LIST OF ABBREVIATIONS

Avg Average
C.f Conversion Factor
Diln Dilution
FDA Food and Drug Administration
HPLC High Performance Liquid Chromatography
ICH International Conference Of Harmonization
LC Liquid Chromatography
LC-MS Liquid Chromatography-Mass Spectroscopy
LOD Limit of detection
LOQ Limit of quantitation
NMT Not more than
NLT Not less than
ODS Octyl deactivated silane
ppm Parts Per Million
RSD Relative Standard Deviation
RT Retention time
RP-HPLC Reverse Phase High Performance Liquid Chromatography
SPL Sample
STD Standard
SD Standard Deviation
US FDA United States Food and Drug Administration
UV Ultra-Violet
µl Micro liter
Wt Weight
%w/w Percent weight per weight
% v/v Percent volume per volume
IS Internal Standard
HPTLC High Performance Thin Layer Chromatography
GC Gas Chromatography
IR Infrared Spectroscopy
TLC Thin Layer Chromatography
nm Nanometer
min Minute
UPLC Ultra Performance Liquid Chromatography
 CONTENTS

CHAPTER PAGE
TITLES
NO NO

1. INTRODUCTION 1

2. LITERATURE REVIEW 30

3. OBJECTIVE AND PLAN OF WORK 41

4. DRUG PROFILE 42

5. MATERIALS AND INSTRUMENTS USED 47

6. METHOD DEVELOPMENT AND OPTIMIZATION 49

7. METHOD VALIDATION 59

8. CHROMATOGRAMS 69

9. RESULTS AND DISCUSSION 87

10. SUMMARY AND CONCLUSION 90

11. BIBLIOGRAPHY 91
Chapter1 Introduction

1. INTRODUCTION

Analytical chemistry is the science to analyze morphologies, compositions, and quantities

of analytical targets. These analytical results have played critical roles from theunderstanding of
basic science to a variety of practical applications, such as biomedical applications, environmental
monitoring, quality control of industrial manufacturing, and forensic science, to name a few
(P.D.Sethi,2001).
Modern analytical chemistry is dominated by instrumental analysis. There are so many
different types of instruments today that it can seem like a confusing array of acronyms rather than
a unified field of study. Many analytical chemists focus on a single type of instrument. Academics
tend to either focus on new applications and discoveries or on new methods of analysis. An effort
to develop a new method might involve the use of a tunable laser to increase the specificity and
sensitivity of a spectrometric method. Many methods, once developed, are kept purposely static
so that data can be compared over long periods of time. This is particularly true in industrial quality
assurance (QA), forensic and environmental applications. Analytical chemistry plays an
increasingly important role in the pharmaceutical industry where, aside from QA, it is used in
discovery of new drug candidates and in clinical applications where understanding the interactions
between the drug and the patient are critical.

Types
Traditionally, analytical chemistry has been split into two main types, qualitative and
quantitative:

Qualitative
• Qualitative inorganic analysis seeks to establish the presence of a given element or
inorganic compound in a sample.
• Qualitative organic analysis seeks to establish the presence of a given functional group or
organic compound in a sample.

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Chapter1 Introduction
Quantitative

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

sample (H.H.Willard,1986).

Most modern analytical chemistry is categorized by two different approaches such as analytical
targets or analytical methods (Sharma.B.K,2005).

By Analytical Targets
• Bioanalytical chemistry
• Material analysis
• Chemical analysis
• Environmental analysis
• Forensics

By Analytical Methods
• Spectroscopy
• Mass Spectrometry
• Spectrophotometry and Colorimetry
• Chromatography and Electrophoresis
• Crystallography
• Microscopy
• Electrochemistry

Traditional analytical techniques

• Titration
• Gravimetry
• Inorganic qualitative analysis

Instrumental Analysis
• Spectroscopy
• Mass Spectrometry
• Crystallography
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Chapter1 Introduction

• Electrochemical Analysis
• Thermal Analysis
• Separation
• Hybrid Techniques
• Microscopy

Analytical chemistry research is largely driven by performance (sensitivity, selectivity,

robustness, linear range, accuracy, precision, and speed), and cost (purchase, operation, training,
time, and space).

Analytical chemistry has played critical roles in the understanding of basic science to a
variety of practical applications, such as biomedical applications, environmental monitoring,
quality control of industrial manufacturing, forensic science and so on.

Pharmaceutical Analysis plays a very significant role in quality control of pharmaceuticals

through a rigid check on raw materials used in manufacturing of formulations and on finished
products. It also plays an important role in building up the quality products through in process
quality control. Pharmaceutical analysis is the application of principles of analytical chemistry to
drug analysis. The analytical chemistry may be defined as the science of developing sensitive,
relative and accurate methods for determining the composition of materialsin terms of elements or
compounds which they contain(D.A.Skoog, 1998).
The most important aspect of analysis is quantitative chemical analysis. In the present age,
the physical, chemical and biological analysis, Involve computerized techniques to facilitatebetter
result.

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 Chapter1 Introduction
ANALYTICAL TECHNIQUES:

TECHNIQUES APPLICATIONS
SPECTROSCOPY

Atomic Absorption To analyse alkali and alkaline earth metals in

And Emission Spectroscopy(AAS/AES) dilute solution, natural liquids, and extracts at

trace levels.

Ultraviolet-Visible To analyse molecular (organic) and ionic

species capable of absorbing at UV or Visible
Spectroscopy (UV/Vis)
wavelengths in dilute solutions.

Fourier Transform Infrared To analyse only molecular compounds (organic

compounds, natural products, polymers, etc.).
Spectroscopy (FT-IR)

Fourier Transform Raman To analyse molecular (organic) compounds

which are not responding well in the IR region
Spectroscopy (FT-Raman)
and hence, it is an alternate to IR.

Nuclear Magnetic Resonance To identify and characterize the organic and

inorganic compounds.
Spectroscopy (NMR)

Microwave Spectroscopy To analyse simple gaseous molecules in Far IR

region, to study their stereo chemistry.

Electron Spin Resonance To study the formation and life time of the free
radicals formed in organic reactions and also
Spectroscopy (ESR)
finds applications in biological works.

Molecular Fluorescence To study the molecular and ionic compounds

Spectroscopy in dilute solutions capable of giving

fluorescence, applications in vitamin analysis.

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Chapter1 Introduction

CHROMATOGRAPHY

High Performance Liquid To separate and analyse complex mixtures or

Chromatography (HPLC) solutions which include liquids and solids of

both organic and inorganic origins.

Gas Chromatography (GC) To separate and analyse mixtures of volatile

organic compounds, solvent extracts and gases.

THERMAL ANALYSIS

Thermo gravimetric Analysis To study the mass changes of materials like

polymers, glasses, ceramics, etc., such as
(TGA)
evaporation, decomposition gas absorption, de-
sorption, dehydration, etc.

Thermo mechanical Analysis To study the expansion coefficient of

composite and laminate materials.
(TMA)

Differential Thermal Analysis To study the exothermic and endothermic

behavior of clay materials, ceramics, ores, etc.
(DTA)

Differential Scanning To study the glass transition temperature, curing

process of the thermo set polymers and heat of
Calorimetric (DSC)
melting of thermoplastic polymers.

X-RAY TECHNIQUE

X-Ray Fluorescence (XRF) To identify the elements and their valence

states present in the surface of the materials.
Spectrometry and

X-Ray Photoemission

Spectrometry (XPS)

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Chapter1 Introduction

X-Ray Diffractometry (XRD) To study the crystalline properties of solid

substances.

MICROSCOPY

Scanning Electron Microscopy (SEM) To study the topography, electronic structure

and compositions of metals, ceramics,
polymers, composites and biological materials.

Transmission Electron To study the local structures, morphology, and

dispersion of multicomponent polymers, cross
Microscopy (TEM)
sections and crystallizations of metallic alloys,
semiconductors, microstructure of composites,
etc.

Scanning Probe Microscopy To study the hardness and topography of

(SPM) materials like ceramics, polymers, composites,

etc., on a nano-scale range.

ELECTRO-CHEMICAL TECHNIQUES

Polarography To study and determine metals, metal

complexes and organic compounds in trace
levels.

Capillary Electrophoresis (CE) To study and characterize biologically active

compounds like proteins, amino acids and
other bio-molecules.

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Chapter1 Introduction

MISCELLANEOUS TECHNIQUES

Total Organic Carbon Analyzer To monitor pollutants in environmental studies

by determining the carbon contents of the trace
(TOC)
compounds.

Elemental Analyzer (CHN/S) To estimate percentage compositions of

elements like carbon, hydrogen, nitrogen and
sulphur present in newly synthesized organic
compounds, pharmaceuticals, etc.

Polarimetry To analyse and quantitative optically active

compounds like sugar.

Circular Dichroism (CD) and To get the structural information of optically

active Compounds like, amino acids, proteins,
Optical Rotatory Dispersion
etc.
(ORD)

Vibrational Circular Dichroism Same as above but in the IR region. VLD

measurement is employed to study the
(VCD) and Vibrational Linear
molecular orientations of thin polymer films.
Dichroism (VLD)

Mass Spectrometry (MS) To identify the organic compounds. Often used

as detectors with HPLC and GC.

Laser Light Scattering System In the study of macromolecules like polymers,

gels, proteins,etc., for determining molecular
(LLIS)
mass & size and their associations.

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Chapter1 Introduction

CHROMATOGRAPHY

Introduction: Chromatography (from Greek: chroma, colour and: "grafein" to write) is the
collective term for a family of laboratory techniques for the separation of mixtures. It involves
passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the
analyte to be measured from other molecules in the mixture and allows it to be isolated(P.D.Sethi,
2001).

Chromatography may be preparative or analytical. Preparative chromatography seeks to separate

the components of a mixture for further use (and is thus a form of purification). Analytical
chromatography normally operates with smaller amounts of material and seeks to measure the
relative proportions of analytes in a mixture. The two are not mutually exclusive.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD

HPLC was introduced commercially in 1969 and since then it has undergone extensive
modifications and innovation which lead to its emergence as the foremost analytical tool for
quantitative analysis. HPLC is a type of liquid chromatography that employs a liquid mobile phase
and a very finely divided stationary phase. In order to obtain a satisfactory flow rate liquidmust be
pressurized to a few thousands of pounds per square inch.

The rate of distribution of drugs between stationary and mobile phase is controlled by
diffusion process. If diffusion is minimized, a faster and effective separation can be achieved.
The technique of high performance liquid chromatography is so called because of its improved
performance when compared to classical column chromatography. Advances in column
technology, high pressure pumping system and sensitive detectors have transformed liquid column
chromatography into high speed, efficient, accurate and highly resolved method ofseparation.

For the present study a combination of two drugs Amlodipne and Telmisartan wasselected
for their estimation. The HPLC method was considered the choice of estimation, since

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Chapter1 Introduction

this method is the most powerful of all chromatographic and other separative methods. The HPLC
method has enabled analytical chemist to attain great success in solving his analytical problems.

The HPLC is the method of choice in the field of analytical chemistry, since this method is
specific, robust, linear, precise, and accurate and the limit of detection is low and also it offers
the following advantages.

• Speed (many analysis can be accomplished in 20 minutes or less)

• Greater sensitivity (various detectors can be employed)

• Improved resolution (wide variety of stationary phases)

• Reusable columns (expensive columns but can be used for many analysis)

• Ideal for the substances of low volatility

• Easy sample recovery, handling and maintenance

• Instrumentation leads itself to automation and quantitation

• Precise and reproducible

• Calculations are done by integrator itself

• Suitable for preparative liquid chromatography on a much large scale.

Principle of separation in HPLC

The principle of separation in normal phase mode and reverse phase mode is adsorption.
When a mixture of components is introduced in to a column, they travel according to their relative
affinities towards stationary phase. The component which has more affinity towards the adsorbent
travels slower. The component which has less affinity towards stationary phase travels faster. Since
no two components have the same affinity towards the stationary phase, the components are
separated.

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Chapter1 Introduction

The HPLC system is shown in below fig

Schematic Representation of HPLC:

Solvent container ⎯ Pump ⎯ Damping unit ⎯ Injection port
|
Column
|
Recorder ⎯ Detector
|
Effluent

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Chapter1 Introduction

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. 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.
Isocratic and Gradient elution
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.

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 Chapter1 Introduction

SAMPLE INTRODUTION SYSTEM

Two means for analyte introduction on the column are injection in to a flowing stream and a
stop flow injection. These techniques can used with a syringe or an injection valve. Automatic
injector is a microprocessor-controlled be 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.
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 μm size) packed in such a way that highestnumbers of
theoretical plates are possible.
Silica (SiO2 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.
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, suchmaterials 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 siloxanelinkages 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

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 Chapter1 Introduction
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.

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(Beckett and Stenlake,2001).

Bulk property detectors

Solute property detectors

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.
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.

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Chapter1 Introduction
Types of HPLC techniques
i) Based on Modes of Chromatography

(a) Normal Phase chromatography

(b) Reverse Phase chromatography

ii) Based on principle of separation

(a) Adsorption chromatography

(b) Ion exchange chromatography

(c) Ion pair chromatography

(d) Size exclusion chromatography

(e) Affinity chromatography

(f) Chiral phase chromatography

iii) Based on elution technique

(a) Isocratic separation

(b) Gradient separation

iv) Based on the scale of operation

(a) Analytical HPLC,

(b) Preparative HPLC.

1) Normal Phase Chromatography:

The term normal phase refers to a system where a stationary phase is polar and mobile phase
is a relatively non polar liquid (hexane, benzene, chloroform, etc). In this mode most probably
used stationary phase is silica gel. The silica structure is saturated with silanol groups at the
end and ‘OH’ groups attached to silicon atoms are the active binding sites. Best separating
compounds include plasticizer, dyes, steroids, amines, alkaloids, alcohols, phenols, aromatic
and metal complexes.
2) Reversed Phase Chromatography:
A most popular mode for analytical and preparative method for separation of compounds of
interest in chemical, biological, pharmaceutical, food and biomedical sciences. One common

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Chapter1 Introduction

stationary phase is silica which has been treated with RMe2SiCl, where R is a straight chain
alkyl group such as C18H37 or C8H17. The retention time is therefore longer for molecules which
are more non-polar in nature, allowing polar molecules to elute more readily. Retention Time
(RT) is increased by the addition of polar solvent to the mobile phase and decreased by the
addition of more hydrophobic solvent. In this mode the stationary phase is a non polar
hydrophobic packing with octyl or octadecyl functional group bonded to the silica gel and
mobile phase is a polar solvent. An aqueous mobile phase allows the use of secondary solute
chemical equilibria (such as ionization control, ion suppression, ion pairing and complexation)
to control retention and selectivity.
The polar compound gets eluted first in this mode and non-polar compounds are retained for
a larger time. Since most of the drugs and pharmaceuticals are polar in nature, they are not
retained for longer time and eluted faster. The different columns used are octadecyl silane or
C18, C8, C4 etc (In order to increase the polarity of the stationary phase). Hence by varying
the organic moiety in the silianization reagent (dimethyl chlorosilane derivatives) different
stationary phase of polarities can be realized (R.Synnder,1997).
3) Adsorption Chromatography
In adsorption chromatography, the mobile phase containing the dissolved solutes passes
over the surface of the stationary phase. Retention of the component and their consequent
separation depends on the ability of the atoms on the surface of stationary phase to remove the
solutes from the mobile phase and adsorb them temporarily by means of electrostatic forces.
Usually silica or alumina is utilized as the adsorbent with relatively non-polar solvents such as
hexane as the mobile phase in normal phase adsorption whereas in reversed phase adsorption non
polymer beds with relative polar solvents such as water, acetonitrile methanol are used as mobile
phase.

4) Partition chromatography
In partition chromatography an inert solid material such as silica gel or diatomaceous earth
serves to support a thin layer of liquid which is the effective stationary phase. As the mobile phase
containing the solutes passes in close proximity to this liquid phase, retention and separation occurs
due to the solubility of the analytes in the two fluids as determined by their partition coefficients.
The method suffers from disadvantage due to some solubility of stationary

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Chapter1 Introduction

phase in the mobile phase. Hence precautions must be taken to limit dissolution of stationary phase.

5) Ion exchange chromatography

In ion exchange chromatography the stationary phase consists of a polymeric resin matrix
on the surface of ionic functional groups, eg., carboxylic acids or quaternary amines, have been
bonded chemically. As the mobile phase passes over this surface, ionic solutes are retained by
forming electrostatic chemical bonds with the functional groups. The mobile phase used in this
type is always liquid.

6) Size Exclusion Chromatography

In size exclusion chromatography the stationary phase is a polymeric substance containing
numerous pores of molecular dimensions. Solutes whose molecular size is sufficientlysmall will
leave the mobile phase to diffuse into the pores. Large molecules which will not fitinto the
pores remain in the mobile phase and are not retained. This method is mostly suitable for the
separation of mixtures in which the solutes vary considerably in molecular size. The mobile phase
in this type may be liquid or gaseous.

Isocratic flow and gradient elution

With regard to the mobile phase, a composition of the mobile phase that remains constant
throughout the procedure is termed isocratic.

In contrast to this is the so called "gradient elution", which is a separation where the
mobile phase changes its composition during a separation process. One example is a gradient in
20 min starting from 10 % Methanol and ending up with 30 % Methanol. Such a gradient can be
increasing or decreasing. The benefit of gradient elution is that it helps speed up elution by
allowing components that elute more quickly to come off the column under different conditions
than components which are more readily retained by the column. By changing the composition of
the solvent, components that are to be resolved can be selectively more or less associated with the
mobile phase. As a result, at equilibrium they spend more time in the solvent and less time in the
stationary phase, and therefore they elute faster.

VARIOUS METHODS OF QUANTITATIVE ANALYSIS IN HPLC

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Chapter1 Introduction

Quantitication involves the measurement of peak height or peak area.To determine the
concentration of a compound, the peak area or height is plotted Vs the concentration of the
substance For well resolved peaks, both peak height and area are proportional to the
concentration.Five different calibration methods used in quantitative analysis are,

• Calibration by Standards

• Normalized peak area

• External standard addition method

• Internal standard addition method and

• Standard addition method

Calibration by Standards

Calibration curves for each component are prepared from pure standards, using identical
injection volumes of operating conditions for standards and samples. The concentration
of solute is read from its curve if the curve is linear.
X = K x area.

Where, X = Concentration of solute.

In this evaluation method only the area of the peaks of interest is measured. Relative
response factors must be considered when converting areas to volume and when the response of
a given detector differs for each molecular type of compounds.

Normalized Peak Area

The area present of any individual peak is referred to as the normalized peak area. The
technique of normalized peak area is actually not a calibration method, since there is no
comparison to known amounts for any peak in the chromatogram.

External Standard Calibration

The most general method for determining the concentration of an unknown sample is to
construct a calibration plot using external standards. Standards are prepared at known
concentrations. A fixed volume of each standard solution is injected and analyzed, and the peak
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Chapter1 Introduction
responses are plotted Vs concentration. The standard solutions are referred to as external standards,
since they are prepared and analyzed in separate chromatograms from those of the unknown
samples. Unknown samples are then prepared, injected and analyzed in exactly the same manner.

Internal Standard Calibration

In this technique a known quantity of the internal standard is chromatographed and area
versus concentration is ascertained. Then a quantity of the internal standard is added to the raw
sample prior to any sample pretreatment or separation operations.

The peak area of the standard in the sample run is compared with the peak area when the
standard is run separately. This ratio serves as a correction factor for variation in sample size, for
losses in any preliminary pretreatment operations, or for incomplete elution of the sample. The
material selected for the internal standard must be completely resolved from adjacent sample
components, must not interfere with the sample components and must never be present in samples.

Method of Standard Addition

A calibration standard ideally should be prepared in a blank matrix of drug

formulation components without the drug substance or an animal without added compound
usually can be used for standard calibration solutions. The method of standard addition is most
often used in trace analysis. In this approach, different weights of analyte(s) are added to the
sample matrix, which initially contains an unknown concentration of the analyte. Extrapolation
of a plot of response found for the standard addition calibration concentration to zero
concentration defines the original concentration in the unspiked sample.

INTRODUCTION TO VALIDATION

Method validation is the process to confirm that the analytical procedure employed for a
specific test is suitable for its intended use. Methods need to be validated or revalidated.
• Before their introduction into routine use
• Whenever the conditions change for which the method has been validated, e.g., instrument
with different characteristics.
• Whenever the method is changed, and the change is outside the original scope of the

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Chapter1 Introduction
method.
Method validation is completed to ensure that an analytical methodology is accurate,specific,
reproducible and rugged over the specified range that an analyte will be analyzed. Method
validation provides an assurance of reliability during normal use, and is sometime referred to
as "the process of providing documented evidence that the method does what it is intended to
do." Regulated laboratories must perform method validation in order to be in compliance with
FDA regulations(FDA,2000).

For method validation, these specifications are listed in USP Chapter <1225>, and can be
referred to as the "Eight Steps of Method Validation," as shown in figure below. These terms are
referred to as "analytical performance parameters", or sometimes as "analytical figures of
merit." Most of these terms are familiar and are used daily in the laboratory. However some may
mean different things to different people. Therefore, in order to continue the discussion of method
validation, it is necessary to have a complete understanding of the terminology and definitions.13

The USP Eight Steps of Method Validation

In response to this situation, one of the first harmonization projects taken up by the ICH
was the development of a guideline on the "Validation of Analytical Methods: Definitions and
Terminology." ICH divided the "validation characteristics" somewhat differently, as outlined in

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Chapter1 Introduction
Figure below

ICH Method Validation Parameters

METHOD VALIDATION

The developed methods were validated by following steps(R.Synder,1997).

• Accuracy
• Precision
• Specificity
• Limit of detection
• Limit of quantitation
• Linearity of range
• Ruggedness and
• Robustness

KEY PARAMETERS OF THE ANALYTICAL METHOD VALIDATION

It is important for one to understand the parameters or characteristics involved in the

validation process. The various performance parameters, which are addressed in a validation
exercise, are grouped as follows.

(1) Accuracy :
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 Chapter1 Introduction

The accuracy of an analytical method may be defined as the closeness of the test results
obtained by the method to the true value. It is the measure of the exactness of the analytical method
developed. Accuracy may often express as percent recovery by the assay of a known amount of
analyte added. Accuracy may be determined by applying the method to samples or mixtures of
excipients to which known amount of analyte have been added both above and below the normal
levels expected in the samples. Accuracy is then calculated from the test results as the percentage
of the analyte recovered by the assay. Dosage form assays commonly provide accuracy within 3-
5 % of the true value. The ICH documents recommend 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 replicated of each concentration).

(2) Precision :

The precision of an analytical method is the degree of agreement among individual test
results when the method is applied repeatedly to multiple samplings of homogenous samples. This
is usually expressed as the standard deviation or the relative standard deviation (coefficient of
variation). Precision is a measure of the degree of reproducibility or of the repeatability of the
analytical method under normal operating circumstances. Repeatability involves analysis of
replicates by the analyst using the same equipment and method and conducting the precision study
over short period of time while reproducibility involves precision study, different occasions,
different laboratories, different batch of reagents, different analysts and different equipments.
Determination of Repeatability :
Repeatability can be defined as the precision of the procedure when repeated by same analyst under
the same operating conditions (same reagents, equipments, settings and laboratory) over a short
interval of time. It is normally expected that at least six replicates be carried out and a table showing
each individual result provided from which the mean, standard deviation and co-efficient of variation
should be calculated for set of n value. The RSD values are important for

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Chapter1 Introduction

showing degree of variation expected when the analytical procedure is repeated several time in a
standard situation. (RSD below 1% for built drugs, RSD below 2 % for assays in finished product).
The ICH documents recommend 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 six determinations at 100 % of the test
concentration).

Determination of reproducibility :

Reproducibility means the precision of the procedure when it is carried out under different
conditions-usually in different laboratories-on separate, putatively identical samples taken from
the same homogenous batch of material. Comparisons of results obtained by different analysts, by
the use of different equipments, or by carrying out the analysis at different times canalso provide
valuable information.

(3) Selectivity and Specificity :

The selectivity of an analytical method is its ability to measure accurately and specifically
the analyte of interest in the presence of components that may be expected to be present in the
sample matrix. Selectivity may be expressed in terms of the bias of the assay results obtained
when the procedure is applied to the analytes in the presence of expected levels of other
components, compared the results obtained when the procedure is applied to the analyte in the
presence of expected levels of other components, compared to the results obtained on the same
analytes without added substances. When the other components are all known and available,
selectivity may be determined by comparing the test results obtained on the analytes with and
without the addition of the potentially interfering materials. When such components are either
unidentified or unavailable, a measure of selectivity can often be obtained by determining the
recovery of a standard addition of pure analytes to a material containing a constant level of the
other components.

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Chapter1 Introduction

Limit of Detection and limit of Quantitation:

(4) Limit of Detection (LOD) :

The limit of detection is the parameter of limit tests. It is the lowest level of analytes that
can be detected, but not necessarily determined in a quantitative fashion, using a specific method
under the required experimental conditions. The limit test thus merely substantiates that the
analytes concentration is above or below a certain level. The determination of the limit of detection
of instrumental procedures is carried out by determining the signal-to-noise ratio by comparing
test results from the samples with known concentration of analytes with those ofblank samples
and establishing the minimum level at which the analyte can be reliably detected. A signal-to-noise
ratio of 2:1 or 3:1 is generally accepted. The signal-to-noise ratio is determinedby dividing the
base peak by the standard deviation of all data points below a set threshold. Limit of detection is
calculated by taking the concentration of the peak of interest divided by threetimes the signal-
to-noise ratio. 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 = 3.3 Sa / b

(5) Limit of quantitation (LOQ):

Limit of quantitation is a parameter of quantitative assays for low levels of compounds in sample
matrices such as impurities in bulk drugs and degradation products in finished pharmaceuticals.
The limit of quantitation is the lowest concentration of analyte in a sample that may be determined
with acceptable accuracy and precision when the required procedure is applied. It is measured by
analyzing samples containing known quantities of the analyte and determining the lowest level at
which acceptable degrees of accuracy and precision are attainable. Where thefinal assessment
is based on an instrumental reading, the magnitude of background response by analyzing a number
of blank samples and calculating the standard deviation of this response. Thestandard deviation
multiplied by a factor (usually 10) provides an estimate of the limit of

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Chapter1 Introduction

quantitation. In many cases, the limit of quantitation is approximately twice the limit of
detection.

LOQ=10 Sa/b

(6) Linearity and range :

The linearity of an analytical method is its ability to elicit test results that are directly (or
by a well defined mathematical transformation) proportional to the analyte concentration in
samples within a given range. Linearity usually expressed in terms of the variance around the slope
of regression line Calculated according to an established mathematical relationship from test
results obtained by the analysis of samples with varying concentrations of analytes. The linear
range of delectability that obeys Beer’s law is dependent on the compound analyzed and the
detector used. The working sample concentration and samples tested for accuracy should be in the
linear range. The claim that the method is linear is to be justified with additional mentionof zero
intercept by processing data by linear least square regression. Data is processed by linear least
square regression declaring the regression co-efficient and b of the linear equation y = ax +b
together with the correlation coefficient of determination r2. For the method to be linear the r2 value
should be close to1. The range of an analytical method is the interval between the upperand
lower levels of the analyte (including these levels) that have been demonstrated to be determined
with precision, accuracy and linearity using the method as written.

(7) Ruggedness :

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 normal test conditions such as
different laboratories, different analysts, using operational and environmental conditions that
may differ but are still within the specified parameters of the assay. The testing of ruggedness is
normally suggested when the method is to be used in more than one laboratory. Ruggedness is
normally expressed as the lack of the influence on the test results of operational and environmental
variables of the analytical method. For the determination of ruggedness, thedegree of
reproducibility of test result is determined as function of the assay variable. This

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Chapter1 Introduction

reproducibility may be compared to the precision of the assay under normal condition to obtain a
measure of the ruggedness of the analytical method.

(8) Robustness:
Robustness is the capacity of a method to remain unaffected by small deliberate variations
in method parameters. The robustness of a method is evaluated by varying method parameters
such as percent organic, pH, ionic strength, temperature, etc., and determining the effect (if any)
on the results of the method. As documented in the ICH guidelines, robustness should be
considered early in the development of a method. In addition, if the results of a method or other
measurements are susceptible to variations in method parameters, these parameters should be
adequately controlled and a precautionary statement included in the method documentation.

(9) System Suitability Test (SST):

SST is commonly used to verify resolution, column efficiency, and repeatability of the
chromatographic system to ensure its adequacy for a particular analysis. According to the United
States pharmacopoeia (USP) and the International Conference on Harmonization (ICH), SST is an
integral part of many analytical procedure.
Primary SST parameters are most important as they indicate system specificity, precision
and column stability. Other parameter include capacity factor (K) and signal to noise ratio (S/N)
for impurity peaks. The purpose of system suitability test is to ensure that the complete testing
system (including instrument, reagents, column, and analyst) is suitable for intended application
(I.C.H.Q2A,1995).

The USP chromatography general chapter states:

“System suitability tests are an integral part of gas and liquid chromatographic methods. They
are used to verify that the resolution and reproducibility of the chromatographic system are
adequate for the analysis to be done. The tests are based on the concept that the equipment,
electronics, analytical operations and samples to be analyzed constitute an integral system can be
evaluated as such” (USP 25-NF 20,2002).

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Chapter1 Introduction

SYSTEM SUITABILITY PARAMETERS FOR HPLC

❖ Retention (KA)

❖ Resolution (Rs)

❖ Capacity factor (k/)

❖ Selectivity (α)

❖ Column efficiency (N) and

❖ Peak asymmetry factor (As)

i) Retention (KA)

The retention of a drug with a given packing material and eluent can be expressed as
retention time or retention volume, but both of these are dependent on flow rate, column length
and column diameter. The retention is best described as a column capacity ratio (K), which is
independent of these factors. The column capacity ratio of a compound (A) is defined as
V−
Vt − t
K A 0 A 0
A= 0 = 0
V t
Where,

VA = Elution volume of A

V0 = Elution volume of a non retained compound (void volume)

At constant flow rate, retention times (tA and to) can be used instead of retention volumes.

Retention data is sometimes expressed, relative to a known internal standard (B). The ratio of
t A −to 
  is better
retention times (tA / tB) can be used, but the ratio of adjusted retention times −t
 tB o 
when data need to be transferred between different chromatographs.

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Chapter1 Introduction

ii) Resolution (Rs)

The resolution, Rs of two neighboring peaks is defined by the ratio of the distance between
the two peak maxima. It is the difference between the retention times of two solutes divided by
their average peak width. For baseline separation, the ideal value of Rs is 1.5. It is calculated by
using the formula,

Where,

Rt1 and Rt2 are the retention times of components 1 and 2

W1 and W2 are peak widths of components 1 and 2.

iii) Capacity factor (k/ )

Capacity factor, k/ is defined as the ratio of the number of molecules of solute in the
stationary phase to the number of molecules of the same in the mobile phase. Capacity factor is a
measure of how well the sample molecule is retained by a column or TLC plate during an isocratic
separation. The ideal value of k/ ranges from 2 - 10. Capacity factor can be determined by using
the formula,

Vl - Vo
K/=
Vo
Where, V1 = retention volume at the apex of the peak (solute) and V0 = void volume of the system.

The values of k/, individual bands increase or decrease with changes in solvent strength. In
reversed phase HPLC, solvent strength increases with the increase in the volume of organic phase
in the water / organic mobile phase. Typically an increase in percentage of the organic phase by
10 % by volume will decrease k/ of the bands by a factor of 2 - 3.

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Chapter1 Introduction

iv) Selectivity (α)

The selectivity (or separation factor), α , is a measure of relative retention of two

components in a mixture. The ideal value of selectivity is 2. It can be calculated by using the
formula,

V2 - Vo
α =
V1 - Vo
Where, V0 is the void volume of the column and V2 and V1 are the retention volumes of the
second and the first peak respectively.

v) Column efficiency

Efficiency, N, of a column is measured by the number of theoretical plates per meter. It is

a measure of band spreading of a peak. Smaller the band spread, higher is the number of theoretical
plates, indicating good column and system performance. Columns with N ranging from 5,000 to
100,000 plates / meter are ideal for a good system. Efficiency is calculated byusing the formula,
Rt2
N = 16
W2
Where, Rt is the retention time and w is the peak width.

vi) Peak asymmetry factor (As)

Peak asymmetry factor, As can be used as a criterion of column performance. The peak half
width, b, of a peak at 10 % of the peak height, divided by the corresponding front half width, a,
gives the asymmetry factor.

As = b/a

Data elements required for assay validation (Gurdeep Chatwal, 2002).

It is not always necessary to evaluate every analytical performance parameter, as different

test methods require different validation schemes. The Most common categories of assays for
which validation data should be required are as follows:

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 Chapter1 Introduction

i) Quantitation of major components or active ingredients.

ii) Determination of impurities or degradation compounds.
iii) Determination of performance characteristics

Category-I: Analytical methods for quantitation of major components of bulk drug substances or
active ingredients (including preservatives) in finished pharmaceutical products.
Category-II: Analytical methods for determination of impurities in bulk drug substances or
degradation compounds in finished pharmaceutical products. These methods includes quantitative
assays and limit tests.
Category-III: Analytical methods for determination of performance characteristics (e.g.
dissolution, drug release).
The type of method and its intended use indicates which parameters are required to be
investigated. They are illustrated in the following Table.
Data elements required for assay validation

Analytical Assay category-II Assay

Assay
Performance Category-III
Category-I Quantitative Limit Test
Parameter

Accuracy Yes Yes * *

Precision Yes Yes No Yes

Specificity Yes Yes Yes *

LOD No No Yes *

LOQ No Yes No *

Linearity & range Yes Yes No *

Ruggedness Yes Yes Yes *

*may be required, depending on the nature of specific test

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Chapter2 Literature review

2. LITERATURE REVIEW

1. A. Mohammadi, et.al.,(2009) studies A stability-indicating high performance liquid

chromatographic (HPLC) assay for the simultaneous determination of atorvastatin and amlodipine
in commercial tablets. A simple, rapid, precise and accurate isocratic reversed-phase stability-
indicating HPLC method was developed and validated for the simultaneous determination of
atorvastatin (AT) and amlodipine (AM) in commercial tablets. The method has shown adequate
separation for AM, AT from their associated main impurities and their degradation products.
Separation was achieved on a Perfectsil Target ODS-3, 5μm, 250 mm × 4.6 mm i.d.
column using a mobile phase consisting of acetonitrile–0.025 M NaH2PO4 buffer (pH 4.5) (55:45,
v/v) at a flow rate of 1 ml/min and UV detection at 237 nm. The drugs were subjected to oxidation,
hydrolysis, photolysis and heat to apply stress conditions. The linearity of the proposed method
was investigated in the range of 2–30 μg/ml (r = 0.9994) for AT and 1–20 μg/ml (r = 0.9993)
for AM. The limits of detection were 0.65 μg/ml and
0.35 μg/ml for AT and AM, respectively. The limits of quantitation were 2 μg/ml and 1 μg/ml for
AT and AM, respectively. Degradation products produced as a result of stress studies did not
interfere with the detection of AT and AM and the assay can thus be considered stability- indicating
.

2.Thanikachalam Sivakumar , et.al.,(2010)developed Simultaneous analysis of amlodipine and

atorvastatin in pharmaceutical formulations, Statistical experimental design and Derringer's
desirability function were applied to develop an improved RP-HPLC method for the simultaneous
analysis of amlodipine and atorvastatin in pharmaceutical formulations. Four independent factors
were considered: acetonitrile content in the mobile phase; buffer pH; buffer concentration; and
flow rate. The preliminary screening step was carried out, to identify the significant factors
affecting the analysis time response. Then central composite design wasapplied for a response
surface study, in order to examine in depth the effects of the most important factors. Subsequently,
Derringer's desirability function was employed to simultaneously optimize the six responses:
retention factor of first peak; two resolutions; and three retention times, each having a different
target. This procedure allowed deduction of two separate optimum conditions, intended for the
analysis of quality control and plasma samples, within the experimental domain. The predicted
optimum for the quality control samples was :
Department of pharmaceutical analysis,PIPER Page 30
Chapter2 Literature review

methanol-acetonitrile-15 mM K2HPO4 buffer (pH 5.33) (10:42.08:47.92, v/v/v) as the mobile

phase and 1.12 mL/min as the flow rate. The method using this optimized condition showed higher
sensitivity and shorter analysis time than the previously published reports. The optimized assay
condition was validated according to International Conference on Harmonization guidelines.

3.K Raja Rajeswari, et. al., (2008) developed RP-HPLC method for the simultaneous
determination of Atorvastatin and Amlodipine in tablet dosage form, A simple, precise, accurate,
and rapid HPLC method has been developed, and validated for the determination of atorvastatin
and amlodipine simultaneously, in combined tablet dosage form. The mobile phase used was a
mixture of acetonitrile and 0.03M phosphate buffer pH 2.9 (55:45% v/v). The detection of
atorvastatin and amlodipine was carried out on dual g absorbance detector at 240 nm and 362
nm, respectively. Results of the analysis were validated statistically, and by recovery studies.
The proposed method can be successfully used to determine the drug contents of marketed
formulation.

4. DA Shah, KK Bhatt, et. al., (2008) developed RP-HPLC determination of atorvastatin calcium
and amlodipine besylate combination in tablets, A simple, specific, accurate and precise reverse-
phase high-performance liquid chromatographic method was developed for the simultaneous
determination of atorvastatin calcium and amlodipine besylate in tablet dosage forms. A
phenomenex Luna C-18, 5 µm column having 250 × 4.6 mm i.d. in isocratic mode,with mobile
phase containing methanol: acetonitrile: 50 mM KH 2 PO 4 (20:50:30; pH 3.5) was used. The flow
rate was 1.0 ml/min and effluent was monitored at 240 nm. The retention time of atorvastatin
calcium and amlodipine besylate was 7.6 min and 3.2 min respectively. The linearity for
atorvastatin calcium and amlodipine besylate was in the range of 5-120 µg/ml and 5-100 µg/ml
respectively. The proposed method is accurate, precise, specific and rapid for simultaneous
estimation of atorvastatin calcium and amlodipine besylate in tablets.
5. MR Khan, et. al. ,(2005) developed Simultaneous spectrophotometric determination of
atorvastatin calcium and amlodipine besylate in tablets .Two simple, accurate and precise methods
for simultaneous estimation of atorvastatin calcium and amlodipine besylate in combined dosage
form have been described. First method employs formation and solving of

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Chapter2 Literature review

simultaneous equations using 245 nm and 363 nm as two analytical wavelengths. Second is dual
wavelength method, which uses the difference of absorbance value at 259.9 nm and 354 nm for
estimation of atorvastatin calcium and absorbance at 363 nm for amlodipine besylate. Fifty percent
methanol was used as solvent, in which atorvastatin calcium and amlodipine besylate shows
linearity in the range of 0-40 µg/ml and 0-20 µg/ml, respectively. Standard deviation was
<1.5 in the assay of tablets. Methods were validated as per ICH norms and accuracy, precision,
repeatability and robustness was found to be with in the acceptable limit.

6.R Sahu , et. al.,(2009) developed Simultaneous spectrophotometric determination of amlodipine

besylate and atorvastatin calcium in binary mixture, A rapid, simple, accurate and precise UV
Spectrophotometric method using simultaneous equation was developed for the simultaneous
determination of amlodipine besylate and atorvastatin calcium in a binary mixture. In the proposed
method, the signals were measured at 238.2 and 246.6 nm corresponding to the absorbance
maxima of amlodipine besylate and atorvastatin calcium in methanol, respectively. Linearity was
observed in the concentration range of 5-30 µg/ml for both the drugs. Concentration of each drug
was obtained by using the absorptivity values calculated for both the drugs at two wavelengths,
238.2 and 246.6 nm and solving the simultaneous equations. The method was validated statistically
and recovery study was performed to confirm the accuracy of the method. Laboratory prepared
synthetic mixture was successfully analyzed using the developed method.

7.P Mishra, et. al.,(2009) developed Simultaneous estimation of atorvastatin calcium and
amlodipine besylate from tablets. The present communication deals with the development of a
new, simple, specific, sensitive, rapid and economical procedure for simultaneous estimation of
atorvastatin calcium and amlodipine besylate in a combined dosage form. The method is based
on the native ultraviolet absorbance maxima of the two chemotherapeutic agents. As both
compounds do not interact chemically in methanol, two wavelengths 246 nm for atorvastatin
calcium and 360 nm for amlodipine besylate were used. Both the drugs obeyed Beer's law in the
concentration range .

8. Riahi, et.al.,(2006) developed Spectrophotometric Simultaneous Determination of Atorvastatin and

Amlodipine from their Combined Drug Products. The simultaneous spectrophotometric determination
of Atorvastatin calcium (ATV) and Amlodipine besylate (AML) in tablets in the presence of the

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Chapter2 Literature review

overlapping spectra was accomplished with the derivative spectrophotometry (DS) and the partial
least squares (PLS) approaches without using any chemical pre-treatment. In this study, the
concentration model was based on the absorption spectra in the range of 220-400 nm for 25
different mixtures of ATV and AML. The calibration curve was linear over the concentration range
of 10-160 and 3-33 μg mL-1 for ATV and AML, respectively. These two methods were tested by
analyzing the synthetic mixtures of the above drugs and they were applied to the real samples,
containing two commercial pharmaceutical preparations of the subjected drugs. Acomparative
study was carried out using the experimental results obtained from the two analytical
methodologies. The root mean squares differences (RMSD) with PLS and DS were 0.6618 and
1.2555 for ATV and 0.1589 and 0.3490 for AML, respectively.

9. Suresh Kumar G.V, et. al., (2010) developed a validated reverse phase HPLC method for
Simultaneous Estimation of Telmisartan and Atorvastatin calcium in Tablet dosage form. The
chromatographic separation was achieved on (Waters Symmetry C18, 250 mm x 4.6 mm, 5μ)
analytical column. A mixture of ammonium acetate (0.02 M, pH 4.0 adjusted with glacial acetic
acid) and acetonitrile in ratio (40:60 v/v) was used as the mobile phase with a flow rate of
1.0ml/min. UV detection was at a wavelength of 254 nm.Validation parameters: linearity (>
0.999). Intraday precision (R.S.D. 0.24- 0.45%) and interday precision (R.S.D.is 0.49 - 0.81), LOD
for telmisartan is 0.7 and the LOQ is 2.1and all the validation parameters are within the acceptance
range.

10. SB Wankhede, et. al.,(2007) developed a RP-HPLC method for simultaneous estimation of
telmisartan and hydrochlorothiazide in tablet dosage form. Chromatography was performed on a
ODS Hypersil C18 (25 cm×4.6 mm I.D) column from thermo in isocratic mode with mobile phase
containing acetonitrile: 0.05 M KH2PO4 ; pH 3.0 (60:40). The flow rate was 1.0 ml/min and the
eluent was monitored at 271 nm, by UV detection.Validation parameters: resolution is 9.620,
capacity factor is 4.21 for telmisartan, tailing factor- 1.647, Number of theoretical plates/column-
3420 for telmisartan .The plot of peak area Vs. concentration was found to be linear in the range
of 4.10-20.48 µg/ml for telmisartan, correlation coefficient for calibration curve of both telmisartan
and hydrochlorothiazide, (r= 0.99). The mean recoveries of telmisartan and hydrochlorothiazide
were 99.48% ± 0.52 and 99.13% ± 0.72, respectively. The low values ofstandard deviation for
analyst to analyst variation (±0.44 for telmisartan and ± 0 .37 for

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Chapter2 Literature review

hydrochlorothiazide) and intra-day variation (1.18 for telmisartan and ± 0.24 for
hydrochlorothiazide) indicated the robustness of the method.

11. Gao Ling, et. al.,(2010) established an RP-HPLC method for the determination of Telmisartan
in tablet dosage form A Shim-pack C-18 column with UV detection at 298nm was used. The
mobile phase was methanol: water (including 2.5% phosphate buffer, pH 5.8) in the ratio of (80:20)
was selected and the flow rate was set to 1.0 ml·min-1. The plot of peak area vs. concentration was
found to be linear in the range of 5.0-50 µg/ml, correlation coefficient for calibration curve (r=
0.9999). The average recovery was 101.7% and RSD 1.2% (n=5).

12. LI Xin-chun, et. al.,(2009) established a HPLC-FLU method for determination of telmisartan
in human plasma. Acetonitrile: water: acetic acid: triethyl amine (60:40: 0.4: 0.3; pH 4.2) was used
as the mobile phase; the flow rate was 1 ml·min-1. The excitation and emission wavelength was
295 nm and 385 nm, respectively. Internal standard method quantified telmisartan concentration
in plasma. Prior to analysis, the plasma sample was pretreated easily through one- step extraction.
Linear relationship was observed from 2.5- 120.0 µg/l, the detection limit was
1.25 µg/l. The relative recovery rate of three concentration samples were 98.02%, 102.13%,
100.57%; and the precise were 4.06%, 6.17%, 2.38%, respectively.

13. LIU Mei-liang, et. al.,(2008) developed an RP HPLC method for the determination of
telmisartan in the tablets Diamonsil TM C18 (5 μm, 4.6 mm×250 mm) is used as a stationary phase,
Acetonitrile - 0.5% phosphoric acid (pH 6.0, adjusted by triethylamine) (52:48) is used as mobile
phase with flow rate of 1 ml/min UV detection was at a wavelength of 297 nm .The content of
telmisartan tablet was counted by external standard method. The linear range was
28.16 - 140.8 µg/l (r= 0.9999).The average recovery was 100.56%, and RSD=0.22%.

14. Jing Nie, et. al.,(2006) developed a method for the determination of telmisartan in rat tissue by
in-tube solid –phase micro extraction coupled to HPLC. A poly (meth acrylic acid-ethylene glycol
dimethacrylate, MAA-EGDMA) monolithic capillary was used for the direct and on-line
extraction of telmisartan from Sprague-Dawley rat tissue (heart, kidney, and liver) homogenates.
The tissue homogenates were simply diluted with a mixture of phosphate buffer (pH 2) /ACN
(90:8 v/v), and then injected for extraction only after centrifugation and filtration. Coupled to
HPLC with fluorescence detection. The method was linear over the range of 1.25-1500 ng/g for

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Chapter2 Literature review

telmisartan in heart and kidney, 12.5-15 000 ng/g in liver with correlation coefficients over 0.9992.
The detection limits were found to be in the range from 0.24 to 1.8 ng/g. RSDs for intra- and inter-
day ranged from 1.2 to 8.1%.

15. Leena R Bhat, et. al.,(2007) developed a simple, selective, and precise validated RP-HPLC
method for simultaneous determination of Telmisartan and Hydrochlorothiazide in Pharmaceutical
Formulation. The mobile phase consisted of methanol and acetonitrile (70:30 v/v) at a flow rate
of 1 ml/min and the wavelength of detection was 270 nm. Rabeprazole was used as an internal
standard. The retention times of Telmisartan, hydrochlorothiazide and rabeprazole were 1.79 ±
0.01, 2.80 ± 0.01, and 3.19 ± 0.01 minutes, respectively.

16. Ganesh M, et. al.,(2005) developed an isocratic RP-HPLC.method for the quantification of
telmisartan in the pharmaceutical formulation. Stainless steel analytical column Inertsil ODS-3V
(5mum, 15 cm x 0.46 cm) is used as stationary phase. The system was operated at ambient
temperature (25 +/-2 C) ; acetonitrile : water : tetrahydrofuran : perchloricacid (250:740:10:1) is
the mobile phase and the flow rate is 1 ml/min. ultraviolet detection at 245 nm has been used for
the determination. The calibration curve for telmisartan was linear over the tested concentration
range of 50%, 75%, 100%, 125% and 150% with reference to the label claim and a correlation
coefficient of 0.999. The intra- and inter-run precision and accuracy re sults were 98.07 to
100.34 with the %RSD of 0.71% and tailings factor 1.07.
17. Min zhang, et. al.,(2009) developed a method for Biocompatible in-tube solid-phase
microextraction coupled to HPLC for the determination of angiotensin II receptor antagonists
(candesartan, losartan, irbesartan, valsartan, telmisartan), in human plasma and urine. A poly
(methacrylic acid-ethylene glycol dimethacrylate, MAA-EGDMA) monolithic capillary was used
for the in-tube solid-phase microextraction (in-tube SPME) of several angiotensin II receptor
antagonists (ARA-IIs) from human plasma and urine. Coupled to HPLC with fluorescence
detection, this on-line in-tube SPME method was successfully applied for thedetermination of
candesartan, losartan, irbesartan, valsartan, telmisartan, and their detectionlimits were found to be
0.1-15.3 ng/mL and 0.1-15.2 ng/mL in human plasma and urine,respectively. The method was
linear over the range of 0.5-200 ng/mL for telmisartan, with correlation coefficients being above
0.9985 in plasma sample and above 0.9994 in urine sample. The method reproducibility was
evaluated at three concentration levels, resulting in the R.S.D.

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Chapter2 Literature review

<7%. The poly (MAA-EGDMA) monolithic capillary was demonstrated to be robust.

18. Yogesh Gupta, Alankar shrivastava,(2009) developed An RP-HPLC method has been
developed for the simultaneous estimation of ramipril and telmisartan in tablet dosage forms, using
UV-detector. The developed method was validated as per ICH guidelines and specificity, linearity
& range, accuracy, precision and robustness was performed. Specificity was determined by
comparing the results obtained by running the placebo solution with that of standard and method
was found to be specific due to no interference between placebo peaks and drugs peaks. Linearity
range was found to be 4 to 16 µg/ml and 32 to 128 µg/ml of ramipril and telmisartan respectively.
The method was found to be linear in the range of 4 to 16 µg/ml and 32 to 128 µg/ml for ramipril
and telmisartan respectively. In the linearity study, regression equation and coefficient of
correlation for ramipril and telmisartan were found to be (y = 924480x - 151831, r = 0.9997) and
(y = 2901878.3558x + 3803877, r = 0.9996) respectively. This newly developed method was
successfully utilized for the simultaneous estimation of ramipril and telmisartan in pharmaceutical
tablet dosage forms.
19. Suresh Kumar Gv,Rajendraprasad Y,(2009) studies
A simple, precise and accurate reversed‐phase liquid chromatographic method was developed
and validated for simultaneous estimation of Telmisartan and Amlodipine in tablet formulations.
The chromatographic separation was achieved on (Waters symmetry C18 250mm x 4.6mm,5µm)
analytical column with mobile phase consisting mixture of Potassiumdihydrogen phosphate (0.0
2M, pH 3.0 adjusted with ortho‐phosphoric acid) and acetonitrile
in ratio (60:40 v/v) at flow rate of 1.5ml/min and detector wavelength 237 nm.The retention time
of Amlodipine andtelmisartan was found to be 3.5 and 8.1 minutes respectively. The validation
of the proposed method was carried out for its specificity,linearity, accuracy, precision, limit of d
etection and quantification for both Telmisartan and Amlodipine.The developed method can be u
sed for routine quality analysis of titled drugs in combination in tablet formulation.
20. Aniruddha R. Chabukswar, et. al.,(2010) develops Method describes an HPTLC method for
the simultaneous determination of Telmisartan and Amlodipine Besylate from tablet dosage form.
This employes a precoated silica gel 60 F254 (0.2 mm thickness) on aluminium sheets and a mobile
phase Ethyl acetate: 1, 4 Dioxane: Methanol: 25% Ammonia in the ratio of 15:1.5:3:1.5v/v, having
chamber saturation for 30 min at room temperature. The developing chamber was
run upto 8cm. The Rf values was found to be 0.16 and 0.33 for Telmisartan and Amlodipine

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Chapter2 Literature review

respectively. The plate was scanned and quantified at 323nm. The linear detector response was
observed between 100 µg/ml to 500 µg/ml and 200 µg/ml to 1000 µg/ml for Telmisartan and
Amlodipine respectively. The method so developed was validated for its accuracy and precision.
The LOD and LOQ were found to be 0.025, 0.0747µg/ml and 0.0236, 0.0714µg/ml, respectively
for Telmisartan and Amlodipine. The recovery was carried out by standard addition method. The
Average recovery was found to be 100.38% and 100.24% for Telmisartan and Amlodipine
respectively.
21. Safeer K. , anbarasi B. et. al.,(2010) develops A simple, specific, accurate and precise reverse
phase high pressure liquid chromatographic method has been developed for the simultaneous
determination of Amlodipine and Hydrochlorothiazide from combined dosageform by reverse
phase C18 column (Phenomenex C18, 5μ, 250mm x 4.6mm). The sample was analysed using
Triethylamine: Acetonitrile: Methanol in the ratio of 50:25:25(pH adjusted to 3.0 with
Orthrophosphric acid) as a mobile phase at a flow rate of 2.0ml/min and detection at 235nm. The
retention time for Amlodipine and Hydrochlorothiazide was found to be 6.631 and
2.183 min respectively, and recoveries from combined dosage form were between 98 and 102%.
The method can be used for estimation of combination of these drugs in combined dosage form.
22. Santaji Uttam nalwade , et. al.,(2011) develops A simple, precise and rapid stability-
indicating ultra-performance liquid chromatography (UPLC) method was developed for the
simultaneous quantitative determination of Telmisartan, Amlodipine besylate and
Hydrochlorothiazide from their innovative poly pill combination drug product, with the presence
of degradation products. It involved a 100 mm x 2.1 mm, 1.7 µm C-18 column. The separation
was achieved on simple gradient method. The mobile phase A contains a mixture of sodium
perchlorate buffer pH 3.2(0.053M): acetonitrile in the ratio 90:10, v/v, and mobile B contains a
mixture of sodium perchlorate buffer pH 3.2(0.053M): acetonitrile in the ratio 20:80, v/v. The flow
rate was 0.6 mL min-1 and column temperature was maintained at 55°C.The gradient program
(T/%B) was set as 0/5, 1.2/5, 1.6/40, 4/40, 4.1/5 and 4.5/5. The detector wavelength was 271 nm
for Hydrochlorothiazide and telmisartan and 237 nm for Amlodipine. The retention times of
Telmisartan, Amlodipine, and Hydrochlorothiazide are 3.6 minutes, 3.2 minutes and 0.9 minutes;
respectively. The total runtime was 4.5 minutes within which three active compounds and their
degradation products were separated. The described method was validated with respect to system
suitability, specificity, linearity, precision and accuracy. The precision of the assay

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Chapter2 Literature review

method was evaluated by carrying out six independent assays of T, A and H (0.032 mg mL of T,
0.004 mg mL of A, 0.01 mg mL of H). The accuracy of the method was evaluated in triplicate at
three concentration levels, i.e. 50%, 100% and 150% of target test concentration (0.64 mg mL-1
of T, 0.08 mg mL-1 of A, 0.2 mg mL-1 of H) The described method was linear over the range, 16
to 48 µg mL for T, 2 to 6 µg mL A and 5 to 15 µg mL for H. The method is fast and is suitable for
high-throughput analysis of the drug and one can analyze about 250 samples per working day,
facilitating the processing of large-number batch samples.
23. R. Vijayamirtharaj , J. Ramesh, et. al.,(2010) Studies The present research work deals with
the development of RP-HPLC method for the determination of Telmisartan and Atorvastatin
calcium in bulk and in formulation using UV detector. Selected mobile phase was a combination
of Acetonitrile: Buffer (0.01M Potassium dihydrogen phosphate) 65:35 PH4.00 (adjusted with
Orthophosphoric acid) and the wavelength selected was 250nm. The flow rate was kept at 2.0
ml/min, and the injection volume was 10µl. The sepration was performed at ambient temperature.
Retention time of Telmisartan and Atorvastatin calcium was found to be 3.72 and
6.14 minutes respectively. Linearity of the method was found to be 319-480 µg/ml for Telmisartan
and 86-130 µg/ml for Atorvastatin calcium. The correlation co-efficient of Telmisartan was found
to be 0.9998 and the correlation co-efficient of Atorvastatin calcium was found to be 0.9999.
Accuracy of the method was determined through recovery studies by adding known quantities of
standard drug to the pre analyzed test solution and was found to be 98.92-
100.02 for Telmisartan and 99.93-100.96 for Atorvastatin calcium respectively The system
suitability parameters such as theoretical plates and tailing factor were found to be 6347, 1.652
and 9720, 1.394 respectively for Telmisartan and Atorvastatin calcium. This method was validated
according to ICH guidelines.
24. N. R. Vekariya, G. F. Patel , et. al.,(2009) develops A rapid, selective and stability indicating
high performance thin layer chromatographic method was developed and validated for
simultaneous estimation of telmisartan and amlodipine besylate in pharmaceutical dosage forms.
The method employed TLC aluminium plates precoated with silica gel 60F254 as the stationary
phase. The solvent system consisted of tetrahydrofuran: dichloroethane: methanol: ammonia
solution (3.0:1.0:0.5:0.2 v/v). This system was found to give compact spots for both telmisartan
(Rf value of 0.22 ± 0.02) and amlodipine besylate (Rf value of 0.45 ± 0.02). Spectrodensitometric
scanning-integration was performed at a wavelength of 326 nm. The

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polynomial regression data for the calibration plots showed good linear relationship with r2 =
0.9993 in the concentration range of 1200 – 7200 ng for telmisartan and 400 – 1400 ng for
amlodipine besylate with r2 = 0.9996. The method was validated for precision, accuracy,
ruggedness and recovery. The minimum detectable amounts were found to be 149.41 ng and
53.07 ng for telmisartan and amlodipine besylate, respectively.
The limits of quantitation were found to be 452.78 ng for telmisartan and 160.83 ng for
amlodipine besylate. Statistical analysis proves that the method is reproducible and selective for
the simultaneous estimation of telmisartan and amlodipine besylate. 25. Mohammad Younus, et.
al.,(2010) develops A simple, specific, accurate and precise Reverse Phase High Performance
Liquid Chromatographic method was developed for simultaneous estimation of Amlodipine
Besylate (AB), Valsartan (VAT) and Hydrochlorothiazide (HTZ) in tablet dosage form on RP C-
18 Column (Hypersil 250*4.6 mm) using Acetonitrile: Mixed Phosphate buffer (6.8 pH) (55:45)
(buffer was prepared by mixing the equal proportions of 0.01M Potassium dihydrogenphosphate
and 0.001M Dipotassium hydrogenphosphate. pH was adjusted with Orthophosphoric acid) as
mobile phase. The flow rate was 1.0 ml/min and effluent was monitored at 237nm. The retention
time for VAT, HTZ and AB was found to be as 2.28, 2.99 and 4.57 respectively. Proposed method
was validated for Precision, Accuracy, Linearity range, Robustness and Ruggedness.
26. P Kalaiselvi, et. al.,(2005) develops,A simple, precise, accurate and rapid HPTLC method has
been developed, and validated for the determination of Pioglitazone HCl and Telmisartan
simultaneously, in combined dosage form (15mg and 20mg). Pioglitazone HCl and Telmisartan
was chromatographed on silica Gel 60F254 TLC plate using Toluene: ethyl acetate: methanol
(7:2:1v/v) was used as the mobile phase. The Rf value of Pioglitazone HCl and Telmisartan was
0.56±0.04 and 0.23±0.02 and scanned at 254nm using camag TLC scanner 3. The applicability
of the method for simultaneous determinations of Pioglitazone HCl and Telmisartan was verified
by the determination of these compounds in marketed tablets. Results of the analysis were
validated statistically, and by recovery studies (99.47-101.36% and 99.40 -102.37%). The
recovery and RSD values were with in limits given in ICH guidelines, method developments
indicates that the suitability of proposed for the routine determination of these compounds in
tablets. The validation parameters; linearity the range 30-150 ng/spot and 40-200ng/spot.
(r=0.9995 and r=0.9996) sensitivity. The limit of detection was found to be 15ng/spot and
20ng/spot. The limit of quantification was found to be 45ng/spot and 60ng/spot respectively. The

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accuracy ((99.88% and 99.33%) and reproducibility were found to satisfactory. The proposed
method can be successfully used to determine the drug contents on marketed formulation.
27.Amrish Sharma, et. al.,(2010) develops A new and simple reverse phase HPLC method was
developed for the determination of amlodipine in its pharmaceutical dosage form. The mobile
phase used acetonitrile, methanol, KH2PO4 was in the ratio of 250: 250: 500. Buffer solution was
prepared by dissolving 0.05m KH2PO4 + 0.1 % H3PO4 adjunct with triethylamine. The separation
was achieved on hypersil C-18 column, phenomenex Gemini (250×4.6mm) and 5µ particle size
with rheodyne injector. The flow rate was 1ml/min and UV detection at 238nm. Theretention time
for amlodipine was 9 min. The linearity coefficient of Amlodipine was 0.99% and the percentage
recoveries for amlopdipine was 100.02% respectively. The proposed method was accurate, precise
and linear within the desiredrange. This method can be successfully employed for the quantitative
analysis of amlodipine.

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Chapter3 Objective and plan of work

3. OBJECTIVE AND PLAN OF WORK

The drug analysis plays an important role in the development of drugs, manufacturing and
therapeutic use. Pharmaceutical industries rely upon quantitative chemical analysis to ensurethat
the raw material used and the final product obtained meets the required specification. The literature
review indicates there are several analytical methods have been reported for estimation of these
drugs as individual or in combination with other drugs, and also several analytical methods for the
determination of Simultaneous estimation of Amlodipine Besylate and Telmisartan by HPTLC,
UPLC and UV in dosage formulation and its bioanalytical applications.

The objective of this work is to develop and the estimation of Amlodipine Besylate and
Telmisartan in formulations. So an attempt was made to develop and validate a simple, precise,
accurate, linear and rapid. RP-HPLC method is done as per as ICH guidelines for the estimation
of Amlodipine and Telmisartan in pure pharmaceutical dosage forms and to apply the developed
method to determine the validation of compounds. Hence, the present study work to develop the
simple, rapid, accurate, precise and validated methods for the estimation in combination dosage
forms.

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Chapter4 Drug profile

4. DRUG PROFILE

AMLODIPINE BESYLATE

STRUCTURE:

CHEMISTRY:

2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-
pyridinedicarboxylic acid,3-ethyl,5-methyl ester besylate. mono benzene sulphonate

a) Molecular formula : C20H25CIN2O5. C6H6O3s

b) Molecular weight: 567.1

APPEARANCE: It is odorless, white crystalline powder

SOLUBILITY : It is slightly soluble in water and sparingly soluble in ethanol, soluble in

methanol, and acetonitrile.

CATEGORY: Long-acting calcium channel blocker.

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MECHANISM OF ACTION:

Amlodipine is a dihydropyridine calcium antagonist (calcium ion antagonist or slow-channel

blocker) that inhibits the transmembrane influx of calcium ions into vascular smooth muscleand
cardiac muscle. Experimental data suggest that amlodipine binds to both dihydropyridineand
nondihydropyridine binding sites. The contractile processes of cardiac muscle and vascular smooth
muscle are dependent upon the movement of extracellular calcium ions into these cells through
specific ion channels. Amlodipine inhibits calcium ion influx across cell membranes selectively,
with a greater effect on vascular smooth muscle cells than on cardiac muscle cells.

Pharmacodynamics:

Hemodynamics: Following administration of therapeutic doses to patients with hypertension,

NORVASC produces vasodilation resulting in a reduction of supine and standing blood pressures.
These decreases in blood pressure are not accompanied by a significant changein heart rate
or plasma catecholamine levels with chronic dosing. Although the acute intravenousadministration
of amlodipine decreases arterial blood pressure and increases heart rate in hemodynamic studies
of patients with chronic stable angina, chronic oral administration of amlodipine in clinical trials
did not lead to clinically significant changes in heart rate or blood pressures in normotensive
patients with angina.

Pharmacokinetics and Metabolism:

After oral administration of therapeutic doses of NORVASC, absorption produces peak plasma
concentrations between 6 and 12 hours. Absolute bioavailability has been estimated to be between
64 and 90%. The bioavailability of NORVASC is not altered by the presence of food.

Amlodipine is extensively (about 90%) converted to inactive metabolites via hepatic metabolism
with 10% of the parent compound and 60% of the metabolites excreted in the urine.. Elimination
from the plasma is biphasic with a terminal elimination half-life of about 30–50 hours. Steady-
state plasma levels of amlodipine are reached after 7 to 8 days of consecutive daily dosing.

DOSE: 2.5mg, 5mg, 10mg.

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BRAND NAMES: NORVASC

ADVERSE EFFECTS:

CNS : hypoesthesia, neuropathy peripheral, tremor, vertigo.

CVS :Arrhythmia, bradycardia, hypotension,peripheral ischemia.

GIT : anorexia, constipation, pancreatitis, diarrhea, dyspepsia.

TELMISARTAN

STRUCTURE:

CHEMISTRY:

Telmisartan is chemically 2-[4-[[4-methyl-6-(1-methylbenzimidazol-2-yl)-2-

propylbenzimidazol-1-yl] methyl] phenyl] benzoic acid

a) Molecular formula: C33H30N4O2

b) Molecular weight: 514.617

APPEARANCE: Telmisartan is a white solid powder.

MELTING POINT:261-2630C

SOLUBILITY: It is insoluble in water, methanol, Acetonitrile.

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Chapter4 Drug profile

CATEGORY: Angiotensin II receptor antagonist, used in the treatment of hypertension.

MECHANISM OF ACTION:

It inhibits vasoconstricting effects and blocks aldosterone-producing effects of

angiotensin II at various receptor sites, including vascular smooth muscle and adrenal glands.
Telmisartan is an angiotensin II receptor blocker (ARB), shows high affinity for the
angiotensin II, type I (AT-I) receptors, has a long duration of action and has longest half life of
any ARB. In addition to blocking the rennin- angiotensin system, it also acts as selective
modulator of peroxisome proliferator-activated receptor-γ (PPAR-γ), a central regulator of
insulin and glucose metabolism. It is believed that telmisartan dual mode of action may provide
protective benefits against the vascular and renal damage caused by diabetes and CVS diseases.
It has binding capacity 3000 times greater for AT-I than AT-II receptors

PHARMACOKINETICS:

The bio availability of telmisartan is about 42-100%; ≥ 99.5% of the drug is plasma protein
bound. It undergoes minimal hepatic metabolism and the t1/2 is found to be 24 hrs. It is highly
bound to plasma proteins (> 99.5%), mainly albumin and α1-acid glycoprotein and the binding is
not dose-dependent. Minimally metabolized by conjugation to form a pharmacologically inactive
acylglucuronide; the glucuronide of the parent compound is the only metabolite that has been
identified in human plasma and urine. The cytochrome P450 isoenzymes are not involved in the
metabolism of telmisartan. It is excreted through faeces.

DOSE: 20mg, 40mg, 80mg.

BRAND NAMES:

TELMINORM, TELMA, TELDAY, PRITOR, MICARDIS

DRUG INTERACTIONS:

Amiloride - Increased risk of hyperkaliemia

Digoxin - Telmisartan increases the effect of digoxin

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Chapter4 Drug profile

Drospirenone - Increased risk of hyperkaliemia

Lithium - The ARB increases serum levels of lithium
Potassium - Increased risk of hyperkaliemia
Spironolactone - Increased risk of hyperkaliemia
Triamterene - Increased risk of hyperkaliemia

ADVERSE EFFECTS:
CNS : dizziness, headache, fatigue
CVS : chest pain, peripheral edema, hypertension
ENT : sinusitis, Pharyngitis
GIT : nausea, vomiting, diarrhea, dyspepsia, abdominal pain
GUT : urinary tract infection
Musculoskeletal : myalgia, back and leg pain

Respiratory : cough, upper respiratory infection

Other : pain, flu or flulike symptoms.

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Chapter5 Materials and instruments used

5. MATERIALS AND INSTRUMENTS USED

Drug samples
Amlodipine Besylate and Telmisartan were generously given by Lyka laboratories,
Mumbai.
Drug name Purity
Amlodipine 99.80%
Telmisartan 99.65%
Tablets used
Brand: TWYNSTA
Each tablet contains:
Amlodipine Besylate – 5mg and
Telmisartan 40mg
Chemicals and solvents used
 Water for HPLC
 Acetonitrile HPLC grade
 Disodium hydrogen phosphate AR grade
 Methanol AR grade
 Distilled water
Instruments used
 Shimadzu electronic balance
 Elico pH meter
 Soltec ultra sonicator
 Millipore – solvent filtration unit
 Labindia-3000, UV-visible spectrophotometer.
➢ 1 cm matched quartz cells.
 Shimadzu Isocratic HPLC system with following configurations,
➢ LC-10AT Vp series, Isocratic solvent delivery system (pump).
➢ Rheodyne 7725i injector with 20 µl loop.
➢ spinchrome data station.
➢ Analytical column: Phenomenex – Luna, C18 (250 x 4.6 mm i.d., 5µ).
➢ UV-Visible – SPD 10AVp series detector.

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PREPARATION OF MOBILE PHASE:

Preparation of Buffer

3.8954g of disodium hydrogen phosphate and 3.4023g of potassium dihydrogen

phosphate in 1000 ml volume of distilled water and pH is adjusted to 4 with orthophosphoric acid.

Mobile- phase

580 ml of acetonitrile and 420 ml of buffer were added in a beaker to give 1000ml.
Finally the pH was adjusted to 4.0 by adding orthophosphoric acid.

Mobile-Phase Ratio

Buffer: Acetonitrile (42: 58 % V/V)

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Chapter6 Method devolpment and optimization

6. METHOD DEVELOPMENT AND OPTIMIZATION

The method development by RP-HPLC method for the estimation of Amlodipine Besylate and
Telmisartan is

• Development of suitable mobile phase.

• Optimization of the chromatographic conditions.
• Selection of suitable detection of wavelength.
• Preparation of standard calibration curve of Amlodipine and Telmisartan Assay of
pure mixed standards and formulation.
• Validation of the developed method.

The chromatographic parameters for the development and optimization for the Amlodipine
Besylate and Telmisartan are

SOLUBILITY

Solubility of drugs in different solvents at different pH conditions which is useful while selecting
diluents for standard solution and extraction solvents for test solutions. According to Indian
pharmacopoeia and literature review collected Amlodipine Besylate and Telmisartan are very
Slightly soluble in water, Acetonitrile. Sparingly soluble in alcohol. Then it was checked for
different dilutions of Acetonitirile and Disodium hydrogen Phosphate buffer and finally at P H - 4
the mixture was chosen for present work.

STABILITY
Stability of drug with storage conditions .This helps to adopt suitable and adequate precaution
while handling drug substances and its solutions.

SELECTION OF CHROMATOGRAPHIC METHOD FOR SEPARATION

Depending on nature of sample (ionic or neutral molecule), its solubility and molecular weight,
chromatographic method is to be selected. The drugs are polar in nature and so reverse phase
chromatographic technique were selected for the present study.

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Chapter6 Method devolpment and optimization

DETECTION OF WAVELENGTH (λmax)

Selection of detection of wavelength is a critical step in the analytical method. The conditions for
the development of the assay method for the choice of detection wavelength was based on the
scanned absorption for Amlodipine Besylate and Telmisartan The spectrum was scanned over the
range of 200 – 400 nm and was obtained by measuring the absorption of 0.01mg/ml solution of
Amlodipine and Telmisartan and in methanol prepared from stock solution. The spectrum was
obtained by using 1cm quartz cell using water as reference solution. λmax of Amlodipine besylate
was 361 nm and λmax of Telmisartan was 225nm. Hence for simultaneous estimation 236nm was
selected.

UV Spectrum of Amlodipine

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Chapter6 Method devolpment and optimization

UV Spectrum of Telmisartan

UV Spectrum of Amlodipine Besylate and Telmisartan

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 Chapter6 Method devolpment and optimization

6.1 METHOD DEVELOPMENT TRIALS

Five trials on composition of buffer and organic phase were done to decide the ultimatecomposition
of mobile phase.

TRIAL – 1

The Trial 1 was performed by operating HPLC with acetonitrile and phosphate buffer
80:20 % v/v with flow rate of 1.5 ml/min.

Chromatogram No – 1

The retention time of both peaks was 8.007 and 9.480 tailing occurred hence further trials
were carried out.

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TRIAL – 2

The trial 2 was performed by operating HPLC with Acetonitrile and phosphate buffer
ratio 75:25% v/v with flow rate of 1ml/min.

Chromatogram No – 2

The retention time was 3.740 and 10.807, peaks were improved tailing factor was
remained slightly more than 2.

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 Chapter6 Method devolpment and optimization

TRIAL -3
The trial 3 was performed by operating HPLC with acetonitrile and phosphate buffer 70:30
%v/v with flow rate of 1 ml/min.

Chromatogram No – 3

The peaks are good but impurity peaks were obtained and retention time was less2.633 and
5.600

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 Chapter6 Method devolpment and optimization

TRIAL-4

The trial 4 was performed by operating HPLC with Acetonitirile and phosphate buffer65:35
%v/v with flow rate of 1.0ml/min.

Chromatogram No – 4

The peaks are good, but retention time of both peaks was less than 2, retention time was
less3.701 and 4.686.

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Chapter6 Method devolpment and optimization

TRIAL -5

The trial was performed by operating HPLC with Acetonitirile and phosphate buffer (pH –4)
58:42 %v/v with flow rate of 1.0ml/min.

Chromatogram No – 5

The peaks of Amlodipine Besylate and Telmisartan were good and retention time of both
peaks was 4.320 and 5.320, there is a good resolution of 3.771.
Among the 5 trials, the 5th trial was selected as the peaks are good with less retention time.

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Chapter6 Method devolpment and optimization

6.2 OPTIMIZED CHROMATOGRAPHIC CONDITIONS

Stationary phase : Phenomenex Luna C18 (250 × 4.6 mm, 5µm)

Mobile phase : Acetonitrile : Phosphate buffer(58:42% v/v)
PH : 4
Flow rate : 1 ml/min.
Column Temperature : Room temperature.
Volume of Injection : 20µl.
Detection of Wavelength : 236 nm.
Run time : 8 min.
Mode of Operation : Isocratic.

PREPARATION OF SOLUTIONS
Preparation of buffer solution:

3.8954g of disodium hydrogen phosphate and 3.4023g of potassium dihydrogen

phosphate in 1000 ml volume of distilled water and pH is adjusted to 4 with orthophosphoric acid.

Preparation of Mobile Phase

Mobile phase is prepared by mixing 580 ml of acetonitrile and 420 ml of buffer
(58:42).

PREPARATION OF STANDARD STOCK SOLUTION:

An accurately weighed quantity of 5 mg of Amlodipine besylate and 40 mg of Telmisartan were
transferred into a 100 ml volumetric flask. Dissolved with 25 ml of mobile phase and diluted to
required volume with mobile phase, having the concentration of 0.4 mg/ml of Telmisartan and
0.05 mg/ml of Amlodipine besylate.

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Chapter6 Method devolpment and optimization

Preparation of Standard Solution

From the standard stock solution 5 ml is pipetted out into 100 ml volumetric flask
and made up the volume with mobile phase, having the concentration of 0.02 mg/ml of Telmisartan
and 0.0025 mg/ml of Amlodipine besylate.

PREPARATION OF SAMPLE SOLUTION

Twenty tablets were weighed and ground to a fine powder. An amount of powder
equivalent to 40 mg of Telmisartan and 5 mg of Amlodipine besylate were weighed accurately and
transferred into a 100 ml volumetric flask containing 25 ml of mobile phase and sonicatedfor
30 min. and diluted to 100 ml with mobile phase, then the solution was filtered through 0.45 µm
membrane filter and 5 ml of filtrate taken into 100 ml volumetric flask and made up to the volume
with mobile phase.
The standard stock solution is diluted to the working concentration equivalent to that ofsample.
20 µl of the standard and sample are injected separately and chromatograms are generated, with
peak area obtained for standard and sample.

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Chapter7 Method validation

7. METHOD VALIDATION

After the method development the method was validated in terms of parameters like
Accuracy, Precision, Specificity, Linearity, Ruggedness, and Robustness, stability etc;

SYSTEM SUITABILITY PARAMETERS

For system suitability, five replicates of standard solutions of Amlodipine and Telmisartan
were injected and studied the suitability parameters like Plate number (N), Resolution (R) and
Relative retention time (α), and Peak symmetry of samples (As) were studied with the help of
standard chromatograms.

Table No: 1.System suitability Parameters

SystemSuitability Amlodipine Besylate Telmisartan

Parameters
Resolution
3.746
Tailing Factor 1.0402 0.918

Number of theoretical 3594 3334

Plates
Retention time 4.348 5.342

LINEARITY AND RANGE: The linearity of calibration curves (analyte to peak area ratio Vs
concentration) in pure solution was checked over the concentration ranges of 2.5-15 µg/ml for the
Amlodipine Besylate and 20-120 µg/ml for Telmisartan respectively. The linearity was evaluated
by linear regression analysis, using least squares method.
The calibration curves were linear in the studied range and equations of the regression analysis
obtained for Amlodipine, and Telmisartan Y = 65.93X + 10.34(r = 0.9999), and Y = 46.22X +
82.08 (r = 0.998) respectively. The mean ± standard deviation (SD) for the slope, intercept and
correlation coefficient of standard curves were calculated.

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 Chapter7 Method validation

The slope, intercept and correlation coefficient values for Amlodipine Besylate were found to
be 65.93, 10.34 and 0.999 respectively.
The slope, intercept and correlation coefficient values for Telmisartan were found to be 46.22,
82.08 and 0.998 respectively.

Table-2: Linearity Data for Amlodipine Besylate and Telmisartan

Concentration of Peak Area of Concentration of Peak Area of
Amlodipine Amlodipine Telmisartan Telmisartan(Mv)
Besylate (µg/ml) Besylate(Mv) (µg/ml)
169.754 987.511
2.5 20

347.523 2031.509
5.0 40

518.159 2899.555
7.5 60

680.211 3800.012
10.0 80

832.696 4717.711
12.5 100

985.623 5552.031
15.0 120

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Figure no.4 Linearity plot for Amlodipine Besylate

AMLODIPINE BESYLATE
1200
y = 65.935x + 10.341
1000
R² = 0.9991
Peak Area

800

600

400

200

0 5 10 15 20
Concentration

Figure no.5 Linearity plot for Telmisartan

TELMISARTAN
6000
y = 46.223x + 82.083
5000 R² = 0.9989

4000
Peak Area

3000

2000

1000

50 100 150

Concentration

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Table .3. Analytical performance Parameters for Amlodipine Besylate

and Telmisartan

Amlodipine
Parameters Telmisartan
Besylate

Linearity range 2.5-15µg/ml 20-120µg/ml

Correlation
0.999 0.998
Coefficient

Slope (m) 65.93 46.22

Intercept 10.34 82.08

ACCURACY
Accuracy expresses the closeness of agreement between the value, which was accepted either
as conventional true value or and accepted reference value (International standard e.g
pharmaceutical standard) and the value found (mean value) obtained by applying the test
procedure a number of times.
To study reliability, suitability and accuracy of the method, recovery studies were carried out,
by adding a known quantity of the standard to the pre analysed sample and recovery study were
done. The recovery was carried out at 80%, 100%, 120% level and the contents were determined
from respective chromatogram .From the results obtained we conclude that themethod was
accurate.

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Chapter7 Method validation
Table No: 4 Recovery studies for Amlodipine Besylate and Telmisartan
S.I
Inj.Sample Spike level Amount Amount % Recovered
No:
Present Recovered

1 80 % 10mcg 9.9765 99.765%

Amlodipine
2 100 % 12.5mcg 12.471 99.77%
Besylate

3 120 % 15mcg 14.9558 99.70%

4. 80 % 80mcg 79.968 99.60%

5 Telmisartan 100 % 100 mcg 99.951 99.99%

6 120 % 120mcg 119.872 99.89%

Table No: 5 Mean Average Recovery of Amlodipine Besylate and Telmisartan for
Accuracy

Mean Recovery of Mean Recovery of

Accuracy level
Amlodipine Besylate Telmisartan

Accuracy 80%
99.6
99.76

Accuracy 100%
99.77 99.99

Accuracy 120%
99.70 99.89

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Chapter7 Method validation

PRECISION:
REPEATABILITY OF INJECTION
The Precision of test method was done by performing assay on five replicate
determination of sample preparation at test concentration level (as per method of analysis) and
calculated relative standard deviation of assay results.
Five injections from standard solutions were injected and the peak areas were obtained
and %RSD was calculated. System precision and method precision were determined.
Table No: 6 System Precision Amlodipine Besylate and Telmisartan

Area of
Area of
Amlodipine
S.NO: Telmisartan
Besylate
(mV)
(mV)

1 804.202 4664.222

2 806.602 4665.943

3 816.968 4562.585

4 815.694 4663.066

5 817.332 4664.558

Mean 812.1596 4642.075

S.D 6.256531 44.65221

R.S.D 0.770357 0.961902

Acceptance Criteria: The RSD should be NMT 2.0%.

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 Chapter7 Method validation
Table No: 7. Method Precision of Amlodipine Besylate and Telmisartan.

Area of Area of
Sample
Amlodipine Besylate Telmisartan
No:
(mV) (mV)

1 833.187 4565.943

2 826.814 4664.222

3 828.435 4572.585

4 811.505 4663.066

5 822.505 4654.558

Mean 824.489 4642.075

S.D 8.20293 44.65221

R.S.D 0.9949105 0.961902

Acceptance Criteria: The RSD should be NMT 2.0%.

ROBUSTNESS

For demonstrating the robustness of the developed method experimental conditions werepurposely
altered and evaluated. The method must be robust enough to withstand such slight changes in
chromatographic conditions and allow routine analysis of the sample. Effect of column temperature,
and Effect of buffer PH were carried out and standard was injected. There was no change in system
suitability parameters.

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Chapter7 Method validation
Table No: 8

Retention time of
Retention time of
Effect Amlodipine
Telmisartan
Besylate

PH (3.8) 4.667 5.633

PH (4.2) 4.660 5.620

Temp(23ºc) 4.567 5.640

Temp(30ºc) 4.260 5.227

RUGGEDNESS

Defined by USP, The Ruggedness is the degree of reproducibility of test results obtained
under a variety of conditions, such as different laboratories, analysts, instruments, environmental
conditions, operators and materials. Ruggedness is a measure of reproducibility of test results
under normal, expected operational conditions from laboratory and from analyst to analyst.
Table no: 9

Area of Amlodipine
Analysts Area of Telmisartan
Besylate

Analyst 1 849.651 4828.953

Analyst 2 828.034 4778.204

LIMIT OF DETECTION
Limit of detection is the lowest concentration of the analyte that can be
detected by injecting decreasing amount, not necessarily quantity by the method, under the stated
experimental conditions. The minimum concentration at which the analyte can be detected is

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Chapter7 Method validation
determined from the linearity curve by applying the formula.
LOD = 3.3 SD/slope.
The lowest concentration of Amlodipine Besylate that can be detected was determined
from standard curve was 0.04791 µg/ml.
The lowest concentration of Telmisartan that can be detected was determined from
standard curve was 0.04496 µg/ml.

LIMIT OF QUANTITATION
Limit of quantitation is the lowest concentration of the analyte in a sample that
can be estimated quantitatively by injecting decreasing amount of drug with acceptable precision
and accuracy under the stated experimental conditions of the method .Limit of quantitation canbe
obtained from linearity curve by applying the following formula.
LOQ = 10 SD/ slope.
The lowest concentration at which peak can be quantified is called LOQ. It was found
to be 0.1452 µg for Amlodipine Besylate and for Telmisartan was found to be 1.3625 µg.

Table No: 10

Sample LOD LOQ

Amlodipine 0.04791µg 0.1452 µg

Besylate

Telmisartan 0.4496µg 1.3625µg.

STABILITY STUDIES:
Stability of the sample and standard used in HPLC method is required for a
reasonable time to generate reproducible and reliable results. The stability of the sample spiked
with drug was subjected to short term stability at room temperature (Initial & after 8 hours).

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 Chapter7 Method validation
Table No: 11 Solution Stability after 8 hours.

Area of Amlodipine Area of

Time
Besylate Telmisartan

Initial 845.034 4826.725

After 8 hours 846.390 4829.735

Deviation 1.356 3.010

ASSAY RESULTS:

PROCEDURE:
Separately Blank, Standard and test preparation was injected into liquid
chromatogram and the areas for major peaks were recorded by using the following
formula.Calculation:

Calculated the amount of Amlodipine Besylate and Telmisartan using the formula.
Sample area × sample dilution × purity of working standard × Average weight×100
Standard area × standard dilution × Label claim
Table No: 12 Assay result for Replicate injection
Area of % of AMLO % of
Inj .No Amlodipine Area of Recovered TELMI
Besylate Telmisartan recovered
1 808.439 4715.886 98.55 98.11
2 820.048 4793.930 99.96 99.73
3 825.462 4771.852 100.62 99.22
Mean 817.983 4760.556 99.71 99.02
S.D 8.697344 40.22954 1.05740 0.82831
%R.S.D 1.06326 0.84506 1.06048 0.83651

RESULT : The mean recovery for assay results was found to be 98 – 101 % for
Amlodipine Besylate as well as Telmisartan.
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Chapter-8 Chromatograms

8.CHROMATOGRAMS

Chromatogram No – 6

BLANK

Chromatogram No – 7

Standard Chromatogram: Amlodipine Besylate

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 Chapter-8 Chromatograms

Chromatogram No – 8

Standard chromatogram: Telmisartan

Chromatogram No – 9

Standard chromatogram: Amlodipine and Telmisartan

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 Chapter-8 Chromatograms

Chromatogram No – 10
Assay 1

Typical Chromatogram of Assay I

Chromatogram No – 11
Typical Chromatogram of Assay II

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Chapter-8 Chromatograms

Chromatogram No – 12

Typical Chromatogram of Assay III

Chromatogram No – 13

Typical Chromatogram of Assay(spl) Chromatogram No – 14

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Chapter-8 Chromatograms

Chromatogram of Assay (spl) II

Chromatogram No – 15

System suitability

Chromatogram No – 16

Linearity 1

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Chapter-8 Chromatograms

Chromatogram No – 17

Linearity 2

Chromatogram No – 18

Linearity 3

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Chapter-8 Chromatograms

Chromatogram No – 19

Linearity 4

Chromatogram No – 20

Linearity 5

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Chapter-8 Chromatograms

Chromatogram No – 21

Linearity 6

Chromatogram No – 22

System Precision -1

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Chapter-8 Chromatograms

Chromatogram No – 23

System Precision -2

Chromatogram No – 24

System Precision -3

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Chapter-8 Chromatograms

Chromatogram No – 25System Precision -4

Chromatogram No – 26

Systemsion – 5

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Chapter-8 Chromatograms

Chromatogram No – 27

Method Precision – 1

Chromatogram No – 28

Method Precision – 2

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Chapter-8 Chromatograms

Chromatogram No – 29

Method Precision – 3

Chromatogram No – 30 Method Precision – 4

Chromatogram No – 31

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 Chapter-8 Chromatograms

Method Precision – 5

Chromatogram No – 32Accuracy -1

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 Chapter-8 Chromatograms

Chromatogram No – 33Accuracy -2

Chromatogram No – 34

Accuracy -3

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Chapter-8 Chromatograms

Chromatogram No – 35

Robustness (Column Temp)

Chromatogram No – 36

Robustness (Column Temp)

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Chapter-8 Chromatograms

Chromatogram No – 37

Robustness (PH _ 3.8)

Chromatogram No – 38

Robustness (PH _ 4.2)

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 Chapter-8 Chromatograms

Chromatogram No – 39Ruggedness (analyst 1)

Chromatogram No – 40Ruggedness (analyst 2)

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 Chapter-8 Chromatograms

Chromatogram No – 41

Stability Studies (0 hrs)

Chromatogram No – 42

Stability Studies (8 hrs)

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Chapter-9 Results And Discussion

8. RESULTS AND DISCUSSION

After several trails with various solvents, mobile phase system composed of acetonitrile
and phosphate buffer of PH 4.0 in the proportion of 58: 42 v/v. respectivelywas chosen
for the simultaneous estimation of Amlodipine Besylate and Telmisartan and in
combined dosage form by RP-HPLC. This mobile phase composition offered maximum
resolution for the drug at the detection wavelength of 236nm.The column used was C18
phenomenex luna (250 × 4.6mm) with flow rate of 1.0 ml/min and UV Detection was
carried out.
The chromatogram of Amlodipine Besylate and Telmisartan reference standard were
presented in chromatogram 7 and chromatogram - 8 respectively. The individualpeaks
of Amlodipine Besylate and Telmisartan were identified by knowing the retention time
4.320 and 5.320 minutes respectively.
Estimation of Amlodipine Besylate and Telmisartan in tablet dosage forms by
RP-HPLC method was carried out using optimized chromatographic conditions. The
standard and sample solutions were prepared. The chromatograms were recorded. The
peak area ratio of standard and sample solutions was calculated. The results of analysis
shows that the amount of drugs was in good agreement with the label claim of the
formulation. The tablet shows percentage purity values ranging from 99.80 % for
Amlodipine Besylate and 99.65 % for Telmisartan respectively.
The Linearity for the both drugs, From the calibration curve constructed by plotting
concentration vs. peak area, it was found that there exists a linear relationship in the
concentration range 2.5 to 15 µg/ml and 20 to 120 µg/ml for Amlodipine Besylate and
Telmisartan , with 0.999 and 0.998 as the value of correlation coefficient for the both
drugs respectively as (Table 2) & (Fig 3).these are within Limit.
System suitability studies were carried out in which the resolution between the peaks,
tailing factor and number of theoretical plates was found and are presented.( Table 1).
For System Precision studies, the standard solution was prepared at working
concentration and analysis was carried for five replicated injections. The percentage
relative standard deviation (% RSD) was calculated for the peak areas for Amlodipine
and Telmisartan and it was found and presented in (Table 6).and it is not more than
2.0%.

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Chapter-9 Results And Discussion

The acceptance criterion of method precision was found to be RSD NMT 2.0% and
the Method Precision for Amlodipine and Telmisartan shows 0.9949 and 0.9619. For
the Accuracy of the method was determined by performing recovery studies at 80%,
100%, 120%. The recovery study was carried out and results were expressed in terms
of the percentage recovery range found to be within the limit and the data was presented
in (Table 4 and 5).
The Robustness of the method was by changing the parameters like buffer pH and
changing the column temperature and the result was found to be with in limit. The
method shows no in the system suitability and precision parameters.
Ruggedness was determined by preparing six individual samples in replicate injections
using different analyst, different column, different system.
The Limit of Detection (LOD) and Limit of Quantitation (LOQ) Of the developed
method were determined by injecting progressively low concentrations of the standard
solutions using the developed RP-HPLC method .The LOD is the smallestconcentration
of the analyte that gives a measurable response (signal to noise ratio of 3).The detection
limit (LOD) was found to be 0.04791 µg/ml for Amlodipine Besylateand 0.4496 µg/ml
for Telmisartan respectively.
The LOQ is the smallest concentration of the analyte, which gives response that can be
accurately quantified (signal to noise ratio of 10).The quantitation limit (LOQ) was
found to be 0.1452 µg/ml for Amlodipine Besylate and 1.3625 µg/ml for Telmisartan
respectively.
The Stability studies was carried on sample and standard solutions on initial day
and after 8 hours for the estimations of Amlodipine Besylate and Telmisartan and
were considered stable for 8 hrs.
The Linearity, precision, accuracy, specificity, repeatability of measurement of peak
area as well as repeatability of sample applications are validated as per ICH guidelines
and the results are shown in the table below.

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Chapter-9 Results And Discussion

Observation
Parameter Limits AMLO TELM
S.NO

No No
1 Specificity No interference interference interference
System precision 0.7703 0.9619
Method precision RSD NMT 2.0% 0.9949 0 .9619
2
Correlation
Linearity range coefficient NMT – 0.999 0.998
3 0.999

Accuracy % Recovery 99.70-99.77 99.60-99.99

4 range 98 –10 2 %
Signal noise ratio
Limit of Detection should be more than 0.04791 0.4496
5 3:1

Limit of Signal noise ratio

Quantitation should be more than 0.1452 1.3625
6 10:1
Asymmetry factor
7 NMT 2% 1.85 1.06
Number of
8 Theoretical Plates NLT 2000 3594 3334
Robustness No effect on No effect on
Change in column system system
temperature, suitability suitability
9 Change in buffer parameters parameters
pH

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 Chapter10 Summary and conclusion

9. SUMMARY AND CONCLUSION

RP-HPLC method was developed and validated as per ICH guidelines for the estimation
of Amlodipine and Telmisartan.
Simultaneous Estimation of Amlodipine and Telmisartan were carried out by RP- HPLC using
Phosphate buffer (PH 4.0): Acetonitrile (42:58) and column Phenomenex Luna C-18(250*4.6 mm,
5um) as a stationary phase and peak was observed at 236 nm which was selected as a wavelength
for quantitative estimation. After the development of the method, it was validated for specificity,
linearity, precision, accuracy, robustness and ruggedness studies.
The system suitability parameter also reveals that the values within the specified limit for
the proposed method. Theoretical plate for Amlodipine was found to be 3594, for Telmisartan it
was found to be 3334.
The precision of the System and Method were checked and found to be within limits.
This indicates that the method is precise.

From the linearity studies, the specified range for Amlodipine and Telmisartan was found to
be 20% to 120%. It was evaluated by the visual inspection of the plot of Peak area vs. Concentration.
From the results shown in the accuracy table, it was found that recovery value of pure drug
were between 98.0 % to 102% which indicates that the method is accurate and also reveals that
commonly used excipients and additives present in the pharmaceutical formulations were not
interfering in the proposed methods .
The ruggedness of the method was checked by different analysts and found that the results
were nearly same which indicates that the method is rugged.

The robustness of the method was checked by varying PH change in buffer solution, variation in
temperature and found that the system suitability parameters were within limit at all variable
conditions, hence the method is robust.

Based on the results observed, it was concluded that proposed method can be used for
routine analysis of Amlodipine and Telmisartan.

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Chapter-11 Bibliography

10. BIBLIOGRAPHY

1. Amrish Sharma, Mukul Tailang, Bhaskar Gupta and Ashish Acharya, RP HPLC
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publishers and distributors, New Delhi (India), 2001,3-137.

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