SUBMriTEO FOR THE AWARD OF THE DEGREE OF IBottor of $I|ilo

QUANTITATIVE ANALYSIS OF DRUGS IN PURE FORM AND IN PHARMACEUTICAL PREPARATIONS

USING ANALYTICAL TECHNIQUES

T H E S I S SUBMriTEO FOR THE AWARD OF THE DEGREE OF

IBottor of $I|ilo£ioplip

CHEMISTRY

iv T A L A T g A Y O O M

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH ( INDIA)

2000

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( Ace, No.

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The work embodied in the thesis comprises five chapters. The

first chapter describes a general literature survey of the

subject matter.' In order to illuminate an otherwise defunct

period, it is customary to credit ancient scholars right from

the past history of medicinal chemistry. For example, Ipecac,

from Brazillian Cephaelis species, was used as an

antiamebic medication. Iôwever, based on superstitions &

misunderstanding of natural phenomena, their observations

and interpretations must be regarded as purely heuristic.

' The research on drugs with major therapeutic action is

introduced with the mention of earlier agents. Based on

structural reactivity, bacterial and antibacterial properties, a

vast multitude of drugs are observed in the present period. The

role of physico-analytical and organic chemistry in drug

designing is worth mentioning which provides the best

solution of the problem developed in biological- environment

such as unrelated factors, loss of drug by adsorption or

incomplete absorption.

The impurities associated with drugs from many sources are

therefore a subject of great attention by both manufacturer and

regulatory agencies. The requirements for quality, safety and

purity warrants careful thought over analytical chemists.

The classif icat ion of enormous drugs based on their

chemotherapeut ic action is important which requires a special

at tention to be elaborated.

The capital , basic and accessory analytical propert ies have

been well explained and their goals and relat ionships

establ ished in hierarchical manner.

Various quality improving act ivi t ies to ensure the rel iabi l i ty

of analytical data are discussed. Quality management needs of

research laborator ies , where drug analysis is carried out on

non-rout ine basis are well emphasized.

Due to avai labi l i ty of sub-standard medicine to the general

public in developing countr ies , it s crucial to assay drug

samples in formulat ions. Dealing with this concern, we made

some successful attempts in this direction. The recommended

methods are val idated following current guidel ines.

The second chapter describes the analysis of a series of

penici l l ins e.g. amoxycil l in, ampicil l in, c loxaci l l in ,

benzylpenic i l l in , carbenici l l in & becampici l in in

pharmaceut ical preparat ions using volumetric and

spectrophotometr ic methods, s imultaneously as well as

separately. In simultaneous method, the determination is based

on a single stage hydrolysis of penici l l ins by dilute sodium

hydroxide solution under specific experimental condi t ions.

The degraded products containing sulphur derivat ives are

neutral ized by dilute hydrochlor ic acid to prevail a pH~2 to be

conducive for redox ti tration using potassium iodate as t i trant.

A coloured product containing sulphonic acid is formed at the

end point in organic layer. Spectrophotometry is performed

after separat ing the organic layer and measuring the

absorbance of red-purple colour at 520nm.

The t i t r imetr ic method using dilute hydrochloric acid for the

est imation of degraded products of antibiotics obtained after

the hydrolysis by dilute sodium hydroxide has been found to

be advantegeous over the old Andrews t i t r imetr ic method. In

this method strong hydrochloric acid solution is required for

the quant i ta t ive reduction of iodate to iodine monochloride

complex. Using a dilute hydrochloric acid solution, it is

possible to reduce quanti ta t ively iodate to free iodine because

the degraded products of antibiot ics contain sulphur

derivat ives which can be oxidized by a very mild oxidizing

agent. The half reaction (E°) of iodate in strong hydrochlor ic

acid and in dilute acid respect ively is described below:

IO'3 + 6 H ' + 2Cl"+4e"

2IO"3 + 12H"+ lOe'

" • ICl ' s + 3H2O E ' ^ = 1 . 2 3 V

- • I2 + 6H2O E"=1.19V

The direct spectrophotometr ic method for the determinat ion of

degraded products of ant ibiot ics is performed under similar

experimental condit ions, except that excessive iodate ( ImL) is

used for the redox reaction. The mechanism of degraded

products is established in the light of TLC, UV and IR

spectroscopic studies. The stoichiometr ic ratio between the

analyte (penici l l in) and t i trant (KIO3) is evaluated.

The resul ts obtained for the degraded products by thin

layer chromatography conform to the presence of 4-

hydroxymethylene oxazolone. This observation is in agreement

with previous studies performed under similar condi t ions.

The reaction mechanism is further aided by conducting IR

study of the degraded products . The formation of a broad band

(1500-1350cm' ' ) in spectrum-B is at tr ibuted to the presence of

4-hydroxymethylene oxazolone. Besides, a strong peak at

835cm"' in spectrum-C is at tr ibuted to the presence of

sulphonic acid formed quant i ta t ively.

The third chapter describes two spectrophotometr ic methods

for the determinat ion of a non-steroidal anti- inflammatory

agent, i.e. diclofenac sodium in the visible region of spectrum.

In the first method, the formation of charge-transfer complex

with chlorani l ic acid is utalized to carryout est imation. The

resul t ing predominant chloranil ic acid radical anion is an

intermediate molecular associat ion compound that exhibits a

character is t ic absorption maximum at 530nm. The second

method is based on the reduction of Iron(III) by diclofenac &

subsequent complexation with 1,10-phenanthroline to form a

reddish chelate complex, which shows A,max. at 500nm. The

reduction of Iron(III) and the formation of I ron(II ) -1 ,10-

phenanthrol ine complex depends on pH. The development of

colour is instantaneous in phosphate buffer solution in the pH

range 6-8. These two methods are satisfactori ly applied for

diclofenac content estimation in pharmaceut ical preparat ions .

In fourth chapter, UV spectrophotometr ic studies are carried

out to develop two different method3 for the analysis of

c isapride. In first method, Boyland-Sims oxidation is

performed for the purpose of its est imation. This involves the

t reatment of cisapride with peroxydisulphate in alkaline

medium on heating to yield a yel low coloured product having

Xmax. at 308nm. This reaction product is stable enough and

permit est imation with good reproducibi l i ty . In second method

the oxidimetr ic nature and high solution stabi l i ty of Ce(IV)

are uti l ized for the quanti tat ive determination of cisapride.

The acidic oxidation of the drug s tudUd with Ce(IV) yields a

coloured product having A,max. • • a t ^ 9 n m. Beens^'iaw obeyed in

\ { Ace. No ) n

the two methods that are described is in the range 10-

100mgmL"'and 20-120mgmL"' respectively.

The last chapter describes a kinetic spectrophotometric

determination of nalidixic acid in bulk, and its

phamaceuticals. The interaction of nalidixic acid with ferrous

ammonium sulphate yields a coordination compound, which

possess X,max. at 420nm. The intensity of colour requires forty

minutes to get stablized. It is therefore decided to perform the

kinetic study, which is sensitive, rapid and less time

consuming. The various reaction parameters evaluated conform

to the proposed mechanism. The kinetic studies reveal first

order reaction with respect to nalidixic acid.

QUANTITATIVE ANALYSIS OF DRUGS IN PURE FORM AND IN PHARMACEUTICAL PREPARATIONS

USING ANALYTICAL TECHNIQUES

T H E S I S SUBMITTED FOR THE AWARD OF THE DEGREE OF

Battot of ^dilosfopiip IN

CHEMISTRY

•* \ %

\

BY

TALAT QAYOOM

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH ( INDIA)

2000

T5388

THESIS

D e d i c a t e d

To m y P a r e n t s

Saidul Zafar Qureshi M.Sc, Ph.D.,C.Chem., MRIC ((London)

Professor of Aniytical Chemistry

Phone Off. 0091-571 - 703515 Res. 0091-571 - 400534

E-mail:- [email protected]

ANALYTICAL RESEARCH LABORATORY DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALIGARH-202002 (INDIA)

Dated.

(EFrttftratF

This is to certify that the thesis entitled "Quantitative Analysis of Drugs in

pure form & in Pliarmaceutical Preparations using Analytical Techniques"

is the original work carried out by IVIr. Talat Qayoom under my supervision and is

suitable for the award of Ph.D degree in Chemistry.

(Dr. Saidul ZararOQureshi) Professor & Chairman

mailto:[email protected]

ACKNOWLEDGEMENTS

Many ideas and encouragements from several sources have gone into carrying out the present work. There are many who need to be acknowledged, but there are few who deserve special acknowledgement at a personal level.

With pr ivi lege, I express indebtedness to my supervisor Professor Saidul Zafar Qureshi Chairman, Department of Chemistry for his analytical thinking and ceaseless efforts, which made this work progressive. It was due to his organized methodology that developed in me a sense of sinceri ty and self-confidence. This made me familiar with related fields, which has really broadened the horizons.

I would like to accord special thanks to my esteemed teacher Dr. Nafisur Rehman for his feverest help during my research programme. He generously and graciously dispensed with innovative ideas in the form of suggest ions.

With pleasure, 1 express grat i tude to my colleagues Dr. Murad, Dr. Khyer, Abdullah. S. Ahmad, Nasrul Hoda, Najmul Hejaz and Nadeem Ahmad. 1 extend thanks to my friends Atta Ahmad, Sadat Kar, Anees Ahmad, Hassan Mohusein and Omar Basudan for always being alongside me through thick and thin.

A substantial amount of credit goes to my only sister, who put up with my long absence during iny studies.

The research facili t ies of the department deserve the highest level of acknowledgement, that made charming an otherwise somber and a wil ly-nil ly si tuation.

I am grateful to all the members of Anglo Computers for their herculean efforts in typing this thesis.

(Talat Qayoom)

We raise to degrees of wisdom whom We please; but overall endued with knowledge is One, the All-knowing.

(AL-Quran)

CONTENTS

Page No.

CHAPTER!

General Introduction 1-54

References 55-67

CHAPTER-2

Simultaneous Spectrophotometric and Volumetric Determination 68-109

of Penicillins in Drug Fornuilations: Reaction Mechanism in the

Base Catalysed Hydrolysis Followed by Oxidation with lodate in

Dilute Acid Solution.

CHAPTER-3

Spectrophotometric Determination of Diclofenac Sodium in Drug 110-131

Formulations.

CHAPTER-4

Ultraviolet Spectrophotometric Studies for the Estimation of 132-153

Cisapride in Pharmaceutical Dosage Forms.

CHAPTERS

Kinetic Spectrophotometric Method for the Determination of 154-172

Nalidixic acid.

LIST OF TABLES

Table No. Page No.

1.1 Validation of analytical methods-international definitions. 6

1.2 List of the six natural penicillins. 41

1.3 Type of R-groups in penicillins. 42

2.1 Volumetric determination of amoxycillin, ampicillin and 82

cloxacillin using potassium iodate as titrant.

2.2 Volumetric determination of benzylpenicillin, carbenicillin and 83

becampicillin using potassium iodate as titrant

2.3 Spectrophotometric determination of amoxycillin, ampicillin 84

and cloxacillin after separating the extracted coloured complex

form organic layer formed at equilibrium point using potassium

iodate as titrant.

2.4 Spectrophotometric determination of benzylpenicil l in, 85

carbenicillin & becampicillin after separating the extracted

coloured complex form organic layer formed at equilibrium

point using potassium iodate as titrant.

2.5 Spectrophotometric determination of amoxycillin, ampicillin 86

and cloxacillin after separating the extracted coloured complex

from organic layer formed by adding excess of potassium iodate.

2.6 Spectrophotometric determination of benzylpenicil l in, 87

carbeni.cillin, becampicillin after separating the extracted

coloured complex from organic layer formed by adding excess

of potassium iodate.

2.7 Maximum tolerance amount of excipients in the determination 88

of penicillins.

2.8 Determination of penicillins in pharmaceutical preparations by 89-90

the recommended method.

2.9 Treatment of analytical data using statistical approach for 91

various penicillins.

3.1 Test of reproducibility of the proposed methods for the 119

determination of diclofenac sodium.

3.2 Determination of diclofenac sodium in pharmaceuticals & their 120

comparison with the reference method & statistical treatment

of the analytical data.

3.3 Maximum tolerance amount of the foreign substances in the 121

determination of diclofenac sodium.

4.1 Maximum tolerance amount of excipients in the analysis of 141

cisapride.

4.2 Test of precision for the determination of cisapride by 142

recommended methods.

4.3 Results obtained for the determination of cisapride in 143

pharmaceutical preparations by proposed methods and

statistical comparision with reference method.

5.1 Values of absorbance at different concentrations of nalidixic 162

acid, keeping the cone, of ferrous ammonium sulphate constant.

5.2 Result obtained by standard addition procedure. 163

5.3 Maximum tolerance amount of interferences added in the 164

analysis of nalidixic acid.

5.4 Statistical comparision of results obtained by proposed & 165

reference methods.

5.5 Calibration data for nalidixic acid. 166

LIST OF FIGURES

Figure No. Page No.

1.1 Goals of analytical chemistry & their relationships. 8

1.2 Analytical tetrahedra. 9

1.3 Relationships among basic analytical properties. 10

1.4 Relationship between the precision & the cone, level of 11

analytes.

1.5 Distortion of the analytical tetrahedra. 13

1.6 General reaction sequence of the total synthesis of penicillin. 43

1.7 Structure of amoxycillin (a) & dicloxacillin (b). 45

2.1 Overconsumption of iodate at the end point in the recommended 77

procedure.

2.2 Andrews titration. 78

2.3 Possible degradation pathways of penicillin. 92

2.4 Pure amoxycillin, Spectrum-A. 93

2.5 Degraded product of amoxycillin in base catalysed hydrolysis, 94

Spectrum-B.

2.6 Degraded base hydrolysed product of amoxycillin treated with 95

O.IM hydrochloric acid, followed by titration against KIO3,

Spectrum-C.

2.7 Effect of temperature on the colour stability of penicillins. 96

2.8 Effect of time on the colour stability of penicillins. 96

2.9 Effect of varying volume of sodium hydroxide for penicillins. 97

2.10 Effect of varying volume of potassium iodate for penicillins. 97

2.11 Effectofvarying volume of hydrochloric acid for penicillins. 97

2.12 Absorption spectra of the reaction product of penicillins. 98

2.13 Relation between volume of titrant (KIO^) & the concentration 99

of penicillins in reference with the table 2.1.

2.14 Relationbetween volume of titrant (K103)& the concentration 99

of penicillins in reference with the table 2.2.

2.15 Calibration curves of penicillins in reference with the table 2.3 100

2.16 Calibration curves of penicillins in reference with the table 2,4 100

2.17 Calibration curves of penicillins in reference with the table 2.5 101

2.18 Calibration curves of penicillins in reference with the table 2.6 loi

2.19 Best straight line equation in the determinatin of penicillins in 102

reference with the table 2.1.











3.1 Formation of chloranilic acid radical anion. 122

3.2 Absorption spectrum of charge-transfer complex formed by 123

chloranilic acid-diclofenac sodium.

3.3 Effect of time on the colour stability of reaction product. 124

3.4 Effect of varying volume of chloranilic acid. 124

3.5 Calibration graph of diclofenac sodium. 125

3.6 Jobs plot for the reaction of diclofenac sodium with chloranilic 126

acid.

3.7 Absorption spectra with Fe^^&Fe-'^ 127

3.8 Effectofvarying volume of 1,10-phenanthroline. 128

3.9 Effectofvarying volume of Fe^". 128

3.10 Calibrationgraphof diclofenac sodium. 129

4.1 Structure of Cisapride. 132

4.2 Absorption spectrum of cisapride-persulphate reaction 144

product in alkaline medium.

4.3 Effect of persulphate on the reaction product. 145

4.4 Effect of sodium carbonate on the reaction product. 145

4.5 Effect of heating time on the reaction product. 146

4.6 Effect of temperatrue on the reaction product. 146

4.7 Calibration graph of cisapride. 147

4.8 Absorption spectrum of cisapride-Ce (IV) reaction product. 148

4.9 Effect of cerium (IV) on the reaction product. 149

4.10 Effect of nitric acid cone, on the reaction product. 149

4.11 Calibration graph of cisapride. 150

5.1 Tentative reaction mechanism of nalidixic acid with ferrous 159

ammonium sulphate.

5.2 Absorption spectra of nalidixic acid-ferrous ammonium sul- 167

phate reaction product.

5.3 Plot of absorbance vs. time for nalidixic acid, keeping the ^^^

ferrous ammonium sulphate cone, constant.

5.4 Calibration graphs for nalidixic acid (a) Initial reaction rate (R) 1 9

vs. concentration (C); (b) log R Vs. log C.

5.5 Jobs plot for the reaction of nalidixic acid with ferrous ammo- 1 0

nium sulphate.

RESEARCH PUBLICATION(S)

I. Simultaneous Spectrophotometric & Volumetric Determination of

Amoxycillin, Ampicillin and Cloxacillin in Drug Formulations;

Reaction Mechanism in the Base Catalysed Hydrolysis Followed by

Oxidation with lodate in Dilute Acid Solution.

J.Pharm.Biomed.AnaL, 21(1999) 473-482.

Chapter-1

(genera fOnfrodvtcBon

With the growing global awareness in health hazards and

environmental pollut ion, Analytical chemistry has played a

key role to unveil its causes and day by day it has broaden

its spectrum with the concerned situation. It is extensively

and intensively responsible for analytical measurements on

variety of materials such as drug analysis , microbial

analysis of food, water, cosmetics, agricultural products,

environmental spi^cies forensic sampling and products

affecting public health and welfare where quali tat ive, semi

quali tat ive and quantitative analysis on macro to micro scale

are performed. In recent decades analytical chemistry has

reached to the pinnacle of precession with the modern

sophist icated instrumentation techniques which make

possible to elucidate the microstructure of molecular

species, determining antibiotic residues in milk and tissues

of food producing animals, detection of complex

hydrocarbons, studies of rare and artificial radioactive

elements and to obtain substances in the highest state of

purity. Moreover, with the advent of digital computer the

precision is further enhanced. However, it has always

conceived the basic phenomenon of its revelation despite the

change in instrumentat ion. Analytical excellence has been a

great boon for chemists, microbiologis ts , entomologists and

other scientis ts , working in the mult i -discipl inary areas of

research.

Medicinal chemistry has taken place in many branches of

chemistry and biological science. The earlier name of

pharmaceutical chemistry reflects that pharmacists working

in their laboratories focussed on the extraction and

purif ication of naturally occurring drugs. In 1876 the

pharmacologis t Buchheim wrote that "the mission of

pharmacology was to identify the active substances with the

drug, to find out chemical propert ies responsible for their

action and to prepare synthetic analogs that were

more effective" [1] . The pharmacologists latter became

preoccupied with other objectives, such as " to study the

change brought about the drug in the organism and then to

explore the possible influence of such changes upon

pathological condit ion" [1], while the chemist took over the

role of isolation and chemical identification of biologically

active consti tuents from medicinally important plants.

Drug design has also been aided by the understanding of

biochemical metabolism and biosynthesis , and also by the

stat ist ical analysis of some relat ionship of physical

propert ies of chemicals and their biological performance.

This progress has begun to elevate medicinal chemistry to a

science in its own right. The fashion in which the drug is

searched changes, from time to time which is dictated by

contemporary medical, economical , sociological , and

sometimes even poli t ical considerat ions. In 1890

ant iprotozoal chemotherapy set in motion and the early

synthetic antimaterials intermingled with the development of

hypnotics and anti- inflammatory drugs. Then followed the

feverish discovery of ant ibacterials and ant ibiot ics , while in

pharmacodynamic area the potent analget ics ,

ant ih is taminics , vitamins and new hormones moved to the

fore. The post world war 11 period featured three major

breakthroughs in drug research; (i) the anti tuberculous

agent, (ii) the steroid hormones and contracept ives , (iii) the

ant ipsychot ic , anxiolytic and antidepressant psychopharma-

cological drug.

The application of increasingly sophist icated methods of

structural analysis like spectroscopy, isotopic labeling,

automated quantitative analysis , separation by

chromatography and their parti t ion procedures opened

avenues of study of minute amounts of biochemicals .

Hahnemann,^ the founder of homeopathy, believed that,

"Drug's solution should be diluted to the point of practically

omitting their content of active components because high

concentrat ion of drugs produce toxic react ions ." Thomson

stated that "all the diseases arc the effect of one general

cause and may be removed by one general remedy" [2] . Paul

Ehrlich, a German physician and immunologist turned

chemist is the first true exponent drug research as we know

it today [3].

During the period 1940 to 1960, the drug development,

introduct ion and pharmaceutical manufacturing entered their

"golden age". Very large proport ion of the important and

innovative new drugs in medical use were introduced during

this period. Public expectat ions of new "miracle drugs" led

to increased funding of medicinal research, especial ly by

National Insti tute of Health in the United States and by a

number of wealthy private foundations in several countr ies .

It is important to recognize that all drugs contain impuri t ies .

There impurit ies result from many sources; like from raw

materials and reagents as reaction by-products , and through

degradation during manufacture and storage. Since, the

impuri t ies can have safety and efficiency implicat ions, they

are, therefore, the subject of considerable attention by both

the manufacturers and regulatory agencies. Impurit ies can be

classified into three groups, namely organic, inorganic and

residual solvents. Organic impurit ies may arise from starting

mater ia ls , intermediates and synthetic by-products , or from

reagents and catalysts , or as a consequence of degradation.

Inorganic impurit ies may result from reagents , l igands or

catalysts , as heavy metals or inorganic salts. Residual

solvents are inevitable in drug substances since without

solvents, the synthetic chemistry, purification and

generation of the desired crystal morphology would be

impossible .

Qualif ication is defined as "the process of acquiring and

evaluating data which establish the biological safety of the

individual impurity or a given impurity profile at the levels

specif ied." Thus, the pharmaceutical analyst and

toxicologist must work hand in hand throughout the pre-

chemical and clinical development programme in order to be

able to set meaningful specification requirements . The

methods used for impurit ies measurements , determinat ions

and qualif ications are addressed in more detail in the

coming pages. The pharmaceutical analyst must give careful

thought to the analytical technology. Especial ly in the

development phases, it may be necessary to util ize methods

with high selectivity, including sophist icated techniques.

The importance of qualifying impurity profiles as also

relevant to the developmental scientists to ensure

considerat ion is given to the impurit ies present in batches

being used in safety studies, although there are

opportuni t ies to carry out metabolic studies to help in the

qualif ication processes.

The validation of analytical methods used in bioavai labi l i ty ,

bioequivalence and pharmacokinet ic studies in humans and

animals has been the subject of discussion in recent years

[4-8] . It has been agreed that the key for evaluation of

method rel iabi l i ty and overall performance are; (i) analyte

s tabi l i ty; (ii) method select ivi ty; (iii) limit of quanti ta t ion;

(iv) accuracy and precision; (v) relat ionship between

response and concentrat ion; (vi) recovery; and (vii)

ruggedness . Before an analytical method can be used for

routine analysis , it must first be demonstrated that the

method fulfills certain performance criteria. When this has

been documented, the method is said to be validated. The

first and one of main difficulties for the pract icing

analyt ical chemist is to decide exactly which parameters

should be measured and to set the performance criteria,

which have to be fulfilled before a method can be said be

val idated. Once the parameters have been fixed, it must be

shown that they meet performance criteria. How to do this is

the second difficulty that is faced by the analyst . However,

for a number of years analytical chemists in the

pharmaceut ical and biomedical field have learnt how to

demonstrate that their methods have a performance that can

guarantee reliable results after the advent of good laboratory

pract ice. We know this exercise as validation of analytical

methods. In the field of drug analysis , it is very clear that

the above definitions cover the entire field of analytical

chemistry from bioanalysis to substance and product

analysis . This is an important observation, as the same

principle should apply whatever type of sample is to be

analyzed. There are a number of definitions given by several

internat ional organizations [Table 1.1]. Most of them are

congruent .

Table 1.1

Validation of analytical methods-international definitions |9 | .

Organization

lUPAC

ILAC

WELAC

ICH

ISO

Appl i cab i l i t y

Worldwide

Worldwide

Europe

Europe, Japan, USA

Worldwide

Remarks

Pharmaceutical

products

Lacks selectivity

and specificity

ABBREVIATIONS:

lUPAC-International Union of Pure and Applied Chemistry; ILAC-International Laboratory Accreditation Conference; WELAC-Western European Laboratory Accreditation Corporation; ICH-International Conference on Harmonization; ISO-International Organization for Standardization;

The quality of the analytical information produced

in a laboratory depends on the quality of the information

available to managers as they define the analytical problem,

design the analytical process and adjust it features so that the

results produced meet the required objectives. It is important

to have a clear understanding of analytical properties such as

accuracy and representativeness as well as their relationship

to quality in the analytical process and to the results. These

two facets of analysis are frequently dealt with

inconsistently, resulting confusion in laboratory work and in

communication with clients or services users.

Types of analyt ical p rope r t i e s

Quality in the analytical results, the primary goal of

analytical chemistry, relies heavily on two capital analytical

properties; accuracy and representativeness. Accuracy means

consistency between the results obtained and the actual

concentration of the analyte in the particular sample,

representativeness refers to consistency between the results

and the analysed sample as well as between the results and

the definition of the analytical problem. Capital properties

rely on another group of analytical properties that we will

call basic properties. They determine the quality of the

analytical process both inside and outside the laboratory

during sampling. Basic properties are sensitivity, selectivity,

precision and sampling. The analytical process is also limited

by seemingly less significant accessory propert ies. These

properties, however, often have major practical implications.

They include expeditiousness, cost-effectiveness and

personal-related considerations, all of which affect laboratory

output. Figure 1.1 shows two essential components of

analytica-l quality and displays capital, basic and accessory

analytical properties in a hierarchical manner. It also outlines

the generic objectives of present day analytical chemistry;

obtaining larger amounts of analytical information of higher

quality by expending less material, time and human resources,

minimizing risks; and incurring the smallest expenses

possible [10]. Whereas capital analytical properties are

associated with results, basic and accessory properties are

related to the analytical methodology used.

Goals Quality

Rcsulls < Accuracy RepcseiUalivencss

/

Properties

apital

t i t t Obtaining more and better information

CA a S

a. *-

• ^

in n n n

^ M ft n

o 3

GO w 3

13

g-era

Basic

\ Analytical Process Expeditiousness

Cost-effectiveness

Accesson'

Minimizing analysis time, expenses labour, and hazards

Personnel Safetv/conifort

Figure 1.1 Goals of analytical chemistry and their

relationships to analytical quality and

analytical properties.

Analytical tetrahedra

The most straightforward and intuitive form of graphically

displaying analytical properties involves two tetrahedra that

share a vertex and can be of varying sizes relative to each

other [Fig. 1.2]. This illustration provides a useful means of

distinguishing among the three principle groups of analytical

properties, establishing a hierarchy, and determining

symbiotic and opposing relationship among them. Figure 1.2

also includes sampling, which determines representativeness.

Precision Cost-effectiveness

w o a. &

s v> 3 n </> w

Personnel Safelv/coinforl

Figure 1.2 Analytical tetrahedra.

The vertices of main tetrahedron (left) depict sensitivity,

selectivity and precision; one of its faces represents accuracy,

which relies on these three basic properties. This tetrahedron,

together with representativeness of sampling, has the greatest

statistical weight on analytical results and is connected with

the objectives of obtaining more and better information. The

secondary tetrahedron (right) has vertices depicting the

accessory analytical properties; one of its faces represents

laboratory productivity. This tetrahedron is related to the

objective^ of minimizing time, costs, labour and hazards.

Relationship among basic analytical properties

Figure 1.3 depict the opposing relationships among the three

basic analytical properties. Enhancing one usually can be

10

achieved only at the expense of the other two. The central

equilateral triangle represents a balanced situation and can be

distorted to an isosceles or scalene triangle, depending on

which property is favoured. The most salient relationships

among the three properties are discussed below.

Figure! .3 Relationships among basic analytical properties.

Prec i s ion and Sens i t iv i ty

It is well known that precision decreases as analyte

concentration decreases. Figure 1.4 shows an example of the

results from intercomparison of food analysis experiments in

which the coefficient of variation increases exponentially

with decreasing analyte concentration [ I I ] . Consequently, a

higher sensitivity method will also be highly variable when

applied to trace and ultratrace determinations. Incorporating

11

an analytical separation technique to indirectly enhance

sensitivity and facilitate the determination of analyte

concentrations below the detection or quatification limit of

the analytical method used, complicates preliminary

operations and increases the time spent by the researcher. In

addition, precision is diminished.

(0

50

10

? '0 T JO

I "> I 0 o -10

J - 2 0

1-30 3 -40

-50

- t o

-

/ / y'

^ '"^ 'N^^

N -; N

. i . I 1.,, 1 1_

Izz

^

AnQ|yt»conctnlroliOn

Figure 1.4 Relationship between the precision and the cone,

level of analytes to be determined.

Sensi t ivi ty and Selectivity

Not withstanding their seemingly distant natures, sensitivity

and selectivity are also related and not always in an opposite

manner. In fact, the difference between the additive and

multiplicative interferences lies in whether the perturbation

concerned affect the slope (sensitivity) of the calibration

curve. The more sensitive the method used to determine a

given analyte, the more a sample can be diluted to decrease

the concentration of interferences and hence minimize or

eliminate their perturbations. The method is widely known

and exploited in the analysis of clinical/biological samples;

sensitive methods call for high initial dilutions (1 :10-

12

1:10,000) to minimize the typical perturbations of

niacromolecules. Sensitivity and selectivity can be enhanced

by similar mechanisms [12]. Newly emerging analytical

laboratory instruments tend to incorporate these mechanisms

to enhance their basic analytical properties. The use of non-

chromatographic continuous separation techniques and multi

channel detectors are good examples in this content.

Selectivity and Precision

The uncertainty inherent in the mechanisms described above

for enhancing selectivity is related to the complexity of the

analytical process. When several such mechanisms come into

play, the interminate error arising from each is seen in the

final results, and precision is diminished. Thus if the

selectivity of the methodology in question arises from the

detector used, the analytical process is simpler and higher

precision is achieved. Appropriate chemometric or simple

mathematical treatments increase selectivity. Univariate

calibration and differential kinetic methods [13,14] are two

good choices. As a rule the use of these methods result in

poor precision-even poorer than that obtained from analytical

separation techniques. However, one can use these techniques

to avoid some stages of the analytical process.

Accuracy and accessory analy t ica l p roper t i e s

Designing an analytical method by which to achieve high

accuracy means that high levels of the basic analytical

properties must be obtained at the expense of accessory

properties. As can be seen in Fig. 1.5a, the main tetrahedron is

13

('0

2? >

I 9 0

CO

distorted when the area of triangular accuracy face in

increased. Whereas the secondary tetrahedron is considerably

shrunk by shifting these forces pointing towards the main

tetrahedron This situation reflects the priorities among the

capital, basic and accessory analytical properties

Precision (b) Cost-cffcclivcncss

Personnel Scife(\/comfort

1

Sensitivity Personnel Safety/comfort

Figure I 5 Distortion of the analytical tetrahedra for

boosting (a)accuracy, (b)accessory analytical

propeities

The opposite situation [Figure 1 5b] occurs when temporal,

economic and /oi personal aspects related to laboratory

productivity prevail upon the quality of the results. Obtaining

immediate analytical results in response to an urgent demand

( e g , in the clinical field) or controlling a process in near

real time entails sacrificing some accuracy Thus some recent

applications rely on FT-IR spectroscopy to obtain accurate,

reproducible results in as short time as possible with

minimum sample handling and manual involvement [15]

Occasionally, a timely "yes or no" reply to indicate whether a

given parameter is above or below the tolerated level is more

14

important than obtaining an accurate analytical result a few

hours latter. Sensors [16] and screening test based on

immunoassay [17] are additional good examples.

Accessory analytical properties

The accessory properties at the vertices of the productivity

triangular face of the secondary analytical tetrahedron also

exhibit opposing relationships. The imposed or advisable

prevalence of one or two of these properties distort the

equilateral triangular face and hence disturbs the balance

among them. When an analytical process is designed to

enhance one of the three properties viz; cost-effectiveness,

expeditiousness, or personal safety/comfort-the other two

sides of the resulting productivity triangular face are

markedly shortened. If two properties rather than one are

favoured; the side of the remaining property is contracted.

Quality Compromise

Meeting the demands imposed on the information produced by

an analytical laboratory requires that a compromise be made

between the two components of the analytical

chemistry/quality binomial: external quality, which refers to

the properties of the monitored product or service, and

internal quality, which deals with the analytical process and

the results produced. External quality may result from

compliaiice with legislation or quality requisites for a

production process. It directly affect the analytical quality,

which depends on capital, basic and accessory analytical

properties. From a strictly theoretical point of view, the first

15

situation in accessory properties can be considered ideal-at

least one should reach the balanced situation depicted in

F ig l .2 . However the facts may justify this situation depicted

for accessory properties for the analytical laboratory to meet

the demands posed by the analytical problem derived from the

economic or social problem addressed.

Quality management in drug analysis covers a wide range of

quality improving activities designed to ensure the reliability

of the analytical data. These activities include ensuring that

the sample are properly collected and preserved prior to

analysis, that the analysis is carried out using the appropriate

technique, and that the results are properly recorded and

reported. Emphasis is given on the quality management needs

of research laboratories where drug analysis is carried out on

non-routine basis.

Analyt ica l Techniques

Drug analysis is taken during various phases of

pharmaceutical development, such as formulation and

stability studies, Qc and toxicological and pharmacological

testing in animals and man. In hospitals, drug analysis is

performed on patient 's samples in support of clinical trials

(bioavailabili ty and pharmacokinetic studies) and in

monitoring therapeutic drugs and drugs of abuse [18-21]. All

these investigations require reliable and validated analytical

methods in order to measure drugs in complex media such as

formulations and biofluids. Because of their selectivity,

sensitivity and overall versatility, techniques such as GC,

HPLC, superficial fluid chromatography (SFC) and capillary

16

electrophoresis coupled with selective detectors are

frequently used to analyse multicomponent drug mixture.

Immunoassays because of their speed and simplicity are

mainly used in drug monitoring in patients. Spectroscopic

(UV,IR, AAS) and titrimetric techniques are routinely used

in QC work, and TLC & more recently near infrared (NIR)

spectrometry has been used for the rapid identification of

impurities and degradation products in pharmaceuticals.

Personne l

The quality of the analytical results depends on both

instrument capability and the professional expertise of the

user, and therefore for reliable results instruments must be

used by an adequately qualified trained operators using

clearly written standard operating procedures. A detailed job

description should be available and the required level of

qualifications and experience set for each post.

E q u i p m e n t and C h e m i c a l s

When choosing new equipment it is important to ensure that it

is fit for its intended purpose and its performance is up to

specification [22]. Other things to be considered are cost,

ease of use, quality after sales-service and health and safety

aspects. There should be clear unambiguous, written

procedures for the operation of the instruments. They should

give corrfect readings within their specified tolerance limits

when checked at appropriate intervals by standard procedures

using reference materials. All standards and calibrators used

must be traceable to nationally accepted standards. Minor

17

preventive maintenance operations should be established &

followed, such as keeping pH electrodes in a wet condition or

storing chromatography columns according to supplier 's

instructions. Special attention should be given to the quality

of the laboratory glassware and reagents & standards.

Reference standards should be prepared according to written

procedures. For quantitative work, be sure to use good quality

glassware and analytical reagents and solvents, because other

grades can contain impurities. Drug substances in pure form

can be obtained from the suppliers or be requested from the

manufacturer. Due considerations should be given to the safe

storage and disposal of chemical and solvents used in the

laboratory. Finally, as a matter of good housekeeping, one

should keep laboratories clean, with equipment uncluttered

and benches tidy [23].

Method Development

Good planning is essential in the selection and development

of methods for drug analysis. It is necessary to be able

quantify the active components in the sample rapidly with

acceptable precision, accuracy and reliability within the cost

and other constraints. It often happens that during product

development or any other long-term work, analytical methods

evolve. A logic and systemic approach is required in method

selection and development. This can be outlined in four basic

steps; (a) 'generate background data, (b) review the literature

(c)develope the method (optimize the experimental

conditions) and (d) validate the performance of the method

before using it routinely. Before starting any work, all the

18

initial information on the drug (physicochemical properties

such as solubility, dissociation constant & UV adsorption)

and the dosage form or the intended dosage form (strength,

preservatives, types of container, stability etc.) should be

collected. For bio-analytical work, adequate information

about the sample, subject and dosage, including co

administered drugs should be obtained. All this information is

useful in setting up the analytical method. Bio-analyt ical

methods for pharmacological and tdxicological studies must

be selective and sensitive enough to follow the absorption,

distr ibution and elimination of the drug. Sensit ivi ty is

par t icular ly important for paediatr ic or small animal

samples since their availabil i ty may be l imited. Further

move biofluid assays must be accurate and precise in order

to reveal differences between dosage form or subjects

(human volunteers , patients or animals) .

Although experience is required in method development

work, guidance can be obtained from l i terature sources

special izing in method development [24-31] . Alternat ively,

one can use one of the commercial ly available software

packages to speed up chromatographic method development,

par t icular ly HPLC [32-33] . These systems are developed to

assist the chromatographer in the selection & optimization

of condit ions during method development. With the

assis tance of these systems, maximum information can be

extracted from a minimum number of experiments . The main

advantage of expert systems is that they provide immediate

availabil i ty of an expert opinion through an inherent

19

knowledge base, and respond to the users inquiry when

presented with a problem.

Va l ida t ion

Once the analytical method has been developed, it is

validated before or during its use. Validation of the method

establ ishes that its performance character is t ics are adequate

for the intended use. In pharmaceutical industry, validation

of analytical method is required in support of product

registrat ion appl icat ions.

Many of the principles , procedures and requirements of

val idat ion are common to the majority of analytical

methods. Validation is performed by conducting a series of

experiments using specific condit ions of the method.

Validat ion does not imply that method is free from error. It

only confirms that it is suitable for the purpose. Any

modification to the method during its use require its

reval idat ion. Some revalidat ion may also be required when

transferring the method between laboratories or when

changes are made in the manufacturing process of the drug.

Once the method has been developed and val idated, it is

fully documented and approved for the use. It should be

described in sufficient detail to allow any analyst to use it

without difficulty.

Ruggedness

In addition to the various scientific parameters , another

useful parameters to consider during validation is the

ruggedness or robustness of the analytical method. The

20

ruggedness of a method is its ability to remain unaffected by

small, unintentional changes in the experimental conditions,

such as; temperature, mobile phase composition and pH, or

different source^s of reagents and chromatographic columns.

These occur when a method is used over a long period or is

transferred to another location. Since the drug development

work can last for many years and be conducted at more than

one site, the ruggedness of the method is critical so that it

can be transferred between laboratories and used by

different workers.

System Suitability tests

Once the method is in routine use, check or system

suitability test (SST) should be run each time the method is

used, to confirm that it is performing satisfactorily. Since

the instrument to instrument variations can have a

significant effect on the assay, SSTs provide added

assurance that on specific occasion the method is giving

acceptable results. SSTs should not be confused with good

method validation, which is carried out at the method

development stage. SSTs on the other hand, check the

performance of a given system on a given day prior to

analysis. SSTs can detect normal changes in the equipment

during normal usage. If the method is robust enough, it

should not fail such tests. SSTs are particularly useful in

routine QC work & stability studies.

21

Method compar i son

The performance of a newly developed or modified method

can be assessed by comparing the results obtained by it with

those found with a reference method of known accuracy and

precis ion using linear regression analysis [34-37], A

reasonable number of samples evenly spaced over a

concentrat ion range of interest are analysed by both the

candidate method and the reference method. However it is

not always possible to conduct method comparison studies

simply because a suitable reference method is not routinely

avai lable, or is costly requiring special facil i t ies.

Solu t ions & Reagents

Standard solution of drugs in water or methanol are used

during many stages of analysis , such as calibration and

val idat ion. In bioanalytical work, although stock solutions

can be prepared in water or methanol, standard solution for

cal ibrat ion and other experiment should be prepared by

dilution of the stock solution with a relevant biological

fluid, since aqueous solution differ greatly from the

biological matrix. Drugs and reagents are sorted in such a

way to maintain their integri ty. In general solutions of drugs

& chemical are more stable at low temperatures (4or-20°C)

than at room temperature. All solutions must be clearly

labeled with preparation and expiry dates. Stock solution of

photo-decomposable substances should be stored in amber

colored containers and those of very unstable and volati le

substances should be freshly prepared.

22

Samples

Sampling is often considered to be a weak link in the quality

chain part icularly in drug bioanalysis , and is often the major

contr ibutor to measurement error. Samples should be

homogeneous and representat ive of the original material .

The sample taking should be carried out in accordance with

the approved procedures. It is essential that the samples are

collected in a suitable container at the correct time in

relat ion to the dose correctly labeled and preserved under

appropriate conditions [38-41]. The laboratory must have a

rel iable system of documentation of samples from receipt in

the laboratory to the disposal of the reminder. Samples must

be unpacked in a suitable area taking into account any safety

requirements . If the entire sample received by the laboratory

is not init ially required, it should be divided into sub-

samples, taking precautions to avoid contamination and

losses through spil lages. This practice is useful in case a

repeat assay is required at a latter stage.

Analys is

All analyses should be carried out in accordance with

writ ten procedures. Assays should preferably be performed

in duplicate each time using a separate portion of the sample

rather than a repeat determination on final solut ions. A

complete repeat analysis gives confidence in the results and

serves to check on the homogeneity of the sample and the

random variat ion in the instrumental response [42]. The

laboratory should maintain records of the results as a matter

of professional policy. The test performed, results and

23

relevant observations should be written down in a laboratory

notebook and corresponding copies of the instrument traces

stored. The notebook should serve as a record of each days

work so that each step in the analysis can be traced at a

latter stage if required.

Internal & External Quality Control

Internal QC is usually applied to drug bioanalysis to

monitor assay performance with regard to accuracy &

precision [43]. This is done by analyzing specially prepared

QC samples along-side a batch of samples and plotting the

results of QC samples on a QC chart [44-46]. Like

calibration standards, QC samples are prepared by adding

known amounts of drug in the biofluid. Usually, two

concentrations of QC samples, one near the top and the

other near the bottom of the calibration range, a third QC

samples in the middle of the range are used. For an assay

with a wide calibration range, a third QC sample in the

middle of the range can be tested. If the method is working

satisfactorily, the results of the QC sample should fall

within the pre-set limits, typically + 10% of the known

values.

If a laboratory offers drug analysis as a part of a routine

service, it is useful for such a laboratory to participate in a

an external QC scheme (preficiency testing scheme)

involving many laboratories, provided the drugs in question

are included in such a scheme. In an external QC scheme,

identical samples are periodically sent to the participants

for assay, and the scheme coordinator after receiving the

24

results reports the labora tory ' s performance together with a

summary of results from all par t ic ipants . Market

improvement in analytical standards over a period is

consistent finding with many quality assessment schemes.

Only certain groups of drugs, such as ant iconvulsant ,

ant idepressants and benzodiazepines , are monitored by

external quality assessment scheme. However, regular

par t ic ipat ion in such schemes is an effective way to measure

the competence of laboratory to undertake certain analyses.

Acc red i t a t i on

In addition to proficiency testing schemes, increasing

at tention is being given to external quality assessment

schemes by which a laboratory has to prove its competence

to an independent third party by par t ic ipat ing in a

recognized external quality assessment scheme. Laboratory

Accredi tat ion is a formal recognit ion by an authorized body

such as National Measurement Accredi tat ion Services

(NAMAS) that the labora tory ' s quality system is in place

and meets the required standards to carry out specific tests

or specific types of tests. Accreditat ion is normally awarded

following successful assessment of the l abora tory ' s system

by NAMAS officers and is subject to periodic review. Much

emphasis is placed on documentat ion, cal ibrat ion of

equipment, staff competence and training and traceabil i ty of

s tandard-mater ia ls . Accredi tat ion gives the laboratory and is

customers added confidence in the quality of resul ts .

25

L a b o r a t o r y Aud i t

Cost considerat ions prevent laboratories from jo in ing a

proficiency or accreditat ion scheme. In this si tuation, it is

useful to conduct an in house audit of the labora tory ' s

quali ty management system at appropriate intervals say at

least once a year. Auditors should preferably be independent

of the activit ies of the laboratory that they are going to

audit . Audit should cover all aspects of the laboratory

pract ice from sample collection to report ing results , and not

jus t analytical performance. It should be sufficiently

probing to highlight any drawback in the service. The use of

audit is the most reliable way of identifying problems in the

management of the laboratory. Quality management in drug

analysis incorporates all measures taken to ensure the

rel iabi l i ty of analytical data, starting from obtaining a

satisfactory sample, analyzing it correctly with a val idated

method, to reporting the results , with all procedures well

documented and periodical ly reviewed. By implementing an

effective quality system, a significant improvement is made

to the overall performance of the laboratory engaged in drug

analysis .

All the drugs in their nature are divided into inorganic and

organic. Both can be prepared from natural sources, or

art if icially, i.e. synthet ical ly. The enormous number of

drugs used in medicine can be classified into two way;

26

1. Pharmacological Classification

In this section, drugs are divided on the basis of their action

on an organism (the heart, lymphatic system, stomach,

intest ine etc.) . Accordingly, drugs are divided into such

groups as norcot ics , soporifics, analgesics , diuret ics and

local anaesthet ics .

2. Chemical Classification

Here, the drugs are divided according to their chemical

structure and propert ies regardless of their pharmacological

action. It is beyond the scope of this thesis to discuss all the

type of drugs, however, few important classes of compounds

used as drugs are discussed below.

Alkaloids

Alkaloids are substances mostly of plant and rarely of

animal origin. In medicine, alkaloids are successfully used

as drugs in the treatment of cardiovascular , nervous,

al imentary trace and many other diseases [47]. They are

organic substances of basic nature because their molecules

always contain nitrogen. The alkaloids are chiefly tert iary

amines, and only some of them are secondary amines or

derivatives of te t rasubst i tuted ammonium bases. Such class

of drugs can be grouped into the following heads.

(i) Pyridine & Piperidine derivatives ( l ibel ine & its

companions) .

(ii) Tropane derivatives (atropine, hyoscyamines, cocaine

etc.) .

27

(iii) Quinoline derivatives (quinol ine, quidine etc.)

(iv) Isoquinoline derivatives (opium)

(v) Indole derivatives (physost igmine, reserpine etc.)

(vi) Imidazol derivatives (pi locarbine, omeperazole) .

(vii) Purine derivatives (caffeine, theobromine, theophyll in

etc.)

An t ib io t i c s

Antibiot ics are very important class of pharmaceut icals

consist ing of ant ibecter ia ls , ant i - infect ives, antifungals,

ant iparasi t ics and ant imicrobials . Antibiot ics are organic

compounds derived from or produced by living organisms,

which are capable, in small concentrat ions, of inhibi t ing the

life processes of micro-organisms [48].

The chemistry of antibiotics is so varied that a chemical

classif ication is of little value. However, some antibiot ics

are the products of similar mechanism in different organisms

and that these structurally similar products may exerts their

act ivi t ies in a similar manner. A number of ant ibiot ics have

in common a macrolide s tructure, a large lactum ring.

Erythromycin, Oleandomycin are included in this family.

Tetracycl ine family represents a group of compounds very

closely related chemically. Streptomycins, kanamycins,

neomycins, paramomycins and gentamycins are included into

a family of compounds containing closely related amino

sugar moit ies . The antifungal ant ibiot ics , nystat ins and

amphotericins are examples of a group of conjugated

28

polyenes. The pencillins and cephalosporins are antibiotics

derived from amino acids. A large number of polypeptide

compounds that exhibit antibiotic action include bacitracine,

tyrothricins and polymixins.

Analgesics

A drug which brings about insensibility to pain without loss

of consciousness is known as analgesic. There are numerous

drugs which, in addition to possessing distinctive pharma

cologic activities in other area may also possess analgetic

properties [49]. The analgetic property may have direct or

indirect effect. Such class of drugs are grouped as

sedatives.(e.g. barbutrates), muscle relaxants, tranquilizers,

(e.g. meprobamate), morphine and related compounds, Some

of them been quantitatively analysed in pharmaceutical

preparation [50-53].

Central Nervous System Depressants

The agents that produce depressant effects on the central

nervous system [54] as their main pharmacological action,

include the general anesthetics, hypnotic sedatives, skeletal

muscle relaxants, tranquilizing agents and anticonvulsants.

The general anesthetics (e.g. ether) and hypnotic sedatives

(e.g. phenobarbital) produce generalized or non-selective

depression on C.N.S function. Many sedatives if given in

large doses produce anesthetic effect. Certain analgesics

also produce depression on central nervous system.

Central Nervous System Stimulants

29

Central nervous system is a complex network of sub-units

which acts as conducting pathways between peripheral

receptors and effectors, enabling one to respond to his

environment. Drugs which have in common the property of

increasing the activit ies of various portions of the central

nervous system are called central nervous system stimulants

[55] .

Respiratory Stimulants and Analeptics

Carbonic acid is a most effective respiratory stimulant.

Analeptic agents used to lessen narcosis brought about by

excess of depressant drugs often stimulate a variety of nerve

centers as well as many analeptic drugs are also predecessor

drugs because their st imulation of the vasomotor center

produce an increase in vasoconstr ic t ion. There are many

drugs (e.g. cocaine, a troprine, amphetamines, ephidrine etc.)

of varying pharmocologic classes, which in addition to their

desired effects, elicit a pronounced st imulatory effect on

central nervous system.

Cardiovascular Agents

Agents used for their action on the heart or on other parts of

the vascular system to modify the total output of blood for

the distr ibution to certain parts of the circulatory system,

are called cardiovascular agents. This class include

cardiotonic drugs, vasodiala tors , hypotensive drugs,

ant ihypercholes tero- lemic drugs and s c l e r o s i n g agents

[56]. Certain drugs have a direct action on cardiovascualr

system, and in addition, they affect certain const i tutents of

30

blood. The later include ant icoagulants , the hypoglycemic

agents, the thydroid hormones and the anti thyroid drugs.

Analysis of Drugs

The methods for analyzing medicinal agents are divided into

physical , chemical, physio-chemical and biological ones.

Physical methods of analysis involve the studying of the

physical propert ies of a substance. They include the

determination of solubili ty, t ransparency and the degree of

turbidity, density or specific gravity, melting, boiling and

freezing points.

Chemical methods used for the analysis of drugs are based

on chemical reactions. They include the determination of

ash content, the hydrogen ion concentrat ion, (pH) of

medium, and characteris t ic numerical indices of oils and

fats. Quanti tat ive and quali tat ive analysis of drugs are

generally carried out with respect to functional groups.

Physio-chemical methods are used to study the physical

phenomenon that occurs as result of chemical reaction.

Among the physio-chemical methods of analysis are,

photometry including photo-colorimetry and spectrophoto

metry, electrochemical and chromatographic methods.

Recently, various kinds of chromatography (column, paper,

thin layer, gas, gas-l iquid) and photometeric methods based

on the absorption of light by the analyte have found their

use in pharmaceut ical analysis . NMR and mass spectrometry

with gas chromatography are the most important analytical

methods used for the analysis of drugs.

31

Biological methods of analysis characterize the

pharmaceut ical effect of the drug. Biological tests are

conducted on animals and less frequently on separate

isolated organs or parts of organs of animals. The effect of

drugs is expressed in A.U, (Activity units) and is

determined by the comparison with the activity of a

reference substance. The biological methods are very

sensi t ive. Alkaloids, vitamins and antibiot ics still reveal

activity when diluted millions of t imes.

Photocolorimetr ic methods of analysis are based on

measuring the absorption of non-monochromatic light by

coloured compounds, in the visible part of the spectrum. If

the analytes are colourless, they are converted into coloured

compounds by reaction with suitable reagents. In the visual

method, called colorimetry, the intensity of the colour of

analyte solution is compared to that of standard solution if

the concentrat ion of substance is known.

In recent years pharmaceutical analysis has been

character ized by the use of methods such as

f lamephotometry and differential spectrophotometry in the

quali tat ive analysis of drugs. These methods enable one to

determine the large amount of individual components of a

mixture because the absorbance of the analyte solution is

measured not relative to the pure solvent (or a solution of

reagent) but relative to a reference solution containing a

known amount of the analyte.

Magne t ic resonance imaging (MRI) has achieved an

amazing level of success in clinical medicine as a non-

32

invasive diagnostic tool. Moreover, NMR spectroscopy is

favoured by chemists as a povêrful technique for molecular

structure determination. It therefore seems natural that these

clearly related techniques should merge to yield non

invasive, spatially localized NMR spectroscopy for

determining molecular structure and concentrat ion in vivo.

Considerable effort has been directed towards developing in

vivo NMR spectroscopy as a clinical diagnostic tool. Less

commonly, in vivo NMR spectroscopy has been used to

monitor drugs as well as other xenobiotic agents directly in

both human and animal studies. The magnitude of the

pharmacologic or toxic effect of a drug is expected to

depend on the concentrat ion at the receptor sites, which are

generally located in the tissue cells of the target organ [57].

Measuring drug levels in plasma is a reasonable method for

monitoring drug therapy. It is also relat ively non-invasive

and inexpensive, and a wide range of analytical techniques

including high-resolut ion NMR spectroscopy [58], can be

used for quantitation or identification of drug metabol i tes .

NMR spectroscopy can, in principle measure drug

concentrat ion in tissue in vivo and can probe drug

metabolism if metaboli te resonances are resolved. Because it

is non-invasive, NMR spectroscopy can be used repet i t ively

on the same individual permitt ing pharmacokinet ic and

longitudinal studies.

C a p i l l a r y e l ec t rophores i s , with its two major modes,

capil lary zone electrophoresis (CZE) and micellar

e lectrokinet ic capillary chromatography (MEKC), is gaing

33

acceptance in the pharmaceutical field for the determination

of drugs in formulation [59,60] . Sampling preparat ion

involving dissolution and dilution of the formulations in the

running buffer is perfectly adequate.

Chemi luminescence is a powerful tool for drug analysis

since its detection limits are extremely low and its

instrumentat ion very simple and of low cost. In combination

with derivatizat ion techniques in order to increase

sensi t ivi ty, it has a wide range of appl icat ions. The

excellent sensit ivity and the versat i l i ty of the

chemiluminometr ic methods of analysis are the main reason

for the recent surge of interest in CL. The introduction of

flow injection analysis and liquid chromatography with

chemiluminometr ic detectors is responsible for the plethora

of original research work devoted to this area of chemical

analysis .

Multiple linear regress ion (MLR) is the most common

choice for mult icomponent determination which allows the

easy resolution of complex mixtures provided the

contr ibut ion of each mixture component to overall spectrum

can be determined from the spectra of the pure components

used for calibration [61,62]. While rather limited in scope,

the method provides good results if background noise is

fairly low and all the mixture components are known, absorb

appreciably, and do not interact with one another. When one

or more of these conditions is not met and some analyte

absorbs weakly at a high concentrat ion of the analyte

contr ibut ions to the mixture, absorbance are rather

34

desperate, a different mathematical procedure is required.

Foremost among these is partial least-squares regression

(PLSR) [63-65] , which is also used with other analytical

tecniques where interaction between analytes are quite

strong (e.g. in IR and NIR spectroscopy).

In Cap i l l a ry e l e c t r o c h r o m a t o g r a p h y ( C E C ) , a liquid

mobile phase is driven through a stat ionary phase in a

packed capil lary column by the electro-osmotic flow (EOF)

generated by a large difference in potential across the

column. There is no pressure drop, and it follows that the

flow profile is rectangular and that small diameter part icles

may also be employed. Higher column efficiencies can

therefore, be obtained than in pressure driven HPLC. While

much interest has been shown in the theory and mechanism

of CEC, its development and eventual widespread use in the

pharmaceutical industry will only come about if CEC is

shown to have advantages over more conventional HPLC in

routine use. CEC can be applied with considerable

advantages in the analysis of pharmaceutical compounds.

Separat ions are efficient, repeatable and reproducible . As

long as the pressure is pressurized and the column

temperature controlled, quanti tat ive analysis can be carried

out with electrokinetic injection. CEC has been applied to

the separation of several pharmaceutical compounds; a

mixture of diastereoisomers, not previously separated on a

chiral HPLC column, was separated by CEC [66],

Der iva t ive s p e c t r o p h o t o m e t r y presents greater selectivity

than does normal spectrophotometry and so offers a

35

convenient solution to the problem of resolving spectral

overlap in the analysis of multicomponent system [67-68].

Peak-to-peak and base-line measurement (generally referred

to as graphical measurements) and zero-crossing

measurements are the most common techniques used to

prepare analytical working curves. The zero-crossing

method involves the measurement of the absolute value of

the total derivatives spectrum at an abscissa value

(wavelength) corresponding to zero-crossing point of the

derivatives spectrum of the interfering component. At this

wavelength, the amplitude of the derivative signal of one of

the two components passes through zero; measurement of

the value of derivative spectrum of mixture, made at the

zero-crossing point of the derivative spectrum of one of the

components is therefore a function only of the concentration

of the other component [69].

Difference specti ophotometery has proved particularly

useful in the determination of medicinal substances by

eliminating specific interference from degradation products,

co-formulated drugs and also the non-specific irrelevant

absorption from the formulation matrix. Its advantages for

selective analysis have been described by several workers

[70-71]. The technique involves the reproducible alterations

of the spectral properties of the absorbance difference

between two solutions provided that the absorbance of the

other absorbing substance is not affected by the reagents

used to alter the spectral properties. Simple aqueous acids,

alkalis & buffers are most frequently used for inducing

spectral alterations since many drugs are weak acids or

36

bases whose state of ionization and absorptivity depends on

the pH of the solution. [72].

Flow injection analysis (FIA) is characterized by its

simplicity, speed and the use of in-expensive equipment. Its

results are accurate and precise, and there are clear

advantages in terms of the short time required for each

assay. The usefulness of the (Fl) method for routine analysis

has been shown in a large number of determinations

developed for clinical, pharmaceutical, food and

environmental analyses.

High performance thin-layer chromatography (HPTLC) is

particularly attractive as an analytical tool as it is the

fastest chromatographic technique and flexible enough to

analyse different kinds of samples. It is an off-line

technique where every stage of the analyses can be

visualized. HPTLC depends on the object, scanning is

performed in reflectance and its transmittance made by

adsorbance or by fluoresence. It can be used in

pharmaceutical, medical, biological/clinical areas.

Electrochemical methods are characterized by high

sensitivity, selectivity and accuracy. Analytical sensitivity

attainable even exceeds pg./mL level and analysis in the

nanogram or subnanogram range is possible.

Electrochemical methods have been extensively developed

and each basic electrical parameter namely current (i),

resistance (r) and voltage (v) has been utilized alone or in

combination of analytical purposes. Electrochemical

methods have entered a new phase since development of

37

electrochemical detectors for HPLC. These are amperometric

or coulometric detectors which measure the current

associated with the oxidation or reduction of solute.

Pharmaceutical drugs containing groups such as phenolic,

amino, heterocyclic nitrogen, keto and aldehyde undergo

oxidation at characteristic potential. Since all compounds do

not undergo oxidation at a particular potential applied, this

increases the selectivity of the technique. Electrochemical

detection is often used where ultraviolet detectors do not

have sufficient sensitivity.

Conventional UV spectrophtometeric methods are simple,

quick, economical and usually do not require an elaborate

preparation step prior to assay. However, a UV method is

not adequate when two or more drugs with similar UV

spectra are present in the sample, or excipients or

decomposition product exhibits UV interferences, as the

case with the dissolution testing of phenytoin capsules [73].

The principle and the application of the kinetic methods

have been reviewed [74]. Essentially, kinetic method relay

on measurements of concentration changes (detected via

signal changes) in a reactant with time after the sample and

the reagents have been mixed. The use of kinetic methods in

micellar media [75] is another approach to kinetic-based

determinations that have proved useful in drug analysis.

Micelles increase the reaction rate (through micellar

catalysis) and may additionally increase the sensitivity and

selectivity for the analyte. On the other hand, new kinetic

chemometric (kinetometric) approaches such as the Kalman

filter has been developed and applied to the simultaneous

38

determinat ion of various compounds of pharmaceutical

interest .

The analysis of drug entai ls , essential ly the following;

(i)the quanti tat ive, quali tat ive and relative bioact ivi ty assay

of pure and impure drugs in different matr ices; (ii)

e lucidat ion of the mechanism of action of drugs at the

molecular level; (iii) clinical evaluat ion, including

assessment of site of action (in vivo) and side effects, and

methods of assaying development of resis tance of cells to

drug action.

Methods of quanti'âtive and quali tat ive analysis of drugs are

important in all three aspects. Quanti tat ive drug

measurement require to be of high reproducibi l i ty , precision

and accuracy should be efficient. Drug abuse also leads to a

need for quali tat ive and quanti tat ive analysis of drugs, but

because dosage forms are of high purity and precisely of

known composit ion the limits of detection are not so

demanding as in clinical medicine and reasonably large

quanti t ies of sample are usually available.

The methods currently available may be broadly divided into

two categories; (1) classical microbiological methods [76]

and modifications there of ;(2) physiochemical techniques

[77].

Microbiological assays may be considered as variants of

chemical assays in which a living organism or t issue is the

reagent responding to the substance under invest igat ion. The

39

classical techniques can be classified into (a) diffusion

methods and (b) dilution techniques.

Respirometr ic methods depend on the ability of a drug to

inhibit formation of carbon dioxide by either direct or

indirect action. Radioactively labeled carbon dioxide, and

indicator methods can be used, but the indicators should not

interfere by complexing with the drug.

Turbidimetr ic methods measure the inhibit ion of growth

photometr ical ly . The growth is assumed to be inversely

proport ional to the drug concentrat ion. Errors can be caused

by variat ion in inocula, inoculum volume errors & light

absorption by the drug.

In dilution methods, several dilutions of the drug are

incubated with an inoculum of test organisms, and the

lowest concentrat ion of drug, producing complete inhibit ion

of growth is taken as the inhibitory concentrat ion.

Numerous variation of these techniques continue to be

reported, and routinely practised in clinical and

pharmaceut ical laboratories . One of the primary reason for

this is that the structure of the drug need not be known. The

methods are also usually cheap and simple and do not need

elaborate apparatus, but are slow, have rather poor

reproducibi l i ty and are open to subjective error, and can not

be used quali tat ively.

The availabil i ty of sub-standard medicines to the general

public poses many problems, both cl inically and

economical ly. It is widely believed that such sub-standard

40

preparat ions are readily available in many developing

countries [78-79]. This may be due to poor manufacturing

procedures , poor storage conditions or deliberate

counterfei t ing of branded or generic products . However,

most l i terature reports on this problem do not contain

quanti tat ive information.

As a part of an on-going study looking at the quality of

selected pharmaceut icals in developing countries it is

necessary to assa> samples of the drugs in formulat ions. In

examining the quality of the active ingredient present , it is

of advantage to be able to detect starting materials and

major decomposit ion products if these are present. This may

allow the cause of any lack of quality determined. The

method employed should allow rapid determination of the

drug from the dosage forms with little sample pretreatment.

In addit ion the method should be validated following current

guidel ines [80-81] and in view of this a method is required

which is as simple as possible so that the val idat ion

requirement could be adhered to.

Antibiotics

Antibiot ics are defined as compounds that have inhibitory

activity against micro-organism, eukaryotic cells , viruses

that are produced by secondary metabolism of living

organism.s, and that possess a variety of widely different

s t ructures . In 1929, Fleming discovered a mould of the

penici l l ium species, which inhibited the growth of certain

bacteria. The antibiot ics cover a wide range of compounds

of different chemical structures. Penicill in is a name given

41

to the mixture of natural compound having the molecular

formula C9H11N2O4SR, and differing only in the nature of R.

There are at least six natural penici l l ins (Table 1.2).

Tab le 1.2

Chemical name

Pen t -2 -eny lpen ic i l l in

Benzylpen ic i l l in

p -Hydroybenzy lpen ic i l l i n

n -Hepty lpen ic i l l in

n - A m y 1 p e n 1 c 1111 n

phenoxymethy lpen ic i l l in

Other name

Penic i l l in- I or F

Penic i l l in- I I or G

Penic i l l in -III or X

Penic i i l in - IV or K

Dihydro-F-pen ic i l l in

Penic i l l in -V

R

-CH2CH = CHCH2CH3

-CH2C6H5

-CH2C6H4OH (1,4)

-(CH2)6CH3

-(CH2)4CH3

-CH20C6H,

The penici l l ins are strong monobasic acids, i.e., they are

known to form salts The penicil l ins get hydrolyzed by hot

dilute inorganic acids, and one carbon atom is eliminated as

carbon dioxide, and two products in equimolar proport ion

are obtained, one being an amine (Penici l lamine) , and the

other an aldehyde, (Peni l loaldehyde) . All the pencil l ins

yield same type of amine, but the aldehydes formed are

different. It is these aldehydes which contain the R group.

The R group in commonly available penici l l ins is given in

table 1.3.

42

Table 1.3

Penicillin R-group

Ampicillin

Amoxycillin

Cloxacillin

Benzylpenicillin

v\ //

H0< A //

v\ //

CH-

NH2

T NH2

• c — c -N C

0 TH3

A // CH2 —

43

The first successful synthesis of penici l l in was carried out

by Sheehan et al., (1957,1959) , after sixteen years in the

preparat ion of totally synthetic penici l l ins . The general

reaction sequence is outlined in F ig l . 6 .

R \ ,

-NCHCHO

COjCMe,

\

R' -NCH-

C02CMe3

HSCMe,

NHjCHOjH

S CH,

COjH

\

R ' -NCH- ^

COjCMe^ HN CH,

COjR'

ii'yl iici

\ .NCH-

CO,ll

\

SOCI, COjIV

0

r N-

CH,

Hj-Pd

CO^R'

R-^TIT

or OH"

S . .CH3

f CH,

COjH

^x,_^^N. -N 'l ^^J, ^^^ ^N-C,H,CH,SOjNH-

R'= CH3, QHjCHj

Fig 1.6 Total Syiillicsis of penitilliii juialogs.

44

The two commonly used semisynthet ic P-lactum antibiotics

are ampicil l in (oc-aminobenzylpenicill in) and amoxycil l in

(oc- amino-p-hydroxybenzylpenic l l in) . These are widely used

for the treatment of bacterial infections. The analysis of

these aminobenzylpenci l l ins is carried out by most of the

analytical techniques. They include flourimetry [82],

spectrophotometry [83-84], e lectroanalyt ical [85] chromato

graphic [86-87] and microbiological[88] methods. The

spectrophotometr ic determinat ions for the assay of

ampicil l in and amoxycil l in are based on equilibrium

methods, which involve a previous step where an acid [83-

84] or base [89] hydrolyses the P- lactum ring. About 20min

to 2hrs. are required for such treatments, so the equilibrium

determinat ions are slow and hinder applicat ions to routine

analysis .

Ampicil l in and cloxacil l in, due to the presence of P-lactum

and thiozol idine rings possess similari ty in their s tructures,

and a method for their analysis in combined preparat ions

must be designed to achieve a high degree of selectivi ty [90]

but only liquid chromatographic methods are selective. A

broader spectrum of antibacterial activity is provided by

penici l l in in combinations and may be advantageously

prescribed in cases of (3-lactamase producing strains.

Synergist ic activity are reported for some pencill in

combinat ions against gram-negative bacteria [91-92].

Amoxycil l in (Fig.1.7a) and dicloxacil l in (Fig.1.7b) also

possess synergy against clinical isolates of P-lactamase

producing & non-producing strains [93].

45

HO o CH-CONH.

NH2

(a)

H H

O ^

-N-

S .CH3

CH3

CO2H

i / S . .CH3

Figure 1.7 (a & b)

Pharmaceutical preparations containing above mentioned P-

lactum antibiot ics are readily available in the market. A

variety of analytical procedures are recommended for the

determinat ion of amoxycil l in, which include spectro-

photometr ic measurement of the copper chelate [94] or the

formation of penici l lenic acid by treatment with mineral

acids & copper (II) [95]. The t i tr imetric procedure is

recommended in British pharmacopoeia [96], for

amoxycil l in in bulk form using standard mercury (II) nitrate

and potent iometr ic detection of the equivalent complex, and

a colorimetr ic one using immidazole mercury reagent at

325nm in case of capsules containing the drug. Two

chromatographic procedures are reported for the assay of

ampici l l in in the presence of another penici l l in [97-98] . The

first method involves the estimation of ampicil l in embonate

and amoxycil l in embonate [97], The reverse phase

separation column is used at pH~7 with detection at 240nm.

Amoxycil l in is used as the internal standard for ampicillin

while phenoxymethylpenici l l in that for amoxycil l in.

Amoxycil l in and embonic acid are determined

s imultaneously while separate analysis is required for

ampicil l in and embonic acid. Another method involves

reverse phase-18 determination of ampicil l in and cloxacillin

46

in capsules [98]. Simple dilution with sulphamethoxazole

solution is made for sample preparation and determination is

achieved at 254n;n. Other methods for chromatographical ly

determining a single penici l l in include the determination

and monitoring of azlocil l in during bulk drug analysis [99],

benzylpenici l l in determination by capil lary zone

electrophoresis [100] and a collaborative study of

determining penicil l in V in tablets [101].

Several papers have described the use of enzyme selective

electrodes for determining penici l l ins [102-103] . The effect

of pH and buffer concentrat ion on the response of an enzyme

membrane coated wire electrode is also described [102].

Spectrophotometr ic methods continue to enjoy populari ty for

the determination of a variety of penici l l ins . The first

derivative mode of operation is found to be simple,

accurate , and precise for the determination of amoxycil l in

and clavulanic acid in pharmaceutical preparat ions[ 104].

Second derivative spectroscopy has been applied to

quanti tat ion of ampicil l in and dicloxaci l l in [105], &

ampicil l in and cloxacil l in [106]. Both method by Morelli are

suitable for the analysis of these P-lactums in

pharmaceut ical preparat ions. A forth-derivative spectro

scopic approach has been described for the determination of

4-hydroxyphenoxymethylpenici l l in in phenoxymethyci l l in

samples [107]. A second derivative spectroscopic method

described the determination of ampicil l in & cloxacil l in

[108]. The method consists of dissolution of the formulation

in water followed by scanning the spectra between 260 nm

and 330 nm. Derivative measurements are obtained at 269nm

47

and 284nm for ampicill in and cloxacil l in respect ively. The

hydrolysis of ampicill in and cloxacil l in followed by direct

spectrophotometr ic determination at 273nm has been

described [109]. The method is found to be specific to

ampicil l in in the presence of cloxacil l in & linear from 5-35

|j,gmL"'.

A variety of techniques have been evaluated for the

quantif ication of amoxycill in alone or in combination with

other penici l l ins [1 lO-l 12], Micellar electrokinetic capillary

chromatography has been found to be a useful approach for

identifying degraded products in aged amoxycil l in [110]. In

addit ion, the reaction of amoxycil l in with acetylacetone-

formaldehyde has served as the basis for a simple

spectroscopic method [111]. Beer" law is obeyed from 10-

100)j.gmL"', and no interference is observed in mixtures

containing cloxacil l in excipients . The simultaneous

flourometric determination of amoxycill in and clavulanic

acid is possible using a stopped-flow kinetic approach

[1 12]. The method involves the reaction of the analytes with

acidic Ce (IV) and detection at ' '365nm. Another

spectroscopic method for the ampicil l in is carried out using

ni t robenzene as colorimetric reagent [113]. An

electrochemical method for quantifying P-lactum antibiot ics

and their hydrolysis products is described [114]. Cyclic

voltametry at the water /ni t robenzene interface in four

electrode system is used. Hydrolysis products are

character ized across UV- vis spectrum.

48

Diclofenac sodium

Diclofenac sodium is the most commonly used, non-steriodal

ant i- inflammatory drug. It has potent analgesic, antipyretic

and anti- inflammatory actions, with minimum adverse

effects. It is an ideal drug for anthrit ic pateints , requiring

long term therapy. Rheumatic conditions are recalci trant

disorders which cause immense anxiety, loss of mobili ty,

dexteri ty and sleep to its pat ients , even leading to

depressions. Non-steriodal anti- inflammatory drugs

especial ly the sustained release forms have proved to be

highly efficacious in tackling chronic rheumatoid condit ions

associated with pain and inflammations. In chronic

condit ions, patient compliance's assume a critical role

especial ly where prolonged drug treatment is required. The

challenge to the pharmaceutical industry is to developed

dosage forms that ensure once daily dosing while

maintaining steady and constant therapeutic level of the

drug within the pat ient ' s system.

Different analytical methods have been reported in the

scientific l i terature for the determination of diclofenac in

plasma, urine, synovial fluid and pharmaceutical

preparat ions . Several anti- inflammatory drugs including

diclofenac have been quantified in different dosage forms by

capil lary zone electrophoresis and micellar electrokinetic

capi l la ry 'chromatography [115]. The experiments have been

performed without specific sample pretreatment . The

samples are directly dissolved in the running buffer and the

internal standard method has been used for assays with

49

respect to standard solutions. Diclofenac with other drugs

has been determined by flow injection analysis with

spectrophotometr ic detection [116]. The method has offered

good advantage with respect to short start-up time,

simplicity of instrumentat ion & high sampling rate. The

spectrophotometr ic determination of trace amounts of

diclofenac has been carried out by l iquid-l iquid extraction

using arcidine yellow with a flow system [117]. The

determinat ion of diclofenac in the range of 3-80}4.mgL'' is

achieved with a sampling frequency of 40 sample h' . The

spectroflourometric determination of diclofenac in

pharmaceut ical tablets and ointments has been described

[118]. It involves excitat ion at 287nm of an acid solution of

the drug, and measurement of the fluorescences intensity at

362nm. A second derivative spectrophotometr ic method has

been developed [119] for the determination of degraded

products of diclofenac sodium in gel ointment. The

ampli tudes in the second derivative spectra at 260nm and

265nm are selected to determine oxidol. The diclofenac and

codeine phosphate in diclofenac tablets has been determined

[120] by RP-HPLC at the wavelength of 250nm. Another RP-

HPLC method for the simultaneous estimation of diclofenac,

paracetamol & chloroxazone in formulations has been

described [121]. The method is carried out on zorbax C8

colunm at the detection wavelength of 220nm.

Spectrophotometr ic methods for the determination of

diclofenac in pharmaceutical preparat ions have been

described [122]. The methods are based on the oxidation of

drug using reagents viz; eerie ammonium sulphate,

50

dibromodimethylhydant ion, N-bromosuccinimide &

potassium bromate to yield coloured products with maximum

absorbances at 450, 385, 385 and 495nm respect ively.

Diclofenac sodium in Ganmaotong tablets has been

determined by first derivative spectrophotometry [123] at

292nm. A simple and precise HPTLC method has been

developed for the simultaneous determination of diclofenac

and paracetamol [124]. Voltametric propert ies of diclofenac

sodium on the glassy carbon electrode have been described

and the determination concentrat ion of diclofenac with

differential pulse voltmetry are revealed [125]. The

sensit ive anodic current of diclofenac sodium on 0.93V is

observed. Diclofenac sodium in Ganmaotong tablets has

been determined by forming a green complex with copper

acetate in chloroform in a specified pH range [126].

Diclofenac sodium also has been determined in dosage forms

[127] by spectrophotometry based on the formation of

ni troso derivatives followed by alkal inizat ion to yield a

stable yellow chromophore which exhibits A-max at 378nm.

C i s a p r i d e

Cisapride is a benzamide and a recently developed

prokinet ic drug. It decrease receptive relaxation in the upper

portion of stomach and increases antral contract ions . The

pylorus and duodenum are relaxed, while the tone of the

lower esophageal splinter is enhanced. These effects

combine to accelrate the emptying of gastric contents and to

reduce reflux from the duodenum and the stomach into the

esophagus. Cisapride is believed to increase colonic motility

51

and can cause diarrhea. The prokinetic action of cisapride is

blocked by atropine and may involve the release of

myenteric acetylcholine. Cisapride appears to be devoid of

dopaminergic blocking activity and it does not influence

the concentrat ion of prolactin in plasma or cause extra

pyramidal symptoms. Recently some reports of serious

ventr icular arrhythmia's and death have appeared among

patients who took antifungal/macrolide ant ibiot ics while on

cisapride. Blood level of cisapride was raised due to

inhibi t ion of CYP 3A4 by erythromycin, ketoconazole etc.

Used alone has not caused such arrhythmia 's . A warning has

been issued to use cisapride cautiously in patients receiving

macrol ides / imidazoles.

Cisapride has been always determined in dosage forms and

biological media. An HPLC method is described for the

quanti tat ive analysis of cisapride in human plasma [128].

Cisapride together with internal standard are analysed

isocrat ical ly on a reversed phase column and eluted drugs

are detected with UV-Vis detector at 276nm. A stability

indicat ing HPTLC method is used for the determination of

cisapride and related impurit ies in tablets [129]. Beer ' s law

follows in the concentrat ion range 2.5-15 j imgL' ' . A single

dose crossover study of two conventional formulations of

cisapride tablets is carried out in twelve healthy human male

volunteers [130]. Blood samples are collected at time

intervals and plasma cisapride is determined using RP-HPLC

with UV detection. Three spectrophotometr ic methods (A-C)

for the assay of cisapride in pure and dosage forms are

described [13 1]. Method A is based on coupling reaction of

52

cisapride with 3-methyl-2-benzothiazol inene hydrazone-HCl

in presence of FeCh to form a coloured species with X-max.

at 565nm. Method B is based on the oxidation of cisapride

with Fe^^ and subsequent chelation of Fe " with 1,10-

phenanthrol ine. Method C is based on the formation of a

coloured charge transfer complex with chlorani l ic acid

having Xmax at 555nm. Another reversed phase liquid

chromatographic method is reported for the determinat ion of

cisapride in pharmaceutical dosage forms [132]. A method is

also proposed [133] for the polarographic determinat ion of

cisapride in which the electroactive product is obtained by

ni t rat ion with potassium nitrate in sulphuric acid. The 3s-

detection limit is 1.8x10" M and the relat ive standard

deviation is 1%.

N a l i d i x i c acid

Nalidixic acid is an antibacterial agent that has been

extensively used in the treatment of Gram-negative urinary

tract infections. It is commercially available for the

medicinal use and has also found application as an effective

aid in the control of chicken infection. Nal idixic acid

inhibits the replication of DNA in bacteria, without

immediately affecting RNA or protein synthesis in sensit ive

bacteria. Its toxic effects in animals are limited to inhibit ing

mitochondrial DNA replicat ion at concentrat ions that do not

affect nuclear DNA. Nalidixic acid being a synthetic drug is

excreted together with inactive metaboli tes , in the urine. It

is bacter icidal to most of the Gram-negative bacteria that

cause common urinary tract infections, except for

53

pseudomonas strains. Resistance to nalidixic acid develops

readily but is apparently not transferable from one organism

to another.

Nal idixic acid is given orally because it is absorbed from

the gastrointest inal tract. A large proportion of the drug is

bound to plasma proteins and penetrat ion into the tissue in

poor. The plasma half-life is about 1.5 hours, and the major

route of excretion of both parent compound and metaboli tes

is via the kidney. The metaboli tes of nal idixic acid are

bacter iological ly inactive, but sufficient unmetabolised

compound is excreted into the urine to make nal idixic acid

useful in the treatment of urinary tract infections. The toxic

effect of nalidixic acid include nausea and vomit t ing, skin

rashes and photosensi t ivi ty reactions and central nervous

system effects such as dizziness and visual disorders.

Nalidixic acid can displace warfarm from protein binding

sites and can prolong the clotting time, or cause

naemorrhage, if these drugs are given together. From

analyt ical point of view several methods have been proposed

for the assay of nalidixic acid in biological samples and

commercial formulations. The luminescence character is t ics

of the inclusion complex of nalidixic acid with y-

cyclodextr in has been investigated [134]. The study involves

the characterizat ion of inclusion process involved and

development of a spectroflourimetric method for the

determinat ion of this drug. The calibrat ion range has been

found to be 0.1-2.0 ngmL' ' . A TLC procedure has been

developed for the quanti tat ive monitoring of the degradation

of nal idixic acid in solutions irradiated with high pressure

54

mercury lamp [135]. High performance TLC combined with

densi tometry enabled accurate and reproducible quanti tat ion

of nal idixic acid in the presence of its degraded products .

Another HPTLC method has been described for the

simultaneous determination of nalidixic and metronidazole

in pharmaceutical preparat ions [136]. Linear response is

observed in the range of 20-120ng and 40-140ng for

metronidazole and nalidixic acid and respect ively. Capillary

e lectrophoresis has been applied to the separation of

nal idixic acid and other quinolones of first and second

generation [137]. The same system is applied for the

quanti ta t ive determination of these quinolones in tablets and

capsules without specific sample pretreatment. An HPLC

assay for nalidixic acid has been developed. Reverse- phase

chromatography [138] is conducted using mobile phase of

ammonium acetate, methanol & acetronitr i le and detection at

254nm.

55

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Chapter-2

Simuftaneous ^joectrofofiotometric and Vofumetric Uetermination of IPenicillins in Urua "Tormufations: Reaction Is/lechamsm in me "Base Catafijsed 1-[ijdrofijsis "Toffowed hij Oxidation wim Oodate in Uifute Acid Solution.

68

I n t r o d u c t i o n

A large number of methods for the analysis of ampicil l in,

amoxycil l in and cloxacil l in and related antibiot ics are

described [1-3] . Emphasis has been given on

spectrophotometr ic and t i t r imetr ic methods, because they

are simple and easily manageable to the third world. The

volumetr ic t i t rat ions are carried out by some oxidants prior

to a base or acid catalysed reaction of ant ibiot ics . The

degraded hydrolyzed products of ant ibiot ics are t i trated

quanti tat ively against oxidants such as Hg " [4] and iodate

solut ions [5]. It is observed that when hydrolyt ic condit ions

are changed from moderate concentrat ion of alkali or acid to

a weak or using the selective pH region [6] , a different

pathway of antibiotics mechanism results . The t i t r imetr ic

method [4] described by alkaline or enzymic hydrolysis

followed by ti tration with Hg(l l) might suffer a setback. The

use of nitric acid to neutral ize the excess alkali before

performing the t i trat ion may oxidize thiozol idine ring of the

penici l l in molecule and therefore could give erroneous

resul ts . The authors themselves reported better results with

enzyme hydrolysis than base hydrolysis .

In direct t i tration of antibiot ics [5], with potassium iodate

solution, strong acidic condit ions are prevailed. The acid

plays a ,dual role, first it brings about acid hydrolysis ,

secondly it makes quanti tat ive oxidation t i t rat ion feasible.

Two procedures are adopted [5]. In the first, the end point is

detected by a colour change in carbon tetrachloride layer

from colourless to brown or deep red and in the second dye

69

Amaranth is used to detect change in colour from deep red

to pale yellow, as the drug is destroyed by an excess of

iodate in aqueous medium at the neutral izat ion point.

The determination of amoxycil l in and flucloxacil l in in

mixtures and pharmaceutical preparat ions using 3,5-dinitro-

benzoic acid as a reagent is described [7] . Extractive

spectrophotometr ic determination of benzylpenici l l in is

described in pharmaceutical preparat ion, based on the

formation of a yellowish red colour with potassium iodate in

acidic medium [8]. The colour is stable for half an hour and

Beer's law is obeyed in the concentrat ion range 0.05-0.5

mgmL"'. Another such method for benzylpenici l l in sodium

in 10% potassium nitrate solution containing sulphuric acid

involves a colour reaction with 2 ,3 ,5- t r iphenyl te t razol in

chloride [9]. The coloured product in extracted with

enthanol having ^tmax. 400nm. A selective colorimetr ic

method is described for the analysis of ampici l l in in its

reaction with p-dimethylaminocinnamaldehyde in the

presence of te t ra-chloroacet ic acid to form a stable red

sch i f f s base [10]. The coloured product is detected at

Xmax. 501nm and obeyed Beer's law in the range 10-50

/.igmL. The formation of deep blue complexes are utalized

for the micro-quant i ta t ive determination of ampicil l in and

cloxaci l l in in pure form, mixture and in pharmaceut ical

preparat ion [11]. The reaction is carried out with

bromothymol blue (BTB) at 25°C and 50°C respect ively.

Beer's law is valid in the concentrat ion range 0.4-18 and

0.4-20 mgmL' of ampicil l in and cloxacil l in respect ively.

70

The determination of amoxycil l in and f lucloxacil l in by

HPLC in biflocin injection is described [12]. The mobile

phase is phosphate buffer (pH-6.5) and the valid

concentrat ion range is 0.25-2.0 mgmL•^ Another HPLC

method is carried out for the simultaneous determination of

ampicil l in and dicloxacil l in in pharmaceutical preparation

[13]. The complexation of amoxycil l in and carbocystein

with Ni(I l ) is used for spectrophotometer ic determiantion

[14]. A flow injection analysis is described for the

determinat ion of amoxycil l in and ampicil l in [15].

Ti t r imetr ic method for the assay of selected ant ibiot ics in

non-aqueous media is carried out by conductometeric

t i t rat ion [16]. A spectrophotometr ic method for the

simultaneous determination of amoxycill in and dicloxacil l in

in pharmaceutical preparat ions is described [17]. The assay

of ampici l l in in presence of cloxacil l in in pharmaceut ical

preparat ions , which involves the reaction of hydrolysed

ampicil l in with fromaldehyde in acidic phosphate and

subsequent measurement of the absorbance at 373nm is

described [18]. Copper( l l ) acetate has been

used as a complexing agent for the spectrophotometr ic

determinat ion of benzylpenici l l in in pharmaceutical

preparat ions [19].

In this chapter, we described the base catalysed hydrolysis

of ant ibiot ics in dilute sodium hydroxide solution.

The hydrolysis product of antibiot ics which contains

sulphur derivatives e.g., -C-S-C group, -S-H group are

quant i ta t ively oxidized to sulphonic acid by t i t rat ing them

71

against potassium iodate solution as oxidant, in dilute

hydrochlor ic acid to prevail conducive atmosphere of pH~ 2.

The chemistry of this reductant system is described which

shows the observed stoichiometr ic value of thiol group to

iodate 1:2, which is consistent with theoret ical value. The

point of difference of our finding to that of previously

described by Andrews ti tration [20] have been high-l ighted.

The volumetric results are compared to spectrophotometr ic

determinat ions . The pathway and the oxidation mechanism

of hydrolysis products of antibiot ics have been proposed in

the light of IR, TLC and spectrophotometr ic s tudies.

EXPERIMENTAL

Reagents: All the experiments were performed with

analytical reagent grade chemicals using double dist i l led

water. Stock solution of ant ibiot ics , ImgmL"' were prepared

by dissolving the pure and standard drugs in water and

stored in a well-dark and closed container to avoid direct

contact with light. Hydrochloric acid standard solution

( l .OM), potassium iodate and sodium hydroxide of O.IM

each were prepared in disti l led water.

Apparatus: A Bauch and Lomb spectronic-20D'^ and

spectronic-1001 spli t-bean spectrophotmeter (Milton Roy

Co.) with 1cm path length quartz cells and a controlled

temprature water bath (NSW-133 India) were used. IR

spectra of the isolated products were recorded on a

spectrometer (Shimadzu IR-408, Japan) with KBr disc.

72

Procedure I:- S imu l t aneous determination of Antibiotics

by volumetric titration against potassium iodate and

spectrophotometry of coloured complex in organic layer.

Aliquots of 2.5 mL of O.IM sodium hydroxide were added

into conical flasks followed suitable volumes of each of the

standard antibiotic solutions, ranging between 0.2-2.0mL of

amoxyci l l in , ampicil l in, carbenici l l in and Becampici l l in ,

0.1-2.5mL cloxacil l in and 0.2-2.3mL of benzylpenici l l in .

The contents were shaken and placed in a water bath at 80°C

for 10-1 Sminutes. After completion of the heat treatment,

the mixture was cooled to room temperature, followed by

0.5mL of O.IM hydrochloric acid and 5mL carbon

te t rachlor ide . The mixture content were t i trated against

potassium iodate with intermittent shaking till the colour

changed from colourless to deep red in non-aqeous layer.

For sepctrophotometr ic determination, the non-aqueous

coloured layer was removed using 50mL separat ing funnel

and dried over sodium sulphate. Absorbance was measured

at 520nm against the blank, containing all the species

except drug. This was the idea behind simultaneous

determinat ion, that is performing two different methods at a

time in the same mixture content.

Procedure II: Determination of Antibiotics by Spectro

photometric method .

To a known volume of antibiotic solution ranging

between amoxycill in (0.4-3.5mL), ampicil l in (0.4-2.8mL),

c loxaci l l in (0.1-2.5mL), benzylpenici l l in (0.1 -2.9mL),

73

carbenici l in (0.2-2.3mL) and becampici l l in (0. l -2 .4mL), 2.5

mL of sodium hydroxide was added in a conical flask. The

reaction mixture was kept for 10-15min.on a water bath at

80°C. After cooling to room temperature , l.OmL of

potassium iodate, 0.5mL of hydrochloric acid and 5mL of

carbon tetrachloride were added to the reaction mixture. The

solution was shaken vigorously till a deep red colour was

obtained in organic layer. It was determined

spectrophotometr ical ly by the recommended procedure 1.

Dosage forms: Tablets & Capsules

Twenty tablets were weigh ed and grinded to a fine powder.

An accurately weighed portion of this powder equivalent to

lOOmg is taken in a lOOmL standard flask. Similarly the

contents of the capsules were weighed and dissolved

equivalent to lOOmg in 0.1 l i tre. The mixture was shaken for

about 30minutes to ensure a homogeneous solution. The

residue was removed through whattman No . l filter paper and

the washing were taken into final volume of 0.1 l i tre.

R E S U L T S AND D I S C U S S I O N

Mechanistic approach:

This involves UV,1R, TLC and molar s toichiometric studies.

The hydrolysis of ant ibiot ics is catalysed by a base using

O.IM sodium hydroxide solution at pH~13 in a single stage

reaction. It is carried out in a water bath at 80°C for 15

minutes with intermittent shaking result ing in different

degraded products which contain sulphur derivat ives, e.g

74

-S-S-(disulphide) and -S-H(thiol) groups. These degraded

products when subjected to redox ti tration against potassium

iodate as oxidant in dilute hydrochloric acid solution, they

are quanti tat ively converted to sulphonic acid with

some intermediate species viz; penici l lenic acid(IIb) and

sulphoxide(IIIb) . The degradation pathway is more precisely

i l lustrated in figure 2 3 -

The results obtained by performing thin layer

chromatography(TLC) with Rf = 0.46 in solvents (v/v)

comprising, 66% n-butanol, 17% glacial acetic acid and 17%

water, suggested the presence of 4-hydroxymethylene

oxazolone. This observation is in agreement with previous

studies [6], which confirms the existence of la, (Rf = 0.42 in

the same solvent system). Our studies of the degraded

products of penici l l ins at pH~13 involving a single step

reaction using water bath at 80°C for 15 minutes show one

Xmax at 240nm. This at tr ibutes the existence of one of the

three species viz; Ib(i) or Ib(ii) or Ib(i i i) . Moreover the

formation of penici l loic acid Ib(i) from basic hydrolysis of

penici l l in is well established [21]. Another Xm&x at 270nm

is obtained when time interval is extended to 12 hours, thus

confirming the establishment of 1(a). The previous studies

show the existence of 1(a) as a degraded product of

ant ibiot ics in the pH range 13-14 with UV spectroscopic

analysis showing Xmax at 297.5nm. The slight difference in

the Xmax. value may be at tr ibuted to different experimental

condit ions of hydrolysis , such as temperature , time of

hydrolysis and pH etc.

75

To carryout IR studies of ant ibiot ics , three different spectra

were taken for,

(i) Pure amoxycil l in, Spectrum-A (Fig.2.4) .

(ii) Degraded in base catalysed hydrolysis , Spectrum-B

(Fig. 2.5)

(i i i) Degraded base hydrolysed products t reated with O.IM

hydrochlor ic acid followed by t i trat ion against KIO3,

Spectrum-C (Fig. 2.6).

The comparison of spectrum (A) and (B), revealed that the

character is t ic bai>ds for different groups at specified

frequencies in spectrum (A) are found (i) missing, (ii) the

formation at different frequencies or (iii) remained

unchanged. A broad flat portion (3450-2600cm"') same as it

was found in spectrum (A), at tr ibuted to primary al iphatic

amine N-H (3450-3250cm"') , carboxylic (3300-2500cm"')

and S-H (2600-2500cm' ' ) groupings.

The bands are found missing in the range (1800-1500cm"')

and 1350-lOOOcm"') result ing the formation of bands at

860cm"', 680cm"' and in the range (1500-1350cm"') . The

identi t ies of thiozoiidine and secondary amine groups with

different group frequencies lying in the range (1800-

1510cm"') are completely swept out. The second range of

missing band (1350-1000cm"') includes the groups e.g. CH3-

S, ester etc. The two missing ranges are revealed by a

descending and ascending portion of spectra-B respect ively.

The formation of a broad band (1500-1350cm"') is at tr ibuted

76

to the presence of 4-hydroxymethylene oxazolone ( la) ,

which is revealed by the group frequency C-0 stretching and

0-H deformation vibration (1440-1395cm' ' ) . The ascending

portion (1350cm"' onwards) of spectrum-B further describes

the hydrolysis of rest of the groups present. A strong peak at

860cm"' may be at tr ibuted to S-0 (870-810cm"') of

sulphonic acid. The peak at 680cm"' suggests to disulphide

-S-S, C-S (705-570cm' ' ) and sulphoxides (>S=0, C-S-0 at

730-690 c m ' ) after their partial oxidation to sulphonic acid

in semiquanti ta t ive manner as the sulphur compounds

especial ly containing the group S-H, S-S are oxidized by

mild oxidant or even atmospheric oxygen [22]. The spectrum

-C, which is obtained after completing the t i tration against

iodate in dilute acidic condit ions, gave the final product

corresponding to strong peak at 835cm' ' , a t t r ibuted to the

presence of sulphonic acid quanti ta t ively.

The recommended procedure 1 describes s imultaneous

volumetr ic determination of penici l l in against potassium

iodate as oxidant in dilute acid medium. The deep-violet

colour extracted in carbon tetrachloride at the equivalence

point is stable for two hours and has been studied

spectrophotometr ical ly . The hydrolysis product of

ant ibiot ics which contained sulphur derivatives i.e., -C-S-C

(lb), -S-H groups are quanti tat ively oxidized to some

intermediate species and finally to sulphonic acid whose

identi t ies have already been discussed in IR studies . The

hydrolysis by dilute sodium hydroxide solution has been

carried out slowly at a constant temperature 80°C with

77

intermit tent shaking. The excess of base is neutral ized by

adding hydrochloric acid (0.5 mmoles) so that the reaction

mixture attains a pH~2. This provides a conducive

environment of HjOîon concentrat ion for carrying out

quanti tat ive t i tration of degraded products against potassium

iodate. The reaction is summarized as follov/s;

6Q

HS-R+3H2O -• RS03H+6H'+ 6e"] x 2 Step I

I0"3 + C1" 6 H ' +4e" > ICI + 3H3O'] x3

In the first step reaction a quanti tat ive oxidation of thiol to

sulphonic acid followed by reduction of IO3' to ICI takes

place which may be regarded as the reaction at equivalence

point, but without any colour change. This shows that the

s toichiometr ic value calculated theoret ical ly should be of

2mol. of thiol group to 3mol of iodate (1:1.5) which is

inconsis tent with experimental value 1:2 [23]. However, the

system still involves more iodate ions to make the end point

visible due to its reduction to iodine monochloride and

finally to free iodine. This is described in step II reaction

^3^.4c«pT;«^d diagramatical ly: "^"^""^ ''" IO3(+5) mdilute HCl

reduction

10-3 + Cr + 6Fr+4e ^ ICI+3H2O Step II

M:55Q9 '^ Quantitafttese oxitfiltrbn ot penicillin PenecillinIOi(l 1 5)

-. i ; itfiltibn ^ JpWr^nd point

Increases

True end point

Penicillin 10", (1 ly

ylCU+l) • More IO3 involeved in theSyite*-..

Overconsuniption of IO3

' I: (0 00)

Fig .2 .1 . Over consumption of iodate at the end pt. in

recommende^d procedure.

78

iCl dissociation

-> I'+cr

hydrolysis > H O l + H " r+HsO

over consumption , 5 H 0 I • 2 l 2 + IO-3 + H " + 2 H 2 0

of iodate

In the step II, an overconsumption of iodate takes place

[24], thereby increasing the mole ratio of iodate from 1.5 to

2mol which is consistent with the exper imental ly observed

stoichiometry of Imol thiol to 2mol iodate. A stable red

violet colour of free iodine in organic layer indicates the

equivalence point. It is observed that further addition of

iodate solution beyond the end point results no

decolourizat ion in mixture content. This means that a

quanti tat ive oxidat ion-reduct ion in dilute hydrochloric acid

has taken place in the system. The titrant iodate is reduced

first to iodine monochloride and finally to free iodine to

show a visible end point. The E° of iodate in dilute

hydrochloric acid (1.19V) is less than the E° in Andrews

t i trat ion (1.23V). The E° (1.19V) is high enough to oxidize

the degraded products of antibiot ics containing derivat ives.

In Andrews t i trat ion strong hydrochloric acid (3-9M)

solution is required for reduction of iodate to iodine

monochloride (ICI2") complex.

Penicillin complclely consumed -4

10-3 (+5) Strong HCl (3-9 M)

IO"3+6H*+2Cr+4e" ^ IClz+SHaO E° = 1 23V

T IC1'2(+1)

In presence of penicillin

• 12(0 00) 2ICl"2+2e- ^ I2+4CJ-£"= I06V

Figure 2.2 Andrews t i t rat ion

79

During the course of t i trat ion with reducing agent, the

presence of free iodine in the system also results from the

further reduction of ICI2' complex as given above. When the

reducing agent has been consumed, the free iodine is t i trated

to form iodine monochloride complex. The end point is

marked by the disappearance of the last free iodine. (A point

of difference from our observation given in Step II).

2I2 +IO".v + 6H"+ lOCr -> 51Cr2+3H20

The above conditions prevail , if the concentrat ion of the

hydrochlor ic acid at the end point is at least 3M. The iodate

reacts quanti tat ively with reducing agents such as Kl,l2,

AS203,N2H4 and SO2 to iodine monochloride [25]. In many

other cases the concentration of hydrochloric acid is not

cri t ical , but for Sb( l l l ) , it is 2.5 to 3.5M; that is the

optimum acidity for reasonably rapid reaction varies from

one reactant to another reactant [20] .

The recommended procedure 1, which describes the use of

dilute hydrochloric acid is applied successfully to the

simultaneous quanti tat ive reduction of iodate to free iodine

at the equivalence point, especial ly in a system which

contains the degraded products of ant ibiot ics with sulphur

derivat ives which can be oxidized by very mild oxidizing

agents [22] . The half reaction of iodate in dilute acid

solution with E° [25] can be proposed as under.

2IO"3 + 12H"+10e" -^ I2 + 6H2O E° = 1.19V

The method described earlier [5] for the direct t i t rat ion of

ant ibiot ics by iodate solution in a high concentrat ion of acid

is based on Andrews method. The reduction of iodate to

iodine-monochlor ide complex involving electron change of

80

four and oxidation of thiol group from penici l lamine (IV) to

sulphonic acid(VI) and that of aldehyde from

peni l loaldehyde (111) to the carboxylic acid(V) are

accompanied with electron changes of six and two

respect ively.

The overall s toichiometry has been computed by summing

up two oxidation products with a total electron change of

eight which is consistent with their experimental

s toichiometr ic ratio 2mol iodate and Imol penici l l in . This is

revealed by the following react ion:

Penicill in + [V] +[V1] + 8e"

10"3 + 6 H ' + 2 C r + 4e" -^ ICl'z + 3H20]x2

However, if the above oxidation reduction is considered

separately it reflects a different aspect as given below:

H-S (IV) -> H03S"(Vl)+6e"]x2

I0"3 +4e" -» I d s ' ] X 3 (i)

-CHO (111) -^ COOH (V) + 2e"]x2

I0 '3 + 4e" ^ I C l 2 " ] x i (ii)

In reaction (i), the stoichiometry of 2mol penici l l in /H-S to

3mol iodate and in (ii) 2mol penici l l in/-CHO to Imol

iodate are observed, which is inconsistent with their

experimental ly stoichiometric ratio of iodate to penici l l in

2 :1 .

We have tried to elaborate the above reaction system taking

place in two fold manner i.e. potassium iodate is reduced to

iodine monochloride in the presence of high concentrat ion

of acid (12moldm' ' ' ) . During the cource of t i t rat ion as long

as the reductant is present in the system, iodine

monochloride is further reduced to free iodine. The stage of

81

titration can be regarded as the first step, when equivalence

point can be declared theoretically which immediately

vanishes due to further reaction of iodine with iodate. The

second step of titration is started with free iodine to yield

iodine monochloride complex, the disappearance of deep red

violet colour to colourless resulting the declaration of

equivalence point, followed by an over consumption of

iodate which increases the number of moles of iodate,

approximately to experimental value.

The effect of interfering substances in the determination of

penicillin are shown in Table2.7. This study shows the

maximum tolerance of interfering substances in the

minimum amount of penicillin. The validity of the proposed

method was tested by successive determination of penicillin

in pharmaceutical preparation. The results obtained by the

recommended method are presented in table2.8. The

treatment of analytical data using statistical approach for

the determination of all the penicillins using different

concentrations have been presented in Table2.9. The best

straight line equation with variables x and y and

incorporating the slope and intercepts for each antibiotic is

computed.

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ei t «

A. Degraded products; single stage hydrolysis of penicillin at pH ~ 13

92

R-CONH-HC HC^ "^C-CCHbb

OC- N— penicillin

OH

-CH-CO2H

At SOT , (15 minutes)

H H

R - C O - N H - C C"" ""CCCH^b

H H s

R-CO-N-CH2—C^ ^C(CH,)2

-CO2

HOOC HN CH-COOH

penicilloicacid

HN- - CH-COOH

Ib(i) - H 2 O

penillonicxid.

Ib(ii)

HS. N-

OH

4-hydiioxymethylene (oxazole-5(4H)-one

la

N- I Airoxidation Conversion into NH-CH — > species; Ub,mb,

R' C C

pencillenic add

Ib(iii)

COOH Seniquartilative pyt.

B. Oxidized Products: Obtained titrimstrically using IO3" as oxidanL I2/IQ3*

0 II

R-S-

OH

Sulphonic add

IVb

O O oxidation „ 11 H r. oxidation M R—S—S—R M

Sulphoxide

fflb

N C=

c c

. I NH-CH

I COOH

penidllatiic acid (disulphide)

R - S - S - R Db

Fig 2.3 The possible degi-adation pathways of penidllin; Followed by oxidation with iodate ions.

93

(*/>)33NViilHSNV)Ji

94

o o S o O

(•».)3DNtlllWSN>rBl

95

o o o O

«0 o o

(X)33NVi imSNVUi

0.4 n 96

70

D-

•

M

X

•

- A = amoxycillin

- B = ampicillin C = cloxacillin

D = benzylpenicillin

E = carbenicillin

F = becampicillin

80 90 100

Temperature (°C)

Fig. 2.7. Effect of temperature on the colour stability of penicillins, >.max. = 520nm.

0.5 1

0.4

S 0.3-1 c (0

w o (/> < 0.2

0.1 •

• • X X X X

• • a • 1 A — A

• A = amoxycillin

-a— B = ampicillin

-A— c = cloxacillin

X D = benzylpenicillin

X E = carbenicillin

• F = becampicillin

10 15 20

Time (min.)

Fig. 2.8. Effect of time on the colour stabilityof penicillins, A,max. = 520nm.

97

z J— _—-•

B

•

A • •

•

A

• 0

•

8 A • •

•

A • •

•

A • •

•

— • — • — •

A = amoxycillin A C = cloxacillin *— E = carbenicillin

-•— B = ampicillin -X— D = benzylpenicillin -•— F = becampicillin

I

25 I

35 05 1 15 2 25 3 35 4

Volume of NaOH (mL) Fig. 2.9. Effect of varying volume of sodium hydroxide at 80°C,

Xmax. = 520nm.

- 1

45

• • • * • » X X X K M M A A A A A A

« X X X K S

• • • • • « • - • - • -• • •

—••— A = amoxycillin

—^-~ C = cloxacillin

—*— E = carbenicillin

- • — B = ampicillin

—H— D = benzylpenicillin

—»— F = becampicillin

1 5 2 25 3

Volume Of KI03(mL)

35 45

Fig, 2.10. Effect of varying volume of potassium iodate at 80°C, ; max. = 520nm.

0) u c (0 Si b . o (/) n <

04

0 3 "

02

01 ••— A = amoxycillin B = ampicillin C = cloxacillin D = benzylpenicillin

—*— E = carbenicillin —•— F = becampicillin

I I I I I I I • I •

0 01 02 03 04 05 06 07 08 09 1

Volume of Hcl (mL) Fig. 2.11. Effect of varying volume of hydrochloric acid at 80°C,

A,max. = 520nm.

98

0 9 -

0 8 -

07

06

0) u c TO

o V)

<

05

04

03

02

01

400 I -

440 I

480 520 560 — I

600

Wavelength (nm)

Fig. 2.12. Absorption spectra of the reaction product of penicillins at SO'C.

0.5

0.4

%"'

o i 0.2

"o >

0.1 •

99

lli

• A = amoxycillin

B B = amplcillin

A c = cloxacillin

1 2

Amount of Drug (mg)

Fig. 2.13. Relation between volume of titrant (KIO3) and the concentration of penicillins in reference with the table 2.1.

0.3

0.2

c ra

O a> E 3 •5 0-1

-•- A = benzylpenicillin

-B— B = carbenicillln

-A- C = becampicillin

0 1 2 3

Amount of Drug (mg)

Fig. 2.14. Relation between volume of titrant (KIO3) and the concentration of penicillins in reference with the table 2.2.

100

• A = amoxycillin

- • - B = amplclllin

—*r- c = cloxacillin

Amount of Drug (mg)

Fig. 2.15. Calibration curves of penicillins in reference with the table 2.3.

-•— A = benzylpenicillin ~o— B = carbenicillin ~A—C = becampicillin

Amount of Drug (mg)


101

-•— A = amoxycillin • B = ampiclllin A C = cloxacillin

Amount of Drug (mg)

Fig. 2.17. Calibration curves of penicillins in reference witli the table 2.5.

0.8

0.7

-•— A = benzylpenicillin -B— B = carbenicillin -A— c = becampicillin

Amount of Drug (mg)


0.5 102

0 .4 -

0.3

O 0 .2 -

E

0 1 2

Amount of amoxycillin (mg)

0.5

0.4 -

5 0-3 -J

O 0-2 •

E = 0.1 H

y

0 1 2 3

Amount of ampicillin (mg)

0,4 1

E 0.3

'4-> C 2 •S 0.2 -o

I 0.H I

0 1 2

Amount of cloxacillin (mg)

Fig. 2.19. Best straight line equation in the determination of penicillins in reference with the table 2.1.

0.5

;j 0.4-1

c 0.3

1 0.2

0-K

103

2 0 1

Amount of carbenicillin (tng)

c (0

0.5

0.4-

0.3-

0,2-o a

I 0.1-1

1 1 1

0 1 2 3

Amount of benzylpenicillin (mg)

0.4

0.3

2 0.2

o * E 0.1-I I

0 1 2 3

Amount of becampicillin (mg)


u re J3

0.5 •

0.4

0.3

| o .

0.1 •


104

0.5 n

0.4-

g 0.3 ^ a n 8 0.2 H X) <

0.1

0 JL — I 1 1

0 1 2 3


0.6

0.5

c re •e 0.3 o (0 < 0.2-

0.1 -

0 0 1 2 3

Amount of cloxaciJIin (mg)


0) u c n € o I

0.6-]

0.5-

0.4-

0.3-

0.2-

0.1 •

n

(

•

•

D

•

1

1

«

•

2 3

Amount of carbenicillin (mg)

105

0.5 n

0.4

0.3 -J 8 0.2 §

o c n o

<

0.1 -

0 1 2 3


0 4 - ,

0 3

0.2

0.1

0 - t

0 1 2 3



106 0.6

0,5 H

« 0.4 o

1 0 . 3 o iS 0.2-^ <

0.1

0 ~l

4 0 1 2 3


0) u c 10

o

<

1.11

1

0.9

0.8

0.7-

0.6-

0.5-

0.4

0.3

0.2

0.1

0

0


9) U C ra

JQ k. o w n <

0.9 1

0.8

0,7

0.6

0.5

0.4

0.3

0.2

0.1

0

Amount of cloxacillin (mg)


o u

n JQ

0,3

0.2

O XI 0.1 -<

0 1 2 3

Amount of carbenicillin (mg)

107

a> o c «

o w SI

<

0.4

0.3-

0.2

0.1 -

0 1 2 3 4


0) o c ra Xi o w <

0.6 1

0.5

0.4-

0.3-

0.2

0.1 -I

0 1 2 3



108

REFERENCES

[ I ] A.G. Davidson and J.B. Stenlake, . 99(1974) 476.

Anlyst

[2] J .Talegaonkar and K.S. Boparia, Talanta. 29(1982)

525.

[3] U.Forsman, Anal. Chim. Act. 93 (1977) 153.

[4] B.Karlberg and U.Forsmaan, Anal. Chim Acta.

83(1976) 309.

[5] J.K. Grime and B.Tan, Anal. Chim. Acta. 105 (1979)

361 .

[6] J.L. Longridge and D.Timms, J.Chem. Soc. (B) (1971)

852.

[7] A.S. Amin, An. Quim. 89(7-8) (1993)726.

[8] A.S.Khan and I. Ahmad, Indian Drugs. 33(2) (1996) 71

[9] V.K. Shormanov and E.P. Duritsyn, Farmatsiya. 44(4)

(1995) 31 .

[10] P. Parimoo, K. Padma, R.J. Babu and K. Ravishankar,

East. Pharm. 38 (456) (1995) 115.

[ I I ] Y.M. Issa and A.S. Amin, Egypt. J. Pharm. Sci. 36 (1-

6) (1995) 235.

[12] Fenxi Ceshi Xuebao. 14(3) (1995) 87; Chem. Abstr.

123 (1995) 237991V.

[13] K.A. Al-Rashood, J. Liq. Chromatogr. 18(2) (1995)

2457.

[14] G.G. Pargaonkar and S.G. Kashedikars , Indian Drug.

31(12) (1994) 590.

[15] M.S. Garcia, C.S. Pedreno, M.I. Albero and V.

Rodenas, J.Pharm. Biomed. Anal. 12(12) (1994) 1585.

i09

[16] K. Esma, K. Filnat, K.Adnan and A.M. Abdul Kadir,

J. Pharm. Biomed. Anal. 13(12) (1995)1453.

[17] M.Ezzat and A. Moety, J. Pharm. Biomed. Anal. 9(2)

(1991) 187.

[18] A.O. Akanni and J.S.K.Ayim, J. Pharm. Biomed Anal.

10(1) (1992) 43.

[19] U.Saba, Analyst. 111(1986)1180.

[20] L.W. Andrews, J.Amer. Chem. Soc. 25(1903) 756.

[21] M.A. Carroil and J.E. Zarembo, J. Antibiot . 30(1977)

811 .

[22] D.R. John and M.C. Caserio, " Basic Principles of

Organic Chem" 2"'' edn. (1977) P. 745.

[23] El-Sebai A. Ibrahim, S.M. Rida, Y.A. Baltagy and

M.M.Abd El-Khalek, J.Drug. Res. (Egypt) . 6(1973) 13.

[24] I.M. Kolthoff and R. Belcher, "Volumetric Analysis" ,

Vol. I l l , (19S7) p. 450.

[25] I.M. Kolthoff, E.B. Sandell , E.J. Merhan and

Bruckenstein, "Quanti tat ive Chem. Anlysis ." 4'' edn.,

(1969) p. 819, 1159.

Chapter-3

ShBctrohhotometrk Ueferminafion of Dkfofenac Sodium in Urua "Tormufations.

no

Introduction:

Diclofenac sodium is the sodium salt of [o-(2,6

dichloroanilino) phenyl] acetic acid and is an antipyretic,

analgesic&well-tolerated non-steriodal anti-inflammatory

agent with potent activity in the treatment of rheumatic

diseases. The assay of diclofenac sodium is carried out by

most of the analytical techniques. A method for the

simultaneous determination of diclofenac sodium and its

hydroxy metabolite has been described [1] by cappillary

column gas chromatography with electron capture detection

which follows extractive alkylation. The method is also

applied to analyze urine samples of volunteers treated with

single doses of diclofenac sodium. A high performance

liquid chromatographic assay has been proposed [2] for the

determination of a number of anti-inflammatory drugs

including diclofenac in plasma. The samples in this method

are prepared by adding acetronitrile and perchloric acid to

200).iL plasma. Diclofenac sodium with other drugs is

quantified in the supernatant procedure using a mobile phase

of perchloric acid-acetronitrile and a detecting wavelength

of 254nm. The method is also applied to the cross -sectional

study of medication compliances. Another sensitive HPLC

procedure for the assay of diclofenac and its

monohydroxylated metabolites in biological fluids has been

described [3]. Diclofenac is assayed in plasma at

concentration down lOngmL'', while in combination with its

monohydroxylated metabolites it is assayed in urine, after

chemical analysis at concentration down to 200 ngmL"'. A

liquid chromatographic method has been developed [4] for

I l l

the determination of diclofenac and related materials in

formulations and raw materials. The method resolves ten

related compounds with limits of quantitation 0.2% or less.

Pharmacokinetics of diclofenac sodium has been also

studied [5] by developed high performance liquid

chromatography in serum and urine using acetaminophen as

internal standard, a reversed phase C-18 column and

ethanol: water as mobile phase. A spectroflurometric

determination of diclofenac sodium in bulk and

pharmaceutical preparations has been described [6]. The

method is based on the hypersensitive property of the

fluorescent probe ion Eu"" , at 616nm. The interaction with

diclofenac, which contains a carboxylic group, makes the

transition allowed and enhances the intensity of its

flourescence emission. A spectrophotometric method based

on the fixation of diclofenac on sephadex QAEA-25 resin as

solid phase has been described [7]. The absorbance of solid

phase is measured at 281nm and 400nm. A multifactor

optimization techniques has been successfully applied to

develop new spectrophotometric method in which diclofenac

is analysed and determined as its Fe(III) complex [8]. A

four-variable two level factorial design is used to

investigate the significance of each variable and interaction

between them. A colorimetric method for the determination

of diclofenac sodium is based on the formation of coloured

charge-transfer complex with chloranil [9]. The complex is

formed in EtOH showing Xmax. at 545nm and concentration

ranges between 0.08-0.9 mgmL''. Another charge-transfer

study of diclofenac for its determination has been carried

112

out with p-benzaquinone and chloranil [10]. In ehtnol

medium the reaction occurs readily with p-benzaquinone,

while the reaction with chloranil is feasible in alkaline

medium. Spectrophotometric methods for the determination

of diclofenac sodium in its formulations have been described

[11]. The first method involves the oxidation of drug studied

by ammonium persulphate in alkaline medium and coupling

with dimethylaminobenzaldelyde reagent. The resultant

solution is a yellow coloured product with its analytical

wavelength at 450nm. In second method the diclofenac has

been treated with sodium hypobromite in presence of

cetrimide in alkaline medium. The yellow coloured solution

is determined colorimetrically at 460nm. Another

spectrophotometric method has been described [12]which

involves the treatment of diclofenac with sodium nitrite in

presence of hydrochloric acid, producing a yellow colour

having /\,max. at 390nm. Two more spectrophotometric

methods have been described [13] for the determination of

diclofenac sodium. In the first method diclofenac reduces

Iron(III) to Iron (II), which subsequently forms complex

with 2,2-bipyridine. The chelate complex formed posses

maximum absorbance at 520nm. In the second method the

diclofenac forms ion pair complex with methylene blue in

presence of phosphate buffer which is extracted with

chloroform. The complex formed shows maximum

absorbance at 640nm and the graph of absorbance versus

concentration is linear in the range 5-40^lgmL"^ An

extractive spectrophotometric determination of some anti

inflammatory 4rugs including diclofenac in pharmaceutical

113

preparations has been described [14]. It is based on the

formation of a chloroform soluble ion-association complex

between the drugs studied and methylene violet at pH~7.6.

The present work describes the colorimetric methods for the

determination of diclofenac sodium in bulk and commerical

dosage forms. In first method determination is achieved

through the formation of stable donor-acceptor complex with

chloranilic acid. The resulting intensely reddish chloranilic

acid radical anion possesses a characteristic absorption

maximum at 530rm. The second method is based on the

reduction of Iron(IIl) by diclofenac and subsequent

complexation of Iron(II) with 1, lO-phenanthroline, having

maximum absorbance at 500 nm. The possible interferences

by various excipients have been checked out. Application of

the suggested methods to representative dosage forms are

presented and compared with the reference method.

E X P E R I M E N T A L

Apparatus Method-I

A Spectronic-20D^ (Split-beam Milton Roy Co)

spectrophotometer, with Icm path length quartz cell was

used.

Reagents

All reagents used were of analytical-reagent grade and the

solvent of spectroscopic grade. A 0.1% chloranilic acid

.solution in 1,4-dioxane and the diclofenac sodium solution

of ImgmL'' concentration in methanol were used.

114

General procedure for the calibration:

-1 Accurately measured volumes equivalent to 0.1-1.80 mgmL

diclofenac sodium was transferred into a series of lOmL

standard flasks. Then 2.5mL of chloranilic acid solution

were added to each flask. The contents were mixed well and

made up to volume by a solvent mixture of 1,4-dioxane and

methanol made in the ratio 2:8. The absorbance was

measured at 530nm against the reagent blank prepared

simultaneously without diclofenac solution.

Procedure for the assay of dosage forms

About ten tablets were finely powered. An amount of the

tablet powder equivalent of 50mg of the diclofenac sodium

was weighed accurately and treated as described above for

the standard drug solution. Filtration was performed in

instantaneous where insoluble matter remained during

preparation of the sample solutions.

RESULTS AND DISCUSSION

A chloranilic acid solution in 1,4-dioxane is yellow. The

spectrum of 0.1% chloranilic acid in 1,4-dioxane possess a

strong band at 440nm. Addition of diclofenac sodium to this

solution causes an immediate change in the absorption

spectrum, with a new characteristic band at 530nm (Fig 3.2),

believed to be due to chloranilic acid radical anions.

Reaction involved

Chloranilic acid in organic solvents exist in un-ionized form

and acts as a 7i-acceptor in similar manner to other quinones

115

[15,16]. Therefore, the addition of diclofenac sodium results

in the formation of charge-transfer complex of n-7i and n-n*

type. The complex is believed to be intermediate molecular

association compound for which in appreciable polar

solvents, complete transfer of charge takes place, producing

the corresponding radical ion, which is responsible for the

production of intense absorption band at 530nm. This

predominant chroinogen with chloranilic acid is the reddish

radical anion, which is formed by the dissociation of an

original donor-acceptor (DA) complex.

D + A^=T b—A ^=r D '+A • DA Complex (radical ions)

The dissociation of the DA complex is promoted by the high

ionizing power of solvent. Diclofenac acts as an electron

donor and hence forms charge-transfer complex with TC-

acceptors. The donated electrons originate from the nitrogen

atom and the aromatic ring forming n-n and n-K* complex

respectively as shown in Fig. 3.1.

Optimization of variables:

To develop a quantitative method based on this reaction, a

search was conducted for most effective 7r-acceptor.

Chloranilic acid in 1,4-dioxane readily reacts with

diclofenac sodium, forming a stable reddish chloranilic acid

radical anion. 1,4-dioxane proved to be most suitable

solvent. Other organic solvents were not suitable because

the complex formed in these solvents either had low

absorbance or precipitated on dilution. The reaction was

instantaneous and the product remained stable for forty

116

minutes (Fig. 3.3) permitting quantitative determination to

be carried out with good reproducibility.

Performance characteristics:

Under the proposed experimental conditions, there was a

linear relationship between absorbance and final

concentration over the range 10-180|agmL'^ of drug (Fig.

3.5) with corresponding molar absorbitivity of 1.5x10"

LmoT'cm"'. The jobs plot (Fig. 3.6) showed the molar ratio

of 1:2 between diclofenac and the chloranilic acid. The

reproducibility of the method was determined by the

analysis of various samples covering the recommended

concentration range for diclofenac sodium. The recoveries

show, the method is reproducible, accurate and precise

(Table 3.1).

Application to dosage forms

Results for determination of diclofeanc sodium in different

dosage forms are also presented and show good agreement

with those of the reference method [12]. This conclusion

was confirmed by statistical analysis, showing no significant

difference in accuracy and precision between the proposed

and the reference methods (Table 3.2).

Apparatus Method-II

A Spectronic-20D'^ spectrophotometer, with 1cm cell, and a

water bath (NSW-133) India was used during heat treatment

and the colour development.

117

Reagents:

All the reagents used were of analytical grade.

A 0 .1% ferric ammonium sulphate (w/v) solution and O.OIM

EDTA solution were prepared in O.OOIM sulphuric acid and

conductivity water respectively. A O.IM solution of 1,10

phenanthroline was prepared in ethnol. A mixed phosphate

buffer (pH= 6.8) was used.

Aqueous 0.1% (w/v) pure diclofenac solution was used for

the preparation of calibration curve.

Preparation of the calibration curve

Mixed up l.OmL of buffer and 2mL of Iron (111) solution in

lOmL volumetric flasks. Added sample solutions containing

0.05-1.0mgL"' of diclofenac, followed by 0.5mL of 1,10-

phenanthroline. Heated for 25 minutes on a boiling water

bath for the complete development of colour, then added

l.OmL of EDTA solution, dilute up to the mark with

distilled water, and measured the absorbance of the coloured

solution against the reagent blank at 500nm.

The proposed method was employed for diclofenac content

estimation in commercial pharmaceutical preparations. A

comparison of the reference method was also performed

(Table 3.2).

Ten tablets were crushed and finely powdered. An

accurately weighed amount equivalent to 50mg of diclofenac

was stirred in 50mL conductivity water. The residual solid

was filtered on Whattman No-42 paper and washed with

118

water. The filtrate was made up to 50mL in a standard flask.

The determination was carried in the same way as for the

pure drugs in the calibration procedure.


The absorption spectrum of the blank Iron (II) and

Iron (111) complexes are shown in the Figure3.7. The

maximum absorbance for both A and B is observed at

500nm. The shapes of the absorption spectra are quite

similar for A and B.

The l ron(l l ) - l , 10-phenanthroline complex shows an intense

red colour and is more stable than its corresponding ferric

complex. The ferric complexes are shown to get reduced by

reducing agents, which is the basis of present work. The

reduction of Iron(lll) by diclofenac and the formation of

Iron(lI)-l , 10- phenanthroline complex depends on pH. The

development of colour is instantaneous in mixed phosphate

buffer solution in the pH range 6 to 8. The photo-reduction

of the ferric complex is avoided by adding a chelating agent

such as ethylenediamine tetra-acetic acid to mask the

excessive ferric ions after the colour development. Beer's

law is obeyed in the concentration range 5-100|agmL''

(FigS.lO) with the molar absorbitivity of 4x 10^1moL''cm''.

Some excipients with the dosage forms are found not to

interfere (Table3.3). Paracetamol being an oxidant, interfers

with the test, so the method can not be used for the

determination of diclofenac in presence of paracetamol. The

method is accurate and sensitive with respect to the

microgram range of determination and higher molar

absorbitivity, than are the earlier methods.

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0.4 • 124

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125

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126

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127

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129

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130

REFERENCES

[1] W.Schneider and P.H. Degen, J. Chromatography. 217

(1981) 263.

[2] S.G. Owens, M.S. Roberts and W.T. Friesen, J.

Chromatography. I l l (1975) 293.

[3] J .Godbil lon, S. Gauron and J.P. Metayer, J.

Chromatography. 338 (1985), 151.

[4] Beaulieu. N, Lbvering. E.G, Lefrancois J. and Ong.H,

J. Assoc, off. Anal. Chem. 73(5) (1990) 698.

[5] Said. S.A, and Sharaf. A.A, Arznemit te l forschung.

31(12) (1981) 2089-92.

[6] Carreira. L.A, Rizk. M, el-Shabrawy Y, Zakhari . N.A.

and Toubar. S.S, J. Pharm. Biomed. Anal. 13(11)

(1995) 1331-7.

[7] M.L. Fernandez. Cordova, P. Ortega. Barrales and

A.Molina. Daiz, Anal. Chim. Acta. 369 (1998) 263-68.

[8] S.Agatonovic-Kust in, L.J. Zivanovic, M. Zecevic and

D. Radulovic, J. Pharm. Biomed. Anal. 16 (1997) 147-

53.

[9] El-Sadak. M, Bark. M, Khier. A.Adil , Egy. J. Pharm.

Sci. 24(1-4) (1988) 367-79.

[10] Shen-Hui Zhang, Jain-ZhangFeng, and Shen-yung

Tung, Fenxi kexue Xuebao. 12(3) (1996) 194-7.

131

[11] K.Shivram, B.V. Kamath, and V.P. Arya. East Pharm.

34(402) (1990) 119-20.

[12] Y.K. Agrarwal, V.P. Upadhyay and S.K. Menon, Ind. J.

Pharm. Sci. 50 (1988) 58.

[13] Y.K. Agrarwal and K.Shivramchandra, J. Pharm.

Biomed. Anal. 2(9) (1991) 97-100.

[14] C.S.P. Sastry, A.S.R. Prassad.Tipirneni , and M.V.

Suryanarayana, Analyst . 114 (1989) 513-15.

[15] Alaa. S. Amin, Gamal.O. El-Sayed, and Yousry. M.

Issa, Anlyst. 120(4) (1995) 1189-93.

[16] M.Abdel-Hedy Elsayed and Suraj. P. Agarwal, Talanta.

29 (1982) 535-37.

Chapter-4

%{ftraviofet Sheotrohôiomeirk Studies for fke "Estimation of Cisapride in IPfiarmaceuticaf Uosaae orms.

132

Introduction

Cisapride is a gastrointest inal s t imulat ing agent, effective in

the relief of gastrointest inal or esophagus disorders and in

the promotion of gastric emptying of gast rointes t inal

moti l i ty [1]. It is a prokinet ic agent believed to facil i tate

acetyl choline release from the myenteric plexus of the gut

[2] . It is chemically known as (+) c is -4-amino-5-chloro-N-

[ 1 - [ 3- f lourophenoxy )propyl ] - 3- methoxy -4 -p iper idyl ] -

2-methoxy benzamide (Figure 4.1).

Fig 4 . 1 . Cisapride

The drug is official in the British Pharmacopoeia [3] . The

official method for the assay cisapride involves the t i trat ion

with perchloric acid and the determination of end point by

potent iometry . Three spectrophotometr ic methods for the

determinat ion of cisapride are described [4]. The methods

are based on the coupling of the diazotized drugs with the

reagents such as chromotropic acid, pholoroglucinol and N-

(-1-naphthyl) ethylene diamine dihydrochloride to give

coloured products having maximum absorbances at 530nm,

450nm and 540nm respect ively. A colorimetr ic procedure for

the est imation of cisapride in dosage forms has been

desci ibed [5]. The method is based on the reaction of

133

cisapride with para-dimethylamine c innamaldehyde in

presence of t r ichloroacet ic acid in methanol forming a

sch i f f s base with ;^max. at 525nm. An HPLC method for the

determinat ion of cisapride in tablets and oral suspensions in

the presence of methyl polysi loxane is developed and

val idated [6]. A reversed phase Cig Bandapak column and

mobile phase comprising methanol and water were used. The

drugs were detected and assayed at 275nm. Another HPLC

method for the assay of cisapride in plasma is described [7].

Alkal inised samples of plasma are extracted with l.OmL of

propanol in chloroform. Cisapride and internal standard are

detected by flourescence monitoring at 295nm (exci ted) and

350nm (emission) . Cisapride levels in human plasma are

determined by RP-HPLC method [8] in which carbamazepine

is used as internal standard. Cisapride and the internal

standard are extracted from the plasma with

dichloromethane and detected by UV detector at 370nm. A

flourimetric method for the determination of cisapride in

pharmaceut icals [9] under continuous and discont inuous

condit ions is based on the fact that cisapride exhibits an

emission spectrum with a maximum at a wavelength of

355.2nm and excitat ion at 310nm. A spectrophotometr ic

method for the determination of cisapride is based on its

reaction with p-dimethylaminocinamaldhyde in phosphoric

acid solution to form an orange chromogen [10] with

maximum absorbance at 546.5nm and obeying Beer's law

in the range 160-560ngmL"' . A UV Spectrophotometr ic

method for the determination of cisapride in pharmaceut ical

dosage forms is developed [11] with its concentrat ion range

134

between 2.0-20|igmL''. Two more methods are developed for

the determination of cisapride [12]. In the first method the

drug is reacted with N-dimethylaminocinnnamaldehyde in

presence of sulphuric cid to form Schiff base having A,max.

at 520nm and obeying Beer's law in the range 0.8-12)igmL' .

In the second method, the drug solution reacts with folin-

cocalteu reagent in presence of sodium carbonate forming a

blue chromogen with Xmax. at 880nm and linearity in the

concentration range 0.4-8.0)j,gmL"'. The diazotization

followed by coupling with N-(l-naphthyl) ehtylenediamine

dihydrochloride or thiocol is studied with the aim of

standardizing the methadol of cisapride pharmacentical

analysis [13]. Reaction products with N-(l-napthyl)

ethylenediamine has an absorption maximum at 535nm and

products with thiocol at 495nm. The development of a

spectrophotometric method based on the charge transfer

formation of cisapride with iodine and catechol is reported

[14]. Four extractive spectrophotometric methods for the

assay of cisapride in pure and dosage forms based on the

formation of chloroform soluble ion-associates under

specified experimental conditions are described [15]. Four

acidic dyes viz; Suprachen violet 3B, Erioglaucine A,

Napthalene Bluel2 BR and Tropaelin 000 are utalized.

Another spectrophotometric method for the determination of

cisapride [16] in dosage forms is based on the oxidation of

drug with nitrous acid followed by treatment with cresol fast

violet acetate and measurement of the absorbance.

In this chapter, two different procedures for the

determination of cisapride are reported which are based on

135

redox reaction followed by spectrophotometric studies. In

the first method, cisapride is treated with peroxydisulphate

in presence of alkili. A yellow coloured product is obtained

on heating, which has A,raax. 308nm. The second method

involves acidic oxidation of cisapride with Ce(IV) to

produce coloured product having Xmax, at 289nm. The

methods are simple, accurate and can be satisfactorily

applied to representative pharmaceutical formulations.

EXPERIMENTAL

Instrumentat ion Method-I

DU-40 spectrophotometer (Beckman) was used for

absorbance measurements. Controlled temperature water

bath NSW-133 was used for heat treatment & colour

development.

Reagents

All chemicals used were of analytical or pharmaceutical

grade. Pure cisapride was obtained as a gift sample from

Sigma Laboratories Mumbai (India) and prepared (ImgmL'')

in dimethylformamide (DMF). Potassium persulphate and

Sodium carbonate of O.IM each were prepared in double

distilled water.

Calibrat ion

Aliquots of 0.1-l.OmgmL"' of the standard solution of

cisapride were pipetted into a series of lOmL volumetric

flasks. Optimum results were obtained after the addition of

2.0mL of potassium persulphate and l.OmL of sodium

136

carbonate. The mixture was heated on a water bath at 85-

90°C for ten minutes to complete the reaction. After cooling

to room temperature and dilution to lOmL with distilled

water, the absorbance of the yellow coloured product was

measured against the reagent blank at 308nm.

Application of the proposed method

About ten tablets were grinded to a fine powder. The tablet

powder equivalent to 50mg of cisapride was weighed

accurately and treated as described above. The insoluble

matter remained during preparation of sample solution &

was filtered off.


The reaction mix,ture of cisapride and peroxydisulphate in

alkali yields a yellow coloured product on heating having

>.max. at 308nm (Fig 4.2), whereas the reagent blank shows

Xmax. at 236nm. This type of reactions between aromatic

amines and persulphate ions in aqueous base have been

already cited in the literature [17-20]. Such reactions are

appropriately named as Boyland-Sims oxidation. The

mechanistic approach is believed to be an electrophilic

displacement by the peroxide oxygen [21]. This involves the

electrophilic attack by the persulphate ion on the nitrogen of

the neutral amine at ortho orientation to from aryl

hydroxylamine-0-sulphonate. This intermediate is then

assumed to rearrange to the 0-aminoaryl sulphate. The intial

attack at nitrogen is consistent with the work in other

systems [22-25]. The postulated intermediate is more readily

137

oxidized than the start ing material . The yield of o-aminoaryl

sulphate is believed to fall in strong alkali , a fact

a t t r ibutable to more ready attack by persulphate on the

dianion of the intermediate formed.

Study of the proposed experimental condit ions.

To obtain optimum condi t ions , the effect of different

volume ratio of persulphate reagent and sodium carbonate

are studied. It was observed that 2.0mL of O.IM persulphate

(Fig. 4.3) and l.OmL of O.IM sodium carbonate (Fig. 4.4)

gave the optimum results for the complete oxidation of

c isapr ide. Under the optimum experimental condit ions the

product is found to be stable after ten minutes of heating

(Fig. 4.5) on a water bath at 85-90°C (Fig. 4.6) Beer 's law is

obeyed over a narrow range from 10-100iJ,gL' (Fig.4.7) with

the molar absorbit ivi ty of 4.63x10^ Lmol ' ' cm"'.

The reproducibi l i ty of the procedure is studied by running

various repl icate samples, each containing different amount

of drug in the final solution (Table 4.2). In order to check

the validi ty of the proposed method, the resul ts are

compared with standard reference method [14]. The results

are presented in the (Table 4.3). A study of possible

interference caused by common diverse ions in dosage forms

has been performed. The results are summarized in (Table

4.1).

The proposed method is sensit ive and rel iable at the

recommended concentrat ion range. The stabil i ty of the

colour is high and is not affected by moisture. Hence, the

138

proposed method can be recommended for the routine

analysis of cisapride in drug control laboratories.

Reagents Method-II

The solution for cisapride (ImgmL' ') was prepared in

dimethylformamide and Ce(IV) Stock solution (0.4M) was

prepared in 2M nitric acid.

Calibration Procedure

To a series of lOmL standard flasks, appropriate volumes of

standard solution of cisapride between 0.2-1.2mgmL"' were

placed, followed by 1.5mL of Ce(IV) solution. The contents

were mixed well and allowed to stand at room temperature.

The volume was completed with dimethylformamide and the

absorbance meaurements were recorded at 289mm against

the reagent blank.

Assay of dosage forms

Ten tablets were grinded and powdered. The content of

tablet power equivalent to 50mg of cisapride is dissolved in

dimethylformamide, It is filtered instantaneously and

proceeded as described under " Calibration Procedure".


Ammonium eerie nitrate is a versatile oxidimetric reagent.

Its high oxidation potential and excellent solution stability

promoted us to use this reagent for the quantitative

determination of cisapride. The absorption spectrum for

Ce(IV) solution in 2M nitric acid shows ;\.max. at 357nm,

139

whereas its reaction product with cisapride gives absorption

maximum at 289nm (Fig.4.8), thus indicating a

hypsochromic shift with respect to ?iniax. of the cerium (IV)

solution. The condit ions for the production of analyt ical ly

useful measurements from Ce(IV) oxidation of cisapride

were optimized so as to achieve maximum and reproducible

absorbance measurements . The Ce(IV) concentrat ion and the

nature and concentrat ion of the acid present have a marked

influence on the analytical signal and were invest igated to

obtained maximum absorbance reading. The effect of cerium

(IV) concentrat ion for I.O mL of cisapcide is shown in

(Figure 4.9). Nitric acid was the most effective medium for

the sensit ive measurement of cisapride, and cerium (IV)

concentrat ion of 2.73x10"^ molL ' ' was chosen for further

s tudies . For the given concentrat ion of cer ium(IV), 2moIL' '

nitric acid produced maximum absorbance (Fig .4 .10) , hence

this concentrat ion was chosen for the analyt ical procedure.

Under the selected experimental condit ion described above,

the cal ibrat ion graph was linear in the concentrat ion range

20-120|.igmL' of cisapride (Fig.4.11) . The regression

equation evaluated for the cal ibrat ion graph is

A=0.21x+0.019, with the correlat ion coefficient of 0.9989.

In order to apply suggested method for the analysis of

pharmaceut ical dosage forms, the influence of commonly

used excipients and addit ives was invest igated and the

resul ts obtained are presented in Table 4 . 1 . Under the

react ion condit ions used, the foreign substances tested did

not interfere with the proposed method.

140

The precision of the method was determined by the percent

error for ten multiple analyses of a series of solutions. The

accuracy of analysis was tested by applying the

recommended procedure (Table 4.2). The recoveries of the

different amounts tested were determined from the standard

reference graph and found to be satisfactory.

The proposed spectrophotometric method has been

successfully applied for the determination of cisapride in

pharmaceutical dosage forms. The results obtained for this

procedure were evaluated against the reference method [14]

as shown in (Table 4.3). The proposed method has the

advantage of being simple, accurate and sensitive for

estimation of cisapride in pure form and formulations.

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147

0.2 0.4 0.6 0.8

Amount of Cisapride (mg)

Fig. 4.7. Calibration graph) of cisapride at (85-90°C), Xmax =308 nm.

1 5n

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150

02 04 06 08 1

Amount of Cisapride (mg)

12 14

Fig. 4.11. Calibration graph of cisapride, Xmax = 289 nm.

151

R E F E R E N C E S

[1] J. Robert Sims and W.Slivka. PCT Inst. Appl. W09501 ,

803 (cl. A61K37/54) , 19 January 1995, US Appl. 87,

590, 06July 1993, 21pp.

[2] F.Onat, B.Yegen, K.Berkman and S.Oktay, Gen.

Pharmacol . 26(1994) 1253.

[3] British Pharmacopoecia , Vol .1 , HMSO, London, 1995,

P.1746.

[4] Sast ry .C.S.P, Srinivas.Y and Subba. Rao.P.V, Indian J.

Pharm. Sci. 58(4) (1996) 169-71.

[5] Par imoo.P, Padma.K, Jagadesh.Babu. R and

Ravishankar K, East Pharma. 39(457) (1996)159-60.

[6] Gaba. Sangeeta, Badwa. Nagesh and Sethi. P.D, East

Pharma. 38 (453) (1995) 157-158.

[7] Preechagoon. Y and Charles .B, J. Chromatogr. Biomed.

Appl. 670 (1) (1995) 139-43.

[8] Arafat. Tawfiq, Shami. Munzar, Kaddomi. Amal,

Badwan Adnan and Yassein. Mohamed, Alexandria . J.

Pharm. Sci. 9(1) (1995) 1-3.

[9] Gonzalez Martin M.l, Gonzalez. Perez. C and Blanco

Lopez. M. A, Anal. Lett. 27 (9) (1994) 1713-18.

[10] Barbhai . A.J, Mahadik. R.K, Moore H.N. and Panzade

P.D, Indian Drugs. 3 6 ( 1 0 ) ( 1 9 9 9 ) 665-666.

152

[11] Bhatia M.S, Kashedikar .S.G and Chaturvedi .S. C,

East Pharma. 42 (504) (1999) 131-132.

[12] Mohan. Y. Rame, Srinivas. J .S, Avadhanula. A. B and

Anjaneyulu Y, East Pharma. 40 (480) (1997) 119-21.

[13] Honda. Akimi. Mori, Magalhaes and Joae. Farnendes ,

Rev. Farm. Bioquim. Univ. Sao Paulo. 33 (2) (1997)

111-115.

[14] Krishnakumar. K.R, and Raju. R, Indian. J. Pharm. Sci.

58(1) (1996) 39-40.

[15] C.S.P. Sastry, Y. Srinivas, P.V. Subba. Rao, Talanta.

44 (1997) 517-526.

[16] Sastry. C.S.P, Srinivas. Y and Subha. Rao. P.V, J. Inst.

Chem (India) . 68 (4) (1996) 108-109.

[17] E. Boyland, D. Manson and P. Sims, J. Chem. Soc.

3623 (1953).

[18] E. Boyland and P. Sims, J. Chem. Soc. (1954) 980.

[19] P. Sims, J. Chem. Soc. (1958) 44.

[20] E. Boyland and P. Sims, J. Chem. Soc. 4198 (1958).

[21] N. Venkatasubramanian and A. Sabesan, Cand. J.

Chem. 47 (1969) 3710.

[22] D.B. Denney and D.Z. Denny, J. Amer. Chem. Soc.

82 (1960) 1389.

153

[23] K.M. Ibne-Rasa and J.O. Edwards, J. Amer. Chem.

Soc. 84 (1962) 763.

[24] K.M. Ibne-Rasa, C.G. Lauro and J.O. Edwards,

J. Amer. Chem. Soc. 85 (1963) 1165.

[25] D.M. Graham and R.B. Mesrobian, Can. J.Chem.

41 (1963) 2938.

Chapter-5

%inetk SpecfropftofomeMc TAetfôdfor ifê

Uetermination of Nafiaipdic acid.

154

Introduction

Nalidixic acid ( l - e thy l - l , 4 -d ihydro -7me thy I -4 -oxo- l , 8 -naph-

thyr id ine-3- Carboxylic acid) is an ant ibacter ial agent which has

been occasional ly used intravenously for the t reatment of gram-

negative urinary tract infections since 1963. Renal failure is

common amongst these pat ients and therefore it is necessary to

monitor the plasma concentrat ion in order to prevent toxic or, on

the other hand subtherapeutic levels . The major metabol i te ,

hydroxynal idixic acid is in vitro also active against Gra:m-

negative micro-organisms. Nal idixic acid has also been used for

the prevent ion and treatment of infectious diseases in fish.

Concern has arisen as to the presence of drug residues in fish

t issues, and demand for a rapid, simple and sensit ive analyt ical

method for determining them has increased. In man nal idixic acid

is converted to the 7-hydroxymethyl metaboli te which is further

oxidized to generate the dicarboxylic acid.

A variety of methods have been described for the assay of

nalidixic acid in biological media and dosage forms. A high

pressure liquid chromatographic method has been developed for

the s imultaneous assay of nal idixic acid and hydroxynal idixic

acid in human plasma and urine [1]. The procedure measures

both the compounds in the same sample. Chromatograms from

control plasma and control urine yield a small blank which do

not interfere with chromatograms from processed s tandards. A

gas chromatographic method has been described for the

determinat ion of nal idixic acid in plasma [2]. The method is

sensit ive enough to determine therapeutic levels in l.OmL

155

plasma. Quantitation is carried out by means of both peak height

measurements and peak area measurements. A method for the

simultaneous determination of nalidixic acid and its analogues in

cultured fish has been developed by high performance liquid

chromatography [3]. The method also involves UV and

flourometric detection, and Sep-pak Cu cartridges as a clean-up

step. A selective TLC- densitometric method has been described

by means of which nalidixic acid is determined in duplicate with

good precision at therapeutic levels in a 100-}a,L plasma sample

[4]. A high performance liquid chromatographic method for the

analysis of nalidixic acid in plasma and urine has been described

[5]. The statistical evaluation of this assay technique shows

acceptable accuracy and precision at concentrations 2.0|j,gmL'' of

plasma or 29.0)igmL'' of urine for samples augmented with the

drug. A differential pulse polarographic method has been

proposed for the determination of nalidixic acid and its

metabolite in urine [6]. The method involves a simple extraction

which is followed by controlled-potential analysis at the mercury

pool electrode and at the HMDE. Another high performance

liquid chromatographic method has been described [7] for the

analysis of nalidixic acid and its two metabolites in human

plasma and urine following the oral administration of a

therapeutic dose in human. The applicability of this method to

pharmacokinetic studies of nalidixic acid in humans has also

been demonstrated. A gas chromatographic method has been

established for the quantitative analysis of nalidixic acid [8].

The method is based on the derivatization of nalidixic acid with

diazomethane, and 5-a-cholestane is used as an internal

156

Standard. Quantitation is achieved by measuring peak height

ratios. The improved simplicity, specificity & accuracy of the

method has been demonstrated for the quantitation of nalidixic

acid in tablets. A high performance liquid chromatographic

analysis of nalidixic acid and its hydroxymetabolite in plasma

with an anion exchange system has been reported [9]. Proposed

experimental parameters have also been studied. Another HPLC

assay of nalidixic acid in plasma after alkylation with methyl

iodide has been performed [10]. A packed column supercritical

fluid chromatography has been employed [11] for the assay of

nalidixic acid and metronidazole in bulk and combined dosage

forms employing mebendazole as the internal standard. The

validation of the method of assay of drugs has been done by the

internal standard method 8L linear range for both the drugs has

been 10-60|j.gmL'V A pH-induced difference spectrophotometric

method for the simultaneous determination of metronidaole and

nalidixic has been developed [12] in presence of each other as

well as the excipients. The presence of identical isobestic points

indicate the non-interference of excipients in the absorption at

respective wavelengths. A spectrophototric method has been

developed for the simultaneous determination of nalidixic acid

and metronidazole in pharmaceutical preparation [13] using

multicomponent mode of instrument. Another method for the

simultaneous determination of these drugs in two component

tablets and suspensions is based on the dual wavelength

spectrophotometry [14] without any prior separation. Two more

methods have been developed for the simultaneous determination

of nalidixic acid & metronidazole. The methods used

simultaneous equation and Q-analysis for their determination

157

[15]. The absorbance maxima of two drugs and their isobestic

point are used as analytical wavelength. Another

spectrophotometric procedure using multicomponent mode of

analysis has been developed for the simultaneous determination

of nalidixic acid and metronidazole in their dosage forms [16].

The method is based on the measurement of UV absorbance

maxima of the two drugs.

The literature survey for the assay of nalidixic acid has revealed

that, a few methods are available in the visible range of

spectrometry. Besides, the unstability of its reaction product

with ferrous ammonium sulphate at maintained temperature

(50+0.2°C) made us to develop a kinetic method which permits

sensitive determination within a short limit of time. The study

shows that the interaction between nalidixic acid and ferrous

ammonium sulphate is first order reaction with respect to

nalidixic acid. The proposed method can be successfully applied

to the commercial dosage forms.

EXPERIMENTAL

Instrumentation

DU-40 Spectrophotometer (Beckman) was used to record the

spectra and Spectronic-ZOD"" (Milton Roy Co.) was used for

absorbance Vs. time measurements. A temperature controlled

water bath (NSW-133 India) was used to maintain a constant

temperature (50 + 0.2"C).

158

Reagents

All the Chemicals used were of analytical or pharmaceutical

grade. Stock solution of 5.0xlO'^M ferrous ammomium sulphate

was prepared in 1.0X10"' 'M sulphuric acid. Pure nalidixic acid

(Himedia Labs. India) ImgmL"' was prepared in methanol.

Calibration Procedure

In 10ml volumetric flasks, the final concentration of standard

pure solution of nalidixic acid between 0.2-0.8mgmL'' were

placed, flowed by 1.5mL of ferrous ammonium sulphate. The

flasks were immersed in a water bath maintained at 50+0.2°C.

Each flask was taken out at a time interval of five minutes,

cooled at room temperature and diluted upto the mark with

distilled water. The contents were transferred to the

spectrophotometric cell and the absorbance-time curve at 420nm

was recorded against the blank containing all the constituents of

sample except the drug. Calibration graph was prepared by a plot

of logR (Initial rate of reaction) Vs. logC (nalidixic acid

concentration).

Analysis of nalidixic acid in tablets.

The above proposed method was used for the estimation of

nalidixic acid in tablets. About ten tablets were accurately

weighed and grinded into fine powder. An amount of the tablet

power equivalent to lOmg of nalidixic acid was weighed and

treated as described for the calibration procedure. The insoluble

matter was filtered instantaneously during the preparation of

sample solutions.

159


The pure nalidixic acid solution in methanol shows two ?^max. in

the ultravoilet region at 247nm and 338nm.A yellow coloured

chromogen is formed; when ferrous ammonium sulphate is added,

having A,max at 420nm (Fig.5.2),which indicates a bathochromic

shift with respect to A,max. of nalidixic acid.

The reaction product is unstable for forty minutes and

hence the kinetic approach is preffered over conventional

spectrophotometry, where unstability of colour preclude their

efficacy. The kinetics of the formation of reaction product at

maintained temperature (50+0.2°C) is studied at 420nm.The

increase of absorbance is measured as a function of time.

The redox reaction between nalidixic acid and ferrous ammonium

sulphate leads to the formation of a coordination compound. The

reduction of nalidixic acid results in hydrogenation of ethylene

bond in azinone ring. The oxygen atoms, because of lone pair act

as ligands and form coordinate bonds with Iron (III) as shown in

Figure 5.1.

Et . Me. N^ HH

H ^ P J ' " reairangement

•COOH

Fig.5.1 Tentative reaction mechanism of nalidixic acid with

ferrous ammonium sulphate.

To study the rate of reaction with respect to nalidixic acid

(keeping the ferrous ammonium sulphate cone, constant) at

160

50+0.2°C, absorbance versus time observat ions are recorded

(Table 5.1). A plot of absorbance versus time (t) gives straight

lines (Fig 5.3) whose slopes are used as a measure of reaction

rate for solut ions containing different concentrat ion of nalidixic

acid. The kinet ic studies with respect to nal idixic acid gave first

order plots . Job's method of cont inuous variat ion establ ished that

molar s toichiometr ic ratio of reaction between nalidixic acid and

ferrous ammonium sulphate is 1:1 (Fig 5.5). Calibrat ion graphs

constructed by plott ing initial rate of react ion(R) Vs.

concentra t ion (C) or by logR Vs. logC (Fig 5.4(a,b)) show a

linear re la t ionship with respect to the concentrat ion of nalidixic

acid in the range 20-80|j,gmir'. The curve is also used to calculate

order of react ion and found to be equal to +1.0. Validity of the

proposed kinetic method was checked by running different

concentrat ions of nal idixic acid, and the results obtained are

presented in Table 5.2. The optimum reaction temperature was

50+0.2°C, lower temperature showed inaccurate resul ts and the

reaction was incomplete . On the other hand, higher temperature

led to the decomposi t ion of reaction product. Some common

excipients such as lactose, starch, fructose, glucose and sucrose

which are usually present with dosage forms show no

interference in determinat ion and the maximum tolerance amount

was calculated using the procentual change in the absorbance of

nal idixic acid measured value. The results are summarized in

(Table 5.3).

The proposed kinetic method has been successfully applied to the

determinat ion of nal idixic acid in commercial dosage forms. The

results are s tat is t ical ly compared with those obtained with the

161

reference method [17] and are presented in Table 5.4. The

calculated 't' values suggested no significant difference between

the two methods in terms of precision & accuracy. Hence the

recommended method is at par with conventional spectrometric

methods in terms of its reproducibi l i ty and the time required for

the analysis .

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Fig. 5.2. Absorption spectra of nalidixic acid-ferrous ammonium sulphate reaction product at 50+0.2°C.

168

10 15 20 25

time (mjn.)

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Fig. 5.3. Plot of Absorbance Vs time for nalidixic acid, keeping ferrous

ammonium sulphate cone, constant (2.55x10"^ mol L' ) at 50+ 0.2 °C .

Nalidixic acid concentrations are; (a) 8.6x10"^ (b) 1.72x10'' (c) 2.15x10"^

(d) 2.58x10"^ and (e) 3.43x10^ molL\

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170

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171

REFERENCES

[1] L. Shargel , R.F. Koss, A.V.R. Grain and V.J. Boyle, J.

Pharm. Sci. 9, 62 (1973) 1452.

[2] H.Roseboom, R.H.A. Sorel, H. Lingeman and R, Bouwman,

J. Chromatography. 163 (1979) 92-95.

[3] Masakazu Horie et. al, J. Chromatography. 402 (1987) 301

308.

[4] H.K.L. Hundt and E.C. Barlow, J. Chromatography. 223

(1981) 165-172.

[5] F.H. Lee, R. Koss, S.K. O'Neil, M.P. Kullberg, M.McGrath

and J .Edelson, J. Chromatography. 162 (1978) 145-152.

[6] W.J.Vanoort , R.H.A. Sorel, D. Brussee, S.G. Schulman, P.

Zuman and J. Denhartigh, Anal. Chimca. Acta. 149 (1983)

175-191.

[7] G. Cuisinand, N. Ferry, M. Seccia, N. Bernard & J.Sassard,

J .Chromatography. 181 ( 1 9 8 0 ) 3 9 9 - 4 0 6 .

[8] Hsin-Lung Wu, Erumichi Nakagawa & Toyozo Uno, J.

Chromatography. 157 (1978) 297-302.

[9] R. H. A .Sorel, A. Hulshoff and C. Snelleman, J.

Chromatography. 221 (1980) 129-137.

[10] R.H.A. Sorel and Roseboom, J. Chromatography. 162

(1979) 461-465.

172

[11] Videsh R. Bari, U.J. Dhorda and M. Sundaresan, Anal

Chimca. Acta. 376 (1998) 221-225.

[12] P. Parimoo, C.V.N. Prasad and R. Vineeth, J. Pharm.

Biomed. Anal. 14 (1996) 389-393.

[13] G.P. Jadhva, H.N. More and K.R. Mahadik, Indian. J.

Pharm. Sci. 60(4) (1998) 246-48.

[14] R. Paliwal, D.K. Jain, R.S. Gaud and P. Trivedi, Indian. J.

Pharm. Sci. 60(3) (1998) 140-143.

[15] G.P. Jadhva, H.N. Moore and K.R. Mahadik, Indian Drugs.

35(8) (1998) 475-480.

[16] R. Paliwal, D.K. Jain and P. Trivedi, Indian Drugs, 35(3)

(1998), 165-167.

[17] Shingbal D.M, S. Sarjyotishi, Indian Drugs. 28 (1989) 569.

Reprinted from

JOURNAL OF

PHARMACEUTICAL AND BIOMEDICAL

ANALYSIS

Journal of PliarmatcuUcrfl and Biomedical Aiialyiib 21 (1999) 473-482

Simultaneous spectrophotometric and volumetric determinations of amoxycillin, ampicillin and cloxacillin in

drug formtilations: reaction mechanism in the base catalysed hydrolysis followed by oxidation with iodate in dilute acid

solution

Saidul Zafar Qureshi *, Talat Qayoom, Marud I. Helalet Dcimiiiiiciil of Cliciiinn \ AinihiKdl Rccanli L<ihiii<ii<ir\\ Ahviiili Un\liiii I'niivisin 1lii;,iili-2()2(l()2 (I /') liului

ELSEVIER

Journal of Pharmaceutical and Biomedical Analysis ELSEVIER 21(1999)473-482

lOURNALOr

PHARMACEUTICAL AND BIOMEDICAL

ANALYSIS

www.elsevier.com/locate/jpba

Simultaneous spectrophotometric and volumetric determinations of amoxycillin, ampicillin and cloxacillin in

drug formulations: reaction mechanism in the base catalysed hydrolysis followed by oxidation with iodate in dilute acid

solution

Saidul Zafar Qureshi *, Talat Qayoom, Marud I. Helalet Department of Chemistry, Analytical Research Laboratory, Aligarh Muslim University, Aligarh-202002 (U.P.), India

Received 16 October 1998; received in revised form 29 January 1999; accepted 5 May 1999

Abstract

A method for the analysis of degraded products of amoxycillin, ampicillin and cloxacillin in drug formulations, obtained as a result of their base hydrolysis is described. Simultaneous spectrophotometric and volumetric determinations of the antibiotic is based on the neutralization of the degraded product by dilute hydrochloric acid to get a pH ~ 2 to be conducive for redox titration using potassium iodate as titrant. A red purple colour is developed in carbon tetrachloride at the end point. Spectrophotometry is done after separating the organic layer and measuring the absorbance of red-purple colour at X,„.„ 520 nm. The pathways of different degraded products and their oxidation mechanism is described on IR, TLC and UV spectroscopic studies. © 1999 Elsevier Science B.V. All rights reserved.

Keywords: Amoxycillin; Ampicillin; Cloxacillin; Spcclropholoniclric and volumclric dclcrminalion; Simullancous dclcrmination; Pure and drug Ibrmulations

1. Introduction ^^^ carried out by some oxidants prior to a base or acid catalysed reaction of antibiotics. The de-

A large number of methods for the analysis of S'"'' ' ' hydrolysed products of antibiotics are ti-ampicillin, amoxycillin and cloxacillin and related ^'''^^'^ quantitatively against oxidants such as antibiotics are described [1 -3] . Emphasis has been " § ' ' ["^ ^"'^ '"'^^'^ solutions [5]. It is observed given on spectrophotometric and titrimetric meth- ^^'^^ *^^" hydrolytic conditions are changed from ods, because they are simple and easily manage- moderate concentration of alkali or acid to a

able to the third world. The volumetric titrations ^ . ' , f °' " ' ' "^ '^' f •"'= .?'. P " '^^l^"^. ^^^' ^ diiierent pathway of antibiotics mechanism re-suits. The titrimetric method [4] described by al-

• Corresponding author. Tel; +91-571400515. kaline or enzymic hydrolysis followed by titration

0731-7085/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: 50731-7085(99)00205-8

http://www.elsevier.com/locate/jpba

474 S.Z. Qureshi et at. /J. Pham. Biomed. Anal. 21 (1999) 473-482

with Hg(II) might suffer a setback. The use of nitric acid to neutralize the excess alkali before performing the titration may oxidize thiazolidine ring of the penicillin molecule and therefore could give erroneous results. The authors themselves reported better results with enzymic hydrolysis than alkaline hydrolysis.

In the direct titration of antibiotics [5], with potassium iodate solution, strong acidic conditions are prevailed. The acid plays a dual role, firstly it brings about acid hydrolysis, secondly it makes quantitative oxidation titration feasible. Two procedures are adopted [5]; in the first, the end point is detected by a colour change in the carbon tetrachloride layer from colourless to brown or deep red and in the second one, dye Amaranth is used to detect change in colour from deep red to pale yellow, as the dye is destroyed by an excess of iodate in aqueous medium at the neutralization point.

The present manuscript describes the base catalysed hydrolysis of antibiotics in dilute sodium hydroxide solution. The hydrolysis product of antibiotics which contains sulphur derivatives, eg. -C-S-C groups, -S-H groups are quantitatively oxidized to sulphonic acid by titrating them against potassium iodate solution as oxidant, in dilute hydrochloric acid to prevail conducive atmosphere of pH ~ 2. The chemistry of this reductant system is described which shows the observed stoichiometric value of thiol group to iodate 1:2 which is consistent with theoretical value. The point of difference of our findings to that of previously described by Andrews titration [12] have been highlighted. The volumetric results are compared to spectrophotometric determination. The pathway and the oxidation mechanism of hydrolysis products of antibiotics have been proposed in the light of IR, TLC and spectrophotometric studies.

2. Experimental

2.1. Reagents

All the experiments were performed with analytical reagent grade chemicals using double dis

tilled water. Stock solution of antibiotics, 1 mg ml ~' were prepared by dissolving the pure and standard drugs (Himedia laboratories Bombay India) in water and stored in a well-dark and closed container to avoid direct contact with light. Hydrochloric acid standard solution (1.0 M), potassium iodate and sodium hydroxide of 0.1 M each were prepared in distilled water.

2.2. Apparatus

A Bauch and Lomb Spectonic -200"^ and Spec-tronic-1001 spUt beam spectrophotometer (Milton Roy Co.) with 1 cm path length quartz cells and a controlled water bath (NSW-133 India) were used. IR spectra of the isolated products were recorded on a spectrometer (Shimadzu IR-408, Japan) with KBr disc.

2.3. General procedure

2.3.1. Procedure I: simultaneous determination of penicillins by-volumetric titration against potassium iodate and spectrophotometry of coloured complex in organic layer

Aliquots of 2.5 ml of 0.1 M sodium hydroxide were added into conical flasks followed by suitable volumes of each of the standard penicillin solutions, containing between 0.2-2.0 mg ml""' of amoxycillin and ampiciUin and 0.1-2.5 mg ml~' of cloxacillin. The contents (pH ~ 13) were shaken and placed in a water bath at 80°C for 10-15 min. After completion of the heat treatment, the mixture was cooled to room temperature, followed by 0.5 ml of 1.0 M hydrochloric acid and 5 ml carbon tetrachloride. The mixture contents were titrated against potassium iodate with intermittent shaking. The end point was detected by a colour change from colourless to deep red colour in non-aqueous layer. For spectrophotometric determination, the nonaqueous layer was removed using a 50 ml separating funnel, dried over anhydrous sodium sulphate and measured absorbance at 520 nm against the blank, containing all the species except drug.

S.Z. Qureshi et al. /J. Pharm. Biomed. Anal. 21 (1999) 473-482 475

2.3.2. Procedure II: determination of penicillins by spectrophotometric method

To a known volume of amoxycillin solution ranging between 0.4-3.5 ml, ampicillin 0.4-2.8 ml and cloxacillin 0.1-2.5 ml, 2.5 ml of sodium hydroxide were added in a conical flask. The reaction mixture was kept for 10-15 min on a water bath at 80°C. After cooling to room temperature, 1 ml of potassium iodate, 0.5 ml of hydrochloric acid and 5 ml carbon tetrachloride were added to the reaction mixture. The solution was shaken vigorously till a deep red colour was obtained in the organic layer. It was determined spectrophotometrically by the recommended procedure I.

2.4. Assay of formations

For the determination of penicillins in tablets and capsules, the above methods were used with no modification. About twenty tablets were grinded into finely divided powder. 100 mg of powder was accurately weighed and transferred into a 100 ml standard flask. Similarly the contents of the capsules were weighed and dissolved equivalent to 100 mg in 0.1 1. The solution was well shaken for about 30 min to ensure a homogeneous solution. The residue was removed through Whatman No. 1 filter paper and the washings were taken into a final volume of 0.1 I followed by the recommended procedures I and II.

3. Results and discussion

The mechanism of penicillins in 0.1 M sodium hydroxide resulting in different degraded products can be rationalized in the light of IR, UV spectroscopic studies, TLC and molar stoichiometrics molar ratio of iodate to penicillin titration. The possible degradation pathways are illustrated in Fig. 1.

3.1. TLC and spectrophotometric observations

The presence of la has been suggested on the basis of the results obtained by TLC (Rf= 0.46 in a solvent system made v/v and comprising, 66%

n-butanol, 17% glacial acetic acid, 17% water). This observation is in agreement with previous studies [6], which confirms the existence of 4-hy-droxymethylene oxazolone la, (/?/=0.42 in the same solvent). Our studies of hydrolysis in O.IM NaOH at pH ~ 13 involving a single step reaction using water bath at 80°C, show one X,„„ at 240 nm, at reaction time of 15 min. This attributes the existence of one of the three species viz; lb (i) or lb (ii) or lb (iii). Moreover the formation of penicilloic acid lb (i) from basic hydrolysis of penicillin is well established [7]Tables 1-3. On extending the time interval to 12 h, another X a at 270 nm is obtained, which further confirms the establishment of 1(a). The previous studies show the existence of 1(a) as degraded product of antibiotics in the pH range 13-14 with UV spectroscopic analysis: X ^ x 297.5. The slight difference in "K^.^^ value may be attributed to different experimental conditions of hydrolysis, such as temperature, time of hydrolysis and pH etc.

3.2. IR studies

Three different spectra were taken, (i) pure amoxycillins, spectrum-A (Fig. 2) (ii) degraded in base catalysed hydrolysis, speclrum-B (Fig. 3) (iii) degraded base hydrolysed product treated with 0.1 M hydrochloric acid followed by titration against KI03, spectrum-C (Fig. 4).

The spectrum (B), compared with spectrum (A), revealed that the characteristic bands for different groups at specified frequencies in spectrum (A) are found, (i) missing, (ii) the formation at different frequencies or (iii) remained unchanged. A broad flat portion (3450-2600 cm~') same as it was found in spectrum (A), attributed to primary aliphatic amine N-H (3450-3250 cm" ' ) , car-boxylic (3300-2500 c m " ' ) and S-H (2600-2500 c m " ' ) groupings.

3.3. Missing and formation of new bands

The bands are found missing in the range (1800-1500 c m - ' ) and (1350-1000 cm" ' ) resulting the formation of bands at 860 cm~' , 680cm-' and in the range (1500-1350 cm" ' ) .

476 SZ Qureshi et al /] Pharm Biomed Anal 21 (1999) 473-482

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S.Z. Qureshi et al /J Pharm. Biomed. Anal. 21 (1999) 473-482 479

The identities of thiozolidine and secondary amine groups with different group frequencies lying in the range (1800-1510 cm~') are completely swept out. The second range of missing band (1350-1000 cm"') includes the groups e.g. CHj-S, ester etc. The two missmg ranges are revealed by a descending and ascending portion of spectra-B respectively. The formation of a broad band (1500-1350 cm"') is attributed to the pres

ence of 4-hydroxy-methylene oxazolone (la), which is revealed by the groups frequencies C-0 stretching and 0-H deformation vibration (1440-1395cm~'). The ascending portion (1350 cm~' onwards) of spectrum B further describes the hydrolysis of the rest of the groups present. A strong peak at 860 cm"' may be attributed to S-0 (870-810 cm-') of sulphonic acid. The weak peak at 680 cm " ' suggests to disulphide -S-S, C-S

A Degradation products, Suê Stage hydrolysis at pH~ 13

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4-hydroxy methylene oxaxole-5(4H)-one

la

penctllemc acid

Ib(m)

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spectrophotometncally using lOo' as Oxidant ^ i I lyiOa'

o II

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SuJphomc acid IVb

O O oxidation 11 11 . . < R - S - S - R °'"'^^°"

Sulphoxide Illb

C-CH-, -C=\ • = \

NH-CH

pencillenic acid (disulphide)

R-S—S-R

lib

- 1 2

Fig 1 The possible degradation pathways of pencilline, followed by oxidation with lodate ions

480 S.Z. Qureshi et at. /J. Pharm. Biomed. Anal. 21 (1999) 473-482

4000 3100 1000 MOO 2000 1*00 l«00 1700 WOO 1S00 HOO 1300 )200 1100 1000 «00 (00 700

WAVENUMBER CM'*

Fig. 2. (i) Pure amoxycillin, spectrum-(A).

4000 3S00 3oS30 It'oO lOOO 1100 1*00 1700 t«00 ttOOUOO 1300 »00 1100 *000 100 ioO 700 WAVENUMBER CM'*

Fig. 3. (ii) Degraded in base catalysed hydrolysis, spectrura-(B).

S.Z Qureshi et al /J. Pharm. Biomed Anal. 21 (1999) 473-482 481

(705-570 cm" ') and sulphoxides ( > S = O, C-S-O at 730-690 cm"') after their partial oxidation to sulphonic acid in a semiquantitative manner as the sulphur compounds especially containing the group S-H, S-S are oxidized by mild oxidant or even atmospheric oxygen [8]. The spectrum -C, which is obtained after completing the titration against iodate in dilute acid conditions, gave the final product corresponding to strong peak at 835 cm"', attributed to the presence of sulphonic acid quantitatively.

The recommended procedure I describes simultaneous volumetric determination of penicillin against potassium iodate as oxidant in dilute acid medium. The deep-violet colour extracted in carbon tetrachloride at the equivalence point is stable for 2 h and has been studied spectrophotometri-cally. The hydrolysis product of penicillins (amoxycillin, ampicillin and cloxacillin) which contained sulphur derivatives, i.e. -C-S-C (lb), -S-H groups are quantitatively oxidized to some intermediate species and finally to sulphonic acid whose identities have already been discussed in IR studies. The hydrolysis by dilute sodium hydrox

ide solution has been carried out slowly at a constant temperature of 80°C with intermittent shaking. The excess of base is neutralized by adding hydrochloric acid (0.5 mmoJes) so that the reaction mixture attains a pH ~ 2. This provides a conducive environment of HjO" to concentration for carrying out quantitative titration of degraded products against potassium iodate. The reaction is summarized as follows;

.6e-[HS-R + 3H20^ RSO3H -H 6H+65] X 2 Step I

[IO3- +C\- -H6H++4e-->ICl-h3H30+]x3

In the first step reaction a quantitative oxidation of thiol to sulphonic acid followed by reduction of lOf to to ICl takes place which may be regarded as the reaction at equivalence point, but without any colour change. This shows that the stoichiometric value calculated theoretically should be of 2 mol of thiol group to 3 mol of iodate (1:1.5) which is inconsistent with experimental value 1:2 [9]. However, the system still involves more iodate ions to make the end point visible due to its reduction to iodine monochlo-

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5 401-

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20

10 •

4000 ISOO 3000 2S00 2000 ISOO | |00 1700 1(00 ISOO UOO 1300 UOO 1100 tOOO >00 (00 700 WMEMUM&CR CM**

Fig 4. (m) Degraded base hydrolyzed treated with I 0 M hydrochloric acid, followed by titration against KIO3, spectrura-(C)

482 S.Z. Qureshi el a!. /J. Pharm. Bwmed. Anal. 21 (1999) 473-482

ride and finally to free iodine. This is described in step II reaction which is given below:

IO3- + 6H+ + CI- + 4e -"""-^nci + 3H,0 ), —..dissociation-

ici -• r + C1-

I-+H30'^''^''^'^H0I + H +

5HOI°^"^°"4'-''"°"2l2 + IO3- + H- + 2H,0 ofioddte

In the step II, an over consumption of iodate takes place [10], thereby increasing the mole ratio of iodate from 1.5 to 2 mol which is consistent with the experimentally observed stoichiometry of 1 mol thiol to 2 mol iodate. A stable red violet colour of free iodine in organic layer indicates the equivalence point. It is observed that further addition of iodate solution beyond the end point results no decolourization in mixture content. This means that a quantitative oxidation-reduction in dilute hydrochloric acid has taken place in [the system. The titrant iodate is reduced first to iodine monochloride and finally to free iodine to show a visible end point. The E" of iodate in dilute hydrochloric acid (1.19 V) is less than the E" in Andrew titration (1.23 V). The E° 1.19 V is |high enough to oxidize the degraded products of antibiotics containing derivatives. I In Andrev/s titration a strong hydrochloric acid (3-9 M) solution is required for reduction of iodate to iodine monochloride (IClf) complex.

IO3 + 6H++2CI- -h4e--ÎCU- +3H,0

E° = 1.23 V

During the course of titration with reducing agent, the presence of free iodine in the system also results from the further reduction of IClj" complex as given below:

2ICl2-+2e-^l2 + 4Cl- E°= 1.06 V

When the reducing agent has been consumed, the free iodine is titrated to form iodine monochloride complex. The end point is marked by the disappearance of the last free iodine. (A point of difference from our observation given in Step II).

2I2 4- IO3- + 6H+ -I- lOCl - -> 5ICI2" + 3H2O

The above conditions prevail if the concentra

tion of the hydrochloric acid at the end point is at least 3 M. The iodate reacts quantitatively with reducing agents such as KI, I2, AS2O3, N2H4 and SO2 to iodine monochloride [11]. In many other cases the concentration of hydrochloric acid is not critical, but for Sb(III), it is 2.5-3.5 M; that is, the optimum acidity for reasonably rapid reaction varies from one reactant to another reactant [12].

The recommended procedure I, which describes the use of dilute hydrochloric acid is applied successfully to the simultaneous quantitative reduction of iodate to free iodine at the equivalence point, especially in a system which contains the degraded products of antibiotics with sulphur derivatives which can be oxidized by very mild oxidizing agents [10]. The half reaction of iodate in dilute acid solution with E° [II] can be proposed as under.

2IO3- + 12H+ -H lOe - -* P -f- 6H2O

E° = 1.19 V

Acknowledgements

The research facilities at the Department of Chemistry, AMU Aligarh are highly acknowledged.

References

[1] A.G. Davidson, J.B. Stenlake, Analyst 99 (1974) 476. [2] J, Talegaonkar, K..S. Boparai, Talanta 29 (1982) 525. [3] U. Forsman, Anal. Chim. Acta. 93 (1977) 153. [4] B. Karibcrg, U. Forsman, Anal. Chim. Acta. 83 (1976)

309. [5] J.K. Grime, B. Tan, Anal. Chim, Acta. 105 (1979) 361. [6] J.L. Longridgc, D. Timms, J. Chcm. Soc. B (1971) 852. [7] M.A. Carroill, J.E. Zarcmbo, J. Antibiol. 30 (1977) 811. [8] D.R. John, M.C. Cascrio, Basic Principles of Organic

Chemistry, 2nd cdn, 1977, p. 745. [9] El-Scbai A. Ibrahim, S.M. Rida, Y.A. Baltagy, M.M.

Abd El-Khalek, J. Drug. Res. (Egypt) 6 (1973) 13. [10] I.M. Kolthoff, R. Belcher, Volumetric Analysis, vol. Ill,

1957, p. 450. [II] I.M. Kolthoff, E.B. Sandell, E.J. Merhan, S. Brucken-

stein. Quantitative Chemical Analysis, 4th edn, 1969, pp. 819,1159.

[12] L.W. Andrews, J. Amer. Chem. Soc. 25 (1903) 756.

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