Development and Validation of Analytical Methods for Quantification of Some Novel Drugs in Formulation.

(1)

1

DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS FOR QUANTIFICATION OF SOME NOVEL DRUGS IN FORMULATION

THESIS

Submitted to

The Tamilnadu Dr. M. G. R. Medical University

Guindy, Chennai-600032, Tamilnadu, India

In partial fulfillment of the requirement for the award of the degree of

DOCTOR OF PHILOSOPHY

(Faculty of Pharmacy)

Submitted by

A. MEENA, M. Pharm.,

Under the guidance of

Prof. V. VAIDHYALINGAM, M. Pharm, Ph.D.,

VELS COLLEGE OF PHARMACY CHENNAI

(2)

2

CONTENTS

S.no.

Title

Page no.

I

INTRODUCTION

1

1.1 Drug Profile

20

II

AIM AND OBJECTIVE OF THE STUDY

30

III

REVIEW OF LITERATURE

31

IV

MATERIALS AND METHODS

42

4.1 Reference Standards

42

4.2 Reagents and Instruments

43

4.3 Pharmaceutical Formulation Used

45

4.4 Development and Validation of Analytical Techniques

46

V

RESULTS AND DISCUSSION

102

VI

SUMMARY AND CONCLUSION

122

(3)

3 ABBREVIATIONS

% Percentage

max Maximum wave length

mM Millimole

µg Microgram

ACN Acetonitrile

APZ Alprazolam

AR Analytical grade

ASN Aspirin

BP British Pharmacopeia

CIL Cilnidipine

CLN Cilastatin sodium

et al And others

g Gram

H3PO4 Phosphoric acid

HCl Hydrochloric acid

HPLC High Performance liquid Chromatography

HPTLC High Performance Thin Layer Chromatography

ICH International conference on Harmonization

IMI Imipenem

IP Indian Pharmacopeia

ISM Isosorbide Mononitrate

KH2PO4 Potassium dihydrogen Phosphate

(4)

4

LOQ Limit of quantification

min Minutes

ml Millilitre

NaOH Sodium hydroxide

NAP Naproxen

ng Nanogram

nm Nanometer

Rf Retention factor

RP-HPLC

Reverse Phased High Performance Liquid

Chromatography

RSD Relative Standard Deviation

RT Retention time

SD Standard Deviation

TEL Telmisartan

TLC Thin Layer Chromatography

USP United States Pharmacopeia

v/v Volume by volume

(5)

5

LIST OF TABLES

Table No. Title

Formulation 1 : Naproxen and Esomeprazole magnesium

1 Linearity range (UV Spectrophotometry)

2 Analytical performance (UV Spectrophotometry) 3 Recovery Data (UV Spectrophotometry)

4 Repeatability (UV Spectrophotometry) 5 System Suitability Test (RP - HPLC) 6 Linearity Range (RP -HPLC)

7 Statistical Data for precision ( RP- HPLC ) 8 Accuracy /Recovery Data ( RP - HPLC ) 9 Stability of analytical solutions (RP - HPLC) 10 Robustness Data ( RP - HPLC )

11 Linearity data (HPTLC )

12 Analytical performance parameter ( HPTLC ) 13 Precision Data ( HPTLC )

14 Accuracy Data (HPTLC )

Formulation 2 : Telmisartan and Cilnidipine

15 Linearity range ( UV Spectrophotometry)

16 Analytical performance ( UV Spectrophotometry) 17 Recovery Data ( UV Spectrophotometry)

(6)

6

19 System Suitability Test (RP - HPLC) 20 Linearity Range (RP -HPLC)

21 Statistical Data for precision (RP- HPLC) 22 Accuracy /Recovery Data ( RP - HPLC ) 23 Stability of analytical solutions (RP - HPLC) 24 Robustness data( RP-HPLC)

25 Linearity data ( HPTLC )

26 Analytical performance parameter (HPTLC) 27 Precision Data ( HPTLC )

28 Accuracy Data ( HPTLC )

Formulation 3 : Alprazolam and Propranolol hydrochloride

29 Linearity range (UV Spectrophotometry)

30 Analytical performance (UV Spectrophotometry) 31 Recovery Data ( UV Spectrophotometry)

32 Repeatability (UV Spectrophotometry) 33 System Suitability Test (RP – HPLC) 34 Linearity Range (RP -HPLC)

35 Statistical Data for precision ( RP- HPLC ) 36 Accuracy /Recovery Data ( RP - HPLC ) 37 Stability of analytical solutions (RP - HPLC) 38 Robustness Data ( RP - HPLC )

(7)

7

Formulation 4 : Cilastatin sodium and Imipenem

43 Linearity range ( UV Spectrophotometry)

44 Analytical performance ( UV Spectrophotometry) 45 Recovery Data ( UV Spectrophotometry)

46 Repeatability ( UV Spectrophotometry ) 47 System suitability Test ( RP - HPLC ) 48 Linearity Range ( RP -HPLC )

49 Statistical Data for precision (RP- HPLC) 50 Accuracy /Recovery Data ( RP - HPLC ) 51 Stability of analytical solutions ( RP - HPLC ) 52 Robustness data( RP-HPLC)

53 System Suitability Test ( RP - HPLC )

Formulation 5 : Isosorbide Mononitrate and Aspirin

54 Linearity Range ( RP -HPLC )

55 Statistical Data for precision ( RP- HPLC ) 56 Accuracy /Recovery Data ( RP - HPLC ) 57 Stability of analytical solutions ( RP - HPLC ) 58 Robustness Data ( RP - HPLC )

(8)

8

LIST OF FIGURES

Figure:1 Schematic procedure for HPTLC method development

Figure:2 Chromatogram of Naproxen and Esomeprazole magnesium (Trial )

Figure:3 Chromatogram of Telmisartan and Cilnidipine

Figure:4 Chromatogram of Propranolol hydrochloride and Alprazolam

Figure:5 Chromatogram of Cilastatin sodium and Imipenem

Figure:6 Chromatogram of Isosorbide Mononitrate and Aspirin.( Trial )

Figure:7 UV spectrum for Naproxen max

Figure:8 UV spectrum for Esomeprazole magnesium max

Figure:9 Overlain spectrum of Naproxen and Esomeprazole magnesium.

Figure:10 Linearity graph of Naproxen and Esomeprazole magnesium

Figure:11 Calibration curve of Naproxen

Figure:12 Calibration curve of Esomeprazole magnesium

Figure:13 Chromatogram of standard (I) Naproxen and Esomeprazole magnesium

Figure:14 Chromatogram of standard (II) Naproxen and Esomeprazole magnesium

Figure:15 Chromatogram of standard (III) Naproxen and Esomeprazole magnesium

Figure:16 Chromatogram of standard (IV) Naproxen and Esomeprazole magnesium

Figure:17 Chromatogram of standard (V) Naproxen and Esomeprazole magnesium

Figure:18 Linearity of Naproxen (RP-HPLC)

Figure:19 Linearity of Esomeprazole magnesium (RP-HPLC)

Figure:20 Chromatogram of Naproxen and Esomeprazole magnesium in formulation

Figure:21 Overlain spectrum of Naproxen and Esomeprazole magnesium

(9)

9

Figure:23 Standard (I) Densitogram of Naproxen and Esomeprazole magnesium

Figure:24 Standard (II) Densitogram of Naproxen and Esomeprazole magnesium

Figure:25 Standard (III) Densitogram of Naproxen and Esomeprazole magnesium

Figure:26 Standard (IV) Densitogram of Naproxen and Esomeprazole magnesium

Figure:27 Standard (V) Densitogram of Naproxen and Esomeprazole magnesium

Figure:28 Linearity for Naproxen (HPTLC)

Figure:29 Linearity for Esomeprazole magnesium (HPTLC)

Figure:30 Densitogram of Naproxen and Esomeprazole magnesium formulation

Figure:31 UV spectrum for Telmisartan max

Figure:32 UV spectrum for Cilnidipine max

Figure:33 Overlain spectrum of Telmisartan and Cilnidipine tablets

Figure:34 Linearity graph of Telmisartan and Cilnidipine tablets

Figure:35 Telmisartan calibration curve

Figure:36 Cilnidipine calibration curve

Figure:37 Standard (I) Chromatogram of Telmisartan and Cilnidipine

Figure:38 Standard (II) Chromatogram of Telmisartan and Cilnidipine

Figure:39 Standard (III) Chromatogram of Telmisartan and Cilnidipine

Figure:40 Standard (IV) Chromatogram of Telmisartan and Cilnidipine

Figure:41 Standard (V) Chromatogram of Telmisartan and Cilnidipine

Figure:42 Linearity curve of Telmisartan

Figure:43 Linearity curve of Cilnidipine

Figure:44 Chromatogram of Telmisartan and Cilnidipine formulation

Figure:45 Overlain spectra of Telmisartan and Cilnidipine

Figure:46 Dimensional spectrum of Telmisartan and Cilnidipine

(10)

10

Figure:48 Standard (II) Densitogram of Telmisartan and Cilnidipine

Figure:49 Standard (III) Densitogram of Telmisartan and Cilnidipine

Figure:50 Standard (IV) Densitogram of Telmisartan and Cilnidipine

Figure:51 Standard (V) Densitogram of Telmisartan and Cilnidipine

Figure:52 Linearity for Telmisartan (HPTLC)

Figure:53 Linearity for Cilnidipine (HPTLC)

Figure:54 Chromatogram of Telmisartan and Cilnidipine formulation

Figure:55 UV spectrum for Alprazolam max

Figure:56 UV spectrum for Propranolol hydrochloride max

Figure:57 Overlain spectrum of Alprazolam and Propranolol hydrochloride

Figure:58 Linearity graph of Alprazolam and Propranolol hydrochloride

Figure:59 Calibration curve of Propranolol hydrochloride

Figure:60 Calibration curve of Alprazolam

Figure:61 Chromatogram of standard (I) Alprazolam and Propranolol hydrochloride

Figure:62 Chromatogram of standard (II) Alprazolam and Propranolol hydrochloride

Figure:63 Chromatogram of standard (III) Alprazolam and Propranolol hydrochloride

Figure:64 Chromatogram of standard (IV) Alprazolam and Propranolol hydrochloride

Figure:65 Chromatogram of standard (IV) Alprazolam and Propranolol hydrochloride

Figure:66 Linearity of Propranolol hydrochloride (RP-HPLC)

Figure:67 Linearity of Alprazolam (RP-HPLC)

Figure:68 Chromatogram of Alprazolam and Propranolol hydrochloride formulation

Figure:69 Overlain spectrum of Alprazolam and Propranolol hydrochloride

Figure:70 Standard (I) Densitogram of Alprazolam and Propranolol hydrochloride

Figure:71 Standard (II) Densitogram of Alprazolam and Propranolol hydrochloride

(11)

11

Figure:73 Standard (IV) Densitogram of Alprazolam and Propranolol hydrochloride

Figure:74 Standard (V) Densitogram of Alprazolam and Propranolol hydrochloride

Figure:75 Linearity for Propranolol hydrochloride (HPTLC)

Figure:76 Linearity for Alprazolam (HPTLC)

Figure:77 Densitogram of Alprazolam and Propranolol hydrochloride formulation

Figure:78 UV spectrum for Cilastatin sodium max Figure:79 UV spectrum for Imipenem max

Figure:80 Overlain spectrum of Cilastatin sodium and Imipenem tablets

Figure:81 Linearity spectrum of Cilastatin sodium and Imipenem tablets

Figure:82 Cilastatin sodium calibration curve

Figure:83 Imipenem calibration curve

Figure:84 Standard (I) Chromatogram of Cilastatin sodium and Imipenem

Figure:85 Standard (II) Chromatogram of Cilastatin sodium and Imipenem

Figure:86 Standard (III) Chromatogram of Cilastatin sodium and Imipenem

Figure:87 Standard (IV) Chromatogram of Cilastatin sodium and Imipenem

Figure:88 Standard (V) Chromatogram of Cilastatin sodium and Imipenem

Figure:89 Linearity curve of Cilastatin sodium

Figure:90 Linearity curve of Imipenem

Figure:91 Chromatogram of Cilastatin sodium and Imipenem formulation

Figure:92 Chromatogram of standard (I) Aspirin and Isosorbide Mononitrate

Figure:93 Chromatogram of standard (II) Aspirin and Isosorbide Mononitrate

Figure:94 Chromatogram of standard (III) Aspirin and Isosorbide Mononitrate

Figure:95 Chromatogram of standard (IV) Aspirin and Isosorbide Mononitrate

Figure:96 Chromatogram of standard (V) Aspirin and Isosorbide Mononitrate

(12)

12

Figure:98 Linearity of Isosorbide Mononitrate (RP-HPLC)

Figure:99 Chromatogram of Aspirin and Isosorbide Mononitrate in formulation

Figure:100 Overlain spectrum of Aspirin and Isosorbide Mononitrate

Figure:101 Dimensional spectrum of Aspirin and Isosorbide Mononitrate

Figure:102 Standard (I) Densitogram of Aspirin and Isosorbide Mononitrate

Figure:103 Standard (II) Densitogram of Aspirin and Isosorbide Mononitrate

Figure:104 Standard (III) Densitogram of Aspirin and Isosorbide Mononitrate

Figure:105 Standard (IV) Densitogram of Aspirin and Isosorbide Mononitrate

Figure:106 Standard (V) Densitogram of Aspirin and Isosorbide Mononitrate

Figure:107 Linearity for Aspirin (HPTLC)

Figure:108 Linearity for Isosorbide Mononitrate (HPTLC)

(13)

1

I.

INTRODUCTION

Analytical chemistry is the study of separation, identification and

quantification of the chemical components of natural and artificial material.

Quantitative analysis gives an indication of identity and purity of the chemical species

in the sample and quantitative analysis determines the amount of one or more of these

components. The separation of components often performed prior to analysis.

Analytical chemistry is also focused on improvement in experimental design,

chemometrics and creation of new measurement tools to provide better chemical

information.

Analytical instrumentation plays an important role in production and

evaluation of new product. Analytical technique is a method that is used to determine

the concentration of a chemical compound or chemical element. There are a wide

variety of techniques used for analysis, from simple weighing to titration to advanced

techniques using highly specialized instruments. The most common techniques used

in analytical chemistry are the following.

 Titrimetry, based on the quantity of the titrant needed to react with the analyte.

 Electroanalytical methods, includes potentiometry and volumetry.

 Spectroscopy based on the differential interaction of the analyte along with

electromagnetic radiation.

 Chromatography in which the analyte is separated from the rest of the sample

so that it may be measured without interference from other compounds

 Microscopy

 Bio-analysis

(14)

2 NEED FOR METHOD DEVELOPMENT

Analytical method development and validation plays important role in the

discovery, development and manufacture of pharmaceuticals. Pharmaceutical

products formulated with more than one drug are referred to as combination products.

These combination products can present daunting challenges to the analytical chemist

responsible for the development and validation of analytical methods.

The aim of method development is to develop and validate analytical methods

like UV, high performance liquid chromatography (HPLC) and High performance

thin layer chromatography (HPTLC) for drug products containing more than one

active ingredient. The official test methods that results from these process can be used

by quality control laboratories to ensure the identity purity, potency and efficacy of

the drug products.

Basic criteria for new method development of drug analysis

 The drug or drug combination may not be official in any pharmacopoeia

 The proper analytical procedure of the drug may not be available in the

literature due to patent regulation.

 Analytical methods may not be available for the drug in the form of a

formulation due to interference caused by the excipients.

 Analytical methods for the quantification of the drug in biological fluids may

not be available.

 The existing analytical procedure may require expensive reagent and solvents.

 It may also be cumbersome extraction and separation procedure and these may

(15)

3 UV SPECTROPHOTOMETRIC METHODS

The technique of ultraviolet visible spectroscopy is one of the most frequently

employed methods in pharmaceutical analysis.

The basis of all the spectrophotometric techniques for multicomponent sample is the

property that at all wavelengths

 the absorbance of the solution is the sum of absorbance of the individual

components or

 the measured absorbance is the difference between the total absorbance of the

solution in the sample cell and that of the solution in the reference cell.

There are various spectrophotometric methods available which can be used for the

analysis of combination samples. The following methods can be used

 Simultaneous equation method

 Derivative spectroscopic method

 Absorbance ratio method [Q- Absorbance method]

 Orthogonal polynomial method

(16)

4 SIMULTANEOUS EQUATION METHOD

If a sample contains two absorbing drugs namely X and Y each of which absorbs at

the λmax of the other, it may be possible to determine both the drugs by the technique

of simultaneous equation method [vierodt’s method]

The information’s required are

i. The absorptivities of X at λ1 and λ2, ax1and ax2 respectively

ii. The absorptivities of Y at λ1 and λ2, ay1and ay2 respectively

iii. The absorbance of the diluted samples at λ1 and λ2, A1 and A2 respectively

iv. At λ1 A1 = ax bcx+ay1bcy 1

At λ2 A2 = ax2bcx+ay2bcy 2

For measurement in 1 cm cell b = 1

Rearrange eq. (2)

A2-ax2Cx

Cy =

ay2

A2ay2-A1ay2

Cx =

Ax2ay1-axay2

criteria for obtaining maximum precision based upon absorbance ratio

A2/A1 ay2/ay1

and ax2/ax1 A2/A1

should lie outside the range 0.1- 2.0 for the precise determination of Y and X

respectively. These criteria are satisfied only when the λ max of the two components

(17)

5 ABSORBANCE RATIO METHOD

The absorbance ratio method is the modification of the simultaneous equation

procedure. It depends on the property that, for a substance which obeys Beer’s law at

all wave length, the ratio of absorbance at any two wave length is a constant value

independent of concentration or path length. Two different dilutions of the same

substance give the same absorbance ratio A1/A2 and this is referred as Q value in USP.

In the quantitative assay of two components in admixture by the absorbance

ratio method, absorbance is measured at two wavelengths. One being a wave length of

equal absorptive of the two components ie, an iso-absorptive point [permarowski et

al., 1961]

The above equation explains the concentration of X in term of absorbance

ratio, the absorbance of a mixture and the absorptive wavelength. Accurate dilution of

the sample solution and of the standard solution of X and Y are necessary for the

accurate measurement of A1 and ax1 respectively.

Geometric correction method and orthogonal polynomial methods involves

mathematical correction procedure which reduces or eliminate the background

(18)

6 DIFFERENCE SPECTROSCOPY

The essential feature of a difference spectroscopy assay is that the measured

value is the difference in the absorption (∆A) between equimolar solutions of the

analyte in different chemical forms which exhibit different spectral characteristics.

The criteria for applying difference spectrometry to the assay of the substance

in the presence of other absorbing substance are that;

 Reproducible changes may be induced in the spectrum of the length by

the addition of one or more reagents.

 The absorbance of the interfering substance is not altered by the

(19)

7 DERIVATIVE SPECTROSCOPY

Derivative spectrophotometry involves the conversion of a normal spectrum to

its first, second or higher derivative spectrum.

The first derivative (D1) spectrum is a plot of the rate of change of absorbance

with wavelength against wavelength ie.,

The second derivative (D2) spectrum is a plot of the curvature of the D0

spectrum against wave length ie.,

d2A

vs 

d2

SOLVENT EXTRACTION METHOD

If the interference from other absorbing substance is large or if its contribution

to the total absorption cannot be calculated, it may be possible to separate the

interfering substance from the analyte by solvent extraction procedure. This is

appropriate for acidic and basic drugs whose state of ionization determines the solvent

(20)

8 HPLC METHOD

High Performance Liquid Chromatography is the most versatile and widely

used type of elution chromatography. The types of high performance liquid

chromatography are often classified by separation mechanism or by the type of

stationary phase.

These include

i. Partition or liquid- liquid chromatography

ii. Adsorption or liquid - solid chromatography

iii. Ion- exchange or ion chromatography

iv. Size exclusion chromatography

v. Affinity chromatography

INSTRUMENTATION

The basic system of HPLC is illustrated below

The mode of operation of this system is isocratic ie., one particular solvent or a

mixture is pumped throughout the analysis and for some determinations the solvent

(21)

9 HPLC instrumentation includes a

i. Pump

ii. Injection

iii. Column

iv. Detector

v. Data system

Pump

High pressure pumps are needed to force solvents through packed stationary

beds.

Mechanical pumps of the reciprocating piston type give a pulsating supply of

mobile phase. A damping device is therefore required to smooth out the pulses so that

excessive noise at high level of sensitivity or low pressure does not distract from

detection of small quantities of sample.

Dual piston reciprocating pumps produce an almost pulse-free flow because

the two pistons are carefully phased so that as one is filling and the other is pumping.

These pumps are expensive than single piston pumps but are of benefit when using a

flow-sensitive detector such as an ultraviolet or refractive index detector.

Injection system

Injection ports are of two basic types

 Those in which the sample is injected directly into the column

 Those in which the sample is deposited before the column inlet and then swept

(22)

10 Column

Typical HPLC columns are 5, 10, 15 and 25 cm in length and are filled with

small diameter (3, 5 or 10 µm) particles. The internal diameter of the column is

usually 4.6 mm. However if pure substances are to be collected then larger diameter

column may be needed.

There are two variants in use in HPLC depending on the relative polarity of

the solvent and stationary phase.

Normal phase HPLC.

Here the column is filled with tiny silica particles and the solvent is non-polar.

Polar compounds in the mixture sticks to the polar silica and the non-polar

compounds will elute quickly.

Reverse Phase HPLC

In Reversed Phase HPLC silica is modified to make it non polar by attaching

long hydrocarbon chains to its surface with either 8 or 18 carbon atoms and the

solvent is polar. Non polar compounds in the mixture are retained by the column and

the polar molecules will travel through the column more quickly.

Detector

The detector of the separated components in the eluate from the column is

based upon the bulk property of the eluate (eg. its refractive index) or the solute

property of the individual components (eg. their ultraviolet absorption, fluorescence

or electrochemical activity). The most commonly used detector in the HPLC analysis

of pharmaceutical substance is described below.

Photometric detectors

These normally operate in the ultraviolet region of the spectrum and are the

most extensively used detector in pharmaceutical analysis. They comprise essentially

(23)

11

measurement and a photomultiplier tube or diode to measure the intensity of

transmitted light.

Photometric detectors are of five principal types.

Single wavelength detector:

The absorbance is measured at the most intense resonance wavelength of

mercury at 254 nm.

Multi-wavelength detector:

It allows a number of monochromatic wavelengths to be selected eg. 206,

226,280, 313,340 or 365 nm.

Variable wavelength detector:

It allows the selection of wavelength between 190-360 nm.

Programmable detector:

It allows the automatic change of wavelength between and during

chromatographic analysis.

Diode assay detector:

These are microprocessor- controlled photodiode array spectrophotometer.

Each diode detects light at a discrete wavelength.

Fluorescence Detector.

These are filter fluorimeter or spectrofluorimeter equipped with grating

monochromator and micro flow cells. Their sensitivity depends on the fluorescence

properties of the components in the eluate. For substance that are fluorescent

fluorescence detector are used. They are more sensitive than photometric detectors.

Refractive index detector

These are differential refractometer which responds to the change in bulk

property of the refractive index of the solution of the components in the mobile

(24)

12 Electro chemical detector

These are based on standard electrochemical principle involving amperometry,

voltametry and polarography. These detectors are very sensitive for substances that

are electroactive, ie., those that undergo oxidation or reduction at a suitable potential.

Data system

The main goal in using electronic data system is to increase analytical

accuracy and precision while reducing operator attention. In routine analysis where no

automation is needed a pre programmed computing integrator may be sufficient. If

higher control levels are desired, more intelligent devices such as data station or

(25)

13

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

HPTLC is a sophisticated instrument technique based on the full capabilities

of thin layer chromatography. The advantage of automation, scanning full

optimization, selective detection principle, minimum sample preparation, hyphenation

etc enable it to be a powerful analytical tool for chromatographic information of

complex mixture of inorganic, organic and biomolecules.

The advancement in terms of better separation and resolution and faster

resolution is due to

 Use of readymade HPTLC plates with optimized adsorbent layers having

small particle size with uniform particle size distribution as the stationary

phase.

 The use of pressure enables faster development of chromatogram even in

complex mixtures.

(26)

14 Stationary phase

It uses HPTLC plates featuring small particles with a narrow size distribution.

HPTLC plates provide improved resolution, higher detection sensitivity and improved

in situ quantification and are used for industrial pharmaceutical densitometric

quantitative analysis. Silica gel with a less polar mobile phase, such as chloroform–

methanol, has been used for NP- HPTLC. Lipophilic C-18, C-8, C-2; phenyl

chemically-modified silica gel phases; and hydrocarbon- impregnated silica gel plates

developed with a more polar aqueous mobile phase, such as methanol–water or

dioxane–water, are used for RP-HPTLC.

Plates are used without pretreatment unless chromatography produces

impurity fronts due to contamination of the plate. Generally methanol is used as a

prewashing solvent.

Mobile Phase:

The selection of mobile phase is based on adsorbent material used as

stationary phase and physical and chemical properties of analyte.

Sample preparation and application:

Proper sample preparation is an important prerequisite for the successful

HPTLC separation. If the analyte concentration is sufficiently high, pharmaceutical

dosage forms can often be simply dissolved in a solvent that will completely

solubilize the analyte and leave excipients or extraneous compounds undissolved to

yield a test solution that can be directly spotted for HPTLC analysis. Several steps

may be necessary such as grinding, sonication, filtration, extraction, centrifugation,

and concentration procedure if low concentration of analyte is present in

(27)

15 Development of Chromatogram:

HPTLC plates are developed in flat-bottom chambers, twin-trough chambers,

or horizontal-development chambers. Separations obtained in HPTLC are affected by

the vapor phase, which depends on the type, size, and saturation condition of the

chamber during development. The interactions of these three phases as well as other

factors, such as temperature and relative humidity, must be controlled to obtain

reproducible TLC separations.

Detection

Detection under UV light is the first choice as it is nondestructive. The layer

can be viewed under long [366nm] and short wave [254nm] UV light.

Chemical Derivatization

Derivatization is necessary in most cases to visualize the analytes of interest.

Derivatization can be performed either by immersing the plates or by spraying the

plates with a suitable reagent. To induce or optimize the derivatization reaction, it

may be necessary to heat the plates.

Quantification

Most modern HPTLC quantitative analysis are performed in situ by measuring

the zones of samples and standards using a chromatogram spectrophotometer usually

called a densitometer or scanner with a fixed sample light beam in the form of a

rectangular slit. Quantitative evaluation is performed with the TLC Scanner 3 using

(28)

16 Validation of Analytical Method

Analytical method validation is the process used to confirm that the analytical

procedure employed for a specific test is suitable for its intended use. Results from

methods validation can be used to judge the quality, reliability and consistency of the

analytical results; it is an integral part of any good analytical practice.

Analytical methods used to be validated or revalidated.

 Before their introduction into routine use

 Whenever the condition changes or which the method has been validated

The USP has published specific guidelines for method validation for compound

evaluation. USP defines right steps for validation.

 Accuracy  Precision  Specificity

 Limit of detection  Limit of quantitation  Ruggedness

 Robustness

The FDA has also published guidelines for the validation of bio-analytical methods.

Wegscheider15 has published procedure for method validation with a special focus on

calibration, recovery experiments, method comparison and investigation of

ruggedness.

Accuracy

The accuracy of an analytical method is the closeness of test results obtained

by that method to the true value.

Precision

The precision of an analytical procedure expresses the closeness of agreement

(29)

17

homogenous sample under prescribed condition. Precision may be considered at three

levels.

 Repeatability

 Intermediate precision

 Reproducibility

The precision of an analytical procedure is usually expressed as the variance, standard

deviation or coefficient of variation of a series of measurements.

Repeatability expresses the precision under the same operating conditions over

a short interval of time. Repeatability is also termed intra- assay precision.

Intermediate precision expresses within laboratory variations different days,

different analysis, different equipment etc.

Reproducibility expresses the precision between laboratories.

Limit of detection.

The detection limit of an individual analytical procedure is the lowest amount

of analyte in a sample which can be detected but not necessarily quantitated as an

exact value.

Limit of Quantitation:

The quantitation limit of an individual analytical procedure is the lowest

amount of analyte in a sample which can be quantitatively determined with suitable

precision and accuracy.

Linearity and Range

The linearity of an analytical procedure is its ability (within a given range) to

obtain test results which are directly proportional to the concentration of analyte in the

(30)

18

The range of an analytical procedure is the interval between the upper and

lower concentration of analyte in the sample for which it has been demonstrated that

the analytical procedure has a suitable level of precision, accuracy and linearity.

Robustness

The robustness of an analytical procedure is a measure of its capacity to

remain unaffected by small, but deliberate variations in method parameters and

(31)

19

STATISTICS IN ANALYTICAL METHOD VALIDATION

The statistics required for the interpretation of validation results include:

 The calculation of the mean, standard deviation, confidence intervals and

relative standard deviation for data sets obtained;

 Regression analysis for evaluation of linearity and accuracy

 Comparative studies

 Validation statistics software package are normally used for the calculation of

(32)

20

I.1 DRUG PROFILE

A. Cilnidipine

Structure:

Molecular formula : C27 H28O2N2

Molecular weight : 492.52

IUPAC Name :

1,4-dihydro-2,6-dimethyl-4-(3-nitropheny -3,5 pyridine dicarboxylic

acid- 2 methoxyethyl(2E)- 3-Phenyl-2-Propenyl ester

Description : Light yellow crystalline powder

Solubility : Soluble in DMSO and ethanol

Therapeutic use : Slow acting calcium channel blocker, antihypertensive and

(33)

21 B. Telmisartan

Structure:

Molecular formula : C30 H30N4O2

IUPAC Name :

2(4-{(4-methyl-6-(1-methyl-1H-1,3benzodiazol-2-yl)-2-propyl-1H-1,3-benzodiazol-1-yl)methyl}phenyl)benzoic

acid

Description : White to slightly yellowish colored powder.

Solubility : Insoluble in water soluble in strong base and sparingly

soluble in strong acid

(34)

22 C. Imipenem

Structure:

Molecular formula : C12 H17N3O4S

IUPAC Name

:

(5R, 6S)-6-((1R)-1-hydroxy ethyl)-3-({2-[(iminomethyl)

amino] ethyl} thio)-7-oxo-1-azabicyclo (3.2.0)

hept-2-ene-2-carboxylic acid.

Description : White solid.

Solubility : Soluble in water

(35)

23 D. Cilastatin sodium

Structure:

Molecular formula : C16H25N2NaO5S

IUPAC Name : (Sodium 7-(2-amino-2-carboxy-ethyl)sulfanyl-2-(2,2

-dimethylcyclopropyl)carbonylamino-hept-2-enoate;

Description : Off white solid.

Solubility : Soluble in water and dimethyl sulfoxide

Therapeutic use : It inhibits human enzyme dehydro peptidase which is

(36)

24 E. Naproxen

Structure:

Molecular formula : C14 H14O3

IUPAC Name : (+)-(S)-2-(6-methoxy naphthalene- 2-yl) propanoic acid

Description : White powder.

Solubility : Soluble in methanol, dimethyl sulfoxide

(37)

25 F. Esomeprazole magnesium

Structure:

Molecular formula : C34H36MgN6O6S2

IUPAC Name :

(T-4)-Bis[5-methoxy-2-[(S)-[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazolato]magnesium

Description : White to slightly coloured powder

Solubility : Soluble in methanol, slightly soluble in water

(38)

26 G. Alprazolam

Structure:

Molecular formula : C17 H13ClN4

IUPAC Name : 8- Chloro-1-methyl-6-phenyl- 4H - s-triazolo

[4,3-α] [1,4] benzodiazepine.

Description : White crystalline powder

Solubility : Soluble in methanol and ethanol

Therapeutic use : Anxiolytic, Sedative, Hypnotic, Skeletal muscle

(39)

27 H. Propranolol hydrochloride

Structure:

Molecular formula : C16H21NO2•HCl

IUPAC Name : 1-(1-naphthyloxy) -2- hydroxy -3-isopropyl amino propane

hydrochloride

Description : White powder

Solubility : Soluble in water

(40)

28 I. Aspirin

Structure:

Molecular formula : C9 H8O4

IUPAC Name : 2-acetoxy benzoic acid

Solubility : Soluble in ethanol, dimethyl sulfoxide and dimethyl

formamide

Therapeutic use : Anti-pyretic, inflammatory, analgesic and

(41)

29 J. Isosorbide Mononitrate

Structure:

Molecular formula : C6 H9NO6

IUPAC Name : 8-nitrooxy-2,6-dioxabicyclo[3.3.0] octan-4-ol

Solubility : Soluble in alcohol, water and Chloroform

(42)

30

II.

AIM AND OBJECTIVE OF THE STUDY

Analytical techniques play a crucial role in new drug development. New drugs or

combinations are often developed for eliciting better clinical response. An appropriate

analytical method is required to study the pharmacokinetic, therapeutic and

toxicological effects of such new entities.

The present work is focused on developing analytical techniques for multi-component

dosage forms without prior separation.

The aim of the study is to develop rapid, sensitive, simple and accurate methods like

UV, HPLC, and HPTLC techniques for the following formulations.

1. Cilastatin sodium and Imipenem

2. Cilnidipine and Telmisartan

3. Naproxen and Esomeprazole magnesium

4. Alprazolam and Propranolol hydrochloride

5. Aspirin and Isosorbide Mononitrate

THE AIM AND SCOPE OF THE PROPOSED WORK ARE AS UNDER

 To develop a simple, rapid and reliable methods by UV, HPLC and HPTLC

for simultaneous determination of selected formulations.

 To validate the developed methods

 The scope is the application of the validated methods for routine analysis

(43)

31

III.

LITERATURE REVIEW

1. Parra A et al., (1993) performed first and second derivative

spectrophotometry for the quantitation of mixture of Imipenem and Cilastatin

sodium compound that have closely overlapping spectral band. Beers law was

obeyed at concentration up to 100 mg/ml for Imipenem in both the first and

second derivative model and up to 75 mg/ml for Cilastatin in the first

derivative mode. Detection limits at the p=0.05 level of significance were

calculated to be 0.4 and 0.52 mg/ml of Imipenem and Cilastatin sodium

respectively in the first derivative mode and in a range of from 0.45 to

0.68mgm/ml for Imipenem in the second derivative mode. The method which

is rapid, simple and does not require a separation step has been successfully

applied to the assay of commercial injections.

2. Forsyth et al., (1994) described a UV spectrophotometric assay to measure

the concentration of the active drug components (Imipenem & Cilastatin) or

primaxin for routine release testing. The assay is based on the use of first order

derivative spectrophotometry. The trough amplitude in the first derivative

spectrophotometric spectra at 243 nm and 318 nm were selected to determine

Cilastatin and Imipenem respectively. A linear relationship (R>0.99) between

the trough amplitude and concentration were demonstrated over the range

14-42 mg/ml for both drug components.

3. L Garcia-Capdevila et al., (1997) developed a rapid and simple HPLC

method for the determination of Imipenem in human plasma. After blood

collection, plasma was separated by centrifugation and immediately stabilized

with 3-morpholino propane sulfonic acid (MOPS) and ethylene glycol (1:1).

(44)

32

ultrafiltration. The mobile phase was boric acid buffer. The Imipenem was

detected at 300nm and Cilastatin co-administered did not interfere. Calibration

curve in human plasma was linear from 0.1 to 100 mcg/ml. The limit of

detection was 0.03mcg/ml. Inter-day precision at 0.1mcg/ml. Only 250 of

plasma were required in our assay. Due to the limited stability of Imipenem,

stability studies in plasma were done to established storage conditions. The

assay was applied to pharmacokinetic studies in patients.

4. Gandhimathi M et al., (2003) developed simple and accurate method for

simultaneous determination of Aspirin and IsosorbideMononitrate in

formulation by reverse phase high pressure liquid chromatography. The

method was carried out on a Thermo quest C-18 Column using a mixture of

water – methanol (water pH Adjusted to 3.4 using dilute phosphoric acid) and

detection was carried out at 215 nm using chlorzoxazone as internal standard.

5. Aparicio I et al., (2006) developed and validated an analytical method for the

simultaneous determination of Imipenem and Sulbactum in mouse plasma.

Analytical determination was carried out by high performance liquid

chromatography with diode array detection. Chromatographic separation was

achieved within 11 min on a C18 column by gradient elution with borate buffer

[0.1mM pH 7.2] and methanol. Imipenem and Sulbactum were monitored at

295 nm and 230 nm respectively.

6. Wankhede SB et al., (2007) described a validated reverse phase high

performance liquid chromatographic method for simultaneous estimation of

Telmisartan and Hydrochlorothiazide in tablet formulation. Chromatography

was performed on a ODS hypersil C- 18 (25 cm x 4.6 mm ID) column from

thermo isocratic mode with mobile phase containing acetonitrile: 0.05 M

(45)

33

monitored at 271 nm. The selected chromatographic conditions were found to

be effectively separate Telmisartan (RT – 5.19 min) and hydrochlorothiazide

( RT - 2.97 min) .

7. GirijaBhavar, VA chatpalliwar (2008) devised a simple, accurate and

precise HPTLC method for estimation of propanol hydrochloride from bulk

drug and tablet formulations. The separation was achieved on TLC plates

using Isopropanol: ethyl acetate: ammonia (1:8.5:0.5, V/V/V) or mobile phase.

The spots so developed were densitometrically scanned at 290nm. The

linearity of the method was found to be within the concentration range of

200-2000 ng/spot. The validation parameters tested in accordance with the

requirements of ICH guidelines, prove the suitability of this method. The

method was successively applied for determination of drug in tablets, wherein

no interference from tablet excipients was observed.

8. Lee et al., (2008) developed a LC-MS /MS method for the analysis of

Cilnidipine in human plasma. A water 2795 liquid chromatography was used.

The stationary phase was capeellpak UG 120 CN column (50X 2 mm, 5 µm

particle sizes). The column temperature was maintained at 40οC. The mobile

phase consist of 10 mM ammonium acetate buffer and methanol (70:30, v/v)

adjusted to pH 5 with acetic acid and was isocratically delivered at 0.2 ml/

min. The injection volume was 10 µl. The total run time was 3 min.

Calibration curves for Cilnidipine were constructed over the range from 0.1 to

30 ng/ml. Correlation coefficient was 0.9999. The limit of quantification was

0.1g/ml.

9. PrabhatAgarwal et al., (2008) developed a accurate, precise and economical

bio analytical method for the simultaneous determination of Imipenem and

(46)

34

10.Shah N N et al., (2009) described a simple, rapid, sensitive and accurate UV

spectrophotometric method for simultaneous estimation of esomeprazole

magnesium and naproxen in dosage form. In (40%v/v) methanol, naproxen

and esomeprazole magnesium showed absorbance maxima at 262 nm and

302nm respectively.

11.Sujana K et al., (2010) explained a simple, selective, precise and stability

indicating RP-HPLC method for analysis for Telmisartan in pure and

pharmaceutical dosage form. The chromatographic conditions comprised of a

reverse phase C-18 column (4.6mm x 150 mm x 3.5 µm) with a mobile phase

composed of buffer and methanol (40:60 % v/v, adjusted the pH to 3 with

ortho-phosphoric acid). Flow rate was 0.5ml/min. Detection was carried out at

230 nm. The retention time of Telmisartan was 2.6 min.

12.Patel VA et al., (2010) described a HPTLC method and validated for the

simultaneous estimation of Telmisartan and Ramipril in a combined dosage

form. The method was carried out in TLC pre-coated silica gel on aluminum

plate 60 F 254 (10 cm x 10 cm, pre-washed by methanol and activated at 60º C

for 5 min. prior to chromatography). The solvent system was Acetone:

Benzene: Ethyl Acetate: Glacial Acetic acid in the proportion of 5: 3: 2: 0.03

(v/v/v/v) with Rf value of Telmisartan and Ramipril was 0.673 and 0.353

respectively.

13.Madhuri et al., (2010) recorded a simple, accurate and precise

spectrophotometric method for simultaneous estimation of Aspirin and

Clopidogrel bisulphate by employing second order derivative zero crossing

method. The second order derivative absorption spectra at 254 nm (zero cross

point of Aspirin) were used for Clopidogrel bisulphate and 216 nm (zero cross

(47)

35

individual constituents under stabilized conditions is very high and ranges

from 99.68% to 100.18%. A good accuracy and precision of simultaneous

determination of Clopidogrel bisulphate and Aspirin were confirmed by

statistical analysis.

14.Chaudhary AK et al., (2010) devised a simple, rapid, accurate and economic

reverse phase HPLC method for the determination of DEHP [di (2-ethyl

hexyl) phthalate] in reconstituting diluents and reconstituted solutions of

Imipenem and Cilastatin for injection. As per requirements of various

regulatory authorities of different countries, DEHP contents need to be

monitored in reconstitution. The method utilizes C18 column (250 x 4.6 nm,

5µ) and a mixture of methanol, propan-2-ol and water as mobile phase. DEHP

was detected at 225nm.

15.Shubhangi M Pawar et al., (2010) described a new, simple, precise and

accurate HPTLC method for simultaneous quantitation of Domperidone

maleate and Naproxen sodium as the bulk drug and in tablet dosage form.

Chromatographic separation of the drugs was performed on aluminum plated

pre-coated with silica gel 60 F254 as the stationary phase and the solvent

consisted of Toluene: Methanol: Acetone (8:2:2, v/v/v). Densitometric

evaluation of the separated zones was performed at 266nm. The two drugs

were satisfactorily resolved with RF values 0.44±0.02 and 0.56±0.02 for

Domperidone and Naproxen respectively. The method was linear in the

concentration range of 20-140 ng/spot for Domperidone and 500-3500

ng/spot for Naproxen

16.Sunil Jawla et al., (2010) developed and validated a simple isocratic HPLC

method for simultaneous determination of Telmisartan and Ramipril in

(48)

36

column, 5m, 250 mm v 4.60mm id with flow rate of 1.5ml/mm using PDA

detection at 210nm. The separation was carried out using a mobile phase

consisting of potassium dihydrogen phosphate buffer having pH 2.8 and

Acetonitrile in the ratio of 60:40 respectively. The retention time for

Telmisartan and Ramipril was found to be 5.7min and 10.8min respectively. A

linear response was observed over the concentration range of 2.5-25.5 ppm

and 3.0-7.25 ppm for the assay of Telmisartan and Ramipril respectively. The

limit of detection and the limit of quantitation for Ramipril were found to be

0.75ppm and 2.5ppm respectively and for Telmisartan were found to be

1.25ppm and 3ppm respectively. The result of analysis was validated

statistically and by recovery studies.

17.RekhaGangola et al., (2011) developed a simple, sensitive, specific and

economic spectrophotometric method for simultaneous estimation of

Hydrochlorothiazide and Telmisartan in tablet dosage form. In dual

wavelength method, Hydrochlorothiazide and Telmisartan were quantified

based on the absorption difference between two points on mixture spectra is

directly proportional to concentration of components of interest. The accuracy

and reproducibility of the proposed method was statistically validated by

recovery studies.

18.Tulja Rani G et al., (2011) developed a new, simple precise and accurate RP-

HPLC method for simultaneous estimation of Propranolol hydrochloride and

Alprazolam in bulk and formulation. Chromatographic separation was

achieved on a waters C-18 column using acetonitrile: water (adjusted to pH

2.3 with ortho phosphoric acid) in the ratio of 60: 40 v/v as mobile phase.

(49)

37

drugs were satisfactorily resolved with retention time values 1.737 min and

3.810 min for Propranolol hydrochloride and Alprazolam respectively.

19.Deepak Kumar Jain et al., (2011) devised a simple, precise, reliable, rapid,

sensitive RP-HPLC method to determine Esomeprazole magnesium and

naproxen in synthetic mixture form. Chromatographic separation was

achieved isocratically on Phenomenex, Luna C-18 column (5µm,

150mmX4.6mm) and acetonitrile: Phosphate buffer (pH7.0) in the ratio of

50:50 (v/v) as the mobile phase, at a flow rate of 0.5ml/min. Detection was

carried out at 300nm. The retention time for naproxen and Esomeprazole

magnesium was found to be 2.67± 0.014 and 5.65±0.09 min respectively.

Parameters such as linearity, precision, accuracy, recovery specificity and

ruggedness are studied as reported in the ICH guidelines. The method was

linear in the concentration range of 5--250µg/ml for Esomeprazole with

correlation coefficient of 0.9999 and 0.998 respectively.

20.Suresh Kumar et al., (2011) described a sensitive, accurate and rapid reverse

phase chromatography coupled tandem mass spectroscopy (LCMS/MS)

method to estimate Telmisartan in human plasma. Detection was made at m/z

513.2/ 469.3 for Telmisartan and 344/193.8 for internal standard using ESI

negative ion spray ionization mode. Analyst 1.51 software was used for the

quantification. The stationary phase was high purity advanced C-18, 50x4.6

mm, 5 µm. The separation method developed produced recovery of 84.67%.

the retention time of analyte and internal standard were 1.38 and 1.41 minutes

21.Vani P et al., (2011) developed and validated a simple, sensitive and precise

high performance liquid chromatographic method for estimating the contents

of Naproxen and Esomeprazole simultaneously in commercial pharmaceutical

(50)

reversed-38

phase column by use of mobile phase consisting of 0.05M potassium

dihydrogem phosphate (pH 7.0) and Acetonitrile (70:30 v/v) at a flow rate of

1.0ml/ min with detection wavelength at 286nm. The linearity range was

100-200 mcg/L for Naproxen and 4-8 mcg/L for Esomeprazole. The recovery

found was more than 99% for both the compound.

22.Vijayakumar G et al., (2011) published a simple, selective, linear, precise

and accurate RP-HPLC method for rapid assay of Telmisartan in serum

samples. Isocratic elution at a flow rate of 1 ml/min was employed on an

Equasil, 250X4.6 mm, 5µ at ambient temperature. The mobile phase consists

of Buffer: Acetonitrile (35:65v/v). The UV detection wavelength was 282 nm

and 20µl sample was injected. The retention time of Telmisartan was 3.32

min.

23.RakeshPrajapati et al., (2011) documented a stability indicating HPLC

method for Alprazolam and Propranolol hydrochloride in combined dosage

form. The method was validated by subjecting the drugs to forced

decomposition under hydrolysis, oxidation, photolysis and thermal stress

conditions prescribed in ICH guidelines. The drugs were successfully

separated from major and minor degradation products on a RP C18 column by

using phosphate buffer (pH 4) acetonitrile: methanol (55: 35: 10 % v/v) as the

mobile phase with detection at 254 nm. The flow rate was 1ml/min.

24.Demetriades JL et al., (2011) reported a procedure for quantification of

Cilastatin in human plasma and urine. The assay involved sample purification

on a C18 extraction cartridge, reversed phase high performance liquid

chromatography with post column derivatization and fluorescence detection.

(51)

39

200 µg/ml in urine. The limits of reliable detection were 0.75 and 2.5µg/ml in

plasma and urine respectively.

25.Neha A Jain et al., (2011) described an accurate and specific and précised

UV Spectrophotometric method for the simultaneous determination Naproxen

and Esomeprazole in a laboratory mixture of these two components. The

method involved the area under the curve (AUC) method in the range 227-237

nm and 296.5- 306.5 nm, formation of simultaneous equation at 232 nm and

301.5 nm respectively, absorption correction method at 232 nm max of

naproxen, 239.2 nm isoabsorptive point of Naproxen & Esomeprazole and

301.5nm for absorption ratio method by using methanol as a solvent.

26.PalavaiSripal Reddy et al., (2011) reported a simple, precise cost effective

and stability indicating RP- HPLC method for Naproxen and Esomeprazole in

pharmaceutical formulations by RP-HPLC separation of naproxen and

esomeprazole achieved on a X terra RP-18 Colum (150X4.6mm, 5 µ) using a

mobile phase consisting of buffer prepared with 0.005 mole of sodium

perchlorate, 5 ml of n- butyl amine in milli-Q grade water with a pH of 8.7

which was mixed with acetonitrile and methanol at a flow rate of 1.5ml / min.

Wave length chosen for detection is 305 nm. The retention times of Naproxen

and Esomeprazole peaks were around 3 and 6 minutes respectively.

27.ChandrakantSojitra et al., (2012) published a rapid, specific, sensitive and

simple high performance liquid chromatography for simultaneous estimation

of Naproxen and Esomeprazole in combined pharmaceutical formulations.

The separation was achieved by SUPELCO 516 C18 column (250 x 4.6 µm,

particle size 5 µm) with mobile phase consisting of 0.01M phosphate buffer

pH 4.5: acetonitrile: Methanol (40: 50: 10 v/v/v, addition of 0.1% triethyl

(52)

40

Retention time of Esomeprazole and naproxen was found to be 4.6 and 3.2

min respectively.

28.Mitul Patel et al., (2012) studied novel, simple, sensitive and rapid

spectrometric method for simultaneous estimation of Naproxen and

Esomeprazole magnesium trisilicate in tablet dosage form. The method

involved solving simultaneous equations based on measurement of absorbance

at two wavelengths 276nm and 302 nm respectively.

29.DeshpandePadmanabbh et al., (2012) devised a new simple high

performance thin layer chromatography (HPTLC) method for determination of

Cilnidipine and Telmisartan in combined dosage form and validated. The

separation was carried out on Merck aluminum plates pre coated with silica

gel 60 F254 using toluene : methanol : ethyl acetate (8:2:1 v/v)as the mobile

phase and the detection was carried out at 260 nm. Results were linear in the

range of 200-1200 ng/band for Cilnidipine and 800-4800 ng/ band for

Telmisartan.

30.Devanand B Shinde, Kiran R Patil et al., (2012) developed a simple, rapid

and precise method for the quantitative simultaneous estimation of

Telmisartan and hydrochloride-thiazide in combined pharmaceutical dosage

form. A chromatographic separation of the two drugs was achieved with an

ACE 5 C18 (250x4.6 mm) analytical column using buffer acetonitrile 55:45

v/v. the buffer used in mobile phase contains 0.1 M sodium perchlorate

monohydrate in double distilled water pH adjusted 3 with trifluro acetic acid.

The detection was carried out at the wavelength of 215 nm using a photodiode

array detector. The correlation coefficient for Telmisartan and

(53)

41

31. Yogeshwari S Bhangale et al., (2012) devised a simple, accurate and precise

densitometry method for the simultaneous estimation of Metoprolol succinate

and Isosorbide Mononitrate in combined capsule dosage form and validated

separation of drug was carried out using methanol. Ethyl acctate: triethylamnie

(6:4:0.1, v/v/v) as mobile phase on precoated silica gel 60 F254 plates. The

densitometric evaluation of band was carried out at 215nm. The retention

factor for Metoprolol succinate and Isosorbide Mononitrate were found to be

0.58 + 0.012 and 0.080 + 0.011 respectively. The method was validated with

respect to linearity, accuracy, precision and robustness as per ICH guidelines.

Results were found to be linear in the concentration range of 1000-7000 ng/

band for Metoprolol succinate and 3000-9000 ng/band for Isosorbide

mononitrate respectively. The method has been successfully applied for the

(54)

42

IV.

MATERIALS AND METHODS

4.1 REFERENCE STANDARDS

The following drug samples were obtained from Lupin Ltd., J.B Chemicals &

Pharmaceutical Ltd, Mumbai, Orchid Pharmaceuticals and Malladi Pharmaceuticals

Chennai. They certified the purity of the drugs.

s.no Sample name Gift sample from

1. Naproxen Lupin Limited, Pune

2. Esomeprazole Lupin Limited, Pune

3. Propranolol hydrochloride Lupin Limited, Pune

4. Alprazolam Malladi Drugs and Pharmaceuticals Ltd. Chennai

5. Aspirin Lupin Limited, Pune

6. Iso-sorbide mono nitrate Lupin Limited, Pune

7. Cilnidipine J.B Chemicals & Pharmaceutical Ltd, Mumbai

8. Imipenem Orchid chemicals and pharmaceuticals Ltd,

Chennai

9. Cilastatin sodium Orchid chemicals and pharmaceuticals Ltd,

Chennai

(55)

43

4.2 REAGENTS AND INSTRUMENTS

REAGENTS

All the solvents used in spectrophotometric analysis were of analytical reagent grade

Potassium di-hydrogen phosphate, methanol, toluene, ortho-phosphoric acid, sodium

hydroxide, formic acid, n- Hexane, ammonium acetate, isopropyl alcohol used in

HPTLC and HPLC were of analytical grade.

Methanol and water used in HPLC were of HPLC grade.

INSTRUMENTATION

The following instruments were used for the purpose of Research work.

UV Spectroscopy:

A Schimadzu spectrophotometer, Model no.1700 with 1 cm matched quartz

cells were used for the experimental work. The absorption spectra of the reference and

test solution were carried out in a 1cm quartz cell over the range of 200-400 mm. A

Schimadzu electronic analytical balance (AUX-220) was used for weighing the

samples. An ultrasonic cleaner was used for sonicating the tablet sample solution.

High Performance Liquid Chromatography:

The analysis of the drugs were carried out on a SCHIMADZU HPLC system

equipped with a reverse phase C18 column (250 x 4.6 mm, 5µm in particle size) a

LC-20 AT prominence liquid chromatography, a 20µl injection loop and SPD-20A

(56)

44

High Performance Thin Layer Chromatography:

Camag HPTLC systems with Camag linomat IV automatic sample applicator

were used for HPTLC methods. Saturation was done on Camag Twin-through

chamber (10x10cm). Camag TLC scanner 3 and Camag Wincats software and Merck

HPTLC plates coated with silicagel 60 F 254 (0.2 mm thickness) on aluminum sheets

were used.

METHODS

UV, HPLC & HPTLC methods were developed and validated for the following

combination of formulations.

i. Cilnidipine and Telmisartan

ii. Cilastatin sodium and Imipenem

iii. Naproxen and Esomeprazole magnesium

iv. Alprazolam and Propranolol hydrochloride

(57)

45

4.3 PHARMACEUTICAL FORMULATION USED

The following drug samples were procured from the local market for the purpose of

developing analytical methods.

1. Cilacart-T

Each tablet contains

Telmisartan - 40mg

Cilnidipine - 10mg

2. Naproxen and Esomeprazole magnesium tablets

(generic tablets obtained from Lupin)

Naproxen - 500mg

Esomeprazole magnesium -20mg

3. Cilanem injection

Powder for intravenous injection

Cilastatin sodium - 250mg

Imipenem - 250mg

4. Nitren

Aspirin - 50mg

Isosorbide mononitrate - 10mg

5. Alprine

Each tablet contain

Propranolol hydrochloride - 10mg

(58)

46

4.4

DEVELOPMENT AND VALIDATION OF ANALYTICAL

TECHNIQUES

4.4.1 DEVELOPMENT AND VALIDATION OF ANALYTICAL

TECHNIQUES FOR SIMULTANEOUS ESTIMATION OF NAPROXEN AND ESOMEPRAZOLE MAGNESIUM

4.4.1.1 Development of UV Spectrophotometry (Simultaneous Equation Method) for Simultaneous Estimation of Naproxen and Esomeprazole magnesium.

Preparation of standard stock solution

A stock solution 1000µg/ml each of Naproxen and Esomeprazole magnesium

were prepared by dissolving separately 100mg of drugs in methanol.

For simultaneous estimation of Naproxen [NAP] and Esomeprazole

magnesium [EOZ], 10µg/ml each of Naproxen and Esomeprazole magnesium were

prepared by diluting appropriate volume of standard stock solutions. The scanning of

the solution NAP and EOZ were carried out in the range of 400-200nm to obtain the

overlain spectrum. Absorbance were measured and absorptivities of standard solution

were calculated at selected wavelength λ,(262nm) and λ2 (304nm) for Naproxen and

Esomeprazole magnesium respectively.

Preparation and analysis of Formulations

Twenty tablets were weighed and crushed to a fine powder. An accurately

weighed powder equivalent to 100mg of Naproxen and 4 mg of Esomeprazole

magnesium was transferred to 100ml volumetric flask and dissolved in about 25ml of

methanol. After the immediate dissolution, the volume was made up to the mark with

(59)

47

Whatmann filter paper No.41 and was diluted to prepare the concentration of

100µg/ml Naproxen and 4µg/ml of Esomeprazole magnesium. Absorbance of this

solution were measured at 262nm (λ max of Naproxen) and 304nm respectively (λ

max of Esomeprazole magnesium) and the amount of drugs present in the average

weight of tablet were obtained by substituting the values in respective simultaneous

equation.

A2 ay1 – A1 ay2

CNAP =

---X2 ay1 – ax1 ay2

A1 ax2 – A2 ax1

CEOZ =

---X2 ay1 – ax1 ay2

Where

i. ax1 and ax2 are absorptivities of NAP at λ1 and λ2 respectively

ii. ay1 and ay2 are absorptivities of EOZ at λ1 and λ2 respectively

iii. A1 and A2 are absorbance of mixtures at λ1 and λ2 respectively

iv. CNAP and CEOZ are concentration of Naproxen and Esomeprazole

magnesium respectively.

Validation of the proposed method

The proposed method was validated for the parameters like linearity, accuracy,

precision and robustness as per ICH guidelines.

(60)

48

Linearity graph was plotted over a concentration range of 5 to 25 µg/ml of

Naproxen and 5-15 µg/ml of Esomeprazole magnesium. The absorbance of the

solutions were measured at 262nm and 304nm against methanol as blank. The

calibration curve was constructed by plotting absorbance versus concentration.

The regression analysis was carried out for the calibration graph to find out

correlation coefficient, y-intercept and slope of the regression line which estimates the

degree of linearity. The correlation coefficient was found to be 0.9984 for Naproxen

and 0.9981 for Esomeprazole magnesium.

Precision

Precision expresses the repeatability of the method under same operating

condition. The repeatability of the analytical method was established by estimating

the assay for six different sample preparation of the same batch and the %RSD were

determined.

Robustness

The robustness of the method was determined by subjecting the method to

slight changes in the spectrophotometric conditions. It was observed that there was no

marked change in the spectrum which demonstrated that the method developed is

robust.

Recovery studies. (Accuracy)

To check the accuracy of the developed method and to study the interference

of formulation additives, analytical recovery experiments were carried out by standard

addition method at 80,100 and 120% level. From the total amount of drug found, the

(61)

49

4.4.1.2 DEVELOPMENT OF RP-HPLC METHOD FOR THE

SIMULTANEOUS ESTIMATION OF NAPROXEN AND

ESOMEPRAZOLE MAGNESIUM

Initial chromatographic conditions were selected based on absorption,

solubilit