Analytical Method Development, Impurity Profiling and

Method Validation of Selected Cardiovascular Drugs

Thesis Submitted to the Bharathidasan University

in partial fulfillment of the requirements for the award of the Degree of

DOCTOR OF PHILOSOPHY IN CHEMISTRY

Submitted By
A. MOHAN

Research Supervisor

Dr. D. SARAVANAN

PG and Research Department of Chemistry

National College (Autonomous)
(Nationally Accredited at ‘A” grade by NACC)
Tiruchirappalli - 620 001
Tamil Nadu, INDIA.

JUNE 2011
Dr. D. Saravanan
Assistant Professor
Department of Chemistry
National College (Autonomous)
Tiruchirappalli - 620 001.

CERTIFICATE

This is to certify that the thesis entitled, “Analytical Method Development, Impurity

profiling and Method Validation for selected cardiovascular Drugs” is a bonafide record of

the work done by Mr. A. Mohan in the Post Graduate and Research Department of Chemistry,

National College (Autonomous), Tiruchirappalli-620 001, and the thesis has not previously

formed in part or fully the basis for the award to the candidate of any degree, associateship,

diploma, fellowship or any other similar title. It is also certified that this thesis represents the

independent work on the part of the candidate.

Date: Supervisor-Guide

( D. Saravanan)
 DECLARATION

I hereby declare that the thesis entitled, “Analytical Method Development, Impurity

profiling and Method Validation for selected cardiovascular Drugs” is a record of the work

done by me under the guidance and supervision of Dr. D. Saravanan, Assistant Professor,

Post Graduate and Research Department of Chemistry, National College (Autonomous),

Tiruchirappalli-620 001.

I also declare that this work has not formed in part or fully the basis for the award of any

other degree or diploma by any university.

(A. Mohan)

Date:
 Acknowledgement

“The act of thanks giving does not exhibit ones sense of gratitude, but the true tendency of lending a

helping hand during emergency and the fact that every work has thousands of hands behind”.

Fact is that every mission needs a spirit of hard work and dedication but it needs to be put on

the right path to meet its destination and in my case this credits goes to my esteemed guide,

Dr. D. Saravanan, Assistant Professor, P.G. & Research Department of Chemistry, National College

(Autonomous), Tiruchirappalli. By virtue of his invaluable scholastic suggestions and constructive

criticism, I have been able to look at things in a better way.

It’s my privilege and honor to extend profound gratitude and indebtedness to the

Management Council of National College (Autonomous), Tiruchirappalli, for accepting me as research

scholar.

I wish to extend my gratitude to Shri K. Ragunathan, Secretary, National College

(Autonomous), Tiruchirappalli, for providing the necessary help and support to carry out my work.

It is a pleasure to record my respectful thanks to Dr. K Anbuarasu, The Principal, National

College (Autonomous), Tiruchirappalli, for providing the necessary support during my research work.

I express my sincere thanks to Prof. K. Lakshmanan, Head of the Department (Chemistry),

National College (Autonomous), Tiruchirappalli, for his support and encouragement.

I express my profound and sincere gratitude to Dr. B.R.Venkataraman, Assistant Professor,

PG & Research Department of Chemistry, Periyar E.V.R.College (Autonomous), Tiruchirappalli, for

his invigorate guidance, felicitous advice, valuable hints with energizing criticism throughout the

course of this dissertation work.

i
 I would like to express my sincere thanks to Dr. A. Jafar Ahamed, Associate Professor, Post

Graduate & Research Department of Chemistry, Jamal Mohamed College (Autonomous),

Tiruchirappalli, for his valuable advice, during the Research.

I would like to thank Dr. Shameela Rajam, (Doctoral Committee Member) Reader, P.G.&

Research Department of Chemistry, Bishop Heber College (Autonomous), Tiruchirappalli, for her

valuable comments, suggestions during committee discussions.

I would like to express my deep and very much special indebtedness to my philosopher and

Guru Dr. B. Sivakumar, Associate Director, Research Center, Orchid Chemicals and Pharmaceutical

ltd., Chennai, for his incessant support during my research work.

I wish to extend my gratitude to the management of Torrent Research Centre, Ahmedabad, for

providing the necessary help and support to carry out my research work.

My precious thanks to my colleague and friends Mr. Sukhdev Singh, Mr. Vikraman,

Mr. Mandhar Kulkarni, Mr. Hariharan Mr. Shanmuga Velu, Dr. Hitesh Patel, Mr. Ananadan and

Mr. Dhayalamurthi for their support to complete my research work.

Last but not the least, present research work is beyond belief without the support of family

members and friends who have contributed in numerous ways to my marathon journey for t his research

work. It is fact that without their constant positive attitude and never ending patience, I could not

have completed of my research work

Finally, I thank the Almighty who has given me the strength and knowledge to complete my

research work.

Arivozhi Mohan

ii
 TABLE OF CONTENTS

CHAPTER 1
INTRODUCTION
1.1 Drugs 1
1.2 Cardiovascular drugs 1
1.3 Impurity profiling 6
1.4 Analytical methods 21
References 41

CHAPTER 2

SCOPE OF THE RESEARCH

2.0 Scope of the Research 46

CHAPTER 3

IDENTIFICATION AND CHARACTERIZATION OF A PRINCIPAL

OXIDATION IMPURITY IN CLOPIDOGREL DRUG SUBSTANCE AND
DRUG PRODUCT
3.1 Introduction 48
3.2 Drug profile 49
3.3 Literature review 51
3.4 Scope of the study 54
3.5 Experimental 55
3.6 Results and discussion 58
3.7 Conclusion 82
References 83

CHAPTER 4

IDENTIFICATION, ISOLATION AND CHARACTERIZATION OF FIVE

POTENTIAL DEGRADATION IMPURITIES IN CANDESARTAN CILEXETIL
TABLETS
4.1 Introduction 84
4.2 Drug Profile 84
4.3 Literature review 85
4.4 Scope of the study 87
4.5 Experimental 88
4.6 Results and Discussion 92
4.7 Formation of impurities 133

iii
 4.8 Validation of analytical method 133
4.9 Conclusion 135
References 136

CHAPTER 5

DEVELOPMENT OF A VALIDATED REVERSED PHASE UPLC METHOD

FOR RELATED SUBSTANCES AND ASSAY OF LACIDIPINE

5.1 Introduction 137

5.2 Drug profile 137
5.3 Literature review 138
5.4 Scope of the study 143
5.5 Experimental 144
5.6 Results and Discussion 151
5.7 Results of method validation experiments 158
5.8 Conclusion 176
References 177

CHAPTER 6

DEVELOPMENT AND VALIDATION OF A DISSOLUTION METHOD FOR

NOVEL FIXED DOSE COMBINATION OF ETODOLAC AND PROPRANOLOL
HYDROCHLORIDE TABLETS BY RP-HPLC
6.1 Introduction 179
6.2 Drug profile 180
6.3 Literature review 182
6.4 Scope of the study 185
6.5 Experimental 186
6.6 Method validation 188
6.7 Results and Discussion 191
6.8 Results of method validation experiments 198
6.9 Conclusion 212
References 213

iv
 LIST OF TABLES

Table 3.1 Marketed brand name list of Clopidogrel Tablets

1
Table 3.2 H and 13C NMR assignment for Clopidogrel bisulphate and Related
compound-D

Table 4.1 NMR assignments for Candesartan cilexetil and impurity-I

Table 4.2 NMR assignments for impurity-II

Table 4.3 NMR assignments for impurity-III

Table 4.4 Crystal data and refinement of Impurity-III

Table 4.5 NMR assignments for Impurity-IV

Table 4.6 NMR assignments for Impurity-V

Table 4.7 Method validation data

Table 5.1 System suitability report

Table 5.2 Summary of forced degradation results.

Table 5.3 LOD and LOQ data

Table 5.4 Summary of Linearity data.

Table 5.5 Accuracy data

Table 5.6 Method precision data

Table 5.7 Intermediate precision data

Table 5.8 Compiled data of Method precision and Intermediate precision

Table 6.1 The outcomes of each trial on various mobile phase compositions

Table 6.2 Solubility Studies of Etodolac and Propranolol hydrochloride in Various

Buffers

Table 6.3 Linearity data of Etodolac

Table 6.4 Linearity data of Propranolol hydrochloride

Table 6.5 Accuracy results for Etodolac

Table 6.6 Accuracy results for Propranolol hydrochloride

v
Table 6.7 Precision data for Etodolac and Propranolol hydrochloride

Table 6.8 Robustness study of the HPLC assay

vi
 LIST OF FIGURES

Fig. 1.1 Anatomy of Heart

Fig. 1.2 Scheme for impurity profile study

Fig. 1.3 Van Deemter plot, illustrating the evolution of particle sizes over the years

Fig. 3.1 Structural formula of Clopidogrel

Fig. 3.2 Structural formula of known Related Compounds of Clopidogrel

Fig. 3.3 Structural formula of Clopidogrel related compound D

Fig. 3.4 Typical chromatogram of Clopidogrel spiked with Impurities as per USP
method

Fig. 3.5 Typical chromatogram of Clopidogrel spiked with Impurities as per

developed method

Fig. 3.6 HPLC chromatogram of Clopidogrel drug substance

Fig. 3.7 HPLC chromatogram of Clopidogrel drug product

Fig. 3.8 HPLC chromatogram of Clopidogrel drug product spiked with related
compound D

Fig. 3.9 UV spectral overlay of Clopidogrel and Related Compound D

Fig. 3.10 MS-MS spectrum of Related compound D (Cl35 isotope)

Fig. 3.11 MS-MS spectrum of Related compound D (Cl37 isotope)

Fig. 3.12 IR spectrum of Related compound D

Fig. 3.13 Mass spectrum of Related compound D

Fig. 3.14 Fragmentation pattern of related compound D

Fig. 3.15 Fragmentation pattern of Clopidogrel bisulphate

1
Fig. 3.16 H NMR Spectrum of Clopidogrel Related compound D
13
Fig. 3.17 C NMR Spectrum of Clopidogrel Related compound D

Fig. 3.18 135 DEPT NMR Spectrum of Clopidogrel Related compound D

vii
Fig. 3.19 Irradiation data (Proton decoupling experiment)

Fig. 4.1 Structural formula of Candesartan cilexetil

Typical HPLC chromatograms of Candesartan cilexetil Tablets - Initial

Fig. 4.2
sample

Fig. 4.3 Typical HPLC chromatograms of Candesartan cilexetil Tablets - 60°C for 2
weeks

Typical HPLC chromatograms of Candesartan cilexetil Tablets-40°C /75%H

Fig. 4.4
for 3 months

Fig. 4.5 The UV spectra of the Candesartan cilexetil and its impurities

Fig. 4.6 Typical Preparative LC chromatogram of Candesartan cilexetil autoclaved

sample

Fig. 4.7 Synthetic pathway of impurities

Fig. 4.8 Structural formula of Impurity-I

1
Fig. 4.9 H NMR Spectrum of Candesartan Cilexetil
13
Fig. 4.10 C NMR Spectrum of Candesartan Cilexetil

Fig. 4.11 135 DEPT NMR Spectrum of Candesartan Cilexetil

1
Fig. 4.12 H NMR Spectrum of Impurity – I
13
Fig. 4.13 C NMR Spectrum of Impurity – I

Fig. 4.14 135 DEPT NMR Spectrum of Impurity – I

Fig. 4.15 Structural formula of Impurity-II

1
Fig. 4.16 H NMR Spectrum of Impurity - II
13
Fig. 4.17 C NMR Spectrum of Impurity – II

Fig. 4.18 135 DEPT NMR Spectrum of Impurity – II

Fig. 4.19 Structural formula of Impurity-III

1
Fig. 4.20 H NMR Spectrum of Impurity – III
13
Fig. 4.21 C NMR Spectrum of Impurity – III

Fig. 4.22 135 DEPT NMR Spectrum of Impurity – III

viii
Fig. 4.23 Single crystal structure of Impurity-III

Fig. 4.24 Structural formula of Impurity-IV

1
Fig. 4.25 H NMR Spectrum of Impurity – IV
13
Fig. 4.26 C NMR Spectrum of Impurity – IV

Fig. 4.27 135 DEPT NMR Spectrum of Impurity – IV

Fig. 4.28 Structural formula of Impurity-V

1
Fig. 4.29 H NMR Spectrum of Impurity – V
13
Fig. 4.30 C NMR Spectrum of Impurity – V

Fig. 4.31 135 DEPT NMR Spectrum of Impurity – V

Fig. 5.1 Structural formula of Lacidipine

Fig. 5.2 Structural formula of Known Impurities of Lacidipine

Fig. 5.3 Typical UPLC chromatograms of Blank and Placebo

Fig. 5.4 Typical UPLC chromatograms of System suitability

Fig. 5.5 Typical UPLC chromatogram of Thermal stressed sample

Fig. 5.6 Typical UPLC chromatogram of LOD (0.05µg/mL)

Fig. 5.7 Typical UPLC chromatogram of LOQ (0.15µg/mL)

Fig. 5.8 Typical UPLC chromatogram of Linearity at 100% level

Fig. 5.9 Typical chromatogram of Unspiked sample used in accuracy

Fig. 5.10 Typical UPLC chromatogram of Accuracy at 100% level

Fig. 5.11 Typical UPLC chromatogram of Accuracy at 120% level

Fig. 5.12 Typical UPLC chromatogram of sample Unspiked

Fig. 5.13 Typical UPLC chromatogram of sample spiked with six impurities

Typical UPLC chromatograms of Robustness study-Column oven

Fig. 5.14
temperature

Fig. 5.15 Typical UPLC chromatograms of Robustness study-Organic phase

Fig. 6.1 Structural formula of Etodolac

ix
Fig. 6.2 Structural formula of Propranolol hydrochloride

Fig. 6.3 UV spectrum of Etodolac and Propranolol hydrochloride

Fig. 6.4 Dissolution profile data

Fig. 6.5 Typical HPLC chromatograms of Blank (Dissolution medium) and Placebo

Fig. 6.6 Typical HPLC chromatograms of Analytes

Fig. 6.7 Calibration curve of Etodolac

Fig. 6.8 Calibration curve of Propranolol hydrochloride

Fig. 6.9 Typical HPLC chromatogram of Linearity solution at 10% level

Fig. 6.10 Typical HPLC chromatogram of Linearity solution at 100% level

Fig. 6.11 Typical HPLC chromatogram of Linearity solution at 150% level

Fig. 6.12 Typical HPLC chromatogram of Accuracy at 10 % level

Fig. 6.13 Typical HPLC chromatogram of Accuracy at 100 % level

x
 LIST OF ABBREVIATIONS

° pulse Degree Pulse

µg/ml Microgram Per Milliliter

µg Microgram

µl/min Micro liter Per Minute

µm Micrometer

µs Micro Second

13
C-NMR Carbon-13 Nuclear Magnetic Resonance

1
H-NMR Proton Nuclear Magnetic Resonance

ADP Adenosine Diphosphate

APIs Active Pharmaceutical Ingredients

ARA II Angiotensin II Receptor Antagonist

bs Broad Singlet

CN Column Cyano Column

CVD Cardiovascular Diseases

db Decibel

DEPT Distortionless Enhancement By Polarization Transfer

DMSO-D6 Dimethylsulphoxide-D6

ESI Electrospray Ionization

ESI-MS/MS Electrospray Ionization Tandem Mass Spectrometry

ET Etodolac

FDA Food And Drug Administration

xi
FDC Fixed Dose Combination

HCTZ Hydrochlorothiazide

HSQC Heteronuclear Single Quantum Coherence

i.d Internal Diameter

ICH International Conference on Harmonization

ICP-MS Inductively Coupled Plasma Mass Spectrometry

IS Internal Standard

LC Liquid Chromatography

LCDP Lacidipine

LMICs Low And Middle-Income Countries

LOD Limit of Detection

LOQ Limit of Quantitation

LRC Long Range Correlation

MHz Megahertz

MRM Multiple Reactions Monitoring

mt Multiplet

ng Nanogram

nm Nanometer

NSAIDs Nonsteroidal Anti-Inflammatory Drugs

PDA Detector Photo Diode Array Detector

PEG Polyethylene Glycol

pg/ml Picogram Per Milliliter

xii
PH Propranolol Hydrochloride

QA Quality Assurance

QbD Quality By Design

RH Relative Humidity

RIA Radioimmunoassay

RRF Relative Response Factor

RRT Relative Retention Time

RSD Relative Standard Deviation

UPLC Ultra Performance Liquid Chromatography

UPLC–ESI- Ultra-Performance Liquid Chromatography–Tandem Mass

MS/MS Spectrometry

USP United States Pharmacopoeia

USP/NF United States Pharmacopoeia /National Formulary

xiii
INTRODUCTION

CHAPTER 1
 Chapter 1

1.1 Drugs

A drug may be defined as a substance meant for diagnosis, cure, mitigation and

prevention, treatment of diseases in human beings or animals, for altering in structure or

function of the body of human beings or animals.[1] Pharmaceutical chemistry[2-6] is a

science that makes use of general laws of chemistry to study drugs i.e. their preparation,

chemical nature, composition, structure, influence on an organism, the methods of quality

control and the conditions of their storage etc. The family of drugs may be broadly

classified as Pharmacodynamic agents and Chemotherapeutic agents. Pharmacodynamic

agents refer to a group of drugs, which stimulate or depress various functions of body so

as to provide some relief to the body in case of body abnormalities, without curing the

disease. They are mainly used in case of noninfectious diseases so as to correct the

abnormal body functions. Non-selective central nervous system modifiers (depressants or

stimulants), adrenergic stimulants and blocking agents, cholinergic and cholinergic

blocking agents, cardiovascular agents, diuretics, antihistaminic agents and

anticoagulating agents are some examples of this group. These agents have no action on

infective organisms, which cause various diseases.

Chemotherapeutic agents are agents, which are selectively more toxic to the

invading organisms without harmful effect to the host. Some of the examples of this

group are organometallic agents, antimalarials, antibacterials, antiprotozoals, antifungal

agents, antihelmentics, antiseptics, antitubercular agents, antineoplastics, etc.

1
 Chapter 1

1.2 Cardiovascular drugs

Heart diseases or cardiovascular diseases (CVD) are the class of diseases that

involve the heart or blood vessels.[7] While the term technically refers to any disease that

affects the cardiovascular system, it is usually used to refer to those related

to atherosclerosis (arterial disease). Heart attacks and strokes are usually acute events and

are mainly caused by a blockage that prevents blood from flowing to the heart or brain.

The most common reason for this is a build-up of fatty deposits on the inner walls of the

blood vessels that supply the heart or brain. Strokes can also be caused by bleeding from

a blood vessel in the brain or from blood clots.

Every year, 17.1 million lives are claimed by the global burden of heart disease

and stroke 82% of which are in the developing world. The number of deaths especially in

low and middle-income countries (LMICs) is alarming and saddening. These deaths could

be prevented through steps such as eating a healthy diet, regular physical activity and

avoiding tobacco. By the year of 2030, almost 23.6 million people will die from CVD,

mainly due to heart diseases and stroke. These are projected to remain the single leading

causes of death. The largest percentage increase will occur in the Eastern Mediterranean

Region. The largest increase in number of deaths will occur in the South-East Asia

Region.

Each year, heart disease kills more Americans than cancer. In recent years,

cardiovascular risk in women has been increasing and has killed more women than breast

cancer.[8] By the time, the heart problems are detected, the underlying cause

(atherosclerosis) is usually quite advanced, having progressed for decades.

2
 Chapter 1

Therefore increased emphasis on preventing atherosclerosis by modifying risk factors,

such as healthy eating, exercise and avoidance of smoking.

Most countries face high and increasing rates of cardiovascular disease.

Cardiovascular medications are used as a means to control or to prevent certain forms

of heart disease. Many people with advanced heart disease may take several of

these drugs, and drug treatment may change if the disease advances or improves. The

reason people may require several types of drugs are because they may have numerous

symptoms or conditions that need control at the same time. Understanding the various

categories of these medications can be helpful.

Types of cardiovascular drugs may be broken into groups depending upon their

action or what they treat. Treatment categories are more difficult to describe since many

of these medications may address several symptoms of heart disease and have more

than one use. Categories that might describe drug actions include the following:

statins, diuretics, anticoagulants, anti-platelet, beta-blockers, digitalis drugs, vasodilators,

calcium channel blockers, and ACE inhibitors.

Cardiovascular diseases (CVD) are a group of disorders of the heart and blood

vessels and include:

Coronary heart disease – disease of the blood vessels supplying the heart muscle.

 Cerebrovascular disease - disease of the blood vessels supplying the brain.

 Peripheral arterial disease – disease of blood vessels supplying the arms and legs.

 Rheumatic heart disease – damage to the heart muscle and heart valves from

rheumatic fever, caused by streptococcal bacteria.

3
 Chapter 1

 Congenital heart disease - malformations of heart structure existing at birth.

 Deep vein thrombosis and pulmonary embolism – blood clots in the leg veins,

which can dislodge and move to the heart and lungs.

In practice, cardiovascular disease is treated by cardiologists, thoracic

surgeons, vascular surgeons, neurologists, and interventional radiologists, depending on

the organ system that is being treated. There is considerable overlap in the specialties, and

it is common for certain procedures to be performed by different types of specialists in

the same hospital.

Yet it would be hard to keep track of every single drug intended to assist in heart

disease because of the plethora that exist, and the intense research existing in this area,

which results in frequent development of new drugs.

4
 Chapter 1

Fig. 1.1 Anatomy of Heart

5
 Chapter 1

1.3 Impurity profiling

Worldwide, impurity profiling (identification and characterization of impurities

associated with drugs or drug products) is increasingly viewed as a valuable and essential

part of quality requirements. Various regulatory authorities like United States Food and

Drug Authority[9] (USFDA), European Directorate of Quality Medicine[10] (EDQM),

Therapeutic Goods Administration,[11] World Health Organization (WHO)[12] and other

health agencies[13-16] are emphasizing on the purity requirements and the identification of

impurities in drug substance and products. A key component of the overall quality of a

pharmaceutical is control of impurities, as their presence, even in small amounts, may

affect drug safety and efficacy. International Conference on Harmonization of Technical

Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines[17-23]

are developed with the joint efforts of regulators and industry representative from the

European Union, Japan and the United States. The guidelines helped to ensure that the

different regions have consistent requirements for the data that should be submitted to the

various regulatory agencies. As per ICH Q3A(R)[18] and ICH Q3B(R)[19] guidelines,

unknown impurities associated with bulk drug and dosage form, greater than the

identification threshold should be identified.

Impurity profiles for pharmaceutical products require a basis for well reasoned

and rational argument that seeks to limit both the number and amount of impurities on

safety grounds. The efficacy, safety and the dosage of drug product will determine the

level of impurities to be present in drug compound. The dose of active substance

administered will vary considerably between products and it is obvious, but frequently

ignored, that this will influence the amount of impurity administered when impurities are

controlled on a percentage or parts per unit basis.[24]

6
 Chapter 1

The toxicological assessor in a regulatory agency cannot necessarily be reassured

on safety, simply by the limitation of an impurity to a low percentage level. Some drugs

are administered by mouth at doses of >30 g/day, so that even 0.01% of an

uncharacterized impurity gives a potential patient exposure of >3 mg daily. Conversely,

the analyst may not need to struggle to achieve a low (e.g. 0.1%) limit for detection,

quantification, validation or reporting, if the daily dose to humans will only be in the

microgram or low milligram range. As a rough guide, the limitation to 1 mg daily oral

intake of an uncharacterized or poorly characterized impurity will probably satisfy a

safety assessment for regulatory purposes.[18, 19]

It is therefore necessary from the safety point of view, to know enough about the

impurity profile (both qualitative and quantitative) to allow a judgment that the impurity

will not pose any concern over safety or will be an acceptable risk factor for treating

serious diseases where there is no other therapy suitable for a particular patient.

Impurity profiling of any pharmaceutical substance is a crucial part of process

development. Hence, it was felt necessary to develop the modern chromatographic and

mass spectrometric methods for qualitative and quantitative determination of impurities in

pharmaceutical dosage forms.

1.3.1 Sources of impurities

Various sources of impurities were reported.[25, 26] They are crystallization-related

impurities, stereochemistry-related impurities, process impurities, residual solvents,

inorganic impurities and impurities arising during storage.

7
 Chapter 1

Degradation of the drug substance is one of the main sources of impurities in both

bulk drug and formulated product. Degradation of the drug substance is caused by

chemical instability of the drug substance under the conditions (e.g., heat, oxidation,

humidity, solvent, pH, light, etc.) of manufacturing, isolation, purification, drying,

storage, transportation, and interactions with other chemical entities in the formulation

(e.g., excipients and coating materials). Chemical stability is an inherent property of a

drug substance and is a reflection of the chemical properties of all functional groups in

the drug molecule.

1.3.2 Identification of Impurities

Identification and characterization of impurities is an analytical activity aiming to

elucidate the chemical structures and the possible mechanisms of formation of unknown

impurities. Because of the complexity and diversity of the impurities in both their origins

and properties, the identification strategies are determined by the specific situations.

A general strategy can be set for the identification and characterization of the

impurity of bulk drug substances by the rational use of analytical techniques. The

schematic use of the methods[27] for impurity profiling of drug substances is shown in

figure 1.2. The impurity profile of a drug substance also includes the identification of the

key impurities in the intermediates and key starting materials of their synthesis. In case

of synthesis related impurities, their mechanism and source of their formation can also be

presented. Impurity profiling also includes identification and determination of residual

solvents and inorganic impurities in the drug substances.

8
 Chapter 1

Fig. 1.2 Scheme for impurity profile study

9
 Chapter 1

Detection of an unknown impurity is the first step in impurity profiling. Typically,

unknown impurities are observed during analysis of intermediates or drug substances for

process control or at release. Once the decision for identification is made, the nature and

origin of the impurity can be assessed, based on when, where, and how the unknown is

initially observed. Depending on the structure of the drug substance, the synthetic

scheme, impurities in the starting material, known process related impurities, formulation

ingredients, and the analytical method that is used for the initial detection of the unknown

impurities; it is possible to evaluate the impurities. The impurities can be identified

predominantly by different methods like reference standard method, separation methods

and isolation methods. The characterization of impurities can be carried out using

different spectroscopic and its hyphenated techniques.

Reference standard method can be adapted by making use of the available

standard of the impurity. If the initial analysis indicates that the observed impurity falls

into this category, the impurity identification turns into practice. This can be achieved by

three experiments. First, analysis of sample is followed by analysis of standard and then

spiked sample with standard by applying any of the chromatographic or spectroscopic

techniques.

1.3.3 Quantitation of Impurities

The following techniques are being regularly used for the quantitation of

impurities and degradation products: High Performance Liquid Chromatography

(HPLC), Gas Chromatography (GC), Thin Layer Chromatography (TLC), High

Performance Thin Layer Chromatography (HPTLC), Capillary Electrophoresis (CE),

Supercritical Fluid Chromatography (SFC), and Gel Permeation Chromatography (GPC).

10
 Chapter 1

Recently Ultra Performance Liquid Chromatography (UPLC) is emerging as a fast

separation liquid chromatographic technique. Since, we have used only HPLC and UPLC

in our present work, we have described these techniques in detail as below.

1.3.3.1 High Performance Liquid Chromatography (HPLC)

HPLC is a chemistry based tool for separation, quantification and analysis of

mixtures of chemical compounds. As a consequence of the enormous development of the

analytical technology in the last two decades entirely new possibilities have been created

for the determination of the purity of drug materials. Nearly all organic impurities are

determined by chromatographic or related methods of which HPLC has been the most

important for over a decade.

HPLC is regarded as the most important analytical method in pharmaceutical

analysis as it provides a number of highly selective variants to resolve almost every type

of separation problem.[28] Derivatization of the drugs prior to analysis is normally not

required. HPLC can be operated in both modes i.e. reverse phase and normal phase mode.

Reverse phase analysis involves use of polar mobile phase (e.g. water, methanol,

acetonitrile, etc.) along with stationary phase like C8, C18, phenyl etc. Normal phase

analysis involves use of non polar solvents (e.g., hexane, dichloromethane, ethyl acetate

etc.) along with silica as a stationary phase. Reverse phase analysis is useful for polar

compounds (e.g., amines alcohols, acids etc.) while normal phase provides separation of

non polar compounds.

In reverse phase liquid chromatography, increasing the molecular size increases

the hydrophobicity of solutes and results in a greater retention volume. This indicates that

the van der Waals volume is an important property in optimization. Increasing the number
11
 Chapter 1

of substituent with n-electrons and hydrogen bonding increases the solubility in water,

i.e., they increase the polarity of the solutes. This indicates that dipole-dipole and

hydrogen-bonding interactions contribute to hydrophobicity. Therefore, these properties

are important in controlling the retention volume in reverse-phase liquid chromatography.

However, the n-electrons of stationary phase materials such as polystyrene gel and the

hydrogen bonding of non-end capped bonded silica gels also contribute to the retention.

Many compounds can be analysed by both the methods. For a preparative scale

separation, normal phase chromatography is suitable due to the easy removal of solvent.

Gradient elution (change in mobile phase composition with respect to time), temperature

and wavelength programming techniques provide valuable information regarding the

undetected components of a given drug. UV absorbing components are easily detected, if

present in sufficient quantity. Multiple wavelength UV detection program is capable of

monitoring several wavelengths simultaneously.

Photo Diode Array (PDA) detectors are used to record spectro-chromatograms

simultaneously. PDA allows simultaneous collection of chromatograms at different

wavelengths during a single run. Following the run, a chromatogram at any desired

wavelength can be displayed. The UV spectrum of each separated peak is also obtained as

an important tool for selecting an optimum wavelength for HPLC analysis. By examining

UV spectrum for a peak from beginning to end, peak purity can be evaluated.

Fluorescence (FL) detectors are exquisitely sensitive and selective, making it ideal

for trace analysis. The detection is based on analyte fluorescence by applying

monochromatic light of desired wavelength for excitation of sample molecule.

12
 Chapter 1

Refractive Index (RI) detectors is considered as a universal detector since the RI is a

physical property of all compounds, any compound can be detected at moderate levels.

Electrochemical (EC) detectors commonly used in HPLC can be classified

according to their operation:

(i) Direct- Current Amperometry (DCA) and

(ii) Conductivity.

If the analyte is EC active, a DCA detector is usually preferred because sample

derivatization and related problems are usually avoided. EC detection can be performed

in either oxidative reductive mode depending on the nature of analyte. Conductivity

detectors are mainly used in ion chromatography.[29]

1.3.3.2 Ultra Performance Liquid Chromatography (UPLC)

UPLC takes advantage of technological strides made in particle chemistry

performance, system optimization, detector design, and data processing and control.

Using submicron (about 2 micron) particles and mobile phases at high linear velocities,

dramatic increase in resolution, sensitivity, and speed of analysis can be obtained. This

new category of analytical separation science retains the practicality and principles of

HPLC while creating a step function improvement in chromatographic performance.

As illustrated in figure 1.3, as the particle size decreases to less than 2.5 µm there

is not only a significant gain in efficiency, but also the efficiency does not diminish at

increased flow rates or linear velocities. By using smaller particles, speed and peak

capacity (number of peaks resolved per unit time) can be extended to new limits, termed

as Ultra Performance Liquid Chromatography or UPLC.

13
 Chapter 1

The promises of the Van Deemter equation cannot be fulfilled without smaller

particles than those traditionally used in HPLC. The design and development of

submicron particles is a significant challenge, and researchers have been active in this

area for some time to capitalize on their advantages.[30-32] Although high efficiency,

non porous 1.5 µm particles are commercially available, they suffer from poor loading

capacity and retention due to low surface area. To maintain retention and capacity similar

to HPLC, UPLC must use novel porous particles that can withstand high pressures. Silica

based particles have good mechanical strength, but can suffer from a number of

disadvantages, which include a limited pH range and tailing of basic analytes. Polymeric

columns can overcome pH limitations, but they have their own issues, including low

efficiencies and limited capacities.

In 2000, a first generation hybrid chemistry that took advantage of the best of both

the silica and polymeric column worlds was introduced, [33, 34] a classical sol-gel synthesis

that incorporates carbon in the form of methyl groups, these columns are mechanically

strong, with high efficiency, and operate over an extended pH range. But, in order to

provide the kind of enhanced mechanical stability required for UPLC, a second
[35]
generation bridged ethane hybrid (BEH) technology was developed . These 1.7 µm

particles derive their enhanced mechanical stability by bridging the methyl groups in the

silica matrix.

Packing 1.7 µm particles into reproducible and rugged columns was also a

challenge that needed to be overcome. Requirements include a smoother interior surface

of the column hardware, and redesigning the end frits to retain the small particles and

resist clogging. Packed bed uniformity is also critical, especially if shorter columns are to

maintain resolution while accomplishing the goal of faster separations.

14
 Chapter 1

Fig. 1.3 Van Deemter plot, illustrating the evolution of particle sizes over the years

15
 Chapter 1

In addition, at high backpressure, frictional heating of the mobile phase can be

quite significant and must be considered[36] with column diameters typically used in

HPLC (3.0 to 4.6 mm). A consequence of frictional heating is the loss of performance due

to temperature induced non uniform flow. To minimize the effects of frictional heating,

smaller diameter columns (1–2.1 mm) are typically used for UPLC. [37, 38]

Faster separations can lead to higher throughput and time savings when running

multiple samples. But, a significant amount of time can also be consumed in developing

the method in the first place. Faster, higher resolution UPLC separations can cut method

development time from days, to hours, or even minutes.

Mass spectrometry (MS) has gained widespread acceptance as an analytical tool

for the qualitative and quantitative analysis of many types of compounds. MS detection is

significantly enhanced by UPLC, increased peak concentrations with reduced

chromatographic dispersion at lower flow rates (no flow splitting) promotes increased

source ionization efficiencies. Jorgenson et al. have shown that higher chromatographic

efficiency, resulting from the use of UPLC, translates into better resolution and higher

peak capacity, which is particularly important for the analysis of peptides and proteins.[39]

The increased resolving power made the resulting data easier to interpret, since

more of the MS peaks consisted of a single compound, and up to a 20 fold improvement

in the quality of the spectral information due to Nano electrospray was obtained.

Lee et al.,[40] used MS detection for the analysis of low molecular weight compounds

similar to those that might comprise a combinatorial library.

16
 Chapter 1

It was demonstrated that, in order to address the very narrow peaks produced by

UPLC, it is necessary to use a very high data capture rate MS such as a TOF or

quadrupole with fast scan rates. Lee et al., also pointed out that, in some instances, related

compounds of the same molecular weight and similar structures could not be

differentiated by MS, necessitating chromatographic resolution on the UPLC time scale.

Plumb et al. have investigated the use of UPLC/MS for the analysis of metabolites[41, 42]

and as a tool for differential metabolic pathway profiling in functional genomics

studies.[43] Their data illustrate the benefit obtained from the extra resolution of UPLC,

both in terms of specificity and spectral quality, revealing new information and reducing

the risk of not detecting potentially important metabolites.

1.3.4 Isolation techniques

It is often necessary to isolate impurities for their structural elucidation and

qualification. Generally chromatographic and non chromatographic techniques are used

for isolation of impurities prior to characterization.[44] A list of techniques that can be

used for isolation of impurities is given below.

(i) Solid phase extraction (ii) Liquid-liquid extraction (iii) Accelerated Solvent

extraction (iv) Supercritical fluid extraction (v) Column chromatography (vi) Preparative

HPLC (vii) Preparative TLC and (viii) Flash chromatography

Since preparative HPLC was used in the present investigation, this technique

alone is described in detail as under

17
 Chapter 1

Preparative HPLC is the most powerful and commonly used technique for

isolation and purification tasks in the pharmaceutical industry.[45] Application of this

technique comes into picture when the identification of impurity cannot be carried with

acceptable certainty by use of simple analytical (chromatographic, spectroscopic and

hyphenated) techniques. In this case, preparative HPLC isolation followed by

spectroscopic (NMR, MS and IR) investigation provides an appropriate solution for

structural elucidation of any unknown impurity.

In order to carry out successful isolation of targeted impurity, an appropriate

analytical LC method needs to be developed for its detection. The HPLC and TLC

information is very important for development preparative HPLC.

Selection of input sample is also important for isolation of impurities. If the

material contains only small amounts of impurities, it is advisable to select crude samples

or mother liquors which are obtained from recrystallization process, as an input sample

wherein the targeted impurities are likely to be high in concentration. Extraction or

desalting procedures are to be adopted to remove any buffers from the collected fraction.

Rotary evaporator or freeze drying technique can be implemented for concentration of

aqueous/organic fractions during work up process to get solid impurities.

1.3.5 Characterization techniques (Spectroscopic and Hyphenated Techniques)

Structural characterization of unknown impurities is a challenging task in

Pharmaceutical Analysis. Ultraviolet (UV), Fourier Transform Infrared (FT-IR), Mass

Spectrometry (MS) and Nuclear Magnetic Resonance Spectroscopy (NMR) are used for

the characterization of synthesized, isolated and degradation impurities.

18
 Chapter 1

FT-IR spectroscopy gives information about the functional groups present in the

molecule and used as a valuable tool in the structure elucidation in combination with

other spectral techniques. UV absorption spectroscopy can characterize those types of

compounds which absorbs UV radiation. Identification is done by comparing the

absorption spectrum with the spectra of known compounds and generally used for

characterizing aromatic compounds and aromatic olefins. It gives useful information

about the presence or absence of unsaturation and the presence of hetero atoms.[46]

Liquid Chromatography-Mass Spectrometry[47] (LC-MS) is an analytical

technique that couples high resolution chromatographic separation with sensitive and

specific mass spectrometric detection. This includes high performance liquid

chromatography (HPLC)-MS, Capillary Electrophoresis (CE)-MS and more recently

Capillary Electro Chromatography (CEC)-MS. The technique is still fast developing,

particularly in the mass spectrometry area, with vastly improved sensitivity and

resolution. It is probably the most powerful technique currently available for

pharmaceutical analysis.

Since impurities and degradants are usually present in relatively small quantities

compared to the drug, an analytical technique capable of separating a mixture containing

highly varied concentrations of analytes with sensitive and specific detection is required.

LC-MS is therefore widely used for this purpose. LC-MS has proved to be an extremely

sensitive and specific technique for the analysis of pharmaceuticals. It plays important

roles in the studies of drug metabolism,[48] discovery of new drug candidates and the

analysis, identification and characterization of impurities and degradants in drug

substances and products. Technical advances in MS continue, with improvements in

sensitivity and resolution. The trend is towards the further development of hybrid
19
 Chapter 1

instruments such as Q-TOF, FT-ICR will become more prominent as developments are

made and instruments become less complex and more available. The likely importance of

proteomics in pharmaceutical development will have implications for MS, leading to the

further requirements for high resolution sequencing. Coupling high-throughput sample

preparation techniques with multiplexed LC-MS-MS will lead to even faster analysis and

the potential of interfacing LC-NMR with MS to give an LC-NMR-MS system will allow

the unequivocal identification of drugs and metabolites. .[49,50] LC-MS-MS in turn gives

very useful information about the structure of the molecules by means of selective

fragmentation. Advance instrumentation techniques like Ion Trap will help in structural

identification by the fragmenting molecule up to MS8. The technical difficulties in linking

micro and capillary separation techniques with nanospray MS are being solved and

advances in this front can be expected. The use of microfluidic systems offers prospects

for miniaturized chip separations and even the possibility of miniaturized mass

spectrometers in the rather more distant future.

NMR is the most widely used technique for structural elucidation of synthesized

and isolated organic molecules. For identification and characterization of drug impurities

modern NMR offers various ranges of experiments[51] like 1H, 13C, DEPT, NOE, COSY,

Homo and Hetero Nuclear Irradiation and LRC (Long Range Correlation experiments).

Structural elucidation of impurities in drug materials mostly involve 1H, D2O Exchange
1
H and 13C experiments, the information obtained from these experiments is sufficient to

ascertain the structure of the unknown impurity in the drug material along with the other

advanced 2D NMR techniques such as Correlation spectroscopy (1H-1H COSY and

1
H-13C HSQC) etc.,[52] The 15
N, 19
F and 31
P in special cases are powerful tools for the

molecules containing the respective elements.

20
 Chapter 1

The introduction of advanced NMR probes especially for on-line coupling to

HPLC greatly reduces the need for preparative isolation of impurities. Stop-flow and

on-flow techniques are used to detect the analytes of interest.[53] HPLC analysis is carried

out in the reverse phase mode using D2O/buffer, acetonitrile based eluents with a higher

injection volume (50-100 µl).

1.4 Analytical methods

Pharmaceutical analysis[54] deals not only with medicaments (drugs and their

formulations) but also with their precursors i.e. with the raw material on which degree of

purity and the quality of medicament depend. The quality of a drug is determined after

establishing its authenticity by testing its purity and the quality of pure substance in the

drug and its formulations. Quality[55] is important in every product or service but it is

vital in medicine as it involves life. Unlike ordinary consumer goods there can be no

“second quality” in drugs. Quality control is a concept, which strives to produce a perfect

product by series of measures designed to prevent and eliminate errors at different stages

of production.

The ability to provide timely, accurate, and reliable data is central to the role of

analytical chemists and is especially true in the discovery, development, and manufacture

of pharmaceuticals. Analytical data are used to screen potential drug candidates, aid in the

development of drug synthesis, support formulation studies, monitors the stability of bulk

pharmaceuticals and formulated products, and test final products for release. The quality

of analytical data is a key factor in the success of a drug development program.

The process of method development and validation has a direct impact on the quality of

these data.

21
 Chapter 1

Analytical method is a specific application of a technique to solve an analytical

problem. The use of instrumentation is an exciting and fascinating part of chemical

analysis that interacts with all areas of chemistry and with many other areas of pure and

applied science. Analytical instrumentation plays an important role in the production and

evaluation of new products and in the protection provides the lower detection limits

required to assure safe foods, drugs, water and air. Instrument or physicochemical

methods are based on the theory of relation between the content and the corresponding

physicochemical and physical properties of the chemical system being analyzed.

Physicochemical methods [56, 57] are used to study the physical phenomena that occur as a

result of chemical reactions. Changes in the system properties are either detected or

recorded through the measurement of current, electrode potential, electrical conductivity,

optical density, refractive index etc., with suitable and sensitive instruments. In

instrument analysis physical property of a substance is measured to determine its

chemical composition. The instrument is only one compound of the total analysis. Often

it is necessary to use several instrumental techniques to obtain the information required to

solve the analytical problem. Instrumental methods may be used by analytical chemists to

save time, to avoid chemical separations or to obtain increased accuracy. The time saving

feature can be realized in routine analysis, or where a considerable number of

determinations are to be made. Most important techniques fit into one of the three

principal areas: spectroscopy, electrochemistry and chromatography.

The analytical method plays a vital role in the dossier submission and it becomes

inevitable to comply with the regulatory requirements. The intricacy of analytical

methods such as assay and related substances remains challenging to the scientists.

Regulatory authority states that the analytical method which is employed for the assay of

22
 Chapter 1

drug substance and drug product should be stability indicating. The stability indicating

assay method may have the advantage of evaluation of degraded components in the

presence of active pharmaceutical substance. The analytical method involved in the

related substances should be sensitive to estimate the synthetic or degraded impurities in

the level of 0.03% and any potential impurities less than 0.03%. The analytical method

for the related substances paves the way for the impurity profiling, elucidation of

degradation pathway, mass balance etc. The dosage forms including the presence of

various excipients pose challenge during the development of assay procedure. Standard

analytical procedures for the estimation of new drug substance for assay, related

substances, dissolution may not be available in Pharmacopoeia; hence it becomes

essential to develop newer analytical methods in such a situation. The estimation of

degraded and synthetic impurities in the presence of the analytes can be analyzed by

HPLC[58], HPTLC, LC-MS, LC-MS/MS, GC[59], GC-MS, CE and CE-MS techniques.

HPLC method is used because of several advantages like rapidity, specificity, accuracy,

precision and ease of automation has the advantage over the other methods.

The chromatographic methods are characterized by high sensitivity, selectivity

and economical consumptions of chemicals. Some of the advantages of HPLC methods

are speed (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 samples), ideal

for the substances of low volatility, easy sample recovery, handling and maintenance,

instrumentation lends itself to automation and quantitation (less time and less labor),

precise and reproducible, calculations are done by software itself and suitable for

preparative liquid chromatography on a much larger scale.

23
 Chapter 1

Specific stability indicating assay method can be defined as a method that is, able

to measure unequivocally the drug in the presence of all degradation products, excipients

and additives, expected to be present in the formulation. The in vitro evaluation of any

drug substance(s) and drug product(s) requires sensitive analytical methods. Dissolution

testing is an important parameter to assess the drug product for its quality control and in

vivo behaviour. The goal of dissolution testing is to ensure the pharmaceutical quality of

product(s). It would therefore, be desirable to develop dissolution test that can assess the

ability of dosage form to release the drug completely and to simultaneously indicate how

a product will perform in vivo. Dissolution testing is a vital parameter in the stability

studies. Stability conditions (accelerated, intermediate and long term stability studies) can

vary the drug content and its release pattern. Stability studies are carried out to predict

the shelf life, container closure system and expiry of the drug product(s).

1.4.1 Method Development

The development of any new or improved method for the analysis of an analyte

usually depends on tailoring the existing analytical approaches and instrumentation.

Method development [60, 61] usually involves selecting the method requirements and on the

type of instrumentation. Today the development of a method for analysis is usually based

on prior art or existing literature, using the same or quite similar instrumentation. It is rare

today that an HPLC based method is developed that does not in some way relate or

compare to existing, literature based approaches. The development of any new or

improved method usually tailors existing approaches and instrumentation to the current

analyte, as well as to the final needs or requirements of the method. Method development

usually requires selecting the method requirements and deciding on what type of

instrumentation to utilize and why. In the development stage, decisions regarding choice
24
 Chapter 1

of column, mobile phase, detectors, and method of quantitation must be addressed. In

this way, development considers all the parameters pertaining to any method. There are

several valid reasons for developing new method of analysis:

a) There may not be a suitable method for a particular analyte in the specific sample

matrix.

b) Existing method may be inaccurate, contamination prone, or they may be

unreliable (have poor accuracy or precision).

c) Existing methods may be too expensive, time consuming or energy intensive, or

they may not be easily automated.

d) Existing methods may not provide adequate sensitivity or analyte selectivity in

samples of interest.

e) Newer instrumentation and techniques may have evolved that provide

opportunities for improved methods, including improved analyte identification or

detection limits, greater accuracy or precision, or better return on investment.

f) There may be a need for an alternative method to confirm analytical data

originally obtained by existing methods for legal or scientific reasons.

Once the instrumentation has been selected, based on the criteria suggested above,

it is important to determine, “analyte parameters” of interest. To develop a method it is

necessary to consider the properties of the analytes of interest that may be advantageous

to establish optimal ranges of analyte parameter values. It is important that method

development is to be performed using only analytical standards that have been identified

and characterized well, and whose purity is already known. Such precautions will prevent

problems in the future and will remove variables when one is trying to optimize or

improve initial conditions during method development.

25
 Chapter 1

Before starting the any method development one has to have knowledge about the

information of the nature of sample, define separation goals, number of compounds

present, chemical structures, molecular weights, pKa values, solubility and UV spectrum

of the compounds. Perhaps maximum method development involves the trial and error

procedures. The most difficult problem usually is where to start, what type of column is

worth trying with what kind of mobile phase. While there are a number of HPLC methods

available to the development chemist, perhaps the most commonly applied method is

reverse phase chromatography method. A typical pharmaceutical compound is considered

to be an active pharmaceutical ingredient (API) of less than 1000 Daltons, either soluble

in water or in an organic solvent.

The water soluble drug is further differentiated as ionic or nonionic which can be

separated by reverse phase. Similarly, the organic soluble drugs can be classed as polar

and non polar and equally separated by reverse phase. In some cases the non polar API

may have to be separated using adsorption or normal phase HPLC, in which mobile phase

would be non polar organic solvent. The other chromatographic modes may need to be

necessary for separation. These include ion exchange, chiral and size exclusion

chromatography. In case of samples like proteins, peptides nuclic acids and synthetic

polymers analysed by using the some special columns or ion pair reagents.

1.4.1.1. General conditions to initiate HPLC method development

Either isocratic or gradient mode may be used to determine the initial conditions

of the separation. In general, one begins with reverse phase chromatography, when the

compounds are hydrophilic in nature with many polar groups and are water soluble. The

organic phase concentration required for the mobile phase can be estimated by gradient

26
 Chapter 1

elution method. For aqueous sample mixtures, the best way to start is with gradient

reverse phase chromatography. Gradient can be started with 5-10% organic phase in the

mobile phase and the organic phase concentration can be increased up to 100%

within 20-30 minutes. separation can then be optimized by changing the initial mobile

phase composition and the slope of gradient according to the chromatogram obtained

from preliminary run. The initial mobile phase composition can be estimated on the basis

of where the compounds of interest were eluted, namely at what mobile phase

composition. Changing the polarity of a mobile phase can alter elution of drug molecules.

The elution strength of a mobile phase depends upon its polarity, the stronger the polarity,

higher is the elution. Ionic samples (acidic and basic) can be separated, if they are present

in the undissociated form. Dissociation of ionic samples may be suppressed by proper

selection of pH. The buffer selected for a particular separation should be used to control

pH over the range of ≈ pKa ± 2.0. The buffer should transmit light at or below 200 nm so

as to allow low UV detection and pH of the buffer should be adjusted before adding

organic. Optimization can be started only after a reasonable has been obtained. A

reasonable chromatogram means that more or less symmetrical peaks on the

chromatogram with detection of all the compounds. The optimized chromatogram is the

one in which all the peaks are symmetrical and are well separated in less run time.

The peak resolution can be increased by using a more efficient column (column

with higher theoretical plate number), which can be achieved by using a column of

smaller particle size, or a longer column in length. These factors, however, will increase

the analysis time. Flow rate does not influence resolution, but it has a strong effect on the

analysis time.The parameter that are affected by the changes in chromatographic

27
 Chapter 1

conditions are Capacity factor (K’), Selectivity (α), Column efficiency (N) and Peak

asymmetry factor or Tailing factor (As)

1.4.1.2. Selection of mobile phase

The selection of the mobile phase mainly based on the solubility and polarity of

the compound. Usually, in RP-HPLC method water and organic solvents are used as the

mobile phase. In NP-HPLC method non polar solvents like Hexane and THF were used.

If the sample contains ionic or ionizable compounds, then use of a buffered mobile phase

to ensure the reproducible results. Under unfavorable circumstances, pH changes as little

as 0.1 pH units can have a significant effect on the separation. On the other hand properly

used buffer allows controlling the pH easily. Buffer works best at the pKa values of its

acid. At this pH, the concentration of the acidic form and the basic form of the buffering

species are equal, and the buffering capacity is maximum. Phosphate has three pKa

values in the range of interest for silica based chromatography at pH 2, pH 7 and

pH 12.32. The pKa of acidic buffer is 4.75. Citrate has three pKa values 3.08, 4.77

and 6.40. Between citrate and phosphate buffers, the entire pH range useful for silica

chromatography can be covered.

In many cases, a silanophilic interaction causes tailing, mainly for the basic

compounds due to ion-exchange interaction. This can usually be reduced or suppressed

by the use of mobile phases modifiers (0.1% v/v triethylamine for basic analyte or 1% v/v

glacial acetic acid for the acidic analyte), or a combination thereof. Whenever buffers or

other mobile phase activities are used, check the solubility in mobile phase. This is

especially true for gradient applications. acetonitrile is the preferred organic modifier in

reverse phase chromatograpy. acetonitrile based mobile phases can give up to two fold

28
 Chapter 1

lower pressure drop that can methanol based mobile phases at equal flow rate. This means

that column efficiency is higher. The elution strength increases in the order methanol,

acetonitrile and tetrahydrofuran. The retention changes by roughly 10% for every 1%

change in the concentration of organic modifier.

1.4.1.3. Role of pH

pH is another factor in the resolution that will affect the selectivity of the

separation in reverse-phase HPLC. In reverse-phase chromatography sample retention

(K’) increases when the analyte is more hydrophobic (nonpolar). Sample retention (K’)

decreases when the analyte is more hydrophilic (polar). Thus when an acid or base is

undergoes ionization it becomes more hydrophilic and less interacting with column

binding sites. When the pH value of the mobile phase equal to the pKa value of the

analyte, it is said to be half ionized, i.e. the concentration of the ionized and unionized

species are equal. As mostly all of the pH caused changes in the retention occur

within ± 2.0 pH unit of the pKa value, it is best to adjust the mobile phase to pH value

atleast ± 2.0 pH unit above or below the pKa to ensure practically 100% unionization of

analyte for retention purpose. Generally at low pH peak tailing is minimized and method

ruggedness is maximized. On the other hand, operating in the intermediate pH offers an

advantage in increased analyte retention and selectivity.

1.4.1.4. Role of buffer

In reverse phase mobile phase pH values are usually between 2.0 and 7.5. Buffers

are needed when an analyte is ionizable under reverse phase conditions or the sample

solution is outside this pH range. Analyte ionisable under reverse phase conditions often

have amine or acid functional group with pKa between 1.0 and 11.0. A correctly chosen

29
 Chapter 1

buffer pH will ensure that the ionisable functional group is in a single form, whether ionic

or neutral. If the sample solution is at pH damaging to the column, the buffer will quickly

bring the pH of the injected solution to a less harmful pH.

If the analyte contain only amine fuctional group buffer selection is easier. Most

amine will be in cationic form at pH value less than 9.0, so any buffer effective at pH 7.0

or lower will work. Buffer at pH 7.0 are used, even though pH of water is 7.0, because

amine retention and peak shapes are pH dependent. As pH is lowered amine retention

time shortens and peak shaps sharpens as the buffer protonates the acidic silanols on silica

surface. Any buffer with pKa less than 7.0 is suitable, but we have found potassium

phosphate at pH 3.0 in the best for amines. In both condition (acidic and alkaline)

potassium phosphate buffer pH 3.0 works well in general is an excellent buffer for

analyte that contain acidic and amine functional groups. The potassium salt works better

than the sodium salt for amines.

1.4.1.5. Selection of column

The HPLC column is the heart of the method, critical performing the separation.

The column must posses the selectivity, efficiency and reproducibility to provide good

separation. Commonly used reverse phases are C18 (octadecyl silane, USP L1), C8

(octylsilane, USP L7), phenyl (USP L11) and cyno (USP L18). They are chemically

different bounded phases and demonstrate significant changes in the selectivity using the

same mobile phase.

During method development selection of column can be streamlined by starting

with shorter column (150, 100 or even 50 mm long). By selecting a shorter column with

an appropriate phase run time can be minimized so that an elution order and an optimum

30
 Chapter 1

mobile phase can be quickly determined. It can also advantageous to consider the column

internal diameter, many laboratories use 4.6 mm i.d. as standard. But it is worth

considering use of 4.0 mm i.d. column as an alternative. This requires only 75% of the

solvent flow that a 4.6 mm column used. Selecting an appropriate stationary phase can

also help to improve the efficiency of the method development. For example, a C8 phase

(reverse phase) can provide a further time saving over a C18 as it doesn’t retain analyte as

strongly as the C18 phase.

1.4.1.6. Role of temperature

Temperature variation over the course of a day has quite significant effect on

HPLC separations. This can even occur in air conditioned rooms. While temperature is a

variable that can affect the selectivity, its effect is relatively small.

1.4.1.7. Role of flow rate

Flow rate, more for isocratic than gradient separation, can sometimes be useful and

readily utilized to increase the resolution, although its effect is very modest. The slower

flow rate will also decrease the column back pressure.

1.4.2 Validation of Analytical Methods

Validation of analytical method is an integral part of product development. The

developed analytical methods are validated in order to demonstrate that it is suitable for

its intended purpose. The analytical methods were validated for the following parameters

in accordance with ICH Harmonized Tripartite Guidelines.[22, 62, 63]

31
 Chapter 1

1.4.2.1 Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement

between the value which is accepted either as a conventional true value or an accepted

reference value and the value found. This is sometimes termed as trueness of the

analytical method. Accuracy of the analytical method is proved by application of the

analytical procedure to synthetic mixtures of the drug product components to which

known quantities of the drug substance to be analysed have been added.

In cases where it is impossible to obtain samples of all drug product components,

it may be acceptable either to add known quantities of the analyte to the drug product or

to compare the results obtained from a second, well characterized procedure, the accuracy

of which is stated and/or defined. Accuracy may be inferred once precision, linearity and

specificity have been established.

Accuracy should be assessed using a minimum of 9 determinations over minimum

of 3 concentration levels covering the specified range (e.g., 3 concentrations / 3 replicates

each of the total analytical procedure). Accuracy should be reported as percent recovery

by the assay of known added amount of analyte in the sample or as the difference

between the mean and the accepted true value together with the confidence intervals.

1.4.2.2 Precision

The precision of an analytical procedure expresses the closeness of agreement

(degree of scatter) between a series of measurements obtained from multiple sampling of

the same homogeneous sample under the prescribed conditions. Precision may be

considered at three levels like repeatability, intermediate precision and reproducibility.

32
 Chapter 1

Precision should be investigated using homogeneous and authentic samples. However, if

it is not possible to obtain a homogeneous sample it may be investigated using artificially

prepared samples or a sample solution. The precision of an analytical procedure is usually

expressed as the variance, standard deviation or coefficient of variation of a series of

measurements.

1.4.2.3 Repeatability

Repeatability expresses the precision under the same operating conditions over a

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

should be assessed using a minimum of 9 determinations covering the specified range for

the procedure (e.g., 3 concentrations /3 replicates each) or a minimum of 6 determinations

at 100% of the test concentration.

1.4.2.4 Intermediate precision

The extent to which intermediate precision should be established depends on the

circumstances under which the procedure is intended to be used. The applicant should

establish the effects of random events on the precision of the analytical procedure.

Typical variations to be studied include days, analysts, equipment, etc., Intermediate

precision expresses within-laboratories variations such as different days, different

analysts, different equipment, etc.

1.4.2.5 Reproducibility

Reproducibility expresses the precision between laboratories (collaborative

studies, usually applied to standardization of methodology).

33
 Chapter 1

1.4.2.6 Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of

components which may be expected to be present. Typically these might include

impurities, degradants, matrix, etc. The procedures used to demonstrate specificity will

depend on the intended objective of the analytical procedure. It is not always possible to

demonstrate that an analytical procedure is specific for a particular analyte. In this case a

combination of two or more analytical procedures is recommended to achieve the

necessary level of discrimination.

1.4.2.7 Detection Limit

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. Several approaches for determining the detection limit are possible, depending on

whether the procedure is a non-instrumental or instrumental. Approaches other than those

listed below may be acceptable.

1.4.2.7.1 Based on Visual Evaluation

Visual evaluation may be used for non-instrumental methods but may also be

used with instrumental methods. The detection limit is determined by the analysis of

samples with known concentrations of analyte and by establishing the minimum level at

which the analyte can be reliably detected.

34
 Chapter 1

1.4.2.7.2 Based on Signal-to-Noise

This approach can only be applied to analytical procedures which exhibit baseline

noise. Determination of the signal-to-noise ratio is performed by comparing measured

signals from samples with known low concentrations of analyte with those of blank

samples and establishing the minimum concentration at which the analyte can be reliably

detected. A signal-to-noise ratio of 3 or 2:1 is generally considered acceptable for

estimating the detection limit.

1.4.2.7.3 Based on the Standard Deviation of the Response and the Slope

The detection limit (DL) may be expressed as

3.3 σ
DL =
S

Where σ = the standard deviation of the response

S = the slope of the calibration curve

The slope(S) may be estimated from the calibration curve of the analyte. The

estimation of slope may be carried out in a variety of ways, for example:

1.4.2.7.4 Based on the Standard Deviation of the Blank

Measurement of the magnitude of analytical background response is performed by

analyzing an appropriate number of blank samples and calculating the standard deviation

of these responses.

35
 Chapter 1

1.4.2.7.5 Based on the Calibration Curve

A specific calibration curve should be studied using samples containing an analyte

in the range of DL. The residual standard deviation of a regression line or the standard

deviation of y-intercepts of regression lines may be used as the standard deviation.

1.4.2.8 Quantitation Limit

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. The quantitation limit is a parameter of quantitative assays for low levels of

compounds in sample matrices, and is used particularly for the determination of

impurities and/or degradation products.

Several approaches for determining the quantitation limit are possible, depending

on whether the procedure is a non-instrumental or instrumental.

1.4.2.8.1 Based on Visual Evaluation

Visual evaluation may be used for non-instrumental methods but may also be used

with instrumental methods. The quantitation limit is generally determined by the analysis

of samples with known concentrations of analyte and by establishing the minimum level

at which the analyte can be quantified with acceptable accuracy and precision.

1.4.2.8.2 Based on Signal-to-Noise Approach

This approach can only be applied to analytical procedures that exhibit baseline

noise. Determination of the signal-to-noise ratio is performed by comparing measured

signals from samples with known low concentrations of analyte with those of blank
36
 Chapter 1

samples and by establishing the minimum concentration at which the analyte can be

reliably quantified. A typical signal-to-noise ratio is 10:1.

1.4.2.8.3 Based on the Standard Deviation of the Response and the Slope

The quantitation limit (QL) may be expressed as

10 σ
QL =
S

Where σ = the standard deviation of the response

S = the slope of the calibration curve

The slope(S) may be estimated from the calibration curve of the analyte. The estimation

of slope may be carried out in a variety of ways for example

1.4.2.8.4 Based on Standard Deviation of the Blank

Measurement of the magnitude of analytical background response is performed by

analyzing an appropriate number of blank samples and calculating the standard deviation

of these responses.

1.4.2.8.5 Based on the Calibration Curve

A specific calibration curve should be studied using samples, containing an

analyte in the range of QL. The residual standard deviation of a regression line or the

standard deviation of y-intercepts of regression lines may be used as the standard

deviation.

37
 Chapter 1

1.4.2.9 Linearity

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

obtain test results which are directly proportional to the concentration (amount) of analyte

in the sample.

1.4.2.10 Range

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

concentration of analyte in the sample (including these concentrations) for which it has

been demonstrated that the analytical procedure has a suitable level of precision, accuracy

and linearity.

1.4.2.11 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 provides an

indication of its reliability during normal usage. The evaluation of robustness should be

considered during the development phase and depends on the type of procedure under

study. It should show the reliability of an analysis with respect to deliberate variations in

method parameters. If measurements are susceptible to variations in analytical conditions,

the analytical conditions should be suitably controlled or a precautionary statement

should be included in the procedure.

38
 Chapter 1

1.4.2.12 Stress degradation methods

The purpose of stability testing is to provide evidence on how the quality of a drug

substance or drug product varies with time under the influence of a variety of

environmental factors such as temperature, humidity, and light, and enables

recommended storage conditions, retest periods, and shelf lives to be established.

Stress testing helps to determine the intrinsic stability of the molecule by

establishing degradation pathways in order to identify the likely degradation products and

to validate the stability indicating power of the analytical procedures used. In principle,

the influence of each factor is first individually explored, then cross-influences are

evaluated. The duration of the study will depend on the studied factor and the sensitivity

of the product to that factor.

1.4.2.12.1 Influence of Temperature

The behaviour of the drug substance under extreme temperatures such as 50° C or

even 70° C should be investigated. The ICH guidelines on stability, recommend a 10°C

increment above the accelerated conditions (40°C).Also sensitivity of drug substance

towards relative humidity (RH) from a dry atmosphere up to a water-saturated

atmosphere (from 10 to 90 percent RH) was investigated. Temperature and humidity will

influence the physical stability.

1.4.2.12.2 Influence of Light

Forced degradation testing evaluates the overall photosensitivity of the material

for method development purposes and/or degradation pathway elucidation. The intensity

39
 Chapter 1

of light and duration of exposure will vary depending on the photosensitivity.

These results will serve as basis to define conditions such as protect drug substance from

light during analysis, handling or storage.

1.4.2.12.3 Influence of pH

The study of the influence of pH will show the susceptibility of hydrolysis of the

drug substance in acidic or alkaline media. As a first approach, the extreme conditions

could be 1 N hydrochloric acid and 1 N sodium hydroxide. Then, when it is evident that

the product is sensitive to pH variations, a step-by-step approach will start to allow the

degradation in relation to pH. The results of these studies will serve to explain or to better

select some conditions for drug substance and drug product manufacture, as well as for

preformulation studies.

1.4.2.12.4 Influence of Oxygen

Oxygen can be a very critical parameter, since it is not always easy to protect the

drug substance or product against oxidation. The stability of the drug substance as a bulk

or in solution under oxygen, air or nitrogen atmospheres, with high contact between the

drug and the atmosphere, can be studied. The results could be aimed at recommending

protections and preliminary limits for residual oxygen.

40
 Chapter 1

REFERENCES

1. R.S. Satoskar, S.D. Bhandarkar and S.S. Ainapure, “Pharmacology and

Pharmacotherapeutics”, 17th edition, Popular Prakashan, Mumbai, India, 2001.

2. D. J. Abraham, “Burger’s Medicinal Chemistry and drug discovery”, 6th

edition, Wiley Interscience, New Jersey, 2007.

3. J.H. Block and J.M. Beale, “Wilson and Gisvold’s Textbook of Organic

Medicinal and Pharmaceutical Chemistry”, 11th edition, Lippincott Williams &

Wilkins, Newyork, 2004.

4. A. Korolkovas, “Essentials of Medicinal Chemistry”, 2nd edition, Wiley

Interscience, New Jersey, 1988.

5. L.L. Brunton, J.S. Lazo and K.L. Parker, “Goodman and Gilman’s The

Pharmacological Basis of Therapeutics”, 9th edition, McGraw-Hill health

professions division, New york, 1996.

6. T.L. Lemke and D.A. Williams, “Foye’s Principles of Medicinal Chemistry”, 6th

edition, Lippincott Williams & Wilkins, Newyork, 2008.

7. A. Maton, “Human Biology and Health” Englewood Cliffs, New Jersey, Prentice

Hall, 1993.

8. D.L. Rainwater, C.A. McMahan and G.T. Malcom, “Arterioscler Thromb Vasc

Biol.”, 19(3), 753–761, 1999.

9. U.S. Food and Drug Administration, “Center for Drug Evaluation and

Research”, www.fda.gov/CDER/GUIDANCE

10. “European Directorate for the Quality of Medicines and HealthCare”,

http://www.edqm.eu/site/Homepage-628.html

41
 Chapter 1

11. Australian government, Department of Health and Ageing, “Therapeutic goods

administration”, www.tga.gov.au

12. World Health Organization, 1211 Geneva 27, Switzerland, www.who.int/en

13. Health Canada, www.hc-sc.gc.ca/dhp-mps/index-eng.php

14. Medicines Control Council, South-Africa, www.mccza.com.

15. The National Health Surveillance Agency (ANVISA), Brazil,

www.anvisa.gov.br/eng

16. Medicines and Healthcare products Regulatory Agency, UK,

www.mhra.gov.uk/index.htm

17. The International Conference on Harmonisation of Technical Requirements

for Registration of Pharmaceuticals for Human Use (ICH), www.ich.org

18. S. Gorog, “Identification and determination of impurities in drugs” vol. 4,

Elsevier, Hungary, 2000.

19. “Impurities in New Drug substance and Products” ICH Q3A (R2) and Q3B

(R2), 2006.

20. “Impurities: Guideline for Residual Solvents”, ICH, Q3C (R3), 2006.

21. “Specifications: Test Procedures and Acceptance Criteria for New Drug

Substances and New Drug Products: Chemical Substances”, ICH, Q6A, 2006.

22. “Stability Testing of New Drug Substances and Products”, ICH, Q1A (R2),

2006.

23. “Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients”

ICH, Q7, 2006.

24. S. Gorog, A. Lauko and B. Herenyi, “J. Pharm. Biomed. Anal.”, 6, 697, 1988.

42
 Chapter 1

25. European Medicines Agency Evaluation of Medicines for Human Use, Committee

for medicinal products, “Guidelines on the limit of genotoxic impurity

CPMP/ICH/174/95”, 1998.

26. S. Ahuja and K. Alsante, “Handbook of Isolation and Characterization of

Impurities in Pharmaceuticals”, 5, Academic Press: San Diego, 2003.

27. N. Rahman, H. Azmi and N. Wu “Accred. Qual. Assur.” 11, 69, 2006.

28. R.N. Rao and V. Nagaraju, “J. Pharm. Biomed. Anal.” 33, 335, 2003.

29. L.R. snyder, J.J. Kirkland and J. L. Glajch, “Practical HPLC Method

Development”, John Wiley, Newyork, 1997.

30. A.D. Jerkovitch, J.S. Mellors and J.W. Jorgenson, “LC-GC North Amer.”, 21, 7,

2003.

31. N. Wu, J.A. Lippert and M.L. Lee, “J. Chromotogr.”, 911, 1, 2001.

32. K.K. Unger, D. Kumar, M. Grun, G. Buchel, S. Ludtke, Th. Adam, K. Scumacher

and S. Renker, “J. Chromatogr. A”, 892, 47, 2000.

33. U.D. Neue, T.H. Walter, B.A. Alden, Z. Jiang, R.P. Fisk, J.T. Cook, K.H. Glose,

J.L. Carmody, J.M. Grassi, Y.-F. Cheng, Z. Lu and R.J. Crowley, “Amer. Lab”,

31, 36, 1999.

34. Y. F. Cheng, T.H. Walter, Z. Lu, P. Iraneta, C. Gendreau, U.D.Neue, J.M. Grassi,

J.L. Carmody, J.E. OGara and R.P. Fisk, “LC-GC”, 18, 1162, 2000.

35. S. Fekete and J. Fekete, “Talanta”, 84, 416, 2011.

36. I. Halasz, R. Endele and J. Asshauer, “J. Chromatogr.” 112, 37, 1975.

37. J.E. MacNair, K.C. Lewis and J.W. Jorgenson, “Anal. Chem.” 69, 983, 1997.

38. L.A. Colon, J.M. Citron, J.A. Anspach, A.M. Fermier and K.A. Swinney,

“Analyst”, 129, 503, 2004.

43
 Chapter 1

39. L. Tolley, J.W. Jorgenson and M.A. Mosely, “Anal. Chem.” 73, 2985, 2001.

40. J.A. Lippert, B. Xin, N. Wu and M.L. Lee, “J. Microcol. Sepn.” 11, 631, 1997.

41. R.S. Plumb, J. Castro-Perez, J.H. Granger, I. Beattie, K. Joncour and A. Wright,

“Rapid Commun. Mass Spectrom”, 18, 2331, 2004.

42. J.C. Perez, R. Plumb and J.H. Granger, “Rapid Commun. Mass Spectrom.” 19,

843, 2005.

43. I.D. Wilson, J.K. Nicholson, J. Castro-Perez, J.H. Granger, B.W. Smith, J. Mazzeo

and R.S. Plumb, “J. Chromatogr. B”, 817, 67, 2005.

44. J.C. Holmes and F.A. Morell, “Appl. Spec.” 11, 86 1957.

45. M. Verzele and C. Dewaele, “Preparative High Performance Liquid

Chromatography”, TEC, Gent: Belgium, 1986.

46. F.A. Settle, “Handbook of instrumental techniques for analytical chemistry”,

Pearson Education, Singapore, 2004.

47. C.K. Lim and G. Lord, “Biol. Pharm. Bull.” 25(5) 547 2002

48. E.J. Oliveira and D.G. Watson , “Biomed. Chromatogr.,” 14, 351, 2000.

49. S.X. Peng, “Biomed. Chromatogr.,” 14, 430, 2000.

50. I.D. Wilson , “J. Chromatogr.,” 892, 315, 2000.

51. G. Tarkanyi, “NMR spectroscopy”, vol. 4, Elsevier, Hungary, 562, 2000.

52. R. E. Hurd, “J. Magn. Reson.” 87, 422, 1990.

53. S. X. Peng, “Biomed. Chromatogr.” 14, 430, 2000.

54. A. H. Beckett and J. B. Stenlake, “Practical Pharmaceutical Chemistry”, Vol. I

and II, CBS Publishers & Distributors, New Delhi, India, 2000.

44
 Chapter 1

55. P. D. Sethi, “Quantitative Analysis of Drugs in Pharmaceutical Formulations”.

3rd edition, CBS Publishers & Distributors, New Delhi, India, 1997.

56. H. H. Willard, L. L. Merrit, J. A. Dean and F. A. Settle, “Instrumental Method of

Analysis”, 7th edition, CBS Publishers & Distributors, New Delhi, India, 1986.

57. R. A. Day and A. L. Underwood, “Quantitative Analysis”, 6th edition, PHI

learning private limited, New Delhi, India, 2009.

58. G. Ramana Rao, S. S. N. Murthy and P. Khadgapathi, “Eastern Pharmacist”, 29

(346), 53 1986.

59. G. Ramana Rao, S. S. N. Murthy and P. Khadgapathi, “Eastern Pharmacist”,

30(353), 35 1987.

60. Ll. Yord, R. Snyder, J.J. Kirkland and J.L. Glajch, “Practical HPLC Method

development”, John Wiley & Sons, INC, U.S.A. 2 nd edition, New York, 1997.

61. S. Ahuja and M.W. Dong, “Handbook of Pharmaceutical Analysis by HPLC”,

Elsevier academic press, 1st edition, Vol. 6, 2005.

62. “Validation of Analytical Procedures: Methodology”, ICH Harmonized

Tripartite Guidelines. (Q2B), 1996

63. “Text on validation of analytical procedures”, ICH Harmonized Tripartite

Guidelines. (Q2A), 1995

45
SCOPE OF THE RESEARCH

CHAPTER 2
 Chapter 2

2.0 SCOPE OF THE RESEARCH

The scope of the present study is to develop and validate analytical methods and

Impurities profiling of the selected cardiovascular drugs such as clopidogrel bisulphate,

candesartan cilexetil, lacidipine and novel fixed dose combination of etodolac and

propranolol hydrochloride. During generic product development of clopidogrel

bisulphate, an unknown impurity to a significant level was observed in the Innovator as

well as in the marketed product. There is no literature available on the same and hence

successful attempt has been made to isolate and characterize the unknown impurity.

Similarly during the generic product development of candesartan cilexetil, many

impurities were observed, in which three impurities were reported in the literature but

impurities were not available for method validation and reference purpose and the routes

of synthesis of impurities are also not available. Hence a successful attempt has been

made for isolation and characterization of five impurities of Candesartan cilexetil of

which three are known impurities and two are unknown impurities. The two unknown

impurities were identified and characterized well by a single crystal XRD as well as

NMR, FT-IR and MS spectral techniques.

Complete literature survey on analytical method of Lacidipine reveals that only

normal phase HPLC method has been used which is more tedious to perform the analysis

and as the expectations are high in the field of analytical research to develop an analytical

method with short runtime. We have taken the challenge to develop a simple isocratic,

46
 Chapter 2

reversed phase method using the latest chromatographic technology of Ultra Performance

Liquid Chromatography (UPLC) and the developed method was successfully validated.

Etodolac and Propranolol hydrochloride fixed dose combination is under clinical

research. Literature search proves that there is no analytical method available for

dissolution which helps the early stages of product development. Hence we developed

and validated the analytical method for this fixed dose combination.

47
 Chapter 3

Identification and characterization of a principal

oxidation impurity in Clopidogrel drug substance

and drug product

 Chapter 3

3.1 INTRODUCTION

Clopidogrel bisulfate, methyl (+)-(S)-α-(2-chlorophenyl)-6,7-dihydrothieno-

[3,2-c]pyridine-5(4H)-acetate sulfate (1:1) (Fig. 3.1), is an oral, thienopyridine class

antiplatelet agent used to inhibit blood clots in coronary artery disease, peripheral

vascular disease, and cerebrovascular disease. It is marketed by Bristol-Myers Squibb and

Sanofi-Aventis under the trade name Plavix, marketed worldwide in nearly 110 countries,

with sales of US$6.6 billion in 2009[1]. It had been the second top selling drug in the

world for few years as of 2007[2] and was still growing by over 20% in 2007. U.S. sales

were US$3.8 billion in 2008.[3] It is also marketed by various leading pharmaceuticals

manufacturers as shown in the table 3.1. Clopidogrel is a prodrug and the mechanism is

irreversible blockade of the adenosine diphosphate (ADP) receptor P2Y12 and is

important in platelet aggregation, the cross-linking of platelets by fibrin. The blockade of

this receptor inhibits platelet aggregation by blocking activation of the glycoprotein

IIb/IIIa pathway. Platelet inhibition can be demonstrated 2 hours after a single dose of

oral clopidogrel, but the onset of action is slow, so that a loading-dose of 300–600 mg is

usually administered.[4]

Clopidogrel is rapidly absorbed after oral administration of repeated doses

of 75 mg clopidogrel (base), with peak plasma levels (appx. 3 mg/l) of the main

circulating metabolite occurring approximately one hour after dosing. The

pharmacokinetics of the main circulating metabolite is linear (plasma concentrations

increased in proportion to dose) in the dose range of 50 to 150 mg of clopidogrel.

Absorption is at least 50% based on urinary excretion of clopidogrel-related metabolites.

Clopidogrel and the main circulating metabolite bind reversibly in vitro to human plasma

48
 Chapter 3

proteins (98% and 94%, respectively). The binding is nonsaturable in vitro up to a

concentration of 110 μg / ml.

3.2 DRUG PROFILE

Clopidogrel is a white to off-white powder and it is practically insoluble in water

at neutral pH but freely soluble at pH 1. It also dissolves freely in methanol, dissolves

sparingly in methylene chloride. It has a specific optical rotation of about +56°. The

empirical formula of clopidogrel is C16H16ClNO2S and its molecular weight is 321.82.

Fig. 3.1 Structural formula of Clopidogrel

49
 Chapter 3

Table 3.1 Marketed brand name list of Clopidogrel Tablets[8]

S.No Brand Name Formulation Strength Manufacturer

1 PLAVIX Tablets 75 mg Sanofi–Synthelabo (France)

2 Plagril Tablets 75 mg Dr. Reddy’s Laboratories Ltd. (India)

3 Clodrel Tablets 75 mg Unichem Laboratories Ltd. (India)

4 Orawis Tablets 75 mg Merck (India)

5 Noklot Tablets 75 mg Zydus Medica (India)

6 Clopigrel Tablets 75 mg USV Ltd. (India)

7 Preva Tablets 75 mg Intas Pharmaceuticals Ltd. (India)

8 Clavix Tablets 75 mg Intas Suprima (India)

9 Clopilet Tablets 75 mg Sun Pharmaceutical Ind. Ltd. (India)

10 Stromix Tablets 75 mg Nicholas Piramal (India)

11 Cloplat-75 Tablets 75 mg IPCA Laboratories Ltd. (India)

12 Deplatt Tablets 75 mg Torrent Pharmaceutical Ltd. (India)

13 Ceruvin 75 Tablets 75 mg Stancare/Reddy (India)

14 Cloplatic Tablets 75 mg Haymann (Uruguay)

15 Plagrel Tablets 75 mg Servimedic (Uruguay)

16 Nefazan Tablets 75 mg Laboratorios Phoenix (Argentina)

17 Talcom Tablets 25 mg Shenzen Salubris (Xi Lin Tai) (China)

18 Clopifran Tablets 75 mg Laboratorios Lufra Farmacos S.A.

19 Clopigrel Tablets 75 mg Noas Farma Uruguay S.A (Uruguay)

50
 Chapter 3

3.3 LITERATURE REVIEW

Agrawal et al.,[5] have developed and validated a stability indicating HPTLC

method for the determination of clopidogrel bisulphate as bulk drug and in

pharmaceutical dosage form. The method employed TLC aluminium plates precoated

with silica gel 60F-254 as the stationary phase. The solvent system consisted of carbon

tetrachloride/chloroform/acetone (6:4:0.15, v/v). Clopidogrel bisulphate was subjected to

acid and alkali hydrolysis, oxidation, photo degradation and dry heat treatment.

Densitometric analysis of clopidogrel bisulphate was carried out in the absorbance mode

at 230 nm. The linear regression data for the calibration plots showed good linear

relationship with r2 = 0.9999/0.001 in the concentration range of 200 - 1000 ng. The mean

value of correlation coefficient, slope and intercept were 0.9999/0.001, 0.0939/0.011

and 8.839/0.99, respectively. The method was validated for precision, accuracy,

ruggedness and recovery. The limits of detection and quantitation were 40 and 120 ng per

spot, respectively. The drug undergoes acidic hydrolysis, basic hydrolysis, oxidative and

thermal degradation. However no data were available on the Unknown impurity of our

interest.

United States Pharmacopeia-32 (USP-32)[6] has enumerated related substance

method for clopidogrel tablets in their monograph, which utilizes liquid chromatography

with Ultron ES-OVM L 57, chiral specific column (4.6 mm × 150 mm) packed

with 5.0 µ particle size. Acetonitrile and potassium phosphate buffer (10 mM) (75:25,

v/v) was used as mobile phase. The flow rate was about 1.0 ml/min and 220 nm used as

51
 Chapter 3

wavelength of detection. When analysis was performed by this method the unknown

impurity under investigation elutes in the void volume of the system. The known related

compounds of clopidogrel were given in figure 3.2.

Clopidogrel Related compound-A

Clopidogrel Related compound-B1

Clopidogrel Related compound-B2 Clopidogrel Related compound-C

Fig. 3.2 Structural Formula of known Related Compounds of Clopidogrel

52
 Chapter 3

Pereillo et al.,[7] have identified and reported that clopidogrel is inactive in vitro

and a hepatic biotransformation is necessary to express the full anti-aggregating activity

of the drug. Moreover, 2-oxo-clopidogrel has been previously suggested to be the

essential key intermediate metabolite from which the active metabolite is formed. In their

paper, they have given the evidence of the occurrence of an in vitro active metabolite

after incubation of 2-oxo clopidogrel with human liver microsomes. This metabolite was

purified by liquid chromatography, and its structure was studied by a combination of

mass spectrometry (MS) and Nuclear Magnetic Resonance (NMR) experiments.

Gomez et al.,[8] have made a comparative study with 18 brands of PLAVIX tablets

containing clopidogrel hydrogensulfate to the innovator drug product for uniformity of

mass, impurity profile, content, dissolution properties and stability. In order to be able to

separate the R-enantiomer of clopidogrel, an enantiospecific liquid chromatographic

method was used to determine the impurities and to perform the assay. The paddle

method was used for dissolution testing. As per the comparative study, most of the brands

were not similar compared to the original drug product: their amount of impurities was

higher, the content of clopidogrel lower, the dissolution profiles different and after three

months under stress conditions in the original packaging, the results for the samples and

the reference were significantly different in most of the cases.

Mitakos and Panderi[9] have developed and validated reversed-phase HPLC,

stability indicating assay method for the determination of clopidogrel in pharmaceutical

dosage forms. The determination was performed on a semi-micro column, BDS C8

(250×2.1 mm i.d., 5 µm particle size); the mobile phase consisted of a mixture of 0.010 M

53
 Chapter 3

sodium dihydrogen phosphate (pH 3.0) and acetonitrile (35:65, v/v), pumped at a flow

rate 0.30 ml/min. The UV detector was operated at 235 nm and naproxen was used as

internal standard. The retention times for clopidogrel and naproxen, which was used as

internal standard, were 3.08 and 6.28 minutes respectively. Calibration graphs are linear

(r better than 0.9991, n = 6), in concentration range 1.00–3.00 µg/ml for clopidogrel. The

intra- and inter-day RSD values were less than 1.96%. Detection and quantitation limits

were 0.12 and 0.39 µg/ml respectively.

3.4 SCOPE OF THE STUDY

Literature survey revealed that four impurities of clopidogrel have been already

identified[5-9] and named as clopidogrel related compound A, positional stereo isomers of

clopidogrel named as clopidogrel related compound B1 and B2 and a chiral isomer of

clopidogrel named as clopidogrel related compound C. It has also been established that

these positional stereo isomers (B1 & B2) are process impurities and other impurities are

formed during the process and also self degradation. Marketed samples of plavix and few

batches of drug substances were analyzed using reported method[6] and found to contain

an unknown impurity. Even though the unknown impurity is formed during oxidation

condition, there is no report on the isolation and characterization. Hence comprehensive

study was undertaken to identify and characterize the oxidative impurity. The new

oxidation impurity is referred to as related compound D.

54
 Chapter 3

3.5 EXPERIMENTAL

3.5.1 Materials and Methods

Clopidogrel bisulphate drug substance was purchased from Dr.Reddys

laboratories Ltd, Hyderabad, India and clopidogrel tablets of brand name plavix

manufactured by Sanofi Pharma Bristol Myers Squibb Inc were used. Potassium

phosphate and ammonium acetate, GR grade were obtained from E. Merck, India.

Methanol and acetonitrile of HPLC grade were obtained from E. Merck, India. Purified

water was collected through Milli-Q water purification system (Millipore, USA).

Dimethylsulphoxide-d6 (DMSO-d6) was purchased from Aldrich Chemical Co., USA, and

all other chemicals used were of analytical grade.

3.5.2 Analytical Method

Analytical method was developed using Waters HPLC system consisting

of Alliance integrated hardware of quaternary solvent delivery module, auto sampler

and PDA detector. Data were processed through Waters Empower software

Version 1.63. Hypersil BDS C8 column (Thermo Electron Corporation) with dimensions

of 250 mm × 4.6 mm internal diameter packed with 5.0 µ particle size was employed

along with gradient conditions for the separations. The gradient involves two mobile

phases consisting of acetonitrile-potassium phosphate buffer (pH 2.3; 10mM) (20:80. v/v)

as solvent A and acetonitrile-potassium phosphate buffer (pH 2.3; 10mM) (80:20. v/v) as

solvent B. The gradient condition was employed with a timed gradient program

of T (min)/%B (v/v): 0.01/0, 5/0, 15/15, 40/30, 45/0, 60/0 for the separations. Flow rate

was kept at 1.0 ml/min and the column eluent was monitored at 220 nm for 60 minutes.

55
 Chapter 3

3.5.3 Preparative HPLC Method

Preparative HPLC system used was a Waters system equipped with W 600

quaternary solvent delivery module Delta prep 2487 dual wavelength UV detector. Data

were processed through Waters empower software. An Xterra MS C18 ODB HPLC

column (Waters, Ireland) with dimensions 100 mm × 30 mm packed with 5.0 µ particle

size was used for preparative work. The gradient conditions were employed for the

separations with a timed gradient program of T (min)/%B (v/v): 01/10, 06/10, 16/95,

17/95, 18/100, 23/100, 28/10, 32/10. Flow rate was kept at 20 ml/min and the column

eluent was monitored at 220 nm and 300nm for about 32 minutes.

3.5.4 Infrared spectroscopy

The IR spectra were recorded in the solid state as KBr as dispersion using

Shimadzu FT-IR 8700 with DRS technique.

3.5.5 Liquid Chromatography-Mass Spectrometry

The isolated compound was dissolved (about 0.05 mg/ml) in methanol containing

0.1% formic acid (v/v) and infused into the ion source by the syringe pump at the rate of

10µl/min. The mass spectrum of the isolated degradation product was acquired on a

Finnegan LCQ instrument from Thermoquest (San Jose CA) in positive spray ionization

(ESI+) mode. The spray potential was set at 5.6kV and the capillary temperature

at 220°C. Mass range was scanned between 100 and 500amu. The mass spectrum was

56
 Chapter 3

also recorded in negative spray ionization (ESI-) mode. The spray potential was set

at 5.6kV and the capillary temperature at 220°C.

3.5.6 Nuclear Magnetic Resonance

1 13
H (400.13 MHz) and C (100.62 MHz) NMR spectra of isolated related

compound D were recorded on an Avance DPX-400 MHz spectrometer Bruker

(Germany). The probe was a 1H/13C 5 mm, 3 axis gradients (x, y, z), optimized for

inverse detection. Spectra were recorded in DMSO-d6 (5-mm tubes) at 300K. Sample

concentration was 0.6 mg in 0.6 ml. The residual protonated resonance of the solvent

(DMSO-d6) was used as an internal chemical shift standard, which was related to

tetramethylsilane with chemical shifts of 2.5 and 39.2 ppm, respectively for 1H and 13C.

Processing of the raw data was performed using Bruker XWinNmr software. The pulse

conditions were 90° pulse, 9.4 µs (attenuation 0db) for 1H and 30° pulse, 11.75µs
13
(attenuation 0db) for C. Gradient pulses used in this study were all shaped to a sine

envelope with 1 ms duration (DQF-COSY, and 1H/13C HSQC). Spectral width was

5431.88 Hz for proton and 18111.66 Hz for carbon.

3.5.7 Preparation of degradation samples of Clopidogrel

3.5.7.1 Acid and base degradation

A solution of clopidogrel bisulphate (500mg) in 50ml of 0.1N hydrochloric acid

was kept at 80°C for 60 minutes. Another sample was prepared in a similar manner by

treating clopidogrel bisulphate (500mg) in 50ml of 0.1N sodium hydroxide.

57
 Chapter 3

3.5.7.2 Peroxide degradation

A solution of clopidogrel bisulphate (500mg) in methanol (0.5ml) and hydrogen

peroxide (5% in water, 5ml) was kept at 60°C for 3 hours. Similarly 10 tablets of

clopidogrel bisulphate were powdered (equivalent to 500mg of clopidogrel bisulphate)

and dissolved in 0.5ml of methanol. To the solution, 5 ml of hydrogen peroxide (5%)

was added and kept at 60°C for 3 hours.

3.5.7.3 Thermal degradation

Clopidogrel bisulphate (1.0g) was moistened with water and was kept in an oven

maintained at 120°C for 24 hours.

3.6 RESULTS AND DISCUSSION

3.6.1 HPLC Method Development and Impurity profiling

An Ultron ES- OVM L 57, chiral specific column (Shinwa chemical industries,

Japan) with dimensions of 150 mm × 4.6 mm i.d packed with 5.0 µ particle size was

employed for separation. Acetonitrile-potassium phosphate buffer (10 mM) (75:25. v/v)

was used as mobile phase and flow rate was kept at 0.8 ml/min with the detection

at 220 nm for 30 minutes. Relative retention time (RRT) of the related compound A, B1,

clopidogrel, B2 and C was found respectively at 0.46, 0.93, 1.0, 1.1 and 2.10. An

unknown impurity was found at about 2.0 minutes (RRT of 0.30) (Fig.3.4).

58
 Chapter 3

As the unknown impurity elutes in the void volume of the system, various buffer

composition, pH, gradients were attempted and found unsuccessful. The main problem

that occurred was peak shape, peak purity and blank interference due to peroxide. Hence,

conventional, cost effective, new method was developed wherein good peak shape and

peak purity were achieved. The new method involves Hypersil BDS C8 column (Thermo

Electron Corporation) with gradient conditions for the separations (Fig.3.5).

During the analysis, it has been observed that the new impurity content in

clopidogrel tablets, were in the range of 0.05 % to 0.07 % (by area percentage) and in

drug substance it ranges from 0.08 % to 0.12 % (by area percentage). Typical

chromatograms of clopidogrel drug substance, drug product and drug product spiked with

the related compound D were shown in figures 3.6 - 3.8. It is a mandatory requirement

from regulatory authorities, to identify and characterize any unknown impurity present in
[7, 8]
it at a level as low as 0.05% . The presence of this impurity in tablets can have a

significant impact on quality and safety of the important drug. The isolation of any

impurity is required to find the response in an analytical method and also to validate the

analytical procedure for its quantitative estimation. Even though the same impurity is

formed during oxidation condition, there is no report on the isolation and characterization.

Hence comprehensive study was undertaken to identify and characterize the oxidative

impurity. The new oxidation impurity in this paper is referred to as related compound D.

The chemical structures of related compound D was shown in the figure 3.3.

59
 Chapter 3

Fig. 3.3 Structural Formula of Clopidogrel related compound D

60
61
Fig. 3.4 Typical HPLC chromatogram of Clopidogrel spiked with Impurities as per USP
Chapter 3

method
 0 .9 0

0 .8 0

0 .7 0

0 .6 0

0 .5 0

AU
0 .4 0

62
0 .3 0

0 .2 0

0 .1 0
R e la t e d C o m p o u n d -B - 3 8 . 5 3 7

R e la t e d C o m p o u n d -D - 1 7 . 6 7 5
C lo p id o g re l - 3 3 . 2 9 3

R e la t e d C o m p o u n d -A - 1 1 . 3 3 8
0 .0 0

0 .0 0 5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0 4 5 .0 0 5 0 .0 0 5 5 .0 0 6 0 .0 0

M in u t e s
Chapter 3

Fig. 3.5 Typical HPLC chromatogram of Clopidogrel spiked with Impurities as per developed

method
 0 .0 5 0

0 .0 4 5

0 .0 4 0

0 .0 3 5

C lo p id o g re l - 3 0 . 1 1 8
0 .0 3 0

- 1 5 .0 7 4

A U
0 .0 2 5

- 9 .1 8 3
D
- 3 5 .3 1 0

A
B

63
0 .0 2 0

c o m p d
0 .0 1 5

c o m p d
c o m p d

0 .0 1 0

R e la t e d

R e la t e d
R e la t e d

0 .0 0 5

0 .0 0 0

- 0 .0 0 5
0 .0 0 5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0 4 5 .0 0 5 0 .0 0 5 5 .0 0 6 0 .0 0

M in u t e s
Chapter 3

Fig. 3.6 HPLC chromatogram of clopidogrel drug substance

 0 .0 5 0

0 .0 4 5

0 .0 4 0

C lo p id o g re l - 3 0 . 1 1 2
0 .0 3 5

0 .0 3 0

AU
0 .0 2 5

64
0 .0 2 0

0 .0 1 5

0 .0 1 0

R e la t e d c o m p d A - 9 . 0 7 3
R e la t e d c o m p d D - 1 5 . 0 6 1
R e la t e d c o m p d B - 3 5 . 2 7 7

6.421
13.230
17.539
0 .0 0 5

4.964
0 .0 0 0

- 0 .0 0 5
0 .0 0 5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0 4 5 .0 0 5 0 .0 0 5 5 .0 0 6 0 .0 0

M in u te s
Chapter 3

Fig. 3.7 HPLC chromatogram of Clopidogrel drug product

 0 .0 5 0

0 .0 4 5

0 .0 4 0

C lo p id o g re l - 3 0 . 0 5 4
0 .0 3 5

0 .0 3 0

AU
0 .0 2 5

65
0 .0 2 0

R e la t e d c o m p d D - 1 5 . 1 1 4
0 .0 1 5

0 .0 1 0

R e la t e d c o m p d A - 9 . 1 3 6
17.526

6.465
13.332

4.997
0 .0 0 5

0 .0 0 0

- 0 .0 0 5
0 .0 0 5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0 4 5 .0 0 5 0 .0 0 5 5 .0 0 6 0 .0 0

Min u te s
Chapter 3

Fig. 3.8 HPLC chromatogram of Clopidogrel drug product spiked with related compound D
 Chapter 3

3.6.2 Analysis of degradation samples by analytical LC

The degradation samples were diluted to the required concentration and analyzed

with analytical LC. In the oxidative conditions, 2% and 7% of related compound D was

found in the drug substance and drug product (plavix) respectively. In other conditions,

formation of related compound D was not noticed. The purity angle of all the impurities

was found less than that of purity threshold and thus the peak purity of the impurities was

confirmed. The spectrum of the related compound D and clopidogrel were extracted from

PDA detector in the range of 210 to 400 nm. The UV spectra are presented in the

figure 3.9. Hence the experiment was used to enrich the impurity. It was also confirmed

that related compound A and C are the degradation products and related compound B 1

and B2 are process impurities based on their trend. The higher level of related compound

D in the drug product reveals that the drug product is more susceptible than that of drug

substance.

3.6.3 Isolation of degradation product(s) by preparative LC

Clopidogrel bisulphate (5g) was treated with hydrogen peroxide (5%, 15ml) and

kept at 80 °C for 3 hours. The aqueous layer was washed with dichloromethane to

remove clopidogrel. The aqueous layer was subjected to preparative LC as described in

the section 3.5.3 and as many as 10 fractions were collected separately. Purity of all these

fractions was analyzed by analytical LC and found to be in the range of 99%. The

fractions were pooled together, 100mg of sulfuric acid was added and the solvent was

evaporated. The resulted solid was reanalyzed on analytical LC and the purity of the

66
 Chapter 3

same was found to be 99% which was good enough for carrying out the spectroscopic

experiments.

3.6.4 Characterization of the degradation product

The isolated related compound D was injected in both the analytical HPLC

methods. The retention time and UV spectrum obtained in the PDA detector matches

with that of targeted impurity. Characterization of the related compound D was

performed using analytical data obtained from CHNS, IR, UV, and Mass, MSn

experiments, 1H /13C NMR spectrum, DEPT and 2D NMR experiments. The Mass,

MS-MS and IR spectrum are given in figures 3.10-3.13.

67
68
Chapter 3

Fig. 3.9 UV spectral overlay of Clopidogrel and Related Compound D

69
Chapter 3

35
Fig. 3.10 MS-MS spectrum of Related compound D (Cl isotope)
70
Chapter 3

37
Fig. 3.11 MS-MS spectrum of Related compound D (Cl isotope)
71
Chapter 3

Fig. 3.12 IR spectrum of Related compound D

 09-Sep-200516:31:56
090905-D02-Clopi Unknown Imp 564 (10.424) Cm (538:624-(85:532+627:1093)) 1: Scan ES+
320 5.17e6
100

72
%
322

323

0 m/z
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Chapter 3

Fig. 3.13 Mass spectrum of Related compound D

 Chapter 3

The isolated related compound D was found as off white powder and it shows UV

absorbance maxima at 299.9 nm which is higher than that of clopidogrel (220 nm).

The +ve ES-MS spectrum of the related compound D showed peaks at m/z 320 and 322
35 37
corresponding to the Cl and Cl isotope respectively. The compound doesn’t form

lithium adduct ion and –ve ES-MS spectrum showed no peaks states that the molecular

ion obtained is positively charged i.e. m/z 320/322 is due to M+. In comparison with

clopidogrel, the related compound D corresponds to 1 atomic mass unit (amu) less which

can be presumed to have similar structure as that of clopidogrel but with short of one

hydrogen atom.

Two daughter ions were obtained at m/z 183 and 155 when the molecular ion

(M+) 320 fragmented in MS/MS experiments. Both the daughter ions were found to

contain chlorine atom as it was confirmed by MS/MS experiments of the chorine isotope

molecular ion (M+) 322 and the m/z values of the daughter ions were 185 and 157. As the

peak intensity ratios are nearly identical, it was confirmed that the eliminated neutral

fragments contains no chlorine atom. Further MS2 experiments of daughter ion (m/z 183)

showed the formation of a fragmentation ion at m/z 155 and MS3 shows the formation of

an ion at m/z 125. Similar experiments were performed with the 37Cl Isotope of daughter

ion of m/z 185 and fragmentation ions of m/z 157 and 127 were found. Based on the

fragmentation data, the structure for related compound D was assigned as shown in the

figure 3.3 and its probable fragmentation pathway is given in the figure 3.14.

73
 Chapter 3

Fig. 3.14 Fragmentation pattern of related compound D

74
 Chapter 3

The fragmentation pathway of related compound D was compared with the

clopidogrel fragmentation pathway given in the figure 3.15.[12] It was found that m/z

value of the clopidogrel daughter ion is 212/214 but the same was not observed in the

case of related compound D. The fragmentation ion obtained from MS2 experiments of

clopidogrel was 183/185 and 155/157 which is similar to that of the daughter ion of

related compound D. Similar fragmentation was not observed in related compound D,

due to the double bond associated with nitrogen atom. However the fragmentation ion

obtained from MS2 experiments of clopidogrel was similar to that of the daughter ion

obtained from related compound D. This was confirmed by MS3 and MS4 experiments.

1
H NMR spectrum of related compound D is slightly different from that of

clopidogrel 1H NMR spectrum and exhibits one hydrogen less than that of clopidogrel.

Comparison of 1H, 13
C and DEPT 135 NMR data with that of clopidogrel (Table 3.2

and 3.3, Fig. 3.16-3.18) shows that the related compound D has one methylene proton less

and one methine proton more than that of clopidogrel. The methine protons at 2nd and 3rd

positions are deshielded to the extent of 0.3, 0.7 ppm respectively.

The methylene protons at 6th and 7th positions are deshielded to the extent of 0.5,

0.4 ppm respectively. The presence of methylene protons at 6th and 7th positions were

confirmed by irradiation (proton decoupling) experiment (Fig.3.19). Irradiation of

protons at chemical shift  ppm (m) affects the multiplicity of protons at  3.94 and

3.80 ppm which further reveals the double bond position. The appearance of one singlet

at  9.19 ppm at 4th position and the deshielding of methine proton at 10th position from

75
 Chapter 3

 5.60 to  6.66 ppm were also noticed. All the above deshielding confirmed that there

was a structural change in the piperidine ring. It was also confirmed by 1H-1H COSY

spectrum. There is no appropriate change in the chemical shift of aromatic ring which
13
confirms the presence of the aromatic rings in the related compound D. C and DEPT

135 NMR spectra results indicate the presence of one methyl carbon, two methylene

carbons, eight methine carbons and five quaternary carbons. Related compound D shows

five quaternary carbons like that of clopidogrel but one methylene carbon less and one

methine carbon more.

Fig. 3.15 Fragmentation pattern of Clopidogrel bisulphate

76
 Table 3.2 1H and 13C NMR assignments for clopidogrel bisulphate and related compound
D

77
Chapter 3
 Chapter 3

The methylene signal at δ 49.62 ppm disappeared and a new methine signal

at  162.51 ppm was observed. The high deshielding chemical shift value of the methine

signal indicates that carbon may be attached to an electronegative atom or attached to

aromatic ring or under the anisotropic influence of an aromatic ring. This led to the

hypothesis of existence of C=N bond in the piperidine ring of related compound D

structure. The position of the double bond was assigned to 4th carbon due the possibility

of extended conjugation explained for the shifting of UV absorbance maxima to the

higher wavelength. This was also confirmed by the deshielding of aliphatic methine

carbon at 10th position to about 5 ppm and the deshielding of one of the quaternary carbon

at 8th position to about 20 ppm. The presence of C=N bond was further confirmed by the

IR characteristic absorption peak at 1469 cm-1.

All the above observations can be explained well on the basis of the proposed

structure (Fig.3.3) with quaternary nitrogen and a double bond. All proton signals were

assigned on the basis of 1H NMR and 1H-1H COSY spectral results. Carbon signals were

assigned on the basis of DEPT135 and 1H-13C HSQC spectral results. Based on the above

spectral data, the structure was characterized as 5-[1-(2-chlorophenyl)-2-methoxy-2-

oxoethyl]-6, 7-dihydrothieno [3, 2-c] pyridin-5-ium with a molecular weight of 320 amu.

78
79
Chapter 3

Fig. 3.16 1H NMR Spectrum of Clopidogrel Related compound D

80
Chapter 3

13
Fig. 3.17 C NMR Spectrum of Clopidogrel Related compound D
81
Chapter 3

Fig. 3.18 DEPT 135 NMR Spectrum of Clopidogrel Related compound

D
 Chapter 3

Fig. 3.19 Irradiation data (Proton decoupling experiment)

3.7 CONCLUSION

The oxidative degradation product, related compound D in clopidogrel drug

substance as well as drug product was isolated by preparative LC and was characterized

by using spectroscopic techniques namely NMR, CHNS, IR, MS and MSn.

82
 Chapter 3

REFERENCES

1. “New products and markets fuel growth in 2005”, IMS Health,

http://www1.imshealth.com/web/content/0,3148,64576068_63872702_70260998_

77974518,00.html. Retrieved 2009-03-02.

2. “Top Ten Global Products – 2007”, IMS Health,

http://www.imshealth.com/deployedfiles/imshealth/Global/Content/StaticFile/Top

_line_Data/Top10GlobalProducts.pdf. Retrieved 2009-03-02.

3. http://drugpatentwatch.com/ultimate/preview/tradename/index.php?query =

PlAVIX.

4. http://www.rxlist.com/plavix-drug.htm

5. H. Agrawal, N. Kaul, A.R. Paradkar and K.R. Mahadik , “Talanta”, 61, 581-589,

2003.

6. United states Pharmacopoeia 30, 1802-1803, 2006.

7. J.M. Pereillo, M. Maftouh, A. Andrieu, M.F. Uzabiaga, O. Fedeli, P. Savi, M.

Pascal, J.M. Herbert, J.P. Maffrand and Claudine Picard, “The American

Society for Pharmacology and Experimental Therapeutics”, Vol. 30, No. 11,

DMD 30, 1288-1295, 2002.

8. Y. Gomez, E. Adams and J. Hoogmartens, “J. Pharm. Biomed. Anal.”, 34, 341-

348, 2004.

9. A.Mitakos and I. Panderi, “J. Pharm. Biomed. Anal.”, 28, 431-438, 2002.

10. “Impurities in New Drug Substances”, Q3A(R2), 2006.

11. “Impurities in New Drug Products”, Q3B (R2), 2006.

12. Witold Danikiewicz and Malgorzata swist, “J. Mass Spectrom.”, 42, 405-406,

2007.

83
 Chapter 4

Identification, Isolation and Characterization of

Five Potential Degradation Impurities in

Candesartan Cilexetil Tablets

 Chapter 4

4.1 INTRODUCTION

Candesartan cilexetil, (±)-1-(cyclohexyloxycarbonyloxy) ethyl-2-ethoxy-1-[[2’-

(1Htetrazol-5-yl) biphenyl-4-yl] methyl] benzimidazole-7-carboxylate (Fig.4.1), is a

nonpeptide antihypertensive drug and used either single or in combination with other

drugs, to treat high blood pressure. In gastrointestinal tract candesartan cilexetil is

converted to candesartan, an angiotensin II receptor antagonist (ARA II) which blocks the

ability of angiotensin II to raise blood pressure by constricting or squeezing arteries and

veins and ultimately leads to a reduction in blood pressure. In also reduced the work of

heart by reducing the pressure against which the heart must pump blood, and is useful in

patients with heart failure.[1-3] It is marketed under the brand name of Atacand and in

combination with a thiazide diuretic drug Hydrochlorothiazide (HCTZ) as Atacand HCT

by Astra Zeneca.

4.2 DRUG PROFILE

Candesartan cilexetil is a white to off-white powder and tt is practically insoluble in

water and sparingly soluble in methanol. Candesartan cilexetil is a racemic mixture

containing one chiral center at the cyclohexyloxycarbonyloxy ethyl ester group.

Following oral administration, candesartan cilexetil undergoes hydrolysis at the ester link

to form the active drug, candesartan, which is achiral. Its molecular weight is 610.67 and

structural formula is

84
 Chapter 4

Fig. 4.1 Structural formula of Candesartan cilexetil

4.3 LITERATURE REVIEW

Stenhoff et al.,[4] have developed a liquid chromatographic method for the

determination of a new effective anti-hypertensive drug candesartan (CV-11974), its

prodrug candesartan cilexetil (TCV-116) and a metabolite, CV-15959 in human plasma

and urine. The assay method comprises liquid–liquid extraction and separation on a

phenyl column with fluorometric detection. The methods gave absolute recoveries

of 70, 83 and 78% for candesartan cilexetil, candesartan and CV-15959, respectively, and

the limit of quantification is 5, 1 and 3 nanomolar of plasma (RSD < 20%), respectively.

This method was applied to plasma and urine samples from biopharmaceutical and

clinical studies in man only.

Ferreiros et al.,[5] have developed a solid phase extraction procedure for the

chromatographic determination of eight antihypertensive compounds, lack of linearity

and reproducibility was observed only for candesartan cilexetil. Due to this fact, they

have performed stability study for this prodrug. It showed that the lack of linearity and

85
 Chapter 4

reproducibility was based on hydrolysis and transesterification processes which occurred

during the drying step after elution with methanol into glass tubes. These phenomena

could be reproduced artificially under basic conditions, which demonstrated the presence

of basic residues in glass tubes. The study of this potential hydrolysis and

transesterification reactions is very important to assure that labile drugs containing ester

groups remain unaffected.

Subba Rao et al.,[6] have developed an isocratic reversed-phase liquid

chromatographic method for the quantitative determination of candesartan cilexetil, used

in the bulk drug and in pharmaceutical dosage forms. The method was also applicable

to analysis of related substances. Chromatographic separation was achieved on

a 250 mm x 4.6 mm, 5 µm particle, CN column with a 50:50 (v/v) mixture of phosphate

buffer, pH 3.0, and acetonitrile as mobile phase. The flow rate was 1.0 ml/min and the

detection wavelength was 210 nm. Resolution of candesartan cilexetil and six potential

impurities was greater than 2.0 for all pairs of compounds. The drug was subjected to

hydrolytic, oxidative, photolytic, and thermal stress and substantial degradation occurred

in alkaline and acidic media and under oxidative and hydrolytic stress conditions. The

major product obtained as a result of basic hydrolysis was different from that produced by

acid hydrolysis and aqueous hydrolysis. The stress samples were assayed against a

reference standard and the mass balance was found to be close to 99.6%. The method was

validated for linearity, accuracy, precision, and robustness.

Kurgan and M. Pesachovich[10] have filled a patent of their invention which

encompasses stable candesartan cilexetil of fine particle size, wherein desethyl-

candesartan (desethyl-CNS) within the stable candesartan cilexetil does not increase to

86
 Chapter 4

more than about 0.1% w/w by HPLC relative to the initial amount of candesartan

cilexetil, when the stable candesartan cilexetil is maintained at a temperature of

about 55°C for at least 2 weeks, methods of making the same and pharmaceutical

compositions thereof. They have also performed an accelerated stability study in Atacand

tablets and found that after two weeks at 55°C, the impurity level of the tablets,

namely 1-N ethyl CNS and 2-Nethyl CNS significantly increased.

4.4 SCOPE OF THE STUDY

Several impurities of candesartan cilexetil have been identified and documented in

[4-6]
the literature . Identification of the impurities and analytical method for quantitation

in drug substances, drug products and plasma samples has been described. Stenhoff et al

have reported the identification of hydroxy candesartan or desethyl candesartan

(metabolite) and candesartan in the plasma samples.[4] Ferreir´os et al have reported

about the base hydrolytic impurities of candesartan cilexetil, i.e.; candesartan and its

transesterification product of methyl ester.[5] Recently Subba Rao et al., established

candesartan, candesartan methyl ester, candesartan ethyl ester, hydroxy candesartan

cilexetil or desethyl candesartan cilexetil, 1N-ethyl candesartan cilexetil and 2N-ethyl

candesartan cilexetil as impurities using an isocratic RP-HPLC method candesartan that is

known to be an active drug and candesartan methyl and ethyl ester are process impurities

and the rest are degradates.

During an accelerated stability studies (40°C and 75% Relative humidity) for

three months and forced degradation study (thermal, 60°C) for two weeks, three

degradates were found to be the same as those reported by Subba Rao et al.,[6]

87
 Chapter 4

Surprisingly, two more unknown degradates were also observed which are found to be

around the reporting threshold (0.05%) as per ICH guidelines.[7, 8] In terms of efficacy

and patient safety, it is important to isolate and identify impurities and/or degradates to

ensure that their presence will not evoke any form of adverse response either

pharmacologic or toxicologic in a patient taking medication. Moreover, reference

materials of all individual impurities were required to validate the method. Hence a

comprehensive study was undertaken to isolate all impurities simultaneously and

characterize them.

4.5 EXPERIMENTAL

4.5.1 Materials

Atacand tablets were purchased from market and candesartan cilexetil were

synthesized at Torrent Research Centre, Gandhi Nagar, India. LC grade acetonitrile,

analytical grade ammonium acetate, acetic acid, spectroscopic grade deuteriated

dimethylsulphoxide-d6 (DMSO-d6) and chloroform (CDCl3) were purchased from Merck,

Darmstadt, Germany. High pure water was prepared by using Millipore Milli-Q plus

water purification system.

4.5.2 Analytical Chromatography

Samples were analysed using Waters Alliance 2695 LC system (Waters

Corporation, Milford, MA, USA) consisting of integrated hardware of quaternary solvent

delivery module, auto sampler and a 2996 photo diode array detector. Data were

processed through Waters Empower software. A gradient analytical method with

88
 Chapter 4

Purosphere Star RP18e column of 150 x 4.6 mm i.d packed with 5 µm particle size was

employed for separation. The gradient involves two mobile phases consisting of

acetonitrile-ammonium acetate (pH 5.0; 20 mM) (10:90. v/v) as solvent A and

acetonitrile- ammonium acetate (pH 5.0; 20 mM) (90:10 v/v) as solvent B. The gradient

program employed with a timed gradient program of T (min)/%B (v/v): 0.01/0, 5/5, 15/35,

35/65, 40/75, 45/100, 55/100, 60/0, 65/0, for the separations. Flow rate was kept

at 1.2 ml/min and the column eluent was monitored at 254 nm for 65 min. The analytical

method was validated as per ICH guidelines for various parameters such as specificity,

precision, linearity, accuracy, and limit of detection, limit of quantitation, ruggedness and

robustness.[9]

4.5.3 Preparative Chromatography

Preparative HPLC was performed in a Waters system equipped with W 600

quaternary solvent delivery module Delta prep 2487 dual wavelength UV detector using an

YMC pack ODS A column of 250 mm × 20 mm i.d packed with 5 µm particle size. Data

were processed through Waters Empower software. Acetonitrile and trifluoroacetic acid

buffer (0.1%) (60:40. v/v) was used as mobile phase and flow rate was kept at 30 ml/min

with the detection at 254 nm for 70 minutes.

4.5.4 Infrared Spectroscopy

The IR spectra were recorded in the solid state as KBr dispersion(DRS technique)

using Bruker FT-IR Tensor 27 (Ettlingen, Germany) in the range of 400 to 4000 cm-1 with a

resolution of 4.0 cm-1.

89
 Chapter 4

4.5.5 Liquid Chromatography-Mass Spectrometry

Pneumatically assisted electrospray ionization (ESI) mass spectrometric analyses

were performed using a single-quadrupole mass spectrometer (Micromass ZQ-2000,

Beverly MA, USA). Operating in the positive ion mode, the spray needle was held at a

potential of 5.6 kV. The skimmer cone potential, which affects the degree of collisional

activation in the source was set to 45 V. The source temperature was maintained

at 150°C. It was also operated in the negative ion mode with -4.5 kV spray needle

potential. Spectra were collected at a rate of 12 points per peak while scanning from

m/z 100 to m/z 1000.

4.5.6 Nuclear Magnetic Resonance

1
H (400.13 MHz) and 13C (100.62 MHz) NMR spectra of isolated impurities were

recorded on an Avance DPX-400 MHz spectrometer Bruker (Silberstreifen, Germany).

The probe was a 1H/13C 5 mm, 3 axis gradients (x, y, z), optimized for inverse detection.

Spectra were recorded in deuteriated solvents (5mm tubes) at 300K. Sample

concentration was 0.6 mg in 0.6 ml. The residual protonated resonance of the solvent was

used as an internal chemical shift standard, which was related to tetramethylsilane with

chemical shifts of 2.5 and 39.2 ppm, respectively for 1H and 13

C. Processing of the raw

data was performed using Bruker XWinNmr software. The pulse conditions

were 90° pulse, 9.4 µs (attenuation 0db) for 1H and 30° pulse, 11.75µs (attenuation 0db)

for 13C. Gradient pulses used in this study were all shaped to a sine envelope with 1 ms

duration (DQF-COSY, and 1H/13C HSQC). Spectral width was 5431.88 Hz for proton

and 18111.66 Hz for carbon.

90
 Chapter 4

4.5.7 X-ray Crystallographic Analysis

The single crystal of Impurity-III was grown by slow evaporation technique using

ethanol solvent system. X-ray diffraction analysis was carried out on a Bruker

Smart Apex CCD diffractometer at room temperature. The crystal was monoclinic,

(space group C2/c) with a unit cell a = 16.3770 (7) Ǻ, b = 8.5928 (4) Ǻ, c =43.7733 (19)

Ǻ,  = 91.150(1) °, V= 6158.7 (5) A3, Z = 8, Density 1.317 mg/m3, Absorption

coefficient (µ) = 0.093 mm-1. CuKα radiation (λ =0.71073 Ǻ). A total of 5399 reflections

with F>2σ (F) gave R = 0.0452 with 412 parameters refined. The structure was

elucidated by direct methods using Siemens SHELXTL PLUS software and final

refinement of the non hydrogen atoms was done by full-matrix least-square refinement.

4.5.8 Characterization of the degradation product

All the isolated impurities were injected in the analytical HPLC. The retention

time and UV spectrum obtained in the PDA detector matched with targeted impurity.

Characterization of all the impurities were performed using analytical data obtained from

IR, UV, Mass experiments, 1H /13C NMR spectrum and DEPT NMR experiments. The

structure of 2N-ethyl oxo candesartan cilexetil is finally confirmed by single-crystal

diffraction analysis.

91
 Chapter 4

4.6 RESULTS AND DISCUSSION

4.6.1 Analysis of degradation samples by analytical LC and LC –MS

The drug product samples kept at an accelerated stability condition for three

months, and exposed directly at 60°C for two weeks were diluted to the required

concentration and analyzed with analytical HPLC method. There were five impurities

found in the range of 0.05% to 0.5%. The purity angle of all the impurities were found

less than that of purity threshold and thus the peak purity of the impurities were

confirmed. The spectra of all the impurities and candesartan cilexetil were extracted from

PDA detector in the range of 210 to 400 nm. The typical chromatographs were given in

the figures 4.2-4.4 and the UV spectra were presented in the figure 4.5. As the analytical

method is compatible with MS, m/z data of all the impurities were generated.

4.6.2 Isolation of degradation product(s) by preparative HPLC

Candesartan Cilexetil (5g) was autoclaved for 3 hours and the material was diluted

to the required concentration and analyzed with analytical HPLC. All the five impurities

were enriched and found in the range of 6 to 40%. The autoclaved material was dissolved

in the mobile phase to get the concentration of 50 mg/ml and a solution (2 ml) was

subjected to preparative HPLC and as many as 25 fractions were collected separately.

The typical chromatogram of preparative HPLC was given in the figure 4.6. Purity of all

these fractions was analyzed by analytical HPLC and found to be in the range of 99%.

The eluent was evaporated and the resultant solution of Impurity-II and III were filtered

individually to separate the solid and washed with chilled water to remove traces of

trifluoro acetic acid. The solid was dried at 60°C under vacuum for 3 hours.

92
 Chapter 4

The impurities from other resultant solutions were isolated using Freeze drier maintained

at 48°C for 8 hours. Thus the isolated impurities were reanalyzed on analytical LC and

the purity of the same was found to be 99% which was good enough for carrying out the

spectroscopic experiments.

93
94
Chapter 4

Fig. 4.2 Typical HPLC chromatograms of Candesartan cilexetil Tablet - Initial sample
95
Chapter 4

Fig. 4.3: Typical HPLC chromatograms of Candesartan cilexetil Tablet - 60°C for 2 weeks
96
Chapter 4

Fig. 4.4 Typical HPLC chromatograms of Candesartan cilexetil Tablet-40°C /75%RH for 3 months
97
Chapter 4

Fig. 4.5 The UV spectra of the Candesartan cilexetil and its impurities
98
Chapter 4

Fig. 4.6 Typical Preparative LC chromatogram of Candesartan cilexetil autoclaved sample

 Chapter 4

4.6.3 Synthesis of Impurities

Candesartan cilexetil (50 g) was dissolved in dichloromethane (250 ml)

at 25-30°C and hydrochloric acid (25%) was added. The mixture was stirred for 24 hours

and the solvent was evaporated. The solid was filtered and washed with chilled water.

Impurity I thus obtained was dried at 60°C under vacuum for 3 hours to give 45 g with

chromatographic purity of 99% by area normalization.

To a suspension of impurity I (40.0 g) in N.N-dimethyl formamide (250 ml)

potassium carbonate (19 g) and ethyl iodide (16.0 g) were added. The mixture was

heated and stirred for 2.5 hours at 70-75°C. The mixture was then cooled and quenched

with water. The solid was filtered and washed with cold water. The material was

analysed and found to have impurity II (30%) and impurity III (40%) in HPLC analysis.

The above solid material (20 g) was subjected to conventional column

chromatography using 2:1 v/v Hexane/ethyl acetate as eluent. The appropriate fractions

were collected separately for impurity-II and III. The combined fractions were

evaporated and the impurities were solidified from the ethyl acetate solution at ~0°C.

White powder was collected by filtration and dried at 60°C under vacuum for 3hours to

give 3.0 g of Impurity II with chromatographic purity of 99% (by area normalization)

and 4.0 g of Impurity III with chromatographic purity of 99% (by area normalization).

Impurity IV and V were synthesized in the similar manner as impurity II and III,

using candesartan cilexetil as a starting material instead of impurity I. The route of

synthesis is represented in the figure 4.7.

99
 Chapter 4

Fig. 4.7 Synthetic pathway of impurities

100
 Chapter 4

4.6.4 Detection of Impurities

The initial study of the tablet shows five impurities at the level of 0.01 to 0.3%.

The stability study of the same tablet shows that all the five impurities were increased to

the level of 0.05 to 0.4%. The same trend was observed during the thermal degradation

studies also. This shows that the molecule is prone to degradation. Those five targeted

impurities were marked as impurity I (RRT: 0.83, MW: 582), impurity II (RRT: 1.14,

MW: 610), impurity III (RRT: 1.26, MW: 610), impurity IV (RRT: 1.36, MW: 638), and

impurity V (RRT: 1.43, MW: 638).

4.6.5 Structural elucidation of Impurity-I

The isolated impurity-I, found as a white powder, shows similar UV absorbance

spectra to parent candesartan cilexetil. In the FT-IR spectrum, a characteristic absorption

band appeared at 1726 cm−1 for -C= O stretching and at 1460 cm−1 for –NH stretching

vibration. The negative ES-MS spectrum of the impurity-I showed peaks at m/z 581

atomic mass unit (amu) and the positive ES-MS spectrum showed peaks at m/z 605 amu

corresponding to the sodium adduct of the impurity. Hence molecular weight of the

impurity–I is confirmed as 582. In comparison with parent, impurity-I is having 28 amu

less which can be presumed to have similar structure skeleton as that of parent but with

short of four hydrogen atoms and two carbon atoms.

In 1H spectrum of the impurity-I in dimethyl sulfoxide-d6 (DMSO-d6), 16 protons

appeared in the up field region ( 1.17–5.34 ppm), 12 protons in the downfield region

( 6.69–7.67 ppm) and two acidic protons appeared at ( 11.56 and 16.23 ppm).

101
 Chapter 4

Deuterium oxide (D2O) exchange experiment showed presence of two exchangeable

protons in the impurity where as only one exchangeable proton was found for the parent.

Comparison of 1H, 13
C and DEPT 135 NMR data (Table 4.1) (Fig. 4.9 to 4.11) shows

that the methylene signal at δ 67.77 ppm and methyl signal at δ 14.40 ppm disappeared in

the impurity-I (Fig. 4.12 to 4.14) spectrum which corresponds to 32 and 33 position in the

parent. This has been supported by mass data of 28 amu less than that of the parent. This

led to the conclusion that ethyl group attached to oxygen atom in benzimidazole group is

hydrolysed, subsequently undergoes keto-enol tautomerism and is stabilized as stable

keto form. This was confirmed by the IR spectrum which reveals the presence of -C= O

stretching, N-H stretching vibration and absence of O-H stretching vibration.

Based on the above spectral data the molecular formula of impurity-I was

confirmed as C31H30N6O6 and the corresponding structure was characterized

as 1-{[(cyclohexyloxy) carbonyl] oxy} ethyl -2-oxo-1-{[2'-(1-H-tetrazol-5-yl) biphenyl-

4-yl] methyl}-1-H-benzimidazole-7-carboxylate. This impurity is referred to as

CNS desethyl (desethyl candesartan cilexetil) in the reported literature (Fig. 4.8).[10]

17
18 16 N 32 N
N
28 OH NH 33
15
29 27 O 24 19 21 N
12
N
1
20 13
26 14 2 7
30 11 8
O 25 O 23 O 22 O 3
31 6
10
9
4
5

Fig. 4.8 Structural formula of Impurity-I

102
 Chapter 4

Table 4.1 NMR assignments for Candesartan cilexetil and impurity-I

CANDESARTAN CILEXETIL IMPURITY I

1 13 1 13
Position H (ppm) C(ppm) DEPT H (ppm) C(ppm) DEPT
(Multiplicity) (Multiplicity)
1 154.79 C 154.89 C
2 140.91 C 138.51 C
3 139.33 C 138.01 C
4 7.31-7.35(dt) 130.47 CH 7.45-7.47(d) 130.52 CH
5 7.55-7.59(dt) 128.26 CH 7.62-7.67(m) 128.99 CH
6 7.55-7.59(dt) 120.47 CH 7.53-7.57(m*) 127.72 CH
7 8.01-8.05(dt) 131.26 CH 7.62-7.67(m) 130.62 CH
8 138.11 C 136.29 C
9 6.90-6.92(d) 124.01 CH 6.98-7.0(d) 128.99 CH
10 6.78-6.80(d) 129.43 CH 6.92-6.94(d) 126.51 CH
11 136.06 C 130.03 C
12 6.78-6.80(d) 129.43 CH 6.92-6.94(d) 126.51 CH
13 6.90-6.92(d) 124.01 CH 6.98-7.0(d) 128.99 CH
14 5.58-5.69(d) 46.69 CH2 5.21-5.34(d) 44.52 CH2
15 157.65 C 140.93 C
16 123.4 C 124.24 C
17 7.13(d) 124.87 CH 7.24-7.27(dt) 122.3 CH
18 6.98-7.02(t) 121.15 CH 7.08-7.12(t) 120.73 CH
19 7.51-7.54(dt) 131.17 CH 7.28-7.30(dt) 113.23 CH
20 115.31 C 113.03 C
21 123.4 C 128.29 C
22 163.08 C 163.44 C
23 6.68-6.72(q) 91.69 CH 6.69-6.73(q) 91.86 CH
24 1.4(d) 19.09 CH3 1.34-1.35(m) 19.03 CH3
25 152.31 C 151.9 C
26 4.21-1.31(m) 77.52 CH 4.54-4.60(m) 76.78 CH
27 1.78 - 1.65(m) 31.26 CH2 1.83(m) 30.74 CH2
28 1.13-1.35(m) 23.46 CH2 1.17-1.31(m) 22.92 CH2
29 1.48-1.49 (m) 24.99 CH2 1.38-1.48(m) 24.54 CH2
30 1.13-1.35(m) 23.46 CH2 1.17-1.31(m) 22.95 CH2
31 1.78-1.65(m) 31.21 CH2 1.64(m) 30.78 CH2
32 4.44-4.52(q) 67.77 CH2 11.56(s) NH
33 1.43(q) 14.4 CH3 16.23(bs) NH
34 13.95(bs) NH

s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad; dt-doublet of triplet

103
104
Chapter 4

Fig. 4.9 1H NMR Spectrum of Candesartan Cilexetil

105
Chapter 4

13
Fig. 4.10 C NMR Spectrum of Candesartan Cilexetil
106
Chapter 4

Fig. 4.11 DEPT 135 NMR Spectrum of Candesartan Cilexetil

107
Chapter 4

Fig. 4.12 1H NMR Spectrum of Impurity - I

108
Chapter 4

13
Fig. 4.13 C NMR Spectrum of Impurity - I
109
Chapter 4

Fig. 4.14 DEPT 135 NMR Spectrum of Impurity - I

 Chapter 4

4.6.6 Structural elucidation of impurity-II

The isolated impurity-II was found as a white powder and shows similar UV

absorbance spectra of parent compound. In FT-IR spectrum a characteristic absorption

band appeared at 1750 cm−1 for -C=O stretching and at 1193 cm−1 for -N–C stretching

vibration. The negative ES-MS spectrum of the impurity-II showed a peak

at m/z 609 amu and the positive ES-MS spectrum showed peaks at m/z 633 amu

corresponding to the sodium adduct of the impurity. Hence molecular weight of the

impurity–II is confirmed as 610. In comparison with its parent, impurity-II has same

molecular weight, which can be presumed to have similar structure as that of parent with

some possible positional isomerism.

In the 1H spectrum of the impurity-II in CDCl3, 21 protons appeared in the up field

region ( 0.84–5.54 ppm), 12 protons in the downfield region ( 6.82–7.64 ppm). One

exchangeable proton ( 10.75 ppm) was observed in the impurity in the D2O exchange

experiment as that of the parent but the chemical shift got shielded from  13.95

to  10.75 ppm. Comparison of 1H, 13

C and DEPT 135 NMR data (Table 4.2)

(Fig. 4.16-4.18) shows that the methylene signal at δ 67.77 ppm disappeared and a new

methylene signal at  45.68 ppm is observed. This led to the hypothesis of possible

rearrangement of ethyl group from benzimidazole to tetrazole. The long range correlation

(LRC) experiment predicts that the ethyl group attached to first “N” atom of tetrazole

group.

110
 Chapter 4

Based on the above spectral data the molecular formula of impurity-II was

confirmed as C33H34N6O6 and the corresponding structure was characterized as

1-{[(cyclohexyloxy) carbonyl] oxy} ethyl-2-oxo-1-{[2'-(1-ethyl-1H-tetrazol-5-yl)

biphenyl-4-yl] methyl}-1H-benzimidazole-7-carboxylate. This impurity is named as

1N-ethyl oxo candesartan cilexetil (Fig. 4.15).

17 33
18 16 N 34 CH3
N
N
28 OH N
15 32
29 27 O 24 19 21 N 12
N
1
20 13
26 14 2 7
30 11 8
O 25 O 23 O 22 O 3
31 6
10
9
4
5

Fig 4.15 Structural formula of Impurity-II

111
 Chapter 4

Table 4.2 NMR assignments for impurity-II

CANDESARTAN CILEXETIL IMPURITY II

1 13 1 13
Position H (ppm) C(ppm) DEPT H (ppm) C(ppm) DEPT
(Multiplicity) (Multiplicity)
1 154.79 C 154.31 C
2 140.91 C 141.17 C
3 139.33 C 137.91 C
4 7.31-7.35(dt) 130.47 CH 7.43-7.55(m) 131.57 CH
5 7.55-7.59(dt) 128.26 CH 7.43-7.55(m) 130.23 CH
6 7.55-7.59(dt) 120.47 CH 7.43-7.55(m) 128.82 CH
7 8.01-8.05(dt) 131.26 CH 7.60-7.64(m) 127.92 CH
8 138.11 C 137.14 C
9 6.90-6.92(d) 124.01 CH 7.06-7.10(d) 128.85 CH
10 6.78-6.80(d) 129.43 CH 7.00-7.02(d) 127.92 CH
11 136.06 C 122.75 C
12 6.78-6.80(d) 129.43 CH 7.00-7.02(d) 127.92 CH
13 6.90-6.92(d) 124.01 CH 7.06-7.10(d) 128.85 CH
14 5.58-5.69(d) 46.69 CH2 5.45-5.54(d) 42.44 CH2
15 157.65 C 156.62 C
16 123.4 C 122.75 C
17 7.13(d) 124.87 CH 7.28-7.30(d) 114.08 CH
18 6.98-7.02(t) 121.15 CH 7.06-7.10(t) 121.18 CH
19 7.51-7.54(dt) 131.17 CH 7.43-7.55(m) 123.85 CH
20 115.31 C 114.28 C
21 123.4 C 122.75 C
22 163.08 C 163.63 C
23 6.68-6.72(q) 91.69 CH 6.82-6.86(q) 91.92 CH
24 1.4(d) 19.09 CH3 1.49-1.50(t) 19.57 CH3
25 152.31 C 152.52 C
26 4.21-1.31(m) 77.52 CH 4.61-4.68(m) 77.68 CH
27 1.78 - 1.65(m) 31.26 CH2 1.71-1.95(m) 31.44 CH2
28 1.13-1.35(m) 23.46 CH2 1.22-1.41(m) 23.6 CH2
29 1.48-1.49 (m) 24.99 CH2 1.44-1.56(m) 25.11 CH2
30 1.13-1.35(m) 23.46 CH2 1.22-1.41(m) 23.63 CH2
31 1.78-1.65(m) 31.21 CH2 1.71-1.95(m) 31.4 CH2
32 4.44-4.52(q) 67.77 CH2 3.40-3.47(q) 45.68 CH2
33 1.43(q) 14.4 CH3 0.84(d) 13.66 CH3
34 13.95(bs) NH 10.75(bs) NH

s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad; dt-doublet of triplet

112
113
Chapter 4

Fig. 4.16 1H NMR Spectrum of Impurity - II

114
Chapter 4

13
Fig. 4.17 C NMR Spectrum of Impurity - II
115
Chapter 4

Fig. 4.18 DEPT 135 NMR Spectrum of Impurity - II

 Chapter 4

4.6.7 Structural elucidation of Impurity-III

The isolated impurity-III was found as white powder and shows similar UV

absorbance spectra of parent as well as Impurity-II. All the spectral data of impurity–III

are similar to that of impurity-II. Comparison of 1H, 13

C and DEPT 135 NMR data

(Table 4.3) (Fig. 4.20 - 4.22) shows that the methine signal corresponds to the first

position at  ppm and got deshielded to  ppm. Similarly the methylene

signal corresponds to 32 position that also got deshielded from  ppm to 

ppm. This led to the hypothesis of possible regio isomerism of ethyl group attached to

another “N” atom of tetrazole group. The existence of tetrazole regioisomers are reported

in the literature.[11] Further this regio isomerism has been confirmed by single-crystal

diffraction analysis (Fig. 4.23) (Table 4.4). The single-crystal diffraction study confirms

that the impurity II and III are regio isomers. Based on the above data the molecular

formula of impurity-III was confirmed as C33H34N6O6 and the corresponding structure

(Fig. 4.15) was characterized as 1-{[(cyclohexyloxy) carbonyl] oxy} ethyl-2-oxo-1-{[2'-

(2-ethyl-1H-tetrazol-5-yl) biphenyl-4-yl] methyl}-1H-benzimidazole-7-carboxylate. This

impurity is named as 2N-ethyl oxo candesartan cilexetil (Fig. 4.19).

33 CH3
17
18 16 N
34 N 32
N
28 OH N
15
29 27 O 24 19 21 N 12
N
1
20 13
26 14 2 7
30 11 8
O 25 O 23 O 22 O 3
31 6
10
9
4
5

Fig. 4.19 Structural formula of Impurity-III

116
 Chapter 4

Table 4.3 NMR assignments for impurity-III

CANDESARTAN CILEXETIL IMPURITY III

1 13 1 13
Position H (ppm) C(ppm) DEPT H (ppm) C(ppm) DEPT
(Multiplicity) (Multiplicity)
1 154.79 C 165.01 C
2 140.91 C 141.56 C
3 139.33 C 139.92 C
4 7.31-7.35(dt) 130.47 CH 7.39-7.50(m) 130.33 CH
5 7.55-7.59(dt) 128.26 CH 7.39-7.50(m) 130.64 CH
6 7.55-7.59(dt) 120.47 CH 7.39-7.50(m) 130.33 CH
7 8.01-8.05(dt) 131.26 CH 7.81-7.83(dt) 129.91 CH
8 138.11 C 126.31 C
9 6.90-6.92(d) 124.01 CH 7.01-7.08(dt) 129.41 CH
10 6.78-6.80(d) 129.43 CH 7.01-7.08(dt) 126.72 CH
11 136.06 C 135.55 C
12 6.78-6.80(d) 129.43 CH 7.01-7.08(dt) 126.72 CH
13 6.90-6.92(d) 124.01 CH 7.01-7.08(dt) 129.41 CH
14 5.58-5.69(d) 46.69 CH2 5.42-5.62(d) 45.67 CH2
15 157.65 C 156.65 C
16 123.4 C 128.68 C
17 7.13(d) 124.87 CH 7.26-7.29(dt) 127.49 CH
18 6.98-7.02(t) 121.15 CH 7.01-7.08(t) 120.94 CH
19 7.51-7.54(dt) 131.17 CH 7.35-7.37(dt) 113.74 CH
20 115.31 C 114.61 C
21 123.4 C 129.53 C
22 163.08 C 163.7 C
23 6.68-6.72(q) 91.69 CH 6.83-6.87(q) 91.89 CH
24 1.4(d) 19.09 CH3 1.42-1.44(d) 19.51 CH3
25 152.31 C 152.53 C
26 4.21-1.31(m) 77.52 CH 4.60-4.72(m) 77.65 CH
27 1.78 - 1.65(m) 31.26 CH2 1.70.1.93(m) 31.04 CH2
28 1.13-1.35(m) 23.46 CH2 1.23-1.31(m) 23.58 CH2
29 1.48-1.49 (m) 24.99 CH2 1.50-1.54(m) 25.1 CH2
30 1.13-1.35(m) 23.46 CH2 1.23-1.31(m) 23.6 CH2
31 1.78-1.65(m) 31.21 CH2 1.70-1.93(m) 31.38 CH2
32 4.44-4.52(q) 67.77 CH2 4.39-4.45(m) 48.08 CH2
33 1.43(q) 14.4 CH3 1.35(t) 14.4 CH3
34 13.95(bs) NH 9.75(bs) NH

s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad; dt-doublet of triplet

117
118
Chapter 4

Fig. 4.20 1H NMR Spectrum of Impurity - III

119
Chapter 4

13
Fig. 4.21 C NMR Spectrum of Impurity - III
120
Chapter 4

Fig. 4.22 DEPT 135 NMR Spectrum of Impurity - III

 Chapter 4

Fig. 4.23 Single crystal structure of Impurity-III

Table 4.4 Crystal data and refinement of Impurity-III

121
 Chapter 4

4.6.8 Structural elucidation of Impurity-IV

The isolated Impurity-IV was found as white powder and shows similar UV

absorbance spectra as that of the parent. In the FT-IR spectrum, a characteristic

absorption band appeared at 1742 cm−1 for -C= O stretching. The negative ES-MS

spectrum of the impurity-IV showed a peak at m/z 637 amu and the positive ES-MS

spectrum showed peaks at m/z 661 amu corresponding to the sodium adduct of the

impurity. Hence molecular weight is confirmed as 638. The mass of impurity-IV is 28

amu higher than the parent, which can be presumed to have similar structure skeleton as

that of the parent with an additional of four hydrogen atoms and two carbon atoms.

In the 1H spectrum of the impurity-I in CDCl3, 26 protons appeared in the up field

region ( 0.88–5.66 ppm), 12 protons in the downfield region ( 6.88–7.74 ppm). D2O

exchange showed absence of any exchangeable proton in the impurity. Comparison of 1H,
13
C and DEPT 135 NMR data (Table 4.5) (Fig. 4.25-4.27)shows that the impurity-IV has

three methyl protons, eight methylene protons, thirteen methine protons and eleven

quaternary carbons. All the spectral data have been compared with the parent and the

presence of an extra methyl group is confirmed. The absence of acidic proton and LRC

experiment confirms that the additional ethyl group attached to the first “N” atom of

tetrazole group. Based on the above spectral data, the molecular formula of impurity-IV

was confirmed as C35H38N6O6 and the corresponding structure was characterized as

1-{[(cyclohexyloxy) carbonyl] oxy}ethyl-2-ethoxy-1-{[2'-(1-ethyl-1H-tetrazol-5-yl)

biphenyl-4-yl] methyl}-1H-benzimidazole-7-carboxylate. This impurity is referred to as

1N-ethyl CNS (1N-ethyl candesartan cilexetil) in the reported literature (Fig. 4.24).[10]

122
 Chapter 4

35
17 CH3 33
18 16 N 34 CH3
N
N
28 O N
15 32
29 27 O 24 19 21 N 12
N
1
20 13
26 14 2 7
30 11 8
O 25 O 23 O 22 O 3
31 6
10
9
4
5

Fig. 4.24 Structural formula of Impurity-IV

123
 Chapter 4

Table 4.5 NMR assignments for Impurity-IV

CANDESARTAN CILEXETIL IMPURITY IV

1 13 1 13
Position H (ppm) C(ppm) DEPT H (ppm) C(ppm) DEPT
(Multiplicity) (Multiplicity)
1 154.79 C 158.82 C
2 140.91 C 142.01 C
3 139.33 C 141.51 C
4 7.31-7.35(dt) 130.47 CH 7.47-7.55(m) 130.64 CH
5 7.55-7.59(dt) 128.26 CH 7.47-7.55(m) 129.94 CH
6 7.55-7.59(dt) 120.47 CH 7.60-7.64(m) 127.54 CH
7 8.01-8.05(dt) 131.26 CH 7.60-7.64(m) 130.31 CH
8 138.11 C 139.98 C
9 6.90-6.92(d) 124.01 CH 6.95-7.01(d) 129.37 CH
10 6.78-6.80(d) 129.43 CH 6.95-7.01(d) 126.63 CH
11 136.06 C 135.87 C
12 6.78-6.80(d) 129.43 CH 6.95-7.01(d) 126.63 CH
13 6.90-6.92(d) 124.01 CH 6.95-7.01(d) 129.37 CH
14 5.58-5.69(d) 46.69 CH2 5.54-5.66(d) 48.62 CH2
15 157.65 C 164.03 C
16 123.4 C 126.29 C
17 7.13(d) 124.87 CH 7.60-7.64(m) 123.93 CH
18 6.98-7.02(t) 121.15 CH 7.14-7.18(t) 120.72 CH
19 7.51-7.54(dt) 131.17 CH 7.72-7.74(dt) 122.55 CH
20 115.31 C 114.66 C
21 123.4 C 131.89 C
22 163.08 C 165.02 C
23 6.68-6.72(q) 91.69 CH 6.88-6.93(q*) 91.75 CH
24 1.4(d) 19.09 CH3 1.54-1.56(m) 19.61 CH3
25 152.31 C 152.57 C
26 4.21-1.31(m) 77.52 CH 3.43-3.49(m) 77.59 CH
27 1.78 - 1.65(m) 31.26 CH2 1.73-1.95(m) 31.4 CH2
28 1.13-1.35(m) 23.46 CH2 1.21-1.43(m) 23.59 CH2
29 1.48-1.49 (m) 24.99 CH2 1.56 & 1.51(m) 25.12 CH2
30 1.13-1.35(m) 23.46 CH2 1.21-1.43(m) 23.59 CH2
31 1.78-1.65(m) 31.21 CH2 1.73 &1.95(m) 31.4 CH2
32 4.44-4.52(q) 67.77 CH2 4.60-4.67(m) 66.75 CH2
33 1.43(q) 14.4 CH3 1.47(m) 14.68 CH3
34 13.95(bs) NH 3.43-3.49(q) 46.92 CH2
0.83(t) 14.39 CH3

s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad; dt-doublet of triplet

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125
Chapter 4

Fig. 4.25 1H NMR Spectrum of Impurity - IV

126
Chapter 4

13
Fig. 4.26 C NMR Spectrum of Impurity - IV
127
Chapter 4

Fig. 4.27 DEPT 135 NMR Spectrum of Impurity - IV

 Chapter 4

4.6.9 Structural elucidation of Impurity-V

Comparison of 1H, 13
C and DEPT 135 NMR data (Table 4.6) (Fig. 4.29-4.31)

shows that impurity-V is similar to that of impurity-IV. The methine carbon at the first

position is deshielded to the extent of 5.2 ppm and the methylene carbon at 34th position

is deshielded to the extent of 4.5 ppm compared with impurity-IV. Similar deshielding

was found between regio isomers of impurity-II and III and it reveals that impurity IV

and V are also another set of regio isomers. Based on the above spectral data the

molecular formula of impurity-V was confirmed as C35H38N6O6 and the corresponding

structure was characterized as 1-{[(cyclohexyloxy) carbonyl] oxy} ethyl-2-ethoxy-1-{[2'-

(2-ethyl-1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-1H-benzimidazole-7-carboxylate. This

impurity is referred to as 2N-ethyl CNS (2N-ethyl candesartan cilexetil) in the reported

literature (Fig. 4.28).[10]

35 H3C 33 CH3
17
18 16 N
34 N 32
N
28 O N
15
29 27 O 24 19 21 N 12
N
1
20 13
26 14 2 7
30 11 8
O 25 O 23 O 22 O 3
31 6
10
9
4
5

Fig. 4.28 Structural formula of Impurity-V

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Table 4.6 NMR assignments for Impurity-V

CANDESARTAN CILEXETIL IMPURITY V

1 13 1 13
Position H (ppm) C(ppm) DEPT H (ppm) C(ppm) DEPT
(Multiplicity) (Multiplicity)
1 154.79 C 164.03 C
2 140.91 C 142.07 C
3 139.33 C 141.19 C
4 7.31-7.35(dt) 130.47 CH 7.35-7.37(dt) 131.56 CH
5 7.55-7.59(dt) 128.26 CH 7.42-7.51(dt) 127.94 CH
6 7.55-7.59(dt) 120.47 CH 7.42-7.51(dt) 128.82 CH
7 8.01-8.05(dt) 131.26 CH 7.81-7.83(dt) 131.53 CH
8 138.11 C 137.91 C
9 6.90-6.92(d) 124.01 CH 7.02-7.04(d) 130.22 CH
10 6.78-6.80(d) 129.43 CH 6.92-6.94(d) 127.67 CH
11 136.06 C 137.42 C
12 6.78-6.80(d) 129.43 CH 6.92-6.94(d) 127.67 CH
13 6.90-6.92(d) 124.01 CH 7.02-7.04(d) 130.22 CH
14 5.58-5.69(d) 46.69 CH2 5.59-5.68(d) 46.92 CH2
15 157.65 C 158.8 C
16 123.4 C 122.77 C
17 7.13(d) 124.87 CH 7.55-7.57(dt) 124.29 CH
18 6.98-7.02(t) 121.15 CH 7.13-7.17(t) 120.92 CH
19 7.51-7.54(dt) 131.17 CH 7.72-7.74(dt) 122.89 CH
20 115.31 C 114.3 C
21 123.4 C 132.16 C
22 163.08 C 164 C
23 6.68-6.72(q) 91.69 CH 6.92(q*) 91.79 CH
24 1.4(d) 19.09 CH3 1.48-1.53(m) 19.64 CH3
25 152.31 C 152.57 C
26 4.21-1.31(m) 77.52 CH 4.59-4.69(m) 77.57 CH
27 1.78 - 1.65(m) 31.26 CH2 1.75 & 1.91(m) 31.4 CH2
28 1.13-1.35(m) 23.46 CH2 1.33 & 1.53(m) 23.62 CH2
29 1.48-1.49 (m) 24.99 CH2 1.44(m) 25.12 CH2
30 1.13-1.35(m) 23.46 CH2 1.26(m) 23.62 CH2
31 1.78-1.65(m) 31.21 CH2 1.91(m) 31.4 CH2
32 4.44-4.52(q) 67.77 CH2 4.59-4.69(q) 66.83 CH2
33 1.43(q) 14.4 CH3 1.43(d) 14.67 CH3
34 13.95(bs) NH 4.37-4.42(q) 42.4 CH2
1.3(t) 13.61 CH3

s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad; dt-doublet of triplet

129
130
Chapter 4

Fig. 4.29 1H NMR Spectrum of Impurity - V

131
Chapter 4

13
Fig. 4.30 C NMR Spectrum of Impurity - V
132
Chapter 4

Fig. 4.31 DEPT 135 NMR Spectrum of Impurity - V

 Chapter 4

4.7 FORMATION OF IMPURITIES

The parent drug molecule undergoes hydrolysis which results in the formation of

Impurity I and ethyl cation. The ethyl cation further reacts with impurity I to form

Impurity II and III which are regioisomers. The content of impurity III is observed to be

more in the stability samples than in that of impurity II. The rationale for this might

be due to the steric hindrances present in the Impurity II. The ethyl cation reacts with

the parent drug molecules leading to the formation of another set of regioisomers

(Impurity IV and V). In this case also impurity V content is more than that of

Impurity IV.

4.8 VALIDATION OF ANALYTICAL METHOD

Analytical HPLC method has been extensively validated for the quantitation of all

the five degradates. Specificity is verified by means of blank, placebo interferences and

forced degradation studies. The results of various validation parameters such as

precision, linearity, accuracy, limit of detection and limit of quantitation were

summarized in the table 4.7. The data are evident that the method met the requirement of

validation. Ruggedness and robustness of the method has also been confirmed. The

relative response factors were established and values given in the table 4.7.

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Table 4.7 HPLC method validation data

Parameters Impurity I Impurity II Impurity III Impurity IV Impurity V

Precision
RSD at 0.5% level 2.35% 0.73% 0.76% 0.82% 3.23%
RSD at LOQ level 2.40% 2.90% 3.30% 1.50% 3.50%

Intermediate Precision
RSD at 0.5% level 1.98% 1.35% 0.96% 0.5% 2.95%

Linearity (LOQ to 1.0%)

Regression
coefficient 0.99972 0.99954 0.99958 0.99978 0.99958

Accuracy (LOQ to 1.0%)

94.5 - 92.3- 96.4 - 91.1 - 97.1 -
Recovery 98.8% 100.6% 106.6% 97.4% 108.0%

Limit of Detection
(LOD) 0.01% 0.01% 0.01% 0.01% 0.01%

Limit of
Quantitation (LOQ) 0.05% 0.05% 0.05% 0.05% 0.05%

Relative Response
Factor* 1.1 1.09 1.03 1.02 0.88

* with respect to candesartan cilexetil

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4.9 CONCLUSION

The five major degradation products of candesartan cilexetil were isolated by

preparative LC and were characterized by using spectroscopic techniques namely IR, UV,

NMR, MS and single-crystal X ray diffraction data. The structures were supported by

synthetic ethylating reaction of candesartan cilexetil and impurity I which allowed the

preparation of four impurities.

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REFERENCES

1. Y. Michihiro and Y. Hirofumi, “Clinical pharmacology”, 93, 175-182, 2000.

2. M. Kenichi, I. Shinji, M. Keiko, E. Shigeru, T. Masayuki, O. Mamoru, S. Hideki,

and K. Masahiko, “Cardiovascular Drugs and Therapy”, 12, 469–474, 1998.

3. www. Rxlist.com

4. H. Stenhoff, P. Lagerstrom and C. Andersen, “J. Chromatogr. B”, 731, 411-417,

1999.

5. N.Ferreiros, S. Dresen, R.M. Alonso and W. Weinmann, “J. Chromatogr.

B”, 855, 134-138, 2007.

6. D.V. Subba Rao, P. Radhakrishnanand, M.V. Suryanarayana and V. Himabindu,

“Chromatographia”, 66, 499-507, 2007.

7. “Impurities in New Drug Substances”, Q3A(R2), 2006.

8. “Impurities in New Drug Products”, Q3B (R2), 2006.

9. “Validation of Analytical Procedures: Text and Methodology”, ICH

Harmonized Tripartite Guidelines. (Q2B), 1996

10. Z. Kurgan and M. Pesachovich, “World intellectual property organization”,

WO 2006/122254 A2, 2006.

11. H. Wolfgang and J. Christine, “Monatshefte fur Chemie”, 123, 1027-1036,

1992.

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Reversed Phase UPLC Method for Related

Substances and

Stability Indicating Assay for Lacidipine

 Chapter 5

5.1 INTRODUCTION

Lacidipine, 3, 5-diethyl 4-{2-[(1E)-3-(tert-butoxy)-3-oxoprop-1-en-1-yl] phenyl}-

2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarboxylate is a calcium channel blocker used

in the treatment of hypertension and atherosclerosis (Fig. 5.1). It also possesses an

antioxidant effect. It is one of the most vascular selective of the dihydropyridines. It has

long duration action because of its high degree of lipophilicity. The active trans form is

used in therapy.[1, 2] Lacidipine undergoes extensive first-pass hepatic metabolism and has

a mean absolute bioavailability of about 10% (range 3–59%). Lacidipine is completely

metabolized in the liver by cytochrome P450 3A4 (CYP3A4) to pharmacologically

inactive metabolites.[2] Lacidipine is available in tablet form and marketed under various

brand names such as Caldine, Lacimen, Lacipil, Midotens and Motens.

5.2 DRUG PROFILE

Lacidipine is White-to-Off-White Crystalline Solid. The solubility and chemical

stability were higher at the neutral pH 6.8. The chemical stability had a tendency to be

decreased at the alkaline solution over pH 8.0-10.0. The drug is not freely soluble in

water, 0.1 M HCl and 0.1 M NaOH solution. Its molecular weight is 455.54 and

structural formula is

Fig. 5.1 Structural formula of Lacidipine

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 Chapter 5

5.3 LITERATURE REVIEW

Ramesh et al.,[3] have developed and validated a high performance liquid

chromatographic (HPLC) method for quantification of lacidipine (LCDP) in rabbit serum.

LCDP and internal standard (IS), felodipine were extracted into n-hexane and

dichloromethane (70:30) solvent system and separated using an isocratic mobile phase, on

an Inertsil C18 column. The effluent was monitored by UV detector at 240 nm and at a

flow rate of 1.0 ml/min. The linearity range of proposed method was 1–500 ng/ml. The

intra-day and inter-day coefficient of variation and percent error values of the assay

method were less than 15% and mean recovery was more than 94 and 95% for LCDP and

IS, respectively and the method was found to be precise, accurate, and specific during the

study. The method was successfully applied for pharmacokinetic study of lacidipine after

application of LCDP micro emulsion gel in rabbits.

Braggio et al.,[4] have reported the automation and validation of the HPLC-

radioimmunoassay (RIA) method for the determination of lacidipine. The solid-phase

extraction step was automated by the introduction of the ASPEC system. A two-column

system was adopted for the HPLC purification. The RIA was converted from

heterogeneous to homogeneous by the scintillation proximity assay system and automated

using an automatic dilution system. All characteristics in terms of accuracy, precision,

specificity, and linearity that resulted similar to the manual version. The quantification

limit was set to 40 pg/ml.

Tang et al.,[5] have developed and validated an Ultra-Performance Liquid

Chromatography–tandem Mass Spectrometry (UPLC–ESI-MS/MS) method which was

for the quantification of lacidipine in human plasma. With nifedipine as an internal

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 Chapter 5

standard, sample pretreatment involved a simple liquid–liquid extraction with

tert-butylmethyl ether of 1 ml plasma. The analysis was carried out on an Acquity TM

UPLC BEH C18 column (50 mm×2.1 mm, 1.7 µm) with a flow rate of 0.28 ml/min.

The mobile phase was 30 mM ammonium acetate buffer–acetonitrile (18:82, v/v, pH 5.5).

The detection was performed on a triple quadrupole tandem mass spectrometer by

multiple reactions monitoring (MRM) mode via electrospray ionization (ESI). Linear

calibration curves were obtained in the concentration range of 0.025–10.000 ng/ml, with a

lower limit of quantification of 0.025 ng/ml. The intra-day and inter-day precision (RSD)

values were below 15% and accuracy was −12.7% to 11.9% at all QC levels.

Pellegatti et al.,[6] have reported a method for the determination of lacidipine, a

new potent antihypertensive dihydropyridine. The method involves solid-phase

extraction, reversed-phase high-performance liquid chromatography and

radioimmunoassay of the collected fraction. The assay provides a limit of detection of

20 pg/ml of plasma, allowing the determination of trough (24 hours) plasma

concentrations. The method was suitable for pharmacokinetic studies in men.

Kharat et al.,[7] have developed and validated a high performance thin layer

chromatographic method that was meant for the estimation of lacidipine. The sample

preparation involved protein precipitation followed by an efficient solid phase extraction

on C18 cartridge. The analytes were isolated from 1 ml of urine and recovered by pure

ethyl acetate solution. The method employed TLC aluminium plate precoated with silica

gel 60F254 as the stationary phase. The solvent system employed consists of

toulene and ethyl acetate [6.5:3.5 v/v]. This system gave a dense and compact spot of the

drug at Rf value of 0.45. The linear regression data for the calibration plots showed good

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linear relationship (r = 0.999) over the concentration range 10–80 ng. Recovery studies

were performed at two different levels. The recovery data reveal that the R.S.D for intra-

day and inter-day analysis at 10 ng was found to be 0.84 and 0.22%, respectively.

Baranda et al.,[8] have developed a high-performance liquid chromatographic

method with electrochemical detection for the simultaneous determination of

five 1,4-dihydropyridines: amlodipine, nitrendipine, felodipine, lacidipine and

lercanidipine. The chromatographic separation was performed on a Supelcosil LC ABZ +

Plus C18 column with a mobile phase consisting of acetonitrile and 10 mM acetate buffer

(72:28, v/v) at a flow rate of 1 ml/min. The temperature was set at 30 ± 0.2 ◦C. The

amperometric detector, equipped with a glassy carbon electrode was operated

at +1100 mV versus Ag/AgCl in the direct current mode. Under these chromatographic

conditions, the drugs eluted in less than 12 minutes. The method is shown to be linear

over the range 4.5–15 µg/ml with a within-day and day-to-day repeatabilities in terms of

R.S.D lower than 15%, an accuracy greater than 98% and detection limits varying from

90 ng/ml (amlodipine) to 1.55 µg/ml (nitrendipine). The method was successfully applied

to commercially available pharmaceuticals with relative errors lower than 5%. The

validity of the method was examined comparing the results obtained with those of HPLC

with photometric detection.

Özkan[9] has developed differential pulse and square wave voltammetry method

for the determination of the antihypertensive drug lacidipine in pharmaceuticals.

Squella et al.,[10] have developed a pulse voltammetric method for determination

of lacidipine using glassy carbon electrode. The repeatability of the measurement

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was 2%. On the basis of the voltammetric response, they have developed HPLC methods

with electrochemical detection. For comparative purposes, they also developed a

spectrophotometric method.

Rossato et al.,[11] have developed HPLC-MS methods in order to characterize the

main biotransformation products of lacidipine, a new antihypertensive drug. Thermospray

and particle beam interfaces were used because of their complementary information. In

fact, the former provided molecular mass indication, while particle beam allowed the

acquisition of typical electron impact and chemical ionization spectra. Chemical

ionization was performed with methane and isobutane as reagent gases.

Filippis et al.,[12] have reported the photostability of lacidipine, a dihydropyridine

drug, they have studied in solutions exposed to UV-A radiations. The effects of the

solvent (ethanol, acetone, dichloromethane), drug concentration and radiation wavelength

on the drug photostability were evaluated. Lacidipine and its photoproducts were

separated by a selective liquid chromatographic (HPLC) method, under normal phase

conditions (CN column), using n-hexane and ethanol 97:3 (v/v) as mobile phase, at a flow

rate of 2.0 ml/min. The main photo degradation products were isolated and characterized

and a photo degradation pathway was proposed for lacidipine in solution.

Belal et al.,[13] have developed a reversed-phase liquid chromatographic method

that was meant for the determination of lacidipine in the presence of its degradation

products. The analysis was carried out using a 150 mm x 4.6 mm i.d., 5 µm particle size

Nucleodur MN-C18 column. Mobile phase containing a mixture of acetonitrile

and 0.02 M phosphate buffer (70:30) at pH = 5.0 was pumped at a flow rate of 1 ml/min

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with UV detection at 254 nm. The method showed good linearity in the range

of 0.06–15 µg/ml with a limit of detection (S/N = 3) of 0.016 µg/ml. The suggested

method was successfully applied for the analysis of lacidipine in bulk and in commercial

tablets with average recoveries of 100.19 ± 0.81% and 100.05 ± 0.69%, respectively. The

results were favourably compared to those obtained by a reference method. The suggested

method was utilized to investigate the kinetics of alkaline, acidic, peroxide and photo-

induced degradation of the drug. The apparent first-order rate constant, half-life times and

activation energies of the degradation process were calculated.

Nozal et al.,[14] have developed a HPLC method for the assay of lacidipine

residues in swabs collected from various surfaces involved in drug manufacture that is

described. The swabbing procedure using two cotton swabs was validated applying a

wipe test. An RP-HPLC method, developed to determine low quantities of the drug in the

presence of its main impurities, was also validated. To remove drug residues from

stainless steel and glass surfaces, the first cotton swab must be soaked preferably in

acetonitrile whereas, on vinyl surfaces better results are obtained using methanol. The

HPLC method selected involves a C12 column, at 40°C, a mixture of acetonitrile and

0.05M ammonium acetate (88:12, v/v) as a mobile phase and UV detection at 282 nm.

Recoveries obtained are strongly dependent on the type of surface tested, being higher on

stainless steel. The surface material has also different influence on the drug stability. The

method was validated over a range of 0.5–100 µg/400 cm2 and had a detection limit of

0.1µg/400 cm2.

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5.4 SCOPE OF THE STUDY

Many methods have been reported for the determinations of lacidipine in the

biological samples[3-7] using HPLC, HPLC-RIA, UPLC-ESI-MS/MS, HPTLC. A method

for the assay of lacidipine which involves the non chromatographic techniques has been

reported[8-10]. Reversed phase HPLC-MS method that can detect lacidipine and its related

metabolites is also available[11]. This method is limited to metabolites and no other

degradation impurities were discussed. Filippis et al.,[12] have discussed exclusively, the

photolytic degradation pathway using normal phase chromatography. A reversed phased

HPLC method is reported in the literature[13], in which forced degradation experiments

were discussed, but impurities were not specified. In addition, method validation

parameters with respect to impurities were not covered. Nozal et al.,[14] discussed a

reversed phase HPLC method for the analysis of lacidipine cleaning validation samples in

the presence of degradation impurities. As the impurities are not well separated, this

method is not suitable for quantitative analysis of impurities. An analytical method that is

reported in British pharmacopeia[15], mainly covers one process impurity and two

degradation impurities. This method is based on normal phase chromatography that is

generally considered as a critical method. However, extra care needs to be taken during

the analysis because of lack of reproducibility of retention times as water or protic

organic solvents change the hydration state of the silica or alumina based

chromatographic media. To the best of knowledge, no single reversed phase UPLC/rapid

analysis method is available for the quantitation of lacidipine and its related impurities

(Fig.5.2) in drug substance and drug product.

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 Chapter 5

Hence a rapid reversed phase UPLC method was developed and validated for

quality control analysis of lacidipine in pharmaceutical preparation. A semi micro

analytical column (2.1 mm i.d) with sub micron particle size (1.7 µ) was used to perform

the experiments. One of the most important advantages of the use of semi micron column

is the reduction of solvent usage as they operate at low flow rates. The reduction in

column diameter from standard 4.6 mm i.d column to a 2.1 mm i.d column and particle

size from 5µ to 1.7µ increase the peak concentration volume, sensitivity and decrease the

run time without compromising the resolution. Three potential degradation product

(impurity B, C and F), and three process related impurities (impurity A, D, and E) were

considered for the current method development and validation. The proposed method is

applicable for routine analysis during release and stability analysis of the drug product

and the drug substance as it conforms to standard validation requirements.

5.5 EXPERIMENTAL

5.5.1 Materials

Lacidipine working standard (99.9%) was gifted from Takeida Pharmaceuticals,

Puducherry, India, whereas its impurities (imp-A 94.8%, imp-B 98.9%, imp-C 98.4%,

imp-D 99.8%, imp-E 99.9%, imp-F 83.5%) were procured from LGC Standards,

Bangalore, India and Simson Pharma, Mumbai, India. Lacidipine tablets (brand name

Motens, 2 mg strength) manufactured by Boehringer Ingelheim, Switzerland were used.

Ammonium acetate GR grade, glacial acetic acid GR grade, methanol and acetonitrile

HPLC grade were obtained from Merck, Mumbai, India. Purified water used was of

Milli-Q grade and all other chemicals used were of analytical grade.

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Fig. 5.2 Structural formula of Known Impurities of lacidipine

5.5.2 LC instrumentation and chromatographic condition

Method development, forced degradation studies and method validation were

performed using Waters Acquity UPLC System (Waters Corporation, Milford, MA,

USA), that consisted of an integrated hardware of a binary solvent manager, a sample

manager and a photodiode array detector. Data were processed using the Waters

Empower software version 1.63. An isocratic analytical method with Waters Acquity

BEH C18, (100 mm×2.1 mm; 1.7 µ) column (Waters Corporation, Milford, MA, USA)

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was employed for the separations. The mobile phase was a mixture of 50 mM

ammonium acetate buffer at a pH of 4.5 with glacial acetic acid and methanol in the

ratio 70:30. The injection volume was 5 µL, the flow rate was kept at 0.25 ml/min and

the column eluent was monitored at 240 nm for 15 minutes and the column temperature was

maintained at 40 °C.

5.5.3 Preparation of standard solutions

A working solution of 50 µg/ml and 0.75 µg/ml of lacidipine was prepared for the

determination of assay and related substances respectively.

5.5.4 Preparation of sample solutions

Twenty tablets were crushed to fine powder using a mortar and a pestle. Sample

powder equivalent to about 25 mg of lacidipine was weighed and transferred to a 50 ml

amber color volumetric flask. About 30 ml of methanol was added, sonicated for 15

minutes, cooled and methanol was added to make up the volume. The solution

contained 500 µg/ml of lacidipine. An aliquot of the solution was filtered through 0.22 µ

nylon membrane (Millipore) prior to injection for related substances test. For drug

potency assay 5 ml of filtrate was diluted to 50 ml with methanol.

5.5.5 Preparation of placebo solution

In-house placebo was prepared which comprises of lactose monohydrate 200 NF

(90%), povidone K30 (1.5%), magnesium stearate NF (1.5 %), titanium dioxide (E 171)

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NF (1.5%), methylhydroxypropylcellulose (1.5%). The placebo solution was prepared as

that of sample solution.

5.5.6 Method development and specificity experiments

For method development and specificity testing, a resolution mixture containing

lacidipine at about 500 µg/ml along with smaller quantities of impurities was prepared in

Methanol. Method development trials were performed by considering the limit of related

substances for the drug substance and drug product. Solutions of individual impurities

were also prepared and injected. Formulation placebo samples were also prepared to

ensure separation of excipients from peaks of interest.

Forced degradation of lacidipine was also performed at 500 µg/ml concentration to

confirm, the stability indicating properties and specificity of the method[16, 17]. The stress

conditions used for the degradation study included sun light, UV light (254 nm),

heat (60°C), humidity (50°C and 90% relative humidity), acid hydrolysis (0.1 M HCl),

base hydrolysis (0.1 M NaOH), aqueous hydrolysis, and oxidation (30% H 2O2).

For studies of the effects of heat, humidity and light the study period was 72 hours

whereas for acid, base, aqueous hydrolysis and oxidation, the samples were refluxed for

an hour. The purity of peaks obtained from stressed samples of lacidipine was checked

using a PDA detector. Assay of stressed samples was performed at 50 µg/ml

concentration and the assay was calculated against a qualified working standard to prove

the mass balance capability of analytical method.

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5.5.7 Limit of detection (LOD) and limit of quantitation (LOQ)

LOD and LOQ assessments were combined into a single set of experiments.

Known quantities of lacidipine and all six impurities were prepared from 0.05 µg/ml

to 0.5 µg/ml. Those samples were injected in the LC, using the developed method and

peak responses were plotted against the concentration. From the slope of linear curve and

relative standard deviation, LOD and LOQ values were established, as per the following

equation.

LOD = 3.3 x Standard deviation of the response / Slope of the calibration curve

LOQ = 10 x Standard deviation of the response / Slope of the calibration curve

Precision study was also carried out at the LOQ level by injecting six preparations

and calculating RSD of the area.

5.5.8 Linearity and Relative response factor (RRF)

For the linearity experiments, solutions of lacidipine and its six impurities at six

concentrations, spanning a range of 0.15 µg/ml (LOQ level) to 6 µg/ml were prepared.

Those samples were injected in the developed method and peak responses were plotted

against the concentration. Regression coefficient, slope and ‘y’ intercept were calculated

for lacidipine and all impurities. From the slope of curve, RRF (Relative Response

factor) of each impurity was calculated using the equation given below.

RRF of Impurity = Slope of impurity / Slope of lacidipine

Linearity samples for the assay method were prepared from lacidipine stock

solution at five concentrations, spanning a range of 25 µg/ml to 75 µg/ml. The linearity

curve was plotted and regression coefficient, slope and ‘y’ intercept were calculated.

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5.5.9 Accuracy

Standard addition and recovery experiments were carried out using a known

amount of the impurity stock solution at five different concentration of 0.15 (LOQ level),

0.75, 2.5, 5.0 and 6.0 µg/ml, that was spiked to a previously analysed sample. Each

concentration level was prepared in triplicates and the mean value of percentage

recoveries for all impurities were calculated and reported. Accuracy of the assay method

was evaluated through similar recovery experiments carried out by spiking the placebo to

analyte and analysing at three different concentration levels 25, 50 and 75µg/ml in

triplicates.

5.5.10 Precision

The system precision for the related substance method was verified by injecting

six replicate injections of standard solution containing lacidipine and its six impurities at

a concentration of 0.75 µg/ml of each. The percentage relative standard deviation (RSD)

for peak area of all the impurities was calculated. Method precision experiments were

conducted in six individual preparations of lacidipine tablet powder at a concentration

of 500 µg/ml spiked with each of the impurities at a concentration of 0.75 µg/ml and the

RSD for area percentage of all impurities was calculated. The intermediate method

precision was evaluated by a different analyst on different days using different instrument

of the same brand. Precision of assay method was evaluated by carrying out six

independent assays of lacidipine tablet at a concentration of 50 µg/ml level against a

qualified working standard. The intermediate precision of the assay method was

evaluated by a different analyst on different days using different instrument of the same

brand.

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5.5.11 Robustness

By deliberate change in experimental conditions the resolution between lacidipine

and all the impurities was evaluated. To study the effect of flow rate on the resolution,

0.02 units were used that changed the flow to 0.23 and 0.27 ml/min. The effect of pH on

the resolution of impurities was studied by varying ±0.2 pH units (at 4.3 and 4.7 buffer

pH). The effect of column temperature on the resolution was studied by studying at two

temperatures (35°C and 45°C). The effect of wavelength was also studied by two

different wavelengths of detection (238 and 242 nm). In all the changed conditions

mentioned above, the components of the mobile phase were held constant. Further the

robustness experiment related to specificity and the effect buffer evaluated by varying the

buffer concentration to 40 mM and 60 mM buffer solutions were prepared. The impact of

organic content is also studied by altering 2% of methanol in the mobile phase.

5.5.12 Solution Stability and Mobile Phase Stability

The solution stability was carried out by placing the method precision solution in

tightly capped amber colour volumetric flask at controlled room temperature for 48 hours.

The sample solutions were analysed at regular interval of 8 hours up to the study period

and the difference in the area percentage of all the impurities was calculated. The mobile

phase stability was also studied out by analyzing the freshly prepared system precision

solution in 8 hours intervals up to 48 hours and the resolutions between each impurity

peak were recorded.

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 Chapter 5

5.6 RESULTS AND DISCUSSION

5.6.1 Method development and optimization

The goal of this work was achieved by separating and quantitating six related

compounds in lacidipine Tablets and its active substances by UPLC. The blend

containing 500 µg/ml of lacidipine and 5 µg/ml of each impurity was prepared in the

methanol. All the impurities of lacidipine were separated using isocratic system on a

Waters Acquity BEH C18, (100 mm × 2.1 mm, 1.7 µ) column with pH 4.5, 50 mM

ammonium acetate buffer and methanol in the ratio 70:30 as the mobile phase. The

impurities were separated well with appropriate resolution. The concomitant quantitation

and analysis in these methods provide significant time-savings advantages in

pharmaceutical analytical laboratory as well as significant decreases in sample analysis

time and solvent consumption compared to the general and conventional HPLC methods.

The main analytical challenge during development of a new method was obtaining

adequate retention and resolution of the polar drug substance and its related substances

using reversed phase system along with a short run time. To achieve this end, various

brands of C18, C8 and Phenyl columns were experimented. As the sample matrix consists

of polar compounds as well as non polar compounds, the C18 column was considered as

the best choice. Waters Acquity BEH C18 (100 mm × 2.1 mm, 1.7 µ) column afforded the

best peak resolution and shortest retention times for the active and all related compounds.

Ion-pairing reagents were also examined but these attempts were unsuccessful. An

ammonium acetate buffer was found more suitable over phosphate buffer due to its

LCMS compatibility and which is useful to identify the mass of the unknown peaks at

later date during time product life cycle management. Higher ratios of methanol and

buffer in the mobile phase were studied and were found unsuitable due to co-elution of

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impurities. When methanol was replaced by acetonitrile, the orders of elution for the

peaks were altered along with a bad peak shape. In addition, impurity C was co eluted

with the principal peak. The sample solution was prepared using methanol to ensure

complete recovery of all the components and the solution stability also assured. For

achieving a good LOD and LOQ values for all impurities, 240 nm wavelength was

chosen and 500 µg/ml was kept as sample concentration for related substance.

In conclusion, an isocratic method for all the components with run time of 15

minutes was successfully developed for the quantitation of lacidipine and all its

impurities. Using the optimized conditions, lacidipine and its impurities were separated

well with a resolution of greater than 2 for the critical pair and the details are provided in

the table 5.1. Typical chromatogram of a blank, placebo and system suitability solution

are provided in the figures 5.3 and 5.4.

Table 5.1 System suitability report

Compound USP USP tailing No. of theoretical plates

resolution factor (USP tangent method)

Impurity E - 1.2 3760

Impurity D 5.7 1.1 5112

Impurity B 3.7 1.1 5488

Impurity A 3.1 1.1 6384

Impurity C 2.4 1.0 7193

Lacidipine 3.2 1.0 7813

Impurity F 2.5 1.0 9138

152
153
Chapter 5

Fig. 5.3 Typical UPLC chromatograms of Blank and Placebo

154
Chapter 5

Fig. 5.4 Typical UPLC chromatograms of System suitability

 Chapter 5

5.6.2 Specificity and Mass balance study

The specificity analysis revealed that UPLC method did not have interference

from the diluent and formulation excipients, as no peaks were found after the void

volume. Lacidipine was found to be stable under stress conditions such as humidity,

water, acid and base hydrolysis. Slight degradation was observed in thermal conditions

leading to the formation of Impurity B (Fig. 5.5). Significant amounts of Impurity B

and C were observed under oxidation, Sun and UV light stress conditions. Small amount

of impurity F was also found under oxidative stress and UV light exposure. Peak purity

results obtained from PDA confirm that the lacidipine and six impurities peak are

homogeneous and pure in all the stress samples. The mass balance results were

calculated for all the stressed samples and found to be more than 99.0% (Table 5.2). The

peak purity and assay of lacidipine were not altered by the presence of its degradation

products and thus confirms that this method is stability indicating.

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Table 5.2 Summary of forced degradation results.

Period of %Assay
% Total Mass balance*
Stress condition Study of
impurities
( hours) Lacidipine

Acid hydrolysis
1 0.7 98.6 99.3
(0.1 M HCl)

Base hydrolysis
1 0.7 99.1 99.8
(0.1 M NaOH)

Oxidation
1 0.5 99.2 99.7
(30% H2O2 )

Water hydrolysis 1 0.9 98.8 99.7

Thermal
72 1.8 97.6 99.4
(60 °C)

UV light
72 13.5 85.6 99.1
254 nm

Sunlight 72 16.2 83.1 99.3

Humidity
72 0.3 99.4 99.7
(50 °C/90% RH)
*: % assay + % impurities + % degradation products

156
157
Chapter 5

Fig. 5.5 Typical UPLC chromatogram of Thermal stressed sample

 Chapter 5

5.7 RESULTS OF METHOD VALIDATION EXPERIMENTS

5.7.1 Limit of detection (LOD) and limit of quantitation (LOQ)

The determined LOD and LOQ values for lacidipine and six impurities are

reported in table 5.3. The obtained values were low and for the practical reasons,

0.05 µg/ml and 0.15 µg/ml were considered as LOD and LOQ respectively. Precision

was carried out at LOQ level for lacidipine along with six impurities and results were

reported in table 5.3. The typical chromatograms of LOD and LOQ experiments were

presented in figures 5.6 and 5.7.

Table 5.3 LOD and LOQ data

Target Area Response

Concentration
(µg/ml) Lacidipine Impurity-A Impurity-B Impurity-C Impurity-D Impurity-E Impurity-F
0.05 3757 3029 1873 2318 2580 1884 222
0.1 6970 5979 3816 4782 5374 3725 932
0.2 13130 11648 7237 9095 10453 7423 1694
0.3 18876 17338 11008 13706 15315 10890 2637
0.4 26064 23457 14663 18332 20569 14679 3658
0.5 31505 28772 17869 22423 25653 17830 4726
1.0 61757 57104 35573 44648 50721 35165 9269
STDEV 387.6 190.0 159.0 166.5 137.7 204.8 106.4
SLOPE 58595.3 54927.1 30963.9 50908.8 49811.9 30298.4 9462.5
LOD (µg/ml) 0.02 0.01 0.02 0.01 0.01 0.02 0.04
LOQ (µg/ml) 0.07 0.03 0.05 0.03 0.03 0.07 0.11

158
159
Fig. 5.6 Typical UPLC chromatogram of LOD (0.05µg/mL)
Chapter 5
160
Chapter 5

Fig. 5.7 Typical UPLC chromatogram of LOQ (0.15µg/mL)

 Chapter 5

5.7.2 Linearity and Relative response factor (RRF)

Linear calibration plot for the related substances method was obtained over the

calibration ranges tested, i.e. 0.15 µg/ml (LOQ) to 6.0 µg/ml for impurities. The

correlation coefficient obtained was greater than 0.999 (Table 5.4) (Fig. 5.8). The result

shows that an excellent correlation existed between the peak area and the concentration of

all the impurities. The linearity of the detector response for assay method was also

determined for lacidipine over the calibration range of 25 to 75 µg/ml and found the value

to be 0.9996. Hence the method is found to be linear in those ranges.

Table 5.4 Summary of Linearity data

Area Response
S.No. % Level
Lacidipine Imp- A Imp- B Imp- C Imp- D Imp- E Imp- F
1 LOQ 6440 6317 4095 4720 5262 3866 1740
2 25 65088 59428 37355 46835 53148 38441 9928
3 50 127163 117128 73740 91116 104183 72859 19647
4 100 249848 230251 145108 180808 204875 147349 38060
5 120 310476 286273 180200 224501 254999 178829 47532
Slope (m) 61214.7 56760.4 32390.2 52885.5 51620.4 32062.5 14079.4
Intercept (C) -11.86 -73.50 -10.13 -283.20 -206.00 209.87 -82.49
Standard error of
4010.35 3715.67 2275.15 2886.86 3421.97 1308.90 724.41
slope and intercept
Regression
0.9992 0.9992 0.9993 0.9993 0.9992 0.9992 0.9990
coefficient

161
162
Fig. 5.8 Typical UPLC chromatogram of Linearity at 100% level
Chapter 5
 Chapter 5

5.7.3 Accuracy

The percentage recovery of lacidipine from a spiked placebo ranged from 99.3

to 100.6%. The percentage recovery of six impurities in lacidipine samples varied

from 99.8 to 106.4% and results are reported in table 5.5. The typical chromatograms

of unspiked sample and Accuracy sample at 100% and 120% were presented in

figures 5.9-5.11.

Table 5.5 Accuracy data

Amount
Recovery (%)
spiked
Lacidipine Imp- A Imp- B Imp- C Imp- D Imp- E Imp- F
µg/ml
0.15 99.3±1.8 106.2±1.0 102.7±2.7 106.4±3.2 103.5±1.9 100.2±3.9 99.8±2.9

0.25 99.9±1.2 104.2±1.5 101.3±2.1 104.2±2.6 99.9±2.3 101.8±2.1 101.2±2.6

0.75 100.2±0.8 104.4±1.7 102.9±2.1 99.9±2.0 102.2±2.6 101.5±0.4 101.3±1.2

6.0 100.6±0.7 103.2±1.2 102.0±1.8 99.8±0.7 100.8±0.7 101.5±0.5 101.6±0.8

163
164
Chapter 5

Fig. 5.9 Typical UPLC chromatogram of unspiked sample used in accuracy

165
Chapter 5

Fig. 5.10 Typical UPLC chromatogram of Accuracy at 100% level

166
Chapter 5

Fig. 5.11 Typical UPLC chromatogram of Accuracy at 120% level

 Chapter 5

5.7.4 Precision

The RSD of assay of lacidipine during the assay method repeatability study

was 0.68% and intermediate precision study was 0.91%. The maximum RSD for the area

of six impurities in the related substance method repeatability study was 2.8% (Table 5.6)

and intermediate precision study was 2.4% (Table 5.7). The overall RSD values suggest

excellent precision of the related substances and assay method (Table 5.8). The typical

chromatograms of method precision analysis are presented in figures 5.12 and 5.13.

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Table 5.6 Method precision data

Impurity-A Impurity-B Impurity-C

S.No. % % %
Peak area Peak area Peak area
w/w w/w w/w
1 251193 0.84 219062 1.36 192418 0.72
2 266587 0.89 225003 1.39 203900 0.76
3 262110 0.88 229143 1.42 199338 0.74
4 262546 0.88 220840 1.37 194489 0.73
5 250129 0.84 219952 1.36 193230 0.72
6 249795 0.84 218147 1.35 192163 0.72
Average 0.86 1.38 0.73
% RSD 2.8 1.9 2.2

Impurity-D Impurity-E Impurity-F

S.No. % % %
Peak area Peak area Peak area
w/w w/w w/w
1 207495 0.81 157521 0.99 38205 0.55
2 215680 0.84 158389 1.00 38052 0.54
3 215732 0.84 157018 0.99 38156 0.54
4 208539 0.81 158857 1.00 38125 0.54
5 207204 0.81 157976 1.00 37965 0.54
6 205403 0.80 157003 0.99 38120 0.54
Average 0.82 1.00 0.54
% RSD 2.1 0.6 0.8

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Table 5.7 Intermediate precision data

Impurity-A Impurity-B Impurity-C

S.No. % % %
Peak area Peak area Peak area
w/w w/w w/w
1 253919 0.86 218611 1.37 202913 0.77
2 261927 0.89 220571 1.39 200700 0.76
3 260181 0.88 221516 1.39 201532 0.76
4 260576 0.89 218164 1.37 201498 0.76
5 263921 0.90 216497 1.36 201037 0.76
6 255680 0.87 218542 1.37 201163 0.76
Average 0.88 1.38 0.76
% RSD 1.7 0.9 0.5

Impurity-D Impurity-E Impurity-F

S.No. % % %
Peak area Peak area Peak area
w/w w/w w/w
1 214651 0.85 156801 1.00 38561 0.56
2 215984 0.86 159640 1.02 38501 0.56
3 217871 0.86 154256 0.99 38513 0.56
4 207364 0.82 155680 1.00 38561 0.56
5 207710 0.82 155701 1.00 38620 0.56
6 206554 0.82 157088 1.01 38578 0.56
Average 0.84 1.00 0.56
% RSD 2.4 1.0 0.0

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Table 5.8 Compiled data of Method precision and Intermediate precision

Percentage w/w

S.No. Impurity-A Impurity-B Impurity-C Impurity-D Impurity-E Impurity-F

MP IP MP IP MP IP MP IP MP IP MP IP

1 0.84 0.86 1.36 1.37 0.72 0.77 0.81 0.85 0.99 1.00 0.55 0.56

2 0.89 0.89 1.39 1.39 0.76 0.76 0.84 0.86 1.00 1.02 0.54 0.56

3 0.88 0.88 1.42 1.39 0.74 0.76 0.84 0.86 0.99 0.99 0.54 0.56

4 0.88 0.89 1.37 1.37 0.73 0.76 0.81 0.82 1.00 1.00 0.54 0.56

5 0.84 0.90 1.36 1.36 0.72 0.76 0.81 0.82 1.00 1.00 0.54 0.56

6 0.84 0.87 1.35 1.37 0.72 0.76 0.80 0.82 0.99 1.01 0.54 0.56

Avg. 0.87 1.38 0.75 0.83 1.00 0.55

% RSD 2.5 1.4 2.6 2.5 0.9 1.8

170
171
Fig. 5.12 Typical UPLC chromatogram of sample Unspiked
Chapter 5
172
Fig. 5.13 Typical UPLC chromatogram of sample spiked with six impurities
Chapter 5
 Chapter 5

5.7.5 Robustness

In all the deliberate varied chromatographic conditions (flow rate, column

temperature, composition of organic solvent, etc.,), the resolution between all pairs of

compounds was greater than 2.0 and tailing factor for lacidipine and its impurities was

less than 1.2. The assay variability of lacidipine was within ±0.8%. The variability in the

estimation of lacidipine impurities in the method precision sample was within 5%.

Overlaid chromatogram of robustness studies was presented in figures 5.14 and 5.15.

5.7.6 Solution Stability and Mobile Phase Stability

The variability in the estimation of lacidipine impurities was within ±5% and

assay was within 1% during solution stability and mobile phase experiments. The results

from solution stability and mobile phase stability experiments confirmed that mobile

phase, standard and sample solutions are stable up to 48 hours.

173
174
Fig. 5.14 Typical UPLC chromatograms of Robustness study-Column oven temperature
Chapter 5
175
Chapter 5

Fig. 5.15 Typical UPLC chromatograms of Robustness study-Organic phase

 Chapter 5

5.8 CONCLUSION

A simple isocratic, reversed phase UPLC method was developed for quantitative

analysis of lacidipine and related substances in a pharmaceutical dosage form that utilizes

a previously unreported set of conditions like gradient and mobile phases, and to effect

the separation without the use of an ion-pair reagent in the mobile phase. The method is

completely validated and was found to be precise, specific, sensitive, linear accurate and

robust. This method can be used to quantitate the amount of related substances for release

and stability of the drug substance and the drug product. The method can also be

successfully used for the assay of lacidipine in the drug substance and the drug products.

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REFERENCES

1. C.R. Lee and H.M. Bryson, “Drugs”, 48, 274, 1994.

2. P. L. McCormack and A.J. Wagstaff, “Drugs”, 63, 2327-2356, .2003.

3. G. Ramesh, Y.V. Vishnu, P.C. Reddy, Y.S. Kumar and Y.M. Rao “Analytica

Chimica Acta”, 632, 278-283, 2009.

4. S. Braggio, S. Sartori, F. Angeri and M. Pellegatti, “J. Chromatogr. B”, 669,

383-389, 1995.

5. J. Tang, R. Zhu, R. Zhao, G. Cheng and W. Peng, “J. Pharm. Biomed. Anal.” 47,

923-928, 2008.

6. M. Pellegatti, S. Braggio, S. Sartori, F. Franceschetti and G.F. Bolelli, “J.

Chromatogr., Bio Medical Anal.”, 573, 105-111, 1992.

7. V.R. Kharat, K.K. Verma and J.D. Dhake, “J. Pharm. Biomed. Anal.”, 28, 789-

793, 2002.

8. A.B. Baranda, R.M. Jimenez and R.M. Alonso, “J. Chromatogr.”, 1031, 275-

280, 2004.

9. S.A. Özkan, “Pharmazie”, 57(7), 503, 2002.

10. J.A. Squella, A.E. Iribarren, J.C. Sturm and L.J. Núñez Vergara, “J.AOAC Int.”,

82(5), 1077, 1999.

11. P. Rossato, M. Scandola and P. Grossi, “J. Chromatogr. A”, 647, 155-166,

1993.

12. P.D. Filippis, E. Bovina, L.D. Ros, J. Fiori and V. Cavrini, “J. Pharm. Biomed.

Anal.”, 27, 803-812, 2002.

13. F. Belal, A. Elbrashy, M. Eid and J.J. Nasar, “Chromatographia”, 69, 1201-

1209, 2009.

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14. M.J. Nozal, J.L. Bernal, J.J. Jimenez, M.T. Martin and F.J. Diez, “J.

Chromatogr. A”, 1024, 115-122, 2004.

15. British Pharmacopeia, Published by the stationery office on behalf of the

MHRA, London, Vol. 2, 1228-1230 and 2828-2829, 2010.

16. ICH, “Stability Testing of New Drug Substances and Products” IFPMA,

Geneva, (Q1AR), 2000.

17. ICH, “Draft Guidelines on Validation of Analytical Procedures, Definitions

and Terminology”, Vol. 60. Federal Register: IFPMA, Switzerland, 11260,

1995.

178
 Chapter 6

Development and Validation of a Dissolution

Method for novel fixed dose combination of

Etodolac and Propranolol hydrochloride Tablets

by RP-HPLC
 Chapter 6

6.1 INTRODUCTION

Etodolac (ET), (±) 1, 8-diethyl-1, 3, 4, 9-tetrahydropyrano-[3, 4-b]-indole-1-

acetic acid (Fig. 6.1), belongs to pyranocarboxylic acid group of nonsteroidal anti-

inflammatory drugs (NSAIDs), primarily used in the therapy of rheumatic diseases and

postoperative pain. The mechanism of action of etodolac is mainly prostaglandin

synthetase enzyme inhibition. Propranolol hydrochloride (PH), (RS)-1-(isopropylamino)-

3-(1-naphthyloxy)propan-2-ol hydrochloride (Fig. 6.2), is a competitive non-selective

β-adrenoreceptor antagonist, it has been used widely to treat various cardiovascular

disorders like hypertension, angina pectoris, cardiac arrhythmias, prophylaxis of

secondary myocardial infarction, migraine, essential tremor, hypertrophic subaortic

stenosis and pheochromocytoma.

The combination of etodolac and propranolol hydrochloride remains a major

objective of research in treating potential diseases like cachexia in cancer patients[1]

improving immune competence and reducing the risk of tumor metastasis[2]. The

serendipitous therapeutic effect of this combination results in simultaneous decrease in

the synthesis of prostaglandins, the endocrine agents which mediate pain and

inflammation and also suppresses excess catecholamine release in cancer patients. As of

now, the combination product of ET and PH is not available globally; several lines of

evidence suggest that the development of this combination would be beneficial for the

above said diseases. The fixed dose combination of ET and PH tablet formulation was

under development at Mother Theresa Post Graduate & Research Institute of Health

Sciences. To understand the drug release characteristic of both drugs in the novel FDC, a

suitable dissolution methodology was found to be a primary need.

179
 Chapter 6

Nowadays in the global regulatory scenario the dissolution development is

considered as a very important part of the product development. Dissolution studies data

are effectively utilized and reviewed by the regulatory agency as a part of the Quality by

Design (QbD) approach. Drug absorption depends on the release of the active ingredient

from the formulation, its dissolution under physiological conditions, and the permeability

across the gastrointestinal tract. Because of the critical nature of the first two of these

steps, in vitro dissolution may be relevant to the prediction of in vivo performance.[3, 4] It

is therefore widely accepted that dissolution testing is a very important tool in the

pharmaceutical industry for providing valuable information to both formulation teams

that design new product and quality control scientists to ensure a lot to lot quality and

consistency within pre-defined specification criteria.[5, 6]

6.2 DRUG PROFILE

6.2.1 Etodolac

Etodolac, (±) 1, 8-diethyl-1, 3, 4, 9-tetrahydropyrano-[3, 4-b]-indole-1-acetic acid,

Etodolac is a White crystalline solid and insoluble in water but soluble in alcohols,

chloroform, dimethyl sulfoxide, and aqueous polyethylene glycol. It has a pKa of 4.65

and an n-octanol water partition coefficient of 11.4 at pH 7.4. It has an empirical formula

of C17H21NO3 and a molecular weight of 287.37

180
 Chapter 6

CH 2CH 3
H
N CH 2CH 3
O

O
HO

Fig. 6.1 Structural formula of Etodolac

6.2.2 Propranolol hydrochloride

Propranolol hydrochloride, (RS)-1-(isopropylamino)-3-(1-naphthyloxy)propan-2-

ol Propranolol hydrochloride is a stable, white and crystalline solid. It is readily soluble in

water and ethanol. It has a pKa value of 9.45. It has an empirical formula of

C16H21NO2.HCl and molecular weight of 295.81.

Cl HO
H O

HN

H3C CH 3

Fig. 6.2 Structural formula of Propranolol hydrochloride

181
 Chapter 6

6.3 LITERATURE REVIEW

Gil et al.,[10] have developed and optimized a novel oral controlled delivery

system for propranolol hydrochloride. The in vitro dissolution profiles of sustained-

release matrix tablets of racemic form were determined and compared with the United

States Pharmacopeia tolerance specifications for Propranolol Hydrochloride Extended-

Release Capsules. They have used the typical Dissolution method enumerated in United

States Pharmacopeia for their studies.

Akbari et al.,[11] have developed buccoadhesive propranolol hydrochloride tablet

formulations for local and systemic delivery of therapeutic peptides and other drugs that

are subjected to first-pass metabolism and are unstable within the rest of the

gastrointestinal tract. The effect of lactose and dicalcium phosphate on dissolution rate,

kinetic of release and adhesion force of buccal adhesive tablets of propranolol HCl were

evaluated. For these studies they have used the method stated in United States

pharmacopeia which utilizes UV Spectroscopy.

Dey et al.,[12] have reported the dissolution and bioavailability of etodolac from

capsules exposed to high relative humidity and temperature that were compared to those

from capsules and evaluated using a USP basket apparatus at 100 rpm with 900 ml pH 7.5

phosphate buffer (0.05 M) at 37°C as mentioned in United States Pharmacopeia.

Özkan et al.,[13] have examined and reported the release of etodolac from various

molecular weight fractions of polyethylene glycol (PEG) solid dispersions. The release

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 Chapter 6

rate of etodolac from the resulting complexes was determined from dissolution studies by

use of USP dissolution apparatus 2 (paddle method). The dissolution rate of etodolac is

increased in all of the solid dispersion systems compared to that of the pure drug and

physical mixtures. The solid dispersion compound prepared in the molar ratio of 1:5 by

the solvent method was found to have the fastest dissolution profile.

Dey et al.,[14] have reported of a bioavailability study conducted in order to

evaluate the relationship between in vitro dissolution and absorption. The computer

program NONLIN was used to model both in vitro drug release and in viva plasma

concentration-time profiles. Based on the results of the pilot study and the kinetic

modeling, optimum target in vitro release rates were identified. Dosage forms exhibiting

these in vitro release profiles were evaluated in a bioavailability and dose proportionality

study over the range of 200-600 mg daily doses. The SR formulations were bioequivalent

to their respective immediate release doses and were dose-proportional. The in vivo

performance was accurately predicted by in vitro dissolution data.

Dongre, et al.,[15] developed and validated a reverse phase liquid chromatographic

(RP-LC) method for the simultaneous determination of etodolac and acetaminophen in

tablet dosage form. The chromatographic separation was achieved on a BDS Hypersil

C18, 100 mm x 4.6 mm, 5 µm column at a detector wavelength of 274 nm using an

isocratic mobile phase consisting of a mixture of 0.05% aqueous orthophosphoric acid

and acetonitrile in the ratio of 50:50 (v/v) at a flow rate of 1.0 ml/min. The retention times

for etodolac and acetaminophen were found to be 1.32 and 4.24 minutes respectively.

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Lee et al.,[16] developed and validated a high performance liquid chromatography

(HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry

(ESI-MS/MS) detection for the determination of etodolac in human plasma, using

indomethacin as an internal standard (IS). Chromatographic separation was performed

isocratically using a Capcellpak MGII C18 column with 65% acetonitrile and 35% water

containing 10 mM ammonium formate (adjusted to pH 3.5 with formic acid). Acquisition

was performed in multiple reactions monitoring (MRM) mode by monitoring the

transitions: m/z 287.99 > 172.23 for etodolac and m/z 357.92 > 139.01 for IS.

The method was validated to determine its selectivity, linearity, sensitivity, precision,

accuracy, recovery and stability. The devised method provides an accurate, precise and

sensitive tool for determining etodolac levels in plasma.

Ververs et al.,[17] reported of a developed analytical method for the simulataneous

determination of propranolol, diltiazem and metabolites of diltiazem in human plasma by

liquid chromatography using a Nucleosil C18 column and UV detection at 238 nm and

at 295 nm. The mobile phase consisted of acetonitrile- methanol- ammonium chloride

(0.04 M)-triethylamine (24:40:36:0.08, v/v). After the four mobile-phase components

were mixed, phosphoric acid (85%, w/w) was used to adjust the pH 6.9. The flow rate

was 1.0 ml/min and imipramine used as internal standard.

Partani et al.,[18] developed and validated a highly selective assay for the

simultaneous determination of conjugated and unconjugated propranolol and its

equipotent hydroxyl metabolite, 4- hydroxy propranolol, in human plasma. The analytes

were simultaneously extracted from 0.3 ml of human plasma using solid phase

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extraction and detected in positive ion mode by tandem mass spectrometry with a turbo

ion spray interface. Deuterium-labeled propranolol and 4-hydroxy propranolol,

propranolol-d7 and 4-hydroxy propranolol-d7 were used as internal standards.

The extraction recoveries were >96 and >64% on an average for propranolol and 4-

hydroxy propranolol, respectively.

6.4 SCOPE OF THE STUDY

The combination tablets were being developed in three dosage strengths, 800/20,

800/40 and 800/80 mg of ET/PH[7]. Analytical methods reported in United States

Pharmacopeia, British Pharmacopeia and other published literatures[8-14], mainly state

about the dissolution method for the individual dosage form of PH and ET. Several LC

and LC-MS methods were published for the determination of both ET and PH

individually, or in combination with other drug substances.[15-18]

Till date as per the best of our knowledge, no analytical method is published for

the drug release (dissolution) study of the novel FDC of ET and PH. Hence it was found

desirable to develop a specific and precise dissolution methodology employing LC

technique, which could be applied for the product development laboratories and also to

ensure the quality of the product in the quality control laboratories. Development,

validation and application of the proposed method for the drug release study of the novel

FDC of ET and PH are described in the following sections.

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6.5 EXPERIMENTAL

6.5.1 Materials

API of ET (100.23 % purity) and PH (99.66 % purity), were gifted from Takeida

Pharmaceuticals, Puducherry, India and tablets and placebo of the novel FDC of ET and

PH were prepared in-house. HPLC grade acetonitrile and methanol were procured from

Merck, Mumbai, India. Buffer components potassium phosphate and sodium hydroxide

were of analytical grade purchased from Merck Mumbai, India. High purity deionised

water was obtained from Millipore; Milli-Q (Bedford, MA, USA) water purification

system was used to prepare the mobile phase and diluent. All dissolution media used in

this study were degassed under vacuum prior to use and stored at 37oC to minimize re-

aeration.

6.5.2 Instrumentation

The HPLC equipment used was Alliance 2695 System (Waters, Milford, MA,

USA), comprising a quaternary solvent delivery module, online degasser, column

thermostat, autosampler, dual wavelength UV detector (2487) and photodiode array

detector (2996). Chromatographic parameters such as peak areas, retention times,

theoretical plates, etc. were calculated using the Empower software, version 2.6.

For all dissolution experiments, LABINDIA DS 8000 dissolution tester, (Mumbai,

India), equipped with a programmable auto sampler was used. For preparation of

standards and mobile phase Sartorius ME 235S analytical balance (Germany) and

EUTECH pH 510 pH meter (Singapore) were used.

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6.5.3 Chromatographic conditions

The chromatographic separations were performed using XTerra RP18, 150 mm X

4.6 mm, 5 µ analytical column, maintained at 25°C, eluted with mobile phase at the flow

rate of 1.0 ml/min. The mobile phase consisted of 40:60 (v/v) acetonitrile / phosphate

buffer pH 5.5 (0.05 M potassium phosphate). The mobile phase was filtered under

vacuum through 0.45 µm nylon membrane filter (Millipore) and degassed ultrasonically

for 30 minutes prior to use. Measurements were made with injection volume 10.0 µl and

ultraviolet (UV) detection at 292 nm. The specificity samples were analyzed using a

photo-diode array (PDA) detector covering the range of 205–350 nm. The total

chromatographic run time was 7.0 minutes.

6.5.4 Dissolution test condition

A calibrated dissolution tester with USP apparatus (I) was used at 100 rpm and

bath temperature maintained at 37 ± 0.5°C. 1000 ml of freshly prepared and degassed

phosphate buffer pH 6.8 (0.05 M) solution was used as dissolution medium. Sample

aliquots of 10 ml were taken, filtered through a 0.45 µm nylon syringe filter. The first

two ml of sample was discarded prior to collecting the sample for analysis. For

dissolution profile testing, samples were taken at 5, 10, 15, 30, 45 and 60 minutes time

points.

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6.5.5 Preparation of standard solutions

ET (8000 µg/ml) standard stock solution was prepared by transferring

about 800 mg of ET working standard to 100 ml volumetric flask, 20 ml of methanol was

added and sonicated for 5 minutes. Then the volume was made up with methanol.

PH (200 µg/ml) standard stock solution was prepared by transferring about 20 mg

of PH working standard to 100 ml volumetric flask, 2 ml of methanol was added and

sonicated for 2 minutes. Then the volume was made up with dissolution medium.

The ET and PH standard stock solutions were combined and diluted with

dissolution medium to prepare standard solutions at concentrations of approximately

800/20, 800/40 and 800/80 µg/ml for 800/20, 800/40 and 800/80 mg strength

respectively.

6.6 METHOD VALIDATION

The in vitro dissolution method developed was validated according to current

guidelines[19, 20]. Specificity, linearity, accuracy, precision and robustness were evaluated.

ET/PH stabilities using test conditions were also evaluated.

6.6.1 Specificity

Specificity was evaluated in placebo samples. The placebo samples consisted of

all the excipients without the active substances. The concentration of excipients in test

sample was based on in-house preparation. The placebo sample was transferred to

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separate vessels (n = 3) filled with 1000 ml of dissolution medium maintained at

37±0.5°C and stirred for 1 hour at 100 rpm using basket (USP Apparatus 1). Aliquots

were collected and analyzed.

6.6.2. Linearity

Linearity of the method was studied through the injection of both ET and PH at

the concentration range of 80–960 µg/ml and 2–120 µg/ml, respectively, with six different

concentration levels in each curve. Dilutions were performed using dissolution medium

from standard stock solution containing 8000 µg/ml of ET and 200 µg/ml of PH. The

peak area versus concentration data were treated by least-squares linear regression

analysis. The correlation coefficient, slope and Y-intercept of the calibration curve were

calculated.

6.6.3. Accuracy

The accuracy of the method was evaluated through the recovery test of known

amounts of ET and PH working standards added to the placebo. Weighed and transferred

accurately 80, 800 and 960 mg of ET along with 10, 80 and 120 mg of PH respectively in

the dissolution medium containing composite placebo mixture. Samples were stirred

at 100 rpm for 1 hour. After that aliquots of each vessel were collected and analyzed.

These studies were performed in triplicate for each level.

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6.6.4 Precision

The precision of the method was demonstrated by intraday and inter day variation

studies. The intra-day study was carried out by injecting six replicate injections of

standard solution and six sample preparations. In the inter day study, six samples were

analyzed by different analyst on different day using different instrument and RSD was

calculated.

6.6.5. Stability studies

Stability of both drugs in the dissolution medium was evaluated using standard

and samples. The standard and sample solution obtained from precision study were

injected at appropriate time intervals up to 48 hours of the respective solution stored at

both ambient and refrigeration conditions. The cumulative RSD of the response was

calculated.

6.6.6. Robustness

The robustness of the method was evaluated by making deliberate minor variation

in the following method parameters like flow rate (±10%), organic solvent in the mobile

phase (±2%), pH of the mobile phase buffer (± 0.2 units) and wavelength (±2 nm).

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6.7 RESULTS AND DISCUSSION

6.7.1 Development and optimization of the chromatographic condition

Spectrophotometry was unsuitable for the combination product owing to the lack

of specificity; moreover, the absorbance of both the drugs interferes with each other

(Fig. 6.3). Therefore, an RPLC method was developed for the simultaneous determination

of ET and PH in the dissolution sample. Interferences from both the analytes were

separated using LC and analysis can still be achieved with UV detection. For HPLC

analysis, ET is strongly retained on reversed-phase columns since it is relatively non-

polar with pKa 4.65. PH is a polar molecule with pKa 9.45. Thus, it is poorly retained on

most of the reversed-phase HPLC columns. Therefore, it is difficult to elute both

compounds efficiently using a simple isocratic system. Our major goal was to develop a

simple, rapid and efficient chromatographic method for two molecules with very different

chemical selectivity.

Trials were performed using C18 stationary phase (4.6 mm, 5 µm) with varying

column lengths from 250 to 150 mm and various brands (Thermo Hypersil C18, YMC-

PackPro C18, Phenomenex C18 gemini, Waters XTerra RP18). Chromatographic

separation using XTerra RP 18, 250 x 4.6 mm, 5 µm column gave higher retention

time 3.6 minutes for PH and 10.6 minutes for ET. The shorter column length (150 mm x

4.6 mm, 5 µm) of same brand has reduced the retention time to 2.3 minutes for PH

and 4.8 minutes for ET with better resolution. In order to further optimize the method,

different trials were carried out with varying acetonitrile composition, buffer pH and the

results were summarized in table 6.1. The experimental data show that pH 3.5 and pH 4.5

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were unsuitable for this combination as the PH elutes in void volume and ET elutes more

than 5 minutes. The trial using phosphate buffer pH 5.5 with acetonitrile in the ratio

of 60:40 (v/v) gave desirable chromatographic separation with better peak shape, tailing

factor, plate count and resolution. As the UV spectrum showed good response at 292 nm

for both components, this wavelength was found suitable for simultaneous determination

of both components.

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Fig. 6.3 UV spectrum of Etodolac and Propranolol hydrochloride

 Chapter 6

Table 6.1 The outcome of each trial on various mobile phase compositions

Propranolo Etodolac Tailing Plate Count

S.No Mobile Phase Ratio l HCl (RT in PH/ET PH/ET
(RT in min) min)
1 pH 3.5 buffer/ ACN 50:50 1.849 6.072 1.1/1.1 4852/7226

2 pH 3.5 buffer/ ACN 60:40 2.362 13.823 1.1/1.0 6075/10137

3 pH 4.5 buffer/ ACN 50:50 1.889 5.184 1.2/0.9 5135/5500

4 pH 4.5 buffer/ ACN 60:40 2.437 10.892 1.2/0.8 6088/8494

5 pH 4.5 buffer/ ACN 55:45 2.096 7.175 1.2/0.9 5579/6794

6 pH 5.5 buffer/ ACN 50:50 1.858 3.080 1.2/1.0 4891/6131

7 pH 5.5 buffer/ ACN 60:40 2.389 4.843 1.2/0.9 5808/7638

8 pH 5.5 buffer/ ACN 55:45 2.051 3.701 1.2/1.0 5685/6878

9 pH 6.5 buffer/ ACN 50:50 1.845 2.274 1.2/1.2 4861/6298

10 pH 6.5 buffer/ ACN 60:40 2.369 2.657 1.2/1.2 5239/6108

11 pH 6.5 buffer/ ACN 70:30 4.301 5.981 1.3/1.3 6184/7121

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6.7.2 Evaluation and optimization of the dissolution test

The dissolution test was optimized in terms of dissolution medium, apparatus type

and rotation speed.

6.7.2.1 Study of the dissolution medium

The dissolution media listed in the USP dissolution methods for single entity ET

and PH were phosphate buffer (pH 6.8; 0.05 M), and dilute HCl (pH 1.2) respectively.

Hence solubility studies for both active ingredients in several media in the pH range 1.2

to 6.8 were investigated (Table 6.2) to establish a common dissolution medium. The

dissolution media at pH 1.2 and pH 4.5 were not suitable for the combination tablets as

the ET solubility is low. Phosphate buffer medium pH 6.8 was found to be suitable as

both drugs show good solubility and sink conditions were met. As the absorption happens

from GI tract, therefore nature of the medium should be aqueous-based and pH in the

range of 5-7. The reason is that it represents pH of intestinal environment where most of

the absorption of the drug occurs. Moreover the Tmax of ET and PH were 1 to 4 hours

hence pH 6.8 buffer medium was finalized keeping in mind that this may indicate the bio

relevancy.

Additional experiments were carried out in order to evaluate the stability of ET

and PH in the selected medium. Accurately weighed amounts of both the API were

dissolved in the phosphate buffer at final mass concentrations of 800 mg/l of ET

and 20, 40 and 80 mg/l of PH. The resulting solutions were incubated in a water bath

at 37°C under continuous stirring at 100 rpm. Sampling and HPLC analysis was carried

out at time intervals t = 0, 15, 30, 60 and 120 min. No instability of the analytes were

observed, since the RSD of the obtained peak areas were <1.2% in all cases.

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6.7.2.2 Study of the dissolution condition

Using phosphate buffer as the dissolution medium, experiments were carried out

to compare the performance of paddle versus basket agitation, rotating at 50 rpm

and 100 rpm respectively. The experimental results yielded similar profile in both the

conditions. However basket type was preferred because of the possibility of capsule

formulation development in future. The effect of the rotation speed on the dissolution

profile of ET and PH containing tablets was examined at 50, 75 and 100 rpm. Decrease in

the rotation speed resulted in incomplete release of ET in 60 minutes. The percent release

of three strengths was analysed using basket at 100 rpm and the results were presented in

figure 5.4.

Table 6.2 Solubility Studies of Etodolac and Propranolol hydrochloride in Various

Buffers

pH Medium Etodolac Propranolol HCl

(mg/ml) (mg/ml)
pH 1.2 0.1 N HCl 0.058 17.325

pH 4.5 0.05M Ammonium 0.053 0.140

acetate
pH 6.8 0.05M Potassium 2.884 33.130
dihydrogen phosphate

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Fig. 6.4 Dissolution profile data
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6.8 RESULTS OF METHOD VALIDATION EXPERIMENTS

6.8.1 Specificity

No interfering peaks were observed at the retention time of the principal peak of

both the analytes, due to placebo mixture and dissolution medium and the typical

chromatograms are presented in figures 6.5 and 6.6.

Fig. 6.5 Typical HPLC chromatogram of Blank (Dissolution medium) and Placebo

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Fig. 6.6 Typical HPLC chromatogram of Analytes
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6.8.2. Linearity

Linearity of the method was confirmed by preparing ET and PH in the analytical

range of 80 to 960 µg/ml and 2 to 120 µg/ml respectively. Excellent correlation between

analyte peak area and concentration of the drug was observed with r ≥0.999 for all

standard curves with the slope of 4416 and 5482 and Y-intercept at +20318 and -400.6 for

ET and PH respectively. The corresponding data, calibration curve and chromatograms

were reported in tables 6.3-6.4 and figures 6.7-6.11

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Table 6.3 Linearity data of Etodolac

S.No. Theoretical conc.(µg/ml) Actual conc. (µg/ml) Peak area

1 80 80.02 369714
2 160 160.04 729947
3 400 400.11 1789587
4 640 640.18 2838076
5 800 800.22 3574942
6 960 960.26 4250005
Correlation coefficient 0.99997

Fig. 6.7 Calibration curve of Etodolac

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Table 6.4 Linearity data of Propranolol hydrochloride

S.No. Theoretical conc.(µg/ml) Actual conc. (µg/ml) Peak area

1 2 1.97 9120
2 10 9.85 55801
3 20 19.7 107419
4 40 39.4 214340
5 80 78.8 432328
6 120 118.2 647432
Correlation coefficient 0.999986

Fig. 6.8 Calibration curve of Propranolol hydrochloride

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Fig. 6.9 Typical HPLC chromatogram of Linearity solution at 10% level

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Fig. 6.10 Typical HPLC chromatogram of Linearity solution at 100% level

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Fig. 6.11 Typical HPLC chromatogram of Linearity solution at 150% level
Chapter 6
 Chapter 6

6.8.3 Accuracy

The accuracy was demonstrated by the recovery of known amounts of ET and PH

to the dissolution vessels. Recoveries from 95.0 to 105.0% of the added amounts are

recommended in dissolution tests. Individual recoveries of ET and PH ranged from 99.3

to 101.7% and 96.4 to 101.1% respectively [Tables 6.5 and 6.6], corroborating the

accuracy of the method. The typical chromatogram of Accuracy at 10% and 100 % level

is presented in figures 6.12 and 6.13.

Fig. 6.12 Typical HPLC chromatogram of Accuracy at 10 % level

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Fig. 6.13: Typical HPLC chromatogram of Accuracy at 100 % level
Chapter 6
 Chapter 6

Table 6.5 Accuracy results for Etodolac

%* Amount Amount % Recovery Average %RSD

Level Added Recovered %
(µg) (µg) Recovery
1 81.25 82.65 101.7
10% 2 81.76 81.51 99.7 100.79 1.02
3 83.92 84.72 101.0
1 799.54 796.91 99.7
100% 2 801.51 805.37 100.5 100.58 1.0

3 801.73 814.48 101.6

1 959.32 958.31 99.9
120% 2 960.84 953.69 99.3 99.70 0.4
3 960.53 960.00 99.9

Table 6.6 Accuracy results for Propranolol hydrochloride

% Amount Amount % Recovery Average %RSD

Level Added Recovered %
(µg) (µg) Recovery
1 10.85 10.46 96.4
50% 2 10.57 10.31 97.6 97.96 1.82
3 10.39 10.38 99.9
1 80.57 80.47 99.9
100% 2 80.26 80.22 99.9 100.27 0.6

3 79.82 80.62 101.0

1 120.84 119.83 99.2
150% 2 119.53 120.88 101.1 100.00 1.0
3 121.66 121.30 99.7

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6.8.4 Precision

System precision: The system precision of the method was evaluated by

performing six replicate injections of the combined standard solution. The peak area RSD

was ≤ 0.9% for ET and ≤ 0.8% for PH. This reproducibility was considered acceptable.

Method and intermediate precision: The intra day precision were calculated by

injecting six samples of all strengths. The RSD of the area for both the analytes was

≤ 3.3%. The same parameter was performed in a different day by a different person to

evaluate inter day precision. The RSD of the area for both the analyte was ≤ 3.0%.

Results for the intra day and inter day precision were summarized in table 6.7,

demonstrating repeatability and reproducibility of the developed method.

Table 6.7 Precision data for Etodolac and Propranolol hydrochloride

%R.S.D
Strength Etodolac Propranolol HCl
#
*MP IP MP IP
800/20 2.2 1.8 3.3 3.0
800/40 2.8 3.0 2.9 2.8
800/80 1.9 1.0 1.9 2.0
*MP – Method Precision and #IP – Intermediate Precision

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6.8.5 Stability studies

The standard and sample solution were monitored for stability up to 48 hours and

found to be stable, when stored at ambient as well as refrigeration condition. The

cumulative RSD of the ET standard and sample solution was found to be ≤ 0.9%

and ≤ 1.2% respectively, at both the storage conditions. The cumulative RSD of the PH

standard and sample solution were found to be ≤ 0.9% and ≤ 2.2% respectively, at both

the storage conditions.

6.8.6. Robustness

To ensure the insensitivity of the HPLC method to minor changes in the

experimental conditions it is important to demonstrate robustness of the method. None of

the alterations caused significant change in resolution between ET and PH, peak area

RSD, USP tailing factor, peak width or theoretical plates and percentage recovery

(Table 6.8).

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Table 6.8 Robustness study of the HPLC assay

Chromatographic parameters Percent recovery (±S.D.)

ET PH

Optimal conditionsa 99.3 (±0.5) 100.5 (± 0.3)

Variation of the mobile phase flow rate

Flow rate = 1.1 ml/min 98.6 (±0.6) 99.6 (±0.6)

Flow rate = 0.9 ml/min 100.2 (±0.8) 100.6 (±0.7)

Variation of the ACN:Buffer ratio

40.8:59.2 100.5 (±0.9) 100.2 (±0.5)

39.2:60.8 98.3 (±0.6) 99.0 (±0.5)

Variation of the mobile phase pH

pH 5.3 99.3 (±1.0) 98.8 (±0.8)

pH 5.7 98.2 (±0.5) 99.1 (±0.7)

Variation of wavelength

294 nm 97.8 (±0.5) 98.5 (±1.1)

290 nm 99.6 (±0.9) 99.6 (±0.3)

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6.9 CONCLUSION

A analytical method had been developed and validated for etodolac and

propranolol hydrochloride, a fixed dose combination tablet which determines the

dissolution profile of both components simultaneously using phosphate buffer pH 6.8

and USP Apparatus 1 (basket) at 100 rpm. This circumvents the problem previously

described with the USP single entity dissolution media, and provides the optimum

dissolution conditions for this product. Also, an HPLC method was developed to measure

concomitantly, etodolac and propranolol hydrochloride in the dissolution samples, thus

eliminating the need for separate HPLC methods for the etodolac and propranolol

hydrochloride components.

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REFERENCES

1. G.S. Bhattacharyya, P.K. Julka, S. Bondarde, R. Naik, A. Ranade, N. Bascomb and

N. Rao, “J. Clin. Oncol.”, 28, e18059, 2010.

2. M. Benish, I. Bartal, Y. Goldfarb, B. Levi, R. Avraham, A. Raz, and S.B. Eliyahu,

“Ann. Surg. Oncol.”, 15, 2042-2052, 2008.

3. F. Gudrun, “Drug Inf. J.”, 35, 865-874, 2001.

4. P.D. Tzanavaras, A. Verdoukas and T. Balloma, “J. Pharm. Biomed. Anal.”, 41,

437-441, 2006.

5. Food and Drug Administration, “Guidance for Industry. Dissolution Testing of

Immediate Release Solid Oral Dosage Forms”, US Department of Health and

Human Services/Food and Drug Administration/Center for Drug Evaluation and

Research, Rockville, MD, 1997.

6. M. Siewert, J. Dressman, C.K. Brown and V.P. Shah, “AAPS Pharm. Sci.

Technol.”, 4, Article 7, 2003.

7. http://www.clinicaltrial.gov/ct2/show/NCT00888797

8. US Pharmacopoeia, 32nd ed., NF-27, 2, 2335-2337 and 3, 3426-3432, 2009.

9. British Pharmacopoeia, 3, 2564-2566, 2007.

10. E.C. Gil , A.I. Colarte , B. Bataille, J.L. Pedraz, F. Rodriguez and J. Heinamaki,

“Inter. J. Pharmaceutics”, 317, 32-39, 2006.

11. J. Akbari, A. Nokhodchi, D. Farid, M. Adrangui, M.R. Siahi Shadbad and M. Saeedi,

“Il Farmaco”, 59, 155-161, 2004.

12. M. Dey, R. Enever, M. Kraml, D.G. Prue1, D. Smith and R. Weierstall, “Pharm.

Research”, 10, 1295-1300, 1993.

13. Y. Özkan, N. Doganay, N. Dikmen and A. Isimer, “Il Farmaco”, 55, 433-438, 2000.

213
 Chapter 6

14. M. Dey, R. Enever, M. Marino, J. Michelucci, D. Smith, R. Warner and R.

Weierstall, “Inter. J. Pharmaceutics”, 49, 121-128, 1989.

15. V.G. Dongre, S.B. Shah, G.S. Bayes, M. Phadke and V.K. Jadhav,

“Chromatographia”, 69, 1019-1023, 2009.

16. H.S. Lee, I.M. Kang, H.W. Lee, J.H. Seo, J.H. Ryu, S.J. Choi, M.J. Lee, S.Y. Jeong,

Y.W. Cho and K.T. Lee, “J. Chromatogr. B”, 863, 158-162, 2008.

17. F.F.T. Ververs, H.G. Schaefer, J.F. Lefevre, L.M. Lopez and H. Derendorf, “J.

Pharm. Biomed. Anal.”, 8, 535-539, 1990.

18. P. Partani, Y. Modhave, S. Gurule, A. Khuroo and T. Monif, “J. Pharm. Biomed.

Anal.”, 50, 966-976, 2009.

19. ICH, “Validation of Analytical Procedures: Text and Methodology”, Geneva,

(Q2R1), 2005.

20. US Pharmacopoeia, 32nd ed., NF-27 <1092>, “The Dissolution Procedure:

Development and Validation”, US Pharmacopoeial Convention, Rockville, MD,

2009.

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