ANALYSIS OF DRUGS IN PHARMACEUTICAL FORMULATIONS

ABSTRACT

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

Battor of $t|iIo!Bioptip IN

OHEMISTRY

BY

NADEEM AHMAD KHAISI

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2003

The thesis entitled ''Analysis of Drugs in Pharmaceutical Formulations" is

comprised of five chapters. The first chapter describes a general literature survey of the

subject matter. The very relevant matters include, (i) a brief discussion on analytical

chemistry, its relation with our daily life from old age, role played in the field of

pharmaceutical and biomedical analysis, (ii) various types of analytical techniques and

procedures involving sophisticated instruments which are in use now a days, (iii) the

validation of the developed method and (iv) the statistical treatments adopted during the

data analysis to ensure and enforce the validity of method.

It is well known that human shows briskness in every era of evolutions and

development, which led to the birth of analytical chemistry. It deals with the study of the

intrinsic properties of the materials to discover their valueable applicability or to warn the

concerned one with the possibility of harm from them. Unlike, other areas of studies in

science, there are different properties, which heavily influence the results of the analytical

methodology at each and every step, thus having direct impact on the results. These

important properties have been discussed. By the time, the demands of the biochemical

sciences and the advancement of physical sciences opened the door of instrumentation in

the field of analytical chemistry. The different types of well-established analytical

techniques have been discussed.

Before or during the development of a method, it is necessary to be very careful

of certain important things. These concepts have been discussed briefly. Once the

analytical method is adjusted, it is necessary to decide its suitability for the intended

purpose. This is known as the method validation. Brief discussion of the validation and

TUESI o 2

its components has been presented. When deciding for validation of the method, the role

of statistical analysis can not be ignored. It is the only way to get the most conclusive

results from the mathematical data obtained during the analysis. It also helps to decide

with the progress of the work at each step. A discussion of the statistical analysis has

been given. A brief literature and classification of the concerned pharmaceuticals also

presented.

The second chapter describes kinetic spectrophotometric method for the

determination of silymarin in pure form and in pharmaceutical formulations. The

method is based on the oxidation of the drug with potassium permanganate at pH

7.0±0.2. The reaction is followed spectrophotometrically by measuring the

decrease in the absorbance at 530 nm. Under the optimum experimental

conditions, a calibration curve was constructed by plotting logarithm of initial

rate of reaction (log v) versus logarithm of silymarin concentration (log C),

which was linear over silymarin concentration range of 18-50 | g ml'^ The linear

regression analysis using the method of least square was made to evaluate the

slope, intercept and correlation coefficient. The linear regression equation and

correlation coefficient are log v = 1.024 log C + 3.235 and r = 0.9997 which

indicates an excellent linearity. The confidence limits for the slope and intercept

of the line of regression were calculated and found to be 1.024±3.96x10'" and

3.235±1.67xl0"\ respectively. This indicated the high reproducibility of the

proposed kinetic method. The proposed method was compared with the reference

method.

THESIS

The third chapter deals with the four simple, sensitive and accurate

spectrophotometric methods for the determination of nifedipine in pharmaceutical

formulations. These methods are based on the formation of ion-pair complexes of amino

derivative of the nifedipine with bromocresol green (BCG), bromophenol blue (BPB),

biomothymol blue (BTB) and eriochrome black T (EBT) in acidic medium. The coloured

products are extracted with chloroform and measured spectrophotometrically at 415 nm

(BCG, BPB and BTB) and 520 nm (EBT). Beer's law was obeyed in the concentration

range of 5-32.5, 4-37.5, 6.5-33.0 and 4.5-22.5 \xg ml" with molar absorptivity of 6.41 x

10^ 4.85 X 10^ 5.26 x 10 and 7.69 x 10 L mol" cm" and relative standard deviation of

0.82, 0.72, 0.66 and 0.68 % for BCG, BPB, BTB and EBT methods, respectively. These

methods have been successfully applied for the assay of drug in pharmaceutical

formulations. No interference was observed from common pharmaceutical adjuvants.

Statistical comparison of the results with the reference method shows excellent

agreement and indicates no significant difference in accuracy and precision.

The fourth chapter describes two simple, sensitive and economical

spectrophotometric methods for the determination of amiodarone hydrochloride

in pure form and commercial dosage forms. Method A is based on the charge

transfer complexation reaction of amiodarone base with p-chloranilic acid in

dioxan- dichloromethane. The coloured product is measured at 535 nm. Method

B also utilizes the charge transfer complexation reaction of amiodarone base with

2,3- dichloro 5,6- dicyano 1,4- benzoquinone (DDQ) in acetonitrile medium,

resulting in the formation of DDQ radical anion, which absorbs maximally at 570

THESIS

nm. Beer's law was obeyed in the concentration range of 10 - 360 and 2 - 6 5 |j,g

ml" for methods A and B, respectively. The molar combining ratio, association

constant and optimum assay conditions were studied. The proposed methods

have, been successfully applied to the determination of amiodarone in

commercial dosage forms. The results are validated statistically and compared

with the reference method.

The fifth chapter deals with the two simple and sensitive spectrophotometric

methods for the determination of verapamil hydrochloride in pure and commercial

dosage forms. The first method is based on the oxidation of the drug with potassium

metaperiodate in sulphuric acid medium to give coloured product, which absorbs

maximally at 425 nm. The second procedure involves the formation of coloured

chloroform extractable ion-pair complex of the drug with tropaeolin 000 at pH 4.0

showing absorption peak at 400 nm. Under tlie optimized experimental conditions, Beer's

law is obeyed in the concentration range of 0.00-187.5 and 2-30 |j,g ml"' for methods A

and B, respectively. Both the methods are highly reproducible and have been successfully

applied to the commercial dosage forms. Results of analyses were validated statistically

and through recovery studies. Statistical comparison of the results with the reference

method shows excellent agreement and indicates no significant difference in accuracy

and precision.

ANALYSIS OF DRUGS IN PHARMACEUTICAL FORMULATIONS

l (

T H E S I S \ - ^ \ \ SUBMITTED FOR THE MilKR^ OF THE DEGREE OF

Bottor of $l)ilo£(opt)p

C H E M I S T R Y

BY

NADEEM AHMAD KHAN

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2003

^ s ^

Fed in Compnter

^2ad Libra v.

•/'

/Wtifl-rt. " i ^ ^

0 9 J - i 2005

T5852

v- V/<!k, .

THESIS

DR. NAFISUR RAHMAN Reader

Analytical Research Laboratory Department of Chemistry Aligarh Muslim University Aligarh-202 002 (U.P.) INDIA Tel.:+91-571-2703515 (Office) E-mail: cht17nr@yahoo.co.in

(Date iN&^^mUv 01 tooS

Certificate

T^is is to certify tHat the thesis entitted "JLnaCysis of<Drugs In

(PharmaceuticaC TormuCations" is the originaC wor^ of

Mr. O^adeem JLhmad an, carried out under my supervision and

is suitaBleforsuSmissionforthe award of the degree of<Doctorof

<PhHbsophy in Chemistry.

Na l4h,u^/^^^J^ VlA.-A^

(Ufafisur <l(ahman)

A.CK9^O'WL<E(DGT.9A(E0fT

I Bring all the •praises aitcC commendation to the MmigHty for-providing me

with strength during the xvor^ of my research, which heCped me to overcome the

trouBCes in the wearisome journey.

It wiCf Be hard to find out the words to than^ my sage supervisor,

(Dr. U^afisur <Rfihman, for his excellent guidance, constant support and trust

throughout the course of my research wor^

I am than^Cto the CHairman, department of CHemistry, JlGgarh MusRm

"University, for providing researchfaciCities.

I dufy ac^nowtedge the ^nd cooperation of my friends and coCkagues for

their nd heCp, vaCuaBk suggestions and encouragement.

I eoq^ress my deep sense to my parents whose Blessing have aCways served as a

sheet anchor against the mundane odds arid inspired me to complete my mission.

I egress my deep than^C to my younger Brother and sisters, for the

acQustment they made during my studies.

C^adeemjihmad%lian)

CONTENTS

List of publications I

List of figures H

List of tables V

Chapter 1 General introduction 1

References 50

Chapter 2 Kinetic spectrophotometric method for the determination of 69 silymarin in pharmaceutical formulations using potassium permanganate as oxidant

References 86

Chapter 3 Extractive spectrophotometric methods for the determination 89 of nifedipine in pharmaceutical formulations using bromocresol green, bromophenol blue, bromothymol blue and eriochrome black T

References 120

Chapter 4 Validated Spectrophotometric Methods for the 126 Determination of Amiodarone Hydrochloride in Commercial Dosage Forms using p-chloranilic acid and 2,3 -dichloro 5,6 - dicyano 1,4 - benzoquinone

References jgg

Chapter 5 Quantitative Spectrophotometric Methods for Determination of 159 Verapamil Hydrochloride in Drug Formulations Using Potassium Metaperiodate and Tropaeolin 000

LIST OF PUBLICATIONS

[1]. Kinetic spectrophotometric method for the determination of silymarin in pharmaceutical formulations using potassium permanganate as oxidant; Pharmazie, Govi Verlag GmbH, 2003 (In press).

[2]. Extractive spectrophotometric methods for the determination of nifedipine in pharmaceutical formulations using bromocresol green, bromophenol blue, bromothymol blue and eriochrome black T; IL Farmaco (Elsevier), (Accepted).

[3]. Validated Spectrophotometric Methods for the Determination of Amiodarone Hydrochloride in Commercial Dosage Forms using p-chloranilic acid and 2,3 -dichloro 5,6 - dicyano 1,4 - benzoquinone; Anal. Sci., The Japan Society for Analytical Chemistry, 2003 (Accepted).

[4]. Quantitative Spectrophotometric Methods for Determination of Verapamil Hydrochloride in Drug Formulations Using Potassium Metaperiodate and Tropaeolin 000; AAPS PharmSci, American Association of Pharmaceutical Sciences, 2003 (Communicated).

II

Figures

Fig. 2.1.

Fig. 2.2.

Fig. 2.3.

Fig. 2.4.

Fig. 2.5.

Fig. 3.1.

Fig. 3.2.

Fig. 3.3.

Fig. 3.4.

Fig. 3.5.

Fig. 3.6.

Fig. 3,7.

Fig. 3.8.

Fig. 3.9.

1 Fig. 3.10.

LIST OF FIGURES

Captions

Absorbance-time curves: KMn04, 4.8 xlO" M and silymarin (a) 3.731 X 10- M; (b) 4.146 x 10' M; (c) 6.219 x 10'^ (d) 8.292 X 10- M; (e) 10.365 x 10" M

Absorbance-time curves : silymarin, 10. 365 x 10' M and KMn04 (a) 3.00 x 10"* M ; (b) 2.10 x 10" M; (c) 1.50 x 10" M; (d)9.00xlO-^M.

Calibration curve for the determination of Silymarin.

Error in the determination of the concentration of silymarin using statistical analysis of standard calibration data.

Variation of the confidence limit at (a) p= 0.05 and (b) p= 0.01 level of significance in the form of uncertainty percentage on the concentration of silymarin.

Absorption spectra: (a) Amino derivative of nifedipine-BCG complex; (b) Amino derivative of nifedipine-BPB complex; (c) amino derivative of nifedipine-BTB complex; (d) amino derivative of nifedipine-EBT complex.

Job's plot for drug-dye (1.443 x 10^ M) system: BCG.

Job's plot for drug-dye (1.443 x 10"* M) system: BPB.

Job's plot for drug-dye (1.443 x 10^ M) system: BTB.

Job's plot for drug-dye (1.443 x 10^ M) system: EBT.

Eftect of the concentration of HCI: 31 jig mp' drug + 7 ml of 0.025% BCG.

Effect of the concentration of HCI: 33 |ig ml"' drug + 7ml of 0.025% BPB.

Effect of the concentration of HCI: 33 ug mP' drue + 7 ml of 0.025% BTB. ^

Effect of the concentration of HCI: 22.5 ^g ml"' drug + 7 ml of 0.025% EBT. /mioi

Influence of the volume of 0.025% dve- BCG

Page No.

74

75

78

80

81

93

94

95

96

97

101

102

103

104

106

Ill

Influence of the volume of 0.025% dye: BPB. 107

Influence of the volume of 0.025% dye: BTB. 108

Influence of the volume of 0.025% dye: EBT. 109

Calibration curve for determination of nifedipine with BCG. I l l

Calibration curve for determination of nifedipine with BPB. 112

Calibration curve for determination of nifedipine with BTB. 113

Calibration curve for determination of nifedipine with EBT. 114

Absorption spectra of (a) 2.30 X lO''' M p-chloranilic acid (b) 132 0.36 |ag ml'' amiodarone with 1.15 X 10" M p-chloranilic acid in dioxane- dichloromethane.

Molar ratio plot for amiodarone - p - chloranilic acid complex; 133 amiodarone concentration, 2.933 X 10^ M.

Absorption spectra of (a) 8.81 X 10" M DDQ in acetonitrile 136 (b) 65 tag ml"' amiodarone with 1.06 X 10' M DDQ in acetonitrile.

Fig. 4.4. Molar ratio plot for amiodarone -DDQ complex; amiodarone 137 concentration, 2.933 X 10"* M.

1 \A\\D] 1 140 Fig, 4.5. Plot of versus -'• --"- •'-X—jjr for 1:1

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

3.11.

3.12.

3.13.

3.14

3.15

3.16

Fig.3.17.

Fig.

Fig.

Fig

4.1.

4.2.

4.3.

^x complex of amiodarone - p- chloranilic acid.

1 \A\\D] 1 141

of amiodarone -DDQ.

Fig. 4.7. Influence ofthe volume ofO.08% p-chloranilic acid. 143

Fig. 4.8. Influence ofthe volume of 0.08% DDQ. 145

Fig. 4.9. Calibration curve for determination of amiodarone (method A). 147

Fig. 4.10, Calibration curve for determination ofamiodarone (method B). 148

Fig. 4.11. Plot ofthe variation ofthe confidence limit at 95% confidence 150 level for method A, presented as uncertainty percentage on the concentration ofamiodarone.

IV

Fig. 4.12. Plot of the variation of the confidence limit at 95% confidence 151 level for method B, presented as uncertainty percentage on the concentration of amiodarone.

Fig. 5.1. Absorption spectrum of the reaction product of Verapamil 164 hydrochloride with potassium metaperiodate.

Fig. 5.2. Job's plot for the reaction of verapamil hydrochloride with 165 potassium metaperiodate (each 5. 09 x 10' M).

Fig. 5.3. Absorption spectrum of the reaction product of 168 Verapamil hydrochloride with tropaeolin 000.

Fig. 5.4. Job's plot for the reaction of verapamil hydrochloride with 169 Tropaeolin 000 (each 1.02 x 10" M).

Fig. 5.5. Effect of heating time. 172

Fig. 5.6. Effect of the volume of 0.2% potassium metaperiodate. 173

Fig. 5.7. Effect ofthe volume of 10 moir'H2S04. 174

Fig. 5.8. Effect of the volume of 0.05% tropaeolin 000, 176

Fig. 5.9. Effect of pH: 30 jig ml'' drug + 2.5 ml of 0.05% tropaeolin 000 177 + 5.0 ml buffer of different pH values.

Fig. 5.10. Calibration curve for determination of verapamil hydrochloride 179 (method A).

Fig. 5.11. Calibration curve for determination of verapamil hydrochloride 180 (method B).

Fig. 5.12. Error in the determination of verapamil hydrochloride using 184 statistical analysis of standard calibration data with potassium metaperiodate.

Fig. 5.13. Error in the determination of verapamil hydrochloride using 185 statistical analysis of standard calibration data with tropaeolin 000.

Tables

Table 1.1.

Table 1.2.

Table 1.3

Table 1.4

Table 1.5.

Table 1.6.

Table 2.1.

Table 2.2.

Table 2.3.

Table 3.1.

Table 3.2.

Table 3.3.

Table 3.4.

LIST OF TABLES

Captions

Assay of drugs in drug formulations by Spectrophotometric procedures.

Maximum permitted standard deviation for assay determinations in dependence on the number of repetitions and the specification range

Requirements for different calibration modes with relevant parameters.

Validation characteristics normally evaluated for the different types of test procedures and the minimum number of determination required

Approaches to demonstrate accuracy according to ICH.

Approaches for determining the detection and quantitation limit

Precision test of the proposed kinetic method.

Determination of silymarin in pharmaceutical formulations by standard addition method

Determination of silymarin in dosage forms by the proposed kinetic method and reference method.

Analytical characteristics of the proposed methods.

Determination of nifedipine in pharmaceutical formulations by standard addition method.

Comparison of the proposed methods with existing spectrophotometric methods for the assay of nifedipine in pharmaceutical formulations.

Determination of nifedipine in dosage forms by the proposed methods and reference method.

Page No.

12

27

31

32

33

37

83

84

85

115

116

118

119

VI

Table 4.1.

Table 4.2.

Table 4.3.

Table 4.4.

Table 4.5.

Table 5.1.

Table 5.2.

Table 5.3.

Table 5.4.

Association constant, K; molar absorptivity, z\ from the 142 Ross and Labes plots for the complex and calculated free energy, AG.

Analytical characteristics of the proposed methods. 146

Test of precision and accuracy of the proposed methods. 153

Standard addition method for the determination of 154 amiodarone in dosage forms.

Comparison of the proposed methods with the reference 155 method.

Optical Characteristics and statistical data of the of the proposed methods.

181

Evaluation of the accuracy and precision of the proposed 186 methods.

Standard addition method for the determination of verapamil hydrochloride in dosage forms.

Comparison of the two proposed methods with the reference method.

187

189

CHapter -1 Qenerat Introduction

Medicine is a very ancient art, and drugs have been used in days of antiquity as

far back as history can take us. The early explorers found the South American hidians in

possession of Cinchona bark from which Pelletier and Caventou extracted the

antimalairal alkaloids, quinine. The Roman Naturalist Galenus (131-200 A.D) said that

proper herb mixture could be used for all conceivable health defects. In Galenus

apothecary shop, herbs and some metallic drugs (copper and zinc ores, iron sulfate,

cadmium oxide) were found in abimdance, and a semblance of assaying was maintained

by insistence on "pure" drugs that is the right variety and age of botanical specimens. The

intimation of dosage levels can be seen in his contention that powerfiil narcotics reUeve

pain but may also cause death in excess. In 1646, the French physician Guy Palin (1602-

1672) suggested to a medical student; "Above all flee books on chemistry, in quorum

lectione oleum et Perdes" . This attitude changed as exact laws of chemistry were

formulated as the physiological action of well defined compounds become amenable to

quantitative study and as the active principles of ancient - herbal drugs were extracted,

purified and fitted into stoichiometric laws [1].

hi the mean time, new medicines were discovered and old ones improved.

Withering (1741-1799) introduced digitalis (purple foxglove) as a therapeutic agent for

dropsy, and almost the same material is used in the treatment of cardiac edema today.

Van Swieten (1700-1772) taught the advantages of corrosive sublimate (mercuric

chloride) over older mercurial preparations in "Fevers". Opium was dispensed and

studied by de Quincey (1785-1859) "to tranquilize all irritations of the nervous system, to

stimulate the capacity of enjoyment and to sustain the else drooping animal energies" [2].

Phenacetin, a pain killing drug, was the first pharmaceutical product

developed by the German chemical company, Friedrich Bayer & Co; which, in

1888, licensed May and Baker to manufacture and market it in the United

Kingdom. Higher standards for the preparation of pharmaceutical ingredients had

been set, following the 1858 Medical Act's stipulation that the General Medical

Council (UK) should produce a list of medicines and compounds and manner of

preparing them together with true weights and measures by which they are to be

prepared and mixed [3]. The first British Pharmacopoeia was published in 1864

and thereafter pharmaceutical and fine chemical manufacturers laid greater

emphasis on meeting the standards it set. In 1890, Jesse Boot established an

analytical laboratory and its staff were involved mainly for analyzing proprietory

medicines of competitors in order that Boots should develop new and / or

cheaper formulations [4].

Analj^ical Chemistry is the science and art of determining the

compositions of materials in term of the elements or compounds contained in

them. Historically, the development of analytical methods has followed closely

the introduction of new measuring instruments. Among the many unprecedented

and fundamental changes occurred in the Chemistry during the past century

perhaps the most notable was the increase in the use of sophisticated instruments

and techniques. Researchers in Pharmaceutical Chemistry by the applications of

these techniques are adding a lot in the development of new drugs, which are

improving the life expectancy but also presenting new challenges.

Analytical Chemistry in pharmaceutics is indispensable, without the use of

analytical techniques, it is impossible for the chemists or the researchers to

develop a new approach to solve the problems of quality control and in

ascertaining optimum experimental conditions to obtain a good yield of products.

Development of new pharmaceutical product from naturally occurring

substances undergoes a nimiber of steps from laboratory to semi scale synthesis,

pilot plant scale up, efficacy and toxicology testing, clinical trials and finally full

scale production. In order to safeguard public health, there should be constant

monitoring of impvirities present in the form of inorganic and organic compounds

in the pure drugs and in pharmaceutical formulations. From the last few decades,

Analytical researchers continuing their efforts to produce high quality products

by improving the existing methods of analysis. A very large number of examples

can be cited where analytical chemistry find its applications to drug analysis in

pharmaceutical preparations and biological samples. [5-7].

In pharmaceutical industry, the quality of the manufactured drug and its

formulations must be carefully controlled. Slight changes in composition or in

the purity of drug itself can affect therapeutic values. Therefore, it is necessary to

establish the properties and therapeutic value of a drug before it is approved and

made available in the market. Establishment of the permissible level of dosage of

drug requires the determination of its composition, toxicity and its metabolite at

various stages.

The availability of sub-standard medicines to the general public possesses

many problems, both clinically and economically. It is widely believed that such

sub-standard preparations are readily available in many developing countries [8-

13]. This may be due to poor manufacturing procedures, poor storage conditions

or deliberate counterfeiting of branded or generic products. Therefore, it is

important to recognize that the drugs may contain impurities. These impurities

may result from many sources like raw materials and regents, as reaction by

products, and through degradation during manufacture and storage. Since the

impurities can have safety and efficacy implications, and are therefore the subject

of considerable attention by both manufacturers and regulatory agencies.

Impurities can be classified into three groups, namely: organic, inorganic and

residual solvents. Organic impurities may arise from starting materials,

intermediates and synthetic by-products or from reagents and catalyst or as a

consequence of degradation. Inorganic impurities may result from reagents,

ligands or catalysts as heavy metals or inorganic salts. Residual solvents are

inevitable in drug substance since without solvents, purification and generation

of the desired crystal morphology would be impossible. However, since residual

solvents also arise in excipients and occasionally in the manufacture of drug

products, it was decided to draft a separate guideline to address appropriate

levels. A key component of the guideline, and a fundamental concept is

qualification. Qualification 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 level(s) specified. Thus, the pharmaceutical analyst and

toxicologist must work hand in hand throughout the pre-clinical and clinical

development programme in order to be able to set meaningful specification

requirements. The pharmaceutical analyst must give the usefiil thoughts to the

analytical methods, especially in the development phases. Therefore, it is an

important task for the pharmaceutical analyst to choose an analytical technique

with modem instrimientation, which provides the best solutions to the problems.

The most commonly used analytical techniques in pharmaceutical analysis

are described below. The chromatographic methods include paper

chromatography, thin layer chromatography, column chromatography, capillary

electrochromatography, high performance liquid chromatography, high

performance thin layer chromatography and gas chromatography. The

electrochemical methods mainly used for this purpose are polarography,

volatammetry and amperometry. The radiotracer techniques include mainly the

isotopic dilution and the activation analysis. The spectroscopic techniques used

for this purpose are UV-visible spectrophotometry, derivative

spectrophotometry, difference spectrophotometry, infirared spectroscopy, flame

photometry, fluorometry and nuclear magnetic resonance.

There are number of analysis that could not have been accomplished in

any other ways except the use of chromatographic techniques. High performance

liquid chromatographic methods have been successfully applied in drug analysis

and have now appeared in the dissolution monographs of pharmacopoeias for the

assay of drugs in dissolution fluids [14-16]. It is becoming increasingly popular

because of its sensitivity and specificity in multicomponent analysis and widely

used for the identification of drugs in plasma and drug formulations [17-37].

High performance thin layer chromatographic technique is flexible enough

to analyze different kind of samples. It is an Off - Line technique whose every

stage of analysis can be visualized. It can be used in pharmaceuticals, biomedical

and environmental analysis. A review paper by Renger on contemporary thin

layer chromatography and high performance thin layer chromatography in

pharmaceutical quality control [38,39] discussed development and validation of

methods. Advantages stated include simplicity of handling and performance;

flexibility and short analysis time; the ability to analyze complex or crude

samples with minimimi sample clean up; wide variety of layers; mobile phase;

operational aspects; and detection reagents; no obligation for elution; ability to

evaluate the entire chromatogram stored on the plate with a variety of techniques

and parameters without time constraints; simultaneous but independent

development of multiple samples and standards on each plate, leading to high

sample throughput and increased reliability of results (in-system calibration); and

robustness, allowing easy transfer and adoption of methods. Several drugs have

been investigated by high performance thin layer chromatography in

pharmaceutical preparations [40-49].

Capillary electrochromatography is a method in which a liquid mobile

phase is driven through a stationary phase in a packed capillary column by the

electro-osmotic flow generated by a large difference in potential across the

column [50-53]. A number of papers have appeared that discuss various aspects

of this technique, including one that considers the use of open-tubular capillaries,

packed capillaries and monolithic capillaries [54].

Capillary electrophoresis technique continues to grow in popularity

especially in areas where they can provide complementary informations to the

other more established methodologies or advantages in selectivity and efficiency

[55]. Two of the areas where usage has increased the most are in assessing

enantiomer purity of pharmaceutical via the addition of chiral additives [56,57],

and in determining the physiochemical information about the analyte. Another

important aspect of this technique is that flow volumes are small, and as such,

coupling it to a mass spectrometer is relatively easy [58,59]. A consideration that

is especially important for a technique to gain acceptance in the pharmaceutical

industry is validation [60,61]. The results generated in electrophoresis technique

are comparable to HPLC and GC in terms of accuracy, precision, linearity and

range, specificity and ruggedness.

Flow injection analysis is widely used as analytical technique [62,63] and

has several advantages: (1) reduced reagent consumption, (2) high sampling

frequency and (3) safety in applying toxic reagents because the whole analysis

proceeds in a closed system. An additional advantage observed in flow -

injection analysis is increased selectivity when the analyte is accompanied by

8

more slowly reacting components. Flow injection analysis has been applied in

the analysis of several pharmaceutical compounds [64-73].

Electrochemistry can be used at the early stage of the drug researches for

screening the pharmacological activity of a homologous series of newly

synthesized molecules. There are several examples in the literature showing the

relationship between the electrochemical data and the pharmacological properties

of drug [74-77]. Electrochemical behavior of cefetamet sodium has been studied

using dc, ac, and differential pulse polarography, cyclic voltammetry and

constant potential electrolysis [78]. Likewise, the electrochemical oxidation of

cefotaxime at activated glassy carbon, platinum, and carbon paste electrodes has

been examined [79]. Square wave voltammetry has been used to determine

sulphamethoxypyridazine and trimethoprim simultaneously in mixtures [80].

The antihypertensive drug doxazosin in urine and formulations was

determined by means of both, differential pulse and square wave adsorption

stripping voltammetry at a mercury drop electrode [81]. The potential of electro

analytical techniques for pharmaceutical analysis, covering a broad spectrum of

electrochemical techniques, was reviewed by Kauffmann et al. [82].

Analytical absorption spectroscopy in ultraviolet and visible region of

electromagnetic spectrum is widely used for quantitative analysis in the field of

pharmaceutical and biomedical analysis. The scope of absorption spectroscopy is further

extended by use of colour reactions often with concomitant increase in sensitivity and or

selectivity. Such reactions are used to modify the spectrum of an absorbing molecule, so

that it could be detected in visible region well separated from other interfering

components. Chemical modification is also useful for transforming non-absorbing

molecule to a stable derivative possessing significant absorption spectral behaviour.

A particular advantage of UV Spectrophotometry is that it does not destroy the

sample. After recording the ultra violet spectrum, one can reextract the solution and

recover the absorbing substances for subsequent analytical procedures, which is in sharp

contrast to gas chromatographic and immunological procedures. UV- spectrophotometric

methods are simple, quick, economical and usually do not require 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 interference, as in case with dissolution testing of Phenytoin capsules [83].

When a beam of light is passed through material, the incident radiation (IQ) will

always be more intense than the convergent radiation (I). Attenuation of the incident

radiation may be attributed to (1) reflections at the interfaces between air and cell wall

and sample and cell wall, (2) scattering by any suspended particles or dust in the sample

and (3) absorption of radiation by the sample, in normal applications light absorption is

the primary factor reducing the incident radiation.

Fluorescence can often increase the apparent intensity of the emergent radiation,

which may result in inaccurate absorption measurements. Effect of fluorescence can be

reduced by chemically inhibiting fluorescence, locating the sample some distance from

the detector, or using appropriate cutoff filters to block the fluorescence occurring at

longer wavelengths than the incident radiation. When using monochromatic light, the

10

fraction of the radiation absorbed by the substances, ignoring losses due to reflections and

scattering, will be function of the concentration of the substance in the light path and the

thickness of the sample. Mathematically this function can be defined as: A = Log [lo/I] =

ebc, where A is defined as absorbance of the sample, "e" is wavelength dependent

proportionality factor called molar absorptivity, "b" is the light path length through the

sample and "c" is the concentration of the sample in appropriate units. The expression A

= ebc is known as Lambert-Beer's law or Beer's law.

The linearity of Beer's law is subject to some restrictions. Concentrations may not

always be directly proportional to absorbance. Chemical and physical interactions and

instrumental limitations can produce deviations, particularly at high concentrations or

high absorbance. Sample scattering, fluorescence, decomposition, and saturation are

examples of sample-related problems affecting linearity. Hydrogen bonding, ion-pair

formation, solvation and chemical reactions can cause incorrect calculations of sample

concentration as the solvent concentration is varied. Instrument factors that can effect

apparent deviation firom linearity are stray light, resolution and both absorbance and

wavelength calibration. Most of the chemical and instrument effects described above will

result in suppression of absorbance as concentration is increased. To prevent errors.

Beer's Law plots should be constructed for each sample system with at least three points

within the expected concentration range of the unknowns.

Simple colorimetric and UV methods continue to be popular for carrying out

single component assay on a variety of formulated products. Representative examples of

some of the many assays that have been published are given in Table 1.1. Colorimetric

11

methods based on charge transfer reaction of certain n - acceptors have been successfiilly

utilized in pharmaceutical analysis. The u-acceptors such as p-Chloranilic acid,

dichlorophenyl-indophenyl, 2,3-dichloro 5,6-dicyano p-benzoquinone,

tetracyanoethylene, tetrachlorobenzoquinone, p-benzoquinone, p-nitrophenol,

tetracyanoquinodimethane [140-141] have been used to carry out spectrophotometric

assay of certain cardiovascular drugs such as amlodipine besylate [89,90], verapamil

hydrochloride, atenolol [96], acebutalol hydrochloride, carazol, propranolol

hydrochloride [142] and lisinopril [115].

Acid dyes, solochrome dark blue, solochrome black T, bromothymol blue,

bromophenol blue, bromocresol green, bromocresol purple, sunset yellow, tropaeolin 000

and indigo carmine form ion-pair complexes with basic drugs which were quantitatively

extracted into chloroform. Colorimetric methods based on dye pairing continue to be

important and have been used to assay compoimds such as amlodipine besylate [143-

145], verapamil hydrochloride [146], phenothiazine derivative [147] and famotidine

[148].

Derivative Spectrophotometry is an analytical technique of great utility for

extracting both qualitative and quantitative information from spectra composed of

unresolved bands. In recent years, the introduction of electronic differentiation by a

microcomputer interfaced with the spectrophotometer makes possible the plotting of the

first, second or higher order derivatives of a spectrum with respect to wavelength.

12

Table 1.1. Assay of drugs in drug formulations by Spectrophotometric procedures.

Name of Drug

Acetaminophen

Alendronate

Amoxycillin

Amlodipine besylate

Ascorbic acid

Aspirin

Astemizol

Reagents used

p-xylenol

Potassium hexacyanoferrate (HI) and

m-cresol

FeCla

KIO3

Tris- (o-phenanthroline) iron (II)

Ninhydrin

2,3-Dichloro 5,6-dicyano 1,4-

benzoquinone

Ascorbic acid

l-Chloro-2, 4-dinitro benzene

NaOH

p-Chloranilic acid

Suprachen Violet 3B

Tropaeolin 000

Iron (III) and 1,10-Phenanthroline

Amax(nm)

635

260

520

510

590

5S0

530

380

302

540

590

500

515

References

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[90]

[91]

[92]

[93]

[94]

[94]

[94]

13

Acetic acid & N-bromosuccinimide 540

Atenolol

Benidipine

hydrochloride

Bromazepam

Chlorhexidine

Cisapride

HCl & Celestine blue

Iodine

Chloranil

2,3-Dichloro-5, 6-dicyanol, 4-

benzoquinone

Bromanil

Tetracyanoethylene

Ciprofloxacin

p-Chloranilic acid

Methanol

Mohr salt

Alizarin yellow G.

Chromotropic acid

Phloroglucinol

p-Chloranilic acid

540

Folin - Ciocalteau reagent & NaOH 720

Glycine buffer and Azocarmine G 540

365

440

470

450

450

7,7,8,8-Tetracyanoquinodimethane 420

535

238

584

405

530

450

520

[95]

[95]

[95]

[95]

[96]

[96]

[96]

[96]

[96]

[96]

[97]

[98]

[99]

[100]

[101]

[101]

[120]

14

Desipramine

Diltiazem

Hydrochloride

Doxorubicin

hydrochloride

L-Dopa

Etidocaine

Famotidine

Flurazepam

Gentamicin

Imipramine

Levopoda

Lisinopril

Tetracyanoethylene

Anunonium metavandate

Diclofenac Sodium Tris buffer

Sodium metavandate

Bromocresol green

Bromothymol blue

Bromophenol blue

Ferric ammonium sulphate

NaOH

Bromocresol green

Alkaline KMn04

Ninhydrin

HCl

Ninhydrin

Ammonium metavandate

Cerium (IV) nitrate

l,Fluoro-2, 4-dinitro benzene

535

618

284,305

750

415

415

415

[102]

[103]

[104]

[105]

[106]

[106]

[106]

510 [107]

300

625

610

590

230

400

618

510

356

[108]

[109]

[110]

[111]

[112]

[113]

[103]

[114]

[115]

15

Loperamide

hydrochloride

Loratadine

Meclozine

hydrochloride

Methyl dopa

Mefloquine

hydrochloride

Mefenamic acid

Menadione

Metoprolol tartarate

Chloranil

Iodine

Bromothymol Blue

Bromophenol Blue

Naphthol Blue Black

Bromophenol Blue

Chromotrope 2B

Chromotrope 2R

Cerivim (IV) nitrate

HCL

p-Dimethylaminocirmamaldehyde

NaOH

Ethylacetoacetate

3-Methyl 2-benzothiazolinone

hydrazone

Tetracyanoethylene

7,7,8,8-Tetracyanoquinodimethane

346

295

414

415

627

415

540

528

550

222

665

450

550

625

450

420

[116]

[117]

[117]

[117]

[117]

[118]

[119]

[119]

[114]

[120]

[121]

[122]

[123]

[124]

[125]

[125]

16

Nalidixic acid

Nifedipine

Nimisulide

Nitrazepam

Nizatidine

Nortriptyline

Hydrochloride

Oxprenolol

Piroxicam

Salbutamol

Trimethoprim

Thyroxine sodium

Tenoxicam

Verapamil

hydrochloride

Persulphate in alkaline medium

4-(Methyl amino phenol) and

K2Cr207

BCMn04

Iminodibenzyl

3-Aminophenol

Ethylacetoacetate

Alkaline KMn04

3-Methyl 2-benzothiazolinone

hydrazone

Cerium (TV) in H2SO4

Alizarin

2,6-Dichloroquinone chlorimide

7,7,8,8-Tetracyanoquinodimethane

Persulphate in alkaline medium

Nitrous acid

Alizarin

Chloramine-T

320,390

525

530

600

470

482

610

619

480

538

602

842

355

420

538

425

[126]

[127]

[128]

[129]

[129]

[130]

[131]

[132]

[133]

[134]

[135]

[135]

[136]

[137]

[138]

[139]

17

The derivative method has found applications not only in UV-spectrophotometry

but also in infrared [149], atomic absorption, flame spectrophotometry [150,151], and

fluorimetry [152,153]. The use of derivative spectrometry is not restricted to special

cases, but may be of advantage whenever quantitative study of normal spectra is difficult.

Its advantage is that the differentiation degrades the signal-to-nose ratio, so that some

form of smoothing is required in conjunction with differentiation [154].

dA For a single-peak spectrum, the first-derivative is a plot of the gradient — of the

UA

absorption envelope versus wavelength and features maximum and minimum; the vertical

distance between these is the amplitude, which is proportional to the analyte dA

concentration; theoretically, — is zero at max for the band in the normal spectrum. The dX

d^A second-derivative spectrum, —r- versus wavelength, has two maxima with a minimum

dX

between them, at X,max of the normal absorption band [155]. In principle, both peak-

d'^A heights (measured from —^= 0) are proportional to the analyte concentration but the

dX

amplitude can also be measured by the so-called tangent method, in which a tangent is

drawn to the maxima and amplitude is measured vertically from tangent to the minimum

[156]. The differentiation discriminates against broad bands, emphasizing sharper

features to an extent that increases with increasing derivative order, because for bands

(Gaussian or Lorentzian) the amplitude Dn of the nth derivative is related to the n* power

of the inverse of the band width, W, of the normal spectrum [157]:

18

D„a

Thus, for two bands A and B of equal absorbance but different width, the

derivative amplitude of the sharper band (A, for example) is greater than that of the

broader (B) by a factor that increases with increasing derivative order [158,159]:

For the reason, the use of derivative spectra can increase the detection sensitivity

[160-162] of minor spectral features. For quantitative analysis, if Beer's law is obeyed for

normal spectrum, the following equation can be obtained:

= Ic

dX" dA"

Where A = Absorbance, e = molar absorptivity, / = cell path- length and C =

concentration of the analyte and this forms the basis of analytical determinations [163].

Derivative Specti'ophotometry has been applied for quantification of drugs in

pharmaceutical preparations [164-171].

Difference spectrophotometry is a technique, which requires high potentiometric

accuracy and linearity. Small spectral changes in solutions with high initial absorbance

are often measured. This technique has been extensively used in the determination of

drugs by eliminating specific interference from degradation products, coformulated drugs

[172], and also the non-specific irrelevant absorption from the formulation matrix

[173,174]. It provides a sensitive method for detecting small changes in the environment

of chromophores. In general, two solutions at the same concentration are directly

19

compared. One solution is selected as sample, the second as the reference. All coinmon

spectral features to the two solutions cancel out. Only bands, which have been displaced

in one solution relative to the other because of environmental differences, are recorded.

Another emerging trend has been the increasing use of near-infrared (NIR)

spectroscopy in the quality assurance and in process analysis of pharmaceutical products.

Both qualitative and quantitative aspects of NIR spectroscopy as well as uses related to

validation of procedures that employ it are discussed [175-177].

Other important emerging technologies in drug development are proteomics [178]

combinatorial chemistry [179], the application of molecularly imprinted media [180,181],

the use of light scattering particle size analysis for flocculated suspensions [182], and the

profiling of monomeric and dimeric impurities in bulk pharmaceutical batches by

^^FNMR[183].

The number of applications of kinetics to analytical chemistry continues to grow

at a rapid pace. Kinetic methods are good choices for drug analysis as they permit the

sensitive and selective determinations of many drugs within a few seconds with no

sample pretreatment is many cases. Kinetic methods, because of its superiority to the

other colorimetric methods due to the formation of unstable colours in some of these

colorimetric ones, are among the first choices of the researchers in analytical chemistry.

Moreover, the instrumentation required is generally very simple. The principles and

applications of the kinetic methods have been reviewed in papers [184-192] and books

[193,194]. Essentially kinetic methods rely on measurements of concentration changes

(detected via signal changes in a reactant which may be the analyte itself) with time after

20

the sample and reagent have been mixed. The sample and reagent can be mixed manually

or automatically, only slow enough reactions tolerate manual mixing and, even so, they

may be better handled automatically, not only to obtain more rapid and reproducible

results, but also to increase the reaction rate in some cases. Kinetic automatic techniques

are generally based on open system, among the most popular of which is stopped flow

(SF) system and the continuous addition of Reagent (CAR) Technique [195-198]. Several

drugs have been determined by using CAR technique Math photometric [199-200] and

fluorimetric [201] detection. The application of micellar systems to kinetic methods was

the subject of a review by Cai and Co-workers [202]. The discussion included

characteristic of micellar catalysis, micellar modification of properties of system, and

application of micelles in multicomponent determinations. CuUen and Crouch [203], have

described the application of multivariate calibration techniques to multicomponent

kinetic based determinations. These techniques are mandatory for fast reactions but can

also be extended to slow reactions. In both cases, the kinetic curve (variation of the

analytical signal with time) can be recorded immediately. The slope of the initial straight

portion of the kinetic curve gives the reaction rate, which is proportional to the analyte

concentration (initial rate method). The fixed time method [204], also frequently used to

determine the concentration involving the measurement of the signal (the value of which

depends on the analyte concentration) at a preset time.

The determinations can be implemented by using combinations of the SF

technique with (a) fluorescence polarization immunoassay (FPIA), (b) micelle-stabilized

room temperature phosphorimetry and (c) a sensitized energy transfer fluorescence

21

reaction, among other, all are novel strategies which have so far yielded excellent results

in drug analysis. On the other hand, the CAR technique has been extended to

chemiluminescence (CL) reactions as CAR chemiluminescence spectrometry (CARCL),

a new approach that has proved outstanding for the analysis of drugs and other substances

of analytical mterest. Moreover, new kmetic Ghemomettic (kinetometric) approaches

[189] such as the Kalman filter have been developed and applied to the simultaneous

determination of various compounds of pharmaceutical interest.

Over the last few years the term chemometrics has been increasingly used. The

organic chemist S. Wold supposedly coined this term in 1972 when submitting a grant

proposal for the application of statistical methods to chemical data. Together with

American analytical chemist B.R. Kowaiski, Wold formed the international

chemometrics society. Subsequently there have been several major reviews, [205-208]

ACS symposia [209,210], a National Bureau of standards conference, [211] a Nato

School, [212], a text book [213] and a series of monographs [214] devoted to

"Chemometrics". Recently two international journals devoted to Chemometrics have

been established [215,216]. A large variety of clustering methods [217] have been

developed, originally as aid to pharmaceutical and clinical researchers. The ability of

modem analytical instruments helps a lot to acquire large amounts of data rapidly. In a

modem laboratory, measurements are cheap compared with samples: many parameters

(e.g.. Chromatographic and Spectroscopic peak intensities) can be obtained from each

sample. Hence new approaches are needed to interpret what may be described as large

multivariate data matrices, involving specialized statistical and computatioual rricthods,

22

clearly we can say that chemometrics involves the application of statistical methods to

analytical data.

Method Validation

Method validation is an important component in determining the reliability and

reproducibility of analytical method and is a reqiiirement of any regulatory submission

[218], According to the European Union directives in referring to the Good

manufacturing Practice and Quality control of drugs [219-221], validation must be

applied not only in manufacturing process but also in analysis and control methods. The

validation of analytical procedures used for raw materials and a finished product is

gathered in the Pharmaceutical Products and Multistate Register [222-224] also and more

recently it has been reviewed for ICH Harmonized Tripartite Guideline [225,226]. As far

as the validation characteristics are concerned, if the analytical method is described in

Pharmacopoeia, it will be proceeded as follows: (a) with regard to raw materials assay,

the validation will not be necessary, although it must be checked before its routine use,

enclosing information about linearity, sensitivity, precision and selectivity, (b) with

regard to the finished product assay, it is necessary to carry out a brief validation to

demonstrate that the precision, selectivity and accuracy are appropriate.

Validation and Strategy

The overall validation strategy consists of four components, which are the

prevalidation, validation proper, study proper and statistical analysis. These components

23

constitute the platform to evaluate the reliability and ruggedness of an analytical method.

Prevalidation

Prior to initiating the validation proper, a prevalidation is performed by the

primary analyst. This component provides the analyst an opportunity to obtain some

practical experience v^th the methods and helps to identify the optimum conditions. It is

recommended that the following studies be conducted prior to initiating the validation

proper. The appropriate peak response to use in quantitating drug concentrations should

be selected first. The optimum standard curve range and the number of calibrators should

be established. The appropriate regression model which best fits the data is then selected.

The extraction scheme and its recovery should be optimized to give insight into

the limit of quantitation and to help determine if the extraction procedure is reproducible.

If possible, potential compounds that could interfere with the method should be

evaluated. The remaining system suitability parameters are then investigated by

optimizing the other factors.

Validation Proper

The validation proper consists of four analytical runs generated on separate days.

Each validation run emulates the analytical conditions of the study proper and its

expected run time. This strategy helps the analyst develop a daily routine to use during

the study proper and anticipate potential assay problems. Suggestions for additional

samples to be analyzed during each validation run include samples fortified with

24

metabolites and potentially interfering compounds to identify response behaviour and

ensure assay specificity. Stability samples to begin generating information pertinent to

sample processing and storage. From the data generated, specific analytical parameters

are reported, including linearity, accuracy, precision, sensitivity and recovery. From each

standard curve; the slope, intercept, correlation coefficient and variance are monitored.

Statistical analysis are used to determine with in and between-run variance and to

demonstrate how the method can be expected to perform on a daily basis. Based on the

mitial quality control data concentration results, acceptance criteria are established.

Subsequent analytical runs are monitored during the study proper usmg these acceptance

criteria to determine if the data generated are valid.

Acceptance criteria for validation parameters

Using the ICH guidelmes as a basis [227,228], it is the responsibility of the

analyst to select for the given individual test procedure relevant parameters and

appropriate acceptance criteria and to design the experimental studies accordingly. These

acceptance criteria can often be derived from specification limits.

As a general rule, the standard deviation of the analytical procedure should be

lower than 1/6 of the specification range. A detailed approach taking the number of

repeated determinations into account is based on confidence intervals [229,230]. Basic

specification limits (BL) include the variability of the manufacturing process and

represent the final limits (SL) if an error-free analytical procedure is used. Describing the

analytical variability as a normal distribution of the experimental results, confidence

25

intervals can be constructed as a representation of the result probability for a given

number of replicates. The combination of the basic limit and the limit (upper or lower) of

the 95% confidence interval (one sided) then represents the overall specification limit

(Eq. 1).

SL = BL±t„_, 095^^ Eq.(l)

It must be taken into account that this calculation is based on the true standard

deviation whereas the standard deviation obtained in a validation study is only a random

estimate which also displays a variability. A reliable experimental determination of the

true value requires repeated intermediate precision studies, but it can be estimated from

the statistical (z^) distribution of standard deviations. The upper 95% confidence limit of

this distribution (UL) will represent the maximum value for the true standard deviation

(Eq. 2). For six experimental values, it can be approximated by twice the experimental

standard deviation [230].

UL(S,^e) = S,^,x U ^ Eq.(2) V Z "-' J0.95

Rearranging Eqs. (1) and (2) gives the maximum permitted experimental standard

deviation for the given specification limits, i.e. the acceptance limit for the validation. For

drug substances, due to the presence of impurities, these are the lower limits. As the

required standard deviation is dependent on the number of repetitions in the assay,

adjustments are possible (Table 1.2.). Thus, the number of repeated determinations can

also be fine-tuned according to the results of the validation. In case of sufficiently wide

26

specification limits compared to the analytical variability, this allows an efficiency

optimization of the analytical procedure.

While in the case of a drug substance assay the basic limits are defined mainly by

the sum of impurities, in the case of drug products, often only estimation is possible. For

simple dosage forms, the same variability may be expected for analytics and

manufacturing. It should be noted that the estimation of the true standard deviation would

result in a maximum value and, therefore, reduce the required acceptance limit.

Consequently, in critical cases an experimental determination (using at least four to six

series) might be reasonable to obtain an estimate for the true standard deviation. If

specification limits are not yet defined or implied due to safety requirements, Eq. (1) can

be used directly to calculate the lunits. For the true standard deviation, the upper limit of

the experimental determination (Eq. 2) can be calculated or twice this value as an

approximation. Alternatively, a soundly based intermediate precision may be used as

estimation for the true standard deviation.

With respect to impurity detenninations, the ICH reporting threshold of 0.05%

[231] can be regarded as the required quantitation limit for unknown impurities, which

would guarantee a reliable quantitation at the specification limit of 0.1%. If other limits

are required due, for example, to safety considerations, the above-mentioned approach

can be applied. Statistical tests such as the student's Mest or the evaluation of 95%

27

Table 1.2. Maximum permitted standard deviation for assay determinations in dependence on the number of repetitions and the specification range

Drug product (%) Drug product (%) Drug product (%)

Specification range 95-105 95-105 98-102

Basic limit (lower) 97.5 (estimated) 99.0 (estimated) 99.5 (sum of impurities)

Niraiber of repetitions Acceptance limit for experimental standard deviation in validation (n=6)

2 0.28 0.45 0.17

3

4

6

0.74

1.06

1.44

1.19

1.7

2.3

0.45

0.64

0.86

28

confidence intervals should only be carefully (directly) applied as acceptance criteria

because they test for statistical differences. Due to sometimes abnormally small

variabilities in the analytical series, differences are identified as significant which are of

no practical relevance [232]. In addition, when comparing independent methods for the

proof of accuracy, different specificities can be expected which add a systematic bias,

thus increasing the risk of the aforementioned danger.

In addition, the power of these tests increases with the number of determinations.

For practical purposes, a sufficient agreement between two results (two means or a mean

and a nominal value, e.g. recovery) is completely adequate. The acceptance criteria can

be derived from previous experience or calculated based on specification limits and

statistical considerations. For example, assuming an assay procedure with specification

limits from 95 to 105%, a recovery range from 98 to 102% would be acceptable.

The acceptance criteria and limits should be defined before starting the validation

and included in a protocol. After the validation studies, they will serve as a basis for the

evaluation.

Validation characteristics

When performing validation studies, the whole analytical procedure including all

the steps of the sample preparation should be applied, as far as possible. In contrast, the

term 'method' should be restricted to the mode of analytical determination alone (e.g.

capillary electrophoresis, (reversed phase) chromatography and spectrometry). Allowed

exceptions to the written procedure concern the number of repetitions as the number of

29

determinations for the various validation characteristics is described in the ICH guideline

(Table 1.2) and the repetitions in the procedure may be fine-tuned based on the validation

results. Such an adjustment may also be used for the final calibration mode.

As far as possible, the analytical procedure should be independent of the actual

equipment used provided that the equipment has been appropriately qualified. This must

be taken into account for the validation studies.

Specificity

There has been some controversy regarding the technical term for this validation

characteristic, i.e. specificity versus selectivity [233]. In contrast to an isolated test

procedure, in pharmaceutical analysis the sum of various control tests and hence there

combined specificity is used for the overall batch evaluation. A very pragmatic definition

describes selectivity as the (physical) separation of substance mixtures with, for example,

chromatography and electrophoresis, i.e. the determination of the analyte in addition to

other substances. The individual determination of an analyte in the presence of other

substances, (i.e. without significant influence of other substances or classes of

substances) is defined as specific by, for instance, mass spectrometry, NMR, infrared,

fluorescence or UV spectrometry, electrochemical detection and titration [234].

With respect to chromatographic techniques, specificity can be demonstrated by a

sufficient separation of the substances present. For the assay, appropriate separation

means an adequate resolution between the main peak and the impurity and placebo peaks,

which need not to be separated fi-om each other. The required resolution is strongly

30

dependent on the difference in the size of the corresponding peaks as well as on their

elution order [235].

Linearity I range

A linear dependence of the signal and the analyte concentration is certainly the

most convenient case and widely used in pharmaceutical analysis. The requirements and

relevant parameters for the various calibrations are given in Table 1.3.

It should be noted that in most cases only a qualitative statement is needed. For

example, if a single-point calibration (external standard) is aimed at, the requirements are

a linear response flmction and the zero intercept. Care should be taken to remain within

the linear range of the individual detector used.

Accuracy

The ICH guideline recommends the demonstration of accuracy over the whole

working range (Table 1.4). However, if only a narrow range is required (e.g. assay or

impurities with a low specification limit), a six-fold determination at a 100% test

concentration as described for the precision studies may also be used. Several approaches

discussed in the ICH guidelines are given in Table 1.5. If the analytical test to be

validated is compared with another procedure or applied to a reference substance, the

probably different specificities must be taken into account. Therefore, statistical tests

should be performed only if the systematic bias based on these differences can be

quantified and thus corrected or are negligible. Otherwise, the comparison should be

31

Table 1.3 Requirements for different calibration modes with relevant parameters

Quantitation Requirements Relevant parameters

Single-point calibration External standard Linear function

Non-significant Ordinate intercept

Homogeneity of variances"

Multiple-point calibration Linear, unweighted Linear function

Linear, weighted

•Non-linear

100%-method(area normalisation for impurities):

Homogeneity of variances^

Linear function

Continuous function

For main peak: Linear function

Non-significant Ordinate intercept

Homogeneity of Variances"

For impurities Linear Function

Standard error of slope (residual standard deviation), Sensitivities (relative standard deviation, graph), residual analysis, statistical tests (vs. quadratic regression)

Inclusion of zero in confidence interval of the ordinate intercept, magnitude of the intercept (as percent of the signal at 100% test concentration)

F-test of the variances at the lower and upper limit of the range

Standard error of slope (residual standard deviation), sensitivities (relative standard deviation, graph), residual analysis, statistical tests (vs. quadratic regression)

F-test of the variances at the lower and upper limit of the range

Standard error of slope (residual standard deviation), sensitivities (relative standard deviation, graph), residual analysis, statistical tests (vs. quadratic regression)

Appropriate equation

Standard error of slope (residual standard deviation), sensitivities (relative standard deviation, graph), residual analysis, statistical tests (vs. quadratic regression)

Inclusion of zero in confidence interval of the ordinate intercept, magnitude of the intercept (as percent of the signal at 100% test concentration)

F-test of the variances at lower and upper limit of the range

Standard error of slope (residual standard deviation), sensitivities (relative standard deviation, graph), residual analysis, statistical tests (Vs. quadratic regression)

May be presumed for a limited range (factor 10-20).

32

Table 1.4. Validation characteristics normally evaluated for the different types of test procedures and the minimum number of determination required

Validation characheristics

Specificity

Linearity

. Range

Accuracy

Precision

Repeatability '

Intermediate precision reproducibility'^

Detection limit

Quantitation limit

Minimum number

5 concentrations

9 determinations over 3 concentrations levels (e.g. 3x3)

6 determinations at 100% or 9 determinations over 3 concentrations levels (e.g.3x3)

2 series

Test procedure

Identity

Yes

No

No

No

No

No

No

No

Impurities

Quantitative

Yes

Yes

Yes

Yes

Yes

Yes

No

Yes

Limit

Yes

No

No

No

No

No

«

Yes

No

Assay^

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No Including dissolution content/Potency.

^Lack of specificity of one analytical procedure could be compensated by other supporting analytical procedure. "Intermediate precision sufficient for submission. May be needed in some cases.

33

Table 1.5. Approaches to demonstrate accuracy according to ICH

Drug substance

Drug Product

Impurities (quantitative)

Application of the analytical procedure to a reference

material

Comparison of the results with those of a second, well

characterized procedure.

Application of the analytical procedure to Drug

product synthetic mixtures of drug product

components

Spiking of analyte to drug product

Comparison of the results with those of a second, well

characterized procedure.

Spiking of the impurity to drug substance

or product

Comparison of the results with those of a second well

characterized procedure.

34

performed, as a qualitative verification of plausibility or an acceptable maximum

difference should be defined. Spiking experiments for recovery investigations should be

performed as closely to the authentic conditions as possible so that possible interferences

between the analyte and matrix can be recognized. This ranges, for example, from the

direct preparation of a drug product with various contents of active ingredient to which

the whole analytical procedure is applied to the addition of a drug substance stock

solution to a placebo solution.

For the quantitation of the analyte, the same calibration mode as described in the

final test procedure must be used. Again, statistical tests should be used carefully,

especially with complex matrices and low concentrations of impurities. Alternatively,

acceptable deviations from the theoretical recovery of 100% can be defined based on the

application, experiences or general statistical considerations.

Precision

In addition to the ICH precision levels (Table 1.4), it is advisable to determine the

system precision (injection repeatability) either by repeated determinations of the same

test solution or firom double determinations of each test solution used for repeatability

(Eq. 3).

S''=\~t(\.-\2)' Eq.(3)

Repeatability, also termed as intra-assay precision, refers to the precision obtained

under the same operating conditions over a short interval of time by applying the whole

35

analytical procedure to the sample. Intermediate precision refers to within laboratory

variations. The extent of investigations will depend on the intended use of the analytical

procedure. Repeatability and intermediate precision can be calculated by an analysis of

variances [236,237]. The difference between the precisions levels as well as their

absolute magnitude indicate the robustness of the anal3^ical procedure. For the evaluation

of the suitability (compatibility with specification limits) the intermediate precision can

be regarded as the relevant parameter. From the standard deviation, the repeatability limit

can be calculated (Eq. 4) which represents the maximum permitted difference between

two repeated measurements.

^ = ^«-i,o.95^^^'^*'2.8x5 Eq. (4)

In this way, a straightforward verification is possible if the degree of scattering

imder the actual conditions is comparable to the validation. By this parameter, the

precision obtained during the validation studies using a larger number of data (thus

increasing the reliability) is linked to the routine analyses without requiring a larger

nimiber of experimental data. Reliable estimate of the standard deviation is required to

calculate appropriate repeatability limits.

Most statistical tests and calculations are based on the assumption that the

experimental values are only influenced by random variability (i.e. that they are normally

distributed). Data, which do not fulfill these assumptions (e.g. due to so called 'gross

errors', weighing, dilution, or by problems with the instrument, etc.) will affect the

results. Such values can be identified by statistical outlier tests (e.g. according to Dixon

or Grubbs [236]) in order to eliminate them before performing further calculations.

36

Detection and quantitation limit

Several approaches are given in the ICH guideline to determine the detection and

quantitation limits (Table 1.6). Generally, they are based either on the analysis of blanks

or on the scattering (variability) of the analytical signals in the low concentration range.

Using the blank procedures, the corresponding calculation value is multiplied by the

factors of 3.3 and 10 for the detection and quantitation limits, respectively. The

calculation value may represent the signal of the blank, the standard deviation of the

blank or of the intercept of a calibration line (corresponding to an extrapolated blank). In

the latter tv/o cases, the analytical signal is transformed by the slope of the calibration

line into a concentration [228]. Using the calibration line directly, the aforementioned

factors (3.3 and 10) can be multiplied by the ratio from the residual standard deviation

and the slope (corresponding to the standard error of slope) [228]. Limits calculated or

extrapolated by these procedures should be verified by the analysis of samples in the

corresponding concentration range [228]. This additional verification is already included

in other procedures, which make direct use of the scattering around the calibration line by

means of the 95% prediction interval around the regression line [238,239]. The

quantitation limit can also be obtained directly from precision studies. For this approach.

37

Table 1.6. Approaches for determining the detection and quantitation limit [228]"

Approach Detection limit Quantitation limit

Visual evaluation Minimum level detectable Minimum level quantifiable

Signal-to- noise 3: lor2 :1 10:1

Standard deviation of the 3.3 X a/S lOxa/S

response and the (a) slope (S)

^Verification with suitable number of samples. *" Standard deviation of the blank, residual standard deviation of the calibration line, or standard deviation of the intercept.

38

decreasing analyte concentrations are analyzed repeatedly. The coefficient of variation

(relative standard deviation) is plotted against the corresponding concentration. If a

predefined limit for the coefficient of variation is exceeded (e.g. 10 or 20%), the

corresponding concentration is established as the quantitation limit [240,241].

Study Proper and Statistical Analysis

Daily standard curves are generated to determine the sample concentrations. All

calibrators and quality control samples are analyzed in duplicate. Sample concentrations

are based on single determination. The quality control sample sequence is carefully

monitored for systematic errors. For each standard curve, the slope, intercept, variance,

correlation coefficient and the interpolated calibrated concentration are reported.

Acceptance of the assay results are determined by monitoring the quality control

results. If the interpolated concentrations are v ithin the control charts confidence limits,

established during the method validation, the data are considered valid. Upon completing

a study proper and accepting the analytical runs, the quality control results are

incorporated into their respective data basis to update their confidence limits.

Statistical calculations of the validation parameters and certain others of common

interest

Linear Calibration

Linearity is often tested by using correlation coefficient, r. this quantity, whose

39

full title is "Product-moment Correlation Coefficient", is given by

i:h-^){y.-y)] r = -

i:{^.-^i\L[y.-y)" \ii

Where the points on the graph are {x^,y^), (xz.jVa) {^nV.) (^^'^^J' ^ ^ x

and y are as usual, the mean value of x, and y, respectively. Value of r should be as:

- l < r < + l .

Least square Line

The slope, b, and intercept, a, of the imweighted least square lines are found from.

6 = -YX{^.-x){y,-y)]

/ _ \ 2 E(^'-^)

a = y-bx

Errors and confidence limits

Random errors of the slope and intercept of the regression line involve the

preliminary calculation of Sy/x, which is given by

40

ll/2

Sy/x = Jl{y'-y>f

n-2

In this equation each y, value is measured signal value from the analytical

instruments, while the corresponding y, is the value of y on the fitted straight line of the

same value of x. After the determination of Sy/x, the standard deviation of the slope, Sb,

and the standard deviation of the intercept, Sa can be determined from:

S, 'ylx

S(-,-?)' 1/2

^a - ^y/x

E--

«Z(^.-^)'

1/2

These standard deviations can be used to estimate the confidence limit for the true

slope and intercept values. The confidence limit for the slope are given by b + ts^ , where

the value of "t" is chosen at the desired confidence level and with n-2 degree of freedom.

Similarly, confidence limits for the intercept are given by a±tSi,. Standard

deviation of S^^, can be calculated by using equation:

-11/2

s. = y/n

1 + - + 1 (yo-yf

" b'Zi-,-^f

41

Where XQ and y^ are the value of single concentration and its instrument signal.

In a typical analysis, the value of y^ might be obtained as the mean of m observations of

a test sample, rather than as a single observation.

ylx s„ = b

1 , 1 , jyo-y) — 1 — I m n b^n-.-^r

1/2

Method of standard additions

Matrix effects on the measured signal demand the use of standard additions. The

concentration of the test sample, x^ is given by the intercept on the x-axis, which is

clearly the ratio of the y-axis intercept and the slope of the calibration line as:

a

Limit of detection and sensitivity

The lowest detectable instrument signal, jv , is given by:

y^=ys+KSs

Where y^ and Sg are respectively, the blank signal and its standard deviation. After yj^

has been established, it can readily be converted into a mass or concentration limit of

detection, c , as:

^L=-KS.

42

CLASSIFICATION OF DRUGS

All the drugs according to their chemical nature can be divided into organic and

inorganic compounds. They can be prepared synthetically (from chemicals) or can be

directly obtained or reconstituted from the natural sources/products. All the drugs having

medicinal importance can be broadly divided into two classes.

Chemical classification

Here the drugs are classified according to their chemical structure and properties

without taking the pharmacological actions vmder consideration, hi this class most of the

drugs are having at least an organic substrate, the fiirther classification is done in the

relevant manner.

Pharmacological classification

The drugs are classified according to their action on the organs (Viz., heart, brain,

lymphatic system, respiratory system, endocrine system, central system, nervous system

etc.) hence these drugs are named like antianginal, narcotics, soporifics, analgesics,

diuretics and anaesthetics etc. Further classification of each group is done according to

the therapeutic/pharmacological specificity Vvith the relevant organ.

43

Hepatoprotective Drugs

The liver is subject to acute and potentially lethal injury by a number of

substances, including phalloidm (the toxic constituent of the mushroom amanita

phalloides), carbon tetrachloride, galactosamine, ethanol and other compounds. The

effect of flavonoids on carbon tetrachloride induced toxicity in isolated rat hepatocytes

has been studied by Perrisoud and Testa [242]. The ability to interfere with carbon tetra

chloride-induced release of aspartate aminotransferase was tested on 55 flavonoid

compounds. The more hydrophilic compounds were observed to inhibit the carbon tetra

chloride-induced toxicity, whereas the more lipophilic derivatives actually potentiated the

toxicity. In a number of countries, silybin and other flavonoids are widely utilized in the

treatment of liver diseases [243].

Antianginals

Angina pectoris is a heart ailment characterized by pressing chest pain that often

radiates to the neck area and arms and shoulders (often towards left side). As a clinical

entity William Heberden first characterized it under the name Pectoris Dolor in 1768,

although its symptoms had been noted as early as 1632 [244-246], in the recent medical

terminology it is referred as the symptom of ischaemic heart disease. For nearly a century

following its first description, there was not much that could be done to relieve an angina

patient of his agony: brandy, ether, chloroform, ammonia and other stimulants and

depressants had been tried, but nothing seems to bring comfort. The breakthrough came

in 1867 when T. Lander Bmnton, a British physician, reported his success with amyl

44

nitrite. In the authoritative but lively journal of medicine, the Lancet, he wrote, "On

pouring from five to ten drops of amyl nitrite on a cloth and giving it to the patient to

inhale, the pain completely disappeared and, generally did not return till its wonted time

next night" [247]. And finally the continuous efforts of the medical researchers, they got

succeeded in knowing the different reasons with this life-threatening ailment and its

therapeutic solutions.

Drugs used in angina pectoris are those that reduce cardiac work and myocardial

need by (a) unloading the heart, (b) dilating the capacitance and resistance vessels, (c)

dilating the coronary arteries and (d) blocking P-adrenoceptors. Anginal pain occurs

when the coronary blood flow is insufficient to meet the hearts metabolic requirements.

The drugs that either improve myocardial perfusion or reduce the metabolic demand, or

both can covmteract it. Several antianginal drugs are available for the treatment of angina.

These include (a) organic nitrates, (b) beta-adrenergic blockers, (c) calcium channel

blockers, (d) potassium charmel activators, (e) antiplatelet drugs and (f) certain others like

diazepam, oxyfedrine etc.

This thesis deals with the quantitative analysis of silymarin, nifedipine,

amiodarone hydrochloride and verapamil hydrochloride in pharmaceutical formulations.

SUymarin

Silymarin i.e. 3,5,7-trihydroxy,2-[3-(4-hydroxy-3-methoxy phenyI)-2-

(hydroxy-methyl)-I,4-benzodioxan-6-yl]-4-chromanone(structure I) is a free

radical scavenger and a mixture of flavonolignans from medicinal plant Silybum

45

marianum, officially listed in Martindale [248] is used in supportive treatment of liver

diseases of different etiology due to its hepatoprotective activity,[249-251] which is

considered to involve antioxidative and the membrane stabilizing effects. The liver plays

an important role in regulation of metabolism of plasma lipoproteins and liver injury is

often reflected as a secondary dyslipoproteinaemia, which may lead to the development

of atherosclerosis, particularly when associated with hypercholesterolaemia. [252]

OH 0

0 . ,CH20H

OCH^

Structure I

Nifedipine

Nifedipine, chemically dimethyl-1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)

pyridine-3,5 dicarboxylate, is the prototype compound of dihydro pyridine class of

calcium channel blockers with peripheral and coronary vasodilator properties. It reduces

peripheral resistance, afterload and blood pressure. It is given by mouth in the

management of hypertension and angina pectoris. In human, nifedipine is rapidly

46

metabolized by oxidative mechanism to dehydronifedipine wliich is further metabolized

to more polar compoimds [253-256] and their structures are given below:

HaCOOC^ ,C00CH3

H3C 1 CH3

H

3HCOOC-

H ^ c ^ ^ r ^

^N02

'COOCH3

CH, H

Nifedipine Dehydronifedipine

3HCOOC-

H ^ C - ^ N ^ ,

'COOCH3

CH,

3HC H C2HCOOCV

/ 3HC

HaC I H

-NO2

'COOCH3

^CH,

Nitroso analogue of dehydronifedipine Nisoiidipine

47

Nifedipine is highly sensitive to photo-oxidation, changing in colour from yellow

to brown upon exposure to light [257]. Nifedipine photodegration products have a little or

no pharmacological activity [258,259]. Several studies in the past have been conducted to

determine the photostability of nifedipine in solution and in the solid state, including

photostability of pulverized nifedipine tablet powder [260-262].

Amiodarone hydrochloride

Amiodarone hydrochloride,[2-n-butyl,3-(3,5-diiodo-4-diethylaminoethoxy

benzoyl)-benzofuran] hydrochloride (structure II) is a potent class III

antiarrhythmic drug [263]. It is used to treat ventricular and supraventricular

arrhythmias, especially when they are resistant to other conventional

antiarrhythmic drugs [264,265]. However, its use is sometimes complicated by

serious adverse effects, including occasionally life threatening pulmonary

fibrosis and hepatitis [266]. Therefore, its concentration in patients treated with

therapeutic doses of the drug vary considerably and therapeutic drug monitoring

of amiodarone hydrochloride may assist in individualizing the dosage regimen.

In human, mono-N-desethylamiodarone is known as the major metabolite

[267]. During long-term therapy, the plasma level of mono-N-

desethylamiodarone is comparable with that of amiodarone hydrochloride, and

this metabolite may contribute to the therapeutic effect of amiodarone

hydrochloride.

48

, ^ ^ ^ -O CH2 CH2- -N . /

Ri

\ R2

o

-c—

0112(01^2)2^113

Structure II

Where Ri= R2 = C2 H5 (amiodarone), Ri = Hi, R2 = C2H5 (desythyl amiodarone),

Ri = R2 = H (di-N-desythyl amiodarone).

Verapamil hydrochloride

Verapamil hydrochloride i.e. (±)-5-[(3,4-dimethoxyphenethyl) methyl amino]-2-

(3,4-dimethoxyphenyl)-2-isopropyl valeronitrile monohydrochloride (Structure III) is

clinically a very useful member of the calcium channel blocker. It is used in the treatment

of supraventricular arrhythmias, angina and hypertension [268-270]. The verapamil

shows conduction through the atrioventricular node, and this slows the increased

ventricular response rate that occurs in atrial fibrillation and flutter. A decrease in both

coronary and peripheral vascular resistense together with a sparing effect on myocardial

intracellular oxygen consumption, and the decrease in peripheral vascular resistance may

explain the antihypertensive effect of verapamil [271-273].

49

CH,0 / / ^ CH3

H3C ,CH3 CH

CH2—CH2—N—CH2—CH2- CH2—C—CN

Hsca

OCH,

Structure III OCH3

50

[1]

[2]

REFERENCES

A. Burger, Medcinal chemistry, vol.1, Interscience publishers. Inc., New York,

1951, plO.

De Quineey, Confessions of an English opium Eater, in the collected Writings of

Thomas de Quineey, Vol. IE, 222, Edinburg, p. 1890.

[3] L.G. Mathews, History of Pharmacy in Britain, London, 1962, p 67.

[4] Roy Porter and Mkula's Terch: Drugs and Narcotis in History, Cambridge

university press, p. 171.

[5] R. K. Gilpin and L. A. Pachla, Anal.chem.. 32 (1991) 1201

[6] R. K. Gilpin and L. A. Pachla, Anal.chem.71 (1999)217R

[7] R. K. Gilpin and L.A. Pachla, Anal.chem..69 (1997)

[8] O. Shakoor, R. B. Taylor, R. R. Moody, Analyst 120 (1995) 2191.

[9] S. Foster, Soc. Sci. Med. 32 (1991) 1201.

[10] P. R. Lee, P. Lurie, M. M. Silverman, M. Lydecker, J. Clin. Epidermiol. 44

(1991)49.

[11] M. T. Ham, Adverse Drug React. Toxicol. Rev. 11 (1992) 59.

[12] S. K. Pandya, Br. Med. J. 297 9 (1988) 117.

[13] M. M. Silverman, M. Lydecker, P. R. Lee, Int. J. Health services 20 (1990) 561.

[14] British Pharmacopoeia, her Majesty stationary office, London, 1993.

[15] The United States Pharmacopoeia XXII edn. The United states pharmacopoeial

convention Inc., Rockville, MD, USA, 1990.

51

[16] The United States Pharmacopoeia, with revision, The National formulary, 19"'

Revision, United States pharmacopoeial Conventions, Inc., Rockville, MD, 2000.

[17] C. K. Lim, J. M. Rideout, J. W. S. Sheldon, J. Liq. Chromatogr. 6 (1983) 887.

[18] E. M. Niazy, H. W. Jun, Anal. Lett. 18 (1985) 1103.

[19] A. T. Kacprowicz, R. 0. FuUinfaw, R. W. Bury, J. Chromatogr. 337 (1985) 412.

[20] J. R. Miksic, M. J. Levitt, L. L. Rombold, Methodol, Anal. Toxicol. 3 (1985) 217.

[21] D. R. Rutledge, A. H. Abadi, L. M. Lopez, J. Chromatogr. Sci. 32 (1994) 153.

[22] J. S. Grundy, R. Kherani, R. T. Foster, J. Pharm. Biomed. Anal., 12 (1994) 1529 .

[23] N. Hayase, Y. I. Itagaki, S. Ogawa, S. Akutsu, S. I. Inagaki, Y. Abiko, J. Pharm.

Sci. 83 (1994) 532.

[24] H. L. Rau, N. Udupa, A. R. Aroor, Indian Drugs 29 (1) (1991) 46.

[25] E. Anzenbacherova, P. Anzenbacher, K. Macck, J. Kvetina, J. Pharm. Biomed.

Anal. 24 (5-6) (2001) 1151.

[26] M. N. Muscara, G. de Nucci, Braz. J. Med. Biol. Res. 26 (1993) 753.

[27] D. Romanova, E. Brandsteterova, D. Kralikova, L. Bozekova, M. Kriska,

Pharmazie 49 (1994) 779.

[28] M. Zhong, H. Wang, J. Zhang, Zhongguo Yiyun Yaoxue Zazhi 15 (1995) 335.

[29] X. Shi, M. Zhong, H. Wang, J. Zhang, Yaown Fenxi Zazhi 15 (1995) 20.

[30] Y. Yazan, B. Bozan, Pharmazie 50 (1995) 117.

[31] M. Garcia, J. J. Arameyona, M. A. Bregante, L. J. Fraile, C. Solans, J.

Chromatogr. B: Biomed. Appl. 693 (1997) 377.

[32] T. Ohkubo, H. Noro, K. Sugawara, J. Pharni. Biomed. Anal. 10 (1992) 67.

52

[33] J. V. Aukunura, U. B. Kompella, G. V. Betageri, J. Liq. Chrometogr. Relat.

Technol. 23 (2000) 565.

[34] K. Ing. Kaninan, C. M. D. Best, R. V. Heijden, A. J. P. Hofte, B. Karabatak, H.

Irth, U. R. Tjaden,, J. V. Greef, R. Verpoorte, J. Chromatogtr. 872 (2000) 61.

[35] T. Aral, J. Chromatogr. B: Biomed. Appl. 717 (1-2)(1998) 295.

[36] E. Tesarova, D. W. Armstrong, J. Chromatgr. Libr. 60 (1998) 197.

[37] C. Pettersson, B. A. Persson, Chromatogr. Sci. Ser. 78 (1998) 669.

[38] B. Renger, J. AOAC Int. 81 (1998) 333.

[39] B. Renger, J. Planar Chromatgr.- Mod. TLC. 10 (1997) 420.

[40] A. N, Campbell, J. Sharma, Acta Chromatographica 13 (2003) 109.

[41] B. Liawruangrath, S. Liawruangrath, Chromatographica 54 (5-6) (2001) 405.

[42] S. A. Shah, I. S. Rathod, B. N. Suhagia, S. S. Savale, J. B. Patel, J.AOAC hit.

84(6) (2001) 1715.

[43]. T. Aburaji, B. I. Amro, K. Aiedeh, M. Abuirjeie, S. Al-Khalil, Pharmazie 55 (10)

(2000)751.

[44] N. Wu, W. Fang, E. Lin, G. Chen, J . Patel, T-M. Chan, B. Pramanik, J. Pharm.

Biomed. Anal. 25 (2001) 663.

[45] S. N. Makhija, P. R. Vavia, J. Pharm. Biomed. Anal. 25 (2001) 663.

[46] H. Mahgoub, A. A. Gazy, F. A. EI-Yazbi, M. A. El-Sayed, R. M. Youssef, J.

Pharm. Biomed. Anal. 31 (2003) 801.

[47] L. I. Bebawy, M. N. El-Kousy, J. Pharm. Biomed. Anal. 20 (1991) 663.

[48] • C. V. Shabadi, B. A. Shelar, A. R. Shelar, Indian drugs 35 (12) (1998) 766.

53

[49] A. P. Argekar, G. J. Sawant, J. Liq. Chromatogr. Relat. Technol. 22 (1991) 2051.

[50] J. H. Knox, I. H. Grant, Chromatographia 24 (1987)135.

[51] J. H. Knox, I. H. Grant, Chromatographia, 32 (1991) 317.

[52] N. W. Smith, M. B. Evans, Chromatographia 41 (1987) 197.

[53] C. Yan, R. Dadoo, H. Zhao R. N. Zare, Anal. Chem. 67 (1995) 2026.

[54] D. Wistuba, V. Schurig, J. Chromatogr: A 875 (2000) 255.

[55] M. Hadey, M. Gilges, J. Senior, A. Shah, P. Camilleri, J. Chromatogr. B: Biomed.

Appl. 745 (1) (2000) 177.

[56] K. Otsuka, S. Terabe, J-Chromatogr. A 875 (1-2) (2000) 163.

[57] Y. K. Jin, A. Staleup, Electrophoresis 19 (12) (1998) 2119.

[58] J. Samskog, M. Wetterhall, S. Jacobsson, K. Markides, J. Mass spectrum. 35 (7)

(2000).

[59] Y. Tanaka, Y. Kishimoto, K. Otsuka, S. Terabe, J. Chramoatogr. A: 817 (1-2)

(1998) 49.

[60] T. Wielgos, P. Turner, K. Havel, J. Capillary Electrophor. 4 (6) (1997) 273.

[61] S. Tonsakasiri, P. L. Massart, Y. Vander Heyder, Anal. Chim. Acta 416 (1)

(2000) 29.

[62] J. Rujieka, E. H. Hansen, in Flow Injection Analysis, Wiley, New York,1981.

[63] M. Varcarcei, M. D. Luque deCastro, in Flow Injection AnaIysis,Principles and

Applications, Horwood, Chichester, 1987.

[64] M. S. Bloomfield, Talanta 580 (2002) 1301.

54

[65] P. Masawat, S. Liawruangrath, Y. Vaneesom, B. Liawruangrath, Talanta 58

(2002) 1221.

[66] S. Garcia, C. Sanchez-Pedreno, I. Albero, C. Garcia, Mikrochim acta 136 (2001)

67.

[67] A. M. Pimenta, A. N. Araujo, M.C.B.S.M. Montenegro, Anal. Chim. Acta 438

(2001)31.

[68] A. Economou. D. G. Themelis, G. Theodoridis, P. D. Tzanavaras, Anal. Chim.

Acta. 463 (2002) 249.

[69] P. D. Tzanavaras, D. G. Themelis, A. Economou, G. Theodoridis, Talanta 57

(2002) 575.

[70] M. E. Palomeque, B. S. F. Band, J. Pharm. Biomed. Anal. 30 (2002) 547.

[71] G. Altiokka, Z. Atkosar, E. Sener, M. Tuneel, J. Pharm. Biomed. Anal. 25 (2001)

339.

[72] F. A. Aly, S. A. Al-Tamini, A. A. Alwarthan, Anal. Chim. Acta. 416 (1) (2000)

87.

[73] A. Danet, L. L. Zamora, J. Martinez Calatayud, J. Flow Injection Anal. 15 (2)

(1998) 168.

[74] J. M. Kauffmann, J. C. Viri, G. J. Patriarche, W. H, Heinman, Analyst 110(1985)

349.

[75] B. Dakova, J. M. Kauffmann, M. Evers, L. Lamberts, G. J. Patriarche,

Electrochim. Acta 35 (1990) 1133.

55

[76] P. Kovcle, J. R. Ames, D. L. Rector, M. Jawdosiuk, M.D. Ryan, Free Rad. Biol.

Med. 6(1989)131.

[77] J. R. Ames, J. Pharm. Sci. 80(1991) 293.

[78] V. Kapetanovic, M. Aleksic, M. Erceg, D. Veselinovic, Farmaco 55 (1) (2000)

13.

[79] N. Yilmaz, I. Biryol, J. Pharm. Biomed. Anal. 17 (8) (1998) 1335.

[80] J. J. Berzas, J. Rodriguez, G. Castaneda, Anal. Chim. Acta 349 (1997) 303.

[81] S. F. de Betono, J. M. Moreda, A. Arranz, J. F, Arranz, Anal. Chim. Acta 329

(1996) 25.

[82] J. M. Kauf&nann, M. Pekle-Novak, A. Nagy, Acta Pharm. Hung. 66 (1996) 57.

[83] V. P. Shah, J. J. Koneeny, R. L. Everett, B. McCullough, A. C. Noorizadeh, J. P.

Skelly, Pharm. Res. 6 (1989) 612.

[84] A. J. Tavakoli, L. Tsan-Zon, Anal. Chim. Acta 443 (2001) 165.

[85] S. Z. Qureshi, A. Saeed, N. Rahman, Chem. Anal. 37 (1992) 227.

[86] K. Jardaranka, J. Ivana, N. Jovan, P. Dusica, M. Vantina, J. Pharm. Biomed. Anal.

28(2002)1215.

[87] M. I. H. Helaleh, N. Rahman, R. M. A. Q. Jamhour, Chem. Anal. (Warsaw) 42

(1997)261.

[88] B. S. Nagaralli, J. Seethamappa, M. B. Melwanki, J. Pharm. Biomed. Anal. 29

(2002) 859.

[89] N. Rahman, M. N. Hoda, Anal. Bioanal. Chem. 31 (2003) 381.

[90] N. Rahman, S. N. H. Azmi, Anal. Sci. 16 (2000) 1353.

56

[91] S. Z. Qureshi, A. Saeed, S. Haq, N. Rahman, Anal. Lett 23 (1990) 995.

[92] A. K. Sanyal, A. J. Dutta, AOAC Int. 79 (1996) 1303.

[93] S. Z. Qureshi, M. A. Khan, Analusis 24 (1996) 190.

[94] C. S. P. Sastry, P. Y. Naidu, Indian Drugs 34 (1997) 140.

[95] C. S. P. Sastry, P. Y. Naidu, Talanta 45 (1998) 795.

[96] S. Hesham, J. Pharm. Biomed. Anal. 29 (2002) 537.

[97] S. P. Agarwal, V. Singhal, A. Prakash, Indian J. Pharm. Sci. 60 (1998) 53.

[98] R. T. Sane, M. Phadke, P. S. Hijii, M. Shah, P. Patel, Indian Drugs 35 (1998) 79.

[99] J. J. B. Nevado, G. C. Penalvo, F. J. Bamado, Anal. Lett. 30 (1997) 2515.

[100] A. V. Sherikar, P. N. Phallke, P. M. Dhadke, Indian Drugs 33 (1996) 272.

[101] C. S. P. Sastry, Y. Sriniva, P. V. Subha Rao, Indian J. Pharm. Sci. 58 (1996) 169.

[102] S. Mostafa, M. U. Sadek, E. A. Alia, J. Pharm. Biomed. Anal. 27 (2002) 133.

[103] W. Misuik, J. Pharm. Biomed. Anal. 22 (2000) 189.

[104] I. Kramaancheva, I. Dobrev, L. Brakalov, A. Andreva, Anal. Lett. 30 (1997)

2235.

[105] N. Rahman, S. N. H. Azmi, Mikrochem. J. 65 (2000) 39.

[106] N. Rahman, S. N. H. Azmi, J. Pharm. Biomed. Anal. 24 (2000) 33.

[107] C. S. P. Sastry, J. S. Y. M. L. Rao, Talanta 43 (1996) 1827.

[108] M. I. H. Helaleh, N. Rahman, R. M. A. Q. Jamhour, E. S. M. Abu Nameh, J.

Pharm. Biomed. Anal. 16 (1997) 269.

[109] N. Silva, E. E. S. Schapoul, J. Pharm. Biomed. Anal. 29 (2002) 749.

[110] N. Rahman, M. Kashif, Anal. Sci. 19 (2003) 9 0 7 . - ^ ' ^ = r ^ r

*"' '^'

57

[111] N. Rahman, M. Kashif, IL Farmaco 58 (2003) 1045.

[112] G. V. Popovic, L. B. Pfendt, Analusis 24 (1996) 259.

[113] P. Frotos, S. Torrado, M. E. Pervez-Lorenzo, G. Fnitos, J. Pharm. Biomed. Anal.

21(2000)1149.

[114] M. I. H. Helaleh, N. Rahman, E. S. M. AbuNameh, Anal. Sci. 13 (1997) 1007.

[115] G. Paraskevas, J. Aha-Politov, M. Koupparis, J. Pharm. Biomed. Anal. 29 (2002)

865.

[116] F. A. El-Yazbi, H. H. Abdine, R. A. Shaalan, J. Pharm. Biomed. Anal. 19 (1999)

819.

[117] Z. A. El. Sherif, A.O. Mohamed, M. I. Walash, F. M. Tarras, J. Pharm. Biomed.

Anal. 22 (2000) 13.

[118] S. J. Rajput, A. G. Vyas, hidian Drugs 35 (1998) 352.

[119] N. T. Abdel-Ghani, A. F. Shoukry, Y. M. Issa, O. A. Wahdan, J. Pharm. Biomed.

Anal. 28 (2002) 373.

[120] A. B. Rao, R. S. K. Murthy, J. Phrm. Biomed. Anal. 27 (2002) 959.

[121] Z. A. El Sherif, M. I. Walash, M. F. El-Tarras, A. O. Osman, Anal. Lett. 30

(1997) 1881.

[122] S. Z. Qureshi, M. I. H. Helaleh, N. Rahman, R. M. A. Q. Jamhour, Aim. Chim.

(Rome) 86 (1996) 509.

[123] M. I. H. Helaleh, N. Rahman, Anal. Sci. 13 (1997) 489.

[124] P. Nagaraj, R. A. Vasantha, H. S. Yathirajan, J. Pharm. Biomed. Anal. 28 (2002)

161.

58

[125] M. A. El-Ries, F. M. Abou Attia, S. A. Ibrahim, J. Pharm. Biomed. Anal. 24

(2000) 179.

[126] M. I. H. Helaleh, S. Z. Qureshi, N. Rahman, R. M. A. Q. Jamhour, Acta Polonie

Pharmaceutical-Drug Research 54 (1997) 111.

[127] N. Rahman, M. N. Hoda, EL Framaco, 000 (2001) 1.

[128] N. Rahman, S. N. H. Azmi, Acta Pharm. 49 (1999) 113.

[129] P. Nagaraja, H. S. Yathirajn, H. R. Arun Kumar, R. A. Vasantha, J. Pharm.

Biomed. Anal. 29 (2002) 277.

[130] M. I. Walash, M. Rizk, A. El- Brashy, Talanta, 35 (11) (1988) 895.

[131] E. M. Hassan, F. Belal, J. Pharm. Biomed. Anal. 27 (2002) 31.

[132] N. A. El-Ragehy, S. S. Abbas, S. Z. EI- Khateeb, J. Pharm: Biomed. Anal. 15

(2001) 143.

[133] F. E. O. Soliman, S. M. Soliman, S. M. Sultan, Mikrochem. J. 57 (1997) 370.

[134] A. S. Amin, J. Pharm. Biomed. Anal. 29 (2002) 729.

[135] G. G. Mohamed, S. M. Khaled, M. A. Zayed, M. Abid H. Hamid, H. Shall, J.

Pharm. Biomed. Anal. 28 (2002) 1127.

[136] S. Z. Qureshi, M.I.H. Helaleh, N. Rahman, R. M. A. Q. Jamhour, Chem. Anal.

(Warsaw) 42 (1997) 65.

[137] S. Z. Qureshi, M. I. H. Helaleh, N. Rahman, R M. A. Q. Jamhour, Chem. Anal.

(Warsaw) 42 (1997) 65.

[138] B. S. Nagaralli, J. Seetharamappa, M. B. Melwanki, J. Pharm. Biomed. Anal. 29

(5) (2002) 859.

59

[139] N. Rahman, M. N. Hoda, Anal. Bioanal. Chem. 374 (2002) 484.

[140] C. S. Xuan, Z. Y. Wang, J. L. Song. Anal. Lett. 31 (1998) 1185.

[141] F. M. Abdel-Gawad. Y. M. Issa, H. M. Fahmy, H. M. Hussein, Mikrochim. Acta

130(1998)35.

[142] M. A. Elsayed, M. Barary, M. Abdel-salam, S. Mohamed, Anal. Lett. 222 (1989)

1665.

[143] K. Sridhar, C. S. P. Sastry, M. N. Reddy, D. G. Sankar, K. R. Srinivas, Anal. Lett.

30(1997)121.

[144] M. N. Reddy, G. T. Rani, K. V. S. P. Rao, D. G. Sanker, K. Sreedhar, Indian J.

• Pharm. Sci. 59 (1997) 188.

[145] L Singvi, S. C. Chattirvedi, India J. Pharm. Sci. 60 (1998) 309.

[146] R. T. sane, M. L. Kubal, V. G. Nayak, V. B. Marlker, V. J. Banawalkar, Lidian

Drugs 22 (1984) 25.

[147] K. Baravaiah, G. Krishnamurthy, Talanta 46 (1998) 665.

[148] A. Z. A. Zuhri, R. M. Shubietah, G. M. Badah, J. Pharm. Biomed. Anal. 21

(1999)459.

[149] I. G. McWilliams, Anal. Chem. 41 (1969) 674.

[150] W. Snelleman, T. C. Rains. K. W. Yee, H. D. Cook, O. Menis, Ibid. 42 (1970)

394.

[151] W. K. Fowler, D. O. Knapp, J. D. Winefodner, Ibid. 46 (1974) 601.

[152] T. C. O'Haver in E. L. Wehiy (ed.), Modem Fluorescence Spectroscopy, Vol. 1,

Plenum Press, New York, 1976, p.65.

60

[153] P. John, I. Soutar, Anal. Chem. 48 (1976) 520.

[154] T. C. O'Haver, T. Begley, Ibid. 53 (1981) 1876.

[155] G. L. Green, T. C. O'Haver, Anal. Chem. 46 (1974) 2191.

[156] A. Schmitt, Tech. Lab. 5 (1978) 1207.

[157] A. F. Fell, G. Smith, Anal. Proc. 19 (1982) 28.

[158] W. L. Butler, D. W. Hopkins, Photochem. Photiol. 112 (1970) 439.

[159] B. A. Gulyaev, F. F. Litvin, Biofizika 15 (1970) 670.

[160] T. C. O'Haver, Anal. Chem. 51 (1979) 91 A.

[161] G. Talsky, L. Mayring, H. Kreuzer, Angew. Chem. Int. Ed. Engl. 17 (1978) 785.

[162] A. F. Fell, D. R. Jarvie, M. J. Stewart, Clin. Chem. 27 (1981) 286.

[163] B. Morelli, J. Pharm. Sci. 77 (1988) 1042.

[164] B. Morelli, J. Pharm. Sci. 84 (1995) 34.

[165] A. Fattah, M. El-Walily, J. Pharm. Biomed. Anal. 16 (1997) 21.

[166] L. Nuevas, R. Gozalez, J. C. Rodriguez, J. Hoogmartens, J. Pharm. Biomed. Anal.

18(1998)579.

[167] A. El-Gindy, A. Ashour, L. Abdel- Fattah, M. M. Shabana, J. Pharm. Biomed.

Anal. 25 (2001) 299.

[168] J. Karpinska, Anal. Sci. 17 (2) (2001) 249.

[169] V. G. Dabbene, M. C. Brinon, M. M. de Bertirello, J. Pharm. Sci. 83 (1994) 1617.

[170] R. J. Forsyth, D. P. Ip, J. Pharm. Biomed. Anal. 12 (1994) 1243.

[171] M. I. Walash, A. M. el-Brashy, M. A. Sultan, Acta. Pharm. Hung. 64 (1994) 5.

[172] T. D. Doyle, F. R. Fazzari, S. Pharm. Biomed. Sci. 63 (1974) 1921.

61

[173] A. M. wahbi, M. Barary, H. Mahgab. M. A. El-Sayed, J. Assoc. Off. Anal. Chem.

68 (1985) 1045.

[174] A.G. Davidson, L. M. M. Mkoji. J. Pharm. Biomed. Anal. 6 (1988) 449.

[175] M. Blanco, J. Coello, H. Iturriaga, S. Maspoch, C.delal Pezuela, Analyst 123 (8)

(1998) 135R.

[176] M. Blanco, J. Coello, A. Eustaquio, H. Itumaga, S. Maspoch, J. Pharm. Sec. 88

(5) (1999) 55.

[177] U. Gnimmisch, Pharma Technol. J. 19 (1) (1998) 69.

[178] S. Steiner, F. A. Witzmann, Electrophoresis 21 (11) (2000) 2099.

[179] L. Van Higfte, B. Mareiniak, N. Froloff, J. Chromatogr. B: Biomed Sci. Appl.

725 (91) (1999) 3.

[180] P. K. Owens, L. Karlsson, E. S. M. Lutz, L. I. Andersson, Trends Anal. Chem. 18

(3) (1999) 146.

[181] B. Sellergren, Trends, Anal. Chem 18 (3) (1999) 164.

[182] A. Bommireddi, C. L. Li, D. Stephens, D. Robinson, E, Ginsburg, Drug Dev. Ind.

Pharm. 24 (11) (1998) 1089.

[183] M. Mistry, M. I. Ismail, R. D. Farrant, M. Liu, J. K. Nicholson, J.C. Lindon, J.

Pharm. Biomed. Anal. 19 (3-4) (1999) 511.

[184] H. A. Mottola, D. Perez-Bendito, Anal. Chem. 68 (1996) 257R.

[185] H. A. Mottola, Analyst, 121 (1996) 381.

[186] D. Perez-Bendito, Quim. Anal. 15 (1996) 266.

[187] H. L. Pardue, Analyst 121 (1996) 385.

62

[188] A. R. Holzwarth, Adv. Photosynth. 3 (1996) 75.

[189] D. Perez-Bendito, M. Silva, TrAc, Trends Anal. Chem. 15 (1996) 232.

[190] D. Perez-Bendito, A. Gomez - Hens, M. Silva, J. Pharm. Biomed. Anal. 14

(1996)917.

[191] M. Lopez Carreto, S. Rubio, D. Perez-Bendito, Analyst, 121 (1996) 33R.

[192] E. V. Albano, Heterog. Chem. Rev. 3 (1996) 389.

[193] D, Perez-Bendito, M. Silva, in kinetic methods in Analytical Chemistry, Ellis

Horwood, Chichester, 1988.

[194] H. A. Mottola, In Kinetic Aspects of Analytical Chemistry, Wiley, New York,

1988.

[195] D. Perez-Bendito, A. Gomez-hens, M. Silva, M. C. Gutierrez, M. Carmona, J.

Pharm. Biomed. Anal. 7 (1989) 1435.

[196] D. Perez-Bendito, M. Silva, A. Gomez -Hens, Trends Anal. Chem. 8 (1988) 302.

[197] M. Marquez, M. Silva, D. Perez-Bendito, Analyst 113 (1988) 1733.

[198] A. Velasco, M. Silva, D. Perez-Bendito, Anal. Chem. 64 (1992) 2359.

[199] M. Marquez, M. Silva, D. Perez-Bendito, Anal. Chim. Acta 237 (1990) 353.

[200] M. Marquez, M. Silva, D. Perez-Bendito, J. Pharm Biomed. Anal. 8 (1990) 563.

[201] M. Marquez, M. Silva, D. Perez-Bendito, Anal. Lett. 22 (1989) 2485.

[202J R. Cai, H. Huang, H. Liu, Fenxi Huaxue 25 (1997) 476.

[203] T. F. CuUen, S. R. Crouch, Mikrochim. Acta 126 (1997) 1.

[204] E. M. Hassan, F. Belal, J. Pharm. Biomed. Anal. 27 (2002) 31.

[205] B. R. Kowalski, Anal. Chem. 52 (1980) 112R.

63

[206] I. E. Frank, B. R. Kowalski, Anal. Chem. 54 (1982) 232R.

[207] M. F. Delaney, Anal. Chem. 56 (1984) 261 R.

[208] L. S. Ramos, K. R. Beebe, W. P. Carey, E. Sanchez, B. C. Erickson, B. R.

Wilson, L. E. Wangen, B. R. Kowalski, Anal. Chem. 58 (1986) 294R.

[209] B. R. Kowalski, Editor, "Chemometrics Theory and Application," ACS

Symposium Series No. 52, American Chemical society, Washington, DC, 1977.

[210] D. A Kutz, Editor, "Chemometrics Estimators of Sampling, Amount and Error,"

ACS Symposium Series No. 284, American Chemical society, Washington, DC,

1985.

[211] C. H. Spieigelman, R. L. Walters, J. Sacks, Editors, J. Res. Natl. Bur. Stand.

Special issue, 90 (6) (1985) 391.

[212] M. A. Sharaf, D. L. lUman, B. R. Kowalski, "Chemometrics", Wiley, New York,

1986.

[213] B. R. Kowalski, Editor, "Chemometrics, Maths and Statistics in Chemistry,"

Reidel, Dordrecht, 1984.

[214] D. Bawden, editor, "Chemometrics Series", Research Studies Press, Latch Worth.

[215] D. L. Massart, Editor-in-chief, P. K. Hopke, C. H. Spiegelman, W. Wegscheider,

Editors, R. G. Brereton, R. E. Dessy, Associate Editors, Chemometrics and

Intelligent Laboratory Systems, Elsevier, Amsterdam.

[216] B. R. Kowalski, Editor-in-chief, S. D. Brown, B. G. M. Vandeginste, Associate

Editors, Journal of Chemometrics, Wiley, Chichester.

64

[217] D. L. Massart, L. Kaufman, "Interpretation of Analytical data by the use of cluster

Analysis," Wiley, New York, 1983.

[218] Food and Drug Administration, Draft Guideline for the Format and Content of the

Human Pharmacokinetics and Bioavailability Section of an Application, Docket

No. 85 D-0275,1985.

[219] European Union Regulations for the good manufacturing and Quality control of

Medicinal products, 75/319.

[220] European Union Regulations for the good manufacturing and Quality control of

Medicinal products, 89/381.

[221] Eviropean Union Regulations for the good manufacturing and Quality control of

Medicinal products, 91/356.

[222] European Union Multi-State register of Pharmaceutical products, 75/318.

[223] European Union Multi-State register of Pharmaceutical products, 89/341.

[224] ICH Harmonised Tripartite Guideline, CPMP/ICH/281/95.

[225] European Union Multi-State register of Pharmaceutical products, 91/507.

[226] ICH Harmonised Tripartite Guideline, CPMP/ICH/381/95.

[227] International Conference on the Harmonization of Technical Requirements for

the Registration of Pharmaceuticals for Human Use (ICH) Q2A: Validation of

Analytical Methods (Definitions and Terminology) (October 1994).

[228] ICH: Q2B Analytical Validation-Methodology (November 1996).

[229] A. G. J. Daas, J. H. Mc. B. Miller, Pharmeuropa 9.1 (1997) 148-156.

[230] F. J. Van de Vaart, Pharmeuropa 9.1 (1997) 139-143.

65

[231] ICH: Q3A, Impurities in New Drug Substances (March 1995, revised October

1999, Step 2).

[232] R. G. Whitfield, D. W. Hughes, T. P. Layloff, R. R. Cox, G. E. Gressett, P. J.

Jimenez, J. Andersen, R. R. Reck, S. Schniepp, Pharmacopoeial Forum 24 (1998)

7051.

[233] B. A. Persson, J. Vessman, R. D. McDowall. LC-GC International, March 1998,

160.

[234] C. M. Riley, Statistical parameters and analytical figures of merit, in: CM.

Riley, T. W. Rosanske (Eds.), Development and Validation of Analytical

Methods, Elsevier, Oxford, 1996, p. 15.

[235] V. R. Meyer, Chromatographia 40 (1995) 15.

[236] DIN ISO 5725-2: Accuracy (trueness and precision) of measurement methods and

results; a basic method for the determination of repeatability and reproducibility

ofa standard measurement method, 1990.

[237] MVA-Method validation in analytics (PC-software, Windows NT), NO VIA

GmbH, Saarbrucken, Germany (http///www.novia.de).

[238] W. Funk, V. Dammann, C. Vonderheid, G. Oehlmann, Statistische Methoden in

der Wasseranalytik, Verlag Chemie, Weinheim, 1985.

[239] DIN 32 645: Chemische Analytik: Nachweis-, Erfassungsund stimmungsgrenze,

Ermittlung unter Wiederholbedingungen. Begriff, Verfahren, Auswertung. Beuth

Verlag GmbH, Berlin.

66

[240] A'cceptable Methods', Drug Directorate Guidelines, National Health and

Welfare, Health Protection Branch, Health and Welfare Canada, 1992.

[241] EURACHEM Guidance Document No. WDG 2: Accreditation for chemical

laboratories: Guidance on the interpretation of the EN 45000 series of standards

and ISO:IEC Guide 25,1993.

[242] D. Perrissoud, B. Testa, Arzneim-Forsch, Drug res. 36 (1986) 1249.

[243] T. B. Pulvertaf, J. S. Lyons, C. A. S. Wink, R. Soc. Med. Int. Congr. Symp. No.

• 42,1981.

[244] J. C. Krantz," Historical Medical Clamics involving Nev/ Drugs"; The William

Wilkins Company, Baltimore, 1974.

[245] J. C. Krantz, in "Handbook of Experimental Pharmacology", Vol. 40, (Editor

P.dleman); Springer-Verlag, New York, 1975.

[246] D. N. Frantz, in "Remington: The Science and Practice of Pharmacy", A.R.

Gennaro (Editor); 19th Ed., Vol. 11, Mack Publishing Company, Pennysylvania,

1995.

[247] T. L. Bruntan, Lancet 2 (1876) 97.

[248] Martindale The Extra Pharmacopoeia 33rd ed.. Royal Pharmaceutical Society,

London, 2002, p 940.

[249] S. Cavalieri, Gazz Med. Ital. 133 (1974) 628.

[250] H. A. Salmi, S. Sama, Scand. J. Gasteroenterol 17 (1982) 517.

[251] P. Ferenci, J. Hepatol. 9 (1989) 105.

[252] N. Skottova, V. Kreeman. Physiol. Res. 47 (1) (1998) 1.

67

[253] J. Dokladalova, J. A. Tykal, S. J. Coco, P. E. Durkee, G. T. Querela, J. J. Korst, J.

Chromatogr, B: Biomed. Appl. 231 (1982) 451.

[254] F. A. Tucker, P. S. B. Minty, G. A. Mac Gregor, J. Chromatogr. B: Biomed. Appl.

342(1985)193.

[255] W. Snedden, P.G. Fernandez, C. Nath, Can. J. Physiol. Pharmacol.64 (1986) 290.

[256] H. Suzuki, S. Fujiwara, S. Kondo, I. Sugimoto, J. Chromatogr. 341 (1985) 341.

[257] J. A. Berson, E. Brown, J. Am. Chem. Soc. 77 (1995) 447.

[258] K. D. Raemsch, J. Sommer, Hypertension 5 (1983) 18.

[259] W. A. Al-Turk, I. A. Majeed, W. J. M. Murray, D. W. Newton, S. Othman, Int. J.

Pharmaceut.41(1988)227.

[260] Y. matsuda, R. teraoka, I. Sugamato, Int. J. Pharmaceut. 54 (1998) 211.

[261] T. Ohkubo, H. Noro, K. Sugawara, J. Pharm. Biomed. Anal. 10 (1992) 67.

[262] I. A. Majeed, W, J. Murray, D. W. Newton, S. Othman, W. A. Al-Turk, J. Pharm.

Pharmacol. 39 (1987) 1044.

[263] D. P. Zipes, E. N. Piystowski, J. J. Heger, J. Am. Coll. Cardiol. 3 (1984) 1059.

[264] S. Levy, Am. J. Cardiol, 61 (1988) 95A.

[265] T. R. Vobel, P. E. Miller, N. D. Mostow, L. Rakita, Prog. Cardiovasc. Dis. 31

(1989)393.

[266] J. W. Mason, N. Engl. J. Med. 316 (1987) 455.

[267] R. J. Flanagon, G. C. Storey, D. W. Holt, P. B. Farmer, J. Pharm. Pharmacol. 34

(1982) 638.

68

[268] D. N. Franz Cardiovascular drugs. In: A. R., Gennaro ed. Remington: The

Science and Practice of Pharmacy, 19th Edition, Vol. II, Mack Publishing

Company, Pennsylvania 1995, p.966.

[269] Martindale The Extra Pharmacopoeia, 31st Edition, Royal Pharmaceutical

Society, London, 1996, p.961.

[270] M. Fisher, J. Grotta, Drugs 46 (1993) 961.

[271] D. Mc Tavish, E. M. Sorkin, Drugs 46 (1993) 961.

[272] Anonymous, Drugs Ther-BuU. 31 (1993) 81.

[273] R. N. Btogden, P. Benfield, Drugs 51 (1996) 792.

CHapter -2 %inetic spectropHotometric metHocCfor the

determination ofsiCymarin inpharmaceuticaC formulations using potassium permanganate as

oxidant

69

INTRODUCTION

Silymarin i.e. 3,5,7-trihydroxy-2-[3-(4-hydroxy-3-methoxy phenyl)-2-

(hydroxy-methyl)-l,4-benzodioxan-6-yl]-4-chromanone is an important

antihepatotoxic drug wiiich is obtained from the dried fruits of Silybum

marianum and widely used for the treatment of hepatic disorders [1-4]. The

drug is officially listed in Martindale The Extra Pharmacopoeia [5].

The assay procedure listed in the monograph of the Italian

pharmacopoeia describes the UV-Vis spectrophotometric method [6] for

determination of the drug in bulk and pharmaceutical formulations. Several

other methods have been employed to determine silymarin in biological

specimens and/or pharmaceutical formulations such as high performance

liquid chromatography [7-21], thin layer chromatography [22-28], high

performance thin layer chromatography [29], potentiometric titration [30],

diffuse-reflectance fourier transform infrared spectroscopy [31] and UV

spectrophotometry [32,33].

Spectrophotometry as a quantitative analytical tool still belongs to the

most frequently used analytical techniques in pharmaceutical analysis. It

provides practical and significant economic advantages over other methods.

In the literature only few spectrophotometric methods have been reported.

The drug content in pharmaceutical formulations was determined [34], based

on the formation of coloured complex of the drug with 2, 4-dinitrophenyl

hydrazine in the presence of tetramethyl amine hydroxide and quantitatively

measured at 490 nm. Another spectrophotometric method has been developed

70

for the estimation of silymarin based on its reaction with diazotized sulfanilic

acid in alkaline medium to form orange-red coloured chromogen, which

absorbed maximally at 460 nm [35]. Reddy et al. [36] have reported two

spectrophotometric methods for its determination. The first method was based

on the oxidation of the drug by Fe (III) and estimating the reduced Fe (II)

with 1,10-phenanthroline at 510 nm. The other method involved the formation

of a blue complex on treatment with Folin-Ciocalteau reagent in the presence

of NaOH and subsequent determination was done at 740 nm. These

spectrophotometric methods are somewhat tedious and time consuming.

Therefore, there is a need to develop a fast, low cost and selective method for

routine analysis of silymarin in bulk and drug formulations.

The extent of oxidation of organic substrate by potassium

permanganate depends on the pH of the medium. The heptavalent manganese

changes to Mn (VI) in alkaline medium while in neutral and acidic medium,

the permanganate is further reduced forming ultimately Mn (II). This

behaviour 'of permanganate has been exploited to develop a kinetic

spectrophotometric method for assay of nifedipine in drug formulations [37].

In this chapter a fast, simple and sensitive kinetic spectrophotometric

method for the assay of silymarin in bulk and pharmaceutical formulations is

described. The method is based on the oxidation of silymarin by potassium

permanganate at pH 7.0±0.2 and the course of the reaction was followed by

measuring a decrease in absorbance at 530 nm.

71

EXPERIMENTAL

Apparatus

The absorbance was measured on a spectronic 20 D" spectrophotometer

(Milton Roy, USA) with 1 cm glass cells.

Reagents

All chemicals used were of analytical or pharmaceutical grade.

Potassium permanganate, 0.003M was freshly prepared by dissolving 47.41

mg of potassium permanganate in 100 ml of doubly distilled water. The

apparent purity of the potassium permanganate solution was checked

titrimetrically [38].

Test solution

Silymarin (1 mg ml'*) (Micro Labs, Bangalore, India) was prepared in

methanol.

Procedure for silymarin

Kinetic spectropltotometric method for determination of silymarin

Aliquots of 0.18-0.50 ml of silymarin standard test solution were

pipetted into a series of 10 ml standard volumetric flasks. To each standard

flask 1.6 ml of 0.003M potassium permanganate was added and then diluted

to volume with doubly distilled water at 30±1°C. After mixing, reaction

mixture was immediately transferred to spectrophotometric cell and the

72

decrease in absorbance was recorded as a function of time for 20 min at 530

nm. The initial rate of the reaction (v) at different concentrations was

obtained from the slope of the tangent to the absorbance-time curve. The

calibration curve was constructed by plotting the logarithm of the initial rate

of reaction (log v) versus logarithm of the concentration of the silymarin (log

C).

Procedure for determination of silymarin in pharmaceutical formulations

An accurately weighed quantity of the mixed contents of five capsules

or five powdered tablets, equivalent to 50 mg of the drug, was extracted into

50 ml chloroform with shaking and the residue was filtered using Whatman

No. 42 filter paper. The filtrate was evaporated to dryness and the residue was

taken up with methanol and transferred to a 50 ml standard volumetric flask,

diluting to volume. The assay was completed following the recommended

procedure.

RESULTS AND DISCUSSION

The oxidation of organic compounds by permanganate is dependent

upon the pH of the medium. During the course of the reaction the valence of

manganese changes and intermediate ions have been suggested as

participating oxidant. But the species having the main role as potential

oxidant depends on the nature of the substrate and pH of the medium. The

oxidation of silymarin by potassium permanganate at pH 7.0±0.2 was

73

followed spectrophotometrically by measuring the rate of change of

absorbance at 530 nm. The absorbance-time curves are shown in Figs. 2.1.

and 2.2., which are all sigmoid in nature throughout the entire range and

therefore, the initial rates of reaction were determined from the slope of the

initial tangent to the absorbance-time curves. The order with respect to

permanganate was determined by studying the reaction at different initial

concentrations of permanganate with fixed silymarin concentration. Keeping

the silymarin concentration higher than that of the oxidant, the plot of initial

rate [-d[A]/dt] against initial absorbance was linear passing through the origin

suggested that the order of reaction with respect to permanganate at the start

is one. The order with respect to silymarin was evaluated from the

measurement of the rates at several concentrations of silymarin but at fixed

concentration of KMn04 which was also found to be one. The first order

dependence on both permanganate and silymarin in the initial stages leads to

the simple rate expression:

Rate = K [silymarin] [Mn04"]

Silymarin is naturally occurring aryl chromanone (I) related to

flavanone. The flavanones are 2,3-dihydro derivatives of flavones, which are

readily interconvertible. Though the basic skeleton of flavanone and flavone

is the same, the key which distinguishes one structural type from another is

the oxidation level of the various carbons in the heterocyclic ring. The

flavones are in a high state of oxidation where as flavanones are in low state

of oxidation. The oxidation of flavanone with a variety of reagents such as

selenium dioxide [39] or triphenyl methyl carbonium ion [40] has been

74

Time (min)

Fig. 2.1. Absorbance-time curves: KMn04,4.8 xlO" M and silymarin (a) 3.731 X10- M; (b) 4.146 x 10" M; (c) 6.219 x 10" ; (d) 8.292 X 10- M; (e) 10.365 x 10" M

75

Time (min)

Fig. 2.2. Absorbance-time curves: silymarin, 10.365 x 10" M and KMn04 (a) 3.00 x 10"* M; (b) 2.10 x 10"* M; (c) 1.50 x 10"* M; (d)9.00xlO-^M.

76

reported in which flavone is formed. In this study, the same mechanism seems

to be involved. The oxidation of silymarin by permanganate takes place at pH

7.0±02 leading to the formation of chromone (II). Thus, the dehydrogenation

is effected by permanganate and consequently disproportionation between

immediate oxidation states of manganese results in the formation of Mn02.

However no precipitation of MnOa was observed under study time period.

Therefore, the reaction mechanism is presented in scheme 2.1.

Calibration graph and Statistical analysis

Under the optimum experimental conditions, a calibration curve (Fig.

2.3.) was constructed by plotting logarithm of initial rate of reaction (log v)

versus logarithm of silymarin concentration (log C), which showed a linear

relationship over silymarin concentration range of 18-50 \x.g ml''. The linear

regression analysis using the method of least square was made to evaluate the

slope, intercept and correlation coefficient. The linear regression equation and

correlation coefficient are log v = 1.024 log C + 3.235 and r = 0.9997 which

indicates an excellent linearity. The confidence limits for the slope of the line

of regression and intercept were computed using the relation b±tSb and a±tSa

[41], at 95% confidence level and found to be 1.024±3.96xl0"2 and

3.235±I.67x10-', respectively. This indicated the high reproducibility of the

proposed kinetic method. Linearity was also evaluated [42] from the relative

standard deviation of the slope of the calibration line and found to be 1.215.

The limit of detection was established using the equation [43,44]:

77

X o-

o I 5=0 I

u c o E p

u

S

u

u c o c ea

6 a u

78

-4.4 -4.2 -4.1

log of molar concentration

-4.0

Fig. 2.3. Calibration curve for tlie determination of Silymarin.

79

Limit of detection = ^^ x 2 n-2^

where n is the number of standard samples, t is the value of student's t for n-

2 degrees of freedom at 95% confidence level and So = variance. The

variance and detection limit were calculated and found to be 1.100 x 10" ^g

ml"' and 2.823 x 10" ^g ml"' respectively, which confirmed negligible

scattering of experimental data points around the line of regression and good

sensitivity of the method.

The error (Sc) in the determination of a given concentration of

silymarin (C) was established using the equation [45]:

-|l/2

Q _ So

°""b" 1+i^. <>'- >'

n b^(ZC^-nC^)

where y and C are average ordinate and abscissa values, respectively, for n

standard samples. Fig. 2.4. shows the graph of Sc versus the concentration of

silymarin. It is apparent from the graph that the error is reached minimum

when the actual initial rate was equal to the average initial rate in the

calibration graph.

The minimum error of 1.123x10"^ fig ml"' was found in the

determination of about 30 yig ml"' silymarin. The value of Sc also allows

establishing the confidence limit at the selected level of significance for the

determination of unknown concentrations by using the relation, C,±tSc The

results are shown graphically (Fig. 2.5.) in the form of percent uncertaintyfi^. ,oo]

on the concentration [46], at 95% and 99% confidence levels. This is

80

10"" X1.30

I

Concentartion of silymarin (^g ml' )

Fig. 2.4. Error in the determination of tlie concentration of silymarin using statistical analysis of standard calibration data.

81

10-2 X4.5

Concentration of slymarin (ng ml' )

Fig. 2.5. Variation of the confidence limit at (a) p= 0.05 and (b) p = 0.01 level of significance in the form of uncertainty percentage on the concentration of silymarin.

82

a useful way of expressing the confidence limits because the relative

uncertainty can be estimated directly on the concentration over the full range

of the concentration tested.

The reproducibility was established for ten independent analyses of solution

containing 0.40 mg silymarin. The analytical results obtained from the

investigation are summarized in Table 2.1. The relative standard deviation

and mean percent recovery were 0.873% and 100.33%, respectively and

considered to be very satisfactory. The validity of the proposed kinetic

method was checked by applying standard addition method and the results are

reported in Table 2.2.

The applicability of the proposed kinetic method for the determination

of silymarin in dosage forms was examined by analyzing marketed products;

the results were compared to those obtained by the reference method [35] and

are summarized in Table 2.3. The performance of the proposed method was

judged by calculating t- and F-values [47]. At 95% confidence judged level,

the calculated t- and F-values do not exceed the theoretical values indicating

no significant difference between the proposed kinetic method and the

reference method.

83

Table 2.1. Precision test of the proposed kinetic method

Sample No.

1

2

3

4

5

6

7

8

9

10

Mean

Initial rate of reaction (mol r' min'')

11.455x10"^

11.508x10-^

11.376x10"^

11.350x10-^

11.561x10-^

11.534x10-2

11.271x10-2

11.402x10-2

11.324x10-2

11.297x10-2

percent recovery = 100.33 %

Relative standard deviation = •• 0 .833 %

Amount found (Hg per 10ml)

402.97

404.79

400.26

399.37

406.61

405.70

396.68

401.17

398.47

397.57

84

Table 2.2. Determination of silyraarin in pharmaceutical formulations by standard addition method

Preparation Amount taken (Hgml-')

Amount added Total amount found (Hgml-') (Hgml-')

Recovery RSD

Silybin-70 (tablet)

Silvia-70 (tablet)

Limarm-70 (capsule)

Sivylar-70 (capsule)

Levalon-70 (capsule)

10 20

10 20

10 20

10 20

10 20

10 20

10 20

10 20

10 20

10 20

20.03 39.99

20.03 40.02

20.02 40.08

20.03 40.02

20.04 39.99

100.16 99.97

100.18 100.05

100.11 100.20

100.18 100.05

100.20 99.97

0.354 0.343

0.471 0.224

0.585 0.467

0.578 0.224

0.685 0.343

' Three Independent analyses.

o

B

e

•a o

5

u B!

•a

o o< o u

s u .2 Oil

a v> O

C3

S

W

u O

ttH

•"•^

1

^

u

U -P

•s J= 4-*

u 6 u '•5 a c 3

Q ^

041::'

Q tn

i I

•33

c o

C3 Q. U

I u I

0

0

r4

0

en t s

m t ^

-^

0

O N

c<i

0

0 0 CO

o

o 00

85

00 ro CS

( S <o 1-H

00 <N CO

i n ON o

0 0

ON

o\

00 m m o u-i ro

O o o

o o o

o o

en CO

CO o\ o

(S

o o

CO

o

ON ON

o

0 0 ON

o

NO

o o

o NO o

U-I o c3 o

o o CO

o

o o

o

o CO

c5 o

CO

i n o

ON ON O N

O o o o

00 <!i. 00 i ^

ca (in

o

<u

13 3

u

0

r

c _o •3 > u

"3 CO

a u

Cfl

^ CO c a

^-t

a u • 0 a u u •a _c

>

VO

r--ci to

• 3

> 0

g - 0 B 0 U

0 ^ V^ o\ " l - l

, 0 ( M

u 3

A

>

' •5

0 u H

NO en

> u u 0 s u

•0 IS § 0

0 ^ v\ 0

« U, t - i 0

« u 3 d

>

0

86

REFERENCES

[1] S. Cavalieri, Gazz Med. Ital. 133 (1974) 628.

[2] H. A. Salmi, S. Sarna, Scand. J. Gasteroenterol 17 (1982) 517.

[3] P. Ferenci, J. Hepatol. 9 (1989) 105.

[4] K. Hruby, Hum. Toxicol. 2 (1983) 183.

[5] Martindale The Extra Pharmacopoeia 31st ed., Royal Pharmaceutical

Society, London, (1996) p. 994.

[6] Farmacopoeia Ufficale Italiana 9th ed., vol. II, tuto Poligrafico e Zecca

cello Stato-Roma, (1985) p. 1673.

[7] E. M. Martinelli, P. Morazzoni, S. Livio, E. Uberti, J. Liq. Chromatogr.

14(1991)1285.

[8] H. Marcher H, C. Klkuta, J. Liq. Chromatogr. 16 (1993) 2777.

[9] B. Rickling, B. Hans, R. Kramerizyk, G. Krumbiegel, R. Weyhenmeyer,

J. Chromatogr. 135 (1977) 499.

[10] G. Tittel, H. Wagner, J. Chromatogr. 135 (1977) 499.

[11] G. Tittel, H. Wagner, J. Chromatogr. 153 (1978) 277.

[12] V. Querela, N. Tierini, G. Incarnate, M. Terracciano, P. Papetti,

Fitoterapia51 (1980) 83.

[13] V. Querela, N. Pierini, U. Valeavi, R. Caponi, S. Innocenti, S. Tederchi,

Anal. Chem. Symp. Ser. 13 (1983) 1.

[14] M. Steinigen Pharm. Ztg., 128 (1983) 783.

[15] K. H. Law, N. P. Das, J. Chromatogr. 388 (1987) 225.

87

[16] E. Tomov,M. Simova, T. Pangarova, D. Bocheva, F.E. C.S. Int. Conf.

Chem. Biotechnol. Biol. Act. Nat. Prod. 5 (1985) 534.

[17] P. Pashankov, Izv. Durzh. Inst. Kontrol Lck Sredstra 21 (1988) 3.

[18] N. Nikolov, A. Bizhev, P. Pashankov, God. Vissb. Khim - Tekhnol. Inst.

30(1990)203.

[19] V. A. Kurkin, Gaertn. fruits. Rastil. Resur. 32 (1996) 80.

[20] P. Wang, R. Cong, J. Wang, L. Zang, Sepu. 16 (1998) 510.

[21] Y. T. Ding, S. Tian, D. Gu, Z. Suu, Z. Zang, Yaowu Fenxi Zazhi 19

(1999)3048.

[22] L. L. Lin, Taiwan Yao. Hsuch. Tsa Chih. 34 (1982) 53.

[23] N. A. Abdel-Salam, M. A. Abdel-Salam, M. A. Elsayed, Pharmazie 37

(1982) 74.

[24] M. Vanhaelen, R. Vaniiaelen - Fastre, J. Chromatogr. 281 (1983) 263.

[25] B. Heinz, W. Funk, A. Voemel, Gaertn. Proc. Int. Symp. lustrum. HPLC

3rd (1985) 313.

[26] Z. Wu, X. Wang, Zhogguo Yaoke Danue Xuebao 20 (1989) 184.

[27] Q. Lin, J. Huang, P. Xie, Guangdong Yaoxue. Yuan. 14 (1998) 250.

[28] R. Szilajtis-Obeiglo, K. Kowalewska, Herba Pol. 30 (1984) 27.

[29] W. Funk, B. Heinz, H. Michel, C. Vonderhied, GIT-Suppl. 4-6 (1987) 9.

[30] J. Koerbl, F. Janick, M. Tkaczyk, K. Havel, (1986) Czech. CS 234387,

IPC GO IN -027126 Appl. 8314077, 06 Jun 1983.

[31] F. Martinez, T. Adzet, J. Iglesias, Mikrochim. Acta 1 (1991) 105.

[32] C. Lan, Z. Kang, S. Zhang, Z. Wu, R. Yu, Nanjung Yaoxueyuan Xuebao

21 (1983)75.

88

[33] V.V Belikov, Rastit. Resur. 21 (1985) 350.

[34] S. S. Zarapkar, N. S. Kanyawar, S. H. Rane, Indian Drugs 37 (2000) 133.

[35] A. Rajasekaran, M. Kumar, G. Krishnamoorthy, B. Jayabar, Indian J.

Pharm. Sci. 59 (1997) 230.

[36] M. N. Reddy, Y. P. N. Reddy, P. J. Reddy, T. K. Murthy, Asian J. Chem.

13 (2001) 1234.

[37] N. Rahman, S. N. H. Azmi, Acta Pharm. 49 (1999) 113-118.

[38] Vogel's Textbook of Quantitative Chemical Analysis 6th ed., Pearson

Education, Singapore, 2002, p. 420.

[39], P. G. Sammel, Comprehensive Organic Chemistry - The synthesis and

Reactions of Organic Compounds, Volume 4, 1st ed., Pergamon Press,

1979, p. 681.

[40] A. Schonberg, G. Schutz, Chem. Ber. 93 (1960) 1466.

[41] J. N. Miller, Analyst 116 (1991) 3.

[42] S. Torrado, S. Torrado, R. Cadorniga, J. Pharm. Biomed. Anal. 12 (1994)

383.

[43] B.Morelli Analyst 108 (1983) 870.

[44] V. V. Nalimov, The Application of Mathematical Statistics to Chemical

Analysis, Pergamon Press, Oxford, 1963, p. 189.

[45] B. Morelli, Anal. Lett. 20 (1987) 141.

[46] R. Cassidy, M. Janoski, LC-GC 10 (1992) 692.

[47] G. D. Christian, Analytical Chemistry, 4th ed., Wiley, New York, 1986,

p. 79.

Cfjcvpter -3 (Ej(tractive spectropHotometric metfiods for the determination of nifedipine in pHarmaceuticaC

formulations using BromocresoCgreen, SromopHenoC SCue, BromotHymoCBCue anderiocHrome 6[ac^^

89

INTRODUCTION

Nifedipine, chemically dimethyl-1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)

pyridine-3,5 dicarboxylate, is an important calcium cliannel blocker with peripheral

and coronary vasodilator activity [1-5]. The drug is frequently used as

antihypertensive and potent arterial vasodilator in the treatment of angina pectoris and

various other cardiovascular disorders [6, 7]. The drug and its formulations are

official in USP [8] and BP [9], which recommended HPLC and non-aqueous titration

for its assay, respectively.

The drug has been determined by a variety of analytical techniques such as

HPLC [10-31], reversed phase HPLC [32-34], HPTLC [35], gas chromatography [36-

53], micellar electrokinetic chromatrography [54], electroanalytical methods [55-60],

flow injection analysis [61], mass spectrometry [62] and UV spectrophotometry [63-

65].

The estimation of nifedipine alone was carried out using second-order

derivative spectra [66] of the compound in 0.1 M HCl whereas first derivative spectra

were utilized for its assay in combined dosage forms [67,68]. The methanolic solution

of the drug reacts with 4-dimethylaminobenzaldehyde in H3PO4 resulting in the

formation of yellow coloured product, which forms a basis for its determination at

380 nm [69]. Two spectrophotometric methods have been recommended, one is based

on the formation of blue coloured complex with Folin Ciocalteau reagent [70] and

second method involves the charge transfer complex formation with chloranil [71]. A

kinetic spectrophotometric method has also been described based on oxidation of drug

with KMn04 at neutral pH [72]. The other two spectrophotometric methods were

developed which involved the reduction of nifedipine with Zn/NHtCl and Zn/HCl to

90

hydoxylamino derivative and primary aromatic amino derivative, respectively

[73,74]. The hydoxylamino derivative was reacted with 4-(methylamino) phenol and

potassium dichromate to give a chromophore, which absorbed maximally at 525 nm.

Thp aromatic amino derivative formed schiffs base with 3,4,5-

trimethoxybenzaldehyde and subsequently determined at 365 nm. So far, no

extractive spectrophotometric method for the assay of nifedipine in pharmaceutical

formulations was reported.

In this chapter, four new extractive spectrophotometric methods for the

determination of nifedipine have been discussed. The methods are based on the

reduction of nitro group of nifedipine by Zn/HCl into primary amino derivative which

forms chloroform extractable ion pair complexes with bromocresol green (BCG),

bromophenol blue (BPB), bromothymol blue (BTB) and eriochrome black T (EBT).

EXPERIMENTAL

Apparatus

A Spectronic 20D* spectrophotometer (Milton Roy, U.S.A.) was used to

measure the absorbance.

An Elico model Li-10 pH meter was used for pH measurements.

Reagents and standards

Preparation of amino derivative of the nifedipine

A 0.05 % solution of reduced nifedipine was prepared by treating a mixture of

12.5 mg of pure nifedipine (J.B. Chemicals and Phannaceuticals Ltd., Mumbai

91

India), dissolved in 3 ml ethanol, 1.8 ml of 5M HCl for BTB (2.5 ml for BCG and

BPB; 3.0 ml for EBT required for ion pair formation) and 3.0 g of Zn dust. The

content was allowed to stand for 20 min at 25 ± 1°C. The solution was then filtered

through a Whatmann No. 42 filter paper, the residue was washed with two 5 ml

portions of doubly distilled water. The filtrate and washings were diluted to volume in

a 25 ml standard volumetric flask with doubly distilled water.

0.025 % solutions of bromocresol green (S.d. fine Chem Limited, India),

bromophenol blue (S.d. fine Chem Limited, India), bromothymol blue (S.d. fine

Chem Limited, India) and eriochrome black T (Fluka Chemie A G, Germany) were

prepared by dissolving 25 mg of each dyestuff in 100 ml standard volumetric flask

and diluting to volume with doubly distilled water.

Procedure of calibration curve

Into a series of 50 ml separating funnel 7 ml of BCG (BPB, BTB or ETB)

followed by an appropriate volume of 0.05 % reduced nifedipine (0.10-0.65 ml for

BCG; 0.08-0.75 ml for BPB; 0.13-0.66 ml for BTB and 0.09-0.45 ml for EBT) were

placed and mixed well. Then 10 ml of chloroform was added to each funnel. The

contents were shaken for 2 min and allowed to separate the two layers. The

absorbance of the organic layer was measured at 415 nm for BCG, BPB, BTB ion-

pair complexes and 520 nm for EBT ion-pair complex against a reagent blank

prepared similarly in each case. The calibration curve was constructed, in each case,

by considering the absorbance measured at seven concentration levels of nifedipine.

The amount of the drug was computed either from calibration curve or from

regression equation. The colour of the complexes were stable for at least 2 hours.

92

Procedure for the assay of drug in dosage forms

An amount of the tablet or capsule equivalent to 12.5 mg of nifedipine was

weighed accurately, and extracted into 25 ml chloroform with shaking. Filtration

through a Whatmann No. 42 filter paper was performed. The filtrate was evaporated

to dryness under vacuum and the residue was dissolved in 3 ml ethanol and converted

into amino derivative following the procedure given under the head "preparation of

amino derivative of the nifedipine" and then subjected to the recommended procedure

for the determination.

RESULTS AND DISCUSSION

Nifedipine contains -NO2 group, which is reduced to amino derivative by zinc

dust and HCl. In the present study, the reduced nifedipine possessing primary

aromatic amino group is protonated in acidic medium, which forms ion-pair

complexes with each of the acid dyes such as BCG, BPB, BTB and EBT. The ion-

associated complexes are quantitatively extracted with chloroform. The absorption

spectra are shown in Fig. 3.1. which revealed that the ion-pair complexes with BCG

(BTB or BPB) and EBT absorbed maximally at 415 and 520 nm, respectively. The

reagent blanks prepared under similar conditions showed no absorption.

Composition and formation constant of ion-pair complexes

The stoichiometry of ion-pair complexes of the reduced drug with each of the

dyestuffs was established following the method of continuous variations [75]. The

results (Figs. 3.2., 3.3., 3.4. and 3.5.) indicated that the molar ratio of the drug to

93

0.7

0.6 -

0.5 -

^ 0.4 ^ a .o 1 . o m < 0.3

0.2

0.1

0.0 -r

350 400 450 500

Wavelength (nm}

550 600

Fig. 3.1. Absorption spectra: (a) Amino derivative of nifedipine-BCG complex; (b) Amino derivative of nifedipine-BPB complex; (c) amino derivative of nifedipiae-BTB complex; (d) amino derivative of nifedipine-EBT complex.

94

0.6

Mole fraction of drug

Fig. 3.2. Job's plot for drug-dye (1.443 x 10" M) system: BCG.

95

0.35

0.2 0.4 0.6

Mole fraction of drug 0.8 1.0

Fig. 3.3. Job's plot for drug-dye (1.443 x 10" M) system: BPB.

96

0.30

0.00 U 0.4

Mole fraction of drug

Fig. 3.4. Job's plot for drug-dye (1.443 x 10" M) system: BTB.

97

o o c n Si

o (0

<

0.2 0.4 0.6

Mole fraction of drug

Fig. 3.5. Job's plot for drug-dye (1.443 x 10 M) system: EBT.

98

dyestuff in each compound is 1:1. The formation constants [76,77] were also

calculated and found to be 2.91 x 10^ 3.32 x 10^ 5.82 x 10 and 5.54 x 10 for

complexes with BCG, BPB, BTB and EBT, respectively.

On the basis of literature background and our experimental findings, the

reaction mechanism is proposed and given in scheme 3.1.

Optimization of the reaction conditions

The optimum conditions for quantitative estimation of the drug were

established via a number of preliminary experiments.

Effect of concentration of hydrochloric acid

The influence of the concentration of HCl on the reduction of the nifedipine

and subsequent ion-pair formation of amino derivative of the drug with various

dyestuffs has been studied. The results are shown in Figs. 3.6., 3.7., 3.8. and 3.9. It is

apparent from figure that the absorbance of ion-pair complexes with BCG (or BPB),

BTB and EBT was found to be constant when the reduction was done in the range of

0.47-0.53 M, 0.34-0.40 M and 0.56-0.64 M HCl, respectively. For the most efficient

extraction of ion-pair with chloroform the optimum value was fixed at 0.50 M, 0.36

M and 0.60 M HCl for ion-pair formation with BCG (or BPB), BTB and EBT,

respectively.

Effect of dye concentration

The effect of dye concentration on the intensity of the colour at the selected

wavelength and constant nifedipine concentration was critically examined using

99

[A]

HjCOOC

HaC^ ^ N ^ ^CH;

HjCOOC

nifedipine

H3C.

COOCH3 H3COOC ^

NH2

amino derivative of nifedipine protonated

Vr' OH HO,

B r ^ ^ * > ^ ^ C C ^ " * * < ^Br Br T ^ \ ^ C H 3

^ A v ^ S O j

bromocresol green (lactoid ring)

(quinoid ring)

HaC^ . N ^ ^CH;

HjCOOC COOCH3 B r - ^ ' ^ * ^ ^ C * ' ' ^ f ^ B r "

r^^ CH3

Br

H3COOC

nifedipine(amino derivative)-bromocresol green complex

Br Br Br

[B]

SO3H

bromoplienol blue (lactoid ring) (quinoid ring)

HaC

H3COOC

Br Br

C ^-^^ "-Br

so7

H3C,

HaCOOC-

Cr nifedipine (amino derivative)-bromophenol blue complex

100

[C]

H3C01

C jH , C3H7

CH3 HO,

COOCH3 Bi

NH3 CH3

H3COOC

C3H7 C3H7

bromothymol blue nifedipine (amino derivative)-bromothynnol blue complex

PI HjCOOC-

nifedipine (amino denvative)-eriochrome black T complex

Scheme 3.1.

101

0.2 0.3 0.4 0.5

Concentration of HCI (M)

0.6 0.7

Fig. 3.6. Effect of the concentration of HCI: 31 ^g ml"' drug + 7 ml of 0.025% BCG.

102

0.3 0.4 0.5

Concentration of HCI (M)

Fig. 3.7. Effect of the concentration of HCi: 33 ^g ml" drug + 7ml of 0.025% BPB.

103

0) u c n Si o tn n <

0.1 0.2 0.3 0.4

Concentration of HCI (M)

0.5

Fig. 3.8. Effect of the concentration of HCI: 33 \xg ml * drug + 7 ml of 0.025% BTB.

104

0.0 0.1 0.2 0.3 0.4 0.5 0.6

Concentration of HCI (M)

0.7 0.8

Fig. 3.9. Effect of the concentration of HCI: 22.5 ^g ml"' drug + 7 ml of 0.025% EBT.

105

different milliliters of the reagent (0.025 %). The results indicated (Figs. 3.10., 3.11.,

3.12. and 3.13) that the maximum absorbance, in each case, was found with 6.0 ml of

the reagent, beyond which absorbance become constant. Therefore, 7.0 ml of each

dyestuff was used for ion-pair formation throughout the experiment.

Choice of organic solvent

A number of organic solvents such as chloroform, carbon tetrachloride,

dichloromethane, benzene and toluene were examined for extraction of the ion-pair

complex in order to provide an applicable extraction procedure. Chloroform was

preferred for its selective extraction of ion-pair complex from the aqueous solution.

Shaking time of 0.5-4.0 min. provided a constant absorbance and hence, 2.0 min was

used as an optimum shaking time throughout the experiment. The ion-pair complexes

were quantitatively recovered in one extraction only and were also stable for at least 2

hours.

Effect of excipients

A systematic study of the effect of excipients was performed, following the

proposed procedures for a 10 ml sample system., by adding a known amount of

excipients to the fixed nifedipine concentration (22.5 \i.% ml"'). The results revealed

the fact that no significant interference was observed from the excipients such as

glucose, fructose, sucrose, lactose and starch commonly present in pharmaceutical

formulations. However, the drug content from the powdered tablets or capsules were

extracted into chloroform which completely eliminates the common excipients found

in drug formulations.

106

o o c n

o lA

<

Volume of BCG (ml)

Fig. 3.10. Influence of the volume of 0.025% dye: BCG.

107

0.50

Volume of BPB (ml)

Fig. 3,11. Influence of the volume of 0.025% dye: BPB.

108

o u c RI a h. o to

<

Volume of BTB (ml)

Fig, 3.12. Influence of the volume of 0.025% dye: BTB.

109

0) u c

o (0

<

Volume of EBT (ml)

Fig. 3.13. Influence of the volume of 0.025% dye: EBT.

110

Analytical data

Calibration graphs (Figs. 3.14., 3.15., 3.16. and 3.17.) were constructed, by

measuring the absorbance at seven concentration levels, which showed linear

response of absorbance in relation to concentration of nifedipine over the range of

5.0-32.5, 4.0-37.5, 6.5-33.0 and 4.5-22.5 |ag ml'' for BCG, BPB, BTB and EBT

methods, respectively. Regression analysis of calibration graphs indicated linear

relationship with negligible intercepts. Table 3.1. summarizes the analytical

parameters, molar absorptivities and the results of statistical analysis of the

experimental data: regression equations computed from calibration graphs along with

standard deviation of slope (Sb) and intercept (Sa), confidence interval of slope (tSb)

and intercept (tSa) on the ordinate. The detection limits [78,79] were found to be 1.06,

0.09, 0.11 and 0.20 [ig ml"' for BCG, BPB, BTB and EBT methods, respectively. The

small value of variance, further, suggested least scatter of experimental data points

around the line of regression.

The repeatability of the proposed procedures was checked by performing ten

replicate determinations of nifedipine at concentration levels of 20 and 30 fig ml''

with BCG (or EBT) and BPB (or BTB), respectively. The relative standard deviations

(RSD %) and recoveries were found to vary over the range of 0.66-0.82 % and 99.8-

100.7 %, respectively.

The accuracy of proposed methods was demonstrated by recovery

experiments, which were carried out by adding a fixed amount of pure drug to the

preanalyzed dosage forms. The analytical results obtained are summarized in Table

3.2.

111

Concentration (ng ml ')

Fig. 3.14. Calibration curve for determination of nifedipine witli BCG.

112

o u c n J3 u O V)

<

Concentration ( g ml )

Fig. 3.15. Calibration curve for determination of nifedipine with BPB.

113

0)

u • c n o in n <

-1 Concentration (^g ml )

Fig. 3.16. Calibration curve for determination of nifedipine witli BTB.

114

Concentration (fxg ml )

Fig. 3.17. Calibration curve for determination of nifedipine with EBT.

u

115

Da in T t

^ o y—t

X c?. ^ t

o I—1

X •—« cs + T o

X o o r-4 fS II <

T o •—1

X o o cs tN

7 o I—1

X o oo • * .

*-H

1 o J—i

X •o o so c

% X

f-H

CN CN

1 o »-H

X o q ri

T o t—l

X 00 ts 00 ri

OS o\ Ov OS d

*o

O f-H

X oo "Tj m

o ts d

oo VO d

' rJ d rH

o

t3

o cu o

.ci

o u

• • ^

."'

u M

« u "w u

1 o

X m oo

o

c

O m

>/1 O

q 5; •* <n *d >ri

U

X

q c 1

>n vd

O

X VO CN vi

X o q *-H

II <

o

X o o

+ T o I—< X o cs

II

<

U

X

0 0

-Si E 2 ca

CL,

6 c

£

*1

1

o X o t q ^

T 1

o «- X o o t in

T' b f—1

X >n vo •*. f—<

r< 1 o *-H

X in

»—» "O -H

I o »—( X o q f*i

tn 1 O *—t

X m 9-^

t>_ f^

o\ 0\ OS OS d

1 O f-H

X OS

»—; in

0 0

o ^

T o

T o t o »-H f -H »—< i -H O <"*

o CN

X o o 0 0 i X o o I

X CN

o o o o

X

00 OS o

m CO Os

. o CN 0 0

O <7\

o X o t—)

11

<

T o t-H

X o o f-H

• *

T o X o o ri 00

T o X n t — <

»-H

CN

V o •-H

X OS •<3-SO

<—<

"? o r—t

X o o • *

T o t—(

X 00 CS q i-H

OS o C3s OS d

ws 1 o r—( X o o r-

so q i-H

CN OO d

oo q d o

-" T "E 60

3 .*J

_s s cd

"*"• » u u 23

' 1

o E J,

>, '> Q. u. O

JD CO

O

c c

• ^

cr u

g 'w

t3) Ui

u.

1 —s ca

o. u o

vl5 00 ' i i O

c u 'o

u o o

c

u Ui

u o o

^

§

> u

E ^J ^

:= 2 u u c .3 >

c o

H -s

e >

u c C3

4 2

a)

.2 o s

s is 8 S "a

o p o o in Z,

O o o r; u. p .

g c •« r f"" ^ U J •£ •° t; •« + o ° « ca w ^ u II "3 S U ^ 5 i3 § o -is -4-» r* C ^ * j . = — O O u D . sis

^ 'S ^

^ O " P

116

-a o .a a> B a

r:

a

d o 1-1

ja "3 S u

s u s u R

B

-a

e o c o '( a

u *-

Q

H

CO vP

O vP

a I. 60

I

< 3

§

ca

CS o

en

o

d

c^ c>

d

CO

d

d o

d

CS

d en

d

d

5 d

d

en d cs V5 d o rn d

es d

d ON

d CS

d

C<1

d o

d CS

d o

d

o> J J

l -

o K; nt •o

L H

o

;

CS

o 0 0 CS

o o

o m •* o

CO

o o m o

o

• *

o

CS

o 00 CM

o C S CO

o •—< CO

o

•—* o c^ CO

o

o

o

^ q d o '"' ^ o o\ o\

< o d o '^ • *

q d o

c^ q d o i - ^

CS o d o *"•

• C3S O N

o>

I—C

q d o

T t

q d o ' ^ ON OS CJN

o\ o d o '"' o

d o

rr> q d o '"' T ( -

o\ OS OS

t OS OS cr\

»-H

q d o

• < *

o d o 1—I

•* o d o

CS o d o '"' OS crs OS 0 \

f<i o d o *—1

C<1 o d o *—< TT OS OS OS

»—< o d o

o o o

o o o i n OS 0 \ C7\

0 ^ CJs

m o o o

CO

o o o CO T—«

o o »—< CS CS o o

O S O N

CJN

0\

o o o f-H

CJN

o o o

o o o

o o C?N CS

o o o

CO

o o r—<

o o o

o o ^ i n --I

CO 3

03 U

c4

•a <

o o — in «

CO 3 60

C 1) •a c u D. O •a c

03

u 03

u

117

The percent relative standard deviations (0.12-0.62 %) can be considered to be very

satisfactory.

The performance of the proposed methods was compared with that of other

existing UV-Visible spectrophotometric methods (Table 3.3.). It is evident from the

table that the proposed methods are more sensitive then the other reported method due

to their higher molar absorptivities and present better accuracy with narrow linear

dynamic range. The methods are found to be simple and can compete with other

existing spectrophotometric methods in determining drug In lower concentrations.

The order of performance of the proposed methods is EB'I>BCG>BTB>BPB.

The proposed methods have been successfully applied to the determination of

nifedipine in commercial tablets and capsules purchased locally. The results (Table

3.4.) obtained by the proposed methods were compared by BP method [9]. The

calculated student's t-value and F-values did not exceed the theoretical ones at 95 %

confidence level. Therefore, there is no significant difference between the proposed

methods and BP method.

118

a

a o >>

w

.2 Oi

•O O

PS • 4 - 1

a B w 'C •<-» u & o o a o la u u o. a

o • 4 - 1

s ^

« .S

'- S

«« es

o u

i « u

Is H

o c

Qi

Q

> o

5: J .

CQ 3

6

E

C u 00

1 o

00 pi x: x; js H H H

o o 0

»•-<

0

0 00

0 0 0

CO

0

0

•n

OS

0

cs 0 0

00 OS

00

OS OS OS

t--

OS

0

Ti 00 0\

t -OS OS

I — <

0 0

1 0 \ OS OS

0 0

1 Os Os OS

OS

•—1

1 00 OS OS °^ s

o o

§ ^

X

>* so

X

00

•<t

X

"O

X

VO t -

t M-

0 00

0

0 0

0

0 <r

0 • *

0

•n

V I

CS en

0 T l

X-1

0 T f

0

r<1

<n vo

tn

irt T f

0 m •n

m vo c

0 00 o i

0

• m

0 0 • *

<o 12 " !2 o •^ v i

u

rt 00 c ca c

6 .5

•5 0-

- 0

s c 0 JD

> i

X 0

0

E

1

• < a ;

m"

• 0

-a

1 c (U j=i Q Ci

'E (3

.E

9 •4

3 CQ <D M

03 D . w 0 (-•

a, •0 a C3

"o c ca

j 3

W

> u

Q

•5 E S 0 '•B •0 c cd

"3 c 0 J3 Q . 0

E

u

4

c u u u. bO

•3 U Ui

0 0 £ 2 ca

3

3 "o c u •a 0

s s 03

0 p

3 "o 1. x: 0 E s CQ

u E o o o *n w

119

ON

n o .£3

•*•>

a B « u a u ^

^ "o E

1-1

« 3

u -a a a VI

o .£3

•a u V) O Oi o u

• • *

>^ .Q

s

e<3 en O

ID

•a ,4>

a

« B E

p

H

w

pa

CQ

Da

o o CQ

> o o u

c o

H CQ W

oa

o u pa

H pa w

m pa

pa

o o CQ

r-^

£ b u > o u Di

H CQ W

CQ H c:

CQ CQ

O o CQ

--

ri

m

m o

en

CO

• *

•O

'^

o

*—<

i n

ri

o ( S

W-l

^

0 \

" fn

(S

o

o d o

o o 0 0

OS

o o

•a 1 -

ca

O o

o o

o d o 1A1 OS OS OS

OS OS

OS

as

OS

d

o

o d o

d

OS

d

V I

d

d

tN d

oo rn d

^ d

d

00 O C^

d

CO

d CO Csl

d

d oo

d

o CO d

"0 d

0 0

d o\

d

OS so d

Csl

d

CO

d

d

CO

d

CO

d

o o o

o o

0 \ OS

o vri

-" oo

CO

CS

t-~

0 0 CO r - <

^ H

*—<

CO

OS

d

0\ o

t s

q d o

d

CO

vo CO

d m <n

o o

o OS OS OS

OS

o 0 0

OS

o\

2 ^ ci '•-' C3

cd "O

<

_o 3 CO

a. cd U

I

d

o o

00

o ts

d

CO

d

1^ 00

CO

d CO CO

o

i n

d

m CO

O

00 tS

o

CO

d

o o q d o

OS

o d o

o d o

'o

U

<N ^ M . 2

5 ij w u o <u o y "> C fi

^ «J -a ill S 8 8 C "n ^ U Ov O^ Ci" * j -tJ

« a <a

120

REFERENCES

[I] Martindale The Extra Pharmacopoeia, 33rd ed., Royal Pharmaceutical

Society, London, 2002, p. 940.

[2] A. Fleckenstein, H. Tritthart, J. H. Doering, K. Y. Byon, Arzneim.-Forsch

(Drug Res.) 22 (1972) 22.

[3] P. D. Henry, Amer. J. Cardiol. 46 (1980) 1047.

[4] D. Murdoch, R. N. Brogden, Drugs 46 (1993) 961.

[5] R. J. Miller, Science 235 (1987) 46.

[6] P. H. Stone, E. M. Antman, J. E. Muller, E. Braunwald, Ann. Inter. Med. 93

(1980) 886.

[7] J. N. Delgado, W. A. Remers, Wilson and Gisvold's Text Book of Organic

Medicinal and Pharmaceutical Chemistry, 9 Ed., J. B. Lippincott Company,

Philadelphia, 1991 p.554.

[8] The United States Pharmacopoeia, 24th ed. Rockville, MD, USA, 2000

pp.1182.

[9] British Pharmacopoeia, Her Majesty Stationary Office, London, Vol. I, 1993

p.449.

[10] P. G. Pietta, A. Rava, P. Biondi, J. Chromatogr. 210 (1981) 516.

[II] C. H. Kleinbloesen, J. Van Harten, P. Van Brummelen, D. D. Breimer, J.

Chromatogr. B: Biomed. Appl. 308 (1984) 209.

[12] G. R. Rao, S. Raghuveer, C. M. R. Srivastava, Indian Drugs 22 (1985) 435.

121

[13] Z. Wang, X. Tang, J. Hou, C. Pan, X. Wei, Shenyang Yaoke Daxue Xuebao

19 (2002) 38.

[14] C. Giachetti, P. Poletti, G. Zanolo, Chromatogr. Commun. 10 (1987) 654.

[15] N. Beaulieu, N. M. Curran, S. J. Graham, R. W. Sears, E. G. Levering, J. Liq.

Chromatogr. 14(1991)1173.

[16] T. Ohlcubo, H. Noro, K. Sugawara, J. Pharm. Biomed. Anal. 10 (1992) 67.

[17] Y. Jiang, Y. Wang, W. Zhong, L. Zhang, Y. Zhang, D. Zhang, E. Zhang,

Fenxi Huaxue 20 (1992) 822.

[18] I. Niopas, A. C. Daftsios, J. Pharm. Biomed. Anal. 32 (2003) 1213.

[19] Z. Sha, H. Gao, W. Sun, D. Tian, Zhongguo Yiyao Gongye Zazhi 24 (1993)

176.

[20] F. Barbato, B. Cappelo, L. Grumetto, P. Morrica, Farmaco 48 (1993) 417.

. [21] C. Liu, G. Chen, Yaowu Fenxi Zazhi 13 (1993) 314.

[22] J. S. Grundy, R. Kherani, R. T. Foster, J. Pharm. Biomed. Anal. 12 (1994)

1529.

[23] A. F. M. K. El-Walili, Bull. Fac. Pharm. 34 (1996) 65.

[24] Yeong-Shun Gau, R. Rhei-Long Chen, S. W. Sun, Chin. Pharm. J. 49 (1997)

41.

[25] M. AbdoUahi, M. Pirali, M. Karimi, M. Shahriarian, A. Shafiee, Daru, J. Fac.

Pharm. 7(1999) 1.

[26] P. Thongnopnua, K. Viwatwongsa, J. Pharm. Biomed. Anal. 12 (1994) 119.

[27] J. Dokladalova, J. A. Tykal, S. J. Coco, P. E. Durkee, G. T. Querela, J. J.

Korst, J. Chromatogr. B: Biomed. Appl. 231 (1982) 451.

122

[28] K. Miyazaki, N. Kohri, T. Arita, J.Chromatogr. B: Biomed. Appl. 310 (1984)

219.

[29] H. Potter, M. Hulm, J. Pharm. Biomed. Anal. 61 (1988) 15.

[30] E. Mikami, S. Yamada, Y. Fujii, N. Kawamura, J. Hayakawa, lyakuhin

Kenkyu23 (1992)491.

[31] Y. Zhang, Z. Liu, Y. Guo, X. Gu, Zhongguo Yiyuan Yaoxue Zazhi 21 (2001)

152.

[32] G. Horvai, A. Hrabeczy-Pall, V. Hor/ath, I. Klebovich, Mickrochim. Acta.

113 (1994) 171.

[33] Q. Liu, Q. Lin, Zhonghua Yixue Jianyan Zashi 18 (1995) 22.

[34] M. Zaater, E. Hasan, N. Najib, Pol. J. Pliarmacol. 52 (2000) 307.

[35] V. B. Patravale, V. B. Nair, S. P. Gore, J. Pharm. Biomed. Anal. 23 (2000)

623.

[36] B. J. Schmid, H.E. Perry, J. R. Idle, J. Chromatogr. 425 (1988) 107.

[37] K. Akira, S. Baba, S. Aoki, Chem. Pharm. Bull. 36 (1988) 3000.

[38] S. Higuchi, Y. Shiobara, Biomed. Mass. Spectrom. 5 (1978) 220.

[39] R. Testa, E. Dolfmi, C. Reschiotto, G. Secchi, P. A. Biondi, IL Farmaco 34

(1979) 463.

[40] P. Jakobsen, O. L. Pedersen, E. Mikkelsen, J. Chromatogr. 162 (1979) 81.

[41] Y. Qin, H. Liu, X. He, H. Zhuang, Shanghai Dier Yike Daxue Xuebao 20

(2000) 512.

[42] S. Kondo, A. Kuchiki, K. Yamamoto, K. Akimoto, K. Takanashi, N. Awata,

I. Sugimoto, Chem. Pharm. Bull. 28 (1980) 1.

[43] M. T. Rosseel, M. G. Bogaert, J. Chromatogr. 279 (1983) 675.

123

[44] S. R. Hamann, R. G. Mc AUister Jr., Clin.Chem. 29 (1983) 158.

[45] F. A. Tucker, P. S. B. Minty, G. A. Mac Gregor, J. Chromatogr. B: Biomed.

Appl. 342 (1985) 193.

[46] G. Pabst, D. Lutz, K. H. Molz, W. Dahmen, H. Jaeger, Arzneim-Forschung,

Drug. Res. 36 (1986) 256.

[47] K. S. Patrick, E. J. Jarvi, A. B. Stranghn, M. C. Meyer, J. Chromatogr. 495

(1989)123.

[48] B. Marciniec, E. Kujawa, M. Ogrodowczyk, Pharmazie. 47 (1992) 502.

[49] J. S. Ellis, S. C. Monkman, R. A. Seymour, J. R. Idle, J. Chromatogr. B:

Biomed. Appl. 621 (1993) 95.

[50] A. Jankowski, I. Z. Siemion, S. Lamparczyk, J, Chromatogr. A: 668 (1994)

469.

[51] J. Martens, P. Banditt, F. P. Mayer, J. Chromatogr. B: Biomed. Appl. 660

(1994)297.

[52] J. Tu, J. Peng, J. Xin, G. Liu, Zhonggno Yiyuan Yaoxue Zazhi 15 (1995)

197.

[53] Y. Qin, H. Liu, X. He, H. Zhuang, Q. Lu, Shanghai Dier Yike Daxue Xuebao

20 (2000)512.

[54] A. E. Bretnall, G. S. Clarke, J. Chromatogr. A: 700 (1995) 173.

[55] J. A. Squella, E. Bamafi, S. Pema, L. J. Nunez-Vergara, Talanta 36 (1989)

363.

[56] P. Tompe, A. Halbauer-Nagy, Acta. Pharm.Hung. 60 (1990) 130.

[57] M. M. Ellaithy, P. Zuman, J. Pharm. Sci. 57 (1992) 191.

124

[58] A. El-Jammal, J. C. Vire, G. J. Patriarche, O.N. Palmeiro, Electroanalysis 4

(1992) 57.

[59] Z. Senturk, S. A. Ozkan, Y. Ozkan, J. Pharm. Biomed. Anal. 16 (1998) 801.

[60] V. Dumitrescu, V. David, A. Pavel, Rev. Chim. 52 (2001) 317.

[61] P. Richter, M. I. Toral, G. Quiroz, P. Jaque, Lab. Rob. Autom. 9 (1997) 255.

[62] T. Kumazawa, K. Sato, H. Seno, O. Suzuki, Hochudoku 11 (1993) 128.

[63] S. P. Vyas, S. K. Goswami, Indian Drugs 30 (1993) 342.

[64] G. Yuan, B. Zhu, Zhongguo Yiyao Gongye Zazhi 27 (1996) 171.

[65] S. Mallick, B. K. Gupta, S. K. Ghosal, East. Pharm. 41 (1998) 129.

[66] P. Umapathi, Int. J. Pharm. 108 (1994) 11.

[67] A. F. M. El-Walily, Acta Pharm. Hung. 67 (1997) 89.

[68] A. F. M. EL-Walily, J. Pharm. Biomed. Anal. 16 (1997) 21.

[69] K. R. Mahadik, G. B. Byale, H. N. More, S. S. Kadam, J. Inst. Chem. 63

(1991)218.

[70] C. S. P. Sastry, R. Chintalapati, R. Venkateswarlu, J. Inst. Chem. 69 (1997)

187.

[71] A. Y. Golcu, S. Serin, Sci. Pharm. 66 (1998) 341.

[72] N. Rahman, S. N. H. Azmi, Acta Pharm. 49 (1999) 113.

[73] N. Rahman, M. N. Hoda, IL Farmaco 57 (2002) 435.

[74] A. B. Karadi, K. U. M. Ravi, M. Shobha, S. A. Raju, East. Pharm. April

(2000)117.

[75] W. C. Vosburgh, G. R. Cooper, J. Am. Chem. Soc. 63 (1941) 437.

[76] W. Likussar, D. F. Boltz, Anal. Chem. 43 (1971) 1265.

[77] K. Momoki, J. Sekino, H. Sato, N. Yamaguchi, Anal. Chem. 41 (1969) 1286.

125

[78] B. Morelli, Analyst 108 (1983) 870.

[79] V. V. Nalimov, "The Application of Mathematical Statistics to Chemical

Analysis", Pergamon Press, Oxford, 1963, p.l89.

CHapter -4 VaCidatecfSpectropHotometnc MetHodsfor tde (Determination ofJLtniocCarone HydrocHCoricCe in CommerciaC(Dosage ^orms using p-cHlhraniCic

acicCand2,3 -dicfiCoro 5,6- dicyano 1,4 — Benzaquinone

126

INTRODUCTION

Amiodarone hydrochloride, 2-butyl, 3-benzofliranyl 4-[2-diethyl amino

ethoxy]-3-5-diiodophenyl methanone hydrochloride is an important

antiarrhythmic drug [1-3], used only in the treatment of documented life-

threatening recurrent ventricular dysrhythmias. It undergoes hepatic

metabolism to produce a major metabolite, desythyl amiodarone, which can

approach the concentrations of the parent drug during chronic therapy. The

molecular formulae of amiodarone and its metabolites are given as:

< ^ ^

O

II -c- -O CHz CH, N

/ ' ^

\ R2

^0 CH2(CH2)2CH3 -j

Where Ri= R2= C2 H5 (amiodarone)

Ri = HI , R2 = C2 H5 (desythyl amiodarone)

R] = R2 = H (di- N- desythyl amiodarone)

British Pharmacopoeia [4] describes potentiometric titration and

spectrophotometric method for the assay of amiodarone hydrochloride in pure

form and pharmaceutical formulations, respectively. The different analytical

techniques that have been reported for the determination of cited drug in

biological fluids and pharmaceutical formulations include high performance

liquid chromatography [5-11], high performance thin layer chromatography

[12], liquid chromatography [13-14], infrared spectroscopy [15], fluorimetry

127

[16] and electrochemical methods [17-19]. In the literature only few

spectrophotometric methods have been reported. Sastry et al. [20] have

described two spectrophotometic methods for the determination of

amiodarone hydrochloride based on its reaction with a known excess of iodine

or ammonium molybdate and estimating the unreacted iodine or reduced

ammonium molybdate with metol - sulfanilic acid or potassium thiocyanate.

The reaction [21] between amiodarone and citric acid-acetic anhydride

reagent yielded an intensely blue coloured product and absorbance was

measured at 580 nm. The drug content in pharmaceutical formulations was

also determined spectrophotometrically [22] using the charge transfer

complexation reaction of drug with chloranil and iodine at 500 and 290 nm,

respectively. The extractive spectrophotometric methods [23] have also been

utilized based on extractable ion-pair complexes of the drug with tropaeolin

00, tropaeolin 000 and wool fast blue at 420, 480 and 580 nm, respectively.

A survey of literature reveals that the existing spectrophotometric

methods [20-23] dealing with the estimation of amiodarone in drug

formulations suffer from some disadvantages such as narrow linear dynamic

range requires heating or extraction and long analysis time. Therefore, the

development of fast, simple, sensitive, low-cost and selective methods is the

need of the day especially for a routine quality control analysis of

pharmaceutical products containing amiodarone hydrochloride.

The charge transfer reaction of certain n- acceptors such as p-

chloranilic acid [24-26] and 2,3-dichloro5,6-dicyanol,4-benzoquinone [27-

31 ] has been successfully utilized in pharmaceutical analysis.

128

This chapter describes two rapid and simple visible spectrophotometric

methods for the determination of amiodarone hydrochloride by exploiting its

basic nature and electron donating property. The methods (A and B) are based

on charge transfer complexation reaction of amiodrone base with p-chloranilic

acid and 2,3-dichloro5,6-dicyanol,4-benzoquinone(DDQ) in dioxane-dichloro

methane and acetonitrile media, respectively. The proposed methods were

validated statistically.

EXPERIMENTAL

Apparatus

Spectral runs and absorbances were recorded on a spectronic 20 D"

spectrophotometer (Milton Roy Company, USA) with 1 cm matched glass

cells.

Reagents and Standards

Amiodarone hydrochloride was kindly provided by Troikaa

Pharmaceuticals Ltd. India and was used as received. Commercial dosage

forms of amiodarone hydrochloride such as Aldarone (Zydus Alidac),

Amiodar (Cardicare), Duron (Samarth Pharma) and Eurythmic (Troikaa) were

purchased from local market.

All other reagents used were of analytical grade. Solutions of p-

chloranilic acid (0.08%; Fluka, Switzerland) and DDQ (0.08%; Fluka,

129

Switzerland) were prepared in dioxane and acetonitrile, respectively. An

aqueous solution of sodium carbonate (0.5M, Merck, India) was prepared in

doubly distilled water.

The standard solutions of amiodarone base (2.0 mg ml'' and 0.5 mg

mr') were prepared in dichloromethane.

Recommended Procedures

Preparation of amiodarone base:

To prepare amiodarone base solution, 25.0 ml of 0.2% (2.0 mg ml')

amiodarone hydrochloride and 50.0 ml of 0.5 M NaiCOs solutions were

transferred into a 250 ml separating funnel. The contents of the separating

funnel were mixed well and shaken for 1 min. The two layers were allowed to

separate. The dichloromethane layer was dried over anhydrous sodium sulfate

and diluted to 25 ml with dichloromethane. A working standard solution of

amiodarone base (0.05%) was prepared on further dilution.

Method A:

Aliquots of 0.2%. amiodarone base solution (0.025 - 0.90 ml) were

pipetted into a series of 5.0 ml standard volumetric flasks. 1.5 ml of 0.08% p-

chloranilic acid was added to each flask and diluted to volume with

dichloromethane. The coloured product formed immediately and remained

stable for 1.5 h. The absorbance was measured within the stability period (2-

90 min) after dilution at 535 nm against the reagent blank prepared

simultaneously.

130

Method B:

Aliquots of 0.05% amiodarone base corresponding to 10 - 325 p-g were

pipetted into a series of 5.0 ml standard volumetric flasks. To each flask, 1.5

ml of 0.08% DDQ was added and completed to volume with acetonitrile. The

absorbance was measured at 570 nm within the stability period of 3 - 40 min

against the reagent blank prepared similarly, omitting the drug.

Procedure for determination of commercial dosage forms

Ten tablets were powdered, mixed thoroughly and weighed accurately

to an amount equivalent to 200 mg of amiodarone hydrochloride. It was

stirred well with dichloromethane and filtered through Whatmann No. 42

filter paper. The residue was washed with dichloromethane for complete

recovery of the drug. The filtrate and washings were diluted to volume in a

100 ml standard flask. The amiodrone hydrochloride solution was converted

into amiodrone base following the recommended procedure given under the

head "preparation of amiodarone base solution". It was further diluted

according to the need and then analyzed following the recommended

procedures.

RESULTS AND DISCUSSION

The spectrum of p-chloranilic acid in the dioxan-dichloromethane

mixture exhibits an absorption band at 440 nm. The addition of amiodarone

base to this solution causes an immediate shift with a new characteristic band

131

at 535 nm (Fig. 4.1.). The band may be attributed to the formation of

p-chloranilic acid radical anion. The p-chloranilic acid in dioxane-

dicholoromethane medium exists in unionized form and acts as n -acceptor in

a similar marmer to quinines [32]. Some hydrochloride salts of amines do not

possess a lone pair of electrons and hence, unable to react with a or 71-

acceptors. Similarly, amiodarone hydrochloride does not react with p-

chloranilic acid owing to non-availability of n-electrons. In order to establish

the assay procedure for amiodarone hydrochloride, it is necessary to convert

amiodarone hydrochloride solution into amiodarone base solution. Due to the

same reason, amiodarone hydrochloride was dissolved in dichloromethane

and shaken with a 0.5 M aqueous sodium carbonate solution, resulting in the

formation of amiodarone base in the dichloromethane layer. The addition of

p-chloranilic acid to amiodarone base (n-donor) results in the formation of a

charge transfer complex of the n -T: type. This compound is believed to be an

intermediate molecular association complex, which dissociates in dioxane-

dichloromethane solvent, producing p-chloranilic acid radical anion. The

mole ratio method [33] has been applied to establish the stoichiometric ratio

of amiodarone base to p-chloranilic acid and was found to be 1:1 (Fig. 4.2.).

This indicated the presence of one n-donating center in the amiodarone base

for charge transfer complexation reaction. On the basis of literature

background search and our experimental findings, a reaction mechanism is

proposed (Scheme 4.1.).

DDQ is TT-acceptor, which reacts instantaneously with basic

nitrogenous compounds to form charge transfer complexes of the n-7r type.

132

350 400 450 500 550

Wavelength (nm)

600 650 700

Fig. 4.1. Absorption spectra of (a) 2.30 X 10' M p-ciiloranilic acid (b) 0.36 |ig ml'* amiodarone with 1.15 X 10" M p-chloranilic acid in dioxan- dichloromethane.

133

0.25

0.20 -

0.15 0) u c n o tn

< 0.10

0.05

0.00 0.00 0.25 0.50 0.75 1.00 1.25 1.50

Molar ratio of p-chloranilic acid : drug

1.75 2.00

Fig. 4.2. Molar ratio plot for amiodarone - p - chloranilic acid complex; amiodarone concentration, 2.933 X 10" M.

134

^ ^ ^ ^

O CH2(CH2)2CH3 I

amiodarone

C2H5

0—CH2--CH2—N: +

C2H5

n-n complexation

HO

CI

CI

OH

p-chloranillc acid

<^^^

C2H5

0—CH2—CH2--N I

O CH2(CH2)2CH3 ^1

n - 71 complex

r . ^ ^ ^

in dichloromethane • dioxan solvent

C2H5

0 ^CH2(CH2)2CH3 I

O—CH2—CH2—Nt +

C2H5

Scheme 4.1.

p - chloranilic acid radical anion (coloured species)

135

The absorption spectrum of DDQ in acetonitrile shows a characteristic band

peaking at 350 nm. The addition of amiodarone base to this solution causes an

immediate change in the absorption spectrum with new characteristics bands

peaking at 435, 540 and 570 nm (Fig 4.3.)- These bands may be attributed to

the formation of DDQ radical anions, which probably resulted through the

dissociation of the donor-acceptor complex in a highly polar solvent like

acetonitrile. In order to avoid the maximum interference from the reagent

blank, the absorbance measurements were made at 570 nm. The mole ratio

method (Fig. 4.4.) suggested a donor to acceptor ratio of 1:1, confirming the

presence of one n-donating center in the amiodarone base molecule. The

reaction sequence for the formation of DDQ radical anion is shown in Scheme

4.2.

The molar absorptivities and association constants for amiodarone-p-

chloranilic acid and amiodarone - DDQ reaction products were calculated using

Ross and Labes equation [34].

[A][D] 1 _ j _ 1 ^ 1

[A] + [D] ^ A;,^° KS;, [A] + [D] Ej,

where [A] and [D] are the molar concentrations of the acceptor and donor,

respectively, A / ' ^ and sx are the absorbance and molar absorptivity of the

complex at the specified max- K is the association constant of the complex.

The values of association constant (K) and molar absorptivity (ex) were

obtained from the plot of 1 against [A] [D] i

136

n 1 1 1 I T — ^ — I 1 r

300 340 380 420 460 500 540 580 620 660 700

Wavelenth (nm)

Fig. 4.3. Absorption spectra of (a) 8.81 X10"* M DDQ in acetonitrile (b) 65 xg ral"^ amiodarone with 1.06 X 10" M DDQ in acetonitrile.

137

2.00

0.50 0.75 1.00 1.25

Molar ratio of DDQ : drug

1.50 1.75 2.00

Fig. 4.4. Molar ratio plot for amiodarone -DDQ complex; amiodarone concentration, 2.933 X 10" M,

138

. ^ ^ ^ ^

^O ^CH2(CH2)2CH3

amiodarone

N =

N= '

n-n complexation

CoH 2rl5 N=

0 CH2(CH2)2CH3 ^1

O—CH2—CHz—N:-

2rl5 H='

CI

CI

n-7t complex

in acetonitrite solvent

C2H5

O ^CH2(CH2)2CH3^I

O—CH2—CH,—Nt + 1 C2H5

N =

N =

DDQ radical anion (coloured species)

Scheme 4.2.

139

(Figs. 4.5. and 4.6.). The free energy was also computed. The results are given

in Table 4.1.

Optimization of variables '

The spectrophotometric properties of the coloured species formed with

p-chloranilic acid and DDQ as well as the different parameters affecting the

colour development were extensively studied. The optimum conditions for the

assay procedures (method A and B) have been established by studying the

reactions as a function of the concentration of reagent, nature of the solvent

and the stability of the coloured species.

Effect of the concentration of p-chloranilic acid

For method A, the effect of the volume of 0.08% p-chloranilic acid was

studied over the range of 0.2 -1,5 ml in a solution containing 360 [a,g ml"'

amiodarone. The results (Fig. 4.7) revealed the fact that 1.0 ml of p-

chloranilic acid was required to achieve the maximum intensity of the colour.

Therefore, 1.5 ml was used as an optimum value and maintained throughout

the experiment. The reaction gets stabilized within 2.0 min of mixing at room

temperature and absorbance remained constant for a further 90 min.

Effect of the concentration of DDQ

For method B, the only effective variable is the concentration of DDQ.

To study the influence of the volume of 0.08% DDQ, an aliquot of drug

containing 300 |ag was pipetted into a series of 5.0 ml standard volumetric

140

1 0 ' ^ X 1 .1

1.7 1.8 1.9 2.0 2.1 2.2 2.3 xlO^

lA ] + [Z? ]

Fig. 4.5. Plot of ^ _ ^ _ ^ versus r .-, r J-. x — ^ r for 1:1 complex

of amiodarone - p- chloranilic acid.

AD

141

10"'^x3.9

2.3 x10^

[A]+[D]

Fig. 4.6. Plot of

amiodarone -DDQ.

[A][D] 1 versus , • . • " • , , x

[ ] + [D] , AD for 1:1 complex of

142

Table 4.1. Association constant, K; molar absorptivity, ej. from the Ross and Labes plots for the complex and calculated free energy, AG

Parameter Amiodarone -p-chloranilic acid Amiodarone - DDQ complex complex

3.855 X 10

7.502 X lO'

-15.251

K(lmor')

E X (1 mol'' cm"')

AG (KJ mor')

1.107X10^

1.421 X 10

-17. 953

143

0.2 0.4 0.6 0.8

Volume of p-chloranillic acid (ml)

Fig. 4.7. Influence of the volume of 0.08% p-chloranilic acid.

144

flasks followed by varying volumes of 0.08% DDQ (0.1-1.5 ml) and diluted

to volume with acetonitrile. Fig. 4.8 shows that the highest absorbance was

obtained with 1.0 ml of 0.08% DDQ. Further addition of DDQ caused no

change in the absorbance and hence, 1.5 ml was selected as an optimum

volume for all determinations. The intensity of the colour formed on mixing

the reagent reached maximum within 3 min. and was stable up to 35 min. at

room temperature. Therefore, it is recommended that the absorbance be

measured within this time period.

Analytical data

Table 4.2. summarizes the analytical parameters such as Beer's law

limit, calibration equations obtained by least square treatment of calibration

data, coefficient of correlation, variance, confidence limit for slope and

intercept . The high value of correlation coefficient suggested the excellent

linearity of both the calibration graphs (Figs 4.9. and 4.10.). The values of

confidence limit at 95% confidence level for slope and intercept of the

regression lines pointed towards high reproducibility of the proposed

methods. Test of significance of the intercept of the regression lines showed

that the experimental intercepts, a, for method A and B do not differ

significantly from the theoretical value of zero. For this justification, a

simplified method was used to calculate the quantity from the relation t = a/Sa,

[35] and its comparison with the tabulated data from the t-distribution. The t-

value for method A and B were found to be 0.056 and 0.596, respectively,

which did not exceed the tabulated t- value ( t = 2.365, when v = 7 ) at 95%)

145

o o c a

£1 o in Si

<

Volume of DDQ (ml)

Fig. 4.8. Influence of the volume of 0.08% DDQ.

146

Table 4.2. Analytical characteristics of the proposed methods

Parameters Method A Method B

> ax (nm)

Beer's law limit ((xg ml'')

Molar absorptivity (I mol'' cm"')

Regression equation*

s\ ±ts%

s\ ±ts\ Correlation coefficient (r)

Variance (So'')

Detection limit (ng ml'')

535

10-360

1.421 X 10

A = 3.515X

6.260X10'^

1.480X10'^

2.862 X 10"*

6.769X10-*

09999

1.048X10'*

1.091

A = 3.515 X 10' + 2.076 X 10''C A = 2.734 X 10' + 1.100 X 10' C

570

1-65

7.502 X 10

A = 2.734 X

4.587X10"^

1.085 X 10"'

1.191 X 10"

2.816X10"^

0.9999

6.408 X lO"''

0.161

'With respect to A = a+bC, where a = intercept, b=slope and C= concentration. Standard deviation of the intercept. 'Confidence interval of the intercept. ''standard deviation of the slope. 'Confidence interval of the slope.

147

<u o c ra

o U)

S3 <

100 200 -1

Concentration (fig ml )

300 400

Fig. 4.9. Calibration curve for determination of amiodarone (method A).

148

« o c ni o in n <

-1 Concentration {p.g ml )

Fig. 4.10. Calibration curve for determination of amiodarone (method B).

149

confidence level. It confirmed that intercepts for the proposed methods are not

significantly different from zero. Thus the present methods are fi-ee from

constant errors independent of the concentration of the amiodarone.

The detection limit [36] for method A and B were calculated and found

to be 1.091 and 0.161 ^g ml'', respectively. The small value of the variance

(1.048 X 10" and 6.408 x 10"'' |ig ml'' for method A and B, respectively)

confirmed the small degree of scatter of experimental data points around the

line of regression. Both the detection limit and the slope of the calibration

graphs indicated the good sensitivity.

The error (Sc) in the determination of a given concentration of

amiodarone was calculated by using the following expression [37].

1/2

b

r + _!_+ (A-A) 1

The value of Sc may be used to .establish the confidence limit at the selected

level of significance for the determination of unknown concentrations by

using the relation C, ± tSc. The results are shown in Figs. 4.11. and 4.12. by

plotting percentage uncertainty versus the concentration of amiodarone [38] at

95% confidence level. Hence the confidence limits established for both

methods (A and B) can be used to evaluate the relative uncertainty directly on

the concentration over the full range of the concentration tested.

The reproducibility of the proposed methods were checked by

estimating three different concentration levels within the Beer's law limit in

150

§

120 180 240

Concentration of amiodarone (ng ml" )

360

Fig. 4.11. Plot of the variation of the confidence limit at 95% confidence level for method A, presented as uncertainty percentage on the concentration of amiodarone.

151

o c3

Concentartion of amiodarone (jig ml )

Fig. 4.12. Plot of the variation of the confidence limit at 95% confidence level for method B, presented as uncertainty percentage on the concentration of amiodarone.

152

six replicates. Tlie analytical results obtained from this investigation are

summarized in Table 4.3. The standard deviation, relative standard deviation

and standard analytical error [39] can be considered to be very satisfactory.

The validity of the proposed methods for the determination of drug in

commercial dosage forms was tested by applying standard addition technique.

In this study a known amount of pure amiodarone was added to preanalyzed

commercial dosage forms. The recoveries were obtained in the range of 99.98

- 100.12%. The results are summarized in Table 4.4. The common excipients

present in formulations did not interfere.

The applicability of the proposed methods for the determination of

amiodarone in commercial dosage forms were examined by analyzing

marketed products such as Aldarone (Zydus Alidac), Amiodar (Cardicare),

Duron (Samarth), Eurythmic (Troikaa). The results of the proposed methods

were statistically compared with those obtained by the reference method [21]

and are summarized in Table 4.5. It is evident from the table that the

calculated t and F values [40] are less than the theoretical ones at 95%

confidence level, indicating no significant difference between the methods

compared.

153

Vi

O

VI

o o u.

.a

o

u B U u

•a B cs B O

u

<

Q 5 ^ P i e

"J > o „

Q tzi +1 B 3

,P

i E 1,^

u H ro rr 0)

s es

-a o o a •o

o Q. O

O O m C~- (N CT\ oo c<i en r~ t^ o m "/ ^" o o o o d o d d o

00 vo >n o 00 oo "sl- o ^ ro cs m '^ <N —; O O O d <i d> o S <z>

.-< i n I—• 00 r<l vo OS CS ^^ • * CS ^ -d d d --< d d

^ \o r~ o *—I O O T-*

^ o o o o o o o o o o o o o

r^ >o m o d d +1 +1 +1

t - - OS p o o d> d> ci +1 +1 +1 o

o d

m en •—' in "^ ^ (N o o o o o O Csl cN en

o o en vo

o o o •«f O {N

o o m vo

C/5

u

i T3 O U

CM O

T3 U > «

•a c cs

> u u o c o •a c

03

1

1-i

o •-?

•to

c •a c u

-o .Si >.

- . - § ?

+1 « 8 ^ < H

*U3

154

H .S

en

o

a o i >

R

o S es o

D

:« a S V

u JS • * *

O J3

B

-a La

e -2

E3

00

(J ^

Q CO -H •T3 C 3 O

O

1 O

3

£

C 3 O

E

•a <

<

Q 00

+1

oo o r- o o ^

O OS

o o d d

•o so d d +1 +1 CS CO —I O

d d o o

o o d d +1 +1 •«• (S

o o d d

H

o o CN e n

d d

o o so so

O <N

d d +1 +1

o o o o

CTs O s

d d +1 +1

o o O <N t N en

o o

2 ° o o

o o 7 c-> u. TO

• 0 -

o -n

< on

>, N

(N SO

o • * <N • * o o d d

C-~ 00

d d +1 +1 t-~ o

o o d d o o

O OS oo OS o o d d

rt 00 en m o o

o +1 o o o

so T—1

o +1 o o o o

d d +1 +1 O O

d d en so

+1 +1 CN —I o o d d en so

o o CN en

O cs oo CN en wn

d d

oo en • * o —; CN

d d

o o +1 +1 oo wn t3s O

o o CN en

m CN ^ CN

d d

O en s o O - ^ CN

O SO

c^ ^ d d +1 +1 CN i n

o o C3\ CJs

O o

s o O en m

d d +1 +1

o o O CN " CS

a\ o en >n

d d +1 +1 un m O - H d d O CN r ^ en

o o O fsl - ^ CN

o

I

c3

1) CO o

-— t-, ! = CO

X} .a

CN O t~ m o -—

00 o\ CN • * o o

J3 " CN »—<

d d +1 +1 t> oo o\ o\ OS 0\ Os o^

r » CN

o •-; d d +1 -H OS CJs a\ C3s CN >n

o o •-1 en

o o 1—c en

o o <-i en

o o >—1 en

o o CN en

O en oo ^H en ^

d d

oo o Tj- s o

o o +1 +1 oo i n C3s C3 CJs d OS O

SO Os

en en d d +1 +1 f~ >n OS »-H CTs' d CJs CN ,-• r n

o o O <N ^ CN

o o o o

o CN I

'p 'a'

hi c3£

I I

<n

I •o u u o

ea u

•o u >

•o c CO

u u

c u •a c 1 ^

. • 8 O ^

E L ' S -»

"= § u

l+i ^ g

2 tzi c)

155

u

Pi

u

-a o

1

O O

O o

o o o

OO

d

o o"

r-o o*

VO

o o

o

s o"

o o

VO

o o

r*-i

O

O

o o

O o

o o o

o o CN

0 \

o

- 1 d

<

Q S an >3

TS (U VI O

a c L< O i ID

J 3 • * —

<«-i O a o

y "C es ft B o U i/i -<t <u

H

< o

• ^ 1

'

Crt

c o _rt =3

O U.

ro ^

w <

cs ^ ^

— — o

o

T3 C

?^

o •a

u o o c

o w u o 1) O

"O i n

> tJ

•g C-T « in"

o u

,i^ I

u <

e 3

7N.

E « 2 E

o ^ c ra u >

•a

•-P 03 o^ in S~ OS 2 « II

« "73 o C3

( / ] ( M

o o CO

E CJ

> <

s •a c iS r; ttf < i^

c >

8 o r o

156

REFERENCES

[I] M. Jafari-Fesahaki, M. M. Scheinman, Pacing Clin. Electrophysiol. 21 (1998)

108.

[2] The Indian Pharmacopoeia, III edn., Vol. II, The Controller of Publications,

Government of India, Delhi, 1996, p.825.

[3] The United States Pharmacopoeia, XXIII edn., National Formulary 18, Rockville,

MD, USA, 2000, p.1218.

[4] British Pharmacopoeia, Vol. I, H. M. Stationary Office, London, 1998, p. 1481.

[5] S. J. Weir, C. T. Veda, J. Pharm. Sci. 74 (1985) 460.

[6] R. J. Flanagan, G. C. A. Storey, D.W. Holt, J. Chromatogr. 187 (1980) 391.

[7] J. A. Cervelli, J. Kerkay, K. H. Pearson, Anal- Lett. 14 (1981) 137.

[8] J. F. Brien, S. Jinmo, P. W. Armstrong, Can. J. Physiol. Pharmacol. 61 (1983)

245.

[9] R. J. Flanagan, G. C. A. Storey, D. W. Holt, Methadol. Anal. Toxicol. 3 (1985)

23.

[10] C. P. Hug, S. Costa, S. Nany, J. R. Claude, M. Hamon, Anal. Lett. 24 (1991) 603.

[II] L. Vio, M. G. Mamoto, Farmaco 43 (1988) 365.

[12] D. Yang, T. Fang, Yaowu Fenxi Zazhi 15 (1995) 35.

[13] N. R. Gupta, S. Connelly, Clin. Chem. 30 (1984) 1423.

[14] M. Geoffriau, I. Girard, S. Ferry, P. Toubout, J. Pharm. Clin. 11 (1992) 118.

[15] R. Jensen, E. Penchant, I. Cartague, A. M. Boirac, G. Rovx, Spectroscopy 6

(1988)63.

[16] Y. E. Mohanmad, M. El- Kousy Naglaa, A. M. Bahia, G. M. Nashwa, Bull. Fac.

Pharm. 36(1998) I.

157

[17] J. C. Vire, B. L. Gallo- Hermosa, P. Chatelain, G. J. Patriarche, Analusis 14

(1986) 40.

[18] R. Y. Ball, G. M. Eckert, F. Gutmann, D. K. Y. Wong, Electroanalysis 8 (1996)

66.

[19] R. I. Stefan, H. Y. Aboul- Enien, G. E. Baivlescu, Sens. Actuators 37 (1996) 141.

[20] T. S. Rao, P. S. N. H. Ramachandra Rao, U. V. Prasad, C. S. P. Sastry, Asian J.

Chem. 14 (2002) 217.

[21] T. S. Rao, C. S. P. Sastry, Indian J. Pharm. Sci. Sept-Oct. (2002) 482.

[22] M. Y. Ebeid, N. M. EI-Kousy, B. A. Mousa, N.G. Mohamed. Egypt. J. Pharm.

Sci. 39 (1998) 31.

[23] T. S. Rao, P. S. N ,H. Ramachandra Rao, A.V.S.S. Prasad, C.S.P. Sastry, Oriental

J. Chem. 17(2001)407.

[24] N. Rahman, S. N. H. Azmi, Anal. Sci. 16 (2000) 1353.

[25] N. A. Zakhari, M. Rizk, F. Ibrahim, M. I. Walsh, Talanta 33(1986) 111.

[26] M. Abdel-Hady Elsayed, S. P. Agarwal, Talanta 29 (1982) 535.

[27] E. A. Taha, S. M. Soliman, H. E. Abdellateef, M. M. Ayad, Microchim. Acta 10

(2002) 175.

[28] H. F. Askal, Talanta 44 (1997) 1749.

[29] L. I. Bebawy, N. El-Kousy, J. K. Suddik, M. Shokry, J. Pharm. Biomed. Anal. 21

(1999)133.

[30] P. Y. Khashaba, S. R. El-Shabouri, K. M. Emara, A. M. Mohamed, J. Pharm.

Biomed. Anal. 22 (2000) 363.

[31] N. Rahman, M. N. Hoda, J. Pharm. Biomed. Anal. 31 (2003) 381.

[32] M. A. Slifin, B. M. Smith, B. M. Smith, R. H. Walmsley, Spectrochim. Acta 25A

(1969) 1479.

158

[33] D. T. Sawyer, W. R. Heinemann, J. M. Beebe, in "Chemistry Experimwents for

Instrumental Metliods", John Wiley and Sons, USA, 1984, p.200.

[34] S. D. Ross, M. M. Labes, J. Am. Chem. Soc. 79 (1957) 76.

[35] B. Morelli, Analyst 113 (1988) 1077.

[36] B. Morelli, Analyst 108 (1983) 870.

[37] J. N. Miller, Analyst 116 (1991) 3.

[38] R. Cassidy, M. Janoski, LC-GC 19 (1992) 692.

[39] Vogel's Textbook of Quantitative Chemical Analysis, Sixth edition, Pearson

Education, Singapore, 2002, p. 125.

[40] G. D. Christian, Analytical Chemistry, Fourth edition, John Wiley & Sons, Inc.,

Singapore, 1994, p.35.

CHapter -5 Quantitative SpectropHotometric MetHocCsfor

determination of^erapamidKycfrocHlbncfe in (Drug formulations ZJsing (Potassium Metaperiodate and

TropaeoCin 000

159

INTRODUCTION

Verapamil hydrochloride i.e. (±)-5-[(3,4-dimethoxyphenethyl) methyl

amino]-2-(3,4-dimethoxyphenyl)-2-isopropyI valeronitrile monohydrochloride

is clinically a very useful member of the calcium channel blocker. It is used

in the treatment of supraventricular arrhythmias, angina and hypertension

[1-3].

The drug and its formulations are officially listed in BP [4], which

suggests potentiometric titration method, USP [5] recommends a gas

chromatographic method and IP [6] describes a non-aqueous titration method

for its assay in bulk and dosage forms. The different analytical methods that

are reported for its determination include high performance liquid

chromatography [7-23], high performance thin layer chromatography [24],

liquid chromatography [25,26], gas-liquid chromatography [27,28], capilllary

gas chromatography [29,30], potentiometry [31], potentiometry-

conductometry [32], stripping voltammetry [33], atomic emission

spectrometry [34], mass spectrometry [35] and UV spectrophotometry [36].

Although the above methods have adequate sensitivity to assay lower

concentrations of the drug and hence use of these methods are justified when

the concentration of drug in biological fluids is low. Moreover, these methods

are not easily manageable because of high cost technique. The dru<

concentrations (40-240 mg per tablet) are high enough in pharmaceutical

160

formulations. Therefore, the need for spectrophotometric method is fully

justified.

A review of literature revealed that few spectrophotometric methods

for the assay of verapamil hydrochloride based on charge transfer complexes

with 2,3,5,6,-tetrachloro-p-benzoquinone [37], 2,3-dichloro-5,6-dicyano p-

benzoquinone and 2,5-dichloro-3,6-dihydroxy-p-benzoquinone [38],

chromotrope 2B and chromotrope 2R azodyes [39]; extractable ion-pair

complexes with bromothymol blue, bromophenol blue, bromocresol green,

bromocresol purple and methyl orange [40], solochrome black-T, solochrome

dark blue, fast sulfone FF and solochrome cyanin R [41], erioglaucine and

indigo carmine [42], alizarin red-S [43], picric acid [44] and oxidation of drug

with chloramine T in HCl medium [45].

This chapter describes two simple, sensitive, fast and economical

spectrophotometric methods for the assay of verapamil hydrochloride in pure

and dosage forms. The first method is based on the oxidation of drug with

potassium metaperiodate in sulphuric acid medium. The second method

utilizes the formation of an ion-pair complex of the drug with tropaeolin 000

and subsequent extraction of the yellow colour into chloroform under reaction

conditions used.

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

Apparatus

A Milton Roy Spectronic 200"" spectrophotometer (USA) and an Elico

model Ll-10 pH meter were used for absorbance and pH measurements,

161

respectively. A water bath shaker (NSW 133, India) was used to control the

heating temperature for colour development.

Reagents and Standards

All chemicals and reagents used were of analytical grade or

pharmaceutical grade. All solutions were prepared in doubly distilled water.

Aqueous solutions of potassium metaperiodate (0.2%; Otto chemie, India),

tropaeolin 000 (0.05%, Fluka), and 10 M sulphuric acid (Merck, India) were

prepared.

A buffer solution of pH 4 was prepared by mixing 50 ml of 1.0 M

sodium acetate solution with 39.5 ml of 1.0 M HCl solution and diluted to 250

ml with doubly distilled water [46]. The pH of the solution was adjusted to an

appropriate value with the aid of a pH meter.

Verapamil hydrochloride was kindly gifted by Samarth Pharma Pvt.

Limited, India. The standard solutions of verapamil hydrochloride (2.5 mg

ml' and 0.4 mg ml"') were prepared in doubly distilled water.

Recommended Procedures

Method A

Into a series of boiling test tubes different concentrations of verapamil

hydrochloride (0.00 - 0.75 ml; 0.25%) solution were pipetted. To each test

tube 1.2 ml of potassium metaperiodate and 5 ml of 10 M H2SO4 were added,

mixed well and placed on a water bath maintained at 100±1°C for 18 min. The

solutions were cooled to room temperature and transferred to a 10 ml standard

volumetric flask. The contents were diluted to volume with doubly distilled

162

water. The absorbance was measured at 425 nm within the stability period of

2 hr. against the reagent blank treated similarly.

Method B

Into a series of 50 ml separating funnels different volumes of 0.04%

drug solution containing up to 300 \ig were pipetted. 5 ml of buffer solution

of pH 4 and 2 ml of tropaeolin 000 were added to each separating funnel and

mixed well. The funnels were shaken with 10 ml chloroform for 2 min for

complete extraction and then allowed to separate the two layers. The

absorbance of the chloroform layer was measured at 400 nm against a reagent

blank treated similarly. The colour is stable for at least 3 hr.

Analysis of Pharmaceutical Formulations

Twenty tablets were powdered and mixed thoroughly. An amount

equivalent to 250 mg of verapamil hydrochloride was weighed accurately. It

was stirred with chloroform and filtered through Whatmann No. 42 filter

paper. The residue was washed well with chloroform for complete recovery of

the drug. The chloroform was evaporated to dryness and the left drug was

dissolved in doubly distilled water and diluted to 100 ml with doubly distilled

water. It was further diluted according to the need and then analyzed

following the recommended procedures.

163

RESULTS AND DISCUSSION

Potassium metaperiodate is a well known oxidant and has been used for

the oxidation of p-blockers [47-48] containing amino group. The oxidation

products were arylaldhyde or aryloxyacetaldehyde, and secondary or tertiary

butylamine [47]. In all such studies, the presence of a methyl or methylene

group attached to nitrogen is the basic need [49] so that >N—CH2 linkage in

tertiary amines was cleaved preferentially by an oxidant (potassium

metaperiodate or chloramine T or N-bromosuccinimide) resulting in the

formation of yellow coloured product. The oxidation of verapamil

hydrochloride with potassium metaperiodate was carried out in aqueous

sulphuric acid medium as it fulfills the basic need of having tertiary amine

with >N-CH2 linkage. Therefore, >N-CH2 linkage was cleaved preferentially

resulting in the formation of aryloxyacetaldehyde and secondary amine. The

oxidation product absorbs maximally at 425 nm (Fig. 5.1.). In order to

establish stoichiometric ratio between verapamil hydrochloride and potassium

metaperiodate, the method of continuous variations [50] has been applied and

was found to be 1:1 (Fig. 5.2.). On isolation of the two major products, one

produces a blue colour when treated with a benzene solution of chloranil

confirming the presence of amino group and other product reacts with 2,4,-

dinitrophenyl hydrazine to give yellow coloured product indicating the

presence of aryloxy benzene acetaldehyde. It has been confirmed that iodine

was not evolved during this reaction.

164

0.50

400 450

Wavelength (nm)

550

Fig. 5.1. Absorption spectrum of the reaction product of Verapamil hydrochloride with potassium metaperiodate.

165

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Mole fraction of drug (XQ)

Fig. 5.2. Job's plot for the reaction of verapamil hydrochloride with potassium metaperiodate (each 5. 09 x 10" M).

166

Therefore, based on the literature background and our experhiiental

findings, the reaction mechanism was proposed and given in Scheme 5.1.

Verapamil forms coloured chloroform extractable ion-pair complex in

acidic buffer with tropaeolin 000. The absorption spectrum of the ion-pair

complex, which absorbed maximally at 400 nm, is shown in Fig. 5.3. The

reagent blank under similar conditions showed no absorption. The molar ratio

between verapamil and tropaeolin 000 is established by Job's method [51].

This measurement (Fig. 5.4.) showed that 1:1 complex was formed between

drug and tropaeolin 000.

Verapamil hydrochloride contains tertiary amino group which is

protonated in acid buffer, while sulphonic acid group in tropaeolin 000 is the

only group undergoing dissociation in the pH range of 0.5 - 6.9. Finally the

protonated verapamil hydrochloride forms ion-pair with tropaeolin 000, which

is quantitatively extracted into chloroform. The possible reaction mechanism

is proposed and given in Scheme 5.2.

Optimization of Variables

The optimum conditions for the assay of verapamil hydrochloride were

established via a number of preliminary experiments.

Method A

Effect of heating time

To investigate the optimum heating time for color development, 0.75

ml of 0.25% verapamil hydrochloride was mixed with 5 ml of 10 M

167

CHj H3Q ,CH3

'CH

CH2—CH2-N-CH2—CH2-CH2—C—CN

H2SO4 KIO4

OCH3

OCH3

O CH

C H 3 O — / \ - C H 2 - C - H + C H 3 - N - C H 2 - C H 2 - C H 2 — C - C N

H3CO

OCH3

OCH3

3,4-dimethoxy benzene acetaldehyde

5 (N-methyl amino) 2-(3,4-dimethoxy phenyl)-2 isopropyl valeronitile

Scheme 5.1

168

300 350 400 450

Wavelength (nm)

500 550

Fig. 5.3. Absorption spectrum of the reaction product of Verapamil hydrochloride with tropaeolin 000.

169

Mole fraction of drug (XQ)

Fig. 5.4. Job's plot for the reaction of verapamil liydrochloride with Tropaeolin 000 (each 1.02 x 10'^ M).

170

CHj

HjCO'

0 - / / ^

CH3 H3C ^CHj

CH

CH2—CH2—N- CH2—CH2-CH2— C—CN

protonated verapamil "V^ ^OCHs

OCH3

'O3S—C \ - N = N OH

tropaeolin 000

O - / ^ CH3

HjCO'

H3C ^CH3 CH3 CH I I

CH2— CH2—N— CH2—CH2— CH2—C—CN H

O3S—f V-N=N OH

Verapamil -tropaeolin 000 complex

Scheme 5.2

171

H2SO4 and 1.2 ml of 0.2% potassium metaperiodate into a boiling test tube.

The content of the mixture was heated for 20 min. on a water bath at

100±1°C. The intensity of the color (Fig. 5.5.) was reached to maximum after

17 min of heating and remained constant and therefore 20 min of heating time

was used throughout the experiment.

Effect of potassium metaperiodate

To 0.75 ml of 0.25% verapamil hydrochloride, different volumes (0.5 -

1.5 ml) of 0.2% potassium metaperiodate and 5 ml of 10 M H2SO4 were

added. The reaction mixtures were heated for 20 min on a water bath at

100+1°C. The reaction mixture was diluted to 10 ml with doubly distilled

water and the absorbance was measured against a reagent blank at 425 nm.

The results (Fig. 5.6) showed that the highest absorbance was obtained with

1.0 ml, which remained unchanged with higher amount of potassium

metaperiodate. Thus, 1.5 ml of the reagent was added for colour

measurements.

Effect of the concentration of sulphuric acid

The influence of the yolume of 10 M H2SO4 on the formation of yellow

colour was. studied. This was performed by pipetting 0.75 mi of verapamil

hydrochloride (0.25%), 1.5 ml of potassium metaperiodate and different

volumes (1-5 ml) of 10 M H2SO4. It is evident from Fig. 5.7. that the

maximum absorbance was obtained with 4.7 ml of 10 M H2SO4 above this

172

0) o c

o V)

Si

<

Time (min)

Fig. 5.5. Effect of Heating time.

173

0.7

0.6 -I

0.5

• - • - • •

-r

2

Volume (ml)

Fig. 5.6. Effect of the volume of 0.2% potassium metaperiodate.

174

0) u c (0

J3

o m n

Volume (ml)

Fig. 5.7. Effect of the volume of 10 mol I" H2SO4.

175

volume, the absorbance remains constant. Therefore, 5.0 ml of 10 M H2SO4

was used for all the measurements.

Method B

Effect of the concentration of tropaeolin 000

The effect of the volume of 0.05% tropaeolin 000 was studied in a

series of 50-ml separating funnels containing 30 |J.g ml"' of verapamil

hydrochloride, 5.0 ml of buffer solution of pH 4 and different volumes of the

reagent. 10 ml of chloroform was used to extract the ion-pair complex. It is

apparent from Fig. 5.8. that the maximum absorbance was found with 1.9 ml

of tropaeolin 000, beyond which absorbance was constant. Thus, 2.5 ml of

tropaeolin 000 was used for ion-pair formation throughout the experiment.

Effect of the pH

The influence of pH of the buffer solution of sodium acetate HCl on

the formation of extractable ion-pair complex of the drug with tropaeolin 000

was studied. The results are shown in Fig. 5.9. It is evident form the figure

that the absorbance of the complex with tropaeolin 000 was found to be

constant within the pH range 3.5 - 4.5. Thus, all the absorbance measurements

were made at pH 4.

Choice of organic solvent and time of shaking

A variety of organic solvents such as benzene, toluene, hexane,

chloroform, carbon tetrachloride, ethyl acetate and 1,2-dichloromethane were

176

\J.I -

0.6 -

0.5-

g 0.4 -c cs .o o (A

JO

< 0.3 -

0.2-

0.1 -

0.0 i

/

/

^ V

Volume (ml)

Fig. 5.8. Effect of the volume of 0.05% tropaeolin 000.

178

examined for an applicable extraction procedure, but chloroform was

preferred for its selective extraction of the ion-pair complex from the aqueous

solution.

The time of shaking for complete extraction of ion-pair complex was

studied and found that the absorbance of the extract remains constant between

0.5 - 4.0 min. Thus, 2.0 min shaking time was utilized as an optimum value

throughout the experiment. The ion-pair complex was quantitatively

recovered in one extraction only.

Interference studies

The effect of excipients and additives commonly used in drug

formulations, such as starch, lactose, talc, stearic acid, magnesium stearate,

titanium dioxide, tartrazine and lactic acid was evaluated using the proposed

methods. It was found that the common excipients and additives did not

interfere with the analysis.

Analytical data

Under the optimized experimental conditions, linear calibration graphs

(Figs. 5.10. and 5.11.) were obtained over the concentration ranges 0.00 -

187.5 and 2-30 ]xg ml"' of verapamil hydrochloride with molar absorptivities

of 1.18 X 10^ and 9.82 x 10^ 1 mof'cm"' using method A and B, respectively.

Table 5.1. summarizes the analytical parameters such as Beer's law limit,

179

o u c ns Si o (0 £1 <

.1 Concentration (ng ml )

Fig. 5.10. Calibration curve for determination of verapamil hydrochloride (method A).

180

Concentration (ngml )

Fig. 5.11. Calibration curve for determination of verapamil hydrochloride (method B).

181

Vi

o

-a m tn O O i o u a (U

• * - !

O

o

u «M

•4-> w < C3

< t 3 O

J3

S O.

u

u

O

0)

H

O cs

I

O

O

o

+

o o •*

ro O

O en <N

o

X cs 0 0

O N

'"' X oo cs o »—t

II <

1

X »—( T-H

rn cs

m

O

X m VO

^ i r i

•0-

O

X VO m cs »-H

• ^

o

X

^ cs ON

cs

oo C7\ C:N C3N

o

VO 0 \ rn O

»o

o

X • * 00 cs •—1

cs "51-

cs o

0 0

o o

X 0 0

u en

I

o o

cs

+ •"J-

I

to X

as o

- <

0 0

o\

o 'x

cs cs cs CJs o

0\ ON O N ON

CS

X

o o

S

a,

^^ -s^

a

E

/'*~»S

j : ;

>->

IS 4-» C/3

1

"s ao i — • 4 - *

1 •—< ^ i2

^ "o

a V — ^

>-. •4-»

|> +-» CL,

>-< o en

(33

O

S

C o

-4-»

3 cr u c o t n

(« U Uc

M 1) 1-1

u . IJ3

u C "•J

CZ! 00 C/2 +1 00 +1

•1-H O

5f3 It-c u o o c o

- I -* cd

"o U i L^ o U

S M i

s.—^ -*-» S

• c ^

C o • •-« •h; o (U +-» o Q

o 00

U

o c K)

'u< nS

>

u c o

u u

u. u ^ o o c:

o.

Si • ex i^ u o

o,—. 1 . S

„ o

^ o II a < .2 _ •*-»

* j " >

o u

CO T3 •J h

O " U

J3 O w _

(,_ J3 t4_

5 c « £: .2 & 7i T^ ni

o " u o ^ u •= -a S O I- «J

- o ca T3

C "5 C ^ 00 U <^ <^ ••-J t-J

182

calibration equations obtained by least square treatment of calibration data,

coefficient of correlation, variance and confidence limit for slope and

intercept. The correlation coefficient was found to be 0.9999 and 0.9998 for

method A and B, respectively, indicating the good linearity of both the

calibration graphs and the intercepts are all close to zero. Test of significance

of the intercept of the regression line showed that the experimental intercept,

a, does not differ significantly from the theoretical value, zero. For this, a

simplified method was used to calculate the quantity from the relation t = a/Sa

[52] and its comparison with the tabulated data from the t-distribution. The t-

value for method A and B are 0.200 and 0.190, respectively which do not

exceed the tabulated t-value (t = 2.365, when v = 7) at 95% confidence level.

It confirmed that intercepts for the proposed methods are not significantly

different from zero. Thus the present methods are free from constant errors

independent of the concentration of the verapamil hydrochloride.

The detection limit [53] and variance were found to be 0.772 |a,g ml"',

7.01 X 10"' |ig ml"' and 0.396 ^g ml"', 1.28 x 10" i g ml"' for method A and B,

respectively. The small value of variance obtained for both the methods

pointed towards negligible scattering of experimental data points around the

line of best fit.

The statistical analysis of the calibration data has been used to

calculate error (Sc) in the determination of a given concentration of verapamil

hydrochloride and also provides a way to establish the confidence limits at the

selected levels of confidence to estimate the relative uncertainty of the

183

concentration over the full range of the concentration tested. These

parameters were established by using the relation [54],

^'~ b 1 1 {A-Af 1 + - + - ^

» b'Yic-cf

1/2

Where A and C are average values of the absorbance and concentration,

respectively, for n standard samples. Figs. 5.12. and 5.13. shows the graphs of

Sc versus concentration of verapamil hydrochloride. The minimum error is

obtained on the determination of about 100 and 16 [ig ml' of verapamil

hydrochloride for method A and method B, respectively.

The accuracy and precision of the proposed methods were evaluated by

the replicate analyses at the three different concentration levels. The results

are summarized in Table 5.2. The relative standard deviation and standard

analytical error were found to be 0.130-1.038% and 0.018-0.102 fag mV\

respectively. The results, therefore, indicated that the precision and accuracy

of the proposed methods were satisfactory.

To check the validity of the proposed methods using standard addition

technique, a fixed amount of pure drug was added to the preanalysed drug

formulations. The results are summarized in Table 5.3. The recovery results

were quite satisfactory.

The applicability of the proposed methods were compared with the

reference method [43] by analyzing authentic commercial products of

verapamil hydrochloride such as Calaptin 80 so (Nicholas Piramal), Verap 80

184

0.42

200 .1

Concentration of verapamil hydrochloride (pg ml )

Fig. 5.12. Error in the determination of verapamil hydrochloride using statistical analysis of standard calibration data with potassium metaperiodate.

185

0.210

0.205

0.200

I

1^

A

1^

6 "1 i i 1 i I i I I I r

10 12 14 16 18 20 22 24 26 28 30 32

Concentration of verapamil hydrochloride ( g ml )

Fig. 5.13. Error in the determination of verapamil Iiydrocliloride using statistical analysis of standard calibration data with tropaeolin 000.

185

u

UJ

0.210

0.205 -

0.200

0.195

0.190

0.185 T i 1 1 1 1 1 1 1 T

12 14 16 18 20 22 24 26 28 30 32

Concentration of verapamil hydrochloride (^g ml" )

Fig. 5.13. Error in the determination of verapamil liydrochloride using statistical analysis of standard calibration data with tropaeolin 000.

186

tn TS O

JS

s

o a, o a, (U

o

u

t 3 ' B c«

u « u a u u cs

x: ••-»

o e _o C! S

rj

u o c u "a

o S

on

CO s=>

0 S^

>

00

o"

00 CN O*

00 CN o

• a ^-s

cS E •1-. bO

< +1

c

g a

CN O

0\

o

00 cs CN

0\ (N

o o

o o

CN O

o

o CD

o

00 cs CN

O

+1 CN O

o

00 CN CN

O +1 CN CN

»—( CN —c ;_<

00 CN CN CD +1 CN CN

i n

o o

<N - I ~ ,

CO ~ O O

a, E

T3 O

CD

o

00

o o oo o r-H

CN in

o CJ

o o 1-H

CN

o

o o o

CN O

o

CN

o o*

o ' — ' ' n o ^ = d

ON O C7N c

o d +1 00 ON

CN

o d +1 CN ON CJN

<n' CO *—(

>n o d +1 o o 0<> CN w

6 o •o u

u

U •o u

•a c

>

o

' O oo CN

•a o

c c O 0)

"= 2 c I- O u o

I S •« Ss u . " ''I

•o t ; ^ o ~ w > M CB

i' o •o .5

Q "a ? CO C "

,°? w c

187

188

(Mano Pharma) and VPL Amp. 05/2 ml (Samarth Pharma); the results are

summarized in Table 5.4. The results of the proposed methods were statistically

compared with those obtained by the reference method. It is evident from Table

5.4. that the calculated t- and F-values are less than the theoretical ones [55] at

the 95% confidence level, which indicates no significant difference between the

methods compared.

-a o

u

o .a

m O a. o

o

o a o CO

C3

a, S o U

U IQ ca

H

•ir; ' ^ - ^ ^ (i Si

o C u ; - (

U-i

u ILf (LI

> I

189

>

(L) s ° Pi e^

tL l

o

OS

00 ro

c o

^

3 o C3

00 s?

>

Pi e-

o o

oo o

OS

O ON

OO

o o

w +-•

O OO

_c • * - »

ex, J3

•S a, tn rt

•—< o .-s

'^ ro IT)

O OS >o

(N IT)

o o o 1—(

rf OS OS OS

r—<

0 0

rn

in ON o

m m •*

ro O r<-i

CN

m f~i

o o *"•

r-OS OS OS

0 0 OS 0 0

,—* o vo

CN

o o

oo P-i

o OS

o

•o

o o

H u E :^^

(N fa

O J=i

1 ) •—) C

OS en

B oj

so o

u

>

u o c u •a c o o

0\

• * "

II > u

g -a = J!, g " D. >

M O

190

REFERENCES

[I] • D. N. Franz Cardiovascular drugs. In: A. R., Gennaro ed. Remington: Tlie

Science and Practice of Pharmacy, 19tli Edition, Vol. II, Mack Publishing

Company, Pennsylvania 1995, p.966.

[2] Martindale The Extra Pharmacopoeia, 31 st Edition, Royal Pharmaceutical

Society, London, 1996, p.961.

[3] M. Fisher, J. Grotta, Drugs 46 (1993) 961.

[4] British Pharmacopoeia, Vol. I, H. M. Stationery Office, London, 1998,

p.1353.

[5] The United States Pharmacopoeia, XXIII Edition, National Formulary 18,

The United States Pharmacopoeial Convention, Rockville, MD, USA,

1995, p.1623.

[6] The Indian Pharmacopoeia, III Edition, Vol. II, The controller of

publications, Government of India, Delhi, 1996, 796.

[7] K. Kasturi, D. S. Rao, (Mrs) R. Sundaram, Indian Drugs 21 (1984) 463.

[8] I. Jane, A. McKinnon, R. J. Flanagan, J. Chromatogr. 323 (1985) 191.

[9] E. M. Niazy, H. W. Jun, Anal. Lett. 18 (1985) 1103.

[10]' V. D. Gupta, Ind. Pharm. 11 (1985) 1497.

[II] D. C. Tsilifonis, K. Wilk, R. Reisch Jr, R. E. Daly, J. Liq. Chromatogr.8

(1985)499.

[12] Y. Oda, N. Asakawa, T. Kajima, Y. Yoshida, T. Sato, J. Chromatogr. 541

(1991)411.

191

[13] M. N. Muscara, G. de-Nucci, J. Med. Biol. Res. 26 (1993) 753.

[14] D. R. Rutledge, A. H. Abadi, L. M. Lopez, J. Chromatogr. Sci. 32 (1994)

153.

[15] B. Grijdanus, R. Herder, H. M. Pieterse, D. R. A. Uges,Ziekenhuis farmaie

10(1994)67.

[16] D. Romanova, E. Brandsteterova, B. Kralikova, L. Bozekova, M. Kriska,

Pharmazie49(1994)779.

[17] P. Hubert, J. Crommen, J. Liq. Chromatogr. 17 (1994) 2147.

[18] G. Stagni, W. R. Gillespie, J. Chromatogr. B: Biomed. Appl. 667 (1995)

349.

[19] L. Valvo, R. Alimenti, S. Alimonti, S. Raimondi, F. Foglietta, F. Campana,

J. Pharm. Biomed. Anal. 15 (1997) 989.

[20] M. A. Garcia, J. J, Aramayona, M. A. Bregante, L. J. Fraile, C. Solans, J.

Chromatogr. B: Biomed. Appl. 693 (1997) 377.

[21] Y. Ozkan, N. Yilmaz, S. A. Ozkan, I. Biryol, Farmaco 55 (2000) 376.

[22] W. Wang, Yaowu Fenxi Zazhi 20 (2000) 113.

[23] Y. Yazan, B. Bozan, Pharmazie 50 (1995) 117.

[24] M. F. A. El-Ghany, A. A. Moustafa, B. E. El-Zeany, J. T. Stewart, J. Planar

Chromatogr. Mod. TLC 9 (1996) 388.

[25] S. M. Lankford, S. A. Bai, J. Chromatogr. B: Biomed. Appl. 663 (1995) 91.

[26] A. Gumieniczek, A. Hopkala, J, Liq. Chromatogr. Relat. Technol 24 (2001)

393.

[27] H. G. Hege, Arzneim.-Forsch. 29 (1979) 1681.

[28] U. A. Shukla, P. L. Stetson, W. D. Enminger, J. Chromatogr. 342 (1985)

406.

192

[29] D. J. Hoffmann, J. Higgins, J. Chromatogr. 374 (1986) 170.

[30] M. T. Rosseel, F. M. Belpaire, J. High Resolut. Chromatogr. Chromatogr.

Commun. 11 (1988) 103.

[31] L. Cunningham, H. Preiser, Anal. Chim. Acta 157 (1984) 157.

[32] K. Nikolic, M. Medenica, Pharmazie 44 (1989) 497.

[33] E. A. Kasim, M. A. Ghandour, M. T. El-Haty, M. M. Ahmed, J. Pharm.

Biomed. Anal. 30 (2002) 921.

[34] S. Khalil, A. Kelzieh, J. Pharm. Biomed. Anal. 27 (2002) 123.

[35] G. Remberg, M. Ende, M. Eichelbaum, M. Schomerus, Arzneim.-Forsch.

30(1980)398.

[36] Y. Sun, M. Liu, Yaowu Fenxi Zazhi 4 (1984) 45.

[37] Y. Long, J. Feng, S. Tong, Zhongguo Yiyao Gongye Zashi 24 (1993) 267.

[38] M. A. El-Sayed, M. Barary, M. Abdel-Salam, S. Mohamed, Anal Lett. 22

(1989) 1665.

[39] Y. M. Issa, A. A. Salem, M. S. Bahbouh, Anal. Lett. 30 (1997) 1153.

[40] R. T. Sane, V. B. Malkar, V. G. Nayak, A. D. Nadkarni, Indian Drugs 20

(1982) 78.

[41] R. T. Sane, M. V. Kubal, V. G. Nayak, A. D. Kulkarni, Indian J. Pharm.

Sci. 45 (1983) 88.

[42] R. T. Sane, M. L. Kubal, V. G. Nayak, V. B. Malkar, V. J. Banawalikar,

Indian Drugs 22 (1984) 25.

[43] A. H. Prabhakar, R. Giridhar, V. B. Patel, Indian Drugs 36 (1999) 372.

[44] J. Emmanuel, R. Mathew, East. Pharm. 22 (1984) 133.

[45] N. Rahman, M. N. Hoda, Anal. Bioanal. Chem. 374 (2002) 484.

193

[46] H. T. S. Britton, Solution of known hydrogen-ion concentartion. In:

hydrogen ions. Tripp E H, ed. Vol. I, Chapman and Hall, London, 1942,

p.301.

[47] H. Salomies, J. Halmekoski, Acta Pharm. Fenn. 90 (1981) 243.

[48] H. Salomies, L. Luukkanen, R. J. Knuutila, J. Pharm. Biomed. Anal. 7

(1989); 1447.

[49] S. Dunstan, H. B. Henbest, J. Chem. Soc. (1957) 4905.

[50] W. Likussar, D. F. Boltz, Anal. Chem. 43 (1971) 1265.

[51] W. C. Vosburgh, G. R. Cooper, J. Am. Chem. Soc. 63 (1941) 437.

[52] V. V. Nalimov, The application of mathematical statistics to chemical

anlaysis, Pergmon Press, Oxford, 1963, p. 167.

[53] B. Morelli, Anlayst 108 (1983) 870.

[54] • J. Mendham, R. C. Denney, J. D. Barnes, M. Thomas, Statistics:

, Introduction to chemometrics. In: vogel's textbook of quantitative

chemical analysis. Sixth Edition, Pearson Education, Singapore, 2002,

p.l37.

[55] G. D. Christian, Data handling. In: Analytical Chemistry, Fifth Edition,

John Wiley & Sons, Inc., Singapore, 2001, p.35.

ORIGINAL ARTICLES

Analytical Chemistty Division, Department of Chemisti-y, Aligarh Muslim University, Aligarh (UP.) India

Kinetic spectropliotometric method for tlie determination of silymarin in pliarmaceutical formulations using potassium permanganate as oxidant

N. RAHMAN, N. A. KHAN, S. N. H. AZMI

Received July 02, 2003, accepted My 22, 2003

Dr. Nafisur Rahman, Analytical Chemistry Division, Department of Chemistry, Aligarh Muslim University, Aligarh - 202002(U.P.) India chtl 7nr@yahoo. co. in

Pharmazie 59: I l l - I l l (2004)

Ug^-

A new simple and sensitive kinetic spectrophotometric method for the detemiinatlon of silymarin in pure form and in pharmaceutical fomnulations Is described. The method Is based on the oxidation of the drug with potassium permanganate at pH 7.0 db 0.2. The reaction is followed spectrophotometri-cally by measuring the decrease in the absorbance at 530 nm. The calibration graph Is linear in the 7ange of 1 8 - 5 ( ^ ml"''. The method has been successfully applied to the determination of silymarin In pharmaceutical formulations. Statistical comparison of the results with the reference method shows excellent agreement and Indicates no significant difference in accuracy and precision.

1. Introduction

Silymarin 3,5,7-trihydroxy-2-[3-(4-hydroxy-3-niethoxy phenyI)-2-(hydroxy-methyI)-l,4-benzodioxan-6-yl]-4-chro-manone, is an important antihepatotoxic drug whicli is ob­tained from tlie dried fruits of Silybum marianum and widely used for tlic treatment of hepatic disorders (Cava-lieri 1974; Salmi and Sama 1982; Ferenci etaL 1989; Hruby 1983). The drug is officially listed in Martindale The Extra Pharmacopoeia (Martindale 1996). The assay procedure listed in the monograph of the Italian pharmacopoeia describes the UV-\^s spectrophotometric method (Farmacopoeia 1985) for deteimination of the drug . in bulk and pharmaceutical formulations. Several other methods have been employed to determine silymarin in bio­logical specimens and/or pharmaceutical formulations such as high performance liquid chromatography (Martinelli etal. 1991; Marcher and Kikuta 1993; Rickling etal. 1995; Tittel and Wagner 1977; Tittel and Wagner 1978; Quercia etal. 1980; Quercia etal. 1983; Steinigen 1983; Law and Das 1987; Tomov etal. 1985; Pashankov 1988; Nikolov 1990; Kurkin 1996; Wang et al. 1998), thin layer chromato­graphy (Lin 1982; Abdel-Salam 1982; Vanhaelen and Van-haelen-Fastre 1983;Heinz etal. 1985; Wu and Wang 1989; Lin etal. 1998; Szilarjtes-Obeiglo and Kowalesioska 1984), high performance thin layer chromatography (Funk et al. 1987), potentiomeuic tiu-ation (Koerbl et al. 1983), diffuse-reflectance fourier transform infraied specU'oscopy (Marti­nez et al. 1991) and UV spectrophotometry (Lan et al- 1983^ Belikov 1985). In the literature only few spectrophotometric methods have been reported. The drug content in pharmaceutical formulations was detennined (Zarapkar et al. 2000), based on the formation of a coloured complex of the drug with 2,4-dinitrophenyl hydrazine, in the presence of tetramethyl amincjJiydroxid-\Qualitativc' detemiinations aie done at 490 nm. Another'spectrophotometric method for the csti-

mation of silymarii^based on its reaction with diazotized sulfanilic acid in alkaline medium to form an orange-red coloured chromogen which absorbed maximally at 460 nm (Rajasekaran 1997). Reddy etal. (2001) have reported two spectrophotometric methods for silymarin determination. The first method was based on the oxidation of the drug by Fe(III) and estimating the reduced Fe(II) with 1,10-phenanthroline at 510 nm. The other method involved the formation of a blue complex on treatment with Folin-Cio-calteau reagent in the presence of NaOH and subsequent determination was done at 740 nm. These methods are somewhat tedious and time consuming. Therefore, we develop^ed a fast, low cost and selective method for rou­tine analysis of silymarin in bulk and drug formulations. The extent of oxidation of organic substrate by potassium permanganate depends on the pH of the rnedium. TTie heptavalent manganese changes to Mri(lV).)^n~"aIkaline medium while in neutral and acidic mediurn^the permaii-ganate is further reduced forming ultimately Mn(II). This behaviour of permanganate has been exploited to develop a kinetic spectrophotometric method for assay of nifedi­pine in drug formulations (Rahman and Azmi 1999). The present communication describes a method for the as­say of silymarin in bulk and pharmaceutical formulations. The method is based on the oxidation of silymarin by potas­sium permanganate at pH 7.0 ± 0.2 and the course of the reaction was followed by measuring a decrease in absor­bance at 530 nm.

2. Investigations, results and discussion

The oxidation of organic compounds by permanganate is dependent upon the pH of the medium. During the course of the reaction the valence of manganese changes and in-tennediate ions have been suggested as participating oxi­dants. But which species have the main role as potential oxidants depends on the nature of the substrate and pH of

•' • o'^ . • C- r/.-'/i

Pharmazie 59 (2004) Ut fi' t

ORIGINAL ARTICLES

1.1-1

1.0-

0.9-

0.8-

0.7-CD

H 0.6-

•e 0.5-o | 0 . 4 -

0.3-

0.2-

0.1-

' 1 1 1 • 1

3 4 8 12

Time(min)

1 16

• a • b

- — - • c

• d

1 20

Rg. 1: Absorbance-time curves: KMn04, 4.8 x 10 *M and silymarin (a) 3.731 X 10-5 M; (j,) 4.146 x 10' 'M; (c) 6.219x10-'; (d) 8.292 x 10-5 M; (e) 10.365 x 10"'M

the medium. The oxidation of silymarin by potassium per­manganate at pH 7.0 ± 0.2 was followed spectrophotome-trically at 530 nm. The absorbance-time curves are shown in Figs. 1 and 2, which are all sigmoid in nature through­out the entire range and therefore, the initial rates of reac­tion were determined from the slope of the initial tangent to the absorbance-time curves. The order with respect to permanganate was determined by studying the reaction at different initial concentrations of permanganate with fixed silymarin concentration. Keeping the silymarin concentra­tion higher than that of the oxidant, the plot of initial rate [-d[A]/dt] against initial absori)ance was linear passing through the origin suggested that the order of reaction with respect to permanganate at the start is one. The order with respect to silymarin was evaluated from the measure­ment of the rates at several concentrations of silymarin but at fixed concentration of KMn04 which was also found to be one. The first order dependence on both permanganate and silymarin in the initial stages leads to the simple rate expression:

Rate = K[silymarin] [MnO^] (1)

Silymarin is a naturally occurring aryl chromanone(l) re­lated to flavanone. The flavanones are 2,3-dihydro deriva­tives of flavones, which are readily interconvertible. Though the basic skeleton of flavanone and flavone is the same, the key which distinguishes one structural type from another is the oxidation level of the various carbons in the heterocyclic ring. The flavones are in a high state of oxidation whereas tlavanones are in low state of oxida-

Scheme 1

0.7-1

I

0.6-

0.5-

8 0.4-c

•e 0.3-o

^ 0.2 J

0.1-

0.0-

\

\

\ ^ \ \ , ^ • • N^X"^" • •

^ v ^ ^ " " - • • " • •

' 1 • ' 1 ' 1 •

D 4 8 12

Time (min)

— • — — • — — • — — • —

16

^a • b • c ed

1 20

Fig. 2: Absoibance-lime curves: silymarin, 10. 365 x 10 ' M and KMn04 (a) 3.00 x 10-^ M; (b) 2.10 x 10"^ M; (c) 1.50 x 10"^ M; (d) 9.00 X 10-5 M

tion. The oxidation of flavanone to flavone with a variety of reagents such as selenium dioxide (Sammel 1979) or triphenyl methyl carbonium ion (Schonberg and Schutz 1960) has been reported. In this study, the same mechan­ism seems to be involved. The oxidation of silymarin by permanganate takes place at pH 7.0 ± 02 leading to the formation of chromone(2). Thus, the dehydrogenation is effected by permanganate and consequently disproportio-nation between inmiediate oxidation states of manganese results in the formation of MnOa. However no precipita­tion of Mn02 was observed under study time period. The reaction mechanism is presented in the Scheme. Under the optimum experimental conditions, a caUbration curve was constructed by plotting logarithm of initial rate of reaction (log v) versus logarithm of silymarin concen­tration (log C), which showed a linear relationship over silymarin concentration range of 18-50 ng • ml"'. TTie Un-ear regression analysis using the method of least square was made to evaluate the slope, intercept and correlation coefficienL The linear regression equation and correlation coefficient are log v = 1.024 log C +3.235 and r = 0.9997 which indicates an excellent linearity. The con­fidence Umits for the slope of the line of regression and intercept were computed using the relation b ± tSb and a ± tSa (Miller 1991), at 95% confidence level and found to be 1.024 ± 3.96 x 10"^ and 3.235 ± 1.67 x 10"', re­spectively. This indicated the high reproducibility of the kinetic method proposed. Linearity was also evaluated (Torrado etal. 1994) from the relative standard deviation of the slope of the caUbration line and found to be 1.215.

OH 0

,0. ^CHjOH + Mn04

OH O OH I

+ HO-Mn—0

Chromiinone (1) Chronione (2)

Phannazie 59 (2004) I I I

2032088 Die I'lianua/n. All Ni P3105/2 (DISK) (p_21p/v/p/i04J)2/8(:i7S/ii/i867Sii 3d SIGNA Bc.uli Kcml

ORIGINAL ARTICLES / ' / ' , I, /,'L

1.30

128-

126-

124-

O 122-

>< 1.20-

r 118

HI 1.16-

1.14-

112

1.10

Table 1: Precision test of the proposed kinetic method

10 —r—

20 —T— 30 40 60

Concentration of silymarin (PQ -mV)

Rg. 3: Eiror in the detenninalion of the concentration of silymarin using statistical analysis of standard calibration data

The limit of detection was established using the equation (Morelli 1983, NaHmov 1963):

Limit of detection (.2 r^-lV" 7

(2)

where n is the number of standard samples, t is the value of student's t for n—2 degrees of freedom at 95% confi­dence level and Si = variance. The variance and detection limit were calculated to be 1.100 x 10~* ng • ml~* and 2.823 X 10"^ [ig • ml"' respectively, which confinned neg­ligible scattering of experimental data points around the line of regression and good sensitivity of the method. The error (Sc) in the determination of a given concentra­tion of silymarin (C) was established using the equation (MorelU 1987):

Sc .So • b n b2(2 C2 - nC')

1/2

(3)

Where y and C are average ordinate and abscissa values, respectively, for n standard samples. Fig. 3 shows the

4S-,

4 0

3 5 '

S 3 0 -

o as ^

o <

15-

lo­

os-10

— I — 20 30

— 1 — 40

— I 50

Concentration of silymann (ng -mr^)

Fig 4 Variaiion of the confidence limit ai (a) p = 0 05 and (b) p = 0 01 level of significance in ihe form of uncertainly percentage on the conccniralion of iilymann

Piuuma/.ic 59 (2004) III

Sample No Imiial rate of reaciion (mol t"' mm"')

1

2

3

4

5

6

7

8

9

10

11 4x 10-2

11.5 X 10-^

11.3 X 10-2

n.3 X 10-2

11.5 X 10-2

11.5 X 10-2

11.2 X 10-2

11.4 X 10-2

11.3 X 10-2

11.2X 10-2

402

404

400

399

406

405

396

401

398

397

Mean percent recovery = 100% Relative standard dcviauon = 0 8% ' 2 ^ ;< lo

graph of Sc versus the concentration of silymarin. It is apparent from the graph that the error is reached mini­mum when the actual initial rate was equal to the average initial rate in the calibration, graph. The minimum error of^^^^310^|ig • m r ' was found in the determination of abouTSO ng • ml~^ silymarin. The va­lue of Sc also allows establishing the confidence limit at the selected level of significance for the determination of unknown concentrations by using the relation, C, ± tSc. The results are shown graphically (Fig. 4) in the form of

percent uncertainty ih^ on the concentration at

95% and 99% confidence levels. This is a useful way of expressing the confidence limits because the relative un­certainty can be estimated directly on the concentration over the full range of the concentration tested. The reproducibility was established for ten independent analyses of solution containing 0.40 mg silymarin. The ana­lytical results obtained fi-om the investigation are summar­ized in Table 1. The relative standard deviation and mean percent recovery were 0.87% and 100.3%, respectively and considered to be very satisfactory. The validity of the pro­posed kinetic method was checked by applying standard addition method and the results are reported in Table 2. The applicability of the proposed method for the determi­nation of silymarin in dosage forms was examined by ana­lyzing marketed products; the results were compared to those obtained by the reference method (Rajasekaran et al. 1997) and are summarized in Table 3. The performance of the proposed method was judged by calculating t- and F-values (Christian 1986). At 95% confidence level judged, the calculated t- and F-values do not exceed the theoreti­cal values indicating no significant difference between the proposed kinetic method and the reference method.

Table 2: Determination of silymarin in pharmaceutical formu­lations by the standard addition method

Preparation Amount taken Amount Total amount (Hg ml- ' ) otitied - / found (lie ml'

. f/l^>,fn( I

Recovery RSD ') (%) (%)'

Silybin-70

(tablet) Silvia-70 (tablet) Limann-70 (capsule) Sivylar-70 (capsule) Levalon-70 (capsule)

10 20 10 20 10 20 10 20 10 20

10 20 10 20 )0 20 10 20 10 20

20 0 39 9 20 0 40 0 20 0 40 0 20 0 40 0 20 0 39 9

100 1 99 9

100 1 100 0 100 I 100 2 100 1 100 0 1002 99 9

0 35 0 34 0 47 0 22 0 58 0 46 0 57 0 22 0 68 0 34

Pifi-c intlt.pLnd(.n[ Jtnl\se\

/C

2032088 Die Pliamw/ic Art -N, PT105/2 (DISK) (p_2)p/i /p/i04_02/S678/ |) /]8678u TKI SIGNA Bc.irb Rcml

ORIGINAL ARTICLES

Table 3: Determination of silymarin in dosage fonns by the kinetic method proposed and by the reference metliod

Preparation

Silybin (tablet) Silvia (tablet) Limarin (capsule) Sivylar (capsule) Levalon (capsule)

Labelled amount) (mg/tablct or capsule)

70

70

70

70

70

Proposed 1

Recovery (%)

100 0

100.1

100 0

100.1

99 9

uneuc method

SD

0 13

0 09

0.09

0.12

0 05

RSD

0 33

0.23

0 24

0 30

0.13

Reference method

Recovery (%)

99 8

100.1

99.6

100.0

100.3

SD

0 09

0.06

0.13

0 06

0.10

RSD (%)•

0 23

0 15

0 32

0 15

0.26

0.452

0 205

1 147

0 227

1268

Fcfc

2.010

2 340

1751

3 940

3.830

• Five independent analyses ' Thcorcucal value for t at 95% confidence level is 2 776 = Ihcortacal value for F at 95% confidence level is 639

In conclusion, the proposed method is fast, simple and sen­sitive for the determination of silymarin in bulk and com­mercial formulations with good accuracy and precision. The sensitivity is very high and compares favourably with that of other known methods and, can therefore (6^n}success-fully applied as an alternative to the existing nieEods>,

3. Experimental

3.1. Apparatus

The absotbance was measured on a spectronic 20 D"*" spectrophotometer (Milton Roy, USA) with 1 cm glass cells

3.2. Reagents

All chemicals used were of analytical or pharmaceutical grade Potassium permanganate, 0.003M was freshly prepared by dissolving 47.41 mg of potassium permanganate in 100 ml of doubly disalled water. The apparent purity of the potassium permanganate solution was checked titiimetrically (Vogel 2002)

3.3 Test solution

Silymarin (J mg • ml"') (Micro Labs, Bangalore, India) was dissloved in methanol.

3.4. Procedure

341. Stlymarin

Aliquots of 0 18-0 50 ml of silymarin standard test solution were pipetted info a senes of 10 ml sumtiaid volumelnc flasks To each staniiaid flask 1 6 ml of 0 003 M potassium permanganate was added and then diluted to volume with doubly distilled water at 30 ± 1 °C. After mixing, reaction mixture was immediately transfened to a spectrophotometric cell and the decrease in ab-sorbance was recorded as a funcuon of time for 20 rmn at 530 nm The initial rate of the reaction (v) at different concentrations was obtained from the slope of the tangent to the absorbance-time curve The calibration curve was con­structed by plotung the loganlhm of the mitral rate of reaction (log v) versus logarthm of the concentration of the silymann (log C)

342 Silymann in pharmaceiilicalformulalions

An accurately weighed quantity of the mixed contents of five capsules or five powdered tablets, equivalent to 50 mg of the drug, was extracted into 50 ml chloroform with shaking and the residue was filtered using What­man No 42 filter paper The filtrate was evaporated to dryness and the residue was taken up with methanol and transferred to a 50 ml standard volumemc flask, diluting to volume The assay was completed following the recommended procedure

Acknowledgements The authors are grateful to Professor Shahfiullah, Chairman, Department of Chemistry, Ahgarh Muslim University, Aligarh for providing research facilities Financial assistance provided by Council of Scientific and Indusuial Research (CSIR) New Delhi, India to Syed Ndjmul Hejaz Azmi as Research Associate is gratefully acknowledged The authors wish to express their gratitude to M/s Micro Labs Limited, Bangalore India for s.miplc of pure silj mann

References:

Abdel-Salam NA, Abdel Salam MA, Hsayed MA 1982) Determination of flavanoids in Recinus communis (L.) and Silybum marianum (L.) Gaertn using TLC-differential kinetic method. Pharmazie 37: 74.

Belikov W (1985) Evaluation of the content of flavonone-deiivatives in Silybum marianum (L.) Gaerm fruits. Rastit Resur 21: 350-358.

Cassidy R, Janoski M (1992) Is your calibration curve linear'' LC-GC 10: 692-695.

Cavalieri S (1974) A controlled chnical trial of legalon in 40 pauents. Gazz Med Ital 133: 628-635.

Christian GD (1986) Analytical Chemistry, 4th ed., Wiley, New Yoik, p. 79 -83 .

Ding YT et al (1999) Separation and determination of effective components in compound products of silymarin by HPiX. Yaowu Fenxi Zazhi 19. 304-308

Farmacopoeia Ufficale Italiana (1985) 9th ed., vol. n, tuto Poligrafico e Zecca cello Stato-Roma, p. 1673.

Feienci P etal. (1989) Randomized controlled trial of silymarin treatment in patients with cinrhosis of the liver J Hepatol 9: 105-113.

Funk W elal. (1987) Quantitative HPTLC of silybin-A-comparison to quantitative HPLC. GfT-Suppl. 4 - 6 . 9 - 1 1 .

Heinz E et al. (1985) Quantitative TLC analysis of flavanoids from Sily­bum marianum Gaertn Proc. 3rd InL Symp lustrum. HPLC 3 1 3 -323

Hruby K etal (1983) Chemotherapy of Amanita phalloides poisoning with intravenous silibinin Hum Toxicol 2: 183-195.

Koeibl J etal (1986) Silymann determination in pharmaceutical prepara­tions by potentiometnc titration with iodine Czech CS 234387, IPC GO IN -027126 Appl 8314077, 06 Jun 1983.

Kurkin VA elal (1996) Determination of silybin and total flavolignans m S mananum (L) Gaertn. fruits. Rastil Resur 32- 80-87

Lan C etal (1983) Determmauon of absorptivity of silybin Nanjung Yaoxueyuan Xuebao T.\. 75 -76

Law KH, Das NP (1987) Dual-wavelength absorbance ratio and spectrum-scanning techniqual for idenufication of flavanoids by HPLC. J Chroma-togr388 225-233

Lin LL (1982) Studies on the identification and quantitative determmauon of silymann Taiwan Yao Hsueh Tsa Chih 34- 53-64

Lin Q etal (1998) TLC assay of silybin in lipotropic complex capsule Guangdong Yaoxue Yuan 14 250-252

Man;her H, Kikuta C (1993) Diastereoisomenc separation of free and con­jugated silibinin in plasma by leverse phase HPLC after specific extrac­tion J Liq Chromatogr 16 2777-2789

Martindale The Extra Pharmacopoeia (1996) 31st ed, Royal Pharmaceuti­cal Society, London, p 994

Martinelii EM etal (1991) Liquid chromatographic as'say of silybm in hu­man plasma and unne J Liq Chromatogr 14 1285-1296

Martinez F etal (1991) Diffuse-reflectance founcr-transform infrared spec­troscopic microanaljsis of the fiavonolignans Mikrochim Acta 1 105-114

Miller JN (1991) Basic StatisUcal methods for analytical chemistry pan 2 Calibration and regression methods Analyst 116 3-14

Morelh B (1987) Determination of penicillins in pure form and in pharma­ceuticals Anal Lett 20 141-159

Morelh B (1983) Spectrophotomeuic assay for chloramphenicol and some denvatives in the pure form and in formulations Analyst 108 870-879

Nalimov VV (1963) The Application of Mathematical StaUstics to Chemi­cal Analysis, Pergamon Pres'. Oxford, p 189

Nikolov N et al (1990) God Vissb Khim - Ickhnol Inst 30 203-208

Phaima/,ie 59 (2004) III

20320S8 Die Plinnnazic An.-Nr.P.3105/2 (DISK) (p_21 pzi/pzi04_02/867S/|)7.i867Sii.3d SIGNA Bcnib.: Rcinl

ORIGINAL ARTICLES

Pashankov P (1988) Isocratic high performance liquid chromatography of flavolignans in Siiybum marianum. Izv Durzh Inst Kontrol Lck Sredstra 21:3-10.

Querela V etal. (1980) Identification and assay by HPLC of the flavanoid components of Siiybum marianum in medicinal specialities. Fitoterapia 51: 83-88.

Querela V elal. (1983) Different isomeric and diastereoisomeric products contained in silymarin-Ncw chemical and analytical aspects. Anal Chem Symp Ser 13: 1-13.

Rahman N, Azmi SNH (1999) Method for determination of nifedipine in pure form and in pharmaceutical preparations. ActaPharm49: 113-118.

Rajasekaran A et al. (1997) Spectrophotometric determination of silymarin. Indian J Pharm Sei 59: 230-231.

Reddy MN etal. (2001) Spectrophotometric determination of silymarin. Asian J. Chem. 13 : 1234-1236.

Rickling B etal. (1977) Two HPLC assays for the determination of free and total silibinin diastereomers in plasma-using calcium switching with elecurochemical detection and reveised-phase chromatography with UV detection. I Chromatogr 135-. 499-501.

Salmi HA, Sama S (1982) Effect of silymarin on chemical, fiinctional, and morphological alteration of the liver, a double blind controlled study. Scand J Gasteroenterol 17: 517-521.

Sammel PG (1979) Comprehensive Organic Chemistry - The synthesis and Reaedons of Organic Compounds, Volume 4, 1st ed., Peigamon Press, p. 681-690.

Schonberg A, Schutz G (1960) Dehydrogenation of chromanones with tii-phenyl methyl pcrchlotate. Chem. Ber. 93: 1466.

Steinigen M (1983) Comparidvc assay of silymarin-containing prepara­tions. Pharm. Ztg. 128: 783-785.

Szilajtis-Obeiglo R, Kowalewska K (1984) SpecU-ophotometric method for determination of flavonolignans and silybin in crude drugs and prepara­tions from Siiybum marianum L. (Gaertn.). Herba Pol 30: 27-34.

Tiltel G, Wagner H (1977) High performance liquid chromatography-se-paration of silymarins and their determination in raw extracts of Siiybum marianum Gaertn. J Chromatogr 135: 499-501.

"nttel G, Wagner H (1978) High performance liquid chromatography of silymarin II Quantitative determination of silymarin from Siiybum maria­num by HPLC. J Chromatogr 153: 277-232.

Tomov E et al. (1985) Silymarin contents investigations of S. marianum-seeds by means of HPLC. FE CS Int Conf Chem Biotechnol Biol Act Nat Pro. 5: 534-538.

Torrado S etal. (1994) Comparison of assay methods by second-derivative spectroscopy, colorimetry and fluorescence spectroscopy of salicylic acid in aspirin preparations with a high performance liquid chromatographic method. J Pharm Biomed Anal 12: 383-387.

Vanhaelen M, Vanhaelen — Fastre R (1983) Quantitative determination of biologically active constituents in medicinal plant crude extracts by TLC-densitomeliy.l. Aesculus hippocastanum L. and Siiybum marianum Gaertn. J Chromatogr 281: 263-271.

Vogcl's Textbook of Quantitative Chemical Analysis (2002) 6th ed., Pear­son Education, Singapore, p. 42.

Wang P et al. (1998) Determination of active flavanoids in silymarin. Sepu 16: 510-512.

Wu Z, Wang X (1989) Determination of siiybum in the peel of S. maria­num. Zhogguo Yaoke Danue Xuebao 20: 184.

Zarapkar SS et al. (2000) Colorimetric estimation of silymarin in pharma­ceutical dosage forms. Indian Drugs 37: 133-134.

Phamiazic 59 (2004) I I I