CHAPTER I

1.1 GENERAL INTRODUCTION

In absorptiometric method of analysis, simultaneous determination of the

components of a binary mixture of drugs, whereby the two known absorbing

substances exert mutual interferences at their individual measuring wavelengths,

may be easily carried out by utilizing the theoretical principles inherent in the

absorbance ratio technique. Determination of both absolute and relative

concentration of individual component in such mixture by this technique is

decidedly faster and has certain qualities that warrants its use for analysis of binary

mixture.

Determination of components in a binary mixture using simultaneous(1)

equation technique was first applied by Vierordt almost 120 years ago. The

technique was based on the assumption that if the two components do not react or

interact in any manner with one another and thus neither affects the light absorbing

properties of the other, the total absorbance of the two components in the solution

is the sum of the absorbances which the two substances would have exhibited

invidually if the substances were in separate solutions under similar conditions and

had the same concentrations as in the mixture(2,3). Despite simplicity of the

Vierordt’s method, the method is far more sensitive to wavelength errors because

some of the absorbance measurement will have to be made on the slopes of the

absorbance curves(4) which require greater care specially with wavelength

calibration of spectrophotometer visavis solutions of known concentrations are

required for establishment of numerical coefficients.

The absorbance ratio method was first used in Germany and Hufner(1) was

the originator of the technique. The technique is based on the theoretical criterion

2

that the ratio of two absorbance values determined on the same solution at two

different wavelengths is a constant. Schroeder and his coworkers(5) were perhaps

the first to tabulate the absorbance ratio values for a relatively large number of

substances of chemical importance. Kuratani(6) Hirt et.al.(7) and Bonnier et.al.(8)

pointed out that such ratios can be used to assess the relative concentrations of the

two components in a binary mixture. Moshe IshShalom et.al.(9) compared the

method as described by Hirt et.al.(7) with the Vierordt’s method and concluded

that the former method gave better results. Glenn(10) had developed a modified

Vierordt equation in terms of absorbance ratios which could be determined from

solutions of unknown concentrations and may require absorptivity value at the

max of each component (where dA/d=0).

Pernarowski et.al.(11) used absorbance ratios to determine both relative and

absolute concentrations of the components of binary mixtures and derived an

equation similar to Glenn’s equation(10). However, these author(11) assumed that an

isoabsorptive point as one of the two wavelengths must be chosen to apply their

equations. These authors(11) have also applied ‘Q Analysis’ based on the

relationship between the absorbance ratio values of a binary mixture and the

relative concentration of such a mixture. Relative analysis of binary mixture by

this technique is comparatively faster than the simultaneous equation technique

but, unlike the later method, results are in terms of fraction of total mixture.

Cho and Parnarowski(12) used absorbance ratios to determine absolute

concentration and derived an equation which do not require isoabsorptive point.

The method is based on the use of two absorbance ratio values, one absorptivity

value and the difference at two wavelengths between the absorbance values of a

solution and the values of a reference solution containing one of the two

3

components in the mixture. However, if the difference in absorbance values at the

two specified wavelengths is small, the error in the analysis is likely to be higher.

Drug dissolution study is generally done in acidic media by the use of either

0.1 N HCl or buffer solution. Direct spectrophotometric assay in application of

such study is very much useful because of rapidity, accuracy and simplicity. The

drugs which are acid labile in character, deteriorates quicly in acidic media and the

spectral behaviour changes with time. If the spectra of such drug at different time

intervals show an isoabsorptive point, then by measuring absorbance at the

wavelength of isoabsorptive point, total drug content can be determined

irrespective of acid degradation, which has valuable utilization in the drug

dissolution rate study. Utilization of the procedure has been described by Fedynec

et.al.(13) and the technique have been made official in USP[3] in the dissolution

study of Aspirin Capsules.

Literature reveals that applications of the absorbance ratio technique have

been applied for the simultaneous assay of binary mixtures of Aminophylline

Phenobarbital(14), AnatazolineNaphazoline(15), TrimethoprimSulphamethoxa

zole(1618), Sulphathiazole in presence of Sulphadiazine and Sulphamerazine(19),

SulphacetamideSulphanilamide(20), NifuroximeFurazolidone(21), Primaquine

Amodiaquine(22), Diloxanide furoate in presence of degradation products(23),

AnalginParacetamol and AnalginOxyphenbutazone(24), Morphine in presence of

Pseudomorphine(25), StrychnineBrucine(26,27) and mixture of alkaloids.(28)

In application of zeroorder simultaneous spectrophotometric methods, the

presence of spectral interferences and/or spectral overlapping such as many

originate from batchtobatch differences between the sample and reference

standard or from the pharmaceutical formulation matrix, would certainly lead to

4

erroneous results. The high excipientdrug ratio and high sample weight require

for these formulations result in backgroundirrelevant absorption of a sufficiently

high intensity to possibly prohibit the application of simple spectrophotometric

methods. Irrelevant absorption in spectra may originate from diluents, moistening

agents, binder, lubricants, excipients, adjuvants and preservatives. Although

simultaneous spectrophotometric methods are applicable by eliminating specific

interference from degradation products and coformulated drugs, however, these

methods require special attention in selecting assay parameters and application of

several predetermined factors. Madsen et.al.(29) has made a linear least squares

approach to analyse mixtures of drugs in the presence of known background

absorption by simultaneous spectrophotometry.

The methods aiming at to reduce or eliminate matrix interference in the

assay of coformulated drugs include MortonSturbs(30) correction procedure,

which requires that the irrelevant absorption is linear over the wavelength range of

the absorption band of the drug; Glenn’s method of orthogonal polynomials for

equally spaced intervals(3136); pj method(37) compensation spectrophotometry, in

which the reference solution contains the matrix or sample at the same

concentration present in the sample solution(38,39) difference spectrophotometry(40)

derivative spectrophotometry(41,42); derivativedifference spectrophotometry(4345);

Vidicon Spectrophotometry(46) and trigonometric functions(47).

Difference spectrophotometry is a recently explored spectrophotometric

technique applicable to acidic, basic, and amphoteric drug substances that undergo

reproducible spectral changes due to pH changes or the effect of reagents. Doyle

and Fazzari(48) stated that assay of mixtures of drugs by difference

spectrophotometry, where more than one drug undergo spectral shifts, constitutes a

5

special challenge which can sometimes be met, but the method require meticulous

technique and depend on the fortuitous juxtaposition of an isoabsorptive point of

one compound with a maximum of another. By contrast the method for single

component dosage forms are usually simple and rugged.

Difference spectrophotometry provides an approximation of the ideal

reference solution by employing an aliquot of the sample solution itself as

reference, adjusted by change in pH or other parameters but containing both the

substance being analysed and all extraneous substances at exactly the same

concentrations as the sample. If the pH or other variation causes an alteration in

the spectrum of the sample, the instrument records this as a characteristic

difference spectrum. If other materials present are unaffected by the change in

conditions, their contribution to the total absorbance of each solution will be

identical and their effect will be exactly cancelled. It has been proved useful

particularly in the assay of medicinal substances by eliminating specific

interference from degradation products and coformulated drugs and also

nonspecific irrelevant absorption from the formulation matrix. The technique is

also applied to substances that exhibit a difference in absorbance between the

equimolar solutions which has been induced by the addition of reagents to one or

both of the solutions. The difference spectrophotometric assay of these substances

in samples that also contain other absorbing components may be carried out

provided the absorbances of the interfering substances remain unaltered by the

reagents. Thus, pHinduced and reactioninduced simultaneous spectro

photometric procedures are specific for certain coformulated drugs and

formulation excipients.

6

Some of the formulations such as Cinnamic and Benzoic acids(49,50),

Acetylsalicylic acid SalicylamideAcetaminophenCaffeine(51), Morphine(52,53),

HydrochlorothiazideReserpine(54), Acetaminophen(55), Chlordiazepoxide and

Demoxepam(56), AcetaminophenSalicylamide and Codeine phosphate(57),

Caffeine(58), analgesics(59). Oxyphenbutazone(60), Phenylbutazone(61,62), Tetra

cycline and Oxytetracycline(63) have been analysed by difference

spectrophotometry based on pHinduced spectral changes. Reported methods

based on reactioninduced spectral changes are for Corticosteriods(64,65),

Phenothiazine drugs(66), and Dipyrone(67).

Purely spectroscopic evidence for irrelevant absorption always involves

observed distortion of the contaminated substances absorption curve, of the more

obvious distortions, max diminishes in the presence of impurities whose

absorption decreases with increasing wavelength in the region of max, and

viceversa. On the other hand, irrelevant absorption possessing a constant

contribution at all wavelengths has no effect on max.

The accuracy and specificity of U.V. absorption methods may be

considerably improved by conversion of the normal zeroorder spectrum into a

higher order derivative spectrum. The improved resolution of overlapping

absorption bands and the discrimination in the favour of narrow bands against

broader bands, which are the principal characteristics of derivative

spectrophotometry, can result in the complete elimination of both nonspecific

matrix intereference(6870) and specific interference from coformulated

compounds(71,72). Thus the technique is applicable for determination of single

component drug dosage forms by eliminating irrelevant absorption due to

excipients which interfere in direct spectrophotometric analysis and more

7

successfully for analysis of ingredients of binary component drug formulations for

which absorbance ratio and difference spectrophotometric techniques are not

applicable. The technique was introduced(73) as a useful means of resolving two

overlapping spectral bands of almost coincident wavelengths(74) and eliminating

marix interference in the assay of many drugs(75,76). The principal advantages of

derivative measurements are the improvement in the detectability of minor spectral

features and in quantitative analysis, a potential reduction in error caused by

overalp of the analyte spectral band by interfering bands of unknown/known

and/or variable intensity.

Analytical applications of derivative spectrophotometry has been increasing

in the past few years(7781) by the introduction of commercial spectrophotometers

operating in derivative mode, the recently introduced commercial systems that can

produce a graphic display of the derivative (dA/d) or (d2A/d 2) of the anlog

signal given by the spectrophotometer provide a different approach to these

problems. Methods to generate derivative spectra have included direct quantitation

by digital(82,83) and analog computers(84) or by mechanical modulation of

wavelength dispersion(8588). The basic difference between the mechanical

modulation and computational methods is that the former produces a

representation of derivative of intensity or absorbance with respect to wavelength,

while the latter produces a derivative of intensity or absorbance with respect to

time which is assumed to be directly related to wavelength. Hager(85) and Green

and O’Haver(84) have given lucid description of the manner in which wavelength

modulation methods coupled with tuned amplifiers generate first and second

derivatives. Pardue et.al.(82), Cook et.al.(89,90), Milano et.al.(91) & McDowell

et.al.(97) reported a brief description of vidicon based derivative spectrophotometer

8

in which derivative spectra are generated directly by the spectrophotometer used in

conjugation with a phase sensitive lockin amplifier(90).

Reported pharmaceutical applications of derivative spectroscopy are few

and for the part have been limited to enhancing the spectral features of a drug to

facilitate its identification or quantitization. Successful application of this

technique has been reported for the determination of single component drug

dosage forms containing excipients which interfere with the spectrometric analysis

and simultaneous determination of more than one active ingredient in

multicomponent drug dosage forms, for which both the absorbance ratio and

difference spectrophotometric techniques are not applicable. Such study is of great

help in content uniformity analysis and quantitization of the drugs.

When the irrelevant absorption is not cancelled by difference

spectrophotometery or derivative spectrophotometry, in such a case difference

spectroscopy coupled with derivative spectroscopy known as derivative

difference spectroscopy(D) is sometimes applicable for further compensation of

irrelevant absorption. This will be the major advantage for the application of

derivativedifference spectrophotometry over the use of each technique alone. The

D method unlike the A method can be applied to the assay of drugs after their

spectra have been changed via change in pH or any other chemical reaction. The

main criteria for such application is that spectral change induction, which may be

do not only to pH change but also due to any other chemical reaction such as

complexation(93), condensation(94), and bromination(95), obviously the limitation(96),

of the A method should be considered before using the D method.

Derivative spectrophotometry has been applied for determination of single

component pharmaceutical dosage forms containing excipients(97101); degradation

9

products such as Procaine in presence of 4Aminobenzoic acid(102); 1,4Benzo

diazepins in presence of their acidinduced degradation products(103); Thiamine

and Pyridoxine in aged pharmaceutical formulations(104); Cephalosporins in the

presence of their degradation products(105); Acetaminophen and Phenacetin in

presence of their degradation products(106); determination of respective degradation

products in presence of intact drugs such as Salicylic acid in Aspirin(107,108) and

sulfoxide in Chlorpromazine(109); estimation of drugs based on zero

crossing(110113) technique and simultaneous determination of more than one active

ingredient in multicomponent drug dosage(114118). Derivativedifference spectro

photometry has been reported for the drug formulations such as, Corticosteriods,

Oxytetracycline and Tetracycline(119), Oxazepam or Phenobarbitone and

Dipyridamole(120).

Spectral studies of chromophore in the visible region have been extensively

used in numerous fields and will continue to remain an important field of study as

it involves very simple instrumentation, resulting nevertheless in sensitive and

accurate measurements with the advantages of speed and simplicity. The

limitations of the procedures lies in the chemical reactions characteristic of various

functional groups capable of giving rise to coloured species. In several instances

chemical species do not possess suitable chromogenic properties and may be

converted to an absorbing species or be made to react with an absorbing reagent,

which forms the basis of their analysis. One of the most common studies in the

field in recent time is the use of compounds having phenolic functional groups to

yield spectrophotometrically useful chromogenes through diazocoupling and

nitrosation(121125) reactions.

10

Aromatic hydroxyl compounds having free ortho or para position form

nitroso derivatives in acidic medium. Inamdar and Kadji(126) have reported that

the stability of the chromogen produced in an acidic medium was increased by the

addition of alcohol. Chafetz et.al.(127) had claimed that nitro rather than nitroso

derivative was actually produced. Belal et.al.(121) reported that alkalinization of the

medium stabilizes the chromophore. Coordination of a transition metal with the

polydentate ligand (orthonitroso derivative) results in the formation of a stable

water soluble metal chelate(121). Thus the nitroso derivatives and their metal

chelates offer a good scope for analysis of phenolic compounds. The compounds

which are reported in the literature using the technique of nitrosation and chelation

reaction are Acetaminophen and Salicylamide(123), Oxyphenbutazone,

Ethinyloestradiol(128) and Amoxicillin(129).

Chromatography first discovered by Michael Tswett in 1903(130) is

extensively applied for separation, isolation purification, identification and

quantitation of the compounds of pharmaceutical interest. The various

chromatographic techniques are thinlayer chromatorgraphy (TLC), high

performance thinlayer chromatography (HPTLC), column and paper

chromatography, ionexchange chromatography, gas chromatography (GC) and

highperformance liquid chromatography (HPLC). Compounds having similar

chemical nature often defy separation by other analytical techniques, are identified

and quantitised by the chromatographic techniques even in microgram quantities.

All the chromatographic techniques are based on basic separation principles such

as adsorption, partition and ionexchange.

TLC was first introduced as a procedure for analytical adsorption

chromatography by Stahl(131). The technique is now widely been used in official

11

compendias(13) for limit test of decomposition products and related foreign

substances. Other wide applications, of TLC are isolation and quantitation of

multicomponent mixture of drugs.(132,133)

The real development of adsorption chromatography began in 1931 when

Kuhn and Lederer(134) introduced the method in the preparative chemistry.

Martin and Synge(135) introduced partition chromatography using columns of

silica gel and Consdon et.al.(136) developed paper chromatography. Applications of

column, paper and ionexchange chromatography are available in the official

compendias(137143) and literature.

Gas chromatography (GC) was first applied by Ramsey in 1905 to separate

gaseous mixtures(144). In 1952, James and Martin(145) introduced gasliquid

chromatography based on the suggestion of Martin and Synge(146). Gassolid

chromatography (GSC) and gasliquid chromatography (GLC) are the two

common technqieus. GC has increasing application in the analysis of drugs and

their metabolites. The speed, resolution and sensitivity makes this techniques very

attractive for drug analysis problems dealing with bioavailability, raw material,

and pharmaceutical formulations. In pharmaceutical analysis, GC has been applied

for the assay of the raw material, drug substances, quantitation of drugs in

formulations, and assay of impurities including minor components in the drug

substance.(147148)

Highperformance liquid chromatography (HPLC) or often called

highpressure liquid chromatography is a technique in which separation is

accomplished by partitioning between a mobile solvent and a stationary column

packing material of small uniform particle size (10 µm or less). The combined

advantage of HPLC have led to the very rapid growth in its technology and

12

popularity since it began to develop a separate discipline in the late 1960s. In

recent years, due to the development of HPLC instrumentation the technique is

preferred over gas chromatographic analysis which require derivatization.

In surveying the literature involving HPLC pharmaceutical analysis, one

finds that probably more than 90 per cent of the applications involve reverse phase

liquid chromatography of a bonded phase packing material consisting of silica

support with an organic moiety bonded to it through a siliconoxygen Silicon

carbon covalent bonding system prepared with a functionalized chlorosilane. The

technique of reverse phase chromatography was introduced in 1950 by Howard

and Martin(149) and involves the use of nonpolar stationary phases and polar

eluents. Reverse phase chromatography has developed mainly since the

introduction of chemical bonded stationary phases in 1969 by Halasz and

Sebestian(150). Now very efficient columns packed with chemically bonded phases

on microparticles of silica have been prepared. The advantages of reversedphase

technique are numerous, the most outstanding being the extremely simple

operating conditions. The ease of sample preparation, speed of analysis,

specificity, accuracy and precision associated with this method are the main

advantages. In pharmaceutical analysis HPLC has many useful applications in

isolation and quantitation of multicomponent mixture of drugs and metabolities.

The Taxanes docetaxel (Taxotere) and Paclitaxel (Taxol) are used in the

treatment of cancer. The naturally occurring paclitaxel was first isolated from the

bark of the pacific yew tree (Taxus brevifolia) in the 1960s and gained commercial

approval in December 1992. Docetaxel was first synthesized starting from

10deacetyl baceatin III, a non toxic precursor found in the European yew (Texus

baccate) in 1986(151). Today these drugs have contributed significantly to the

13

treatment of a variety of malignancies such as varian, breast and non small cell

lung cancers, as well as head and neck cancer and some cancers of the digestive

system.(152)

Despite the major befits of these products, patients receiving

chemotherapeutic treatment can experience severe to life threatening side effects

primarily myelosuppression leading to neutropenia. On the other hand under

dosage might result in sub optimal treatment of the cancer. In addition to their

narrow therapeutic range these substances also display highly variable pharmaco

kinetics. Traditionally the dosing of anticancer agents is calculated on the basis of

the patient body surface area. It has been suggested that pharmaco kinetically

guided chemotherapy and dose individualization might lead to a better treatment

outcome.

Although this subject is still under discussion, it is clear that further clinical

studies are necessary to reveal the optimal treatment schedule(153154) for this

purpose validated analytical methods for the quantification of these compounds in

plasma are a necessity.

In plasma paclitaxel and docetaxel highly bound to proteins, with free

fractions generally lower than 10%. This free active, fraction is better related to

the pharmacological and/or toxic effect and measuring free fractions could

therefore be superior to total plasma concentration. The determination of free

fractions is however complicated and time consuming, limiting its use in clinical

practice. Oral fluid as a therapeutic drug monitoring matrix offers some interesting

opportunities.

This matrix can be viewed as a natural ultrafiltrate of plasma, and oral fluid

concentrations often correlate to free drug levels in plasma. In addition

administration of taxaues is based on short infusion duration and patients are often

14

not hospitalized. Under these conditions, monitoring plasma concentrations, which

requires blood sampling by medical personnel is laborious and increases stress on

patients and health care workers. The collection of oral fluid with a collection

device could be performed by the patients, thus not requiring medical personnel or

a hospital visit. To investigate the correlation between oral fluid and plasma

concentrations a method for the quantification of paci taxel and doce taxel in oral

fluid was developed. In the past, methods have already been developed to monitor

decetaxel or paclitaxel concentrations in plasma or serum. Earlier methods using

liquid chromatography with ultraviolet detection suffered from limited sensitivity

and selectivity, due to their relatively low UVabsorbance and nonselective.

UVmaximum (227 nm). As a consequence methods based on liquid

chromatography coupled to mass spectrometry (LCMS) were developed.

Most published methods report the quantification of either docetaxel(155159)

or paclitaxel(160164). The simultaneous analysis was also reported(165). Most

methods used isocratic elution, which minimizes the total run time but does not

provide a column wash. In addition previous methods did not evaluate the ion

suppression by the drug formulations vehicle. Recent paper reported(165) ion

suppression by the drug formulations of docetaxel (Tween 80) and peclitaxel

(Cremophor EL) due to carry over in subsequent runs with an isocratic LC

elution(166). To monitor paclitaxel and decetaxel levels in patient samples, a new

robust method needed to be developed, devoid of matrix effect.

All these literary calls encouraged the author to study simultaneous

spectrophotometric determination of some binary or ternary mixture of drugs using

absorbance ratio, difference spectrophotometric, derivative spectrophotometric,

and derivativedifference spectrophotometric techniques; visible spectrophoto

15

metric determination of some phenolic drugs through nitrosation and subsequent

chelation; application of TLC, paper chromatography and gasliquid

chromatography for identification and quantitation of active ingradients, added

impurities or degradation products; simultaneous determination of some binary

mixture of drugs by column chromatography and HPLC. Literature survey upto

date revealed that no such study has so far been done on the selected formulations,

hence it was considered worthwhile to undertake this project.

1.2 PLANNING OF WORK:

1.2.1 Category of the Drugs Slected:

The drug is defined as any substance or product that is used to modify or

explore physiological systems or pathological states for the benefit of the recipient.

The drugs are first grouped according to their therapeutic action and then

subdivided according to the chemical structure of drugs. Category of the drugs

selected for the work are as enumerated below:

Antineoplastic:

(i) Alkylating Drugs

Procarbazine (Matulane); Dacarbazine (DTIC) Altretamine (Hexalen).

(ii) Purine Antagonists

Mercaptopurine (6MP); Fludarabine Phosphate.

(iii) Pyrimidine Antagonists

Cytarabine (ARAC); Azacitidine

(iv) Plant Alkaloids

Vinblastine (Velban); Vincristine (Oncovin); Etoposide (VP16, Ve

PeSid); Teniposide (Vumon); Paclitaxel (Taxol); Docetaxel (Taxotere);

16

Dicloxacillin Sodium; Epirubicin; Epirubicin HCl; Epirubicin Benzoate;

Mitoxantrone.

(v) Sulphonamides

Sulphamethoxy Pyridazine (SMPZ)

(vi) Diuretic and Antihypertensive

Frusemide; 4chloro5sulphamoylanthranilic acid (CSAA) (Decom

position Product of Frusemide)

It is a potent diuretic, and is also used in the treatment of hypertension.

(vii) Keratolytic

4methyl benzoic acid; 4methyl salicylic acid. They have bacteriostatic

and Fungicidal properties.

(viii) Antimalarial

Pyrimethamine; 5(4chlorophenyl)6ethyl pyrimidine2,4diamine.

(ix) Methyclo thiazide and candesartan cilexetil

(x) Cycloxacillin; (2S, 5R, 6R)6{[3(2chloro phenyl)5methyloxazole

4carbonyl}amino}3,3Dimethyl7oxo4thia1azabicyclo [3.2.0]

heptane2carboxylic acid (C19H18ClN3O5S) and oxacillin; (2S, 5R,

6R)3,3Dimethyl6[(5methyl3phenyl1,2oxazole4carbonyl)

amino]7oxo4thia1Azabicyclo [3.2.0] Heptane2carboxylic acid.

(xi) Moxonidine;4chloroN(Imidazolidin2ylidene)6methoxy2 methyl

pyrimidine5Amine (C9H12ClN5O) and Amlodipine.

(xii) Gatifloxacin and propyphenazone.

1.2.2 Categorisation of work:

In the present work an attempt has been made to develop quick and reliable

methodology for control analysis of the selected drugs, available in formulations

17

as single component, binary or ternary mixtures. Literature survey reveal no such

work, so far has been done on these formulations using the applied techniques.

Hence it was considered worth while to undertake this project. The methodology

applicable to the selected formulations are summarized in table1 and are

categorized in nine different chapters as enumerated below:

ChapterII, SectionA: Simultaneous spectrophotometric analysis, using

absorbance absorbitivity ratio techniques of the following binary mixture of drugs:

(A) Dicloxacillin Sodium Docetaxel, EpirubicinEpirubicin salt Mitoxantrone;

Mercaptopurine; Fludarabine phosphate; cytarabine; Azacitidine; Viblastine; Vin

cristine, Etoposide; Teniposide, Procarbazine; Dacarbazine; Paclitaxel;

Altretamine; Sulphamethoxy Pyridazine; Frusemide; 4chloro5sulphamoyl

anthranilic acid; 4methyl benzoic acid; 4methyl salicylic acid; pyrimethamine;

methyclo, Thiazide, candesartan; cilexetil; cycloxacillin; oxacillin; Moxonidine;

Amlodipine; Gatigloxacin and propyphenazone and thiabenzazone.

ChapterII, SectionB: Spectrophotometric estimation of total sulphonamides,

using isoabsorptive point in a ternary mixture of trisulphadrugs.

ChapterII, SectionC: Determination of isoabsorptive point, of an intact

molecule and its decomposition product of an acid labile drug thia benzazone.

ChapterIII, SectionA: Simultaneous spectrophotometric analysis using

difference absorbance/difference absorbance ratio technique based on pHinduced

spectral changes of the following binary component drug formulations:

Epirubicin/Epirubicin benzoateMitoxantsrone; Mercaptopurine; Fudarabine

phosphate; viblastine; vincristine; Paceitaxel; Altretamine; Sulphamethoxy

Pyridazine in presence of pyrimethamine.

18

ChapterIII, SectionB: Simultaneous spectrophotometric analysis using

difference absorbance/difference absorbance ratio technique based on pHinduced

spectral changes, of a ternary mixture of salicylamide, propyphenazone and

pyrithyldione in presence of caffeine.

ChapterIII, SectionC: Difference spectrophotometric analysis, based on

reactioninduced spectral changes of Frusemide in presence of its degradation

product.

ChapterIV, SectionA: First derivative spectrophotometric analysis of the

following drugs. Dicloxacillin sodiumDocetaxel, Mercaptopuyrine; Epirubicin/

Fudarabinephosphate; DacarbazineEpirubicin; Epirubicinbenzoate; Dacarbazine

Mitoxantrone.

ChapterIV, SectionB: Second derivative spectrophotometric analysis of the

following drugs. ProcarbazineEpirubicin/Epirubicin benzoate; Frusemide in

presence of its degradation products; 4methyl Benzoic acid and 4methyl

salicyclic acid.

ChapterV: Validated spectrophotometric method for simultaneous estimation of

methylothiazide and candesartan, cilexetil in Tablet Dosage form.

ChapterVI: Determination of Degradation product for combination containing

cycloxacillin and oxacillin in capsule dosage form by LCmass spectroscopy.

ChapterVII: Quantitative determination of paclitaxel first order derivative

UVspectrophotometry using area under curve.

ChapterVIII: Simultaneous spectrophotometric estimation of moxonidine and

Amlodipine in tablet dosage form.

ChapterIX: Simultaneous spectrophotometric estimation of gatifloxacin and

proyphenazone in Bulk Drug and in ophthalmic Dosage form.

19

1.2.3 Further Scope of the work:

The present investigations embodied in this thesis still point towards a

further scope for a fertile investigations to develop the fields in pharmaceutical

analysis with the outcome of new formulations.

TABLE 1

THE METHODOLOGY DEVELOPED PERTAINING TO EACH

FORMULATIONS

No. Formulation Method developed

M1 Dicloxacillin sodium

Docetaxel

Absorbance Ratio Spectrophotometry;

Derivative Spectrophotometry

M2 Epirubicin/Epirubicin

saltMitoxantrone

Absorbance Ratio Spectrophotometry;

Difference Spectrophotometry.

M3 Mercapto Purine Epirubincin

Fludarabine phosphate

Absorbance Ratio Spectrophotometry;

Difference Spectrophotometry; Derivative

Spectrophotometry.

M4 CyatarabineAzacitidine Absorbance Ratio Spectrophotometry.

M5 VinblastineVincristine Absorbance Ratio Spectrophotometry;

Difference Spectrophotometry; HPLC.

M6 EtoposideTeniposide Absorbance Ratio Spectrophotometry.

M7 ProcarbazineAltretamine;

Epirubicin and its salt

Absorbance Ratio Spectrophotometry;

Derivative Spectrophotometry

M8 PaclitaxelAltretamine Difference Spectrophotometry; HPLC

M9 Sulphamethoxy pyridazine Difference Spectrophotometry; Derivative

Spectrophotometry

M10 Sulpha Drugs Absorbance Ratio Spectrophotometry.

20

TABLE 1 Contd… No. Formulation Method developed

M11 SalicylamidePropyphenazone

Pyrithyldione

Difference Spectrophotometry; Derivative

Difference Spectrophotometry; HPLC.

M12 Frusemide in presence of

degradation product.

Difference spectrophotmetry; Derivative

spectrophotometry.

M13 DacarbazineMitoxantrone Derivative Spectrophotometry.

M14 4Methyl Benzoic Acid4

methyl Salicyclic Acid

Derivative Spectrophotometry

1.3 APPROACH TO WORK:

In development of the methods detailed under ChapterIIIX, the

investigations were carried out in the following manner:

1.3.1 Study of Physical Parameters:

Some essential physical characteristics of the compounds like solubility,

pKa values, melting and boiling point, polarity, spectral characteristics, etc. were

compiled from literatures.

1.3.2 Choice of Solvents:

Selection of suitable solvents for stock solutions, standard preparations and

sample preparations have been done taking into consideration that all the active

ingredients are easily extractable and stable in the solvent for prolonged time. The

solvent selected are suitable for repeated dilutions under experimental conditions.

1.3.3 Selection of Formulatiions:

In application of simulataneous spectrophotometric analysis, the proportion

of active ingredients to be determined, is more important for accuracy of the

21

results. Thus a 5050 drug mixture would yield for more accurate results than a

94:6 mixtures of the minor component. However, if the absorption intensity of the

minor component is much greater than that of the major component, then the

accuracy and precision can be attained to a satisfactory limit. The formulations

were selected on these considerations.

1.3.4 Possible Interaction Between the Components:

In application of the simultaneous spectrophotometric methods it was taken

into consideration that the components did not form complexes with each other

under the experimental conditions so that the simple sum of the individual

absorbances of the components at a particular wavelength are equal to the total

absorbance of the mixed components of identical concentrations.[34]

1.3.5 For the Absorbance/Absorptivity Ratio Method:

Absorbance media: For selection of suitable absorbance media, spectral

characteristics of all the components present in a particular formulation were

studied in different acidic, basic, netural (water) and organic media. Finally the

solvent was selected which gave well separated position of maximum absorption

peaks or isoabsorptive point with optimum absorption intensity.

Locatioin of Isoabsorptive point (Isobestic point): An isoabsorptive point is

defined as the wavelength at which two dissimilar substances have identical

absorptivity values, the solvent being the same for both substances. For location of

isoabsorptive point, spectrum of the equiconcentration solutions of the two

substances were recorded against the solvent blank. The wavelength at which both

the curve intersected was the isoabsorptive point. Another alternative method

adopted was that the spectrum of a solution of one substance was recorded relative

to equiconcentration solution of another substance, the wavelength at which zero

absorbance was observed represented the isoabsorptive point.

22

Choice of wavelength: When the maximum of both the components are well

separated and have optimum absorptivity values, the wavelengths selected are the

max. of both the components. In applicatioin of isoabsorptive point method and

‘Q curve’ analysis these are usually be the wavelengths at which one of the two

components exhibit maximum absorption and the isoabsorptive point. In selection

of the wavelengths it was taken into consideration that at the selected wavelengths

both the components had optimum absorptivity values, the wavelengths of

measurement were unaffected by irrelevant spectral interferences, and the

combined spectra of the two components did not have steep slope of the curve at

the wavelengths of determination.

‘Q Curve’ plot: In application of simulataneous spectrophotometry using

isoabsorptive point as one of the selected wavelength, ‘Q curve’ was plotted to

determine the applicability and accuracy of the method. Details of such plot are

discussed under Chapter II (2.2.5).

1.3.6 For the Difference Absorbance Method:

Choice of pH/solvent: For application of pHinduced difference

spectrophotometry for the compounds showing bathochromic or hyperchromic

shift together with hypochromic or hypsochromic effect, spectral interference of

each other were eliminated by measuring interalia absorbance of acidic, basic or

neutral solution of identical concentrations.

Plot of absorptivity versus different pH media were studied at a particular

wavelength. For acidic and basic pH media, HCl and NaOH solutions of different

strengths were used and for neutral pH, glass distilled water (pH~7) was used.

Selection of solvent were done on the basis of maximum absorbance

difference (A), isoabsorptive point of zero absorbance and prolonged stability of

23

the components in the medium. The acidic or basic solutions were so selected, that

both are at least two pH units removed from the pKa on opposite sides of this

value and 10% variation of the strength of the acids or bases did not alter the pH

of the medium.

The influence of pH changes on unbuffered sample/standard solutions were

also studied. The final sample solution was found to have a pH~7 in all the cases,

i.e. equal to the ph of the water used for dilution. Therefore, the use of buffered

solution was not considered to have any advantage over the use of water. Thus the

solutions were prepared in water, suitable strength of acid (HCl) and alkali

(NaOH) because of suitability and simplicity.

Isoabsorptive Point: Isoabsorptive point of zero difference absorbance (A=O)

find many uses in the development and selection of optimum analytical conditions.

The conditions are so selected, that at the wavelength of maximum A of one

component, the other components have isoabsorptive point of zeroA so that the

component is determined directly without interference of the other.

Choice of wavelengths: The wavelength was selected, preferably at or near the

maximum A of the components to be determined, at which other component(s)

had isoabsorptive point of ZeroA. In some cases where isoabsorptive point not

lie at or near A maximum, the wavelength of maximum A was selected and the

result was calculated with the vector sum of the A of the other component at that

wavelength.

Irrelevant absorption: Graphs of log A versus were plotted for sample

solution and for the authentic mixture of identical ratio. The graphs were

completely superimposable, indicating that the irrelevant absorbance was

unaffected by pHchange and so the irrelevant absorption were totally nullified.

24

1.3.7. For the Derivative Spectrophotometric Method:

Zerocrossing deterimination: While developing the derivative

spectrophotometric methods for binary component mixtures, solvent systems were

so selected that would have placed the derivativeabsorbance maximum of the

component to be determined at or near the zerocrossing wavelength of the other

i.e. at the isoabsoptive point, where derivativeabsorbance is equal to zero. Thus at

the zerocrossing wavelength the measuringcomponent could be determined by

measuring absolute value of derivative absorbance at that wavelength. For

zerocrossing determination, recorded the derivative absorption spectra of various

concentrations of the components to be determined on the same chart paper using

identical parameters, and suitable zerocrossing wavelengths at zeroderivative

absorbance were selected for the measurements.

Derivative amplitude measurement: The derivative amplitudes were measured

with respect to the derivative zero i.e. from zerocrossing point at derivative zero

to the measuring derivative curve, with positive or negative value.

1.3.8 For the DerivativeDifference Spectrophotometric Method:

Since in the derivativedifference spectrophotometry, the difference spectra

(A) are differentiated with respect to wavelength, thus the zerocrossing

determination and peak amplitude measurement were done as under 1.3.7.

1.3.9 For the visible spectrophotometric method:

Spectral characteristics of the chromophore: Absorption spectra of the

chromophore were recorded in the visible and near UltraViolet range

(350700nm). The wavelength was selected at which sharp maxima with

maximum intensity was obtained.

25

Reagent concentration: To determine appropriate concentration of each reagents,

a fixed volume of the reagent of different concentrations were added to the

reaction mixture having fixed volume and concentration of the other reagent and

the corresponding drug. The colour intensity of the reaction mixtures for each

concentration after complete reaction under a fixed assay parameters was

measured. The concentration of the reagent which produced maximum colour

intensity was taken for analysis.

Effect of temperature: Variation of colour intensity with temperature was studied

at room temperature, as well as by heating on waterbath for different time

intervals. The heating parameters which produced the maximum colour intensity

within shortest time interval, was used for analysis.

Order of addition of reagents and colour development time: Order of addition of

reagents were varied taking a fixed concentration of the reagents and the

corresponding drug. The colour development time to get the maximum colour

intensity for each set of the order of addition was observed. The order, which

produced maximum colour intensity in minimum time interval, was selected for

assay parameters.

Stability of colour: Stability of the coloured species was studied by measuring the

developed colour intensity at different time intervals.

Effect of solvent: The studies on the influence of other water miscible polar

solvents such as methanol, ethanol, isopropanol and tbutanol instead of water

revealed that aqueous medium was the best for maximum colour development.

Cobalt chelates of nitrosoderivative were extractable in chloroform.

Calibration curve and optimum concentration range: Calibration curves were

constructed by measurement of the absorbance developed by known concentration

26

of the constituents under optimum conditions against reagent blank. Straight lines

were obtained conforming following of Beer’s law in the concentration under use.

Sensitivity: Sensitivity of the colour reaction has been expressed as molar

absorptivity (), which is calculated from the equation

= bCA

where, A = absorbance; C = concentration of coloured species

(mol. 11); b = light path length (cm); is expressed

as 1 mol1. cm1.

1.3.10 For the Chromatographic Methods:

Selection of solvents: Selection of solvent for preparation of standard and sample

solutions are based on compatibility with the techniques adopted. Selection of

solvents for mobile phase are based on polarity of the solute and solvents.

Study of impurities/adultration: The methodology concerned with the detection of

impurities and adultration were developed by considering possible existence of

such substances.

Sensitivity of detection: Minimum detectability limit under each experimental

condition were studied.

System suitabiulity test: To ascertion the suitability and effectiveness of the final

operating system for the HPLC determinations, it was subjected to a suitability test

prior to use. Specific data were collected from the replicate injections of the

standard preparations and efficiency, precision, tailing factor, resolution, retention

time, nature of the calibration curves and recovery were studied.

1.3.11 Interference Studies:

In the estimations of therapeutically important drug substances, the effect of

wide range of excipients, diluents, adjuvants, lubricants, binders, preservatives and

27

other coformulated potent compounds usually present in dosage forms, were

studied according to the nature of formulations as follows:

Excipients: Aerosil 400, Dibasic Calcium Phosphate.

Adjuvants: Activated Charcoal, Light Kaolin, Heavy Kaolin and Talc.

Lubricants: Stearic Acid and Magnesium Stearate.

Diluents: Lactose, Starch and Sucrose.

Ointment base: Anhydrous Lanolin, Soft Paraffin, Bee’s Wax.

Moistening agents/binders: Anacia Mucilage, Gelatin, Liquid Glucose.

Preservatives: Methylparaben, Propylparaben, Phenol, Cresol and Sodium

Benzoate.

In the initial interference studies, a fixed concentration of the drug substance

was determined several times by the optimum procedure in the presence of a

suitable (1100 fold) molar excess of the foreign compounds under investigation,

and its effect on the applicability of the method was observed. The foreign

compound was considered to be not interfering, if at those concentrations it

consistently produced an error less than 1 per cent.

1.3.12 Application to Commerical Formulations and Stnadard Mixtures:

The developed methodology were applied to commercial formulations as

well as to synthetic standard mixtures to determine validity and applicability of the

methods.

1.4. STATISTICAL EVALUATION OF RESULTS:

1.4.1 Precision:

The precision or reproducibility of the analytical results were calculated in

terms of Standard Deviation ‘s’.

s = V

NNXX

1

/)(22

28

where, ‘N’ is number of repeated measurements of a quantity

‘X’, and ‘V’ is the Variance about the Mean ‘ X ’

X = (X)/N

For practical interpretation sometimes it is more convenient to express ‘s’ in

terms of per cent of the average of the repeated measure ‘ X ’ used in the

calculation of ‘s’. This is called the per cent Coefficient of Variation (% C.V.) or

per cent Relative Standard Deviation (% RSD)

% C.V. or % RSD = 100Xs

Standard Error is the quantity used to calculate the Limits of Error of a

Mean. For most of the assays a probability level ‘P’ of 0.95 (P’ = 0.05) is taken,

meaning that in 19 cases out of 20, the true result will lie within the limits. When

the standard errors are small, a probability level of 0.99 (P|=0.01) i.e. 99 cases out

of 100 is often used, Thus,

Standard error = s/N

and Limits of error = t s/N

where ‘t’ is the factor available from Students Tbale.

1.4.2 Tests of Significance:

The ttest: The test decides whether the difference between the mean results of a

sample by two different methods are significant or insignificant. If ‘mA’ and ‘mB’

are means of two results of ‘NA’ and ‘NB’ values respectively, then in order to

decide whether the difference (mAmB) is significant

t = (mAmB)

22

)2(

BABA

BABAddNN

NNNN

where dA2 = sA2 (NA1) and dB2 = sB2 (NB1)

29

The value is compared with the critical limiting values of ‘t’ for different

probability levels.

The Ftest (Variance ratio test): This test is used to compare variance values of

two results of a sample by two different methods, and compared with the

theoretical limiting values of ‘F’ for given probability levels.

F = VA/VB = sA2 /sB2

where ‘sA2’ and ‘sB2’ are the standard deviations of two results.

1.4.3 Linear Regression:

To determine best fitting line or curve, regression analysis is performed by

application of method of least squares. The equations of the regression, or least

squares, line is

Y Y = XX

b =

NXXNYXXY

/

/22

where Y & X are the arithmetic mean and ‘b’ is the slope of the line.

Correlation coefficient: Before carrying out a regression calculation, a test is

made to determine whether there is a significant linear correlation between the sets

of figures. This can be done by calculating the correlation coefficient ‘r’.

r =

}]/}{/[{

/2222 NYYNXX

NYXXY

1.4.4 Accuracy:

The accuracy of the recommended procedure was evaluated by comparing

the results of the proposed method with the well established reported method,

using either synthetic mixtures or commercial formulations. In absence of well

30

established methods the recovery results of standard mixtures were evaluated to

determine accuracy.

1.4.5 Percent Recovery Studies:

Recovery studies were performed by adding known quantities of the drug

substance to the preanalysed formulations, using the proposed procedure. To

study per cent recovery, fixed amount of the sample was taken and different levels

of standard solutions were added. Each level were subjected to the desired

repeated measure and total amount of the drug substance was then determined.

Recovery (%) =

100.

..22

xXXN

YXXYN

where X = amount of the drug added in mg per g or mg per ml of

the sample.

Y = amount of the drug found in mg per g or mg per ml of

the sample.

N = total number of observations.

1.4.6 Adherence to Beer’s Law

To study linearity of the methods, Beer’s law curves were plotted and the

concentrations range within which the method followed linearity were determined.

1.5. INSTRUMENTS AND EQUIPMENTS USED:

The following instruments and equipments (R&D Division, Central Indian

Pharmacopoeia Laboratory, Ghaziabad) were used for conducting the

experimental work and SGSIndia Private Ltd., Gurgoan.

i) Beckman 24 UV/Vis, double beam spectrophotometer with recorder.

ii) Hitachi 15020 double beam recording Spectrophotometer

(microprocessor controlled).

31

iii) PerkinElmer Gas Chromatograph, Model 8500, with GP100 Graphics

Printer. (microprocessor controlled).

iv) Waters Liquid Chromatograph, equipped with Data Module M 730, M45

and 6000 A solvent delivery system; Series 440 Absorbance Detecter;

Model U 6 K Universal Injector.

v) Ultrasonic bath (Brasonic 220).

vi) Corning pH meter, Model 7.

vii) Camag TLC Scanner II, with Camag PC Evaluation system (MBC 990)

and Printer Star NL10.

viii) Camag Linomat III, TLC applicator.

ix) Camag Shortware (254 nm) U.V. lamp (Portable).

x) Mettler DL 40 RC Memo Titrator; with GA 40 Printer.

xi) Millipore Filtration Unit.

xii) Remi Centrifuge,Model R8C.

xiii) Mettler H 54 AR Balance.

xiv) Hot Air Oven

xv) Dessicating Cabinet

xvi) Hemilton Syringe (1 µl; 10 µl; 25 µl).

1.6 GENERAL NOTICES:

Reference Standards: The reference standards used for preparation of the standard

solutions were either C.D.L. reference standards or authentic specimens that have

been verified with the official compendia. (obtained from Central Indian

Pharmacopoeia Laboratory, Ghaziabad).

Commercial formulations: All the usually available marketed samples or received

from C.I.P.L. Ghaziabad were used for verification of the methodology.

32

Chemicals and reagents: Unless otherwise specified, all laboratory grade

chemicals and the reagents as spedcified in I.P. [1] were used.

Water: Unless otherwise specified singleglass distilled water was used, (pH~7).

Filtrations: Where it is directed to filter, without further qualification, it is

employed that the solution was filtered through medium porosity sintered glass

funnel using suction.

Centrifugation: Where it is directed to centrifuge, it is implied that the solution

was centrifuged at 3000 r.p.m. at room temperature.

Waterbath: The term ‘water bath’ means a bath of boiling water, unless water at

some specific temperature is indicated.

Temperature: All the temperature measurements are expressed in Celsius

(Centigrade) scale.

Working temperature: Unless otherwise specified all the experiments were carried

out at 25 5C.

Warm: Any temperature between 30 and 40C.

Cool: Any temperature between 8 and 25C.

1.7 ABBREVIATIONS:

g = Gram; mg = Milligram; mcg = Microgram; ng = Nanogram;

l = Litre; ml – Millilitre; µl = Microlitre;

mol = Grammolecular weight (mole)

cm = Centimeter; mm = Millimeter;

psi = Pounds per square inch.

min = Minutes; hr(s) = hour(s)

mp = Melting point; bp = Boiling point

AUFS = Absorbance unit full scale.

33

RT = Retention time; Rf = Chromatographic retardation factor;

hRf = Rf 100

A = Absorbance; A = Difference absorbance

D = Derivative absorbance; D = Derivative difference absorbance

D1 = First derivative absorbance;

D2 = Secondderivative absorbance

max = Wavelength of maximum absorption

DMF = Dimethyl formamide

MeOH = Methanol

EtOH = Ethanol

34

CHAPTERI

REFERENCES

1. L.Heilmeyer, Spectrophotometry in Medicine, Adam Holger Ltd., London

1943, P7, 65.

2. C.N.Reilley and D.T.Sawyer, Experiments for Instrumental Methods,

McGraw Hill Book Company, New York and London.

3. L.G.Chatten, Pharmaceutical Chemistry, Vol. II, Marcel Dekker, New York

and London 1969.

4. M.Ismail & A.L.Glenn, J.Pharm. Pharmacol, 16 (1964) Suppl. 150 T 155T.

5. W.A.Schroeder, P.E.Wilcox, K.N.True Blood and A.O.Dekker, Anal.

Chem. 23 (1951) 1740.

6. K.Kuratani, J. Chem. Soc. Japan Pure Chem. Sect. 72 (1951) 632.

7. R.C.Hirt, F.T.King and R.G.Schmitt, Anal. Chem. 26 (1954) 1270.

8. J.M.Bonnier, and G.De Gaumaris, Bull. Soc. Chim. France 67 (1955).

9. M.Ish,Shalom, J.D.Fitzpatrik and M.Orchin, J. Chem. Educ. 34 (1957) 496.

10. A.L.Glenn, J. Pharm. Pharmacol. 12 (1960) 595608.

11. M.Pernarowski, A.M.Knevel and J.E.Christian, J. Pharm. Sci. 50(11) (1961)

943953.

12. M.J.Cho and M.Pernarowski, J.Pharm. Sci. 59(9) (1970) 13331335.

13. R.C.Fedynec, G.A.Lewis and H.N.E., Stevens, J. Pharm. Pharmacol, 35

(1983) (Supplement) 104P.

14. F.Yokoyama and M.Pernarowski, J.Pharm. Sci. 50(11) (1961) 953, 2957.

15. A.M.Wahbi, H.Abdine, M.A.Korany, and F.A. ElYazb, J.Pharm. Sci.

67(1) (1978) 140141.

16. A.Ghanem, M.Meshali, J.Pharm. Pharmacol. 31 (1979) 122123.

35

17. H.N.Joshi,G.N.Chaudhari, H.P.Tipnis, Indian Drugs 19 (1982) (8) 446450.

18. M.A.Korany, A.M.Wahbi, M.A.Elsayed and S.Mandour, Anal. Lett; 17

(1984) 13731389; Throu. Anal. Abst; 47 (5) (1985) E30.

19. L.J.Dombrowski, R.S.Browing and E.L.Pratt, J.Pharm. Sci. 66 (10(1977)

14131415.

20. A.A.Wahbi, H.Abdine M.Korany and F.ELYazbi, J. Assoc. Off. Anal.

Chem. 62 (1) (1979) 6770.

21. L.Elsayed, S.M.Hassan, K.M.Kelani and H.M.ElFatatry, J.Assoc. Off.

Anal. Chem; 63(5) (1980) 992995.

22. S.M.Hassan, M.E.S.Metwally and A.M.A.Ouf, J. Assoc. Off. Anal. Chem;

66(6) (1983) 14331435.

23. N.M.Sanghavi and S.P.Kulkarni, Indian Drugs. 17(7) (1980) 299303.

24. C.Bagavant and V.Roshani, Indian Drugs 23(6)(1986) 373376.

25. R.R.A.Pride and E.s.Stern, J. Pharm. Pharmacol. 6 (1954) 590.

26. S.El Masry & A.A.M. Wahbi, J.Assoc. off. Anal. Chem. 61(1)(1978)6567.

27. M.Scott, A.Taub and C.Dosi Pianta, J.Am. Pharm. Assoc. Sci. Ed. 45(1956)

232236.

28. R.N.Bhattacharya & A.K.Ganguly, J.Pharm. Pharmacol. 4 (1952) 190196.

29. B.W.Madsen, D.Herbinson, Evans. And J.S.Robertson, J. Pharm. Pharmacol

26(1974) 629636.

30. R.A.Mortan and A.L.Sturbs, Analyst. 71 (1946) 348356.

31. A.L.Glenn, J.Pharm. Pharmacol 15 (Suppl) (1963) 123T130T.

32. H.Abdine, A.M.Wahbi and M.A.Korany, J. Pharm. Pharmacol 23 (1971)

444447.

36

33. H.Abdine, A.M.Wahbi and M.A.Korany, J. Pharm. Pharmacol 24 (1972)

518524.

34. M.A.Korany, M.Bedair and F.A.ElYazbi, Analyst 111 (1986) 4144.

35. A.M.Wahbi and H.Abdine, J. Pharm. Pharmacol. 25 (1973) 6972.

36. I.Agwu and A.L.Glenn, J.Pharm. Pharmacol 19 (Suppl.) (1967) 765.

37. M.A.Korany and R.Haller, J. Assoc. Off. Anal. Chem. 65 (1982) 144.

38. J.H.Jones,G.R.Clark & L.S.Harrow, J.Assoc.Off.Anal. Chem. 34 (1951)135.

39. C.F.Hiskey, Anal. Chem; 33 (1961) 927.

40. T.D.Doyle and F.R.Fazzari, J. Pharm. Sci. 63 (12) 1974) 19211926.

41. T.C.O. Haver and G.L.Green, Anal. Chem. 48 (2) (1976) 312318.

42. J.Traveset, V.Such, R.Gonzalo and E.Gelpi, J. Pharm. Sci. 69(6) (1980)

629633.

43. A.M.Wahbi, M.Barary, H.Mahgoub and A.H.Elsayed; J. Assoc. off. Anal.

Chem; 68(5) (1985) 10461048.

44. F.A.ElYazbi, M.A.Korany, O.A.Razak and M.A.Elsayed, J. Assoc. Off.

Anal. Chem. 69(4) (1986) 614618.

45. F.A.ElYazbi, M.A.Korany and M.Bedair, J. Pharm. Belg. 40(4) (1985)

244248; Throu. Anal. Abst. 48 (7) (1986) E29.

46. A.E.McDowell and H.L.Pardue, J. Pharm. Sci; 67 (1978) 822.

47. A.M.Wahbi, H.Abdine and M.A. Korany, Pharmazie, 33 (1978) 278.

48. T.D. Doyle and F.R. Fazzari, J. Pharm. Sci. 63 (1974) (12), 19211926.

49. M.R.I. Saleh, A.A.M.Habib and N.E.Shaer, J. Assoc. Off. Anal. Chem.

63(6) (1980) 11951199.

50. A.A.M. Habib and M.R.I. Slach, J. Associ off. Anal. Chem. 63 (3) (1980)

11951199.

37

51. N.Shane and M.Kowblansky, J. Pharm. Sci. 57(7) (1968) 12181223.

52. A.M.Wahbi and A.M. Farghaly, J. Pharm. Pharmacol. 22 (1970) 848850.

53. R.V.D. Ronadia, A.L.Bondani & J.D. Coussio, J. Pharm. Sci. 62 (1973)502.

54. H.Abdine, A.M.Elsayed and Y.M.Elsayed, Analyst 103 (1978).

55. M.A.H. Elsayed, S.F.Belal, A.F.M. Elwality and H.Abdine, J. Assoc. Off.

Anal. Chem. 62(3) (1979) 549551.

56. A.G.Davidson, J. Pharm. Sci; 73 (1) (1984) 5558.

57. M.A.H.Elsayed, S.F.Belal, A.F.M.Elwality and H.Abdine, Analyst 104

(1979) 620625.

58. W.P.Ferren and N.A.Shane, J.Assoc. off. Anal. Chem. 57 (1968) 573.

59. N.A.Shane and J.I.Routh, Anal. Chem. 39 (1967) 414.

60. S.M.Amer, S.M.Hassan and M.F.E.Tarro Anal. Lett. Part (B) 13 (18) (1980)

16251634 Throu Anal. Abst. 41 (3) (1981) E44.

61. V.G.Belikov, E.N.Vergeichik, S.Mutsueva, Kh 33 (2), (1984) 4446;

Throu; Anal. Abstr; 47(2) (1985) E31.

62. Z.Berakova, M.Bachrata, M.Blesova and L.Knazko, Farm. Obz; 49 (1980)

157167; Through; Anal. Abst; 39(5) (1980) E40.

63. A.A.M. Wahbi M.B.H. Mahgoub, M.A.H. Elsayed, J. Assoc. Off. Anal.

Chem. 68 (5) (1985) 10451050.

64. S.Gorog, J. Pharm. Sci; 57 (1968) 1737.

65. L.Chafetz, D.C.Tsilifonis, and J.M.Riedt, J. Pharm. Sci; 61 (1972) 148.

66. A.G.Davidson, J. Pharm. Pharmacol; 28 (1976) 795800.

67. M.A.H. Elsayed, H.Abdine and M.E.A. Hamid, Analyst. 104 (1979)

568572.

38

68. J.Traneset, V.Such, R.Gonzalo and E.Gelpi, J.Pharm. Sci; 69(6) (1980)

629633.

69. A.F.Fell, Proc. Anal. Div. Chem. Soc; 15 (1978) 260.

70. T.C.O.Haver, Anal. Chem; 51 (1979) 91A94A.

71. A.F.Fell and G.Smith, Anal. Proc. 19 (1982) 28.

72. A.G.Davidson, H.Elaheik, Analyst 107 (1982) 879.

73. A.T.Giese and C.S.French, Appl. Spectrosc 9 (1955) 78.

74. T.C.O.Haever and G.L.Green, Anal. Chem; 48(2) (1978) 312318.

75. A.F.Fell, Proc. Anal. Div. Chem. Soc; 15 (1978) 260.

76. A.M.Wahbi, M.A.Abounassif, and H.M.G.AlKahtani, Analyst. 111 (1986)

777780.

77. T.C.O.Haver and G.L.Green, Am. Lab; 7 (1975) 15.

78. J.W.Strojek, D.Yates and T.Kuwana, Anal. Chem; 47 (1975) 1050.

79. G.L.Green and T.C.O. Haver, Anal. Chem; 46 (1974) 2191.

80. R.N.Hager, Anal. Chem. 45 (1973) 1131A.

81. E.F.McFarren, R.J.Lisaka and J.H.Parker, Anal. Chem. 42 (1970) 358.

82. H.L.Pardue, A.E.McDowell, D.M.Fast and M.J.Milan, Clin Chem. 21

(1975) 11921200.

83. A.Savitzky and M.J.E.Golay, Anal. Chem; 36 (1964) 1627.

84. G.L.Green, T.C.O. Haver, Anal. Chem; 46 (1974) 2191.

85. R.N.Hager, Anal. Chem. 45 (1973) 1131A.

86. T.C.O. Haver and G.L.Green, Anal. Chem; 48(2) (1976), 312318.

87. W.Snellman, T.C.Rains, K.W.Yee, H.D.Cook and O.Menis, Anal. Chem. 42

(1970) 399.

88. R.C.Elser and J.D.Winefordner, Anal. Chem. 44 (1972) 698.

39

89. T.E.Cook, H.L.Pardue and R.E.Santini, Anal. Chem. 48 (1976) 451.

90. T.E.Cook, R.E.Santini and H.L.Pardue, Anal. Chem. 49 (1977) 871877.

91. M.J.Milani, H.L. Pardue, T.E.Cook, R.E. Santini, D.W.Margerum and

J.M.T.Raycheba, Anal. Chem. 46 (1974) 374.

92. A.E.McDowell and H.L.Pardue, J. Pharm. Sci; 67 (1978) 822.

93. N.A.Abdel Salam, M.A.Hadi and Y.A.Hamid, Acta Pharm. Jugosl, 27

(1977) 8187.

94. N.A.Abdel Salam, M.A.Hadi and S.M.K. Khafagy, Pharmazie, 30 (1975)

402403.

95. Y.A.Mohamed, N.A.Abdel Salam, N.A.Elsayed and M.A.Abdel Salam, Ind.

J. Pharm. 28 (1976) 117119.

96. G.M.Junego and A.L.Glenn, Chemy. Ind. 75 (1956) 813814.

97. A.M.Wahbi and S.Ebel, Anal. Chim. Acta 70 (1974) 5763.

98. M.A.Korany, A.M.Wahbi, M.A.Elsayed and S.Mandour, Farmaco Ed. Pr.

39 (1984) 243252; Throu; Chem. Abst. 101 (1984) 116795w.

99. A.GF.Davidson and S.M.Hassan, J.Pharm. Sci. 73(3) (1984), 413416.

100. M.A.H.Elsayed, S.Belal, A.M.ElWality and H.Abdine, J. Pharm. Sci; 68

(1979) 739741.

101. M.S.Mahrous, M.M.Abdel, Khalek and M.E.AbdelHamid, J. Assoc. Off.

Anal. Chem. 68 (1985) (31), 535539.

102. M.A.Korany, A.M.Wahbi and I.I.Hewala, Arch. Pharm. Chem. Sci. Ed; 12

(1984) 2630; Throu; Chem. Abstr. 101 (1984) 137106n.

103. M.Abdel Hamid, M.A.Korany and M.Bedair, Acta Pharm. Jugosl, 34 (1984)

183190.

104. V.Such, J.Travest and R.Gonzalo, Anal. Chem. 52 (1980) 412419.

40

105. F.A.ElYazbi and M.Barary, Anal. Lett. 52 (1980) 412419.

106. M.A.Korany, M.Bedair, H.Mahgoub and M.A.Elsayed, J.Assoc. Off. Anal.

Chem. 69(4) (1986) 608611.

107. K.Kitamura and R.Majima, Anal. Chem. 55 (1983) (1), 5456.

108. K.Keisuke and M.Ryo, Anal. Chem; 55 (1983) 5456.

109. A.F.Fell and A.G.Davidson, J. Pharm. Pharmacol. 32 (1980) (Suppl.) 97.

110. D.Y.Tobias, J.Assoc. Anal. Chem. 66 (6) (1983) 14501454.

111. M.A.Korany and A.A.ElDin Seif, J.Assoc. off Anal. Chem. 67 (1984)

138141.

112. S.Shibata, M.Furukowa and K.Goto, Anal. Chim. Acta 65 (1973) 4958.

113. S.Shibata, K.Goto and Y.Ishiguro, Anal. Chim. Acta 62 (1972) 305310.

114. M.A.Korany, A.M.Wahbi, S.Mandour and M.A.Elsayed, Anal. Lett; 18

(1B) (1985) 2134.

115. M.E.Mohamed, Anal. Lett; 19 (1986) (1112) 13231339.

116. F.A.ElYazbi, M.A.Korany and M.Bedair, J. Pharm. Belg. 40(4) (1985)

244248; Throu; Anal. Abstr. 48(7) (1986), E29.

117. F.A.ElYazbi, M.A.Korany, O.Abdel Rajak and M.A.Elsayed, Anal. Lett;

18 (1985) (B17); 21272142; Throu, Anal. Abstr. 48(9) (1986) E55.

118. N.A.ElSebakly, A.A.ElDin Seif, and M.A.Korany, J.Pharm. Belg. 41(4)

(1986) 222235. Throu. Anal. Abst. 49 (6) (1987) E11.

119. A.M.Wahbi, M.Barary, H.Mahgoub, and A.H.Elsayed, J. Assoc. Off. Anal.

Chem. 68(5) (1985) 10461048.

120. F.A.ElYazbi, M.A.Korany and M.Bedair, J. Pharm. Belg. 40(4) (1985)

244248 Throu; Anal. Abstr. 48(7) (1986), E29.

41

121. S.F.Belal, M.ElSayed, A.AbdelHady Elwality and H.Abdine, Analyst;

104 (1970) 919927.

122. S.M.Amer, M.M.Ellalitly and M.F.El Tarasse, Pharmazie 37(3) (1982)

182184; Throu; Anal. Abst. 43(4) (1982) E58.

123. A.A.El Kheir, S.Belal and E.L.Shanwani, Pharmazie 40 (1985) (1) 62;

Throu; Anal. Abst. 47(9) (1985) E41.

124. A.Abou Out, M.I.Walash, S.M.Hassan and S.M.ElSayed, Analyst, 105

(1980) (1247), 169173.

125. M.M.Sanghavi, N.G.Jivani and P.D.Mluye, Ind. J. Pharm. Sci; 39 (1977)87.

126. M.C.Inamdar and N.M.Kadji, Ind. J. Pharm. 31 (1969) 79.

127. L.Chafetz,R.E.Daly,H.Schriftman & J.J.Lomner,J.Pharm.Sci.60 (1971) 463.

128. M.M.Amer, M.I.Walash, I.A.Haroun, and F.M.Ashour, J. Pharm. Belg.

33(5) (1978) 297302; Throu; Anal. Abstr. 38(6) (1980) E23.

129. M.G.Rana, H.K.Dhyani, M.R.Patel and B.J.Shah, Ind. Drugs 17 (5) (1980)

142144.

130. M.T.Swett, Proc.Warsaw, Soc.Nat.Sci; 1903, Biol. Section 14 Minute No.6.

131. E.Stahl, Pharmazie 11 (1956) 633.

132. Clarkes, Isolation and Identification Drugs 2nd Ed. The Pharmaceutical

Press, London (1986).

133. Kurt Randerath, Thin layer chromatography, 2nd Ed. Academic Press, New

York (1968).

134. R.Kuhn and E.Lederer, Berdtsch. Chem. Ges. 64 (1931) 1349.

135. A.J.P.Martin and R.L.M.Synge, Biochem. J. 35 (1941) 1358.

136. R.Consden, A.H.Gordon and A.J.P. Martin, Biochem. J. 38 (1944) 224.

137. Pharmacopoeia of India, 3rd Ed. Controller of Publications, Delhi (1985).

42

138. British Pharmacopoeia, University Press, Cambridge 1980.

139. The United States Pharmacopoeia/National Formulary, United States

Pharmacopoeial Convention, Rock Ville 1985.

140. Strain, Chromatographjic Adsorption Analysis, Inter Science Publishers,

Inc. New York (1945).

141. Ion exchange chromatography, F.Harold Walton, Dowden, Hutchinson &

Ross Inc. Pennsylvania.

142. Chromatographic and Electrophoretic Techniques, Ivor Smith Vol.I Inter

Science Publishers, Inc. U.S.A. (1960).

143. Official Methods of Analysis of A.O.A.C. 13th Ed. Association of Official

Analytical Chemists, Benzamin Franklin Station, Washington, 1980 p704.

144. W.Ramsey, Proc. Roy. Soc. A 76 (1905) 111.

145. A.J.James and A.J.P.Martin, Analyst, 77 (1952) 915.

146. A.J.P.Martin and R.L.M.Synge, Biochem. J; 35 (1941) 1358.

147. S.Roger Schirmer, Modern methods of Pharmaceutical Analysis, Vol. III

CRC Press, Inc. Florida.

148. Gas Chromatography, A.B.Little Wood; The Institute of Petroleum, London

(1966).

149. G.A.Haward and A.J.P. Martin, Biochem. J. 56 (1950) 539.

150. I.Halasz and I.Sebastian, Angew Chem. 8 (1969) 453.

151. J.E.Cortes, R.Pazdur, J. Clin Oncol 13 (1995) 26432655.

152. J.Dubois, D.Guenard, F.Gueritte, Expert Opin Ther. Pat. 13 (2003)

18091823.

153. C.H.Smorenburg, A.Sparreboom, M.Bontenbal, G.Stoter, K.Nooter, J.

Verweij, J. Clin Oncol 21 (2003) 197202.

43

154. M.J.Egorin, J. Clin Oncol 21 (2003) 182183.

155. S.Mao, X.Shuai, F.Unger, M.Simona, D.Bu T.Kissel, Int. J. Pharm. 281

(2004) 4554.

156. P.Dharmani, W.Singh Chauhan, U.Palit, Eur. J. Pharmacology, 519 (2005)

27784.

157. C.Immanuel, A.K.Kumar, J.Chromatogr. (B). Biomed. Sci. Appl. 760

(2001) 9195.

158. M.B.Patel, K.M.Patel, O.S.Patel, B.N.Suhagia, A.M.Prajapati, J.Liq.

Chromatgr. Rel. Technol. 30(2007) 245971.

159. J.Turek, M.Reinbers, D.Dreyer, T.K.Kiffmeyer, K.G.Schmidt, H.M.Kwens,

J. Chromatogr. (B) 83 (2006) 7280.

160. S.Sharma, A.Bhandari, U.R.Choudhary, H.Rayapurohit and P.Khandelwal,

Ind. J. Pharm. Sci. 73(1) (2011) 8488.

161. P.Pattanayak, U.P.Chaudhary, P.Sharma, P.Mahapatr, D.K.Setty, Res. J.

Pharm. And Tech. 2 (2009) 2913.

162. S.Mohammed, G.Mohsin, S.Anwar, J.Chromatogr. Sci. 40 (2002) 42931.

163. R.Yanamandra, C.S.Vadla, U.M.Puppala, B.Patro, Y.L.N.Murty and

B.R.Parimi, Jour. of Pharm. Sci.; 74(2) (2012) 116121.

164. S.R.Desai, U.V.Laddi, R.S.Bennur and S.C.Bennur, Ind. Jour. of Pharm.

Sciences 73(4) (2011) 478482.

165. H.B.Lea,T.F.Perata,M.L.Svobodale, J.Chromatogr.(A) 1055 (2004) 22328.

166. P.R.Kalra,R.Sharma,S.C.Chaturvedi,Ind.Jour.of Pharm.Sci.70(2008) 4914.