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
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