
99.59% to 100.70%. The method was validated for accuracy, linearity, sensitivity,
precision, robustness, system suitability Commercial tablet formulation was
successfully analyzed using the developed method and the proposed method is
applicable to routine analysis of determination of Rosuvastatin and Ezetimibe in
pharmaceutical tablet dosage form.
6. Pandya C.B. et.al., Development and validation of RP-HPLC method for

determination of Rosuvastatin Calcium in bulk and pharmaceutical
dosage form (International Journal of Pharmaceutical Sciences Review and
Research Nov-2010)
A simple, specific, accurate, and precise reverse phase high performance

liquid chromatographic (RP-HPLC) method was developed and validated for the
estimation of Rosuvastatin Calcium (RC) in pharmaceutical dosage forms. A Thermo
hypersil reversed phase C- 18, 5 μm column having 100 × 4.6 mm i.d. in gradient
mode, with mobile phase containing HPLC grade Acetonitrile: Potassium dihydrogen
orthophosphate (50: 50 v / v, pH 3) was used. The flow rate was 0.5 ml / min and
effluents were monitored at 243 nm. Chromatogram showed a main peak of RC at
retention time was 3.333 ± 0.004 min. The method was validated for linearity,
accuracy, precision, limit of detection, limit of quantitation, robustness and
ruggedness. The limit of detection and limit of quantitation for estimation of RC was
found to be 0.14 μg / ml and 0.46 μg / ml, respectively. Recovery of RC was found to
be in the range of 98.50-100.17 %. Proposed method was successfully applied for the
quantitative determination of RC in pharmaceutical dosage forms.

7. Radha et. al.,Analytical method development and validation for the

simultaneous estimation of Rosuvastatin and Finofibate in tablet dosage
form by reverse phase high performance liquid chromatography.(Indian
Journal of Research in Pharmacy and Biotechnology)
A new, simple, precise, accurate and reproducible RP-HPLC method for
Simultaneous estimation of Rosuvastatin (ROS) and Fenofibrate (FEN) in bulk and
pharmaceutical formulations was developed. Separation of ROS and FEN was
successfully achieved on a Hypersil C18 (4.6 x 250mm, 6.5 m, Make: Waters) or
equivalent in an isocratic mode utilizing OPA buffer (pH 3.0): Methanol (65:35%v/v)
at a flow rate of 1.2 mL/min and eluate was monitored at 238 nm, with a retention
time of 1.950 and 3.858 minutes for ROS and FEN. The method was validated and the
response was found to be linear in the drug concentration range of 50 µg/mL to 150
µg/mL for ROS and 50 µg/mL to 150 µg/mL for FEN. The values of the slope,
intercept and the correlation coefficient were found to be 2 2 507, 7467 and 0.999 for
ROS and 21157, 16980 and 0.999 for FEN respectively. The LOD and LOQ for
Rosuvastatine were found to be 0.0053, 0.017 respectivly. The LOD and LOQ for
Fenofibrate were found to be 0.00019, 0.00063 respectively. This method was found
to be good percentage recovery for Rosuvastatine and Fenofibrate were found to be
99.00 and 99.00 respectively indicates that the proposed method is highly accurate.
The specificity of the method shows good correlation between retention times of
standard with the sample so, the method specifically determines the analyte in the
sample without interference from excipients of tablet dosage forms. The method was
extensively validated according to ICH guidelines for Linearity, Range, Accuacy,
Precesion, Specificity and Robustness.
8. De Gruyter et. al., Development and validation of a green RP-HPLC

method for the analysis of rosuvastatin: a step towards making liquid
chromatography environmentally benign(June 2017)
The purpose of this study was to formulate and validate a new green high-
performance liquid chromatography-ultraviolet (HPLC)-UV method for quick
quantification of rosuvastatin calcium (ROC) in standard drugs. The results showed a
combination of ethanol:methanol:ethyl acetate (6:3:1 v/v) at a rate of 1.0 ml/min to be
the best for identifying ROC and its separation from its breakdown products. The
identification of ROC was achieved using a NUCLEODUR 150 mm×4.6 mm RP C8

column packed using 5 μm filler as the stationary phase and detection was performed
at 254 nm. The technique developed was checked for linearity, selectivity, accuracy,
precision, robustness and sensitivity as well as specificity. The usefulness of the
proposed process was confirmed by analyzing ROC in a prepared self-
nanoemulsifying drug delivery system (SNEDDS) and over-the-counter products. The
amount of ROC in SNEDDS was found to be 98.38%. The HPLC-UV system we
developed effectively determined the ROC peak along with its breakdown products
which confirmed the stability-indicating property of the projected system. The system
could also be used to compare the solubility of rosuvastatin nanoparticles in standard
drugs. These outcomes indicated that the developed HPLC could be effectively used
for the regular investigation of ROC in standard drugs, various pharmaceutical
formulations and drug release samples.
9. D Kallol Jana et, al., Development and Validation of a new improved RP-
HPLC Method for estimation of Rosuvastatin calcium in Pharmaceutical
dosage form.
A reliable, sensitive, isocratic and simple RP-HPLC was developed and

validated for assay of Rosuvastatin Calcium in tablets and for determination of
content uniformity. Chromatography was achieved by Thermo scientific C8 column,
250 x 4.6mm, particle size 5 µm with flow rate of 1.0ml/min. Detection was monitor
at 248 nm. The mobile phase consisted of methanol : acetonitrile : water (40:40:20,
v/v). Retention time of Rosuvastatin Calcium was found to be 3.427-and an overall
analytical run time of approximately 5 minutes. The method was linear over
concentration range of 140-260µg/ml ( r2 = 0.999). The limit of detection and
quantification was 3.26 and 9.88 respectively. Accuracy (recovery) of Rosuvastatin
Calcium was between 106.58%,100.18% and 102.81 %. The developed method was
validated for accuracy, precision, ruggedness, robustness and stability of solution. The
proposed method is accurate, simple, rapid, precise, time effective, reproducible and
hence can be applied routine quality control estimation of Rosuvastatin Calcium in
tablet dosage form. The results of validation parameters have been validated
statistically and also by recovery studies.

10. Mohamad Ammar Al‐Khayat et, al., Development and validation of RP-
HPLC method for determination of clopidogrel in tablets ( International
Journal of Pharmaceutical Sciences Review and Research · May 2012)
A Simple, sensitive and specific RP‐HPLC method was developed and
validated for the determination of clopidogrel in tablets. Isocratic chromatography
was performed on a C18 column with acetonitrile‐methanol‐phosphate buffer 0.1M
80:10:10 (v/v/v) as mobile phase at a flow rate of 0.9 ml/min. UV detection was
set at 240 nm. The method was validated with respect to accuracy, linearity,
precision, selectivity, and robustness. All the parameters examined met the
currentrecommendations of U.S.P (30) for analytical method validation. The
method can be reliably used for routine quality control analysis and to determine the
clopidogrel content of marketed tablets.

Chapter 3 Drug Profile
DRUG PROFILE
Clopidogrel
Drug name : Clopidogrel
Chemical name : Methyl (2S)-2-(2-chlorophenyl)-2-(6,7-dihydro-4H-
thieno[3,2-c]pyridin-5-yl)acetate
Chemical structure :
Molecular formulae : C16H16ClNO2S

Molecular weight : 321.822 g/ mol
Solubility : Practically insoluble in water at neutral pH but
. freely at pH1
Melting point : 158° C
Indications : Primary prevention of thromboembolism atrial
. fibrillation.
Secondary prevention post-coronary artery bypass grafting.
Mechanism of action: Clopidogrel is an inhibitor of platelet activation and
aggregation through the irreversible binding of its active metabolite to the P2Y12
class of ADP receptors on platelets.
Absorption:
A 75mg oral dose of clopidogrel is 50% absorbed from the
intestine.Clopidogrel can be taken with or without food.
A meal decreases the AUC of the active metabolite by 57%. The active
metabolite of clopidogrel reaches a maximum concentration after 30-60 minutes.

The Cmax was 31.3±13ng/mL for poor metabolizers, 43.9±14ng/mL for

intermediate metabolizers, and 60.8±34.3ng/mL for extensive metabolizers.
Adverse drug reaction:

 Easy bleeding/bruising,
 Stomach upset/pain,
 Diarrhea,
 Constipation
Condraintication:
 Increased risk of bleeding due to clotting disorder.
 Bleeding from the retina.
 Thrombotic thrombocytopenic purpura,.
 Stomach or intestinal ulcer.
Drug interaction :
 Repaglinide
 Stomach acid drugs (proton pump inhibitors) ...
 Nonsteroidal anti-inflammatory drugs (NSAIDs) ...
 Blood thinners.
 Drugs used to treat depression. ...
 Salicylates (aspirin) ...
 Opioids.
Route of elimination:
Approximately 50% of total radioactivity was excreted in urine and
approximately 46% in feces over the 5 days post dosing. After a single, oral dose of
75 mg, clopidogrel has a half-life of approximately 6 hours.
Brand Names:
 Aclotil - Bal Pharma
 Antiplar - Emcure.
 Aplatin-75 - Saga Lab
 Aptogrel - Aurobindo

Rosuvastatin
Drug name : Rosuvastatin.
Chemical name : Bis((3R,5S,6E)-7-[4-(4-fluorophenyl)-2-
. (N methylmethanesulfonamido) -6-(propan-2-yl)pyrimidin-5-yl]-.
. 3,5- dihydroxyhept-6-enoate)
Structure:
Molecular weight : 341.88 g/ mol

Molecular formulae : C22H28FN3O6S
Solubility : Sparingly soluble in water
Melting point : 173°C to 185°C.
Category : HMG-COA Reductase inhibitor.
Biological half life : 19hours
Mechanism of action:
Rosuvastatin is a statin medication and a competitive inhibitor of the enzyme
HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase, which catalyzes the
conversion of HMG-CoA to mevalonate, an early rate-limiting step in cholesterol
biosynthesis. Rosuvastatin acts primarily in the liver, where decreased hepatic
cholesterol concentrations stimulate the upregulation of hepatic low density
lipoprotein (LDL) receptors which increases hepatic uptake of LDL. Rosuvastatin
also inhibits hepatic synthesis of very low density lipoprotein (VLDL). The overall
effect is a decrease in plasma LDL and VLDL.

Side Effects :
 Blurred vision
 Chest pain, discomfort, tightness, or heaviness
 Difficult, burning, or painful urination
 Dry mouth
 Dry skin
 Fruit-like breath odor
 Increased hunger
 Increased thirst
 Increased urination.

Chapter 4 Aim and Plan Work
5. AIM AND PLAN OF WORK

Most of the pharmaceuticals industries rely upon quantitative chemical
analysis to ensure that the raw materials used and the final product thus obtained meet
certain specifications and to determine how much component is present in the final
product.
The number of drug and drug formulations introduced into the market has
been increasing at an alarming rate. Standard analytical procedures for these drugs of
formulations may not be available and if available may not suit to our actual
conditions of use. So it is require to develop newer analytical methods which are
accurate precise specific linear simple and rapid.
The modern methods of choice of assays are High-pressure Liquid
chromatography (HPLC), which requires highly sophisticated equipment, trained
personnel, high purity chemicals and proper maintenance.
Rosuvastatin and Clopidogrel is a drug which is used to prevent heart attack
and stroke in future. According to literature survey few analytical methods were
reported using solvent Acetonitrile : Buffer pH4.3 to the ratio of (40:60). The present
aim is to develop new analytical method to estimate the Rosuvastatin and Clopidogrel
in its combined dosage form. As the drug is polar in nature, it was proposed to select
RP-HPLC method.
Method development includes the development of initial chromatographic
conditions, setting upto and optimization of developed chromatographic conditions
for the assay of Rosuvastatin and Clopidogrel .
The developed method is validated for parameters such as system suitability,
precision, accuracy, linearity, ruggedness and robustness and evaluation of analytical
method validation report generated for the developed methods as per ICH guidelines.

Chapter 4 Aim and Plan Work
PLAN OF WORK
i. Study of physicochemical properties of drug(pH, pKa, solubility and
molecularweight)
ii. Preparation of drug standard and sample,
iii. Optimization chromatographic conditions like,
a. Selection of wavelength
b. Selection of initial separation conditions
c. Nature of stationary phase
d. Nature of mobile phase(pH,solvent strength, solvent ratio and flow

rate)
iv. Study of system suitability parameters,
v. Validation of proposed method by RP-HPLC .
vi. Applying developed method to marketed formulation.

Chapter 5 Materials and Methods
6. MATERIALS AND METHODS

HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY (HPLC)
TECHNIQUE
Materials required
Instruments employed:
1. Digitital balance : Mettler Toledo
2. pH meter : Digital pH meter Lab India model
3. Sonicator : Sonica- spinco biotech
4. Membrane filter : Nylon membrane filter (0.45µ)
5. HPLC : WATERS-LC
a. Software used : Empower 3
b. Detector : UV visible
c. Analytical column: Column YMC PACK (150 × 4.6mm)5µ
d. Tablets brand used: Rozalet 20.
e. Manufacturing : Sun pharmaceuticals pvt,Ltd.

Chemical Required:
Acetonitrile : HPLC grade
Orthophosphoric acid : AR grade
Potassium dihydrogen phosphate : ARgrade
Water : HPLC grade
Optimization of mobile phase:
Separation of both the drugs was tried using the following combination of
mobilephases. The table gives the details of the same (Table no.1)

Table no.1 Method development trails
Serial
Mobile phase Ratio(v/v) Elution of peak
no
1 Acetonitrile : potassium Not proper
50:50
dihydrogenphosphte dihydrate separation (Broad&
pH.6.0 fronting peak )
2 60:40 Not proper separation (Peak

Acetonitrile : potassium
eluted & more
dihydrogenphosphte dihydrate
noise peak )
pH.6.5
3 Acetonitrile : potassium 70:30 Not proper separation

dihydrogenphosphte dihydrate (Theoretical plate LT 2000
pH.4.5
4 Acetonitrile : potassium 40:60 Good separation
dihydrogenphosphte dihydrate
pH.4.3
Out of 4 trails was selected for further studies because when compared to
other trails 4 the trails was found less in retention time due to the ratio or organic
solvent in mobile phase.
Selection of wavelength:
The solvent used Acetonitrile : Potassium dihydrogen phosphate (v/ v) in the
ratio of 60:40. It was seen that 240 nm both compounds have very good absorbance ,
which can be used for the estimation of compounds by HPLC.
Selection of Chromatographic Method

Proper selection of the method depends on nature of the sample (ionic or
ionisable or neutral molecules), its molecular weight, pKa value and stability. The
drugs selected in the present study are polar and so reversed phase or ion exchange
chromatography can be used. The reversed phase HPLC was selected for the initial
separation because of its simplicity and suitability.

From the literature survey and with the knowledge of properties of the selected
drugs, Column YMC pack (150 × 4.6mm)5µ was chosen as stationary phase and
mobile phase with different compositions such as Acetonitrile : potassium
dihydrogenphosphte dihydrate pH.4.3 was used.
From all the data observed, obtained, available the initial separation conditions
were set to work around.
Effect of Ratio of Mobile Phase

Under the chromatographic conditions mentioned above, the different ratios of
mobile phase were tried. The chromatograms were observed for each of the trails, out
of whichAcetonitrile : potassium dihydrogenphosphte dihydrate pH.4.3 (v/ v) in the
ratio of 60:40 was selected as the separation was achieved in minimum retention time.
Effect of pH of mobile phase

Several trials were made using different pH range. The best separation was
achieved with pH to 4.3 .
Effect of flow rate on separation

The mobile phase consisting Acetonitrile : potassium dihydrogenphosphte
dihydrate pH.4.3 (v/ v) in the ratio of 60:40and the chromatograms were recorded at
flow rates of 1ml to 2ml. The sharp peaks were obtained with 1 ml flow rate.
Effect of column (Stationary phase) on separation

At the chromatographic conditions of mixed solutions, combinations of
Rosuvastatin and Clopidogrel were injected and chromatograms were obtained using
Column YMC pack (150 × 4.6mm)5µ was preferred for further studies.
Chromatographic Conditions
The following optimized parameters were used as a final method for the
Simultaneous estimation of Rosuvastatin and Clopidogrel .

Instrument WATERS 2998 UV

Column Column YMC pack (150 × 4.6mm)5µ
Column Oven Temperature 30°C

Wave length 240nm
Flow rate: 1ml/min
Injection Volume 10µl
Runtime 18 minutes
Mode of Operation Isocratic
Mobile Phase Acetonitrile : potassium
dihydrogenphosphte dihydrate pH.4.3
Solvent ratio 40:60 v/v
ASSAY BY RP- HPLC METHOD

Preparation of Buffer:
1.36gms of potassiumdihydrogen phosphate in 1000 ml of water. Adjusted to
pH 4.3
Preparation of Mobile Phase:
Mixed Buffer and acetonitrile to the ratio of (60:40)v/v

Preparation of Diluent:
Mobile phase act as a diluent.
Preparation of Standard Stock solution:

Rosuvastatin :
Weigh and transfer accurately 20.4 mg of Rosuvastatin working standard
into a 100ml clean and dry volumetric flask and make up with 50ml of diluent and
sonicate to dissolve.
Clopidogrel :
Weigh and transfer accurately 75.5 mg of Clopidogrel working Standard into
100ml clean and dry volumetric flask and make up with 50ml of diluents and sonicate
to dissolve.

Sample stock solution:

Weighed and finely powdered not less than 20 tablets. Transferred an
accurately weighed portion of the powder equivalent to 100ml volumetric flask and
make up 50 ml with mobile phase sonicated for 15 minutes and cooled to room
temperature. Make up the volume with HPLC water. Mixed well and filtered
through 0.45 nylon filter paper discarded first few ml of the filtrate.
Procedure:
Injected separately 10 µl of the standard preparation into the equilibrated
HPLC system in five replicate and measure the response of the major peak due to
Rosuvastatin and clopidogrel .
Then injected 10 µl of the sample preparation in duplicate and
measured the response of the major peak due to Rosuvastatin and clopidogrel.
Calculate the content of Rosuvastatin and clopidogrel .
Calculation:
Formula for Calculation of % of Rosuvastatin:
AT Std. wt 100 Avg.wt P

---------X------------X----------X-----------X-------------X100
AS 100 Spl wt L.C 100
Where,
AT = Mean peak area due to Rosuvastatin obtained with Sample solution
AS = Mean peak area due to Rosuvastatin obtained with Standard .
. solution
Std.wt = Weight of Rosuvastatin working standard in mg
L.C = Label claim of Rosuvastatin in mg per tablet
P = Potancy of Rosuvastatin working standard on as is basis
Spl wt = weight of the sample in mg
For Clopidogrel :
Calculate the amount of Clopidogrel by using the following formula:

Formula for Calculation of % of clopidogrel:
AT Std. wt 100 Avg.wt P

---------X------------X----------X-----------X-------------X100
AS 100 Spl wt L.C 100
Where,
AT = Mean peak area due to Clopidogrel obtained with Sample solution
AS = Mean peak area due to Clopidogrel obtained with Standard solution
Std.wt = Weight of Clopidogrel working standard in mg
L.C = Label claim of Clopidogrel in mg per tablet
P = Potancy of Clopidogrel working standard on as is basis
Spl wt = weight of the sample in mg
METHOD VALIDATION BY– HPLC
 SYSTEM SUITABILITY:
System suitability of the method was performed by calculating the parameter
namely, resolution, tailing and number of theoretical plates on the five replicate
injection of standard solution. (Table no.2)
Table No.2 system suitability date
System suitability Rosuvastatin Clopidogrel
parameters
Resolution 26.47
Tailing factor 1.00 1.00
No. of Theoretical 8709 46951
plates

Acceptance criteria:
 Resolution should be NLT 2
 Tailing factor should be NMT 2
 No .of theoretical plates should be NLT 2000.

The system suitability parameter and% RSD for peak areas for five replicate
injection of standard solution was found to be within limits.
QUANTITATIVE ESTIMATION:
Assay preparation:
Pipette out 5 ml from sample stock solution into 10 ml volumetric flask and
made up to the volume with mobile phase. Inject the replicate six preparations and
record the peak area response.
 LINEARITY:
Appropriate aliquots of (2.5,4, 4.5, 5, 5.5 & 7.5ml) two drug combination
were pipette out from the stock solution into a series of 1 0ml volumetric flaks
.The volume was made up the mark with mobile phase to obtain a concentration of
Rosuvastatin (Table no.4 ) and clopidogrel (Table no.5 ) 50%, 80%, 90%,100%,
110% and 150%µg/ml). Inject 10µl of each concentration into HPLC system and
chromatographed under the optimized conditions. Evaluation was performed with the
UV detector set at 240 nm and the peak areas were recorded. (fig.1 & fig.2)
Procedure for preparation of standard stock solution

Weigh and transfer accurately 20.4 mg of Rosuvastatin working standard and
75.5 mg of clopidogrel standard into 50 ml of standard measuring flask with 25 ml of
mobile phase, sonicate for 15 min, cooled to room temperature and diluted to 50ml
with diluents.

ROSUVASTATIN :
Table no.4 Linearity of Rosuvastatin
S.No Levels Conc(µg/ml) Area of Rosuvastatin

1 Level 1 (50%) 150 246327
2 Level 2(80%) 240 395236
3 Level 3(90%) 270 445373
4 Level 4(110%) 300 555587
5 Level 5(150%) 450 751874
Intercept 7123.11
Slope 1697.43
Correlation coefficient 0.99897
Acceptance criteria:
The correlation coefficient should be NLT 0.99 for Rosuvastatin .
LINEARITY OF ROSUVASTATIN
fig.1. Linearity curve of Rosuvastatin

CLOPIDOGREL:
Table No.5 Linearity of Clopidogrel
Area of
Clopidogrel
S.No Level Conc(µg/ ml)
1 Level 1(50%) 215.68 959045

2 Level 2(80%) 242.64 108862
3 Level 3(90%) 269.6 1186052
4 Level 4 ( 110%) 296.56 1263394
5 Level 5 (150%) 323.52 1392973
Y- intercept 7364
Slope 4328
Correlation coefficient 0.999
Acceptance criteria: The correlation coefficient should be NLT 0.99 for Clopidogrel .
fig.2. Linearity curve of Clopidogrel

PRECISION:
System precision:
System precision was done by using Rosuvastatin and Clopidogrel
combination. prepared six times and injected into the HPLC system under the
optimized conditions. (Table no.6)
Table no.6 System precision data
SampleNO. Area Response %Assay
Ros Clo Ros Clo
1 50165.724 141887.759 101.192 101.099
2 501127.704 1038564.301 100.438 100.193
3 500734.437 1040896.008 100.409 100.468
4 501456.902 1042130.55 100.821 100.854
5 499521.597 1043039.13 100.8 101.312
6 504295.255 1045289.45 101.274 101.042
Average 504235.7 1043516 100.82 100.83
%RSD 0.73 0.69 0.36 0.42
Acceptance criteria: The %RSD should be NMT 2.

Method precision:
The precision of an analytical procedure is the degree of agreement among the
individual test result when the procedure is applied repeatedly to multiple sampling of
a homogeneous sample.
Preparation of sample solution:

Pipette out 5 ml from sample stock solution into 10 ml volumetric flask and
made up to the volume of 10 ml with mobile phase. Inject the replicate six
preparations and record the peak area response and calculate. (Table no.7)
Table no.7 Method precision data

SampleNO. Area Response %Assay
Ros Clo Ros Clo
1 502544 1040611 98.8 100.63
2 503537 1039917 98.9 100.56
3 503065 1043915 99.35 100.13
4 506671 1043578 99.2 100.35
5 507042 1046922 100.3 99.15
6 502555 1046153 100.4 99.09
Average 504235.7 1043516 99.49 99.985
%RSD 0.73 0.69 0.55 0.32
Acceptance criteria: The %RSD for six samples preparations should not be
more than 2.0

RECOVERY STUDIES :
The accuracy of the method is determined by recovery experiments. The
recovery is performed by adding a known quantity of Rosuvastatin and Clopidogrel
recovery studies to sample (50%, 100%, and 150%). The solution was filtered and
injectedin to the column. The eluate was detected at 240 nm and the chromatogram
was recorded.
SYSTEM SUITABILITY:
The system suitability for Rosuvastatin and Clopidogrel in the optimized
chromatographic conditions was calculated, all the values were compared with the
standard values given the ICH guidelines found to be compatible.

ROBUSTNESS:
Effect of variation in wave length
A study to establish the effect of variation in wavelength of chromtographic
conditions was conducted. Two wave lengths were 238 nm to 242 nm. Standard
solutions prepared as per test method were injected into HPLC System . (Table no.08
& Table no.09)
From the above study it was established that the allowable variation in
wavelength from 238 nm to 242 nm
Table no 8 Robustness (Altered wavelength Rosuvastatin )
System suitabilty
S.NO Change in Wave Length Retention
( ± 2nm) time
Tailaing Plate count
1 Less 238 nm 6.470 1.00 8458
2 Actual 240 nm 6.501 1.00 8709
3 More 242 nm 6.470 1.00 8755

Table no.9 Robustness (Altered wavelength Clopidogrel )
S.NO Change in Wave Length Retention

System suitabilty
( ± 2nm) time
1 Less 238 nm 13.610 1.00 47073
2 Actual 240 nm 13.593 1.00 46951
3 More 242 nm 13.620 1.00 46758
Effect of variation in flow rate:

A study was conducted to determine the effect of variation in flow rate.
Standard solution prepared as per test method was injected into HPLC system with
flow rate. 1 ml/min to 1.2 ml /min. The system suitability parameters were evaluated
with the flow rates as per test method and found to be within the limits. (Table no.10
& Table no.11)
From this study it was established that the allowable variations in flow rate is from
0.9 ml/ min to 1.1 ml/ min.
Table no.10 Robustness (Altered flow rate Rosuvastatin)
System suitabilty
S.NO Change in Flow rate ml Retention time
1 Less 0.9 ml 7.169 1.01 9197
2 Actual 1.0 ml 6.476 1.00 8709
3 More 1.1 ml
5.870 1.00 7842

Table no.11 Robustness (Altered flow rate Clopidogrel )
System suitabilty
S.NO Change in Flow rate ml Retention time
Plate
Tailaing count
1 Flow rate Less (0.9 ml) 14.438 1.01 46904
2 Flow rate Actual (1.0 ml) 13.593 1.00 8709
3 Flow rate More (1.1 ml) 12.858 1.00 46610
Effect of variation in column temperature:

A study was conducted to determine the effect of variation in column
temperature. A standard solution prepared as per test method was injected into HPLC
system at 30° C column temperature. The system suitability parameters were
evaluated with both the column temperature and found to be within the limits. (Table
no.12& Table no.13)
From this study it was established that the allowable variations is from 28° C to
32° C
Table no.12 Robustness (Temperature change in Rosuvastatin)
S.NO Change in temperture Retention System suitabilty

( ± 2° C ) time Tailaing Plate count
1 Less 2° C 6.618 1.08 7468
2 Actual 30° C 6.476 1.00 8709
3 More 2° C 6.449 1.04 8023

Table no.13 Robustness (Temperature change in Clopidogrel)
System suitabilty
S.NO Change in temperture Retention time
Plate
( ± 2° C )
Tailaing count
13.874 1.11 7468

1 Less 2° C
13.593 1.00 46951

2 Actual 30° C
3 More 2° C
13.591 1.05 42779

Chapter 6 Chromatograms
6. CHROMATOGRAMS
System suitability: compound A as Rosuvastatin and compound B as Clopidogrel
Blank Chromatogram

Standard chromatogram-1: compound A as Rosuvastatin and compound

B as Clopidogrel
Standard chromatogram-2: compound A as Rosuvastatin and compound B

as Clopidogrel.


as Clopidogrel.

as Clopidogrel.


as Clopidogrel.
Method precion Chromatogram:
Chromatogram 1 : compound A as Rosuvastatin and compound B as Clopidogrel

Chromatogram 2 : compound A as Rosuvastatin and compound B as

Clopidogrel

Clopidogrel.


Clopidogrel.
Chromatogram 5 : compound A as Rosuvastatin and compound B as Clopidogrel

Linearity 50% Level Chromatogram: compound A as Rosuvastatin and

compound B as Clopidogrel.
Linearity 80% Level Chromatogram: compound A as Rosuvastatin

and compound B as Clopidogrel.

Linearity 90% Level Chromatogram: compound A as Rosuvastatin and

Linearity 100% Level Chromatogram: Compound A as Rosuvastatin and


Linearity 110% Level Chromatogram: Compound A as Rosuvastatin and

Linearity 150% Level Chromatogram-1: Compound A as Rosuvastatin and


System precion Chromatogram: Compound A as Rosuvastatin and

Recovery studies:
50% Level chromatogram Compound A as Rosuvastatin and

100% Level chromatogram Compound A as Rosuvastatin and compound B

as Clopidogrel.
150% Level chromatogram Compound A as Rosuvastatin and compound B

as Clopidogrel.

Robustness: Change in increased Flow rate.

Chromatogram 1: Compound A as Rosuvastatin and compound B as
Clopidogrel.
Chromatogram 2: Compound A as Rosuvastatin and compound B as Clopidogrel.


Clopidogrel.
Robustness: Change in decreased Flow rate.

Clopidogrel.


Clopidogrel.
Robustness: Change in increased temperaure.

Clopidogrel.


Clopidogrel.

Clopidogrel.
Robustness: Change in decreased temperaure.


Clopidogrel.

Clopidogrel.


Clopidogrel.
Robustness: Change in increased wavelength.

Clopidogrel.


Clopidogrel.

Robustness: Change in decresed wavelength.


Clopidogrel.


Clopidogrel.

Chapter 7 Results and Discussion
7. RESULTS AND DISCUSSION

The working condition for the HPLC method was established for Rosuvastatin
and Clopidogrel and then was applied on pharmaceutical dosage forms. A simple
reverse phase High Performance Liquid Chromatography method has been developed
and subsequently validated.
The separation method was carried out by using a mobile phase consisting of
Acetonitrile : potassium dihydrogenphosphte dihydrate pH.4.3 to the ratio of 40:60
(v/ v).
The deduction was carried out by using UV Visible detector at 240nm. The
column was The Column YMC PACK (150 × 4.6mm)5µ flow rate was selected as 1.0
ml/min.
The retention time of was Rosuvastatin and Clopidogrel found to be 6.4 &
13.5. The asymmetry factor or tailing factor of Rosuvastatin and Clopidogrel was
found to be 1.01 & 1.00, which indicates symmetrical nature of the peak. The number
of theoretical plates of Rosuvastatin and Clopidogrel was found to be 8709 & 46951
which indicates the efficient performance of the column. These parameters represent
the specificity of the method.
From the linearity studies, specified concentration levels were determined. It

was observed that Rosuvastatin and Clopidogrel were linear in the range of 80 % to
120 % for the target concentration by RP - HPLC . The linearity range of
Rosuvastatin and Clopidogrel was found to obey linearity with a correlation
coefficient of 0.9989 & 0.9990 respectively.
The validation of the proposed method was verified by system precision and
method precision by RP-HPLC.
The robustness studies were performed by changing the wavelength, flow rate
and temperature.
The validation of the proposed method was verified by precision. The

percentage of precision was found to be satisfied which represent in results. These are
all comes under the specified limits and passes.

Chapter 7 Results and Discussion
The analytical method validation was carried out by RP-HPLC as per ICH
guidelines which are mentioned below as follows.
Parameters Limit Observation Result

S. no
ROS CLO
System Resolution NLT 2 26.47
1. Passes
Tailing factor NMT 2.0 1.00 1.00
suitability
Precision
0.36 0.42 Passes
System
2. %RSD NMT 2.0
precision
Method
0.55 0.32 Passes
precision
3. Linearity Correlation coefficient
0.9989 0.999 Passes
(R2 )NLT
0.99
4. Recovery 98.0% to 102.0% 99.1% 100.4% Passes
Robustness
Change in
0.30 0.28
wavelength Passes
0.25 0.31
238nm
242nm
Change in flow
0.19 0.27 Passes
rate
0.35 0.26
-0.1 ml
% RSD NMT 2.0
5. +0.1 ml
Change in
0.22 0.30
temperature Passes
0.29 0.27
-0.2
+0.2

Chapter 8 Summary and Conclusion
8. SUMMARY AND CONCLUSION
A RP-HPLC method for ROSUVASTATIN AND CLOPIDOGREL was

developed and Validated in tablet dosage form as pre ICH Guide lines
UV visible Detector and Column C18(240 × 4.6mm)5µ injection of 20µl is

injected and eluted with the mobile phase of Acetonitrile : water and 0.1 ml triethylamine
and 0.5% trifluroacetic acid (75:25 v/v) in the ratio 75:25 which was pumped at a flow
rate of 1.0ml at 288nm. The peak of Rosuvastatin and Clopidogrel was found well
separated within 13min. The developed method was validated for various parameters as
per ICH guidelines like system suitability, linearity, system precision, method precision,
recovery, robustness , LOD& LOQ.
The analytical method validation of Rosuvastatin and Clopidogrel n and by RP-

HPLC method was found to be satisfactory and could be used for the routine
pharmaceutical analysis of Rosuvastatin and Clopidogrel .
FUTURE SCOPE
In the above mentioned RP-HPLCmethod for estimation of Rosuvastatin and

Clopidogrel in combined tablet dosage form, and the run time was found to be within 18
minutes, retention time of Rosuvastatin and Clopidogrel is 6.47 & 13.58 minutes. Hence
the present method is Rapid, Specific, Precise, Accurate, Linear can be used for routine
analysis of these drugs from tablet formulation.

Chapter 8 Bibliography
9. BIBLIOGRAPHY
1. Beckette .A.H, Stenlake.J.B. , Practical pharmaceutical chemistry 4th edition.

Part-II. CBS Publisher & Distributors 2007.page No:157-174
2. ChatwalG.R., and shan anand K; “Instrumenral methods of chemical
analysis’’ 2004; 5th edition, page no.2.567-2.645
3. Danzo.K.Analytical chemistry(Theoretical & metrological Fundamentals)CBS
4. Publishers& distributors 2005. Page no:1
5. D Kallol Jana et, al., Development and Validation of a new improved RP-
HPLC Method for estimation of Rosuvastatin calcium in
Pharmaceutical dosage form.
6. De Gruyter et. al., Development and validation of a green RP-HPLC method
for the analysis of rosuvastatin: a step towards making liquid chromatography
environmentally benign(June 2017)
7. ICH Topic Q2A “Validation of analytical procedures” methodology,
6th Nov 1996.
8. ICH Topic Q2B “Validation of analytical procedures” Text, 6th Nov 1996.
9. ICH Harmonized triplicate guideline text on validation of analytical procedure

recommended for adaptation at September4 of the ICH process on 27 October
1994 bythe ICH steering committee. page no: 1-8
10. Lane ohannesian, Anthony, strecter.J. , Hand book of pharmaceutical analysis.
marcel Dekker publisher 2005 page no:59-60.
11. Mohamad Ammar Al‐Khayat et, al., Development and validation of RP-HPLC
method for determination of clopidogrel in tablets ( International Journal of
Pharmaceutical Sciences Review and Research · May 2012)
12. Mendham.J Denney.R.C. Barnes.JD.Thomas . MJK.Vogels Text book of
quantitative chemical analysis 6 th edition pearson Education;2004.Page No 1
13. Pandya C.B. et.al., Development and validation of RP-HPLC method for
determination of Rosuvastatin Calcium in bulk and pharmaceutical dosage
form (International Journal of Pharmaceutical Sciences Review and Research Nov-
2010)

Chapter 8 Bibliography
14. POOJA PISAL et al., RP-HPLC Method Development and Validation for the
15. Simultaneous Estimation of Rosuvastatin and Ezetimibe in Tablet Dosage
Form (IJPAR | Jan-Mar -2014)
16. Parimoo.P. Pharmaceutical Analysis CBS pulishers & Distributors;2012 Page
no :278-300.
17. Radha et. al.,Analytical method development and validation for the
simultaneous estimation of Rosuvastatin and Finofibate in tablet dosage form
by reverse phase high performance liquid chromatography.(Indian Journal of
Research in Pharmacy and Biotechnology)
18. Ravisankar S; Text book of pharmaceutical analysis; 2017;RX Pulication
Tirunalveli; 3rd edition; page no.18.1-18.15
19. Sethi.P.D, Quanitative analysis of drugs in pharmaceutical forulation, 3rd
edition.CBS Publishers & Distributors.New Delhi 1997, page no. 17-19.
20. Skoog A; James Holler F.Niemen. A, “Principles of instrumental Analysis” 5
th edition2005, page No : 733- 738.
21. www.pubchem.com
22. www. medchemexpress.com
23. www.drugbank.com
24. WebmD.com
25. www.Wikipedia.com 27.www.thermoscientific.com
26. www.hplc.chem.shu.edu
27. www.studyhplc.com
28. www.labcompliance.com

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Chapter1 Introduction

Analytical chemistry studies and uses instruments and methods used to

Qualitative inorganic analysis seeks to establish in the presence of a given

Quantitative analysis seeks to establish the amount of a given element or

1. Titrimetric and Gravimetric methods

Dept of Pharmaceutical Analysis 1 JKKMMRF’S COLLEGE OF PHARMACY

Variables in Quantitative Analysis:

 In homogeneity of the medication

Methods validation should not be a one-time situation to fulfill Agency

Dept of Pharmaceutical Analysis 2 JKKMMRF’S COLLEGE OF PHARMACY

A. High Performance Liquid Chromatography (HPLC)

Separation of the enantiomers can be achieved on chiral stationary phases by

Dept of Pharmaceutical Analysis 3 JKKMMRF’S COLLEGE OF PHARMACY

5. Reversed Phase Chromatography

6. Size Exclusion Chromatography

Dept of Pharmaceutical Analysis 4 JKKMMRF’S COLLEGE OF PHARMACY

Stationary Phases (Adsorbents):

Main adsorbent parameters are:

 Pore size: 70 to 300 A0;

The last parameter in the list represents an adsorbent surface chemistry

Mobile phases (eluents):

In HPLC type, composition of the mobile phase (eluent) is one of the

There are several common properties:

Dept of Pharmaceutical Analysis 5 JKKMMRF’S COLLEGE OF PHARMACY

caution is needed as these buffers absorb below 220 nm.

7. Ionizable compounds in some cases can present some problems when

Dept of Pharmaceutical Analysis 6 JKKMMRF’S COLLEGE OF PHARMACY

Standard HPLC pump requirements are:

Dual Piston Pumps

Dept of Pharmaceutical Analysis 7 JKKMMRF’S COLLEGE OF PHARMACY

Problems in pumping liquids

In liquid chromatography, liquid samples may be injected directly and solid

Dept of Pharmaceutical Analysis 8 JKKMMRF’S COLLEGE OF PHARMACY

Dept of Pharmaceutical Analysis 9 JKKMMRF’S COLLEGE OF PHARMACY

Choice of the Column

Ultraviolet Visible spectroscopic Detectors

Dept of Pharmaceutical Analysis 10 JKKMMRF’S COLLEGE OF PHARMACY

Infrared (IR) 2500 - 50000 nm

In UV detection, one expresses the detector range in absorbance units

Fixed wavelength detectors.

Dept of Pharmaceutical Analysis 11 JKKMMRF’S COLLEGE OF PHARMACY

Refractive index Detector, Deflection detectors, Reflective detectors,

Diode array permits qualitative information to be obtained beyond simple

Dept of Pharmaceutical Analysis 12 JKKMMRF’S COLLEGE OF PHARMACY

The advantages of intelligent processors in chromatographs:

Capacity factor (k´)

The capacity factor is a measure of where the peak of interest is located

Generally the value of k'is > 2.

Dept of Pharmaceutical Analysis 13 JKKMMRF’S COLLEGE OF PHARMACY

Precision/injection repeatability (RSD)

Tailing factor (T)

N=16 (tR/tW)2= L/H

N - Constant for each peak on a chromatogram with a fixed set of operating

Dept of Pharmaceutical Analysis 14 JKKMMRF’S COLLEGE OF PHARMACY

Relative retention (α)

Relative retention is a measure of the relative location of two peaks. This is

Retention volume (Rv)21

Retention volume = Retention time x flow rate (Ravi sankar S, 2006)

Chromatographic test methods use either external or internal standards for

A. An external standard method-- is used when the standard is analyzed on a

Dept of Pharmaceutical Analysis 15 JKKMMRF’S COLLEGE OF PHARMACY

B. With an internal standard method -- compound of known punity that does

The working concentration is the target concentration of the compound of