ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF NEWLY SYNTHESIZED ESTER

PRODRUGS OF ACECLOFENAC Thesis submitted to Bhagwant University, Ajmer

For the award of the degree of

DOCTOR OF PHILOSOPHY

IN

PHARMACEUTICAL CHEMISTRY

BY:

NITI BHARDWAJEnrollment No. BU/Ph.D/Regn.No./PH/276

Under the supervision of

DR. ASIF HUSAIN

Department of Pharmaceutical Chemistry

Faculty of Pharmacy

Jamia Hamdard (Hamdard University)

New Delhi-110062

September 8th, 2015

CERTIFICATE

This is to certify that the work embodied in this thesis entitled “Analytical Method

Development and Validation of newly synthesized ester prodrugs of Aceclofenac”,

has been carried out under the supervision of Dr. Asif Husain, Department of

Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard, New-Delhi, 110062,

by Ms. Niti Bhardwaj, in fulfillment of the requirements of the degree of Doctorate in

Philosophy in Pharmaceutical Chemistry.

Dr. Asif Husain

Department of Pharmaceutical Chemistry

Faculty of Pharmacy

Jamia Hamdard

New-Delhi

DECLARATION

I hereby declare that the thesis entitled “Analytical Method Development and

Validation of newly synthesized ester prodrugs of Aceclofenac” embodies my own

unaided work.

Dated:

NITI BHARDWAJ

DEDICATED TO MY PARENTS AND MY HUSBAND

ACKNOWLEDGEMENT

Research is to see what everybody else has seen, and to think what nobody else has thought.

Completion of this doctoral dissertation was possible with the support of several people. I

would like to express my sincere gratitude to all of them.

First of all, I am extremely grateful to my research guide, Dr. Asif Husain, Sr. Asst.

Professor, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia

Hamdard, New Delhi-110062, for his valuable guidance, scholarly inputs and consistent

encouragement I received throughout the research work. This feat was possible only

because of the unconditional support provided by my Sir. A person with an amicable and

positive disposition, Sir has always made himself available to clarify my doubts despite

his busy schedules and I consider it as a great opportunity to do my doctoral programme

under his guidance and to learn from his research expertise. Thank you Sir, for all your

help and support.

I thank Dr. Sokinder Kumar, Director, R.V.Northland Institute, Dadri, U.P. for letting

me carry out my work at the institute and have been very encouraging and supportive,

and I express my gratitude to him.

I thank Aditya Dutt, Senior Research Fellow at National Institute of Technology, Raipur

for statistical data analysis.

No research is possible without the Library, the centre of learning resources. I take this

time to express my gratitude to all the library staff for their services.

My colleague and also my well wishers, Dr. Aparna Khansili have all extended their

support in a very special way, and I gained a lot from them, through their personal and

scholarly interactions, their suggestions at various points of my research programme. I

also acknowledge my old pals, Shveta, Vertika, Garima, Shikha, Gaurav Anand,

Mukta and many more for their well wishes.

I owe a lot to my parents, who encouraged and helped me at every stage of my

personal and academic life, and longed to see this achievement come true. My father,

Mr. A.K.Sharma, an engineer by profession, had always has a dream of calling his

daughter as doctor will be the happiest person with the completion of the research. He has

been my advisor and a greatest support during the study. I am very much indebted to my

mother, Mrs. Madhurima Sharma who being a homemaker, always encouraged her

daughter to grow as a strong and independent individual. Without her support and love, I

would never able to finish my work. My brother PARAS, being younger to me, he never

stopped encouraging me to finish my work.

I thank my husband Mayank Kool for his support. Without his support and appreciation

this would really be hard to achieve. He was always there for me whenever I need him in

my highs and lows. Lucky to have him as my companion for life. Last but not the least,

my son PRABHAV, who was ignored so much in all this, but he never complained.

Thanks to his Nana- Nani and Divya maami for taking care of him, while I was busy

with my work. I am grateful to all of you. I also thank my parents-in-law and brother-in-

law Mr. Manu Kool for their good wishes and encouragement. How can I forget my cute

little Pia and Preeti bhabhi for being the support system for me.

Thank You almighty for sending me your best wishes in form of such lovely persons

surrounding me and those who were not there with me to share my achievements, my

Nanaji and Sunil Chacha.

NITI BHARDWAJ

LIST OF ABBREVIATIONS

NMR Nuclear Magnetic Resonance

1H-NMR Proton Nuclear Magnetic Resonance

UV Ultraviolet Spectroscopy

IR Infra-red Spectroscopy

PBS Phosphate buffer solution

Mcg/mL Microgram per milliliter

LOD Limit of detection

LOQ Limit of quantization

MP Melting point

NSAIDS Non-steroidal anti-inflammatory drugs

COX Cyclooxygenase enzyme

DAD Diode Array Detection

API’s Active Pharmaceutical Ingredient

BPA 4-Biphenylacetic acid

FTIR Fourier transform infrared spectroscopy

USP United States Pharmacopoeia

IND Investigational new drug

NDA New drug application

MAA Marketing authorization application

i

LIST OF TABLES

Table 1 Stock solution prepared for compound 1 in methanol

Table 2 Stock solution prepared for compound 1 in ethanol

Table 3 Stock solution prepared for compound 1 in DMSO

Table 4 Stock solution prepared for compound 1 in acetone

Table 5 Stock solution prepared for compound 2 in methanol

Table 6 Stock solution prepared for compound 2 in ethanol

Table 7 Stock solution prepared for compound 2 in DMSO

Table 8 Stock solution prepared for compound 2 in acetone

Table 9 Regression analysis of Compound 1

Table 10 Regression analysis of Compound 2

Table 11 Linearity result for Compound 1&2

Table 12 Assay result for Compound 1 &2

ii

LIST OF GRAPHS

Calibration curve 1 Curve for Compound 1 in Methanol

Calibration curve 2 Curve for Compound 1 in Ethanol

Calibration curve 3 Curve for Compound 1 in DMSO

Calibration curve 4 Curve for Compound 1 in Acetone

Calibration curve 5 Curve for Compound 2 in Methanol

Calibration curve 6 Curve for Compound 2 in Ethanol

Calibration curve 7 Curve for Compound 2 in DMSO

Calibration curve 8 Curve for Compound 2 in Acetone

iii

CONTENTS

Contents Page No.

Certificate

Declaration

Acknowledgement

List of Abbreviations i

List of Tables ii

List of Graphs iii

Publications

1. Research Envisaged 1-3

2. Chapter 1: Introduction 4-31

2a. The Prodrug Concept

2b. Application of Prodrug Approach to NSAIDs

2c. Application of Method Development and Validation

3. Chapter 2: Literature Review 32-71

4. Chapter 3: Experimental Work 72-97

4a. Scheme involved

4b. Synthesis of mutual prodrugs of aceclofenac

4c. Analytical determination of synthesized compounds

4d. Scheme involved for method development and validation

of synthesized compounds

4e. Results and Discussions

Synthesis of Mutual Prodrugs

Pharmaceutical Evaluation

Method Development and Validation

5. Summary 98-99

6. Conclusion 100

1. RESEARCH ENVISAGED

The main aim of medicinal research in the recent times has been to develop drugs

with enhanced efficacy, reduced toxicity and side effects. The pharmaceutical

industry and specifically, a medicinal chemist have continued commitment towards

this drug development. Over the years, innovations in new drug therapy has become,

more complex, time consuming, costly, and the practicing medicinal chemists have

been bombarded with surplus new methods and technologies to make the job of drug

discovery more efficient. These include QSAR analysis, rational drug design,

molecular modeling, and structure based design.

Improvement of drug design can be accomplished by biological, physical and/or

chemical means. Of the three, chemical approach, offers the highest degree of

flexibility in altering drug efficacy. When utilizing the biological approach, versatility

is limited because only the route of administration can be altered. In comparison to

the biological approach, physical approach offers a great degree of flexibility for drug

modification, and is commonly referred to as dosage form.

Drug derivatization has been for long recognized as a significant means of producing

better therapeutic agents. These derivatives can be categorized as reversible and

irreversible. Irreversible derivatization usually involves development of new chemical

entities of similar nature of activity, with increased potency or a broad spectrum of

activity. Reversible derivatization generally involves development of drug derivatives

with less undesirable effects, as well as retaining the intrinsic activity of the molecule.

The precaution is taken as to what functional group is modified as indiscriminate

modification that may result in total loss of activity.

Our approach focuses on reversible chemical modification of some Non-Steroidal

Anti-inflammatory drugs, which upon introduction into an appropriate system; revert

to the parent molecules by virtue of enzymatic or non-enzymatic liability.

The NSAIDs are of immense clinical significance but their potentially deleterious

effects on the stomach are eminent. NSAIDs with free carboxylic group produce

1

upper gastrointestinal side effects like gastric irritation, ulceration, bleeding and

perforation. Formation of GI lesions was well thought to be due to direct contact

mechanism. This is probably attributed to the free carboxylic group of the NSAIDs

and to the local inhibition of prostaglandin on gastric mucosa. Another disadvantage

of these drugs is a relatively short plasma life. This leads to short duration of action

and frequent dosing, resulting in pronounced ulcerogenic activity.

Because NSAIDs arbitrarily inhibit both isoforms of cyclooxygenase (COX), they are

associated with side effects like mucosal damage and bleeding due to local inhibition

of the cytoprotective action of prostaglandins on gastric mucosa. Later, selective

COX-2 inhibitors were developed, which were devoid of the undesirable effects of

classic NSAIDs, as they selectively inhibited the induced COX-2 isoform while

sparing inhibition of the COX-1, resulting in superior GI tolerability. But

subsequently it was found that the selective COX-2 inhibitors were associated with

other more severe side effects.

For this reason, various other approaches have been used to minimize side effects of

the conventional NSAIDs which are as follows:

1. Development of enteric coated tablets, which are targeted to release the drug in

the intestine, thereby preventing its direct contact.

2. Development of prodrugs, e.g. nabumetone, a prodrug of naproxen, produces

negligible gastric irritation as compared to the parent drug. Various prodrugs like

ester, amide, glycolamide, glyceride and polymer prodrugs have also been

synthesized.

In search of a solution to this problem, we temporarily masked the free carboxylic

group of NSAIDs by forming its ester. This was achieved by condensation of the free

carboxylic group of the NSAID as well as to give a synergistic anti-inflammatory,

analgesic and antipyretic response.

Structures of the synthesized compounds were established on the basis of IR, NMR

and Mass spectral results. The synthesized compounds were undergone the method

development procedure with the help of UV-spectroscopy. The correlation

coefficients were calculated on the basis of calibration curves prepared.

2

This is a simple, rapid and efficient method development procedure for the mutual

prodrugs of aceclofenac. This method is time efficient and cost effective. The entire

work had been carried out in normal laboratory conditions.

3

CHAPTER-1 INTRODUCTION

INTRODUCTION

2a. The Prodrug Concept:

The term prodrug was introduced by Albert who used “prodrug” or “proagent” to refer to

a pharmacologically inactive compound that is transformed by the mammalian system

into an active substance by either chemical or metabolic means1, 2. Another term drug

latentiation, which implies a time lag element or component, was coined by Harper3, 4.

Later, the concept of prodrug and latentiated drug for solving various problems was

attempted and the definition of drug latentiation was extended to include non–enzymatic

regeneration of parent compounds5. The prodrug approach has emerged as a tool in

overcoming various obstacles to drug formulation and targeting such as chemical

instability, poor aqueous solubility, inadequate brain penetration, insufficient oral

absorption, local irritation and toxicity6. It is justified by the fact that once the barrier to

the use of parent compound has been overcome, these temporary forms can be converted

to the free parent compound that can exert its pharmacological activity.

A prodrug is thus defined as a biologically inactive derivative of a parent drug molecule

that usually requires a chemical or enzymatic transformation within the body to release

the active drug, and possess improved delivery properties over the parent molecule7-9.

Most of the limitations can be overcome by prodrug approach, but after overcoming the

various barriers, the prodrug should rapidly convert into active moiety after reaching the

target site. The awareness that the onset, intensity and duration of drug action are greatly

affected by the physicochemical properties of drug has promoted the emergence of

various theoretical and predictive models for drug design and evaluation10-11. The design

of an efficient, stable, safe, acceptable and aesthetic way to target a drug to its site of

action while overcoming various physical, chemical and social barriers is certainly an

area where the utilization of the prodrug approach holds great potential.

Classification of Prodrugs:

Prodrugs are categorized into four classes. They are given as follows;

(i) Carrier Linked Prodrugs

4

(ii) Tripartite Prodrugs

(iii) Mutual Prodrugs

(iv) Polymeric Prodrugs

1. Carrier linked prodrugs:

Various adverse physicochemical properties of drug can be tailored and side effects can

be minimized by attaching a non toxic carrier group or promoiety to form a new

compound i.e., prodrug, from which the parent drug is regenerated in vivo. Common

example is dipivalyl ester of epinephrine, which enhances the corneal absorption and

inhibits the rapid metabolic destruction of epinephrine. In addition prodrug produces less

cardiovascular side effects 12.

2. Tripartite Prodrugs:

Structures of most prodrugs are bipartite in nature in which parent drug is attached

directly to promoiety. However in some cases bipartite prodrug may be unstable due to

inherent nature of the drug-promoiety bond. This can be overcome by designing a

tripartite prodrug, utilizing a spacer or connector group between the drug and promoiety.

The spacer or connector group must be designed in such a way that the initial activation

is followed by spontaneous cleavage of remaining drug spacer bond under physiological

condition to release parent drug e.g. a model tripartite prodrug P–(N–(tert-butyloxy

carbonyl) lysyl) amido) benzyloxy carbonyl)–P–nitro aniline has been designed in which

N–tert butyloxy carbomyl lysine group is promoiety, P–amido benzyloxy carbonyl group

is spacer group and P–nitro aniline is the drug.

3. Mutual Prodrugs:

Mutual prodrugs are defined as two pharmacologically active agents joined together so

that each acts as a promoiety for the other and vice versa 13. Benorylate is a common

example of this category which is a prodrug of acetyl salicylic acid and paracetamol.

Major advantage associated with this prodrug is in treatment of chronic inflammation at

decreased dose and reduced risk of irritation.

5

4. Polymeric Prodrugs:

In this type, which is also known as macromolecular prodrug, the drug is dispersed or

incorporated into the polymer (both naturally occurring and synthetically prepared)

system without formation of covalent bond between drug and polymer. Example is p–

phenylene diamine mustard is covalently attached to polyamine polymer backbone

polyglutamic acid.

Functional Groups Amenable to Prodrug Design14:

Ideally, the design of an appropriate prodrug structure should be considered at the early

stages of preclinical development, bearing in mind that prodrugs might alter the tissue

distribution, efficacy and the toxicity of the parent drug. Several important factors should

be carefully examined when designing a prodrug structure, including:

Parent drug: which functional groups are amenable to chemical prodrug derivatization?

Promoiety: this should ideally be safe and rapidly excreted from the body. The choice of

promoiety should be considered with respect to the disease state, dose and the duration of

therapy.

Parent and prodrug: the absorption, distribution, metabolism, excretion (ADME) and

pharmacokinetic properties need to be comprehensively understood.

Some of the most common functional groups that are amenable to prodrug design include

carboxylic, hydroxyl, amine, phosphate/phosphonate and carbonyl groups. Prodrugs

typically produced via the modification of these groups include esters, carbonates,

carbamates, amides, phosphates and oximes.

However, other uncommon functional groups have also been investigated as potentially

useful structures in prodrug design. For example, thiols react in a similar manner to

alcohols and can be derivatized to thioethers and thioesters. Amines may be derivatized

into imines and N-Mannich bases.

1. Esters as prodrugs of carboxyl, hydroxyl and thiol functionalities:

Esters are the most common prodrugs used, and it is estimated that approximately 49% of

all marketed prodrugs are activated by enzymatic hydrolysis. Ester prodrugs are most

6

often used to enhance the lipophilicity, and thus the passive membrane permeability, of

water soluble drugs by masking charged groups such as carboxylic acids and phosphates.

The synthesis of an ester prodrug is often straightforward. Once in the body, the ester

bond is readily hydrolysed by ubiquitous esterases found in the blood, liver and other

organs and tissues, including carboxyl esterases, acetylcholinesterases,

butyrylcholinesterases, paraoxonases and arylesterases.

2. Carbonates and carbamates as prodrugs of carboxyl, hydroxyl or amine

functionalities:

Carbonates and carbamates differ from esters by the presence of an oxygen or nitrogen on

both sides of the carbonyl carbon. They are often enzymatically more stable than the

corresponding esters but are more susceptible to hydrolysis than amides. Carbonates are

derivatives of carboxylic acids and alcohols, and carbamates are carboxylic acid and

amine derivatives. The bioconversion of many carbonate and carbamate prodrugs

requires esterases for the formation of the parent drug.

3. Amides as prodrugs of carboxylic acids and amines:

Amides are derivatives of amine and carboxyl functionalities of a molecule. In prodrug

design, amides have been used only to a limited extent owing to their relatively high

enzymatic stability in vivo. An amide bond is usually hydrolyzed by ubiquitous

carboxylesterases, peptidases or proteases. Amides are often designed for enhanced oral

absorption by synthesizing substrates of specific intestinal uptake transporters.

4. Oximes as derivatives of ketones, amidines and guanidines:

Oximes (for example, ketoximes, amidoximes and guanidoximes) are derivatives of

ketones, amidines and guanidines, thus providing an opportunity to modify molecules

that lack hydroxyl, amine or carboxyl functionalities. Oximes are hydrolyzed by the

versatile microsomal cytochrome P450 (CYP450) enzymes, better known as xenobiotic

7

metabolizing enzymes. Oximes, especially strongly basic amidines and guanidoximes,

can be used to enhance the membrane permeability and absorption of a parent drug.

Applications of prodrug approach 15-18:

Prodrug approach has been extensively studied amongst the drug design scientist for a

wide range of applications. It has been successfully applied to encompassing variety of

drugs; various goals achieved not only for correction of pharmacokinetic behavior but

also pharmaceutical, organoleptic, physical and chemical properties of parent drug

compound which enhance the stability and patient compliance improving the efficacy of

therapy.

1. To enhance aqueous solubility: Prednisolone (R=R1=H) and methylprednisolone

(R=CH3, R1=H) are poorly water soluble corticosteroid drugs. To permit aqueous

injection or ophthalmic delivery of these drugs, they must be converted into water soluble

forms. Prednisolone phosphate (R=H, R1=PO3Na2) is prescribed as a water soluble

prodrug for prednisolone that is activated in vivo by phosphatases.

2. Prodrugs for improved absorption and distribution: If the desired drug is not

absorbed and transported to the target site in sufficient concentration, it can be made

more water-soluble or lipid soluble depending on the desired site of action.

Corticosteroids for the topical treatment of inflammatory, allergic and

pruritic skin conditions can be made more suitable for topical absorption by esterification

or acetonidation.For example, both fluocinolone acetonide (R=H) and fluocinonide

(R=COCH3) are prodrugs used for inflammatory and pruritic manifestations. Once

absorbed through the skin , an esterase release the drug.

2B. NSAIDS (Non-steroidal Anti-Inflammatory Drugs):

Non steroidal anti inflammatory drugs (NSAlDs) are used primarily to treat

inflammation, mild to moderate pain and fever. The diverse uses of NSAIDs comprise

the treatment of headache, arthritis, gout, inflammatory arthropathies, dysmenorrhoea,

sports injuries, migraine, post-operative pain, tissue injury, sciatica and rheumatism.

8

NSAIDs structurally consist of an acidic moiety which is represented by a carboxylic

acid group, an enolic group, a hydroxamic acid group and a sulphonamide or tetrazole

ring . The centre of acidity is attached to a planar aromatic or hetero aromatic ring of

NSAIDs. The anti inflammatory activity depends on the acidic centre attached to the

planar aromatic or hetero aromatic ring. The lipophilicity of NSAIDs is due to the

formation of alkyl chain or additional aromatic ring attached to the planar moiety.

NSAIDs are usually used for the treatment of acute or chronic conditions where pain and

inflammation are present. Research continues into their potential for prevention of

colorectal cancer.

NSAIDs are generally used for the symptomatic relief of the following conditions:19-25

Osteoarthritis

Rheumatoid arthritis

Mild-to-moderate pain due to inflammation and tissue injury

Low back pain

Inflammatory arthropathies (e.g., ankylosing spondylitis, psoriatic arthritis,

Reiter's syndrome)

Tennis elbow

Headache

migraine

9

Acute gout

Dysmenorrhoea (menstrual pain)

Metastatic bone pain

Postoperative pain

Muscle stiffness and pain due to Parkinson's disease

Pyrexia (fever)

Ileus

Renal colic

They are also given to neonate infants whose ductus arteriosus is not closed

within 24 hours of birth

Aspirin, the only NSAID able to irreversibly inhibit COX-1, is also indicated for

inhibition of platelet aggregation. This is useful in the management of arterial thrombosis

and prevention of adverse cardiovascular events. Aspirin inhibits platelet aggregation by

inhibiting the action of thromboxane A2.

Mechanism of action:

Most NSAIDs act as nonselective inhibitors of the enzyme cyclooxygenase (COX),

inhibiting both the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)

isoenzymes. This inhibition is competitively reversible (albeit at varying degrees of

reversibility), as opposed to the mechanism of aspirin, which is irreversible inhibition.[49]

COX catalyzes the formation of prostaglandins and thromboxane from arachidonic acid

(itself derived from the cellular phospholipid bilayer by phospholipase A2).

Prostaglandins act (among other things) as messenger molecules in the process of

inflammation. This mechanism of action was elucidated by John Vane (1927–2004), who

received a Nobel Prize for his work (see Mechanism of action of aspirin). COX-1 is a

constitutively expressed enzyme with a "house-keeping" role in regulating many normal

physiological processes. One of these is in the stomach lining, where prostaglandins serve

10

a protective role, preventing the stomach mucosa from being eroded by its own acid.

COX-2 was discovered in 1991 by Daniel L. Simmons at Brigham Young University.

COX-2 is an enzyme facultatively expressed in inflammation, and it is inhibition of

COX-2 that produces the desirable effects of NSAIDs.

When nonselective COX-1/COX-2 inhibitors (such as aspirin, ibuprofen, and naproxen)

lower stomach prostaglandin levels, ulcers of the stomach or duodenum internal bleeding

can result.

NSAIDs have been studied in various assays to understand how they affect each of these

enzymes. While the assays reveal differences, unfortunately different assays provide

differing ratios.26-27

The discovery of COX-2 led to research to development of selective COX-2 inhibiting

drugs that do not cause gastric problems characteristic of older NSAIDs.

Paracetamol (acetaminophen) is not considered an NSAID because it has little anti-

inflammatory activity. It treats pain mainly by blocking COX-2 mostly in the central

nervous system, but not much in the rest of the body.28

However, many aspects of the mechanism of action of NSAIDs remain unexplained, and

for this reason further COX pathways are hypothesized. The COX-3 pathway was

believed to fill some of this gap but recent findings make it appear unlikely that it plays

any significant role in humans and alternative explanation models are proposed.29

NSAIDs are also used in the acute pain caused by gout because they inhibit urate crystal

phagocytosis besides inhibition of prostaglandin synthase.30

Antipyretic activity

NSAIDS have antipyretic activity and can be used to treat fever. 31-33 Fever is caused by

elevated levels of prostaglandin E2, which alters the firing rate of neurons within the

11

hypothalamus that control thermoregulation. Antipyretics work by inhibiting the enzyme

COX, which causes the general inhibition of prostanoid biosynthesis (PGE2) within the

hypothalamus. PGE2 signals to the hypothalamus to increase the body's thermal set point.

Ibuprofen has been shown more effective as an antipyretic than paracetamol

(acetaminophen). Arachidonic acid is the precursor substrate for cyclooxygenase leading

to the production of prostaglandins F, D & E

Classification

NSAIDs can be classified based on their chemical structure or mechanism of action.

Older NSAIDs were known long before their mechanism of action was elucidated and

were for this reason classified by chemical structure or origin. Newer substances are more

often classified by mechanism of action.

Salicylates

Aspirin (acetylsalicylic acid)

Diflunisal (Dolobid)

Salicylic acid and other salicylates

Salsalate (Disalcid)

Propionic acid derivatives

Ibuprofen

Dexibuprofen

Naproxen

Fenoprofen

Ketoprofen

Dexketoprofen

Flurbiprofen

12

Oxaprozin

Loxoprofen

Acetic acid derivatives

Indomethacin

Tolmetin

Sulindac

Etodolac

Ketorolac

Diclofenac (Safety alert by FDA)

Aceclofenac

Nabumetone (drug itself is non-acidic but the active, principal metabolite has a

carboxylic acid group)

Enolic acid (Oxicam) derivatives

Piroxicam

Meloxicam

Tenoxicam

Droxicam

Lornoxicam

Isoxicam

Anthranilic acid derivatives (Fenamates )

Mefenamic acid

Meclofenamic acid

Flufenamic acid

Tolfenamic acid

13

Selective COX-2 inhibitors (Coxibs)

Celecoxib (FDA alert)

Rofecoxib (withdrawn from market)

Valdecoxib (withdrawn from market)

Parecoxib FDA withdrawn, licenced in the EU

Lumiracoxib TGA cancelled registration

Etoricoxib not FDA approved, licenced in the EU

Firocoxib used in dogs and horses

Sulfonanilides

Nimesulide (systemic preparations are banned by several countries for the

potential risk of hepatotoxicity)

Others

Clonixin

Licofelone acts by inhibiting LOX (lipooxygenase) & COX and hence known as

5-LOX/COX inhibitor

H-harpagide in Figwort or Devil's Claw

Chirality

Most NSAIDs are chiral molecules (diclofenac is a notable exception). However, the

majority are prepared in a racemic mixture. Typically, only a single enantiomer is

pharmacologically active. For some drugs (typically profens), an isomerase enzyme in

vivo converts the inactive enantiomer into the active form, although its activity varies

widely in individuals. This phenomenon is likely responsible for the poor correlation

14

between NSAID efficacy and plasma concentration observed in older studies, when

specific analysis of the active enantiomer was not performed.

Ibuprofen and ketoprofen are now available in single, active enantiomer preparations

(dexibuprofen and dexketoprofen), which purport to offer quicker onset and an improved

side-effect profile. Naproxen has always been marketed as the single active enantiomer.

2C. Aceclofenac 34-39

Aceclofenac is a non-steroidal anti-inflammatory drug (NSAID) analog of Diclofenac. It

is used for the relief of pain and inflammation in rheumatoid arthritis, osteoarthritis and

ankylosing spondylitis. The dose is 100 mg twice daily.

It should not be given to people with porphyria or breast-feeding mothers, and is not

recommended for children.

Aceclofenac has higher anti-inflammatory action than conventional NSAIDs. It is a

cytokine inhibitor. Aceclofenac works by blocking the action of a substance in the body

called cyclo-oxygenase. Cyclo-oxygenase is involved in the production of prostaglandins

(chemicals in the body) which cause pain, swelling and inflammation. Aceclofenac is the

glycolic acid ester of diclofenac.

Aceclofenac synthesis15

Synthesis of Aceclofenac Prodrugs: The synthesis of amide prodrugs of aceclofenac

with L-histidine, L-alanine, L-tyrosine and glycine was successfully carried out as per the

synthetic protocol. The structures of histidine conjugated aceclofenac (AC1), alanine

conjugated aceclofenac (AC2), tyrosine conjugated aceclofenac (AC3) and glycine

conjugated aceclofenac (AC4) are shown in the table below. The prodrugs AC2 and AC3

were found to possess good yield of 97 % each whereas AC1 and AC2 showed a yield of

65 % and 45 % respectively. 40

Chemical structure of

aceclofenac prodrugs

Prodrug

Structure and

Molecular Formula

Chemical Name

AC1 C23H22Cl2N4O5 NH Cl Cl

C O CH2 CO O NH CH

H2CCOOCH3NNH C

N-[β-immidazole -3yl-α-

(methyl propionate)]

2[((2,6 dichloro phenyl)

amino phenyl acetoxy

acetamide

AC2 C20H20Cl2N2O5 NH Cl Cl

C O O CH2 CONH CH

COOCH3 CH3

N-(methyl propionate) 2

[((2,6 dichloro phenyl)

amino phenyl acetoxy

acetamide)]

AC3 C26H24Cl2N2O5 NH Cl Cl

C O O CH2 CONH HC

CH2 OH COOCH3

N-[β-(para hydroxy

phenyl propionate)]-2

[(2, 6 dichloro phenyl)

amino phenyl acetoxy

acetamide)]

AC4 C19H18Cl2N2O5 NH Cl Cl

C O O CH2 CONH CH2

COOCH3

N-[methyl acetate]-2

[(2,6 dichloro phenyl)

amino phenyl acetoxy

acetamide)]

16

2D. Method Development and Validation 41-55:

Analytical methods development and validation play important roles in the discovery,

development, and manufacture of pharmaceuticals. Pharmaceutical products formulated

with more than one drug, typically referred to as combination products, are intended to

meet previously unmet patients need by combining the therapeutic effects of two or more

drugs in one product. These combination products can present daunting challenges to the

analytical chemist responsible for the development and validation of analytical methods.

The official test methods that result from these processes are used by quality control

laboratories to ensure the identity, purity, potency, and performance of drug products.

Identification and quantification of impurities is a crucial task in pharmaceutical process

development for quality and safety. Related components are the impurities in

pharmaceuticals which are unwanted chemicals that remain with the active

pharmaceutical ingredients (APIs), or develop during stability testing, or develop during

formulation or upon aging of both API and formulated APIs to medicines. The presence

of these unwanted chemicals even in small amounts may influence the efficacy and safety

of the pharmaceutical products. Various analytical methodologies are employed for the

determination of related components in pharmaceuticals. There is a great need for

development of new analytical methods for quality evaluation of new emerging drugs.

Basic criteria for new method development of drug analysis:

1. The drug or drug combination may not be official in any pharmacopoeias.

2. A proper analytical procedure for the drug may not be available in the

literature due to patent regulations.

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

formulation due to the interference caused by the formulation excipients.

4. Analytical methods for the quantization of the drug in biological fluids

may not be available.

5. Analytical methods for a drug in combination with other drugs may not be

available.

17

6. The existing analytical procedures may require expensive reagents and

solvents. It may also involve cumbersome extraction and separation

procedures and these may not be reliable.

Method validation:

The need to validate an analytical or bioanalytical method is encountered by analysis in

the pharmaceutical industry on an almost daily basis, because adequately validated

methods are a necessity for approvable regulatory filings. What constitutes a validated

method, however, is subject to analyst interpretation because there is no universally

accepted industry practice for assay validation.

Method Development:

Method development should be based on several considerations. It is preferable to have

maximum sample information to make development fast and desired for intended

analytical method application, physical and chemical properties are most preferable as

primary information.

Moreover, separation goal needs to define at beginning so; appropriate method can be

developed for the purpose. An LC method development is very huge area for even

pharmaceuticals with regulatory requirement of international standards. So, prior to

method validation and usage at quality control many aspects need to focus as per ICH

guidelines. Method development can be based on a sample and goals as well as available

resources for chromatography but few basic steps for method development are can be

discussed as given below.

Steps in method development

1. Sample information, define separation goals

2. Sample pre-treatment, need of special HPLC procedure

3. Selection of detector and detector settings

4. Selection of LC method; preliminary run; estimate best separation conditions

18

5. Optimize separation conditions

6. Check for problems or requirement for special procedure

7. Method validation

Sample information

1. Number of compounds present

2. Chemical structure of compounds

3. Chemical nature

4. Molecular weight of compounds

5. pKa Value(s) of compounds

6. Sample solubility

7. Sample stability and storage

8. Concentration range of compounds in sample

9. UV spectra of compounds or properties for detection of compounds

METHOD VALIDATION

Once an analytical method is developed for its intended use, it must be validated. The

extent of validation evolves with the drug development phase. Usually, a limited

validation is carried out to support an Investigational New Drug (IND) application and a

more extensive validation for New Drug Application (NDA) and Marketing

Authorization Application (MAA). Typical parameters recommended by FDA, USP, and

ICH are as follow 56:

1. Specificity

2. Linearity & Range

3. Precision

(A) Method precision (Repeatability)

(B) Intermediate precision (Ruggedness)

4. Accuracy (Recovery)

5. Solution stability

6. Limit of Detection (LOD)

19

7. Limit of Quantification (LOQ)

8. Robustness

Method validation is vast area which includes many validation parameters with different

approaches for different level of requirement based on intended use of analytical method,

criticality and regulatory requirements. Validated method also can give the unpredicted or

unknown problem during the course of routine usage, because validated method has also

limited level of confidence, as method was validated for known or predicted variable

parameters or every method can fail sooner or later. But still after method development it

needs to be validated as per requirement which gives certain level of confidence for its

intended use. A common method validation protocol is followed for all the method

developed during the research project (FDA, ICH Q2A & Q2B, 2005).

1. Specificity

Specificity is the ability of the method to measure the analyte in the presence of other

relevant components those are expected to be present in a sample. The relevant

components might include impurities, degradants, matrix, etc. Lack of specificity of an

individual procedure may be compensated by other supporting analytical procedure(s).

Specificity can also be demonstrated by verification of the result with an independent

analytical procedure. In the case of chromatographic separation, resolution factors should

be obtained for critical separation. Tests for peak homogeneity, for example, by diode

array detection (DAD) or mass spectrometry (MS) are recommended. The evaluation of

the specificity of the method was determined against placebo. The interference of the

excipients of the claimed placebo present in pharmaceutical dosage form is derived from

placebo solution. Further the specificity of the method toward the drug is established by

means of checking the interference of the degradation products in the drug quantification

for assay during the forced degradation study. The peak purity of analyte peak was

evaluated in each degraded sample with respect to total peak purity and three point peak

purity. The peak purity value must be more than 0.999 (for Agilent system) or purity

angle is less than threshold (for Waters system) in every case.

20

Force degradation studies:

These studies are undertaken to elucidate inherent stability characteristics. Such testing is

part of the development strategy and is normally carried out under more severe condition

than those used for accelerated stability studies. Force degradation of the drug substance

can help identify the likely degradation products, which can in turn help establish the

degradation pathways and the intrinsic stability of the molecule and validate the stability

indicating power of the analytical procedures used. The nature of the stress testing will

depend on the individual drug substance and the type of drug product involved.

Examining degradation products under stress conditions is useful in establishing

degradation pathways and developing and validating suitable analytical procedures. So,

as per the guidelines the stress studies for all the drug under investigation are done in the

same conditions, the only difference is in temperature and the time required for each drug

to degrade up to 5-20% level. Usually, the drugs are kept at solution and solid state

stability in the following stability studies:

Solution state stability:

1. Acidic hydrolysis

2. Alkaline hydrolysis

3. Hydrolytic

4. Oxidative degradation

Solid state stability:

1. Thermal degradation

2. Photolytic degradation

2. Linearity and Range

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

results, which are directly proportional to the concentration (amount) of analyte in the

sample. A linear relationship should be evaluated across the range of the analytical

procedure. It is demonstrated directly on the drug substance by dilution of a standard

stock solution of the drug product components, using the proposed procedure. For the

establishment of linearity, minimum of five concentrations are recommended by ICH

guideline. The value of correlation co-efficient (r2) should fall around 0.99.

21

3. 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. Precision may be considered at two levels: repeatability and

intermediate precision. The precision of an analytical procedure is usually expressed as

the variance, standard deviation or coefficient of variation of a series of measurements.

Repeatability: Repeatability study is performed by preparing a minimum of 6

determinations at 100% of the test concentration and analyzed as per the respective

methodology.

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 analyst should establish the effects of random events on the precision of the

analytical procedure. Typical variations to be studied include days, analysts, equipment,

etc. It is not considered necessary to study these effects individually. Here, intermediate

precision of the method is checked by carrying out six independent assays of test sample

preparation on the different day by another person under the same experimental condition

and calculated the % RSD of assays.

4. 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. The evaluation of accuracy has got very prime importance as

it deliberately force the method to extract the drug and impurities at higher and lower

level.

5. Solution stability

Drug stability in pharmaceutical formulations/active pharmaceutical ingredients is a

function of storage conditions and chemical properties of the drug, preservative and its

impurities. Condition used in stability experiments should reflect situations likely to be

encountered during actual sample handling and analysis. Stability data is required to

22

show that the concentration and purity of analyte in the sample at the time of analysis

corresponds to the concentration and purity of analyte at the time of sampling. Stability

of sample solution was established by storage of sample solution at ambient temperature

(25°C) for 24h.

6. Limit of detection

The limit of detection (LOD) for 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. 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 between 3 or 2:1 is generally considered acceptable for

estimating the detection limit. The limit of detection is evaluated by serial dilutions of

analyte stock solution in order to obtain signal to noise ratios of 3:1.

7. Limit of quantization

The quantization 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 limit of quantization (LOQ) is a parameter of quantitative assays for low

levels of compounds in sample matrices. 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 by establishing the minimum concentration at

which the analyte can be reliably quantified. A typical signal-to-noise ratio is 10:1. The

limit of quantification was evaluated by serial dilutions of analyte stock solution in order

to obtain signal to noise ratios of 10:1.

8. 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.

23

In the case of liquid chromatography, examples of typical variations are:

1. Influence of variations of pH in a mobile phase

2. Influence of variations in mobile phase composition

3. Different columns (different lots and/or suppliers)

4. Temperature

5. Flow rate

The factors chosen for all the drugs under investigation were the flow rate, mobile phase

composition, pH of a mobile phase and using different lot of LC column. The observation

shall be summarized and critical parameters shall be listed out in the validation report.

System suitability parameter must be within the limit of acceptance criteria as mentioned

in the method.

10. Advantages of analytical method validation

The advantages of the analytical method validation are as follow:

1. The biggest advantage of method validation is that it builds a degree of

confidence, not only for the developer but also to the user.

2. Although the validation exercise may appear costly and time consuming, it results

inexpensive, eliminates frustrating repetitions and leads to better time management in the

end.

3. Minor changes in the conditions such as reagent supplier or grade, analytical

setup are unavoidable due to obvious reasons but the method validation absorbs

the shock of such conditions and pays for more than invested on the process.

24

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31

CHAPTER 2Review of Literature

PRODRUGS: A CHALLENGE FOR DRUG DEVELOPMENT:

It is estimated that about 10% of the drugs approved worldwide can be classified as

prodrugs. Prodrugs, which have no or poor biological activity, are chemically modified

versions of a pharmacologically active agent, which must undergo transformation in vivo

to release the active drug. They are designed in order to improve the physicochemical,

biopharmaceutical and/or pharmacokinetic properties of pharmacologically potent

compounds. Jolanta B. Zawilska et al describes the basic functional groups that are

amenable to prodrug design, and highlights the major applications of the prodrug

strategy, including the ability to improve oral absorption and aqueous solubility, increase

lipophilicity, enhance active transport, as well as achieve site-selective delivery. Special

emphasis is given to the role of the prodrug concept in the design of new anticancer

therapies, including antibody-directed enzyme prodrug therapy (ADEPT) and gene-

directed enzyme prodrug therapy (GDEPT).1

University of Finland performed certain synthesis of prodrugs with respect to

different functional groups which are summarized below: 2

Drug Functional Groups and Synthesis of Prodrugs

Drug-X-R,

Where X is the functional group in a drug, in which promoiety R can be attached.

X can be NH, CO2H, OH, SH, CONH, SO2NH, C=O

Prodrug should be:

– Inactive and nontoxic

– Easily synthesizable

– Chemically stable outside site of action

--irreversible (parent drug must be regenerated in vivo)

Krise et al performed the synthesis and preliminary evaluation of a novel prodrug

approach for improving the water solubility of drugs containing a tertiary amine group.

The prodrug synthesis involves a nucleophilic substitution reaction between the parent

tertiary amine and a novel derivatizing reagent, di-tert-butyl chloromethyl phosphate,

32

resulting in formation of the quaternary salt. The tertiary butyl groups are easily removed

under acidic conditions with trifluoroacetic acid giving the N-phosphonooxymethyl

prodrug in the free phosphoric acid form, which can subsequently be converted to the

desired salt form. The synthesis was successfully applied to a model compound

(quinuclidine) and to three tertiary amine-containing drugs (cinnarizine, loxapine, and

amiodarone). The prodrugs were designed to undergo a two-step bioreversion process.

The first step was an enzyme-catalyzed rate-determining dephosphorylation followed by

spontaneous chemical breakdown of the N-hydroxymethyl intermediate to give the parent

drug. Selected prodrugs were shown to be substrates for alkaline phosphatase in vitro. A

preliminary in vivo study confirmed the ability of the cinnarizine prodrug to be rapidly

and completely converted to cinnarizine in a beagle dog following i.v administration. 3

Aggarwal R.K et al studied that Ethambutol (EB), isoniazid (INH) and p-amino salicylic

acid (PAS) are potent antitubercular agents having various side effects due to formation

of toxic metabolites. Their study aims towards prevention of these side effects through

mutual prodrug formation. Mutual prodrugs of EB with PAS (PE), PAS with PAS (PP)

and INH with PAS (PI) were synthesized and characterized. Hydrolytic and absorption

studies were performed in SGF (synthetic gastric fluid) and SIF (synthetic intestinal

fluid). In vivo studies were also performed to confirm the release profile of the

synthesized prodrugs. Formation of imide and ester functionalities was confirmed

by IR spectra. In vitro hydrolysis studies in SGF and SIF reveal that these mutual prodrug

conjugates do not hydrolyze appreciably and are absorbed unhydrolyzed. In vivo studies

showed greater serum concentrations of EB, PAS and INH than their concentrations

when given alone and isoniazid concentrations were greater except for PP. Peak plasma

levels were attained after 3h but these levels were reduced 0.6 times. Thus, mutual

prodrugs PI and PE significantly eliminate the problem of fast metabolism, toxicity and

local irritation and reduction of therapeutic doses. In the case of PP only local irritation

could be avoided. 4

33

Redasani et al, prepared glyceride ester derivatives by reacting 1, 2, 3-trihydroxy

propane 1, 3-dipalmitate/stearate with (S)-naproxen as potential prodrugs. The synthesis

was achieved successfully with the aid of N, N’-dicyclohexyl- carbodiimide. These

prodrugs were evaluated for anti inflammatory, analgesic and gastroprotective activity. It

was found that prodrugs synthesized showed less irritation to gastric mucosa as indicated

by ulcer index. The synthesized glyceride esters were found to possess good

pharmacological profile of anti inflammatory and analgesic activity. The aqueous studies

were performed in order to ensure the release of prodrugs. Both prodrugs were found to

stable at acidic pH while underwent hydrolysis at pH 7.4. These findings suggest that the

glyceride prodrugs 6a and 6b might be used as potential biolabile derivatives. 5

Jilani A et al reported the syntheses of 4-aminophenylbenzoxazol-2-yl-5-acetic acid, (an

analogue of a known nonsteroidal anti-inflammatory drug [NSAID]) and 5-[4-

(benzoxazol-2-yl-5-acetic acid) phenylazo]-2-hydroxybenzoic acid (a novel mutual azo

prodrug of 5-aminosalicylic acid [5-ASA]). The structures of the synthesized compounds

were confirmed using infrared (IR), hydrogen-1 nuclear magnetic resonance (1H NMR),

and mass spectrometry (MS) spectroscopy. Incubation of the azo compound with rat

cecal contents demonstrated the susceptibility of the prepared azo prodrug to bacterial

azoreductase enzyme. The azo compound and the 4-aminophenylbenzoxazol-2-yl-5-

acetic acid were evaluated for inflammatory bowel diseases, in trinitrobenzenesulfonic

acid (TNB)-induced colitis in rats. The synthesized diazo compound and the 4-

aminophenylbenzoxazol-2-yl-5-acetic acid were found to be as effective as 5-

aminosalicylic acid for ulcerative colitis. The results of this work suggest that the 4-

aminophenylbenzoxazol-2-yl-5-acetic acid may represent a new lead for treatment of

ulcerative colitis.6

“Prodrugs Approach” is a versatile approach in solving the problems associated with drug

molecules. Diclofenac is a pain reliever in variety of painful conditions but it has some

side effects i.e. absorption, toxicity, distribution, instability, formulation etc. These side

34

effects can be reduced by “Prodrugs Approach”. Kumar et al synthesized some amide

Prodrugs of Diclofenac via acid amine coupling of Diclofenac and ester derivatives of

amino acids using HOBT, NMM and EDC. HCl in dichloromethane medium. These

newly synthesized. Prodrugs were analyzed by NMR and IR spectroscopy. These newly

synthesized Prodrugs were analyzed by NMR and IR spectroscopy. All the compounds

were evaluated for analgesic activity by acetic acid induced writhing and anti-

inflammatory activity by Carragennan Induced Rat hind Paw method.7

Naproxen is widely used for the treatment of arthritic pain. It can induce gastrointestinal

(GI) side effects ranging from stomach irritation to ulceration and bleeding. These

complications are believed to be determined from the combined effect of the blockage of

prostaglandin biosynthesis in the GI tract and direct action of free carboxylic groups.

Prodrugs approach have been proposed to overcome the Non-steroidal anti-inflammatory

drugs (NSAIDs) side effects by masking the carboxylic acid groups via formation of

bioreversible bonds. It has been demonstrated that the production of reactive oxygen

species (ROS) plays an important pathogenic role in gastrointestinal ulceration. Our goal

was to develop a new Naproxen prodrug which integrates the concepts of prodrug and the

beneficial antioxidant effect. For this purpose we designed and synthesized naproxen

esters containing tocopherols. Among the possible antioxidant compounds attention was

on -tocopherol for its potent antioxidant properties and anti-inflammatory activity. In

addition, esterification of phenolic group on chroman ring of tocopherols, to form the

prodrug, is expected to increase the stability to oxidation of the vitamin. Spadaro et al,

reported the synthesis and the in vitro enzymatic and non-enzymatic hydrolysis of two

prodrug esters of naproxen with alpha- and gamma-tocopherols. We also investigated the

oral pharmacokinetic in rabbit and probed the preliminary pharmacological evaluation in

rat. The synthesized prodrugs exhibited anti-inflammatory activity with a strong and

significant reduction in gastrolesivity. 8

Mutual prodrug is a form of prodrug in which two pharmacologically active agents are

attached to each other in such a way that each drug acts as a promoiety/carrier for each

35

other and vice versa. The association may be “synergistic” if the carrier shows the same

biological action as that of parent drug or may provide “additional” benefit if it shows

new pharmacological action which is lacking in parent drug. The mutual prodrug concept

has shown its marked therapeutic gain in case of well-accepted and useful drugs with

minor undesirable properties and in those active compounds that suffer from severe

limitations, like lack of site specificity, poor bioavailability or lack of particular activity.

Now a days Anticancer, cardiovascular, antiviral, antipsychotic and anti-inflammatory

drugs are best utilizing the concept of mutual prodrug designing for their better effect.

Ohlan et al have reviewed mechanism of activation, contribution of mutual prodrug

approach in different therapeutic areas and the development in this field during the last

few decades including a list of patents. They describes various design approaches,

methods of synthesis, pharmacological evaluations for mutual prodrugs, but has also

highlighted the emerging fields of docking studies and their relevance to the

pharmacokinetics of mutual prodrugs. 9

A novel mutual prodrug (MA-P) consisting of mefenamic acid (MA) and paracetamol (P)

has been synthesized by Shah et al as a gastrosparing NSAID, devoid of ulcerogenic side

effects. The structure of synthesized drug was confirmed by elemental analysis, infrared

spectroscopy, 1H NMR spectroscopy and mass spectrometry. The kinetics of ester

hydrolysis was studied by HPLC at pH 2, pH 7.4 as well as in human plasma. The

pharmacological activities (anti-inflammatory, analgesic and ulcerogenic) were evaluated

for the synthesized drug. The ulcerogenic reduction in terms of gastric wall mucosa,

hexosamine and total proteins were also measured in glandular stomach of rats. The

results indicated that MA-P ester has better ulcer index than the parent drug. 10

Novel mutual prodrugs (MPs) of ATRA (all- trans-retinoic acid) and HDIs (histone

deacetylase inhibitors) connected via glycine acyloxyalkyl carbamate linker (AC linker)

or through a benzyl ester linker (1,6-elimination linker) were rationally designed and

synthesized by Gediya et al. Most novel MPs were potent inhibitors of growth of several

hormone-insensitive/drug resistant breast cancer cell lines and the hormone-insensitive

36

PC-3 prostate cancer cell line. The novel MPs exhibited differential antiproliferative

potencies in both MDA-MB-231 and PC-3 cell lines. Whereas (VNLG/124) [4-

(butanoyloxymethyl)phenyl-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-

2,4,6,8-tetraenoate] with a GI 50 of 10 nM was the most potent MP versus the MDA-

MB-231 cells, (VNLG/66) [{ N-[ N-{2-[4-{[3-pyridylmethoxy) carbonyamino]

methyl}phenyl) carbonylamino]phenyl} carbamoylcarbamoyloxy}methyl-3,7-dimethyl-

9-(2,6,6-trimethyl cyclohex-1-enyl)nona-2,4,6,8-tetraenoate] with a GI 50 = 40 nM was

the most potent versus the PC-3 cells. MP exhibited the most benefit because its GI 50 of

10 nM versus MDA-MB-231 cells was remarkably 1085-fold lower than that of parent

ATRA and over 100000-fold lower than butyric acid (BA). 11

MUTUAL PRODRUG CONCEPT:

A therapeutically significant drug may have limited utilization in clinical practice

because of poor organoleptic properties, poor bioavailability, short duration of action,

nonspecificity, incomplete absorption, poor aqueous solubility, high first-pass

metabolism or other adverse effects. There is a great emphasis on research to discover

methods aimed at improving their therapeutic efficacy by minimizing or eliminating

these undesirable properties. Sometimes, an adequate pharmaceutical formulation can

overcome these drawbacks, but often the galenic formulation is inoperant and a chemical

modification of active molecule is necessary to correct its pharmacokinetic

insufficiencies. This chemical formulation process, whose objective is to convert an

interesting active molecule into a clinically acceptable drug, often involves the so-called

'Prodrug design.' Mutual prodrug is a type of carrier-linked prodrug, where the carrier

used is another biologically active drug instead of some inert molecule. Bhosle et al takes

a review of various applications of mutual prodrugs and the developments in this field

during the last few decades.12

Despite the intensive research that has been aimed at the development of NSAIDs, their

clinical usefulness is still restricted by their GI side effects like gastric irritation,

37

ulceration, bleeding, perforation and in some cases may develop into life threatening

conditions. GI lesions produced by NSAIDs are generally attributed to either direct

and/or indirect mechanisms. The direct contact effects result usually from local irritation

produced by free acidic group of NSAIDs and local inhibition of prostaglandin synthesis

in GIT. Indirect mechanism is due to generalized systemic action occurring after

absorption and is demonstrated on intravenous dosing. This problem has been solved by

derivatization of carboxylic function of NSAIDs into ester and amide mutual prodrugs

using amino acids like L-tryptophan, L-histidine, L-glycine as carriers that have marked

antiinflammatory activity of their own. 13, 14, 15

Other analgesic, antiinflammatory drugs like paracetamol and salicylamide have also

been used as carriers to synthesize mutual prodrugs of NSAIDs, the examples of which

are cited below. Benorylate is a mutual prodrug of aspirin and paracetamol, linked

through ester linkage, which claims to have decreased gastric irritancy with synergistic

analgesic action. Glycine methyl ester conjugate of ketoprofen, histidine methyl ester

conjugate of diclofenac, and various conjugates of flurbiprofen with amino acid like L-

tryptophan , L-histidine, phenylalanine and alanine as mutual prodrugs were reported to

have less ulcerogenicity with better antiinflammatory/analgesic action than their parent

drugs. Mutual prodrugs of ibuprofen with paracetamol and salicylamide have been

reported with better lipophilicity and reduced gastric irritancy than the parent drug.

Naproxen-propyphenazone mutual prodrugs were synthesised with an aim to improve

therapeutic index through prevention of GI irritation and bleeding. Esterification of

naproxen with different alkyl esters and thioesters led to prodrugs with retained

antiinflammatory activity but exhibited greatly reduced GI erosive properties and

analgesic potency, but esterification with ethyl piperazine showed that analgesic activity

was preserved whereas antiinflammatory activity was generally reduced.

Propyphenazone, a nonacidic pyrazole with good analgesic and antipyretic activity, was

coupled with naproxen to achieve many advantages related to the synergistic analgesic

effect with reduced gastric irritation. Propyphenazone is converted to its active

38

metabolite, 3-hydroxy methyl propyphenazone, which actually gives the analgesic effect.

Coupling of these two compounds as a hybrid drug or through a spacer as a mutual

prodrug resulted in potent analgesic/antiinflammatory compound with reduced adverse

local effects related to NSAID. 16-20

A more recent strategy for devising a gastric-sparing NSAID involves chemically

coupling a nitric oxide (NO) releasing moiety to the parent NSAID. Studies have shown

that the use of NSAIDs with NO-releasing properties has an improved GI safety. Along

with prostaglandins, NO plays an important cytoprotective role in GI homeostasis and

defence by helping to maintain mucosal blood flow, optimizing mucus gel secretion and

inhibiting activation of pro-inflammatory cells. Synthesis of NO-releasing organic nitrate

esters of several NO-aspirin and NO-flurbiprofen are in clinical trials at present. 4-

Biphenyl acetic acid (4-BPA) is the active metabolite of fenbufen and is twice active as

the parent drug. 4-BPA suffers severe GI side effects on oral administration and hence is

not used for therapeutic purpose. Mutual prodrugs of 4-BPA have been synthesized using

naturally occurring phenolic antioxidants like thymol, guaiacol, eugenol, and other

alcoholic compounds. The antioxidant activity of phytophenols is likely to enhance the

effectiveness of 4-BPA by lowering its ulcerogenic potential. Probenecid and diclofenac

were converted to hydrazide derivatives via their methyl ester by reacting with hydrazine

hydrate. The hydrazide derivatives were further reacted with biphenyl acetic acid. The

hydrazide derivative of naproxen was reacted with p-chlorobenzoic acid to synthesize

their oxadiazole analogue in order to produce mutual prodrug with lower ulcerogenicity

and synergistic action. Mutual prodrug conjugates of flurbiprofen have been reported

with histamine H2 antagonist in order to reduce gastric damage by NSAID. 21

Mutual prodrugs of ketoprofen, ibuprofen, diclofenac and flurbiprofen with an

antiarthritic nutraceutical D-glucosamine have been reported with reduced

gastrointestinal ulcerogenicity, better analgesic/antiinflammatory effects and additional

antiarthritic activity. Glucosamine is used as an antiarthritic drug and nutritional

39

supplement in conditions like joint ache, stiffness, severely restricted movements and

serious pain. It acts as an essential substrate for the biosynthesis of glucosaminoglycans

and the hyaluronic acid backbone needed for formation of proteoglycans found in the

structural matrix of joints. NSAIDs are used for the symptomatic treatment of

inflammation associated with arthritis but are unable to remove the underlying cause of

the disease. Their prolonged use results in GI side effects. When tested in Fruend's

adjuvant-induced arthritis assay, these mutual prodrugs have shown antiarthritic activity,

which was lacking in the parent drugs with comparable antiinflammatory activity and

lowered ulcerogenicity.22

Sulfasalazine23 is the classic example of colon-specific mutual prodrug of 5-

aminosalicylic acid (5-ASA) and sulfapyridine, used in the treatment of ulcerative colitis.

5-ASA and sulfapyridine are linked together by azo linkage, which is reduced only in the

colon by azo reductases secreted by colonic microflora. This releases the active agent 5-

ASA in the colon, having antiinflammatory effect on the colon along with sulfapyridine.

The advantage of this approach is that the cleavage of azo linkage and generation of 5-

ASA prior to the absorption prevents its systemic absorption and helps it to concentrate at

the active site. Sulfapyridine was selected as a carrier in this mutual prodrug design by

taking into account its antibacterial activity, but even though sulfapyridine proved to be a

good carrier for targeting 5-ASA to colon, it gave rise to many side effects resulting from

its systemic toxicity. Therefore, even if according to definition, sulfasalazine is a mutual

prodrug, due to disadvantages of its carrier, it cannot be referred to as a true mutual

prodrug. This led to the development of interesting mutual prodrug of 5-ASA called

olsalazine, which is actually a diamer of 5-ASA, where 5-ASA is linked through azo

linkage to one more molecule of 5-ASA. When it reaches the large intestine, it is cleaved,

releasing two molecules of 5-ASA for every molecule of olsalazine administered. This

design overcomes the drawbacks of sulfasalazine, targets 5-ASA to colon, and fulfils all

requirements of mutual prodrug too. Improvement in the bioavailability of 5-ASA is also

achieved by this design.

40

Nitrous oxide (NO)24 plays a critical role in a variety of bioregulatory processes,

including normal physiological control of blood pressure, neurotransmission, and

microphage-induced cytostatics and cytotoxicity. NO can inhibit metastasis, enhance

cancer cell apoptosis, and assist macrophages to kill tumour cells. Diazenium diolates

are compounds containing the [N (O) NO]- structural unit. It is known to be an excellent

source for controlled release of NO, both in vitro and in vivo. 5-Fluorouracil (5-FU) is

one antitumour agent most frequently used for treating solid tumours like breast,

colorectal, and gastric cancers. It is poorly tumour-selective, so its therapy causes high

incidences of toxicity in the bone marrow, GIT, CNS and skin, which has promoted the

efforts in the development of derivatives aiming at reducing the adverse effects of 5-FU.

Therefore, search for novel prodrug of 5-FU possessing a broad spectrum of antitumour

activity and less toxicity has led to the design of mutual prodrug of 5-FU and diazenium

diolate with methylene or acyloxymethylene as spacers. The prodrug has been

synthesised with an aim to improve tumour selectivity, efficiency and safety.

One more way to direct 5-ASA25 to colon using mutual prodrug concept has been

reported, where 5-ASA is conjugated with ursodeoxycholic acid (UDCA). UDCA is the

bacterial product of chenodeoxycholic acid and has application in gallstone dissolution

and treatment of cholestatic liver diseases. Recent studies have also shown that UDCA

may be beneficial in colonic polyp reduction. It has been shown that UDCA-5ASA is

poorly absorbed from intestine and is targeted to colon, where it is partially hydrolyzed to

UDCA and 5-ASA. While a portion of UDCA-5-ASA escapes bacterial cleavage, part of

the UDCA is absorbed from the colon, enters enterohepatic circulation, is converted into

taurine conjugate by hepatic enzymes and is secreted into the bile. It is postulated that

both 5-ASA and UDCA may exhibit their antiinflammatory and cytoprotective effects in

colon as well as liver. UDCA has also shown to inhibit polyp formation in experimental

rats. As patients with ulcerative colitis are at a greater risk of primary sclerosing

cholangitis (PSC) and as UDCA has been reported to be beneficial in PSC, the

41

enterohepatic circulation of UDCA generated in colon may be cytoprotective to the

hepatocyte in these patients.

Chlorzoxazone [5-chloro-2 (3H)-benzoxazolone] is a centrally active muscle relaxant,

while acetaminophen (N-acetyl-p-aminophenol) exhibits analgesic properties. Owing to

their synergistic effects, these two drugs can be prescribed together. Using this rationale,

a mutual prodrug of chlorzoxazone and acetaminophen has been designed, and its

synthesis and kinetics have been reported.26

Another example of a mutual prodrug with synergistic action is sultamicillin . In the

design of sultamicillin, the irreversible b-lactamase inhibitor sulbactam has been

combined chemically via ester linkage with ampicillin. This design is based on the

rationale that as sulbactam, a beta-lactamase inhibitor with very limited antibacterial

activity in a physical mixture with ampicillin, clearly enhances the activity of the latter

against certain beta-lactamase-producing bacteria, both in vitro and in vivo, the same

phenomenon might hold true when these two drugs are linked chemically. The mutual

prodrug effect produced by sultamicillin results from its having a more efficient oral

absorption than the single agent does. Peak serum concentrations of ampicillin are

achieved that are approximately 3.5-fold those obtained with an equivalent amount of

oral ampicillin. Equimolar concentrations of sulbactam are also provided with both

ampicillin and sulbactum, being widely distributed among various body fluids and

tissues. The pharmacokinetic parameters of the two components are similar, both being

eliminated primarily by renal excretion.

Mutual prodrugs of some antiinflammatory agents were synthesized by Velingkar et al

with the aim of improving the therapeutic index through prevention of gastrointestinal

complications and check the efficiency of release of the parent drug in presence of

spacer. These mutual prodrugs were synthesized by direct condensation method using

dicyclohexyl carbodiimide as a coupling agent and glycine as a spacer. The title

42

compounds were characterized by spectral techniques and the release of the parent drug

from mutual prodrug was studied in two different non-enzymatic buffer solutions at pH

1.2, pH 7.4 and in 80% human plasma. All mutual prodrugs exhibited encouraging

hydrolysis profile in 80% human plasma. Biological activity of title compounds was

studied by carrageenan-induced paw edema method. From the results obtained, it was

concluded that these compounds retain the antiinflammatory action. 27

Mutual prodrugs comprising retinoid and histone deacetylase inhibitors did synthesized,

methods for production of the mutual prodrugs and methods of treatment comprise

administration of the mutual prodrugs. The retinoid include all-trans retinoic acid, 13-cis

retinoic acid, and retinoic acid analogs that have a substitution at C-4. Further, the mutual

prodrugs of the present invention can be used as therapeutic agents for the treatment of

cancer and dermatological diseases and conditions. 28

4-Biphenylacetic acid (4-BPA), the active metabolite of NSAID fenbufen has been

modified by Sharma P D et al using mutual prodrug approach. Number of 4-BPA

derivatives have been synthesised as potential mutual prodrugs by the attachment of

several phytophenols/alcohol as promoieties through ester linkage, directly as well as

through spacers, with the objective of obtaining safer NSAIDs devoid of their

ulcerogenic side effects. The structures of these derivatives have been established on the

basis of spectral analysis.29

Two mutual prodrugs, in which two different anti-cancer drugs are attached to the same

molecule via labile linkages, are synthesized by Menger et al and examined kinetically.

One of the mutual prodrugs loses a drug component under physiological conditions

within an hour, but the other mutual prodrug (having a longer spacer between the two

drugs) is stable to chemical degradation even at higher pH values. Thus, enzymatic

hydrolysis alone will release the two anti-cancer drugs. 30

43

Tuberculosis is one of the most widespread disease in the world, and can be deadly in

patients with AIDS worldwide. The course of treatment is long (3-9) months, often need

combination therapy to decrease microbial resistance, some of these drugs have serious

side effects and undesirable physicochemical properties like, gastric irritation, short T1/2

(pas), hepatic toxicity and extensive metabolism by the liver (INH), through N-acetyl

transferase enzyme. So by using this approach Qasir et al worked on reduction of

gastrointestinal toxicity of PAS and reduction of intestinal acetylation of Isoniazid. This

drug was synthesized by amidation of free amine group of INH with free carboxyl group

of glycine from one side and amidation of carboxyl group of PAS after protection of it,

and amino group of glycine from other side, here amidation was done by using coupling

agent (DCC) using conventional solution method and it was identified by the following

methods: melting point, thin layer chromatography (TLC), infrared spectroscopy (IR) and

the elemental analysis(CHN). The synthesized compound showed better partition

coefficient compared with the original apromoiety (PAS,INH).31

A new mutual prodrug was synthesized by Mustafa F Y for colon targeting in the

treatment of colon cancer associated with constipation. A new mutual prodrug was

synthesized through several steps included amide hydrolysis in a strong acidic medium,

amide synthesis, diazotization and coupling reactions. The stability of this prodrug in HCl

buffer, in phosphate buffer and in rat fecal matter was monitored. The chemical structure

of mutual prodrug was characterized by physical and spectroscopic techniques as FTIR,

UV-Visible and 13C NMR spectra. In colon, the mutual prodrug was proposed to split by

the action of bacterial azoreductase into two N-substituted benzamides, metoclopramide

and declopramide, that constituted two apoptotic agents, also the local application of

metoclopramide on colonic smooth muscles was proposed to enhance their contractions

affords relief of constipation. In vitro kinetic studies in a hydrochloric acid buffer showed

an insignificant release of metoclopramide and declopramide while in a phosphate buffer,

only (9.12٪) release was observed over six hours. In order to confirm the hydrolysis of

mutual prodrug in colon, the release study in a rat fecal matter was monitored over six

44

hours and showed that the hydrolysis was almost complete (90.88٪) with a half-life of

(166.19 min) followed first order kinetics. The prodrug approach that is based on

enzymes specification may offer a new method to improve drug efficacy and reduce side

effects.32

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly used

medications worldwide for the treatment of pain, fever and acute, chronic inflammatory

diseases like arthritis. Although they are effective in the treatment of pain and

inflammation, their routine and long-term administration is limited due to their

gastrointestinal and renal side effects. Most of the natural constituents, polyphenolic

compounds and flavonoids, possess potent anti-inflammatory effect and fewer side

effects. They may be used as the potent anti-inflammatory agents; however, they suffer

from the bioavailability problems. Gupta Kiran et al synthesized a conjugate of

quercetin and meclofenamic acid, on the basis of the merits and demerits of both of these

compounds. The conjugate and the intermediates were characterized by determining

melting point, Rf value by Thin Layer Chromatography, UV Spectrophotometry, Infrared

Spectra, MASS by L.C.M.S. The anti-inflammatory activity of the conjugate was

compared with quercetin and meclofenamic acid by animal study. The probable

mechanism of action for the synthesis was discussed. The in vivo activity of the

conjugate quercetin-meclofenamic acid significantly inhibited paw edema in the first and

second phase, suggesting an inhibitory effect on the release of histamine, serotonin,

kinins and prostaglandins. The improved pharmacological activity may be contributed to

aqueous solubility and comparatively lesser molecular weight of the conjugate. In

conclusion, a mutual prodrug quercetin-meclofenamic acid, associated with anti-

inflammatory activity, was successfully synthesized and characterized.33

Kavitha Kankanala et al introduced a clean and operationally simple method has been

developed for the preparation of mutual prodrug using paracetamol and various

nonsteroidal anti-inflammatory drugs. The methodology involves use of TFAA/H3PO4 in

45

acetonitrile and a variety of mutual prodrugs has been prepared in good yields by using

this single-step C-O bond forming reaction.34

After performing the literature survey for the mutual prodrugs, prodrugs and how the

same were synthesized and characterized analytically, our study has been proceeded

further. Synthesis of mutual prodrugs were carried out and characterized on the basis of

IR, NMR and Mass Spectroscopy. Next step was to develop the method and to validate

the synthesized compounds by UV-Spectroscopy. But, before that the detailed literature

survey was carried out for the work done on the selected drug and on various other drugs

and the method adopted. Our survey reveals the following information on the work done

on method development and validation by UV-Spectroscopy.

METHOD DEVELOPMENT AND VALIDATION:

The primary focus of this study is on general approaches and considerations toward

development of chromatographic methods for separation, identification, and

quantification of pharmaceutical compounds, which may be applied within the various

functions in the drug development continuum.

The number of drugs introduced into the market is increasing every year. These drugs

may be either new entities or partial structural modification of the existing one. There is a

scope, therefore to develop newer analytical methods for such drugs. Analytical methods

development and validation play important roles in the discovery, development, and

manufacture of pharmaceuticals. Pharmaceutical products formulated with more than one

drug, 35 typically referred to as combination products, are intended to meet previously

unmet patients need by combining the therapeutic effects of two or more drugs in one

product. Various analytical methodologies are employed for the determination of related

components in pharmaceuticals. There is a great need for development of new analytical

methods for quality evaluation of new emerging drugs.

46

Basic criteria for new method development of drug analysis 36-42

1. The drug or drug combination may not be official in any pharmacopoeias,

2. A proper analytical procedure for the drug may not be available in the

literature due to patent regulations,

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

formulation due to the interference caused by the formulation excipients,

4. Analytical methods for the quantization of the drug in biological fluids

may not be available,

5. Analytical methods for a drug in combination with other drugs may not be

available,

6. The existing analytical procedures may require expensive reagents and

solvents. It may also involve cumbersome extraction and separation

procedures and these may not be reliable.

The need to validate an analytical or bioanalytical method is encountered by analysis in

the pharmaceutical industry on an almost daily basis, because adequately validated

methods are a necessity for approvable regulatory filings. What constitutes a validated

method, however, is subject to analyst interpretation because there is no universally

accepted industry practice for assay validation.43

An impurity in a drug substance as defined by the International Conference on

Harmonization (ICH) Guidelines44, 36 is any component of the drug substance that is not

the chemical entity defined as the drug substance. Similarly, an impurity in a drug

product is any component of the drug product that is not the chemical entity defined as

the drug substance or an excipient in the drug product 45.

Solvents are inorganic or organic liquids used as vehicles for the preparation of solutions

or suspensions in the synthesis of the drug substance or the manufacture of the drug

product. Since these are generally of known toxicity, the selection of appropriate limits

for these solvents is easily accomplished (ICH Q3C 46on residual solvents).

A specification is defined as a list of tests, references to analytical procedures, and

appropriate acceptance criteria that are numerical limits, ranges, or other criteria for the

tests described 47.

47

It establishes the set of criteria to which a drug substance or drug product should

conform to be considered acceptable for its intended use. “Conformance to

specifications” means that the drug substance and/or drug product, when tested according

to the listed analytical procedures, will meet the listed acceptance criteria 48.

Specifications are critical quality standards that are proposed and justified by the

manufacturer and approved by regulatory authorities as conditions of approval.

The ICH guidelines have been incorporated as law in the EU, Japan and in the US, but in

reality, besides these other countries are also using them. As these guidelines reflect the

current inspectional tendencies, they carry the de facto force of regulation. The ICH

guideline Q1A on Stability Testing of New Drug Substances and Products 49 emphasizes

that the testing of those features which are susceptible to change during storage and are

likely to influence quality, safety and/or efficacy must be done by validated stability-

indicating testing methods. It is also mentioned that forced decomposition studies (stress

testing) at temperatures in 10 °C increments above the accelerated temperatures, extremes

of pH and under oxidative and photolytic conditions should be carried out on the drug

substance so as to establish the inherent stability characteristics and degradation

pathways to support the suitability of the proposed analytical procedures. The ICH

guideline Q3B entitled ‘Impurities in New Drug Products’ emphasizes on providing

documented evidence that analytical procedures are validated and suitable for the

detection and quantization of degradation products 50. It is also required that analytical

methods should be validated to demonstrate that impurities unique to the new drug

substance do not interfere with or are separated from specified and unspecified

degradation products in the drug product. The ICH guideline Q6A, which provides note

for guidance on specifications 51, also mentions the requirement of stability indicating

assays under Universal Tests/Criteria for both drug substances and drug products. The

same is also a requirement in the guideline Q5C on Stability Testing of Biotechnological

Biological Products 52. Since there is no single assay or parameter that profiles the

stability characteristics of such products, the onus has been put on the manufacturer to

48

propose a stability indicating profile that provides assurance on detection of changes in

identity, purity and potency of the product.

Unfortunately, none of the ICH guidelines provides an exact definition of a stability-

indicating method. Elaborate definitions of stability-indicating methodology are,

however, provided in the United States-Food and Drug Administration (US-FDA)

stability guideline of 1987 and the draft guideline of 1998 53.

The requirement is also listed in World Health Organization (WHO), European

Committee for Proprietary Medicinal Products and Canadian Therapeutic Products

Directorate’s guidelines on stability testing of well established or existing drug

substances and products 54. Even the United States Pharmacopoeia (USP) has a

requirement listed under ‘Stability Studies in Manufacturing’, which states that samples

of the products should be assayed for potency by the use of a stability-indicating assay 55.

The requirement in such explicit manner is, however, absent in other pharmacopoeias.

Current ICH guideline on Good Manufacturing Practices for Active

Pharmaceutical Ingredients (Q7A), which is under adoption by WHO, also clearly

mentions that the test procedures used in stability testing should be validated and be

stability-indicating. 56

Method development should be based on several considerations. It is preferable to have

maximum sample information to make development fast and desired for intended

analytical method application, physical and chemical properties are most preferable as

primary information. So, prior to method validation and usage at quality control many

aspects need to focus as per ICH guidelines. Method development can be based on a

sample and goals as well as available resources for chromatography but few basic steps

for method development are can be discussed as given below 57.

Once an analytical method is developed for its intended use, it must be validated. The

extent of validation evolves with the drug development phase. Usually, a limited

validation is carried out to support an Investigational New Drug (IND) application and a

more extensive validation for New Drug Application (NDA) and Marketing

Authorization Application (MAA).58

49

Method validation is vast area which includes many validation parameters with different

approaches for different level of requirement based on intended use of analytical method,

criticality and regulatory requirements. Validated method also can give the unpredicted or

unknown problem during the course of routine usage, because validated method has also

limited level of confidence, as method was validated for known or predicted variable

parameters or every method can fail sooner or later 59. But still after method development

it needs to be validated as per requirement which gives certain level of confidence for its

intended use. A common method validation protocol is followed for all the method

developed during the research project (FDA, ICH Q2A & Q2B, 2005).60-64

Advantages of analytical method validation

The advantages of the analytical method validation are as follow:

1. The biggest advantage of method validation is that it builds a degree of

confidence, not only for the developer but also to the user.

2. Although the validation exercise may appear costly and time consuming, it

result inexpensive, eliminates frustrating repetitions and leads to better

time management in the end.

3. Minor changes in the conditions such as reagent supplier or grade,

analytical setup are unavoidable due to obvious reasons but the method

validation absorbs the shock of such conditions and pays for more than

invested on the process.65-68

Maleque et al developed a rapid, specific and economic UV spectrophotometric method

using a solvent composed of water: methanol: acetonitrile (9:0.5:0.5) to determine the

levofloxacin content in bulk and pharmaceutical dosage formulations. A tap re-

determined λmax of 292nm, it was proved linear in the range of 1.0–12.0 mg/mL, and

exhibited good correlation coefficient (R2=0.9998) and excellent mean recovery (99.00–

100.07%).This method was successfully applied to the determination of levofloxacin

content in five marketed brands from Bangladesh and the results were in good agreement

with the label claims. The method was validated statistically and by recovery studies for

50

linearity, precision, repeatability, and reproducibility. The obtained results proved that the

method can be employed for the routine analysis of levofloxacin in bulks as well as in

the commercial formulations. 69

Artesunate (ART) is a readily available anti malarial in combination therapy. The assay

method has posed a challenge because it does not have a readily recognizable absorption

chromospheres needed for UV spectroscopy. A simple and rapid assay method involving

two reaction steps has been developed by Oklewogu et al for assay of ART in

pharmaceutical formulations. The method involves basic reaction of ethanolic solution of

ART with 0.1N sodium hydroxide and then neutralisation and acidification of this

reaction mixture with 0.1M solution of acetic acid in 20% ethanol. This gave a good UV

spectrum for ART with λmax at 242nm. HPLC analysis of this mixture revealed the

presence of two prominent peaks. The peaks were further identified with HPLC-mass

spectrometer to possibly be those of glycal and furanose acetal. The method was

validated for stability, linearity, accuracy, intra- and inter-day precision and was used to

assay ART in commercial tablets. The method is simple and suitable for the assay of

ART in pharmaceutical formulations.70

Dhartarkar et al developed and validated a simple, sensitive and specific

spectrophotometric method for the determination of Dexibuprofen, a non-steroidal anti-

inflammatory drug (NSAID) in pure form and in pharmaceutical formulations. UV

spectrophotometric method, which is based on measurement of absorption at maximum

wavelength in phosphate buffer pH 6.8, was found to be at 221.8 nm by using 5%

methanol. The developed method was validated with respect to linearity, accuracy

(recovery), precision and specificity. The optimum conditions for analysis of the drug

were established. The drug obeyed the Beer’s law and showed good correlation. Beer’s

law was obeyed in concentration range 0-60 μg/mL having line equation y = 0.046x

+0.017 with correlation coefficient of 0.999. The results of analysis were validated by

recovery studies. The method was found to be simple, accurate, precise, economical and

robust.71

51

A simple and precise spectroscopic method for determination of Imatinib Mesylate in its

bulk and tablet dosage forms has been developed and validated by Patil et al. This

method based upon measurement of light absorption in UV region. The UV spectra of

Imatinib Mesylate showed that maximum absorbance of light was observed at 281 nm

and linearity was observed in the concentration range of 2-28ug/ml with correlation

coefficient 0.999. The proposed method was validated as per ICH Q2 (R1) guidelines for

linearity, accuracy, precision and recovery. The limit of detection (LOD) and limit of

quantization (LOQ) were found to be 0.040468 (μg/mL) and 0.122263 (μg/mL)

respectively by simple UV Spectroscopy.72

A novel, safe and sensitive method of spectrophotometric estimation in UV-region has

been developed by Behera et al for the assay of Paracetamol in its tablet formulation.

The method have been developed and validated for the assay of Paracetamol using

Methanol and water as diluents. Which does not shows any interference in

spectrophotometric estimations. All the parameters of the analysis were chosen according

to ICH [Q2(R1)] guideline and validated statistically using RSD and %RSD along with

neat chromate grams.73

Srivastav et al developed a new, rapid sensitive, simple and cost effective UV method

for the estimation of Rebamipide in bulk as well as in pharmaceutical formulations. The

relative absorbance of Rebamipide was measured in phosphate buffer (pH 7.4) at new

wavelength (λmax 227). The linearity range was found to be 2.5-12.5 μg/mL with

regression, relative absorbance =0.1061X concentration in μg/mL=0.0009 with

regression coefficient 0.9997. The method was tested and validated for various

parameters as per ICH and USP specification. The detection and quantification limit was

found to be 0.73μg/mL and 2.21 μg/mL respectively. The result demonstrates that the

developed procedure is accurate, precise and reproducible (relative standard deviation

<2.0%). Proposed method is applicable for the estimation of Rebamipide in different

dosage forms and results are in good agreement with label claim.74

52

A simple, accurate, sensitive, precise and economical spectrophotometric method has

been developed by Mrudulesh et al for the determination of Asenapine maleate in bulk

drug form. Measurement of ultraviolet absorption at 220nm. The proposed method was

validated statistically. The developed method obeyed Beer’s law in the concentration

range of 2-10 μg/mL. The limit of detection (LOD) and limit of quantization (LOQ) for

estimation of Asenapine maleate were 0.20381μg/mL and 0.61761μg/mL respectively.

The recovery was in the range of 98.8687 to 101.1068 percentages. The method was

validated for several parameters like accuracy, precision as per ICH guidelines. The

values of relative standard deviation and % recovery were found to be satisfactory,

indicating that the proposed method is precise and accurate and hence can be used for the

routine analysis. 75

Vijaylakshmi et al proposed a simple, accurate, precise, reproducible, highly sensitive,

economic spectrophotometric method for the estimation of Finasteride in tablet dosage

form. UV spectrophotometric method is based on measurement of absorption at

maximum wavelength 254 nm. The percent recovery of Finasteride ranged from 98.82 –

102.11% in tablet dosage form. The developed method was validated with respect to

linearity, accuracy (recovery), Precision (inter and intraday variations). Beer’s law was

obeyed in the concentration range of 5 – 25 μg/mL with correlation coefficient of 0.9986.

Results of the analysis were validated statistically and by recovery study. Hence the

developed and validated method can be used for estimation of finasteride in tablets.76

A specific, rapid and simple UV spectrophotometric method with good sensitivity was

developed and validated for the simultaneous quantification of tramadol HCl and

paracetamol in bulk and marketed product by simultaneous equation method. From the

optical characteristics of the proposed methods, Shukla et al found that the λmax of

tramadol HCl and paracetamol was found to 271 nm and 248 nm respectively. Tramadol

HCl and paracetamol obey linearity within the concentration range of 2.5-15 μg/mL and

3-15 μg/mL. The %RSD is less than 2%. The percentage recovery values of pure drug

53

from the pre-analyzed formulations were in between 99-103%. The analysis of the

formulation showed good result in concentration in range of 98-101%. This analytical

method is also applicable in ordinary laboratories and can be adopted for quality control

tests for these drugs in marketed formulation.77

Dey et al developed a simple accurate, precise and cost effective UV‐Vis

spectrophotometric method for the estimation of cefadroxil, a first generation

cephalosporin an anti‐biotic drug, in bulk and pharmaceutical dosage form. The solvent

used was methanol and distilled water (50:50) and the λmax or the absorption maxima of

the drug was found to be 264nm. A linear response was observed in the range of 10‐

50μg/mL with a regression coefficient of 0.9999. The method was then validated for

different parameters as per the I.C.H. (International Conference for Harmonization)

guidelines. This method can be used for the determination of cefadroxil in quality control

of formulation without interference of the excipients. Cefadroxil was subjected to stress

degradation under different conditions recommended by ICH. The samples generated

were used for degradation studies using the developed method.78

For determination of aliskiren in commercial samples, an analytical UV

spectrophotometric method was developed and validate according to ICH guideline by

Sangoi et al. The method was linear in the range between 40 and 100 μg /mL (r2 =

0.9997, n = 7) and exhibited suitable specificity, accuracy, precision, and robustness. It is

simple, it has low cost, and it has low use polluting reagents. Therefore, the proposed

method was successfully applied for the assay and dissolution studies of aliskiren in

tablet dosage forms, and the results were compared to a validated RP-LC method,

showing non-significant difference (P > 0.05).79

Aceclofenac related literature survey :

Susmita A et al developed a simple, accurate, precise and cost effective UV-

spectroscopic method for the estimation of Aceclofenac, a non-steroidal anti

inflammatory drug with good analegesic anti rheumatic activity in pharmaceutical dosage

54

forms. Method: The drug was dissolved in ethanol solvent. The method was validated for

recovery studies and repeatability studies. Results: The λmax or absorption maxima of

the drug was found to be 277.7nm. A linear response was observered in the range of 2-

60μg/mL. The percentage recovery of Aceclofenac was found to be 99%. The percentage

deviation ranges from 0.3 to 1.7. Conclusion: The proposed method can be used for the

estimation of Aceclofenac in quality control of formulation without interference of the

excipients.80

Two simple, precise and accurate visible spectrophotometric methods were developed for

the estimation of Aceclofenac in bulk drug and in pharmaceutical formulations by Bose

et al. They proposed methods which were indirect and based on determination of

aceclofenac after its reaction with both (p-dimethylaminocinnamaldehyde or 3-Methyl-2-

benzothiazolinone hydrazine hydrochloride and measuring the chromogen at the λmax by

658 and 592, respectively. Beers law obeyed in the concentration range of 1-200 μg/ml

for method A and 1-100 μg/ml for method B. The accuracy of the methods was

determined by recovery studies. The methods showed good reproducibility and recovery

with relative standard deviation (in %) less than 2. The methods were found to be simple,

economical, accurate and reproducible and can be used for routine analysis of

Aceclofenac in bulk drug and in pharmaceutical formulations.81

Aceclofenac is a non steroidal anti-inflammatory drug with good analgesic and anti-

rheumatic properties. Various methods for analysis of the same are available but are time

consuming and expensive. Here we have developed a new, precise and simple UV

spectrophotometric method for estimation of aceclofenac from tablet formulation. The

drug obeyed the Beer’s law and showed good correlation. It showed absorption maxima

at 273 nm; in phosphate buffer pH 7.4. The linearity was observed between 0 – 20

mcg/mL. The results of analysis were validated by recovery studies. The recovery was

more than 99%. The method was found to be simple, accurate, precise, economical and

robust.82

55

The present study by Gupta R K et al describes two simple validated spectrophotometric

methods for the quantitation determination of aceclofenac in tablets. The technique was

applied using methanol as a solvent. The zero order spectrum of aceclofenac shows λmax

at 277.2 nm and the determination of aceclofenac were done A1%, 1cm values at 277.2

nm and by comparison with standard at the selected wavelength (Method I) and the first

derivative absorbance values at 261.6 nm when (n=3) (Method II). The drug was found to

obey beer lamberts law over the range of 5-25 μg/mL. Percent recovery values were

found to be 99.01-99.64 by Method I while it was 101.33-101.66 by Method II for two

different marketed formulations. Reproducibility of the spectrophotometric method was

indicated by the SD values. The intraday and interday precision data proved the

ruggedness of the method. The methods were found to be precise, specific, rugged and

can be adopted for routine analysis of the drug.83

Three simple spectrophotometric methods have been developed by Dharamveer et al for

simultaneous estimation of Diacerein and Aceclofenac from tablet dosage form. 0.1M

acidic methanol was used as solvent. First method, Simultaneous equation method,

involves the measurement of absorbances at two wavelengths 256.0 nm (λmax of

Diacerein) and 276.0 nm (λmax of Aceclofenac), Second method is First order derivative

spectroscopy, wavelength selected for quantitation were 250.0 nm for Diacerein (zero

cross for Aceclofenac) and 256.8 nm for Aceclofenac (zero cross for Diacerein) and third

method is Area under curve method, area under curve in the range of 251.0-261.0 nm (for

Diacerein) and 271.0- 281.0 nm (for Aceclofenac) were selected for the analysis. The

linearity lies between 2-30 μg/mL for Diacerein and Aceclofenac for the simultaneous

equation and area under the curve method. For the first derivative method the linearity

range is 5-50 μg/mL for both the drugs. The accuracy and precision of the methods were

determined and validated statically. All the methods showed good reproducibility and

recovery with % RSD less than 1. The proposed methods were found to be rapid,

specific, precise, and accurate and can be successfully applied for the routine analysis of

Diacerein and Aceclofenac in bulk and combined dosage form.84

56

Jumle et al deals with UV spectrophotometric method development and validation for

estimation of Tizanidine and Aceclofenac tablet dosage form by Vierodt’s method and

first order UV derivative spectrophotometry. The Vierodt's method involves

measurement of absorbance at _max of Tizaridine and Aceclofenac at 282 nm

respectively. The linearity of Tizanidine and Aceclofenac was found to be in the range of

1-10 μg/mL respectively. The % recovery of Tizanidine and Aceclofenac was found out

to be 99.2% and 99.69%s respectively. First order CV derivative spectrophotometry (D1

method). The zero crossing method was chosen as Tizanidine could be easily analyzed

without any interference from Aceclofenac and vice-versa. Tizanidine was determined by

measurement of its D1 amplitude at the zero crossing point of Aceclofenac at (270nm),

While Aceclofenac was determined by measurement of its D1 amplitude at zero crossing

point of Tizanidine at (318 nm) The proposed method was validated as per ICH

guidelines.85

A new sensitive, simple, rapid and precise method for simultaneous estimation of

paracetamol and aceclofenac in combined tablet dosage form has been developed by

Nikam et al. The method is based on ratio derivative spectrophotometry. The amplitude

in first derivative of the ratio spectra at 256 nm and 268 nm (minima) were selected to

determine paracetamol and aceclofenac in combined formulation. The method showed

good linearity, accuracy and reproducibility. Results of analysis were validated

statistically and by recovery studies.86

A simple, sensitive, precise, specific and accurate isocratic reversed phase-high

performance liquid chromatography (RP-HPLC) method has been developed by

Ravishanker et al for the quantitative estimation of aceclofenac in pharmaceutical

formulations. RP-HPLC method was developed by using WELCHROM C18 Column

(4.6 X 250mm, 5μm), SHIMADZU LC-20AT prominence liquid chromatography. The

mobile phase consisting of phosphate buffer pH 6.8 and acetonitrile in the portion of

50:50 v/v. Isocratic elutions at a flow rate of 0.5 mL/min was employed. The responses

57

are measured at 278 nm using SHIMADZU SPD-20A prominence UV-Vis detector. The

retention time for aceclofenac was 8.767 min. The method possesses linearity in the

range of 2- 10μg/mL and correlation coefficient was 0.999. The % recovery was within

the range between 99.91% and 101.26%. The accuracy and reliability of the proposed

method was ascertained by evaluating various validation parameters like linearity,

precision and specificity according to ICH guidelines. The percentage RSD for precision

and accuracy of the method was found to be less than 2%. The proposed method was

successfully employed for routine quality control analysis of aceclofenac in bulk samples

and its pharmaceutical formulations.87

Three simple, specific, reproducible methods have been developed by Bhure et al and

validated for the simultaneous estimation of Aceclofenac and Diacerein in

pharmaceutical formulation by UV-Spectrophotometric methods viz; Method I,

Absorbance Correction method, Method II, Simultaneous Equation method and Method

III, Absorbance Ratio method. For development of Method I, wavelengths selected were

277.0 nm λmax for ACE and 341.5 nm λmax for DIA. For method II, wavelengths

selected were 256.5 nm and 277.0 nm for estimation of DIA and ACE respectively, while

for Method III, 277.0 nm λmax for ACE and 267.5 nm an isoabsorptive point of ACE

and DIA. The two drugs follow Beer-Lambert’s law over the concentration range of 10-

50 µg/mL for ACE and 5-25 µg/mL for DIA for all three methods. The percent recovery

of the drugs was found to be nearly 100 % representing the accuracy of the all three

methods. Validation of the proposed methods was carried out for its accuracy, precision,

specificity and ruggedness according to ICH guidelines. The proposed methods can be

successfully applied in routine work for the determination of ACE and DIA in combined

dosage form.88

For determination of aliskiren in commercial samples, an analytical UV

spectrophotometric method was developed and validate according to ICH guideline by

Sangoi et al. The method was linear in the range between 40 and 100 μg mL-1 (r2 =

58

0.9997, n = 7) and exhibited suitable specificity, accuracy, precision, and robustness. It is

simple, it has low cost, and it has low use polluting reagents. Therefore, the proposed

method was successfully applied for the assay and dissolution studies of aliskiren in

tablet dosage forms, and the results were compared to a validated RP-LC method,

showing non-significant difference (P > 0.05).89

Aceclofenac is a non steroidal anti-inflammatory drug with good analgesic and anti-

rheumatic properties. Various methods for analysis of the same are available but are time

consuming and expensive. Shah et al developed a new, precise and simple UV

spectrophotometric method for estimation of aceclofenac from tablet formulation. The

drug obeyed the Beer’s law and showed good correlation. It showed absorption maxima

at 273 nm; in phosphate buffer pH 7.4. The linearity was observed between 0 – 20

mcg/mL. The results of analysis were validated by recovery studies. The recovery was

more than 99%. The method was found to be simple, accurate, precise, economical and

robust. 90

A dissolution method and an analytical procedure by UV spectrophotometry were

developed and validated for evaluation of the dissolution behavior of tablet dosage form

containing aceclofenac and paracetamol as there was no official method available. Four

different commercially available products were selected for this study. The analytical

method developed by UV spectrophotometry was based on the application of Vierodt’s

equation which involved the formation and solving of simultaneous equations at two

wavelengths 243 nm as the λmax of paracetamol and 273 nm as λmax of aceclofenac.

The method was validated according to International Conference on Harmonization

(ICH) guidelines which include accuracy, precision, specificity, linearity, and analytical

range. Thus, the proposed dissolution method can be applied successfully for the quality

control of aceclofenac and paracetamol tablets. 91

A stability indicating RP-UPLC method was developed and validated by Balan et al for

the simultaneous determination of Thiocolchicoside (TCC) and Aceclofenac (ACF) in

59

tablet dosage form. The chromatographic separation was carried out by Thermo Scientific

UPLC Instrument, Accela 1250 Pump, auto sampler with PDA detector, using column

Thermo Scientific hypersil gold C18, (50 x 2.1mm) particle size 1.9μm using 5%

ammonium acetate buffer and methanol in the ratio of 40:60, pH was adjusted to 5 with

ortho phosphoric acid as mobile phase at a flow rate of 250 μL/min with the detection at

276nm. The run times of the TCC and ACF were about 0.697 and 1.125 minutes,

respectively. The detector response is linear from 4.8 μg/mL to 7.2 μg/mL and 63.8

μg/mL to 96 μg/mL concentrations for TCC and ACF respectively. The linear regression

equation was found to be y = 20620x-677.68 (r2 = 0.9996) for TCC and y= 50931x-319.3

(r2 = 0.9997) for ACF. The detection limit and quantification limit was 0.076μg and

0.23μg for TCC and 0.27μg and 0.71μg for ACF. The percentage of assay of TCC and

ACF were about 99.50% and 99.96% respectively. The stability indicating capability was

established by forced degradation experiments. The method was satisfactorily validated

as per the ICH guidelines.92

New simple, precise, rapid and reproducible UV-spectrophotometric method has been

developed by Durgam et al for the estimation of Drotaverine Hydrochloride and

Aceclofenac in both bulk and tablet formulation. Drotaverine and Aceclofenac in

combined tablet formulation were estimated by using the multicomponent mode at 307

nm for Drotaverine and 276 nm for Aceclofenac in their solution in ethanol: distilled

water in the ratio of 50:50 (v/v %), with correlation coefficient of 0.999 for the both the

drugs. The Beer’s law obeyed the concentration range of 4-40 μg/mL for Drotaverine and

5-40 μg/mL for Aceclofenac. Mean recovery of 99.54% for Drotaverine and 98.23% for

Aceclofenac signifies the accuracy of the method. This method was validated with

respect to linearity, accuracy (recovery), precision, Limit of detection (LOD) and limit of

quantification (LOQ) as per ICH guidelines and successfully applied for the estimation of

Drotaverine Hydrochloride and Aceclofenac in commercially available tablet dosage

form.93

No spectroscopic method has been reported for the Simultaneous estimation of

Paracetamol, Aceclofenac and Rabeprazole in Combined Tablet Dosage Formulation.

60

Hence simple, sensitive, reliable and rapid spectroscopic methods have been developed

by Mandhanya et al for the determination of paracetamol, aceclofenac and rabeprazole

in combined tablet dosage form. Determinations were performed on Shimadzu UV-

Visible double beam recording spectrophotometer (Model UV-1700). The linearity of

paracetamol, aceclofenac and rabeprazole was found to be 3- 30μg/mL, 2-20 μg/mL, and

2-20μg/mL respectively. The stability of the solution was found to be 72 hrs. The method

was validated for accuracy, precision, repeatability as per ICH Guidelines. This method

can be used commercially for routine estimation of various compounds in pharmaceutical

dosage forms.94

A simple, economical, precise, and accurate new UV spectrophotometric baseline

manipulation methodology for simultaneous determination of drotaverine (DRT) and

etoricoxib (ETR) in a combined tablet dosage form has been developed. The method is

based on baseline manipulation (difference) spectroscopy where the amplitudes at 274

and 351 nm were selected to determine ETR and DRT, respectively, in combined

formulation and methanol was used as solvent. Both the drugs obey Beer's law in the

concentration ranges of 4–20 μg/mL for DRT and 4.5–22.5 μg/mL for ETR. The results

of analysis have been validated statistically and recovery studies confirmed the accuracy

and reproducibility of the proposed method which were carried out by following the ICH

guidelines.95

An ester-based prodrug N-Hydroxymethylsuccinimide (+) Aceclofenac was synthesized

using N-Hydroxymethylsuccinimide and Aceclofenac as promoiety and its structure

established on the basis of IR, NMR and Mass spectral data. An analytical, rapid, cost-

effective and accurate method using UV-spectroscopy has been developed for the

synthesized prodrug. The value of R square obtained shows that the developed method is

rapid, easy and precise by Husain et al.96

A prodrug of aceclofenac was synthesized using N-Hydroxymethylisatin as promoiety by

Bhardwaj et al. Its structure was established on the basis of modern analytical

61

techniques. An analytical, rapid, cost-effective and accurate method using UV-

spectroscopy has been developed for the synthesized prodrug. The results and calibration

curves obtained with different solvents for the prodrug revealed that the method

developed was precise and accurate.97

Prodrug designing is an important and fruitful area of drug research. Two ester-based

prodrugs96, 97 of aceclofenac were prepared and their analytical method was developed.

The present study focuses on the validation of the method developed for analysis of the

prodrugs. ICH guidelines have been followed to validate the method. Validation included

limits of detection, linearity, range, and assay method. It was found simple, precise, cost

effective and less time consuming.98

A simple, rapid, accurate and precise Reverse Phase HPLC method was developed by

Mahaparale et al for the quantitative estimation of Aceclofenac and Tolperisone

hydrochloride in combined tablet dosage form. Aceclofenac is a non-steroidal anti-

inflammatory drug (NSAID) and Tolperisone hydrochloride is used as centrally-acting

muscle relaxant. The chromatographic separation of both drugs was carried out using 250

x 4.6 mm, i.d 5 μm C-18 column with acetonitrile: 0.05 M Potassium dihydrogen ortho

phosphate pH adjusted to 3.0 with O- Phosphoric acid (60:40 v/v) at the flow rate

1ml/min. The wavelengths for detection of both compounds were made at 273.0 nm

using UV detector. The linearity range was found to be 5-50 μg/mL for Acelofenac and

5-40 μg/mL for Tolperisone hydrochloride. The coefficient of correlation for Acelofenac

and Tolperisone hydrochloride was found to be 0.9995 and 0.9992 respectively. The

retention time for Acelofenac and Tolperisone hydrochloride were 3.40 min and 5.70

min, respectively. The percent recoveries obtained for Acelofenac and Tolperisone

hydrochloride were found to be 99.73 and 99.90 respectively. The method was validated

for linearity, range, precision, accuracy, specificity, selectivity, intermediate precision,

robustness and Suitability.99

62

A new simple, rapid, selective, precise and accurate isocratic reverse phase high

performance liquid chromatography assay has been developed and validated by

Devanaboyina et al for the estimation of Rivaroxaban in tablet formulation. The

separation was achieved by using C-18 column (250x4.6mm, 5μm in particle size) at

ambient temperature coupled with a guard column of silica in mobile phase Acetonitrile:

Methanol: 0.1%Otho phosphoric acid (90:8:2) with the pH value adjusted to 4.06. The

flow rate was 1.5mL/min and the drug was detected by using UV detector at the

wavelength 234nm and the run time was 7min. The retention time was found 3.32

minutes. The percentage of RSD for precision and accuracy of the method was found to

be less than 2%. The method was validated as per ICH guidelines. The proposed method

was found to be accurate, repeatability and consistent. It can be successfully applied for

the analysis of the drug in marketed formulation and could be effectively used for the

routine analysis of the same drug without any alteration in the chromatographic

conditions.100

A simple, economic, selective, precise, and accurate Reverse Phase High Performance

Liquid Chromatography method for analysis of Amitriptyline HCl & Chlordiazepoxide in

tablet dosage form was developed and validated by Neeli et al according to ICH

guidelines. The quantification of the drug was carried out by using YMC Co limited C8

(250 X 4.6 mm, 5μ) column its equivalent in isocratic mode and maintain column at

400C, using mobile phase comprising of Ortho phosphoric Acid : Methanol in the ratio of

50:50 v/v (Adjust pH -2 with Orthophosphoric Acid ), with a flow rate of 1.0mL/min and

the detection wavelength was carried at 253 nm. The retention time for Amitriptyline HCl

& Chlordiazepoxide was found to be 2.502&5.176. The percent assay was found to be

101%&99%. Proposed method was validated for precision, accuracy, linearity & range,

specificity and robustness according to ICH guidelines. The method was successfully

applied to Amitriptyline Hydrochloride and Chlordiazepoxide combination Tablet dosage

form.101

63

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CHAPTER 3 EXPERIMENTAL WORK

EXPERIMENTAL:

PART-1: Synthesis of Ester prodrugs of Aceclofenac

PART-2: Spectral analysis of the compound synthesized

PART-3: Method validation of the compound through UV-Spectroscopy

PART-1:

Step-1: Synthesis of ester prodrugs using N-Hydroxymethylsuccinimide and NSAID

Aceclofenac

Compound 1:

Step A: Synthesis of N-Hydroxymethylsuccinimide (1)

Procedure: A solution of succinimide and formaldehyde in water was refluxed. The

solvent was removed under reduced pressure and the oily residue obtained was

crystallized from ether/ petroleum ether to give a TLC pure crystalline compound with

M.P= 56-58 ° C.

Step-B: Synthesis of compound 1 by reacting N-Hydroxymethylsuccinimide with

Aceclofenac

Procedure: Aceclofenac and compound 1 were reacted in dry pyridine in the presence of

phosphorus oxychloride maintaining a temperature below 5° C. After completion of the

reaction the contents were poured into ice cold water in small portions while stirring. A

solid mass separated out which was filtered, washed with water, dried and crystallized

from methanol to give TLC pure compound with M.P 96° C. Its structure was established

on the basis of IR and 1H- NMR spectral data.

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SCHEME INVOLVED:

Compound 2:

Synthesis of ester prodrug using N-Hydroxymethylisatin and Aceclofenac

Step 1: SYNTHESIS OF N-HYDROXYMETHYLISATIN (2)

Procedure: A suspension of isatin and formaldehyde solution in water was refluxed. The

hot solution was filtered, cooled overnight and the separated product was filtered, dried in

air and crystallized from ethyl acetate to afford a TLC pure compound with M.P- 150-

152 ° C.

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Step-2: Synthesis of compound 2 by reacting N-Hydroxymethylisatin with Aceclofenac

Procedure: Aceclofenac and N-Hydroxymethylisatin were reacted in dry pyridine in the

presence of phosphorus oxychloride maintaining the temperature below 5° C. After

completion of the reaction the contents were poured into ice cold water in small portions

while stirring. A solid mass separated out which was filtered, washed with water, dried

and crystallized from methanol to give pure compound with M.P- 135 ° C. Its structure

was established on the basis of IR and 1H-NMR spectral data.

SCHEME INVOLVED:

74

Part-2: Spectral analysis of the compounds synthesized:

Compound 1: N-HYDROXYMETHYLSUCCINIMIDE (+) ACECLOFENAC

IR spectral data (KBr/vmax cm-1): 3072(Ar C-H), 1683(C=O, ketone),1518(N-H,

aromatic amine), 1312(C-N, aromatic amine) 1301(C-O, ester)1094 (C-Cl)1H-NMR spectral data (δ in ppm): 2.69 (m,4H,2xCH2), 3.91(s, 2H,CH2CO),4.74 (s,2H,

CH2CO),5.42(s,2H, CH2N), 6.23(s,1H, NH),6.84-7.04(m, 3H, ring A),7.24-7.54(m,

4H,ring B).

Compound 2: N-HYDROXYMETHYLISATIN (+) ACECLOFENAC

IR spectral data (KBr/vmax cm-1): 3072(Ar C-H), 1666(C=O, ketone),1515(N-H,

aromatic amine), 1341(C-N, aromatic amine), 1297(C-O, ester),1091 (C-Cl)1H-NMR spectral data (δ in ppm): 3.88(s, 2H,CH2CO),4.22 (s,2H, CH2N),4.77(s,2H,

OCH2CO), 5.80(s,1H, NH),7.05-7.18(m, 3H, ring A),6.87-6.93(m,4H, ring B),7.45-

7.59(m, 4H,ring C).

Part-3: Method Development and validation of the compound through UV-

Spectroscopy

The main objective was to develop and validate the UV-spectrophotometric method for

the mutual prodrugs of aceclofenac as per ICH guidelines

Materials and Methods:

A simple, rapid, accurate, and economical UV-spectrophotometric method has been

developed for the mutual prodrugs of Aceclofenac.

METHOD DEVELOPMENT PROCEDURE The steps of methods development and

method validation depend upon the type of method being developed. However, the

following steps are common to most types of projects:

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● method development plan

● laboratory method development

● generation of test procedure

● methods validation protocol

● laboratory methods validation

● validation report.

A well-developed method should be easy to validate. A method should be developed with

the goal to rapidly test preclinical samples, formulation prototypes, and commercial

samples. As the methods development and validation processes advance, the

information gathered is captured in the design and subsequent improvement of the

method. Ideally, the validation protocol should be written only following a thorough

understanding of the method’s capabilities and intended use. The validation protocol will

list the acceptance criteria that the method can meet. Any failure to meet the criteria will

require that a formal investigation be conducted. The required validation parameters, also

termed analytical performance characteristics, depend upon the type of analytical

method. Pharmaceutical analytical methods are categorized into five general types:

● identification tests

● potency assays

● impurity tests: quantitative

● impurity tests: limit.

● specific tests.

METHOD VALIDATION PROCEDURE

For validation the developed method is subjected to following studies :

» Precision / Reproducibility

» Accuracy

» Linearity

» Specificity / Selectivity

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» Limit of detection

» Limit of quantitation

» Robustness / Ruggedness

The validated method undergoes Quality Control procedures for further evaluation.

EXPERIMENTAL WORK FOR METHOD DEVELOPMENT

PROCEDURE FOLLLOWED:

Compound 1 and 2 was synthesized and analyzed and further gone through the method

development and validation procedure. The procedure which was followed is as follows:-

Compound was taken (100mg), dissolved in methanol and phosphate buffer with pH 7.4

was added. The volume was made upto 100mL. Stock solution was prepared of 1mg/1mL

concentration.

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This stock solution was subjected to further dilutions in various solvents and the

absorbance was observed in order to prepare a calibration curve. The absorbance were

recorded and reported.

Concentration of solvent and Wavelength selection:

Solution of concentration of 10 µg/mL, 100µg/mL was prepared. They were subjected to

scanning from 200-400nm. From the different absorbance values obtained the

wavelength selected for the present work were, 289nm for compound 1 and 293nm for

compound 2.

COMPOUND 1: Wavelength 289nm

TABLE 1: STOCK SOLUTION PREPARED IN METHANOL:

S.NO. CONCENTRATION(µg/mL) ABSORBANCE

1 5 0.013

2 10 0.047

3 15 0.077

4 25 0.140

5 35 0.198

TABLE 2: STOCK SOLUTION PREPARED IN ETHANOL:

S.NO. CONCENTRATION(µg/mL) ABSORBANCE

1 5 0.017

2 10 0.035

3 15 0.068

4 25 0.117

5 35 0.130

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TABLE 3: STOCK SOLUTION PREPARED IN DMSO (Dimethylsulphoxide):

S.NO. CONCENTRATION(µg/mL) ABSORBANCE

1 5 0.014

2 10 0.044

3 15 0.072

4 25 0.145

5 35 0.189

TABLE 4: STOCK SOLUTION PREPARED IN ACETONE:

S.NO. CONCENTRATION(µg/mL) ABSORBANCE

1 5 0.027

2 10 0.055

3 15 0.080

4 25 0.150

5 35 0.130

COMPOUND 2: Wavelength 293nm

TABLE 5: STOCK SOLUTION PREPARED IN METHANOL:

S.NO. CONCENTRATION(µg/mL) ABSORBANCE

1 5 0.107

2 10 0.112

3 15 0.145

4 25 0.168

5 35 0.207

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TABLE 6: STOCK SOLUTION PREPARED IN ETHANOL:

S.NO. CONCENTRATION(µg/mL) ABSORBANCE

1 5 0.098

2 10 0.107

3 15 0.115

4 25 0.135

5 35 0.147

TABLE 7: STOCK SOLUTION PREPARED IN DMSO:

S.NO. CONCENTRATION(µg/mL) ABSORBANCE

1 5 0.024

2 10 0.057

3 15 0.082

4 25 0.125

5 35 0.148

TABLE 8: STOCK SOLUTION PREPARED IN ACETONE:

S.NO. CONCENTRATION(µg/mL) ABSORBANCE

1 5 0.107

2 10 0.119

3 15 0.127

4 25 0.154

5 35 0.178

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CALIBRATION CURVE 1: COMPOUND 1 IN METHANOL

CALIBRATION CURVE 2: COMPOUND 1 WITH ETHANOL

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CALIBRATION CURVE 3: COMPOUND 1AND DMSO (Dimethylsulphoxide)

CALIBRATION CURVE 4 OF COMPOUND 1 AND ACETONE:

82

CALIBRATION CURVE 5 OF COMPOUND 2 AND METHANOL:

CALIBRATION CURVE 6 OF COMPOUND 2 AND ETHANOL:

83

CALIBRATION CURVE 7 OF COMPOUND 2 AND DMSO:

CALIBRATION CURVE 8 OF COMPOUND 2 AND ACETONE:

84

CALCUATIONS AND RESULTS:

The Beer-Lambert Law

Now let us look at the Beer-Lambert law and explore it's significance. This is important

because people who use the law often don't understand it - even though the equation

representing the law is so straightforward:

A=ebc

Where A is absorbance (no units, since A = log10 P0 / P )

e is the molar absorbtivity with units of L mol-1 cm-1

b is the path length of the sample - that is, the path length of the cuvette in which the sample is contained. We will

express this measurement in centimetres.

c is the concentration of the compound in solution, expressed in mol L-1

The reason why we prefer to express the law with this equation is because absorbance is

directly proportional to the other parameters, as long as the law is obeyed.

Carrying out regression analysis using software

Regression is usually carried out using software supplied with the instrument or packages

such as Excel. Many software packages allow a regression analysis to be carried out

without first plotting the data, however it is good practice to produce a plot before

carrying out the statistical analysis.

The correlation coefficient, r

The correlation coefficient, r (and the related parameters r2 and adjusted r2) is a measure

of the strength of the degree of correlation between the y and x values. In Excel output it

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is described as ‘Multiple R’. r can take any value between +1 and –1; the closer it is to 1,

the stronger the correlation. The correlation coefficient is one of the statistics commonly

used in analytical measurement. Unfortunately, it is easily (and frequently)

misinterpreted. The r value is easily misinterpreted because:

• correlation and linearity are only loosely related. The coefficient r is a measure of

correlation not a measure of linearity;

• it is relatively easy to generate data with apparently good correlation. However, a plot

of the data may well reveal that the data would be unsatisfactory for the purposes of

calibration

• for predictions made from the calibration curve to have small uncertainties, r needs to

be very close to 1

• A low r value does not necessarily mean that there is no correlation. There could be a

relationship between the y and x values, but not a linear one

For these reasons, it is essential to plot calibration data, and not just rely on the statistics,

when assessing the fitness-for-purpose of a calibration curve.

The parameters related to r are r2 and adjusted r2. r2 is often used to describe the fraction

of the total variance in the data which is contributed by the line that has been fitted.

Ideally, if there is a good linear relation, the majority of variability can be accounted for

by the fitted line. r2 should therefore be close to 1. The adjusted r2 value is interpreted in

the same way as r2 but is always lower. It is useful for assessing the effect of adding

additional terms to the equation of the fitted line. The r2 value always increases on the

addition of an extra term to the equation, but this does not mean that the extended

equation is necessarily a better fit of the data.

For calculating the value of r square, the equation used is y=mx+c, where, x and y are the

axis and m & c are the gradient and intercept respectively. The values of these correlation

coefficients were calculated for each calibration curve and were stated below:

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TABLE 9: Represent the correlation coefficient of the synthesized compound 1 in

different reagents:S.No. Compound 1 Reagents Equation R2 Value

1 N-Hydroxymethylsuccinimide

(+) Aceclofenac

Methanol Y= mx+ c 0.9987

2 Ethanol 0.7888

3 DMSO 0.897

4 Acetone 0.8359

TABLE 10: Represent the correlation coefficient of the synthesized compound 2 in

different reagents:S.No. Compound 2 Reagents Equation R2 Value

1 N-Hydroxymethylisatin

(+) Aceclofenac

Methanol Y= mx+ c 0.8854

2 Ethanol 0.9877

3 DMSO 1.324

4 Acetone 0.9997

87

SYNTHESIS:

General procedure involved:

Ester prodrug was synthesized by condensing an appropriate NSAID with N-

Hydroxymethylisatin in the presence of phosphorous oxychloride in dry pyridine

according to the scheme presented below:

Ester prodrug was synthesized by condensing an appropriate NSAID with N-

Hydroxymethylsuccinimide in the presence of phosphorous oxychloride in dry pyridine

according to the scheme presented below:

88

MATERIALS

TLC solvent systems used were Benzene: Acetone (9:1) and Toluene: Ethyl

acetate: Formic acid(5:4:1)

Iodine chamber and UV-lamp were used for visualization of TLC spots.

Whatmann paper no. 1 was used for vacuum filtration.

All other chemicals and solvents used were commercially procured from various

chemical units like E.Merck (India) Ltd. and S.D.Fine.

The drugs used in the synthesis of prodrugs were mostly obtained from Swati

Laboratories, Greater Noida.

EQUIPMENTS:

The IR-spectra were recorded in KBr on FT/IR

UV-Spectrophotometer model-Spectrum SP2000UV.

1H-NMR spectrum was recorded on Bruker spectropsin DPX-300MHz with

tetramethylsilane as internal standard in solvent CDCl3.

The Mass spectra were recorded on Jeol JMS-D 300 instrument fitted with a

JMS 2000 data system at 70 eV.

Microanalysis of the compounds was done on Perkin-Elmer model 240

analyzer and the values were found within ±0.4% of the theoretical values.

PURIFICATION OF SOLVENTS:

Distilled ethanol (2L) was poured in a round bottom flask. To it was added

500gm of calcium oxide that was freshly ignited in muffle furnace and

allowed to cool in desiccators. The flask was filled with a double surface

condenser carrying a calcium chloride guard tube. The mixture was

reassembled for downward distillation via a head adaptor to prevent the

89

carryover of calcium oxide in vapor stream. A receiver flask was attached

with side arm receiver adapter, which was protected by means of calcium

chloride guard tube. Absolute ethanol (99.5%) was distilled, gently

discarding the first 20mL of distillate. The same was subsequently stored

in a clean dry Winchester bottle with a well fitted stopper.

Commercial grades of solvents like Chloroform, Methanol, Acetone, and

Phosphorous oxychloride available were suitable for our purpose without

any further purification.

General Procedure for synthesis of mutual prodrugs from Aceclofenac and esters:

COMPOUND 1:

Ester prodrug was synthesized by condensing an appropriate NSAID with

N-Hydroxymethylsuccinimide in the presence of dry pyridine and

Phosphorous oxychloride. The reactants were dissolved in molar ratio

separately in dry pyridine (min. quantity) and the two solutions were mixed

together under ice cold conditions followed by drop wise addition of

phosphorus oxychloride and stirring while maintaining the temperature below

5° C. The reaction was then decomposed by adding into ice cold water. A

solid mass which separated out was filtered, washed with water and

crystallized from suitable solvent or solvent mixture.

90

Synthesis of compound 1 from Aceclofenac and N-

Hydroxymethylsuccinimide:

Aceclofenac 4 mmol;1.417gm

N-Hydroxymethylsuccinimide 4 mmol; 0.516gm

Dry Pyridine 5 mL

Phosphorous oxychloride 0.5 mL

Reaction time(stirring) 8 hrs

Nature of compound White crystalline powder

Melting point 96 ° C

Partition coefficient 3.04

Yield 89%

TLC behavior Single spot in Benzene: Acetone(9:1)

Rf value 0.79

Spectral Data Given below

Spectral data for compound 1

IR spectral data (KBr/vmax cm-1): 3072(Ar C-H), 1683(C=O, ketone), 1518(N-H,

aromatic amine), 1312(C-N, aromatic amine) 1301(C-O, ester) 1094 (C-Cl)1H-NMR spectral data (δ in ppm): 2.69 (m,4H,2xCH2), 3.91(s, 2H,CH2CO),4.74 (s,2H,

CH2CO),5.42(s,2H, CH2N), 6.23(s,1H, NH),6.84-7.04(m, 3H, ring A),7.24-7.54(m,

4H,ring B).

COMPOUND 2:

General Procedure:

The reactants N-Hydroxymethylisatin and Aceclofenac were dissolved in molar ratio

separately in dry pyridine (min. quantity) and the two solutions were mixed together

under ice cold conditions followed by drop wise addition of phosphorus oxychloride and

stirring while maintaining the temperature below 5° C. The reaction was then

91

decomposed by adding into ice cold water. A solid mass which separated out was filtered,

washed with water and crystallized from some suitable solvent or solvent mixture. Its

structure was established on the basis of IR and 1H-NMR spectral data.

Synthesis of compound 2 from Aceclofenac and N-Hydroxymethylisatin:

Aceclofenac 4 mmol; 1.417gm

N-Hydroxymethylisatin 4 mmol; 0.709gm

Dry Pyridine 5 Ml

Phosphorous oxychloride 0.5 mL

Reaction time(stirring) 8 hrs.

Nature of compound Orange red crystalline powder

Melting point 135 ° C

Partition coefficient 4.80

Yield 87%

TLC behavior Single spot in Benzene: Acetone (9:1)

Rf value 0.71

Spectral Data Given below

Spectral data of compound 2:

IR spectral data (KBr/vmax cm-1): 3072(Ar C-H), 1666(C=O, ketone), 1515(N-H,

aromatic amine), 1341(C-N, aromatic amine), 1297(C-O, ester), 1091 (C-Cl)1H-NMR spectral data (δ in ppm): 3.88(s, 2H,CH2CO),4.22 (s,2H, CH2N),4.77(s,2H,

OCH2CO), 5.80(s,1H, NH),7.05-7.18(m, 3H, ring A),6.87-6.93(m,4H, ring B),7.45-

7.59(m, 4H,ring C).

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METHOD DEVELOPMENT RESULTS: (According to ICH guidelines)

Basic criteria for new method development of drug analysis

Name of Drug combination:

Compound 1: N-Hydroxymethylsuccinimide (+) Aceclofenac (Not reported)

Compound 2: N-Hydroxymethylisatin (+) Aceclofenac

Analytical procedure followed:

Compound 1 and 2 was synthesized and analyzed and further gone through the method

development and validation procedure. The procedure which was followed is as follows:-

Compound was taken (100mg), dissolved in methanol and phosphate buffer with pH 7.4

was added. The volume was made upto 100mL. Stock solution was prepared of 1mg/1mL

concentration. This stock solution was subjected to further dilutions in various solvents

and the absorbance was observed in order to prepare a calibration curve. The absorbance

were recorded and reported.

Concentration of solvent and Wavelength selection:

Solution of concentration of 10 µg/mL, 100µg/mL was prepared. They were subjected to

scanning from 200-400nm. From the different absorbance values obtained the

wavelength selected for the present work were, 289nm for compound 1 and 293nm for

compound 2.

Solvents used:

The solvents used for the method development were: Methanol, Ethanol, DMSO and

Acetone.

Sample Pre-treatment: The synthesized compounds were purified from ethanol

as specified earlier.

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Sample information:

1. Number of compounds present: Aceclofenac, N-Hydroxymethylsuccinimide

and N-Hydroxymethylisatin.

2. Chemical structure of compounds: Compound 1:

Compound 2:

3. Chemical nature: Organic in nature. Formed when ester and acidic group of

Aceclofenac is reacted together under specific conditions and reagents. This is a

hydrolysis reaction.

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4. Molecular weight of compounds

5. pKa Value(s) of compounds: specified above for each compound

6. Sample solubility: Both the synthesized compounds are soluble in solvents

such as, methanol, ethanol, acetone and dimethylsulfoxide (DMSO).

7. Sample stability and storage: The compounds obtained after synthesis were

stable at room temperature once obtained in purified form.

8. Concentration range of compounds in sample: 5,10,15,25, and 35 µg/mL,

were used for method development.

9. UV spectra of compounds or properties for detection of compounds: The

method of development by UV-spectroscopy and the calibration curves at different

concentrations were recorded and noted earlier.

METHOD VALIDATION:

Factors involved in the validation procedure are mentioned below. The procedure was

followed according to ICH guidelines, and because of the limited amount of compounds

synthesized, the following factors were considered and procedures were followed

accordingly.

1. Specificity

2. Linearity & Range

3. Precision

(A) Method precision (Repeatability)

(B) Intermediate precision (Ruggedness)

4. Accuracy (Recovery)

5. Solution stability

6. Limit of Detection (LOD)

7. Limit of Quantification (LOQ)

8. Robustness

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EXPERIMENTAL:

Materials and Methods:

All other chemicals and solvents used were commercially procured from various

chemical units like E.Merck (India) Ltd. and S.D.Fine.

For method validation procedure (observing Beer’s law), Spectrum SP2000UV

(UV spectrophotometer) was used.

Whatmann paper no.41 was used for filtration.

Phosphate buffer pH 7.4 (PBS) was used as a dissolution medium for the assay.

Bath sonicator was also used.

Working standard solution:

10 mL of the stock solution was further diluted to 100 mL with PBS to obtain a working

standard solution containing 100mcg/mL.

Linearity and Calibration:

The aliquots working standard solution was diluted serially with sufficient PBS to obtain

the concentration range of 5 – 50 mcg/mL. A calibration curve for aceclofenac prodrugs

was obtained by measuring the absorbance at the λmax of 289 nm (i) and 293 nm (ii).

Statistical parameters like the slope, intercept, coefficient of correlation, standard

deviation, Relative standard deviation, and error were determined. (Table No.11)

Table No. 11: Parameters for Prodrugs 1&2Parameter Prodrug 1 Prodrug 2

Absorption Maxima 289 nm 293 nm

Beer’s Law limit 0-35 μg/mL 0-35 μg/mL

Coefficient of correlation 0.9987 0.9513

Regression Equation Y=0.0072x+0.0184 Y=0.004x+0.003

Y Intercept 0.02523 0.03865

Slope 0.0250 0.0315

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Assay:

Accurately weighed 2 mg of the compound and transferred to 100mL volumetric flask

and made the volume to mark with PBS. This mixture was sonicated in bath sonicator for

45 minutes and filtered through Whatmann filter paper No. 41. Transferred 5 mL of the

filtrate into a 50 mL volumetric flask and made the volume to mark with PBS. Aliquots

of the sample were removed and diluted to 10 mL with PBS to obtain strengths as

2mcg/mL, 4mcg/mL and 6mcg/mL and determined the respective absorbance at 289nm

and 293nm against the PBS as blank. (Table No. 12)

Table No. 12: Assay results of Prodrug 1&2 Drug Detection

Wavelength

Conc.(mcg/mL) % purity of

PBS(standard)

% purity of

Prodrug

Prodrug 1 289 nm 2 98 98

4 100.78 88.08

6 98.7

Mean=98.5%

92.08

Mean=92.78%

Prodrug 2 293 nm 2 100.04 99.41

4 99.20 99.23

6 100.4 100.07

Mean=99.88% Mean=99.57%

The UV scan of standard solution between 200 – 400 nm showed the absorption maxima

at 289nm and 293 nm respectively for both the products. The Beer’s law was verified

from the calibration curve by plotting a graph of concentration vs. absorbance.

Regression analysis showed very good correlation. The calibration plot revealed

intercept for compound 1 at 0.02523 and for compound 2 at 0.03865 which is clear by the

regression analysis equation Y = mX + C. (Where Y is absorbance, m is the slope and X

is the concentration of aceclofenac in mcg/mL) as obtained by the least square method.

The results thus obtained are depicted in Table No.11. The results of analysis for assay

are shown in Table No.12.

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SUMMARY

The work carried out during entire our study includes literature survey of the selected

drug, selecting the scheme which had to be followed, the methods and protocols which

were to be set as per the scheme, working conditions and analyzing the results and

calculations. The drug selected for the work was Aceclofenac, an NSAID. Besides being

an important NSAID, aceclofenac has certain side effects as discussed previously, GI

irritation being the common one. Our study and literature survey gave us some important

work, where the side effects were reduced by synthesizing the prodrugs of aceclofenac.

Various prodrugs of aceclofenac were synthesized and analyzed successfully by various

researchers. Following those research, we tried to synthesize some of the prodrugs which

were never reported with the help of the esters. So we selected two of our moieties,

succinimide and isatin and with the help of formaldehyde, we obtained N-

Hydroxymethylsuccinimide and N-Hydroxymethylisatin respectively. With simple reflux

of few hours of these esters with aceclofenac and potassium oxychloride as catalyst,

prodrugs were obtained.

In order to obtain the compounds in pure form, recrystallization was performed with

methylated spirit. The compounds were undergone various parameters as melting point,

color, odour, solubility, yield etc. Further the elemental analysis was performed and the

results were reported.

The structure of the compounds was confirmed with the help of IR, NMR and Mass

spectroscopy. On the basis of spectral data, the structures were confirmed and the

compounds were kept for further analysis. The prodrugs obtained were of less quantity,

so now we headed towards our method development and validation procedure. Since

none of the standard data was available to us related to our prodrugs, so whatever data

obtained by us was considered to be the standard. The wavelength for each had been

selected from various dilutions.

The method development procedure was followed as per the ICH guidelines with the help

of UV-spectroscopy. For each compound, considering its solubility, four solvents were

used, methanol, ethanol, acetone and DMSO. Various dilutions were made for each

compound and calibration curves were made accordingly. The wavelength recorded for

compound 1 &2 was 289 nm and 293 nm respectively. The results and calibration curves

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obtained gave good results with both the compounds, but the maximum linearity was

obtained with methanol as solvent in compound 1, with correlation coefficient of 0.9987.

Regression equation for each has been determined. The slope, intercept, r2 value were

calculated and calibration curve was drawn for each.

On performing the method development procedure on the prodrugs of aceclofenac, it was

concluded that the method developed was simple, rapid, less-time consuming and cost-

effective. It gave good results with almost all the solvents. After performing the

development procedure, the work proceeded towards validation.

Method validation of both the compounds was done on the basis of ICH guidelines. Due

to the limitation of the compounds, the validation procedure got restricted to few

parameters. The factors which the present research favored were: linearity, specificity,

precision study and limit of detection tests. The method was validated on the basis of the

above tests performed. The above parameters gave precisely good results.

The linearity was achieved, assay performed gave precise results (the % purity achieved

were in the desired range) and LOD (limit of detection) was observed. For validation

procedure, 10 mL of the stock solution was further diluted to 100 mL with PBS to obtain

a working standard solution containing 100mcg/mL. The Beer’s law was verified from

the calibration curve by plotting a graph of concentration vs. absorbance. Regression

analysis showed very good correlation. The calibration plot revealed intercept for

compound 1 at 0.02523 and for compound 2 at 0.03865 which is clear by the regression

analysis equation Y = mX + C. (Where Y is absorbance, m is the slope and X is the

concentration of aceclofenac in mcg/mL) as obtained by the least square method. A

simple, rapid and less time consuming method with accurate results was obtained from

UV-spectroscopy validation as per ICH guidelines.

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CONCLUSION

Prodrugs of aceclofenac (Compound 1 & 2) were synthesized and its structure was

established by IR, NMR, Mass Spectral data & elemental analysis results. It was analyzed

for method development through UV-Spectroscopy. Methanol emerged as the best

solvent with R square value 0.9987 for the prodrug, and gave the very good calibration

curve. Therefore, it could be a simple, rapid, cost-effective and less time consuming

method for the newly synthesized prodrug (2). Method development for both the

prodrugs was performed and it gave good results for newly synthesized prodrugs. The

best result was given by methanol solvent. The synthesis and analysis of the synthesized

compounds were reported in our study in the previous chapters.

As method development procedure, validation studies were also performed for the same,

but due to limited quantity of the compound only few parameters were observed as

regression analysis, limit of detection, assay and linearity.

The validation procedure followed were as per the ICH guidelines. The compounds 1 &2

gave excellent results. Since there were no reference results for this study, so the results

were not compared to any standard.

The linearity was achieved with methanol solvent, assay results were satisfactory and the

limit of detection (LOD), achieved was also satisfactory. Hence we conclude that the

simple, rapid, less-time consuming, cost effective and precise method was developed and

validated by UV-spectroscopy with the prodrugs of aceclofenac.

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