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QUANTITATIVE ANALYSIS OF DRUGS IN PURE FORM AND IN PHARMACEUTICAL PREPARATIONS
USING ANALYTICAL TECHNIQUES
T H E S I S SUBMriTEO FOR THE AWARD OF THE DEGREE OF
IBottor of $I|ilo£ioplip
CHEMISTRY
iv T A L A T g A Y O O M
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH ( INDIA)
2000
/ , 'r' r
( Ace, No.
- t 1 U
The work embodied in the thesis comprises five chapters. The
first chapter describes a general literature survey of the
subject matter.' In order to illuminate an otherwise defunct
period, it is customary to credit ancient scholars right from
the past history of medicinal chemistry. For example, Ipecac,
from Brazillian Cephaelis species, was used as an
antiamebic medication. I^owever, based on superstitions &
misunderstanding of natural phenomena, their observations
and interpretations must be regarded as purely heuristic.
' The research on drugs with major therapeutic action is
introduced with the mention of earlier agents. Based on
structural reactivity, bacterial and antibacterial properties, a
vast multitude of drugs are observed in the present period. The
role of physico-analytical and organic chemistry in drug
designing is worth mentioning which provides the best
solution of the problem developed in biological- environment
such as unrelated factors, loss of drug by adsorption or
incomplete absorption.
The impurities associated with drugs from many sources are
therefore a subject of great attention by both manufacturer and
regulatory agencies. The requirements for quality, safety and
purity warrants careful thought over analytical chemists.
The classif icat ion of enormous drugs based on their
chemotherapeut ic action is important which requires a special
at tention to be elaborated.
The capital , basic and accessory analytical propert ies have
been well explained and their goals and relat ionships
establ ished in hierarchical manner.
Various quality improving act ivi t ies to ensure the rel iabi l i ty
of analytical data are discussed. Quality management needs of
research laborator ies , where drug analysis is carried out on
non-rout ine basis are well emphasized.
Due to avai labi l i ty of sub-standard medicine to the general
public in developing countr ies , it s crucial to assay drug
samples in formulat ions. Dealing with this concern, we made
some successful attempts in this direction. The recommended
methods are val idated following current guidel ines.
The second chapter describes the analysis of a series of
penici l l ins e.g. amoxycil l in, ampicil l in, c loxaci l l in ,
benzylpenic i l l in , carbenici l l in & becampici l in in
pharmaceut ical preparat ions using volumetric and
spectrophotometr ic methods, s imultaneously as well as
separately. In simultaneous method, the determination is based
on a single stage hydrolysis of penici l l ins by dilute sodium
hydroxide solution under specific experimental condi t ions.
The degraded products containing sulphur derivat ives are
neutral ized by dilute hydrochlor ic acid to prevail a pH~2 to be
conducive for redox ti tration using potassium iodate as t i trant.
A coloured product containing sulphonic acid is formed at the
end point in organic layer. Spectrophotometry is performed
after separat ing the organic layer and measuring the
absorbance of red-purple colour at 520nm.
The t i t r imetr ic method using dilute hydrochloric acid for the
est imation of degraded products of antibiotics obtained after
the hydrolysis by dilute sodium hydroxide has been found to
be advantegeous over the old Andrews t i t r imetr ic method. In
this method strong hydrochloric acid solution is required for
the quant i ta t ive reduction of iodate to iodine monochloride
complex. Using a dilute hydrochloric acid solution, it is
possible to reduce quanti ta t ively iodate to free iodine because
the degraded products of antibiot ics contain sulphur
derivat ives which can be oxidized by a very mild oxidizing
agent. The half reaction (E°) of iodate in strong hydrochlor ic
acid and in dilute acid respect ively is described below:
IO'3 + 6 H ' + 2Cl"+4e"
2IO"3 + 12H"+ lOe'
" • ICl ' s + 3H2O E ' ^ = 1 . 2 3 V
- • I2 + 6H2O E"=1.19V
The direct spectrophotometr ic method for the determinat ion of
degraded products of ant ibiot ics is performed under similar
experimental condit ions, except that excessive iodate ( ImL) is
used for the redox reaction. The mechanism of degraded
products is established in the light of TLC, UV and IR
spectroscopic studies. The stoichiometr ic ratio between the
analyte (penici l l in) and t i trant (KIO3) is evaluated.
The resul ts obtained for the degraded products by thin
layer chromatography conform to the presence of 4-
hydroxymethylene oxazolone. This observation is in agreement
with previous studies performed under similar condi t ions.
The reaction mechanism is further aided by conducting IR
study of the degraded products . The formation of a broad band
(1500-1350cm' ' ) in spectrum-B is at tr ibuted to the presence of
4-hydroxymethylene oxazolone. Besides, a strong peak at
835cm"' in spectrum-C is at tr ibuted to the presence of
sulphonic acid formed quant i ta t ively.
The third chapter describes two spectrophotometr ic methods
for the determinat ion of a non-steroidal anti- inflammatory
agent, i.e. diclofenac sodium in the visible region of spectrum.
In the first method, the formation of charge-transfer complex
with chlorani l ic acid is utalized to carryout est imation. The
resul t ing predominant chloranil ic acid radical anion is an
intermediate molecular associat ion compound that exhibits a
character is t ic absorption maximum at 530nm. The second
method is based on the reduction of Iron(III) by diclofenac &
subsequent complexation with 1,10-phenanthroline to form a
reddish chelate complex, which shows A,max. at 500nm. The
reduction of Iron(III) and the formation of I ron(II ) -1 ,10-
phenanthrol ine complex depends on pH. The development of
colour is instantaneous in phosphate buffer solution in the pH
range 6-8. These two methods are satisfactori ly applied for
diclofenac content estimation in pharmaceut ical preparat ions .
In fourth chapter, UV spectrophotometr ic studies are carried
out to develop two different method3 for the analysis of
c isapride. In first method, Boyland-Sims oxidation is
performed for the purpose of its est imation. This involves the
t reatment of cisapride with peroxydisulphate in alkaline
medium on heating to yield a yel low coloured product having
Xmax. at 308nm. This reaction product is stable enough and
permit est imation with good reproducibi l i ty . In second method
the oxidimetr ic nature and high solution stabi l i ty of Ce(IV)
are uti l ized for the quanti tat ive determination of cisapride.
The acidic oxidation of the drug s tudUd with Ce(IV) yields a
coloured product having A,max. • • a t ^ 9 n m. Beens^'iaw obeyed in
\ { Ace. No ) n
the two methods that are described is in the range 10-
100mgmL"'and 20-120mgmL"' respectively.
The last chapter describes a kinetic spectrophotometric
determination of nalidixic acid in bulk, and its
phamaceuticals. The interaction of nalidixic acid with ferrous
ammonium sulphate yields a coordination compound, which
possess X,max. at 420nm. The intensity of colour requires forty
minutes to get stablized. It is therefore decided to perform the
kinetic study, which is sensitive, rapid and less time
consuming. The various reaction parameters evaluated conform
to the proposed mechanism. The kinetic studies reveal first
order reaction with respect to nalidixic acid.
QUANTITATIVE ANALYSIS OF DRUGS IN PURE FORM AND IN PHARMACEUTICAL PREPARATIONS
USING ANALYTICAL TECHNIQUES
T H E S I S SUBMITTED FOR THE AWARD OF THE DEGREE OF
Battot of ^dilosfopiip IN
CHEMISTRY
•* \ %
\
BY
TALAT QAYOOM
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH ( INDIA)
2000
T5388
THESIS
D e d i c a t e d
To m y P a r e n t s
Saidul Zafar Qureshi M.Sc, Ph.D.,C.Chem., MRIC ((London)
Professor of Aniytical Chemistry
Phone Off. 0091-571 - 703515 Res. 0091-571 - 400534
E-mail:- chtl6szq@amu.up.nic.in
ANALYTICAL RESEARCH LABORATORY DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALIGARH-202002 (INDIA)
Dated.
(EFrttftratF
This is to certify that the thesis entitled "Quantitative Analysis of Drugs in
pure form & in Pliarmaceutical Preparations using Analytical Techniques"
is the original work carried out by IVIr. Talat Qayoom under my supervision and is
suitable for the award of Ph.D degree in Chemistry.
(Dr. Saidul ZararOQureshi) Professor & Chairman
ACKNOWLEDGEMENTS
Many ideas and encouragements from several sources have gone into carrying out the present work. There are many who need to be acknowledged, but there are few who deserve special acknowledgement at a personal level.
With pr ivi lege, I express indebtedness to my supervisor Professor Saidul Zafar Qureshi Chairman, Department of Chemistry for his analytical thinking and ceaseless efforts, which made this work progressive. It was due to his organized methodology that developed in me a sense of sinceri ty and self-confidence. This made me familiar with related fields, which has really broadened the horizons.
I would like to accord special thanks to my esteemed teacher Dr. Nafisur Rehman for his feverest help during my research programme. He generously and graciously dispensed with innovative ideas in the form of suggest ions.
With pleasure, 1 express grat i tude to my colleagues Dr. Murad, Dr. Khyer, Abdullah. S. Ahmad, Nasrul Hoda, Najmul Hejaz and Nadeem Ahmad. 1 extend thanks to my friends Atta Ahmad, Sadat Kar, Anees Ahmad, Hassan Mohusein and Omar Basudan for always being alongside me through thick and thin.
A substantial amount of credit goes to my only sister, who put up with my long absence during iny studies.
The research facili t ies of the department deserve the highest level of acknowledgement, that made charming an otherwise somber and a wil ly-nil ly si tuation.
I am grateful to all the members of Anglo Computers for their herculean efforts in typing this thesis.
(Talat Qayoom)
We raise to degrees of wisdom whom We please; but overall endued with knowledge is One, the All-knowing.
(AL-Quran)
CONTENTS
Page No.
CHAPTER!
General Introduction 1-54
References 55-67
CHAPTER-2
Simultaneous Spectrophotometric and Volumetric Determination 68-109
of Penicillins in Drug Fornuilations: Reaction Mechanism in the
Base Catalysed Hydrolysis Followed by Oxidation with lodate in
Dilute Acid Solution.
CHAPTER-3
Spectrophotometric Determination of Diclofenac Sodium in Drug 110-131
Formulations.
CHAPTER-4
Ultraviolet Spectrophotometric Studies for the Estimation of 132-153
Cisapride in Pharmaceutical Dosage Forms.
CHAPTERS
Kinetic Spectrophotometric Method for the Determination of 154-172
Nalidixic acid.
LIST OF TABLES
Table No. Page No.
1.1 Validation of analytical methods-international definitions. 6
1.2 List of the six natural penicillins. 41
1.3 Type of R-groups in penicillins. 42
2.1 Volumetric determination of amoxycillin, ampicillin and 82
cloxacillin using potassium iodate as titrant.
2.2 Volumetric determination of benzylpenicillin, carbenicillin and 83
becampicillin using potassium iodate as titrant
2.3 Spectrophotometric determination of amoxycillin, ampicillin 84
and cloxacillin after separating the extracted coloured complex
form organic layer formed at equilibrium point using potassium
iodate as titrant.
2.4 Spectrophotometric determination of benzylpenicil l in, 85
carbenicillin & becampicillin after separating the extracted
coloured complex form organic layer formed at equilibrium
point using potassium iodate as titrant.
2.5 Spectrophotometric determination of amoxycillin, ampicillin 86
and cloxacillin after separating the extracted coloured complex
from organic layer formed by adding excess of potassium iodate.
2.6 Spectrophotometric determination of benzylpenicil l in, 87
carbeni.cillin, becampicillin after separating the extracted
coloured complex from organic layer formed by adding excess
of potassium iodate.
2.7 Maximum tolerance amount of excipients in the determination 88
of penicillins.
2.8 Determination of penicillins in pharmaceutical preparations by 89-90
the recommended method.
2.9 Treatment of analytical data using statistical approach for 91
various penicillins.
3.1 Test of reproducibility of the proposed methods for the 119
determination of diclofenac sodium.
3.2 Determination of diclofenac sodium in pharmaceuticals & their 120
comparison with the reference method & statistical treatment
of the analytical data.
3.3 Maximum tolerance amount of the foreign substances in the 121
determination of diclofenac sodium.
4.1 Maximum tolerance amount of excipients in the analysis of 141
cisapride.
4.2 Test of precision for the determination of cisapride by 142
recommended methods.
4.3 Results obtained for the determination of cisapride in 143
pharmaceutical preparations by proposed methods and
statistical comparision with reference method.
5.1 Values of absorbance at different concentrations of nalidixic 162
acid, keeping the cone, of ferrous ammonium sulphate constant.
5.2 Result obtained by standard addition procedure. 163
5.3 Maximum tolerance amount of interferences added in the 164
analysis of nalidixic acid.
5.4 Statistical comparision of results obtained by proposed & 165
reference methods.
5.5 Calibration data for nalidixic acid. 166
LIST OF FIGURES
Figure No. Page No.
1.1 Goals of analytical chemistry & their relationships. 8
1.2 Analytical tetrahedra. 9
1.3 Relationships among basic analytical properties. 10
1.4 Relationship between the precision & the cone, level of 11
analytes.
1.5 Distortion of the analytical tetrahedra. 13
1.6 General reaction sequence of the total synthesis of penicillin. 43
1.7 Structure of amoxycillin (a) & dicloxacillin (b). 45
2.1 Overconsumption of iodate at the end point in the recommended 77
procedure.
2.2 Andrews titration. 78
2.3 Possible degradation pathways of penicillin. 92
2.4 Pure amoxycillin, Spectrum-A. 93
2.5 Degraded product of amoxycillin in base catalysed hydrolysis, 94
Spectrum-B.
2.6 Degraded base hydrolysed product of amoxycillin treated with 95
O.IM hydrochloric acid, followed by titration against KIO3,
Spectrum-C.
2.7 Effect of temperature on the colour stability of penicillins. 96
2.8 Effect of time on the colour stability of penicillins. 96
2.9 Effect of varying volume of sodium hydroxide for penicillins. 97
2.10 Effect of varying volume of potassium iodate for penicillins. 97
2.11 Effectofvarying volume of hydrochloric acid for penicillins. 97
2.12 Absorption spectra of the reaction product of penicillins. 98
2.13 Relation between volume of titrant (KIO^) & the concentration 99
of penicillins in reference with the table 2.1.
2.14 Relationbetween volume of titrant (K103)& the concentration 99
of penicillins in reference with the table 2.2.
2.15 Calibration curves of penicillins in reference with the table 2.3 100
2.16 Calibration curves of penicillins in reference with the table 2,4 100
2.17 Calibration curves of penicillins in reference with the table 2.5 101
2.18 Calibration curves of penicillins in reference with the table 2.6 loi
2.19 Best straight line equation in the determinatin of penicillins in 102
reference with the table 2.1.
2.20 Best straight line equation in the determinatin of penicillins in 103
reference with the table 2.2.
2.21 Best straight line equation in the determinatin of penicillins in 104
reference with the table 2.3.
2.22 Best straight line equation in the determinatin of penicillins in 105
reference with the table 2.4.
2.23 Best straight line equation in the determinatin of penicillins in 106
reference with the table 2.5.
2.24 Best straight line equation in the determinatin of penicillins in 107
reference with the table 2.6.
3.1 Formation of chloranilic acid radical anion. 122
3.2 Absorption spectrum of charge-transfer complex formed by 123
chloranilic acid-diclofenac sodium.
3.3 Effect of time on the colour stability of reaction product. 124
3.4 Effect of varying volume of chloranilic acid. 124
3.5 Calibration graph of diclofenac sodium. 125
3.6 Jobs plot for the reaction of diclofenac sodium with chloranilic 126
acid.
3.7 Absorption spectra with Fe^^&Fe-'^ 127
3.8 Effectofvarying volume of 1,10-phenanthroline. 128
3.9 Effectofvarying volume of Fe^". 128
3.10 Calibrationgraphof diclofenac sodium. 129
4.1 Structure of Cisapride. 132
4.2 Absorption spectrum of cisapride-persulphate reaction 144
product in alkaline medium.
4.3 Effect of persulphate on the reaction product. 145
4.4 Effect of sodium carbonate on the reaction product. 145
4.5 Effect of heating time on the reaction product. 146
4.6 Effect of temperatrue on the reaction product. 146
4.7 Calibration graph of cisapride. 147
4.8 Absorption spectrum of cisapride-Ce (IV) reaction product. 148
4.9 Effect of cerium (IV) on the reaction product. 149
4.10 Effect of nitric acid cone, on the reaction product. 149
4.11 Calibration graph of cisapride. 150
5.1 Tentative reaction mechanism of nalidixic acid with ferrous 159
ammonium sulphate.
5.2 Absorption spectra of nalidixic acid-ferrous ammonium sul- 167
phate reaction product.
5.3 Plot of absorbance vs. time for nalidixic acid, keeping the ^^^
ferrous ammonium sulphate cone, constant.
5.4 Calibration graphs for nalidixic acid (a) Initial reaction rate (R) 1 9
vs. concentration (C); (b) log R Vs. log C.
5.5 Jobs plot for the reaction of nalidixic acid with ferrous ammo- 1 0
nium sulphate.
RESEARCH PUBLICATION(S)
I. Simultaneous Spectrophotometric & Volumetric Determination of
Amoxycillin, Ampicillin and Cloxacillin in Drug Formulations;
Reaction Mechanism in the Base Catalysed Hydrolysis Followed by
Oxidation with lodate in Dilute Acid Solution.
J.Pharm.Biomed.AnaL, 21(1999) 473-482.
Chapter-1
(genera fOnfrodvtcBon
With the growing global awareness in health hazards and
environmental pollut ion, Analytical chemistry has played a
key role to unveil its causes and day by day it has broaden
its spectrum with the concerned situation. It is extensively
and intensively responsible for analytical measurements on
variety of materials such as drug analysis , microbial
analysis of food, water, cosmetics, agricultural products,
environmental spi^cies forensic sampling and products
affecting public health and welfare where quali tat ive, semi
quali tat ive and quantitative analysis on macro to micro scale
are performed. In recent decades analytical chemistry has
reached to the pinnacle of precession with the modern
sophist icated instrumentation techniques which make
possible to elucidate the microstructure of molecular
species, determining antibiotic residues in milk and tissues
of food producing animals, detection of complex
hydrocarbons, studies of rare and artificial radioactive
elements and to obtain substances in the highest state of
purity. Moreover, with the advent of digital computer the
precision is further enhanced. However, it has always
conceived the basic phenomenon of its revelation despite the
change in instrumentat ion. Analytical excellence has been a
great boon for chemists, microbiologis ts , entomologists and
other scientis ts , working in the mult i -discipl inary areas of
research.
Medicinal chemistry has taken place in many branches of
chemistry and biological science. The earlier name of
pharmaceutical chemistry reflects that pharmacists working
in their laboratories focussed on the extraction and
purif ication of naturally occurring drugs. In 1876 the
pharmacologis t Buchheim wrote that "the mission of
pharmacology was to identify the active substances with the
drug, to find out chemical propert ies responsible for their
action and to prepare synthetic analogs that were
more effective" [1] . The pharmacologists latter became
preoccupied with other objectives, such as " to study the
change brought about the drug in the organism and then to
explore the possible influence of such changes upon
pathological condit ion" [1], while the chemist took over the
role of isolation and chemical identification of biologically
active consti tuents from medicinally important plants.
Drug design has also been aided by the understanding of
biochemical metabolism and biosynthesis , and also by the
stat ist ical analysis of some relat ionship of physical
propert ies of chemicals and their biological performance.
This progress has begun to elevate medicinal chemistry to a
science in its own right. The fashion in which the drug is
searched changes, from time to time which is dictated by
contemporary medical, economical , sociological , and
sometimes even poli t ical considerat ions. In 1890
ant iprotozoal chemotherapy set in motion and the early
synthetic antimaterials intermingled with the development of
hypnotics and anti- inflammatory drugs. Then followed the
feverish discovery of ant ibacterials and ant ibiot ics , while in
pharmacodynamic area the potent analget ics ,
ant ih is taminics , vitamins and new hormones moved to the
fore. The post world war 11 period featured three major
breakthroughs in drug research; (i) the anti tuberculous
agent, (ii) the steroid hormones and contracept ives , (iii) the
ant ipsychot ic , anxiolytic and antidepressant psychopharma-
cological drug.
The application of increasingly sophist icated methods of
structural analysis like spectroscopy, isotopic labeling,
automated quantitative analysis , separation by
chromatography and their parti t ion procedures opened
avenues of study of minute amounts of biochemicals .
Hahnemann,^ the founder of homeopathy, believed that,
"Drug's solution should be diluted to the point of practically
omitting their content of active components because high
concentrat ion of drugs produce toxic react ions ." Thomson
stated that "all the diseases arc the effect of one general
cause and may be removed by one general remedy" [2] . Paul
Ehrlich, a German physician and immunologist turned
chemist is the first true exponent drug research as we know
it today [3].
During the period 1940 to 1960, the drug development,
introduct ion and pharmaceutical manufacturing entered their
"golden age". Very large proport ion of the important and
innovative new drugs in medical use were introduced during
this period. Public expectat ions of new "miracle drugs" led
to increased funding of medicinal research, especial ly by
National Insti tute of Health in the United States and by a
number of wealthy private foundations in several countr ies .
It is important to recognize that all drugs contain impuri t ies .
There impurit ies result from many sources; like from raw
materials and reagents as reaction by-products , and through
degradation during manufacture and storage. Since, the
impuri t ies can have safety and efficiency implicat ions, they
are, therefore, the subject of considerable attention by both
the manufacturers and regulatory agencies. Impurit ies can be
classified into three groups, namely organic, inorganic and
residual solvents. Organic impurit ies may arise from starting
mater ia ls , intermediates and synthetic by-products , or from
reagents and catalysts , or as a consequence of degradation.
Inorganic impurit ies may result from reagents , l igands or
catalysts , as heavy metals or inorganic salts. Residual
solvents are inevitable in drug substances since without
solvents, the synthetic chemistry, purification and
generation of the desired crystal morphology would be
impossible .
Qualif ication is defined as "the process of acquiring and
evaluating data which establish the biological safety of the
individual impurity or a given impurity profile at the levels
specif ied." Thus, the pharmaceutical analyst and
toxicologist must work hand in hand throughout the pre-
chemical and clinical development programme in order to be
able to set meaningful specification requirements . The
methods used for impurit ies measurements , determinat ions
and qualif ications are addressed in more detail in the
coming pages. The pharmaceutical analyst must give careful
thought to the analytical technology. Especial ly in the
development phases, it may be necessary to util ize methods
with high selectivity, including sophist icated techniques.
The importance of qualifying impurity profiles as also
relevant to the developmental scientists to ensure
considerat ion is given to the impurit ies present in batches
being used in safety studies, although there are
opportuni t ies to carry out metabolic studies to help in the
qualif ication processes.
The validation of analytical methods used in bioavai labi l i ty ,
bioequivalence and pharmacokinet ic studies in humans and
animals has been the subject of discussion in recent years
[4-8] . It has been agreed that the key for evaluation of
method rel iabi l i ty and overall performance are; (i) analyte
s tabi l i ty; (ii) method select ivi ty; (iii) limit of quanti ta t ion;
(iv) accuracy and precision; (v) relat ionship between
response and concentrat ion; (vi) recovery; and (vii)
ruggedness . Before an analytical method can be used for
routine analysis , it must first be demonstrated that the
method fulfills certain performance criteria. When this has
been documented, the method is said to be validated. The
first and one of main difficulties for the pract icing
analyt ical chemist is to decide exactly which parameters
should be measured and to set the performance criteria,
which have to be fulfilled before a method can be said be
val idated. Once the parameters have been fixed, it must be
shown that they meet performance criteria. How to do this is
the second difficulty that is faced by the analyst . However,
for a number of years analytical chemists in the
pharmaceut ical and biomedical field have learnt how to
demonstrate that their methods have a performance that can
guarantee reliable results after the advent of good laboratory
pract ice. We know this exercise as validation of analytical
methods. In the field of drug analysis , it is very clear that
the above definitions cover the entire field of analytical
chemistry from bioanalysis to substance and product
analysis . This is an important observation, as the same
principle should apply whatever type of sample is to be
analyzed. There are a number of definitions given by several
internat ional organizations [Table 1.1]. Most of them are
congruent .
Table 1.1
Validation of analytical methods-international definitions |9 | .
Organization
lUPAC
ILAC
WELAC
ICH
ISO
Appl i cab i l i t y
Worldwide
Worldwide
Europe
Europe, Japan, USA
Worldwide
Remarks
Pharmaceutical
products
Lacks selectivity
and specificity
ABBREVIATIONS:
lUPAC-International Union of Pure and Applied Chemistry; ILAC-International Laboratory Accreditation Conference; WELAC-Western European Laboratory Accreditation Corporation; ICH-International Conference on Harmonization; ISO-International Organization for Standardization;
The quality of the analytical information produced
in a laboratory depends on the quality of the information
available to managers as they define the analytical problem,
design the analytical process and adjust it features so that the
results produced meet the required objectives. It is important
to have a clear understanding of analytical properties such as
accuracy and representativeness as well as their relationship
to quality in the analytical process and to the results. These
two facets of analysis are frequently dealt with
inconsistently, resulting confusion in laboratory work and in
communication with clients or services users.
Types of analyt ical p rope r t i e s
Quality in the analytical results, the primary goal of
analytical chemistry, relies heavily on two capital analytical
properties; accuracy and representativeness. Accuracy means
consistency between the results obtained and the actual
concentration of the analyte in the particular sample,
representativeness refers to consistency between the results
and the analysed sample as well as between the results and
the definition of the analytical problem. Capital properties
rely on another group of analytical properties that we will
call basic properties. They determine the quality of the
analytical process both inside and outside the laboratory
during sampling. Basic properties are sensitivity, selectivity,
precision and sampling. The analytical process is also limited
by seemingly less significant accessory propert ies. These
properties, however, often have major practical implications.
They include expeditiousness, cost-effectiveness and
personal-related considerations, all of which affect laboratory
output. Figure 1.1 shows two essential components of
analytica-l quality and displays capital, basic and accessory
analytical properties in a hierarchical manner. It also outlines
the generic objectives of present day analytical chemistry;
obtaining larger amounts of analytical information of higher
quality by expending less material, time and human resources,
minimizing risks; and incurring the smallest expenses
possible [10]. Whereas capital analytical properties are
associated with results, basic and accessory properties are
related to the analytical methodology used.
Goals Quality
Rcsulls < Accuracy RepcseiUalivencss
/
Properties
apital
t i t t Obtaining more and better information
CA a S
a. *-
• ^
in n n n
^ M ft n
o 3
GO w 3
13
g-era
Basic
\ Analytical Process Expeditiousness
Cost-effectiveness
Accesson'
Minimizing analysis time, expenses labour, and hazards
Personnel Safetv/conifort
Figure 1.1 Goals of analytical chemistry and their
relationships to analytical quality and
analytical properties.
Analytical tetrahedra
The most straightforward and intuitive form of graphically
displaying analytical properties involves two tetrahedra that
share a vertex and can be of varying sizes relative to each
other [Fig. 1.2]. This illustration provides a useful means of
distinguishing among the three principle groups of analytical
properties, establishing a hierarchy, and determining
symbiotic and opposing relationship among them. Figure 1.2
also includes sampling, which determines representativeness.
Precision Cost-effectiveness
w o a. &
s v> 3 n </> w
Personnel Safelv/coinforl
Figure 1.2 Analytical tetrahedra.
The vertices of main tetrahedron (left) depict sensitivity,
selectivity and precision; one of its faces represents accuracy,
which relies on these three basic properties. This tetrahedron,
together with representativeness of sampling, has the greatest
statistical weight on analytical results and is connected with
the objectives of obtaining more and better information. The
secondary tetrahedron (right) has vertices depicting the
accessory analytical properties; one of its faces represents
laboratory productivity. This tetrahedron is related to the
objective^ of minimizing time, costs, labour and hazards.
Relationship among basic analytical properties
Figure 1.3 depict the opposing relationships among the three
basic analytical properties. Enhancing one usually can be
10
achieved only at the expense of the other two. The central
equilateral triangle represents a balanced situation and can be
distorted to an isosceles or scalene triangle, depending on
which property is favoured. The most salient relationships
among the three properties are discussed below.
Figure! .3 Relationships among basic analytical properties.
Prec i s ion and Sens i t iv i ty
It is well known that precision decreases as analyte
concentration decreases. Figure 1.4 shows an example of the
results from intercomparison of food analysis experiments in
which the coefficient of variation increases exponentially
with decreasing analyte concentration [ I I ] . Consequently, a
higher sensitivity method will also be highly variable when
applied to trace and ultratrace determinations. Incorporating
11
an analytical separation technique to indirectly enhance
sensitivity and facilitate the determination of analyte
concentrations below the detection or quatification limit of
the analytical method used, complicates preliminary
operations and increases the time spent by the researcher. In
addition, precision is diminished.
(0
50
10
? '0 T JO
I "> I 0 o -10
J - 2 0
1-30 3 -40
-50
- t o
-
/ / y'
^ '"^ 'N^^
N -; N
. i . I 1.,, 1 1_
Izz
^
AnQ|yt»conctnlroliOn
Figure 1.4 Relationship between the precision and the cone,
level of analytes to be determined.
Sensi t ivi ty and Selectivity
Not withstanding their seemingly distant natures, sensitivity
and selectivity are also related and not always in an opposite
manner. In fact, the difference between the additive and
multiplicative interferences lies in whether the perturbation
concerned affect the slope (sensitivity) of the calibration
curve. The more sensitive the method used to determine a
given analyte, the more a sample can be diluted to decrease
the concentration of interferences and hence minimize or
eliminate their perturbations. The method is widely known
and exploited in the analysis of clinical/biological samples;
sensitive methods call for high initial dilutions (1 :10-
12
1:10,000) to minimize the typical perturbations of
niacromolecules. Sensitivity and selectivity can be enhanced
by similar mechanisms [12]. Newly emerging analytical
laboratory instruments tend to incorporate these mechanisms
to enhance their basic analytical properties. The use of non-
chromatographic continuous separation techniques and multi
channel detectors are good examples in this content.
Selectivity and Precision
The uncertainty inherent in the mechanisms described above
for enhancing selectivity is related to the complexity of the
analytical process. When several such mechanisms come into
play, the interminate error arising from each is seen in the
final results, and precision is diminished. Thus if the
selectivity of the methodology in question arises from the
detector used, the analytical process is simpler and higher
precision is achieved. Appropriate chemometric or simple
mathematical treatments increase selectivity. Univariate
calibration and differential kinetic methods [13,14] are two
good choices. As a rule the use of these methods result in
poor precision-even poorer than that obtained from analytical
separation techniques. However, one can use these techniques
to avoid some stages of the analytical process.
Accuracy and accessory analy t ica l p roper t i e s
Designing an analytical method by which to achieve high
accuracy means that high levels of the basic analytical
properties must be obtained at the expense of accessory
properties. As can be seen in Fig. 1.5a, the main tetrahedron is
13
('0
2? >
I 9 0
CO
distorted when the area of triangular accuracy face in
increased. Whereas the secondary tetrahedron is considerably
shrunk by shifting these forces pointing towards the main
tetrahedron This situation reflects the priorities among the
capital, basic and accessory analytical properties
Precision (b) Cost-cffcclivcncss
Personnel Scife(\/comfort
1
Sensitivity Personnel Safety/comfort
Figure I 5 Distortion of the analytical tetrahedra for
boosting (a)accuracy, (b)accessory analytical
propeities
The opposite situation [Figure 1 5b] occurs when temporal,
economic and /oi personal aspects related to laboratory
productivity prevail upon the quality of the results. Obtaining
immediate analytical results in response to an urgent demand
( e g , in the clinical field) or controlling a process in near
real time entails sacrificing some accuracy Thus some recent
applications rely on FT-IR spectroscopy to obtain accurate,
reproducible results in as short time as possible with
minimum sample handling and manual involvement [15]
Occasionally, a timely "yes or no" reply to indicate whether a
given parameter is above or below the tolerated level is more
14
important than obtaining an accurate analytical result a few
hours latter. Sensors [16] and screening test based on
immunoassay [17] are additional good examples.
Accessory analytical properties
The accessory properties at the vertices of the productivity
triangular face of the secondary analytical tetrahedron also
exhibit opposing relationships. The imposed or advisable
prevalence of one or two of these properties distort the
equilateral triangular face and hence disturbs the balance
among them. When an analytical process is designed to
enhance one of the three properties viz; cost-effectiveness,
expeditiousness, or personal safety/comfort-the other two
sides of the resulting productivity triangular face are
markedly shortened. If two properties rather than one are
favoured; the side of the remaining property is contracted.
Quality Compromise
Meeting the demands imposed on the information produced by
an analytical laboratory requires that a compromise be made
between the two components of the analytical
chemistry/quality binomial: external quality, which refers to
the properties of the monitored product or service, and
internal quality, which deals with the analytical process and
the results produced. External quality may result from
compliaiice with legislation or quality requisites for a
production process. It directly affect the analytical quality,
which depends on capital, basic and accessory analytical
properties. From a strictly theoretical point of view, the first
15
situation in accessory properties can be considered ideal-at
least one should reach the balanced situation depicted in
F ig l .2 . However the facts may justify this situation depicted
for accessory properties for the analytical laboratory to meet
the demands posed by the analytical problem derived from the
economic or social problem addressed.
Quality management in drug analysis covers a wide range of
quality improving activities designed to ensure the reliability
of the analytical data. These activities include ensuring that
the sample are properly collected and preserved prior to
analysis, that the analysis is carried out using the appropriate
technique, and that the results are properly recorded and
reported. Emphasis is given on the quality management needs
of research laboratories where drug analysis is carried out on
non-routine basis.
Analyt ica l Techniques
Drug analysis is taken during various phases of
pharmaceutical development, such as formulation and
stability studies, Qc and toxicological and pharmacological
testing in animals and man. In hospitals, drug analysis is
performed on patient 's samples in support of clinical trials
(bioavailabili ty and pharmacokinetic studies) and in
monitoring therapeutic drugs and drugs of abuse [18-21]. All
these investigations require reliable and validated analytical
methods in order to measure drugs in complex media such as
formulations and biofluids. Because of their selectivity,
sensitivity and overall versatility, techniques such as GC,
HPLC, superficial fluid chromatography (SFC) and capillary
16
electrophoresis coupled with selective detectors are
frequently used to analyse multicomponent drug mixture.
Immunoassays because of their speed and simplicity are
mainly used in drug monitoring in patients. Spectroscopic
(UV,IR, AAS) and titrimetric techniques are routinely used
in QC work, and TLC & more recently near infrared (NIR)
spectrometry has been used for the rapid identification of
impurities and degradation products in pharmaceuticals.
Personne l
The quality of the analytical results depends on both
instrument capability and the professional expertise of the
user, and therefore for reliable results instruments must be
used by an adequately qualified trained operators using
clearly written standard operating procedures. A detailed job
description should be available and the required level of
qualifications and experience set for each post.
E q u i p m e n t and C h e m i c a l s
When choosing new equipment it is important to ensure that it
is fit for its intended purpose and its performance is up to
specification [22]. Other things to be considered are cost,
ease of use, quality after sales-service and health and safety
aspects. There should be clear unambiguous, written
procedures for the operation of the instruments. They should
give corrfect readings within their specified tolerance limits
when checked at appropriate intervals by standard procedures
using reference materials. All standards and calibrators used
must be traceable to nationally accepted standards. Minor
17
preventive maintenance operations should be established &
followed, such as keeping pH electrodes in a wet condition or
storing chromatography columns according to supplier 's
instructions. Special attention should be given to the quality
of the laboratory glassware and reagents & standards.
Reference standards should be prepared according to written
procedures. For quantitative work, be sure to use good quality
glassware and analytical reagents and solvents, because other
grades can contain impurities. Drug substances in pure form
can be obtained from the suppliers or be requested from the
manufacturer. Due considerations should be given to the safe
storage and disposal of chemical and solvents used in the
laboratory. Finally, as a matter of good housekeeping, one
should keep laboratories clean, with equipment uncluttered
and benches tidy [23].
Method Development
Good planning is essential in the selection and development
of methods for drug analysis. It is necessary to be able
quantify the active components in the sample rapidly with
acceptable precision, accuracy and reliability within the cost
and other constraints. It often happens that during product
development or any other long-term work, analytical methods
evolve. A logic and systemic approach is required in method
selection and development. This can be outlined in four basic
steps; (a) 'generate background data, (b) review the literature
(c)develope the method (optimize the experimental
conditions) and (d) validate the performance of the method
before using it routinely. Before starting any work, all the
18
initial information on the drug (physicochemical properties
such as solubility, dissociation constant & UV adsorption)
and the dosage form or the intended dosage form (strength,
preservatives, types of container, stability etc.) should be
collected. For bio-analytical work, adequate information
about the sample, subject and dosage, including co
administered drugs should be obtained. All this information is
useful in setting up the analytical method. Bio-analyt ical
methods for pharmacological and tdxicological studies must
be selective and sensitive enough to follow the absorption,
distr ibution and elimination of the drug. Sensit ivi ty is
par t icular ly important for paediatr ic or small animal
samples since their availabil i ty may be l imited. Further
move biofluid assays must be accurate and precise in order
to reveal differences between dosage form or subjects
(human volunteers , patients or animals) .
Although experience is required in method development
work, guidance can be obtained from l i terature sources
special izing in method development [24-31] . Alternat ively,
one can use one of the commercial ly available software
packages to speed up chromatographic method development,
par t icular ly HPLC [32-33] . These systems are developed to
assist the chromatographer in the selection & optimization
of condit ions during method development. With the
assis tance of these systems, maximum information can be
extracted from a minimum number of experiments . The main
advantage of expert systems is that they provide immediate
availabil i ty of an expert opinion through an inherent
19
knowledge base, and respond to the users inquiry when
presented with a problem.
Va l ida t ion
Once the analytical method has been developed, it is
validated before or during its use. Validation of the method
establ ishes that its performance character is t ics are adequate
for the intended use. In pharmaceutical industry, validation
of analytical method is required in support of product
registrat ion appl icat ions.
Many of the principles , procedures and requirements of
val idat ion are common to the majority of analytical
methods. Validation is performed by conducting a series of
experiments using specific condit ions of the method.
Validat ion does not imply that method is free from error. It
only confirms that it is suitable for the purpose. Any
modification to the method during its use require its
reval idat ion. Some revalidat ion may also be required when
transferring the method between laboratories or when
changes are made in the manufacturing process of the drug.
Once the method has been developed and val idated, it is
fully documented and approved for the use. It should be
described in sufficient detail to allow any analyst to use it
without difficulty.
Ruggedness
In addition to the various scientific parameters , another
useful parameters to consider during validation is the
ruggedness or robustness of the analytical method. The
20
ruggedness of a method is its ability to remain unaffected by
small, unintentional changes in the experimental conditions,
such as; temperature, mobile phase composition and pH, or
different source^s of reagents and chromatographic columns.
These occur when a method is used over a long period or is
transferred to another location. Since the drug development
work can last for many years and be conducted at more than
one site, the ruggedness of the method is critical so that it
can be transferred between laboratories and used by
different workers.
System Suitability tests
Once the method is in routine use, check or system
suitability test (SST) should be run each time the method is
used, to confirm that it is performing satisfactorily. Since
the instrument to instrument variations can have a
significant effect on the assay, SSTs provide added
assurance that on specific occasion the method is giving
acceptable results. SSTs should not be confused with good
method validation, which is carried out at the method
development stage. SSTs on the other hand, check the
performance of a given system on a given day prior to
analysis. SSTs can detect normal changes in the equipment
during normal usage. If the method is robust enough, it
should not fail such tests. SSTs are particularly useful in
routine QC work & stability studies.
21
Method compar i son
The performance of a newly developed or modified method
can be assessed by comparing the results obtained by it with
those found with a reference method of known accuracy and
precis ion using linear regression analysis [34-37], A
reasonable number of samples evenly spaced over a
concentrat ion range of interest are analysed by both the
candidate method and the reference method. However it is
not always possible to conduct method comparison studies
simply because a suitable reference method is not routinely
avai lable, or is costly requiring special facil i t ies.
Solu t ions & Reagents
Standard solution of drugs in water or methanol are used
during many stages of analysis , such as calibration and
val idat ion. In bioanalytical work, although stock solutions
can be prepared in water or methanol, standard solution for
cal ibrat ion and other experiment should be prepared by
dilution of the stock solution with a relevant biological
fluid, since aqueous solution differ greatly from the
biological matrix. Drugs and reagents are sorted in such a
way to maintain their integri ty. In general solutions of drugs
& chemical are more stable at low temperatures (4or-20°C)
than at room temperature. All solutions must be clearly
labeled with preparation and expiry dates. Stock solution of
photo-decomposable substances should be stored in amber
colored containers and those of very unstable and volati le
substances should be freshly prepared.
22
Samples
Sampling is often considered to be a weak link in the quality
chain part icularly in drug bioanalysis , and is often the major
contr ibutor to measurement error. Samples should be
homogeneous and representat ive of the original material .
The sample taking should be carried out in accordance with
the approved procedures. It is essential that the samples are
collected in a suitable container at the correct time in
relat ion to the dose correctly labeled and preserved under
appropriate conditions [38-41]. The laboratory must have a
rel iable system of documentation of samples from receipt in
the laboratory to the disposal of the reminder. Samples must
be unpacked in a suitable area taking into account any safety
requirements . If the entire sample received by the laboratory
is not init ially required, it should be divided into sub-
samples, taking precautions to avoid contamination and
losses through spil lages. This practice is useful in case a
repeat assay is required at a latter stage.
Analys is
All analyses should be carried out in accordance with
writ ten procedures. Assays should preferably be performed
in duplicate each time using a separate portion of the sample
rather than a repeat determination on final solut ions. A
complete repeat analysis gives confidence in the results and
serves to check on the homogeneity of the sample and the
random variat ion in the instrumental response [42]. The
laboratory should maintain records of the results as a matter
of professional policy. The test performed, results and
23
relevant observations should be written down in a laboratory
notebook and corresponding copies of the instrument traces
stored. The notebook should serve as a record of each days
work so that each step in the analysis can be traced at a
latter stage if required.
Internal & External Quality Control
Internal QC is usually applied to drug bioanalysis to
monitor assay performance with regard to accuracy &
precision [43]. This is done by analyzing specially prepared
QC samples along-side a batch of samples and plotting the
results of QC samples on a QC chart [44-46]. Like
calibration standards, QC samples are prepared by adding
known amounts of drug in the biofluid. Usually, two
concentrations of QC samples, one near the top and the
other near the bottom of the calibration range, a third QC
samples in the middle of the range are used. For an assay
with a wide calibration range, a third QC sample in the
middle of the range can be tested. If the method is working
satisfactorily, the results of the QC sample should fall
within the pre-set limits, typically + 10% of the known
values.
If a laboratory offers drug analysis as a part of a routine
service, it is useful for such a laboratory to participate in a
an external QC scheme (preficiency testing scheme)
involving many laboratories, provided the drugs in question
are included in such a scheme. In an external QC scheme,
identical samples are periodically sent to the participants
for assay, and the scheme coordinator after receiving the
24
results reports the labora tory ' s performance together with a
summary of results from all par t ic ipants . Market
improvement in analytical standards over a period is
consistent finding with many quality assessment schemes.
Only certain groups of drugs, such as ant iconvulsant ,
ant idepressants and benzodiazepines , are monitored by
external quality assessment scheme. However, regular
par t ic ipat ion in such schemes is an effective way to measure
the competence of laboratory to undertake certain analyses.
Acc red i t a t i on
In addition to proficiency testing schemes, increasing
at tention is being given to external quality assessment
schemes by which a laboratory has to prove its competence
to an independent third party by par t ic ipat ing in a
recognized external quality assessment scheme. Laboratory
Accredi tat ion is a formal recognit ion by an authorized body
such as National Measurement Accredi tat ion Services
(NAMAS) that the labora tory ' s quality system is in place
and meets the required standards to carry out specific tests
or specific types of tests. Accreditat ion is normally awarded
following successful assessment of the l abora tory ' s system
by NAMAS officers and is subject to periodic review. Much
emphasis is placed on documentat ion, cal ibrat ion of
equipment, staff competence and training and traceabil i ty of
s tandard-mater ia ls . Accredi tat ion gives the laboratory and is
customers added confidence in the quality of resul ts .
25
L a b o r a t o r y Aud i t
Cost considerat ions prevent laboratories from jo in ing a
proficiency or accreditat ion scheme. In this si tuation, it is
useful to conduct an in house audit of the labora tory ' s
quali ty management system at appropriate intervals say at
least once a year. Auditors should preferably be independent
of the activit ies of the laboratory that they are going to
audit . Audit should cover all aspects of the laboratory
pract ice from sample collection to report ing results , and not
jus t analytical performance. It should be sufficiently
probing to highlight any drawback in the service. The use of
audit is the most reliable way of identifying problems in the
management of the laboratory. Quality management in drug
analysis incorporates all measures taken to ensure the
rel iabi l i ty of analytical data, starting from obtaining a
satisfactory sample, analyzing it correctly with a val idated
method, to reporting the results , with all procedures well
documented and periodical ly reviewed. By implementing an
effective quality system, a significant improvement is made
to the overall performance of the laboratory engaged in drug
analysis .
All the drugs in their nature are divided into inorganic and
organic. Both can be prepared from natural sources, or
art if icially, i.e. synthet ical ly. The enormous number of
drugs used in medicine can be classified into two way;
26
1. Pharmacological Classification
In this section, drugs are divided on the basis of their action
on an organism (the heart, lymphatic system, stomach,
intest ine etc.) . Accordingly, drugs are divided into such
groups as norcot ics , soporifics, analgesics , diuret ics and
local anaesthet ics .
2. Chemical Classification
Here, the drugs are divided according to their chemical
structure and propert ies regardless of their pharmacological
action. It is beyond the scope of this thesis to discuss all the
type of drugs, however, few important classes of compounds
used as drugs are discussed below.
Alkaloids
Alkaloids are substances mostly of plant and rarely of
animal origin. In medicine, alkaloids are successfully used
as drugs in the treatment of cardiovascular , nervous,
al imentary trace and many other diseases [47]. They are
organic substances of basic nature because their molecules
always contain nitrogen. The alkaloids are chiefly tert iary
amines, and only some of them are secondary amines or
derivatives of te t rasubst i tuted ammonium bases. Such class
of drugs can be grouped into the following heads.
(i) Pyridine & Piperidine derivatives ( l ibel ine & its
companions) .
(ii) Tropane derivatives (atropine, hyoscyamines, cocaine
etc.) .
27
(iii) Quinoline derivatives (quinol ine, quidine etc.)
(iv) Isoquinoline derivatives (opium)
(v) Indole derivatives (physost igmine, reserpine etc.)
(vi) Imidazol derivatives (pi locarbine, omeperazole) .
(vii) Purine derivatives (caffeine, theobromine, theophyll in
etc.)
An t ib io t i c s
Antibiot ics are very important class of pharmaceut icals
consist ing of ant ibecter ia ls , ant i - infect ives, antifungals,
ant iparasi t ics and ant imicrobials . Antibiot ics are organic
compounds derived from or produced by living organisms,
which are capable, in small concentrat ions, of inhibi t ing the
life processes of micro-organisms [48].
The chemistry of antibiotics is so varied that a chemical
classif ication is of little value. However, some antibiot ics
are the products of similar mechanism in different organisms
and that these structurally similar products may exerts their
act ivi t ies in a similar manner. A number of ant ibiot ics have
in common a macrolide s tructure, a large lactum ring.
Erythromycin, Oleandomycin are included in this family.
Tetracycl ine family represents a group of compounds very
closely related chemically. Streptomycins, kanamycins,
neomycins, paramomycins and gentamycins are included into
a family of compounds containing closely related amino
sugar moit ies . The antifungal ant ibiot ics , nystat ins and
amphotericins are examples of a group of conjugated
28
polyenes. The pencillins and cephalosporins are antibiotics
derived from amino acids. A large number of polypeptide
compounds that exhibit antibiotic action include bacitracine,
tyrothricins and polymixins.
Analgesics
A drug which brings about insensibility to pain without loss
of consciousness is known as analgesic. There are numerous
drugs which, in addition to possessing distinctive pharma
cologic activities in other area may also possess analgetic
properties [49]. The analgetic property may have direct or
indirect effect. Such class of drugs are grouped as
sedatives.(e.g. barbutrates), muscle relaxants, tranquilizers,
(e.g. meprobamate), morphine and related compounds, Some
of them been quantitatively analysed in pharmaceutical
preparation [50-53].
Central Nervous System Depressants
The agents that produce depressant effects on the central
nervous system [54] as their main pharmacological action,
include the general anesthetics, hypnotic sedatives, skeletal
muscle relaxants, tranquilizing agents and anticonvulsants.
The general anesthetics (e.g. ether) and hypnotic sedatives
(e.g. phenobarbital) produce generalized or non-selective
depression on C.N.S function. Many sedatives if given in
large doses produce anesthetic effect. Certain analgesics
also produce depression on central nervous system.
Central Nervous System Stimulants
29
Central nervous system is a complex network of sub-units
which acts as conducting pathways between peripheral
receptors and effectors, enabling one to respond to his
environment. Drugs which have in common the property of
increasing the activit ies of various portions of the central
nervous system are called central nervous system stimulants
[55] .
Respiratory Stimulants and Analeptics
Carbonic acid is a most effective respiratory stimulant.
Analeptic agents used to lessen narcosis brought about by
excess of depressant drugs often stimulate a variety of nerve
centers as well as many analeptic drugs are also predecessor
drugs because their st imulation of the vasomotor center
produce an increase in vasoconstr ic t ion. There are many
drugs (e.g. cocaine, a troprine, amphetamines, ephidrine etc.)
of varying pharmocologic classes, which in addition to their
desired effects, elicit a pronounced st imulatory effect on
central nervous system.
Cardiovascular Agents
Agents used for their action on the heart or on other parts of
the vascular system to modify the total output of blood for
the distr ibution to certain parts of the circulatory system,
are called cardiovascular agents. This class include
cardiotonic drugs, vasodiala tors , hypotensive drugs,
ant ihypercholes tero- lemic drugs and s c l e r o s i n g agents
[56]. Certain drugs have a direct action on cardiovascualr
system, and in addition, they affect certain const i tutents of
30
blood. The later include ant icoagulants , the hypoglycemic
agents, the thydroid hormones and the anti thyroid drugs.
Analysis of Drugs
The methods for analyzing medicinal agents are divided into
physical , chemical, physio-chemical and biological ones.
Physical methods of analysis involve the studying of the
physical propert ies of a substance. They include the
determination of solubili ty, t ransparency and the degree of
turbidity, density or specific gravity, melting, boiling and
freezing points.
Chemical methods used for the analysis of drugs are based
on chemical reactions. They include the determination of
ash content, the hydrogen ion concentrat ion, (pH) of
medium, and characteris t ic numerical indices of oils and
fats. Quanti tat ive and quali tat ive analysis of drugs are
generally carried out with respect to functional groups.
Physio-chemical methods are used to study the physical
phenomenon that occurs as result of chemical reaction.
Among the physio-chemical methods of analysis are,
photometry including photo-colorimetry and spectrophoto
metry, electrochemical and chromatographic methods.
Recently, various kinds of chromatography (column, paper,
thin layer, gas, gas-l iquid) and photometeric methods based
on the absorption of light by the analyte have found their
use in pharmaceut ical analysis . NMR and mass spectrometry
with gas chromatography are the most important analytical
methods used for the analysis of drugs.
31
Biological methods of analysis characterize the
pharmaceut ical effect of the drug. Biological tests are
conducted on animals and less frequently on separate
isolated organs or parts of organs of animals. The effect of
drugs is expressed in A.U, (Activity units) and is
determined by the comparison with the activity of a
reference substance. The biological methods are very
sensi t ive. Alkaloids, vitamins and antibiot ics still reveal
activity when diluted millions of t imes.
Photocolorimetr ic methods of analysis are based on
measuring the absorption of non-monochromatic light by
coloured compounds, in the visible part of the spectrum. If
the analytes are colourless, they are converted into coloured
compounds by reaction with suitable reagents. In the visual
method, called colorimetry, the intensity of the colour of
analyte solution is compared to that of standard solution if
the concentrat ion of substance is known.
In recent years pharmaceutical analysis has been
character ized by the use of methods such as
f lamephotometry and differential spectrophotometry in the
quali tat ive analysis of drugs. These methods enable one to
determine the large amount of individual components of a
mixture because the absorbance of the analyte solution is
measured not relative to the pure solvent (or a solution of
reagent) but relative to a reference solution containing a
known amount of the analyte.
Magne t ic resonance imaging (MRI) has achieved an
amazing level of success in clinical medicine as a non-
32
invasive diagnostic tool. Moreover, NMR spectroscopy is
favoured by chemists as a pov^erful technique for molecular
structure determination. It therefore seems natural that these
clearly related techniques should merge to yield non
invasive, spatially localized NMR spectroscopy for
determining molecular structure and concentrat ion in vivo.
Considerable effort has been directed towards developing in
vivo NMR spectroscopy as a clinical diagnostic tool. Less
commonly, in vivo NMR spectroscopy has been used to
monitor drugs as well as other xenobiotic agents directly in
both human and animal studies. The magnitude of the
pharmacologic or toxic effect of a drug is expected to
depend on the concentrat ion at the receptor sites, which are
generally located in the tissue cells of the target organ [57].
Measuring drug levels in plasma is a reasonable method for
monitoring drug therapy. It is also relat ively non-invasive
and inexpensive, and a wide range of analytical techniques
including high-resolut ion NMR spectroscopy [58], can be
used for quantitation or identification of drug metabol i tes .
NMR spectroscopy can, in principle measure drug
concentrat ion in tissue in vivo and can probe drug
metabolism if metaboli te resonances are resolved. Because it
is non-invasive, NMR spectroscopy can be used repet i t ively
on the same individual permitt ing pharmacokinet ic and
longitudinal studies.
C a p i l l a r y e l ec t rophores i s , with its two major modes,
capil lary zone electrophoresis (CZE) and micellar
e lectrokinet ic capillary chromatography (MEKC), is gaing
33
acceptance in the pharmaceutical field for the determination
of drugs in formulation [59,60] . Sampling preparat ion
involving dissolution and dilution of the formulations in the
running buffer is perfectly adequate.
Chemi luminescence is a powerful tool for drug analysis
since its detection limits are extremely low and its
instrumentat ion very simple and of low cost. In combination
with derivatizat ion techniques in order to increase
sensi t ivi ty, it has a wide range of appl icat ions. The
excellent sensit ivity and the versat i l i ty of the
chemiluminometr ic methods of analysis are the main reason
for the recent surge of interest in CL. The introduction of
flow injection analysis and liquid chromatography with
chemiluminometr ic detectors is responsible for the plethora
of original research work devoted to this area of chemical
analysis .
Multiple linear regress ion (MLR) is the most common
choice for mult icomponent determination which allows the
easy resolution of complex mixtures provided the
contr ibut ion of each mixture component to overall spectrum
can be determined from the spectra of the pure components
used for calibration [61,62]. While rather limited in scope,
the method provides good results if background noise is
fairly low and all the mixture components are known, absorb
appreciably, and do not interact with one another. When one
or more of these conditions is not met and some analyte
absorbs weakly at a high concentrat ion of the analyte
contr ibut ions to the mixture, absorbance are rather
34
desperate, a different mathematical procedure is required.
Foremost among these is partial least-squares regression
(PLSR) [63-65] , which is also used with other analytical
tecniques where interaction between analytes are quite
strong (e.g. in IR and NIR spectroscopy).
In Cap i l l a ry e l e c t r o c h r o m a t o g r a p h y ( C E C ) , a liquid
mobile phase is driven through a stat ionary phase in a
packed capil lary column by the electro-osmotic flow (EOF)
generated by a large difference in potential across the
column. There is no pressure drop, and it follows that the
flow profile is rectangular and that small diameter part icles
may also be employed. Higher column efficiencies can
therefore, be obtained than in pressure driven HPLC. While
much interest has been shown in the theory and mechanism
of CEC, its development and eventual widespread use in the
pharmaceutical industry will only come about if CEC is
shown to have advantages over more conventional HPLC in
routine use. CEC can be applied with considerable
advantages in the analysis of pharmaceutical compounds.
Separat ions are efficient, repeatable and reproducible . As
long as the pressure is pressurized and the column
temperature controlled, quanti tat ive analysis can be carried
out with electrokinetic injection. CEC has been applied to
the separation of several pharmaceutical compounds; a
mixture of diastereoisomers, not previously separated on a
chiral HPLC column, was separated by CEC [66],
Der iva t ive s p e c t r o p h o t o m e t r y presents greater selectivity
than does normal spectrophotometry and so offers a
35
convenient solution to the problem of resolving spectral
overlap in the analysis of multicomponent system [67-68].
Peak-to-peak and base-line measurement (generally referred
to as graphical measurements) and zero-crossing
measurements are the most common techniques used to
prepare analytical working curves. The zero-crossing
method involves the measurement of the absolute value of
the total derivatives spectrum at an abscissa value
(wavelength) corresponding to zero-crossing point of the
derivatives spectrum of the interfering component. At this
wavelength, the amplitude of the derivative signal of one of
the two components passes through zero; measurement of
the value of derivative spectrum of mixture, made at the
zero-crossing point of the derivative spectrum of one of the
components is therefore a function only of the concentration
of the other component [69].
Difference specti ophotometery has proved particularly
useful in the determination of medicinal substances by
eliminating specific interference from degradation products,
co-formulated drugs and also the non-specific irrelevant
absorption from the formulation matrix. Its advantages for
selective analysis have been described by several workers
[70-71]. The technique involves the reproducible alterations
of the spectral properties of the absorbance difference
between two solutions provided that the absorbance of the
other absorbing substance is not affected by the reagents
used to alter the spectral properties. Simple aqueous acids,
alkalis & buffers are most frequently used for inducing
spectral alterations since many drugs are weak acids or
36
bases whose state of ionization and absorptivity depends on
the pH of the solution. [72].
Flow injection analysis (FIA) is characterized by its
simplicity, speed and the use of in-expensive equipment. Its
results are accurate and precise, and there are clear
advantages in terms of the short time required for each
assay. The usefulness of the (Fl) method for routine analysis
has been shown in a large number of determinations
developed for clinical, pharmaceutical, food and
environmental analyses.
High performance thin-layer chromatography (HPTLC) is
particularly attractive as an analytical tool as it is the
fastest chromatographic technique and flexible enough to
analyse different kinds of samples. It is an off-line
technique where every stage of the analyses can be
visualized. HPTLC depends on the object, scanning is
performed in reflectance and its transmittance made by
adsorbance or by fluoresence. It can be used in
pharmaceutical, medical, biological/clinical areas.
Electrochemical methods are characterized by high
sensitivity, selectivity and accuracy. Analytical sensitivity
attainable even exceeds pg./mL level and analysis in the
nanogram or subnanogram range is possible.
Electrochemical methods have been extensively developed
and each basic electrical parameter namely current (i),
resistance (r) and voltage (v) has been utilized alone or in
combination of analytical purposes. Electrochemical
methods have entered a new phase since development of
37
electrochemical detectors for HPLC. These are amperometric
or coulometric detectors which measure the current
associated with the oxidation or reduction of solute.
Pharmaceutical drugs containing groups such as phenolic,
amino, heterocyclic nitrogen, keto and aldehyde undergo
oxidation at characteristic potential. Since all compounds do
not undergo oxidation at a particular potential applied, this
increases the selectivity of the technique. Electrochemical
detection is often used where ultraviolet detectors do not
have sufficient sensitivity.
Conventional UV spectrophtometeric methods are simple,
quick, economical and usually do not require an elaborate
preparation step prior to assay. However, a UV method is
not adequate when two or more drugs with similar UV
spectra are present in the sample, or excipients or
decomposition product exhibits UV interferences, as the
case with the dissolution testing of phenytoin capsules [73].
The principle and the application of the kinetic methods
have been reviewed [74]. Essentially, kinetic method relay
on measurements of concentration changes (detected via
signal changes) in a reactant with time after the sample and
the reagents have been mixed. The use of kinetic methods in
micellar media [75] is another approach to kinetic-based
determinations that have proved useful in drug analysis.
Micelles increase the reaction rate (through micellar
catalysis) and may additionally increase the sensitivity and
selectivity for the analyte. On the other hand, new kinetic
chemometric (kinetometric) approaches such as the Kalman
filter has been developed and applied to the simultaneous
38
determinat ion of various compounds of pharmaceutical
interest .
The analysis of drug entai ls , essential ly the following;
(i)the quanti tat ive, quali tat ive and relative bioact ivi ty assay
of pure and impure drugs in different matr ices; (ii)
e lucidat ion of the mechanism of action of drugs at the
molecular level; (iii) clinical evaluat ion, including
assessment of site of action (in vivo) and side effects, and
methods of assaying development of resis tance of cells to
drug action.
Methods of quanti'^ative and quali tat ive analysis of drugs are
important in all three aspects. Quanti tat ive drug
measurement require to be of high reproducibi l i ty , precision
and accuracy should be efficient. Drug abuse also leads to a
need for quali tat ive and quanti tat ive analysis of drugs, but
because dosage forms are of high purity and precisely of
known composit ion the limits of detection are not so
demanding as in clinical medicine and reasonably large
quanti t ies of sample are usually available.
The methods currently available may be broadly divided into
two categories; (1) classical microbiological methods [76]
and modifications there of ;(2) physiochemical techniques
[77].
Microbiological assays may be considered as variants of
chemical assays in which a living organism or t issue is the
reagent responding to the substance under invest igat ion. The
39
classical techniques can be classified into (a) diffusion
methods and (b) dilution techniques.
Respirometr ic methods depend on the ability of a drug to
inhibit formation of carbon dioxide by either direct or
indirect action. Radioactively labeled carbon dioxide, and
indicator methods can be used, but the indicators should not
interfere by complexing with the drug.
Turbidimetr ic methods measure the inhibit ion of growth
photometr ical ly . The growth is assumed to be inversely
proport ional to the drug concentrat ion. Errors can be caused
by variat ion in inocula, inoculum volume errors & light
absorption by the drug.
In dilution methods, several dilutions of the drug are
incubated with an inoculum of test organisms, and the
lowest concentrat ion of drug, producing complete inhibit ion
of growth is taken as the inhibitory concentrat ion.
Numerous variation of these techniques continue to be
reported, and routinely practised in clinical and
pharmaceut ical laboratories . One of the primary reason for
this is that the structure of the drug need not be known. The
methods are also usually cheap and simple and do not need
elaborate apparatus, but are slow, have rather poor
reproducibi l i ty and are open to subjective error, and can not
be used quali tat ively.
The availabil i ty of sub-standard medicines to the general
public poses many problems, both cl inically and
economical ly. It is widely believed that such sub-standard
40
preparat ions are readily available in many developing
countries [78-79]. This may be due to poor manufacturing
procedures , poor storage conditions or deliberate
counterfei t ing of branded or generic products . However,
most l i terature reports on this problem do not contain
quanti tat ive information.
As a part of an on-going study looking at the quality of
selected pharmaceut icals in developing countries it is
necessary to assa> samples of the drugs in formulat ions. In
examining the quality of the active ingredient present , it is
of advantage to be able to detect starting materials and
major decomposit ion products if these are present. This may
allow the cause of any lack of quality determined. The
method employed should allow rapid determination of the
drug from the dosage forms with little sample pretreatment.
In addit ion the method should be validated following current
guidel ines [80-81] and in view of this a method is required
which is as simple as possible so that the val idat ion
requirement could be adhered to.
Antibiotics
Antibiot ics are defined as compounds that have inhibitory
activity against micro-organism, eukaryotic cells , viruses
that are produced by secondary metabolism of living
organism.s, and that possess a variety of widely different
s t ructures . In 1929, Fleming discovered a mould of the
penici l l ium species, which inhibited the growth of certain
bacteria. The antibiot ics cover a wide range of compounds
of different chemical structures. Penicill in is a name given
41
to the mixture of natural compound having the molecular
formula C9H11N2O4SR, and differing only in the nature of R.
There are at least six natural penici l l ins (Table 1.2).
Tab le 1.2
Chemical name
Pen t -2 -eny lpen ic i l l in
Benzylpen ic i l l in
p -Hydroybenzy lpen ic i l l i n
n -Hepty lpen ic i l l in
n - A m y 1 p e n 1 c 1111 n
phenoxymethy lpen ic i l l in
Other name
Penic i l l in- I or F
Penic i l l in- I I or G
Penic i l l in -III or X
Penic i i l in - IV or K
Dihydro-F-pen ic i l l in
Penic i l l in -V
R
-CH2CH = CHCH2CH3
-CH2C6H5
-CH2C6H4OH (1,4)
-(CH2)6CH3
-(CH2)4CH3
-CH20C6H,
The penici l l ins are strong monobasic acids, i.e., they are
known to form salts The penicil l ins get hydrolyzed by hot
dilute inorganic acids, and one carbon atom is eliminated as
carbon dioxide, and two products in equimolar proport ion
are obtained, one being an amine (Penici l lamine) , and the
other an aldehyde, (Peni l loaldehyde) . All the pencil l ins
yield same type of amine, but the aldehydes formed are
different. It is these aldehydes which contain the R group.
The R group in commonly available penici l l ins is given in
table 1.3.
42
Table 1.3
Penicillin R-group
Ampicillin
Amoxycillin
Cloxacillin
Benzylpenicillin
v\ //
H0< A //
v\ //
CH-
NH2
T NH2
• c — c -N C
0 TH3
A // CH2 —
43
The first successful synthesis of penici l l in was carried out
by Sheehan et al., (1957,1959) , after sixteen years in the
preparat ion of totally synthetic penici l l ins . The general
reaction sequence is outlined in F ig l . 6 .
R \ ,
-NCHCHO
COjCMe,
\
R' -NCH-
C02CMe3
HSCMe,
NHjCHOjH
S CH,
COjH
\
R ' -NCH- ^
COjCMe^ HN CH,
COjR'
ii'yl iici
\ .NCH-
CO,ll
\
SOCI, COjIV
0
r N-
CH,
Hj-Pd
CO^R'
R-^TIT
or OH"
S . .CH3
f CH,
COjH
^x,_^^N. -N 'l ^^J, ^^^ ^N-C,H,CH,SOjNH-
R'= CH3, QHjCHj
Fig 1.6 Total Syiillicsis of penitilliii juialogs.
44
The two commonly used semisynthet ic P-lactum antibiotics
are ampicil l in (oc-aminobenzylpenicill in) and amoxycil l in
(oc- amino-p-hydroxybenzylpenic l l in) . These are widely used
for the treatment of bacterial infections. The analysis of
these aminobenzylpenci l l ins is carried out by most of the
analytical techniques. They include flourimetry [82],
spectrophotometry [83-84], e lectroanalyt ical [85] chromato
graphic [86-87] and microbiological[88] methods. The
spectrophotometr ic determinat ions for the assay of
ampicil l in and amoxycil l in are based on equilibrium
methods, which involve a previous step where an acid [83-
84] or base [89] hydrolyses the P- lactum ring. About 20min
to 2hrs. are required for such treatments, so the equilibrium
determinat ions are slow and hinder applicat ions to routine
analysis .
Ampicil l in and cloxacil l in, due to the presence of P-lactum
and thiozol idine rings possess similari ty in their s tructures,
and a method for their analysis in combined preparat ions
must be designed to achieve a high degree of selectivi ty [90]
but only liquid chromatographic methods are selective. A
broader spectrum of antibacterial activity is provided by
penici l l in in combinations and may be advantageously
prescribed in cases of (3-lactamase producing strains.
Synergist ic activity are reported for some pencill in
combinat ions against gram-negative bacteria [91-92].
Amoxycil l in (Fig.1.7a) and dicloxacil l in (Fig.1.7b) also
possess synergy against clinical isolates of P-lactamase
producing & non-producing strains [93].
45
HO o CH-CONH.
NH2
(a)
H H
O ^
-N-
S .CH3
CH3
CO2H
i / S . .CH3
Figure 1.7 (a & b)
Pharmaceutical preparations containing above mentioned P-
lactum antibiot ics are readily available in the market. A
variety of analytical procedures are recommended for the
determinat ion of amoxycil l in, which include spectro-
photometr ic measurement of the copper chelate [94] or the
formation of penici l lenic acid by treatment with mineral
acids & copper (II) [95]. The t i tr imetric procedure is
recommended in British pharmacopoeia [96], for
amoxycil l in in bulk form using standard mercury (II) nitrate
and potent iometr ic detection of the equivalent complex, and
a colorimetr ic one using immidazole mercury reagent at
325nm in case of capsules containing the drug. Two
chromatographic procedures are reported for the assay of
ampici l l in in the presence of another penici l l in [97-98] . The
first method involves the estimation of ampicil l in embonate
and amoxycil l in embonate [97], The reverse phase
separation column is used at pH~7 with detection at 240nm.
Amoxycil l in is used as the internal standard for ampicillin
while phenoxymethylpenici l l in that for amoxycil l in.
Amoxycil l in and embonic acid are determined
s imultaneously while separate analysis is required for
ampicil l in and embonic acid. Another method involves
reverse phase-18 determination of ampicil l in and cloxacillin
46
in capsules [98]. Simple dilution with sulphamethoxazole
solution is made for sample preparation and determination is
achieved at 254n;n. Other methods for chromatographical ly
determining a single penici l l in include the determination
and monitoring of azlocil l in during bulk drug analysis [99],
benzylpenici l l in determination by capil lary zone
electrophoresis [100] and a collaborative study of
determining penicil l in V in tablets [101].
Several papers have described the use of enzyme selective
electrodes for determining penici l l ins [102-103] . The effect
of pH and buffer concentrat ion on the response of an enzyme
membrane coated wire electrode is also described [102].
Spectrophotometr ic methods continue to enjoy populari ty for
the determination of a variety of penici l l ins . The first
derivative mode of operation is found to be simple,
accurate , and precise for the determination of amoxycil l in
and clavulanic acid in pharmaceutical preparat ions[ 104].
Second derivative spectroscopy has been applied to
quanti tat ion of ampicil l in and dicloxaci l l in [105], &
ampicil l in and cloxacil l in [106]. Both method by Morelli are
suitable for the analysis of these P-lactums in
pharmaceut ical preparat ions. A forth-derivative spectro
scopic approach has been described for the determination of
4-hydroxyphenoxymethylpenici l l in in phenoxymethyci l l in
samples [107]. A second derivative spectroscopic method
described the determination of ampicil l in & cloxacil l in
[108]. The method consists of dissolution of the formulation
in water followed by scanning the spectra between 260 nm
and 330 nm. Derivative measurements are obtained at 269nm
47
and 284nm for ampicill in and cloxacil l in respect ively. The
hydrolysis of ampicill in and cloxacil l in followed by direct
spectrophotometr ic determination at 273nm has been
described [109]. The method is found to be specific to
ampicil l in in the presence of cloxacil l in & linear from 5-35
|j,gmL"'.
A variety of techniques have been evaluated for the
quantif ication of amoxycill in alone or in combination with
other penici l l ins [1 lO-l 12], Micellar electrokinetic capillary
chromatography has been found to be a useful approach for
identifying degraded products in aged amoxycil l in [110]. In
addit ion, the reaction of amoxycil l in with acetylacetone-
formaldehyde has served as the basis for a simple
spectroscopic method [111]. Beer" law is obeyed from 10-
100)j.gmL"', and no interference is observed in mixtures
containing cloxacil l in excipients . The simultaneous
flourometric determination of amoxycill in and clavulanic
acid is possible using a stopped-flow kinetic approach
[1 12]. The method involves the reaction of the analytes with
acidic Ce (IV) and detection at ' '365nm. Another
spectroscopic method for the ampicil l in is carried out using
ni t robenzene as colorimetric reagent [113]. An
electrochemical method for quantifying P-lactum antibiot ics
and their hydrolysis products is described [114]. Cyclic
voltametry at the water /ni t robenzene interface in four
electrode system is used. Hydrolysis products are
character ized across UV- vis spectrum.
48
Diclofenac sodium
Diclofenac sodium is the most commonly used, non-steriodal
ant i- inflammatory drug. It has potent analgesic, antipyretic
and anti- inflammatory actions, with minimum adverse
effects. It is an ideal drug for anthrit ic pateints , requiring
long term therapy. Rheumatic conditions are recalci trant
disorders which cause immense anxiety, loss of mobili ty,
dexteri ty and sleep to its pat ients , even leading to
depressions. Non-steriodal anti- inflammatory drugs
especial ly the sustained release forms have proved to be
highly efficacious in tackling chronic rheumatoid condit ions
associated with pain and inflammations. In chronic
condit ions, patient compliance's assume a critical role
especial ly where prolonged drug treatment is required. The
challenge to the pharmaceutical industry is to developed
dosage forms that ensure once daily dosing while
maintaining steady and constant therapeutic level of the
drug within the pat ient ' s system.
Different analytical methods have been reported in the
scientific l i terature for the determination of diclofenac in
plasma, urine, synovial fluid and pharmaceutical
preparat ions . Several anti- inflammatory drugs including
diclofenac have been quantified in different dosage forms by
capil lary zone electrophoresis and micellar electrokinetic
capi l la ry 'chromatography [115]. The experiments have been
performed without specific sample pretreatment . The
samples are directly dissolved in the running buffer and the
internal standard method has been used for assays with
49
respect to standard solutions. Diclofenac with other drugs
has been determined by flow injection analysis with
spectrophotometr ic detection [116]. The method has offered
good advantage with respect to short start-up time,
simplicity of instrumentat ion & high sampling rate. The
spectrophotometr ic determination of trace amounts of
diclofenac has been carried out by l iquid-l iquid extraction
using arcidine yellow with a flow system [117]. The
determinat ion of diclofenac in the range of 3-80}4.mgL'' is
achieved with a sampling frequency of 40 sample h' . The
spectroflourometric determination of diclofenac in
pharmaceut ical tablets and ointments has been described
[118]. It involves excitat ion at 287nm of an acid solution of
the drug, and measurement of the fluorescences intensity at
362nm. A second derivative spectrophotometr ic method has
been developed [119] for the determination of degraded
products of diclofenac sodium in gel ointment. The
ampli tudes in the second derivative spectra at 260nm and
265nm are selected to determine oxidol. The diclofenac and
codeine phosphate in diclofenac tablets has been determined
[120] by RP-HPLC at the wavelength of 250nm. Another RP-
HPLC method for the simultaneous estimation of diclofenac,
paracetamol & chloroxazone in formulations has been
described [121]. The method is carried out on zorbax C8
colunm at the detection wavelength of 220nm.
Spectrophotometr ic methods for the determination of
diclofenac in pharmaceutical preparat ions have been
described [122]. The methods are based on the oxidation of
drug using reagents viz; eerie ammonium sulphate,
50
dibromodimethylhydant ion, N-bromosuccinimide &
potassium bromate to yield coloured products with maximum
absorbances at 450, 385, 385 and 495nm respect ively.
Diclofenac sodium in Ganmaotong tablets has been
determined by first derivative spectrophotometry [123] at
292nm. A simple and precise HPTLC method has been
developed for the simultaneous determination of diclofenac
and paracetamol [124]. Voltametric propert ies of diclofenac
sodium on the glassy carbon electrode have been described
and the determination concentrat ion of diclofenac with
differential pulse voltmetry are revealed [125]. The
sensit ive anodic current of diclofenac sodium on 0.93V is
observed. Diclofenac sodium in Ganmaotong tablets has
been determined by forming a green complex with copper
acetate in chloroform in a specified pH range [126].
Diclofenac sodium also has been determined in dosage forms
[127] by spectrophotometry based on the formation of
ni troso derivatives followed by alkal inizat ion to yield a
stable yellow chromophore which exhibits A-max at 378nm.
C i s a p r i d e
Cisapride is a benzamide and a recently developed
prokinet ic drug. It decrease receptive relaxation in the upper
portion of stomach and increases antral contract ions . The
pylorus and duodenum are relaxed, while the tone of the
lower esophageal splinter is enhanced. These effects
combine to accelrate the emptying of gastric contents and to
reduce reflux from the duodenum and the stomach into the
esophagus. Cisapride is believed to increase colonic motility
51
and can cause diarrhea. The prokinetic action of cisapride is
blocked by atropine and may involve the release of
myenteric acetylcholine. Cisapride appears to be devoid of
dopaminergic blocking activity and it does not influence
the concentrat ion of prolactin in plasma or cause extra
pyramidal symptoms. Recently some reports of serious
ventr icular arrhythmia's and death have appeared among
patients who took antifungal/macrolide ant ibiot ics while on
cisapride. Blood level of cisapride was raised due to
inhibi t ion of CYP 3A4 by erythromycin, ketoconazole etc.
Used alone has not caused such arrhythmia 's . A warning has
been issued to use cisapride cautiously in patients receiving
macrol ides / imidazoles.
Cisapride has been always determined in dosage forms and
biological media. An HPLC method is described for the
quanti tat ive analysis of cisapride in human plasma [128].
Cisapride together with internal standard are analysed
isocrat ical ly on a reversed phase column and eluted drugs
are detected with UV-Vis detector at 276nm. A stability
indicat ing HPTLC method is used for the determination of
cisapride and related impurit ies in tablets [129]. Beer ' s law
follows in the concentrat ion range 2.5-15 j imgL' ' . A single
dose crossover study of two conventional formulations of
cisapride tablets is carried out in twelve healthy human male
volunteers [130]. Blood samples are collected at time
intervals and plasma cisapride is determined using RP-HPLC
with UV detection. Three spectrophotometr ic methods (A-C)
for the assay of cisapride in pure and dosage forms are
described [13 1]. Method A is based on coupling reaction of
52
cisapride with 3-methyl-2-benzothiazol inene hydrazone-HCl
in presence of FeCh to form a coloured species with X-max.
at 565nm. Method B is based on the oxidation of cisapride
with Fe^^ and subsequent chelation of Fe " with 1,10-
phenanthrol ine. Method C is based on the formation of a
coloured charge transfer complex with chlorani l ic acid
having Xmax at 555nm. Another reversed phase liquid
chromatographic method is reported for the determinat ion of
cisapride in pharmaceutical dosage forms [132]. A method is
also proposed [133] for the polarographic determinat ion of
cisapride in which the electroactive product is obtained by
ni t rat ion with potassium nitrate in sulphuric acid. The 3s-
detection limit is 1.8x10" M and the relat ive standard
deviation is 1%.
N a l i d i x i c acid
Nalidixic acid is an antibacterial agent that has been
extensively used in the treatment of Gram-negative urinary
tract infections. It is commercially available for the
medicinal use and has also found application as an effective
aid in the control of chicken infection. Nal idixic acid
inhibits the replication of DNA in bacteria, without
immediately affecting RNA or protein synthesis in sensit ive
bacteria. Its toxic effects in animals are limited to inhibit ing
mitochondrial DNA replicat ion at concentrat ions that do not
affect nuclear DNA. Nalidixic acid being a synthetic drug is
excreted together with inactive metaboli tes , in the urine. It
is bacter icidal to most of the Gram-negative bacteria that
cause common urinary tract infections, except for
53
pseudomonas strains. Resistance to nalidixic acid develops
readily but is apparently not transferable from one organism
to another.
Nal idixic acid is given orally because it is absorbed from
the gastrointest inal tract. A large proportion of the drug is
bound to plasma proteins and penetrat ion into the tissue in
poor. The plasma half-life is about 1.5 hours, and the major
route of excretion of both parent compound and metaboli tes
is via the kidney. The metaboli tes of nal idixic acid are
bacter iological ly inactive, but sufficient unmetabolised
compound is excreted into the urine to make nal idixic acid
useful in the treatment of urinary tract infections. The toxic
effect of nalidixic acid include nausea and vomit t ing, skin
rashes and photosensi t ivi ty reactions and central nervous
system effects such as dizziness and visual disorders.
Nalidixic acid can displace warfarm from protein binding
sites and can prolong the clotting time, or cause
naemorrhage, if these drugs are given together. From
analyt ical point of view several methods have been proposed
for the assay of nalidixic acid in biological samples and
commercial formulations. The luminescence character is t ics
of the inclusion complex of nalidixic acid with y-
cyclodextr in has been investigated [134]. The study involves
the characterizat ion of inclusion process involved and
development of a spectroflourimetric method for the
determinat ion of this drug. The calibrat ion range has been
found to be 0.1-2.0 ngmL' ' . A TLC procedure has been
developed for the quanti tat ive monitoring of the degradation
of nal idixic acid in solutions irradiated with high pressure
54
mercury lamp [135]. High performance TLC combined with
densi tometry enabled accurate and reproducible quanti tat ion
of nal idixic acid in the presence of its degraded products .
Another HPTLC method has been described for the
simultaneous determination of nalidixic and metronidazole
in pharmaceutical preparat ions [136]. Linear response is
observed in the range of 20-120ng and 40-140ng for
metronidazole and nalidixic acid and respect ively. Capillary
e lectrophoresis has been applied to the separation of
nal idixic acid and other quinolones of first and second
generation [137]. The same system is applied for the
quanti ta t ive determination of these quinolones in tablets and
capsules without specific sample pretreatment. An HPLC
assay for nalidixic acid has been developed. Reverse- phase
chromatography [138] is conducted using mobile phase of
ammonium acetate, methanol & acetronitr i le and detection at
254nm.
55
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Chapter-2
Simuftaneous ^joectrofofiotometric and Vofumetric Uetermination of IPenicillins in Urua "Tormufations: Reaction Is/lechamsm in me "Base Catafijsed 1-[ijdrofijsis "Toffowed hij Oxidation wim Oodate in Uifute Acid Solution.
68
I n t r o d u c t i o n
A large number of methods for the analysis of ampicil l in,
amoxycil l in and cloxacil l in and related antibiot ics are
described [1-3] . Emphasis has been given on
spectrophotometr ic and t i t r imetr ic methods, because they
are simple and easily manageable to the third world. The
volumetr ic t i t rat ions are carried out by some oxidants prior
to a base or acid catalysed reaction of ant ibiot ics . The
degraded hydrolyzed products of ant ibiot ics are t i trated
quanti tat ively against oxidants such as Hg " [4] and iodate
solut ions [5]. It is observed that when hydrolyt ic condit ions
are changed from moderate concentrat ion of alkali or acid to
a weak or using the selective pH region [6] , a different
pathway of antibiotics mechanism results . The t i t r imetr ic
method [4] described by alkaline or enzymic hydrolysis
followed by ti tration with Hg(l l) might suffer a setback. The
use of nitric acid to neutral ize the excess alkali before
performing the t i trat ion may oxidize thiozol idine ring of the
penici l l in molecule and therefore could give erroneous
resul ts . The authors themselves reported better results with
enzyme hydrolysis than base hydrolysis .
In direct t i tration of antibiot ics [5], with potassium iodate
solution, strong acidic condit ions are prevailed. The acid
plays a ,dual role, first it brings about acid hydrolysis ,
secondly it makes quanti tat ive oxidation t i t rat ion feasible.
Two procedures are adopted [5]. In the first, the end point is
detected by a colour change in carbon tetrachloride layer
from colourless to brown or deep red and in the second dye
69
Amaranth is used to detect change in colour from deep red
to pale yellow, as the drug is destroyed by an excess of
iodate in aqueous medium at the neutral izat ion point.
The determination of amoxycil l in and flucloxacil l in in
mixtures and pharmaceutical preparat ions using 3,5-dinitro-
benzoic acid as a reagent is described [7] . Extractive
spectrophotometr ic determination of benzylpenici l l in is
described in pharmaceutical preparat ion, based on the
formation of a yellowish red colour with potassium iodate in
acidic medium [8]. The colour is stable for half an hour and
Beer's law is obeyed in the concentrat ion range 0.05-0.5
mgmL"'. Another such method for benzylpenici l l in sodium
in 10% potassium nitrate solution containing sulphuric acid
involves a colour reaction with 2 ,3 ,5- t r iphenyl te t razol in
chloride [9]. The coloured product in extracted with
enthanol having ^tmax. 400nm. A selective colorimetr ic
method is described for the analysis of ampici l l in in its
reaction with p-dimethylaminocinnamaldehyde in the
presence of te t ra-chloroacet ic acid to form a stable red
sch i f f s base [10]. The coloured product is detected at
Xmax. 501nm and obeyed Beer's law in the range 10-50
/.igmL. The formation of deep blue complexes are utalized
for the micro-quant i ta t ive determination of ampicil l in and
cloxaci l l in in pure form, mixture and in pharmaceut ical
preparat ion [11]. The reaction is carried out with
bromothymol blue (BTB) at 25°C and 50°C respect ively.
Beer's law is valid in the concentrat ion range 0.4-18 and
0.4-20 mgmL' of ampicil l in and cloxacil l in respect ively.
70
The determination of amoxycil l in and f lucloxacil l in by
HPLC in biflocin injection is described [12]. The mobile
phase is phosphate buffer (pH-6.5) and the valid
concentrat ion range is 0.25-2.0 mgmL•^ Another HPLC
method is carried out for the simultaneous determination of
ampicil l in and dicloxacil l in in pharmaceutical preparation
[13]. The complexation of amoxycil l in and carbocystein
with Ni(I l ) is used for spectrophotometer ic determiantion
[14]. A flow injection analysis is described for the
determinat ion of amoxycil l in and ampicil l in [15].
Ti t r imetr ic method for the assay of selected ant ibiot ics in
non-aqueous media is carried out by conductometeric
t i t rat ion [16]. A spectrophotometr ic method for the
simultaneous determination of amoxycill in and dicloxacil l in
in pharmaceutical preparat ions is described [17]. The assay
of ampici l l in in presence of cloxacil l in in pharmaceut ical
preparat ions , which involves the reaction of hydrolysed
ampicil l in with fromaldehyde in acidic phosphate and
subsequent measurement of the absorbance at 373nm is
described [18]. Copper( l l ) acetate has been
used as a complexing agent for the spectrophotometr ic
determinat ion of benzylpenici l l in in pharmaceutical
preparat ions [19].
In this chapter, we described the base catalysed hydrolysis
of ant ibiot ics in dilute sodium hydroxide solution.
The hydrolysis product of antibiot ics which contains
sulphur derivatives e.g., -C-S-C group, -S-H group are
quant i ta t ively oxidized to sulphonic acid by t i t rat ing them
71
against potassium iodate solution as oxidant, in dilute
hydrochlor ic acid to prevail conducive atmosphere of pH~ 2.
The chemistry of this reductant system is described which
shows the observed stoichiometr ic value of thiol group to
iodate 1:2, which is consistent with theoret ical value. The
point of difference of our finding to that of previously
described by Andrews ti tration [20] have been high-l ighted.
The volumetric results are compared to spectrophotometr ic
determinat ions . The pathway and the oxidation mechanism
of hydrolysis products of antibiot ics have been proposed in
the light of IR, TLC and spectrophotometr ic s tudies.
EXPERIMENTAL
Reagents: All the experiments were performed with
analytical reagent grade chemicals using double dist i l led
water. Stock solution of ant ibiot ics , ImgmL"' were prepared
by dissolving the pure and standard drugs in water and
stored in a well-dark and closed container to avoid direct
contact with light. Hydrochloric acid standard solution
( l .OM), potassium iodate and sodium hydroxide of O.IM
each were prepared in disti l led water.
Apparatus: A Bauch and Lomb spectronic-20D'^ and
spectronic-1001 spli t-bean spectrophotmeter (Milton Roy
Co.) with 1cm path length quartz cells and a controlled
temprature water bath (NSW-133 India) were used. IR
spectra of the isolated products were recorded on a
spectrometer (Shimadzu IR-408, Japan) with KBr disc.
72
Procedure I:- S imu l t aneous determination of Antibiotics
by volumetric titration against potassium iodate and
spectrophotometry of coloured complex in organic layer.
Aliquots of 2.5 mL of O.IM sodium hydroxide were added
into conical flasks followed suitable volumes of each of the
standard antibiotic solutions, ranging between 0.2-2.0mL of
amoxyci l l in , ampicil l in, carbenici l l in and Becampici l l in ,
0.1-2.5mL cloxacil l in and 0.2-2.3mL of benzylpenici l l in .
The contents were shaken and placed in a water bath at 80°C
for 10-1 Sminutes. After completion of the heat treatment,
the mixture was cooled to room temperature, followed by
0.5mL of O.IM hydrochloric acid and 5mL carbon
te t rachlor ide . The mixture content were t i trated against
potassium iodate with intermittent shaking till the colour
changed from colourless to deep red in non-aqeous layer.
For sepctrophotometr ic determination, the non-aqueous
coloured layer was removed using 50mL separat ing funnel
and dried over sodium sulphate. Absorbance was measured
at 520nm against the blank, containing all the species
except drug. This was the idea behind simultaneous
determinat ion, that is performing two different methods at a
time in the same mixture content.
Procedure II: Determination of Antibiotics by Spectro
photometric method .
To a known volume of antibiotic solution ranging
between amoxycill in (0.4-3.5mL), ampicil l in (0.4-2.8mL),
c loxaci l l in (0.1-2.5mL), benzylpenici l l in (0.1 -2.9mL),
73
carbenici l in (0.2-2.3mL) and becampici l l in (0. l -2 .4mL), 2.5
mL of sodium hydroxide was added in a conical flask. The
reaction mixture was kept for 10-15min.on a water bath at
80°C. After cooling to room temperature , l.OmL of
potassium iodate, 0.5mL of hydrochloric acid and 5mL of
carbon tetrachloride were added to the reaction mixture. The
solution was shaken vigorously till a deep red colour was
obtained in organic layer. It was determined
spectrophotometr ical ly by the recommended procedure 1.
Dosage forms: Tablets & Capsules
Twenty tablets were weigh ed and grinded to a fine powder.
An accurately weighed portion of this powder equivalent to
lOOmg is taken in a lOOmL standard flask. Similarly the
contents of the capsules were weighed and dissolved
equivalent to lOOmg in 0.1 l i tre. The mixture was shaken for
about 30minutes to ensure a homogeneous solution. The
residue was removed through whattman No . l filter paper and
the washing were taken into final volume of 0.1 l i tre.
R E S U L T S AND D I S C U S S I O N
Mechanistic approach:
This involves UV,1R, TLC and molar s toichiometric studies.
The hydrolysis of ant ibiot ics is catalysed by a base using
O.IM sodium hydroxide solution at pH~13 in a single stage
reaction. It is carried out in a water bath at 80°C for 15
minutes with intermittent shaking result ing in different
degraded products which contain sulphur derivat ives, e.g
74
-S-S-(disulphide) and -S-H(thiol) groups. These degraded
products when subjected to redox ti tration against potassium
iodate as oxidant in dilute hydrochloric acid solution, they
are quanti tat ively converted to sulphonic acid with
some intermediate species viz; penici l lenic acid(IIb) and
sulphoxide(IIIb) . The degradation pathway is more precisely
i l lustrated in figure 2 3 -
The results obtained by performing thin layer
chromatography(TLC) with Rf = 0.46 in solvents (v/v)
comprising, 66% n-butanol, 17% glacial acetic acid and 17%
water, suggested the presence of 4-hydroxymethylene
oxazolone. This observation is in agreement with previous
studies [6], which confirms the existence of la, (Rf = 0.42 in
the same solvent system). Our studies of the degraded
products of penici l l ins at pH~13 involving a single step
reaction using water bath at 80°C for 15 minutes show one
Xmax at 240nm. This at tr ibutes the existence of one of the
three species viz; Ib(i) or Ib(ii) or Ib(i i i) . Moreover the
formation of penici l loic acid Ib(i) from basic hydrolysis of
penici l l in is well established [21]. Another Xm&x at 270nm
is obtained when time interval is extended to 12 hours, thus
confirming the establishment of 1(a). The previous studies
show the existence of 1(a) as a degraded product of
ant ibiot ics in the pH range 13-14 with UV spectroscopic
analysis showing Xmax at 297.5nm. The slight difference in
the Xmax. value may be at tr ibuted to different experimental
condit ions of hydrolysis , such as temperature , time of
hydrolysis and pH etc.
75
To carryout IR studies of ant ibiot ics , three different spectra
were taken for,
(i) Pure amoxycil l in, Spectrum-A (Fig.2.4) .
(ii) Degraded in base catalysed hydrolysis , Spectrum-B
(Fig. 2.5)
(i i i) Degraded base hydrolysed products t reated with O.IM
hydrochlor ic acid followed by t i trat ion against KIO3,
Spectrum-C (Fig. 2.6).
The comparison of spectrum (A) and (B), revealed that the
character is t ic bai>ds for different groups at specified
frequencies in spectrum (A) are found (i) missing, (ii) the
formation at different frequencies or (iii) remained
unchanged. A broad flat portion (3450-2600cm"') same as it
was found in spectrum (A), at tr ibuted to primary al iphatic
amine N-H (3450-3250cm"') , carboxylic (3300-2500cm"')
and S-H (2600-2500cm' ' ) groupings.
The bands are found missing in the range (1800-1500cm"')
and 1350-lOOOcm"') result ing the formation of bands at
860cm"', 680cm"' and in the range (1500-1350cm"') . The
identi t ies of thiozoiidine and secondary amine groups with
different group frequencies lying in the range (1800-
1510cm"') are completely swept out. The second range of
missing band (1350-1000cm"') includes the groups e.g. CH3-
S, ester etc. The two missing ranges are revealed by a
descending and ascending portion of spectra-B respect ively.
The formation of a broad band (1500-1350cm"') is at tr ibuted
76
to the presence of 4-hydroxymethylene oxazolone ( la) ,
which is revealed by the group frequency C-0 stretching and
0-H deformation vibration (1440-1395cm' ' ) . The ascending
portion (1350cm"' onwards) of spectrum-B further describes
the hydrolysis of rest of the groups present. A strong peak at
860cm"' may be at tr ibuted to S-0 (870-810cm"') of
sulphonic acid. The peak at 680cm"' suggests to disulphide
-S-S, C-S (705-570cm' ' ) and sulphoxides (>S=0, C-S-0 at
730-690 c m ' ) after their partial oxidation to sulphonic acid
in semiquanti ta t ive manner as the sulphur compounds
especial ly containing the group S-H, S-S are oxidized by
mild oxidant or even atmospheric oxygen [22]. The spectrum
-C, which is obtained after completing the t i tration against
iodate in dilute acidic condit ions, gave the final product
corresponding to strong peak at 835cm' ' , a t t r ibuted to the
presence of sulphonic acid quanti ta t ively.
The recommended procedure 1 describes s imultaneous
volumetr ic determination of penici l l in against potassium
iodate as oxidant in dilute acid medium. The deep-violet
colour extracted in carbon tetrachloride at the equivalence
point is stable for two hours and has been studied
spectrophotometr ical ly . The hydrolysis product of
ant ibiot ics which contained sulphur derivatives i.e., -C-S-C
(lb), -S-H groups are quanti tat ively oxidized to some
intermediate species and finally to sulphonic acid whose
identi t ies have already been discussed in IR studies . The
hydrolysis by dilute sodium hydroxide solution has been
carried out slowly at a constant temperature 80°C with
77
intermit tent shaking. The excess of base is neutral ized by
adding hydrochloric acid (0.5 mmoles) so that the reaction
mixture attains a pH~2. This provides a conducive
environment of HjO^ion concentrat ion for carrying out
quanti tat ive t i tration of degraded products against potassium
iodate. The reaction is summarized as follov/s;
6Q
HS-R+3H2O -• RS03H+6H'+ 6e"] x 2 Step I
I0"3 + C1" 6 H ' +4e" > ICI + 3H3O'] x3
In the first step reaction a quanti tat ive oxidation of thiol to
sulphonic acid followed by reduction of IO3' to ICI takes
place which may be regarded as the reaction at equivalence
point, but without any colour change. This shows that the
s toichiometr ic value calculated theoret ical ly should be of
2mol. of thiol group to 3mol of iodate (1:1.5) which is
inconsis tent with experimental value 1:2 [23]. However, the
system still involves more iodate ions to make the end point
visible due to its reduction to iodine monochloride and
finally to free iodine. This is described in step II reaction
^3^.4c«pT;«^d diagramatical ly: "^"^""^ ''" IO3(+5) mdilute HCl
reduction
10-3 + Cr + 6Fr+4e ^ ICI+3H2O Step II
M:55Q9 '^ Quantitafttese oxitfiltrbn ot penicillin PenecillinIOi(l 1 5)
-. i ; itfiltibn ^ JpWr^nd point
Increases
True end point
Penicillin 10", (1 ly
ylCU+l) • More IO3 involeved in theSyite*-..
Overconsuniption of IO3
' I: (0 00)
Fig .2 .1 . Over consumption of iodate at the end pt. in
recommende^d procedure.
78
iCl dissociation
-> I'+cr
hydrolysis > H O l + H " r+HsO
over consumption , 5 H 0 I • 2 l 2 + IO-3 + H " + 2 H 2 0
of iodate
In the step II, an overconsumption of iodate takes place
[24], thereby increasing the mole ratio of iodate from 1.5 to
2mol which is consistent with the exper imental ly observed
stoichiometry of Imol thiol to 2mol iodate. A stable red
violet colour of free iodine in organic layer indicates the
equivalence point. It is observed that further addition of
iodate solution beyond the end point results no
decolourizat ion in mixture content. This means that a
quanti tat ive oxidat ion-reduct ion in dilute hydrochloric acid
has taken place in the system. The titrant iodate is reduced
first to iodine monochloride and finally to free iodine to
show a visible end point. The E° of iodate in dilute
hydrochloric acid (1.19V) is less than the E° in Andrews
t i trat ion (1.23V). The E° (1.19V) is high enough to oxidize
the degraded products of antibiot ics containing derivat ives.
In Andrews t i trat ion strong hydrochloric acid (3-9M)
solution is required for reduction of iodate to iodine
monochloride (ICI2") complex.
Penicillin complclely consumed -4
10-3 (+5) Strong HCl (3-9 M)
IO"3+6H*+2Cr+4e" ^ IClz+SHaO E° = 1 23V
T IC1'2(+1)
In presence of penicillin
• 12(0 00) 2ICl"2+2e- ^ I2+4CJ-£"= I06V
Figure 2.2 Andrews t i t rat ion
79
During the course of t i trat ion with reducing agent, the
presence of free iodine in the system also results from the
further reduction of ICI2' complex as given above. When the
reducing agent has been consumed, the free iodine is t i trated
to form iodine monochloride complex. The end point is
marked by the disappearance of the last free iodine. (A point
of difference from our observation given in Step II).
2I2 +IO".v + 6H"+ lOCr -> 51Cr2+3H20
The above conditions prevail , if the concentrat ion of the
hydrochlor ic acid at the end point is at least 3M. The iodate
reacts quanti tat ively with reducing agents such as Kl,l2,
AS203,N2H4 and SO2 to iodine monochloride [25]. In many
other cases the concentration of hydrochloric acid is not
cri t ical , but for Sb( l l l ) , it is 2.5 to 3.5M; that is the
optimum acidity for reasonably rapid reaction varies from
one reactant to another reactant [20] .
The recommended procedure 1, which describes the use of
dilute hydrochloric acid is applied successfully to the
simultaneous quanti tat ive reduction of iodate to free iodine
at the equivalence point, especial ly in a system which
contains the degraded products of ant ibiot ics with sulphur
derivat ives which can be oxidized by very mild oxidizing
agents [22] . The half reaction of iodate in dilute acid
solution with E° [25] can be proposed as under.
2IO"3 + 12H"+10e" -^ I2 + 6H2O E° = 1.19V
The method described earlier [5] for the direct t i t rat ion of
ant ibiot ics by iodate solution in a high concentrat ion of acid
is based on Andrews method. The reduction of iodate to
iodine-monochlor ide complex involving electron change of
80
four and oxidation of thiol group from penici l lamine (IV) to
sulphonic acid(VI) and that of aldehyde from
peni l loaldehyde (111) to the carboxylic acid(V) are
accompanied with electron changes of six and two
respect ively.
The overall s toichiometry has been computed by summing
up two oxidation products with a total electron change of
eight which is consistent with their experimental
s toichiometr ic ratio 2mol iodate and Imol penici l l in . This is
revealed by the following react ion:
Penicill in + [V] +[V1] + 8e"
10"3 + 6 H ' + 2 C r + 4e" -^ ICl'z + 3H20]x2
However, if the above oxidation reduction is considered
separately it reflects a different aspect as given below:
H-S (IV) -> H03S"(Vl)+6e"]x2
I0"3 +4e" -» I d s ' ] X 3 (i)
-CHO (111) -^ COOH (V) + 2e"]x2
I0 '3 + 4e" ^ I C l 2 " ] x i (ii)
In reaction (i), the stoichiometry of 2mol penici l l in /H-S to
3mol iodate and in (ii) 2mol penici l l in/-CHO to Imol
iodate are observed, which is inconsistent with their
experimental ly stoichiometric ratio of iodate to penici l l in
2 :1 .
We have tried to elaborate the above reaction system taking
place in two fold manner i.e. potassium iodate is reduced to
iodine monochloride in the presence of high concentrat ion
of acid (12moldm' ' ' ) . During the cource of t i t rat ion as long
as the reductant is present in the system, iodine
monochloride is further reduced to free iodine. The stage of
81
titration can be regarded as the first step, when equivalence
point can be declared theoretically which immediately
vanishes due to further reaction of iodine with iodate. The
second step of titration is started with free iodine to yield
iodine monochloride complex, the disappearance of deep red
violet colour to colourless resulting the declaration of
equivalence point, followed by an over consumption of
iodate which increases the number of moles of iodate,
approximately to experimental value.
The effect of interfering substances in the determination of
penicillin are shown in Table2.7. This study shows the
maximum tolerance of interfering substances in the
minimum amount of penicillin. The validity of the proposed
method was tested by successive determination of penicillin
in pharmaceutical preparation. The results obtained by the
recommended method are presented in table2.8. The
treatment of analytical data using statistical approach for
the determination of all the penicillins using different
concentrations have been presented in Table2.9. The best
straight line equation with variables x and y and
incorporating the slope and intercepts for each antibiotic is
computed.
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ei t «
A. Degraded products; single stage hydrolysis of penicillin at pH ~ 13
92
R-CONH-HC HC^ "^C-CCHbb
OC- N— penicillin
OH
-CH-CO2H
At SOT , (15 minutes)
H H
R - C O - N H - C C"" ""CCCH^b
H H s
R-CO-N-CH2—C^ ^C(CH,)2
-CO2
HOOC HN CH-COOH
penicilloicacid
HN- - CH-COOH
Ib(i) - H 2 O
penillonicxid.
Ib(ii)
HS. N-
OH
4-hydiioxymethylene (oxazole-5(4H)-one
la
N- I Airoxidation Conversion into NH-CH — > species; Ub,mb,
R' C C
pencillenic add
Ib(iii)
COOH Seniquartilative pyt.
B. Oxidized Products: Obtained titrimstrically using IO3" as oxidanL I2/IQ3*
0 II
R-S-
OH
Sulphonic add
IVb
O O oxidation „ 11 H r. oxidation M R—S—S—R M
Sulphoxide
fflb
N C=
c c
. I NH-CH
I COOH
penidllatiic acid (disulphide)
R - S - S - R Db
Fig 2.3 The possible degi-adation pathways of penidllin; Followed by oxidation with iodate ions.
93
(*/>)33NViilHSNV)Ji
94
o o S o O
(•».)3DNtlllWSN>rBl
95
o o o O
«0 o o
(X)33NVi imSNVUi
0.4 n 96
70
D-
•
M
X
•
- A = amoxycillin
- B = ampicillin C = cloxacillin
D = benzylpenicillin
E = carbenicillin
F = becampicillin
80 90 100
Temperature (°C)
Fig. 2.7. Effect of temperature on the colour stability of penicillins, >.max. = 520nm.
0.5 1
0.4
S 0.3-1 c (0
w o (/> < 0.2
0.1 •
• • X X X X
• • a • 1 A — A
• A = amoxycillin
-a— B = ampicillin
-A— c = cloxacillin
X D = benzylpenicillin
X E = carbenicillin
• F = becampicillin
10 15 20
Time (min.)
Fig. 2.8. Effect of time on the colour stabilityof penicillins, A,max. = 520nm.
97
z J— _—-•
B
•
A • •
•
A
• 0
•
8 A • •
•
A • •
•
A • •
•
— • — • — •
A = amoxycillin A C = cloxacillin *— E = carbenicillin
-•— B = ampicillin -X— D = benzylpenicillin -•— F = becampicillin
I
25 I
35 05 1 15 2 25 3 35 4
Volume of NaOH (mL) Fig. 2.9. Effect of varying volume of sodium hydroxide at 80°C,
Xmax. = 520nm.
- 1
45
• • • * • » X X X K M M A A A A A A
« X X X K S
• • • • • « • - • - • -• • •
—••— A = amoxycillin
—^-~ C = cloxacillin
—*— E = carbenicillin
- • — B = ampicillin
—H— D = benzylpenicillin
—»— F = becampicillin
1 5 2 25 3
Volume Of KI03(mL)
35 45
Fig, 2.10. Effect of varying volume of potassium iodate at 80°C, ; max. = 520nm.
0) u c (0 Si b . o (/) n <
04
0 3 "
02
01 ••— A = amoxycillin B = ampicillin C = cloxacillin D = benzylpenicillin
—*— E = carbenicillin —•— F = becampicillin
I I I I I I I • I •
0 01 02 03 04 05 06 07 08 09 1
Volume of Hcl (mL) Fig. 2.11. Effect of varying volume of hydrochloric acid at 80°C,
A,max. = 520nm.
98
0 9 -
0 8 -
07
06
0) u c TO
o V)
<
05
04
03
02
01
400 I -
440 I
480 520 560 — I
600
Wavelength (nm)
Fig. 2.12. Absorption spectra of the reaction product of penicillins at SO'C.
0.5
0.4
%"'
o i 0.2
"o >
0.1 •
99
lli
• A = amoxycillin
B B = amplcillin
A c = cloxacillin
1 2
Amount of Drug (mg)
Fig. 2.13. Relation between volume of titrant (KIO3) and the concentration of penicillins in reference with the table 2.1.
0.3
0.2
c ra
O a> E 3 •5 0-1
-•- A = benzylpenicillin
-B— B = carbenicillln
-A- C = becampicillin
0 1 2 3
Amount of Drug (mg)
Fig. 2.14. Relation between volume of titrant (KIO3) and the concentration of penicillins in reference with the table 2.2.
100
• A = amoxycillin
- • - B = amplclllin
—*r- c = cloxacillin
Amount of Drug (mg)
Fig. 2.15. Calibration curves of penicillins in reference with the table 2.3.
-•— A = benzylpenicillin ~o— B = carbenicillin ~A—C = becampicillin
Amount of Drug (mg)
Fig. 2.16. Calibration curves of penicillins in reference with the table 2.4.
101
-•— A = amoxycillin • B = ampiclllin A C = cloxacillin
Amount of Drug (mg)
Fig. 2.17. Calibration curves of penicillins in reference witli the table 2.5.
0.8
0.7
-•— A = benzylpenicillin -B— B = carbenicillin -A— c = becampicillin
Amount of Drug (mg)
Fig. 2.18. Calibration curves of penicillins in reference with the table 2.6.
0.5 102
0 .4 -
0.3
O 0 .2 -
E
0 1 2
Amount of amoxycillin (mg)
0.5
0.4 -
5 0-3 -J
O 0-2 •
E = 0.1 H
y
0 1 2 3
Amount of ampicillin (mg)
0,4 1
E 0.3
'4-> C 2 •S 0.2 -o
I 0.H I
0 1 2
Amount of cloxacillin (mg)
Fig. 2.19. Best straight line equation in the determination of penicillins in reference with the table 2.1.
0.5
;j 0.4-1
c 0.3
1 0.2
0-K
103
2 0 1
Amount of carbenicillin (tng)
c (0
0.5
0.4-
0.3-
0,2-o a
I 0.1-1
1 1 1
0 1 2 3
Amount of benzylpenicillin (mg)
0.4
0.3
2 0.2
o * E 0.1-I I
0 1 2 3
Amount of becampicillin (mg)
Fig. 2.20. Best straight line equation in the determination of penicillins in reference with the table 2.2.
u re J3
0.5 •
0.4
0.3
| o .
0.1 •
Amount of amoxycillin (mg)
104
0.5 n
0.4-
g 0.3 ^ a n 8 0.2 H X) <
0.1
0 JL — I 1 1
0 1 2 3
Amount of ampicillin (mg)
0.6
0.5
c re •e 0.3 o (0 < 0.2-
0.1 -
0 0 1 2 3
Amount of cloxaciJIin (mg)
Fig. 2.21. Best straight line equation in the determination of penicillins in reference with the table 2.3.
0) u c n € o I
0.6-]
0.5-
0.4-
0.3-
0.2-
0.1 •
n
(
•
•
D
•
1
1
«
•
2 3
Amount of carbenicillin (mg)
105
0.5 n
0.4
0.3 -J 8 0.2 §
o c n o
<
0.1 -
0 1 2 3
Amount of benzylpenicillin (mg)
0 4 - ,
0 3
0.2
0.1
0 - t
0 1 2 3
Amount of becampicillin (mg)
Fig. 2.22. Best straight line equation in the determination of penicillins in reference with the table 2.4.
106 0.6
0,5 H
« 0.4 o
1 0 . 3 o iS 0.2-^ <
0.1
0 ~l
4 0 1 2 3
Amount of amoxycillin (mg)
0) u c 10
o
<
1.11
1
0.9
0.8
0.7-
0.6-
0.5-
0.4
0.3
0.2
0.1
0
0
Amount of ampicillin (mg)
9) U C ra
JQ k. o w n <
0.9 1
0.8
0,7
0.6
0.5
0.4
0.3
0.2
0.1
0
Amount of cloxacillin (mg)
Fig. 2.23. Best straight line equation in the determination of penicillins in reference with the table 2.5.
o u
n JQ
0,3
0.2
O XI 0.1 -<
0 1 2 3
Amount of carbenicillin (mg)
107
a> o c «
o w SI
<
0.4
0.3-
0.2
0.1 -
0 1 2 3 4
Amount of benzylpenicillin (mg)
0) o c ra Xi o w <
0.6 1
0.5
0.4-
0.3-
0.2
0.1 -I
0 1 2 3
Amount of becampicillin (mg)
Fig. 2.24. Best straight line equation in the determination of penicillins in reference with the table 2.6.
108
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Anlyst
[2] J .Talegaonkar and K.S. Boparia, Talanta. 29(1982)
525.
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[4] B.Karlberg and U.Forsmaan, Anal. Chim Acta.
83(1976) 309.
[5] J.K. Grime and B.Tan, Anal. Chim. Acta. 105 (1979)
361 .
[6] J.L. Longridge and D.Timms, J.Chem. Soc. (B) (1971)
852.
[7] A.S. Amin, An. Quim. 89(7-8) (1993)726.
[8] A.S.Khan and I. Ahmad, Indian Drugs. 33(2) (1996) 71
[9] V.K. Shormanov and E.P. Duritsyn, Farmatsiya. 44(4)
(1995) 31 .
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East. Pharm. 38 (456) (1995) 115.
[ I I ] Y.M. Issa and A.S. Amin, Egypt. J. Pharm. Sci. 36 (1-
6) (1995) 235.
[12] Fenxi Ceshi Xuebao. 14(3) (1995) 87; Chem. Abstr.
123 (1995) 237991V.
[13] K.A. Al-Rashood, J. Liq. Chromatogr. 18(2) (1995)
2457.
[14] G.G. Pargaonkar and S.G. Kashedikars , Indian Drug.
31(12) (1994) 590.
[15] M.S. Garcia, C.S. Pedreno, M.I. Albero and V.
Rodenas, J.Pharm. Biomed. Anal. 12(12) (1994) 1585.
i09
[16] K. Esma, K. Filnat, K.Adnan and A.M. Abdul Kadir,
J. Pharm. Biomed. Anal. 13(12) (1995)1453.
[17] M.Ezzat and A. Moety, J. Pharm. Biomed. Anal. 9(2)
(1991) 187.
[18] A.O. Akanni and J.S.K.Ayim, J. Pharm. Biomed Anal.
10(1) (1992) 43.
[19] U.Saba, Analyst. 111(1986)1180.
[20] L.W. Andrews, J.Amer. Chem. Soc. 25(1903) 756.
[21] M.A. Carroil and J.E. Zarembo, J. Antibiot . 30(1977)
811 .
[22] D.R. John and M.C. Caserio, " Basic Principles of
Organic Chem" 2"'' edn. (1977) P. 745.
[23] El-Sebai A. Ibrahim, S.M. Rida, Y.A. Baltagy and
M.M.Abd El-Khalek, J.Drug. Res. (Egypt) . 6(1973) 13.
[24] I.M. Kolthoff and R. Belcher, "Volumetric Analysis" ,
Vol. I l l , (19S7) p. 450.
[25] I.M. Kolthoff, E.B. Sandell , E.J. Merhan and
Bruckenstein, "Quanti tat ive Chem. Anlysis ." 4'' edn.,
(1969) p. 819, 1159.
Chapter-3
ShBctrohhotometrk Ueferminafion of Dkfofenac Sodium in Urua "Tormufations.
no
Introduction:
Diclofenac sodium is the sodium salt of [o-(2,6
dichloroanilino) phenyl] acetic acid and is an antipyretic,
analgesic&well-tolerated non-steriodal anti-inflammatory
agent with potent activity in the treatment of rheumatic
diseases. The assay of diclofenac sodium is carried out by
most of the analytical techniques. A method for the
simultaneous determination of diclofenac sodium and its
hydroxy metabolite has been described [1] by cappillary
column gas chromatography with electron capture detection
which follows extractive alkylation. The method is also
applied to analyze urine samples of volunteers treated with
single doses of diclofenac sodium. A high performance
liquid chromatographic assay has been proposed [2] for the
determination of a number of anti-inflammatory drugs
including diclofenac in plasma. The samples in this method
are prepared by adding acetronitrile and perchloric acid to
200).iL plasma. Diclofenac sodium with other drugs is
quantified in the supernatant procedure using a mobile phase
of perchloric acid-acetronitrile and a detecting wavelength
of 254nm. The method is also applied to the cross -sectional
study of medication compliances. Another sensitive HPLC
procedure for the assay of diclofenac and its
monohydroxylated metabolites in biological fluids has been
described [3]. Diclofenac is assayed in plasma at
concentration down lOngmL'', while in combination with its
monohydroxylated metabolites it is assayed in urine, after
chemical analysis at concentration down to 200 ngmL"'. A
liquid chromatographic method has been developed [4] for
I l l
the determination of diclofenac and related materials in
formulations and raw materials. The method resolves ten
related compounds with limits of quantitation 0.2% or less.
Pharmacokinetics of diclofenac sodium has been also
studied [5] by developed high performance liquid
chromatography in serum and urine using acetaminophen as
internal standard, a reversed phase C-18 column and
ethanol: water as mobile phase. A spectroflurometric
determination of diclofenac sodium in bulk and
pharmaceutical preparations has been described [6]. The
method is based on the hypersensitive property of the
fluorescent probe ion Eu"" , at 616nm. The interaction with
diclofenac, which contains a carboxylic group, makes the
transition allowed and enhances the intensity of its
flourescence emission. A spectrophotometric method based
on the fixation of diclofenac on sephadex QAEA-25 resin as
solid phase has been described [7]. The absorbance of solid
phase is measured at 281nm and 400nm. A multifactor
optimization techniques has been successfully applied to
develop new spectrophotometric method in which diclofenac
is analysed and determined as its Fe(III) complex [8]. A
four-variable two level factorial design is used to
investigate the significance of each variable and interaction
between them. A colorimetric method for the determination
of diclofenac sodium is based on the formation of coloured
charge-transfer complex with chloranil [9]. The complex is
formed in EtOH showing Xmax. at 545nm and concentration
ranges between 0.08-0.9 mgmL''. Another charge-transfer
study of diclofenac for its determination has been carried
112
out with p-benzaquinone and chloranil [10]. In ehtnol
medium the reaction occurs readily with p-benzaquinone,
while the reaction with chloranil is feasible in alkaline
medium. Spectrophotometric methods for the determination
of diclofenac sodium in its formulations have been described
[11]. The first method involves the oxidation of drug studied
by ammonium persulphate in alkaline medium and coupling
with dimethylaminobenzaldelyde reagent. The resultant
solution is a yellow coloured product with its analytical
wavelength at 450nm. In second method the diclofenac has
been treated with sodium hypobromite in presence of
cetrimide in alkaline medium. The yellow coloured solution
is determined colorimetrically at 460nm. Another
spectrophotometric method has been described [12]which
involves the treatment of diclofenac with sodium nitrite in
presence of hydrochloric acid, producing a yellow colour
having /\,max. at 390nm. Two more spectrophotometric
methods have been described [13] for the determination of
diclofenac sodium. In the first method diclofenac reduces
Iron(III) to Iron (II), which subsequently forms complex
with 2,2-bipyridine. The chelate complex formed posses
maximum absorbance at 520nm. In the second method the
diclofenac forms ion pair complex with methylene blue in
presence of phosphate buffer which is extracted with
chloroform. The complex formed shows maximum
absorbance at 640nm and the graph of absorbance versus
concentration is linear in the range 5-40^lgmL"^ An
extractive spectrophotometric determination of some anti
inflammatory 4rugs including diclofenac in pharmaceutical
113
preparations has been described [14]. It is based on the
formation of a chloroform soluble ion-association complex
between the drugs studied and methylene violet at pH~7.6.
The present work describes the colorimetric methods for the
determination of diclofenac sodium in bulk and commerical
dosage forms. In first method determination is achieved
through the formation of stable donor-acceptor complex with
chloranilic acid. The resulting intensely reddish chloranilic
acid radical anion possesses a characteristic absorption
maximum at 530rm. The second method is based on the
reduction of Iron(IIl) by diclofenac and subsequent
complexation of Iron(II) with 1, lO-phenanthroline, having
maximum absorbance at 500 nm. The possible interferences
by various excipients have been checked out. Application of
the suggested methods to representative dosage forms are
presented and compared with the reference method.
E X P E R I M E N T A L
Apparatus Method-I
A Spectronic-20D^ (Split-beam Milton Roy Co)
spectrophotometer, with Icm path length quartz cell was
used.
Reagents
All reagents used were of analytical-reagent grade and the
solvent of spectroscopic grade. A 0.1% chloranilic acid
.solution in 1,4-dioxane and the diclofenac sodium solution
of ImgmL'' concentration in methanol were used.
114
General procedure for the calibration:
-1 Accurately measured volumes equivalent to 0.1-1.80 mgmL
diclofenac sodium was transferred into a series of lOmL
standard flasks. Then 2.5mL of chloranilic acid solution
were added to each flask. The contents were mixed well and
made up to volume by a solvent mixture of 1,4-dioxane and
methanol made in the ratio 2:8. The absorbance was
measured at 530nm against the reagent blank prepared
simultaneously without diclofenac solution.
Procedure for the assay of dosage forms
About ten tablets were finely powered. An amount of the
tablet powder equivalent of 50mg of the diclofenac sodium
was weighed accurately and treated as described above for
the standard drug solution. Filtration was performed in
instantaneous where insoluble matter remained during
preparation of the sample solutions.
RESULTS AND DISCUSSION
A chloranilic acid solution in 1,4-dioxane is yellow. The
spectrum of 0.1% chloranilic acid in 1,4-dioxane possess a
strong band at 440nm. Addition of diclofenac sodium to this
solution causes an immediate change in the absorption
spectrum, with a new characteristic band at 530nm (Fig 3.2),
believed to be due to chloranilic acid radical anions.
Reaction involved
Chloranilic acid in organic solvents exist in un-ionized form
and acts as a 7i-acceptor in similar manner to other quinones
115
[15,16]. Therefore, the addition of diclofenac sodium results
in the formation of charge-transfer complex of n-7i and n-n*
type. The complex is believed to be intermediate molecular
association compound for which in appreciable polar
solvents, complete transfer of charge takes place, producing
the corresponding radical ion, which is responsible for the
production of intense absorption band at 530nm. This
predominant chroinogen with chloranilic acid is the reddish
radical anion, which is formed by the dissociation of an
original donor-acceptor (DA) complex.
D + A^=T b—A ^=r D '+A • DA Complex (radical ions)
The dissociation of the DA complex is promoted by the high
ionizing power of solvent. Diclofenac acts as an electron
donor and hence forms charge-transfer complex with TC-
acceptors. The donated electrons originate from the nitrogen
atom and the aromatic ring forming n-n and n-K* complex
respectively as shown in Fig. 3.1.
Optimization of variables:
To develop a quantitative method based on this reaction, a
search was conducted for most effective 7r-acceptor.
Chloranilic acid in 1,4-dioxane readily reacts with
diclofenac sodium, forming a stable reddish chloranilic acid
radical anion. 1,4-dioxane proved to be most suitable
solvent. Other organic solvents were not suitable because
the complex formed in these solvents either had low
absorbance or precipitated on dilution. The reaction was
instantaneous and the product remained stable for forty
116
minutes (Fig. 3.3) permitting quantitative determination to
be carried out with good reproducibility.
Performance characteristics:
Under the proposed experimental conditions, there was a
linear relationship between absorbance and final
concentration over the range 10-180|agmL'^ of drug (Fig.
3.5) with corresponding molar absorbitivity of 1.5x10"
LmoT'cm"'. The jobs plot (Fig. 3.6) showed the molar ratio
of 1:2 between diclofenac and the chloranilic acid. The
reproducibility of the method was determined by the
analysis of various samples covering the recommended
concentration range for diclofenac sodium. The recoveries
show, the method is reproducible, accurate and precise
(Table 3.1).
Application to dosage forms
Results for determination of diclofeanc sodium in different
dosage forms are also presented and show good agreement
with those of the reference method [12]. This conclusion
was confirmed by statistical analysis, showing no significant
difference in accuracy and precision between the proposed
and the reference methods (Table 3.2).
Apparatus Method-II
A Spectronic-20D'^ spectrophotometer, with 1cm cell, and a
water bath (NSW-133) India was used during heat treatment
and the colour development.
117
Reagents:
All the reagents used were of analytical grade.
A 0 .1% ferric ammonium sulphate (w/v) solution and O.OIM
EDTA solution were prepared in O.OOIM sulphuric acid and
conductivity water respectively. A O.IM solution of 1,10
phenanthroline was prepared in ethnol. A mixed phosphate
buffer (pH= 6.8) was used.
Aqueous 0.1% (w/v) pure diclofenac solution was used for
the preparation of calibration curve.
Preparation of the calibration curve
Mixed up l.OmL of buffer and 2mL of Iron (111) solution in
lOmL volumetric flasks. Added sample solutions containing
0.05-1.0mgL"' of diclofenac, followed by 0.5mL of 1,10-
phenanthroline. Heated for 25 minutes on a boiling water
bath for the complete development of colour, then added
l.OmL of EDTA solution, dilute up to the mark with
distilled water, and measured the absorbance of the coloured
solution against the reagent blank at 500nm.
The proposed method was employed for diclofenac content
estimation in commercial pharmaceutical preparations. A
comparison of the reference method was also performed
(Table 3.2).
Ten tablets were crushed and finely powdered. An
accurately weighed amount equivalent to 50mg of diclofenac
was stirred in 50mL conductivity water. The residual solid
was filtered on Whattman No-42 paper and washed with
118
water. The filtrate was made up to 50mL in a standard flask.
The determination was carried in the same way as for the
pure drugs in the calibration procedure.
RESULTS AND DISCUSSION
The absorption spectrum of the blank Iron (II) and
Iron (111) complexes are shown in the Figure3.7. The
maximum absorbance for both A and B is observed at
500nm. The shapes of the absorption spectra are quite
similar for A and B.
The l ron(l l ) - l , 10-phenanthroline complex shows an intense
red colour and is more stable than its corresponding ferric
complex. The ferric complexes are shown to get reduced by
reducing agents, which is the basis of present work. The
reduction of Iron(lll) by diclofenac and the formation of
Iron(lI)-l , 10- phenanthroline complex depends on pH. The
development of colour is instantaneous in mixed phosphate
buffer solution in the pH range 6 to 8. The photo-reduction
of the ferric complex is avoided by adding a chelating agent
such as ethylenediamine tetra-acetic acid to mask the
excessive ferric ions after the colour development. Beer's
law is obeyed in the concentration range 5-100|agmL''
(FigS.lO) with the molar absorbitivity of 4x 10^1moL''cm''.
Some excipients with the dosage forms are found not to
interfere (Table3.3). Paracetamol being an oxidant, interfers
with the test, so the method can not be used for the
determination of diclofenac in presence of paracetamol. The
method is accurate and sensitive with respect to the
microgram range of determination and higher molar
absorbitivity, than are the earlier methods.
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Volume of chloranlllc acid (mL)
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125
0.5 1
Amount of diclofenac sodium (mg)
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126
0.25
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mole fraction diclofenac sodium
Fig. 3.6 Jobs plot for the reaction of diclofenac sodium with chloranilic acid, X max. 530nm.
127
1 5 '
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0,9 • 128
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Volume of Fe'' (mL)
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129
1.4
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02 0.4 0.6 0.8
Amount of diclofenac sodium (mg).
—I
12
Fig. 3.10 Calibration graph of diclofenac sodium, X max. = SOOnm.
130
REFERENCES
[1] W.Schneider and P.H. Degen, J. Chromatography. 217
(1981) 263.
[2] S.G. Owens, M.S. Roberts and W.T. Friesen, J.
Chromatography. I l l (1975) 293.
[3] J .Godbil lon, S. Gauron and J.P. Metayer, J.
Chromatography. 338 (1985), 151.
[4] Beaulieu. N, Lbvering. E.G, Lefrancois J. and Ong.H,
J. Assoc, off. Anal. Chem. 73(5) (1990) 698.
[5] Said. S.A, and Sharaf. A.A, Arznemit te l forschung.
31(12) (1981) 2089-92.
[6] Carreira. L.A, Rizk. M, el-Shabrawy Y, Zakhari . N.A.
and Toubar. S.S, J. Pharm. Biomed. Anal. 13(11)
(1995) 1331-7.
[7] M.L. Fernandez. Cordova, P. Ortega. Barrales and
A.Molina. Daiz, Anal. Chim. Acta. 369 (1998) 263-68.
[8] S.Agatonovic-Kust in, L.J. Zivanovic, M. Zecevic and
D. Radulovic, J. Pharm. Biomed. Anal. 16 (1997) 147-
53.
[9] El-Sadak. M, Bark. M, Khier. A.Adil , Egy. J. Pharm.
Sci. 24(1-4) (1988) 367-79.
[10] Shen-Hui Zhang, Jain-ZhangFeng, and Shen-yung
Tung, Fenxi kexue Xuebao. 12(3) (1996) 194-7.
131
[11] K.Shivram, B.V. Kamath, and V.P. Arya. East Pharm.
34(402) (1990) 119-20.
[12] Y.K. Agrarwal, V.P. Upadhyay and S.K. Menon, Ind. J.
Pharm. Sci. 50 (1988) 58.
[13] Y.K. Agrarwal and K.Shivramchandra, J. Pharm.
Biomed. Anal. 2(9) (1991) 97-100.
[14] C.S.P. Sastry, A.S.R. Prassad.Tipirneni , and M.V.
Suryanarayana, Analyst . 114 (1989) 513-15.
[15] Alaa. S. Amin, Gamal.O. El-Sayed, and Yousry. M.
Issa, Anlyst. 120(4) (1995) 1189-93.
[16] M.Abdel-Hedy Elsayed and Suraj. P. Agarwal, Talanta.
29 (1982) 535-37.
Chapter-4
%{ftraviofet Sheotroh^oiomeirk Studies for fke "Estimation of Cisapride in IPfiarmaceuticaf Uosaae orms.
132
Introduction
Cisapride is a gastrointest inal s t imulat ing agent, effective in
the relief of gastrointest inal or esophagus disorders and in
the promotion of gastric emptying of gast rointes t inal
moti l i ty [1]. It is a prokinet ic agent believed to facil i tate
acetyl choline release from the myenteric plexus of the gut
[2] . It is chemically known as (+) c is -4-amino-5-chloro-N-
[ 1 - [ 3- f lourophenoxy )propyl ] - 3- methoxy -4 -p iper idyl ] -
2-methoxy benzamide (Figure 4.1).
Fig 4 . 1 . Cisapride
The drug is official in the British Pharmacopoeia [3] . The
official method for the assay cisapride involves the t i trat ion
with perchloric acid and the determination of end point by
potent iometry . Three spectrophotometr ic methods for the
determinat ion of cisapride are described [4]. The methods
are based on the coupling of the diazotized drugs with the
reagents such as chromotropic acid, pholoroglucinol and N-
(-1-naphthyl) ethylene diamine dihydrochloride to give
coloured products having maximum absorbances at 530nm,
450nm and 540nm respect ively. A colorimetr ic procedure for
the est imation of cisapride in dosage forms has been
desci ibed [5]. The method is based on the reaction of
133
cisapride with para-dimethylamine c innamaldehyde in
presence of t r ichloroacet ic acid in methanol forming a
sch i f f s base with ;^max. at 525nm. An HPLC method for the
determinat ion of cisapride in tablets and oral suspensions in
the presence of methyl polysi loxane is developed and
val idated [6]. A reversed phase Cig Bandapak column and
mobile phase comprising methanol and water were used. The
drugs were detected and assayed at 275nm. Another HPLC
method for the assay of cisapride in plasma is described [7].
Alkal inised samples of plasma are extracted with l.OmL of
propanol in chloroform. Cisapride and internal standard are
detected by flourescence monitoring at 295nm (exci ted) and
350nm (emission) . Cisapride levels in human plasma are
determined by RP-HPLC method [8] in which carbamazepine
is used as internal standard. Cisapride and the internal
standard are extracted from the plasma with
dichloromethane and detected by UV detector at 370nm. A
flourimetric method for the determination of cisapride in
pharmaceut icals [9] under continuous and discont inuous
condit ions is based on the fact that cisapride exhibits an
emission spectrum with a maximum at a wavelength of
355.2nm and excitat ion at 310nm. A spectrophotometr ic
method for the determination of cisapride is based on its
reaction with p-dimethylaminocinamaldhyde in phosphoric
acid solution to form an orange chromogen [10] with
maximum absorbance at 546.5nm and obeying Beer's law
in the range 160-560ngmL"' . A UV Spectrophotometr ic
method for the determination of cisapride in pharmaceut ical
dosage forms is developed [11] with its concentrat ion range
134
between 2.0-20|igmL''. Two more methods are developed for
the determination of cisapride [12]. In the first method the
drug is reacted with N-dimethylaminocinnnamaldehyde in
presence of sulphuric cid to form Schiff base having A,max.
at 520nm and obeying Beer's law in the range 0.8-12)igmL' .
In the second method, the drug solution reacts with folin-
cocalteu reagent in presence of sodium carbonate forming a
blue chromogen with Xmax. at 880nm and linearity in the
concentration range 0.4-8.0)j,gmL"'. The diazotization
followed by coupling with N-(l-naphthyl) ehtylenediamine
dihydrochloride or thiocol is studied with the aim of
standardizing the methadol of cisapride pharmacentical
analysis [13]. Reaction products with N-(l-napthyl)
ethylenediamine has an absorption maximum at 535nm and
products with thiocol at 495nm. The development of a
spectrophotometric method based on the charge transfer
formation of cisapride with iodine and catechol is reported
[14]. Four extractive spectrophotometric methods for the
assay of cisapride in pure and dosage forms based on the
formation of chloroform soluble ion-associates under
specified experimental conditions are described [15]. Four
acidic dyes viz; Suprachen violet 3B, Erioglaucine A,
Napthalene Bluel2 BR and Tropaelin 000 are utalized.
Another spectrophotometric method for the determination of
cisapride [16] in dosage forms is based on the oxidation of
drug with nitrous acid followed by treatment with cresol fast
violet acetate and measurement of the absorbance.
In this chapter, two different procedures for the
determination of cisapride are reported which are based on
135
redox reaction followed by spectrophotometric studies. In
the first method, cisapride is treated with peroxydisulphate
in presence of alkili. A yellow coloured product is obtained
on heating, which has A,raax. 308nm. The second method
involves acidic oxidation of cisapride with Ce(IV) to
produce coloured product having Xmax, at 289nm. The
methods are simple, accurate and can be satisfactorily
applied to representative pharmaceutical formulations.
EXPERIMENTAL
Instrumentat ion Method-I
DU-40 spectrophotometer (Beckman) was used for
absorbance measurements. Controlled temperature water
bath NSW-133 was used for heat treatment & colour
development.
Reagents
All chemicals used were of analytical or pharmaceutical
grade. Pure cisapride was obtained as a gift sample from
Sigma Laboratories Mumbai (India) and prepared (ImgmL'')
in dimethylformamide (DMF). Potassium persulphate and
Sodium carbonate of O.IM each were prepared in double
distilled water.
Calibrat ion
Aliquots of 0.1-l.OmgmL"' of the standard solution of
cisapride were pipetted into a series of lOmL volumetric
flasks. Optimum results were obtained after the addition of
2.0mL of potassium persulphate and l.OmL of sodium
136
carbonate. The mixture was heated on a water bath at 85-
90°C for ten minutes to complete the reaction. After cooling
to room temperature and dilution to lOmL with distilled
water, the absorbance of the yellow coloured product was
measured against the reagent blank at 308nm.
Application of the proposed method
About ten tablets were grinded to a fine powder. The tablet
powder equivalent to 50mg of cisapride was weighed
accurately and treated as described above. The insoluble
matter remained during preparation of sample solution &
was filtered off.
RESULTS AND DISCUSSION
The reaction mix,ture of cisapride and peroxydisulphate in
alkali yields a yellow coloured product on heating having
>.max. at 308nm (Fig 4.2), whereas the reagent blank shows
Xmax. at 236nm. This type of reactions between aromatic
amines and persulphate ions in aqueous base have been
already cited in the literature [17-20]. Such reactions are
appropriately named as Boyland-Sims oxidation. The
mechanistic approach is believed to be an electrophilic
displacement by the peroxide oxygen [21]. This involves the
electrophilic attack by the persulphate ion on the nitrogen of
the neutral amine at ortho orientation to from aryl
hydroxylamine-0-sulphonate. This intermediate is then
assumed to rearrange to the 0-aminoaryl sulphate. The intial
attack at nitrogen is consistent with the work in other
systems [22-25]. The postulated intermediate is more readily
137
oxidized than the start ing material . The yield of o-aminoaryl
sulphate is believed to fall in strong alkali , a fact
a t t r ibutable to more ready attack by persulphate on the
dianion of the intermediate formed.
Study of the proposed experimental condit ions.
To obtain optimum condi t ions , the effect of different
volume ratio of persulphate reagent and sodium carbonate
are studied. It was observed that 2.0mL of O.IM persulphate
(Fig. 4.3) and l.OmL of O.IM sodium carbonate (Fig. 4.4)
gave the optimum results for the complete oxidation of
c isapr ide. Under the optimum experimental condit ions the
product is found to be stable after ten minutes of heating
(Fig. 4.5) on a water bath at 85-90°C (Fig. 4.6) Beer 's law is
obeyed over a narrow range from 10-100iJ,gL' (Fig.4.7) with
the molar absorbit ivi ty of 4.63x10^ Lmol ' ' cm"'.
The reproducibi l i ty of the procedure is studied by running
various repl icate samples, each containing different amount
of drug in the final solution (Table 4.2). In order to check
the validi ty of the proposed method, the resul ts are
compared with standard reference method [14]. The results
are presented in the (Table 4.3). A study of possible
interference caused by common diverse ions in dosage forms
has been performed. The results are summarized in (Table
4.1).
The proposed method is sensit ive and rel iable at the
recommended concentrat ion range. The stabil i ty of the
colour is high and is not affected by moisture. Hence, the
138
proposed method can be recommended for the routine
analysis of cisapride in drug control laboratories.
Reagents Method-II
The solution for cisapride (ImgmL' ') was prepared in
dimethylformamide and Ce(IV) Stock solution (0.4M) was
prepared in 2M nitric acid.
Calibration Procedure
To a series of lOmL standard flasks, appropriate volumes of
standard solution of cisapride between 0.2-1.2mgmL"' were
placed, followed by 1.5mL of Ce(IV) solution. The contents
were mixed well and allowed to stand at room temperature.
The volume was completed with dimethylformamide and the
absorbance meaurements were recorded at 289mm against
the reagent blank.
Assay of dosage forms
Ten tablets were grinded and powdered. The content of
tablet power equivalent to 50mg of cisapride is dissolved in
dimethylformamide, It is filtered instantaneously and
proceeded as described under " Calibration Procedure".
RESULTS AND DISCUSSION
Ammonium eerie nitrate is a versatile oxidimetric reagent.
Its high oxidation potential and excellent solution stability
promoted us to use this reagent for the quantitative
determination of cisapride. The absorption spectrum for
Ce(IV) solution in 2M nitric acid shows ;\.max. at 357nm,
139
whereas its reaction product with cisapride gives absorption
maximum at 289nm (Fig.4.8), thus indicating a
hypsochromic shift with respect to ?iniax. of the cerium (IV)
solution. The condit ions for the production of analyt ical ly
useful measurements from Ce(IV) oxidation of cisapride
were optimized so as to achieve maximum and reproducible
absorbance measurements . The Ce(IV) concentrat ion and the
nature and concentrat ion of the acid present have a marked
influence on the analytical signal and were invest igated to
obtained maximum absorbance reading. The effect of cerium
(IV) concentrat ion for I.O mL of cisapcide is shown in
(Figure 4.9). Nitric acid was the most effective medium for
the sensit ive measurement of cisapride, and cerium (IV)
concentrat ion of 2.73x10"^ molL ' ' was chosen for further
s tudies . For the given concentrat ion of cer ium(IV), 2moIL' '
nitric acid produced maximum absorbance (Fig .4 .10) , hence
this concentrat ion was chosen for the analyt ical procedure.
Under the selected experimental condit ion described above,
the cal ibrat ion graph was linear in the concentrat ion range
20-120|.igmL' of cisapride (Fig.4.11) . The regression
equation evaluated for the cal ibrat ion graph is
A=0.21x+0.019, with the correlat ion coefficient of 0.9989.
In order to apply suggested method for the analysis of
pharmaceut ical dosage forms, the influence of commonly
used excipients and addit ives was invest igated and the
resul ts obtained are presented in Table 4 . 1 . Under the
react ion condit ions used, the foreign substances tested did
not interfere with the proposed method.
140
The precision of the method was determined by the percent
error for ten multiple analyses of a series of solutions. The
accuracy of analysis was tested by applying the
recommended procedure (Table 4.2). The recoveries of the
different amounts tested were determined from the standard
reference graph and found to be satisfactory.
The proposed spectrophotometric method has been
successfully applied for the determination of cisapride in
pharmaceutical dosage forms. The results obtained for this
procedure were evaluated against the reference method [14]
as shown in (Table 4.3). The proposed method has the
advantage of being simple, accurate and sensitive for
estimation of cisapride in pure form and formulations.
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145
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0.4
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0.6 1
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147
0.2 0.4 0.6 0.8
Amount of Cisapride (mg)
Fig. 4.7. Calibration graph) of cisapride at (85-90°C), Xmax =308 nm.
1 5n
148
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0.8 '
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0.6
0.4
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Nitric acid concentration (mol. L' )
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150
02 04 06 08 1
Amount of Cisapride (mg)
12 14
Fig. 4.11. Calibration graph of cisapride, Xmax = 289 nm.
151
R E F E R E N C E S
[1] J. Robert Sims and W.Slivka. PCT Inst. Appl. W09501 ,
803 (cl. A61K37/54) , 19 January 1995, US Appl. 87,
590, 06July 1993, 21pp.
[2] F.Onat, B.Yegen, K.Berkman and S.Oktay, Gen.
Pharmacol . 26(1994) 1253.
[3] British Pharmacopoecia , Vol .1 , HMSO, London, 1995,
P.1746.
[4] Sast ry .C.S.P, Srinivas.Y and Subba. Rao.P.V, Indian J.
Pharm. Sci. 58(4) (1996) 169-71.
[5] Par imoo.P, Padma.K, Jagadesh.Babu. R and
Ravishankar K, East Pharma. 39(457) (1996)159-60.
[6] Gaba. Sangeeta, Badwa. Nagesh and Sethi. P.D, East
Pharma. 38 (453) (1995) 157-158.
[7] Preechagoon. Y and Charles .B, J. Chromatogr. Biomed.
Appl. 670 (1) (1995) 139-43.
[8] Arafat. Tawfiq, Shami. Munzar, Kaddomi. Amal,
Badwan Adnan and Yassein. Mohamed, Alexandria . J.
Pharm. Sci. 9(1) (1995) 1-3.
[9] Gonzalez Martin M.l, Gonzalez. Perez. C and Blanco
Lopez. M. A, Anal. Lett. 27 (9) (1994) 1713-18.
[10] Barbhai . A.J, Mahadik. R.K, Moore H.N. and Panzade
P.D, Indian Drugs. 3 6 ( 1 0 ) ( 1 9 9 9 ) 665-666.
152
[11] Bhatia M.S, Kashedikar .S.G and Chaturvedi .S. C,
East Pharma. 42 (504) (1999) 131-132.
[12] Mohan. Y. Rame, Srinivas. J .S, Avadhanula. A. B and
Anjaneyulu Y, East Pharma. 40 (480) (1997) 119-21.
[13] Honda. Akimi. Mori, Magalhaes and Joae. Farnendes ,
Rev. Farm. Bioquim. Univ. Sao Paulo. 33 (2) (1997)
111-115.
[14] Krishnakumar. K.R, and Raju. R, Indian. J. Pharm. Sci.
58(1) (1996) 39-40.
[15] C.S.P. Sastry, Y. Srinivas, P.V. Subba. Rao, Talanta.
44 (1997) 517-526.
[16] Sastry. C.S.P, Srinivas. Y and Subha. Rao. P.V, J. Inst.
Chem (India) . 68 (4) (1996) 108-109.
[17] E. Boyland, D. Manson and P. Sims, J. Chem. Soc.
3623 (1953).
[18] E. Boyland and P. Sims, J. Chem. Soc. (1954) 980.
[19] P. Sims, J. Chem. Soc. (1958) 44.
[20] E. Boyland and P. Sims, J. Chem. Soc. 4198 (1958).
[21] N. Venkatasubramanian and A. Sabesan, Cand. J.
Chem. 47 (1969) 3710.
[22] D.B. Denney and D.Z. Denny, J. Amer. Chem. Soc.
82 (1960) 1389.
153
[23] K.M. Ibne-Rasa and J.O. Edwards, J. Amer. Chem.
Soc. 84 (1962) 763.
[24] K.M. Ibne-Rasa, C.G. Lauro and J.O. Edwards,
J. Amer. Chem. Soc. 85 (1963) 1165.
[25] D.M. Graham and R.B. Mesrobian, Can. J.Chem.
41 (1963) 2938.
Chapter-5
%inetk SpecfropftofomeMc TAetf^odfor if^e
Uetermination of Nafiaipdic acid.
154
Introduction
Nalidixic acid ( l - e thy l - l , 4 -d ihydro -7me thy I -4 -oxo- l , 8 -naph-
thyr id ine-3- Carboxylic acid) is an ant ibacter ial agent which has
been occasional ly used intravenously for the t reatment of gram-
negative urinary tract infections since 1963. Renal failure is
common amongst these pat ients and therefore it is necessary to
monitor the plasma concentrat ion in order to prevent toxic or, on
the other hand subtherapeutic levels . The major metabol i te ,
hydroxynal idixic acid is in vitro also active against Gra:m-
negative micro-organisms. Nal idixic acid has also been used for
the prevent ion and treatment of infectious diseases in fish.
Concern has arisen as to the presence of drug residues in fish
t issues, and demand for a rapid, simple and sensit ive analyt ical
method for determining them has increased. In man nal idixic acid
is converted to the 7-hydroxymethyl metaboli te which is further
oxidized to generate the dicarboxylic acid.
A variety of methods have been described for the assay of
nalidixic acid in biological media and dosage forms. A high
pressure liquid chromatographic method has been developed for
the s imultaneous assay of nal idixic acid and hydroxynal idixic
acid in human plasma and urine [1]. The procedure measures
both the compounds in the same sample. Chromatograms from
control plasma and control urine yield a small blank which do
not interfere with chromatograms from processed s tandards. A
gas chromatographic method has been described for the
determinat ion of nal idixic acid in plasma [2]. The method is
sensit ive enough to determine therapeutic levels in l.OmL
155
plasma. Quantitation is carried out by means of both peak height
measurements and peak area measurements. A method for the
simultaneous determination of nalidixic acid and its analogues in
cultured fish has been developed by high performance liquid
chromatography [3]. The method also involves UV and
flourometric detection, and Sep-pak Cu cartridges as a clean-up
step. A selective TLC- densitometric method has been described
by means of which nalidixic acid is determined in duplicate with
good precision at therapeutic levels in a 100-}a,L plasma sample
[4]. A high performance liquid chromatographic method for the
analysis of nalidixic acid in plasma and urine has been described
[5]. The statistical evaluation of this assay technique shows
acceptable accuracy and precision at concentrations 2.0|j,gmL'' of
plasma or 29.0)igmL'' of urine for samples augmented with the
drug. A differential pulse polarographic method has been
proposed for the determination of nalidixic acid and its
metabolite in urine [6]. The method involves a simple extraction
which is followed by controlled-potential analysis at the mercury
pool electrode and at the HMDE. Another high performance
liquid chromatographic method has been described [7] for the
analysis of nalidixic acid and its two metabolites in human
plasma and urine following the oral administration of a
therapeutic dose in human. The applicability of this method to
pharmacokinetic studies of nalidixic acid in humans has also
been demonstrated. A gas chromatographic method has been
established for the quantitative analysis of nalidixic acid [8].
The method is based on the derivatization of nalidixic acid with
diazomethane, and 5-a-cholestane is used as an internal
156
Standard. Quantitation is achieved by measuring peak height
ratios. The improved simplicity, specificity & accuracy of the
method has been demonstrated for the quantitation of nalidixic
acid in tablets. A high performance liquid chromatographic
analysis of nalidixic acid and its hydroxymetabolite in plasma
with an anion exchange system has been reported [9]. Proposed
experimental parameters have also been studied. Another HPLC
assay of nalidixic acid in plasma after alkylation with methyl
iodide has been performed [10]. A packed column supercritical
fluid chromatography has been employed [11] for the assay of
nalidixic acid and metronidazole in bulk and combined dosage
forms employing mebendazole as the internal standard. The
validation of the method of assay of drugs has been done by the
internal standard method 8L linear range for both the drugs has
been 10-60|j.gmL'V A pH-induced difference spectrophotometric
method for the simultaneous determination of metronidaole and
nalidixic has been developed [12] in presence of each other as
well as the excipients. The presence of identical isobestic points
indicate the non-interference of excipients in the absorption at
respective wavelengths. A spectrophototric method has been
developed for the simultaneous determination of nalidixic acid
and metronidazole in pharmaceutical preparation [13] using
multicomponent mode of instrument. Another method for the
simultaneous determination of these drugs in two component
tablets and suspensions is based on the dual wavelength
spectrophotometry [14] without any prior separation. Two more
methods have been developed for the simultaneous determination
of nalidixic acid & metronidazole. The methods used
simultaneous equation and Q-analysis for their determination
157
[15]. The absorbance maxima of two drugs and their isobestic
point are used as analytical wavelength. Another
spectrophotometric procedure using multicomponent mode of
analysis has been developed for the simultaneous determination
of nalidixic acid and metronidazole in their dosage forms [16].
The method is based on the measurement of UV absorbance
maxima of the two drugs.
The literature survey for the assay of nalidixic acid has revealed
that, a few methods are available in the visible range of
spectrometry. Besides, the unstability of its reaction product
with ferrous ammonium sulphate at maintained temperature
(50+0.2°C) made us to develop a kinetic method which permits
sensitive determination within a short limit of time. The study
shows that the interaction between nalidixic acid and ferrous
ammonium sulphate is first order reaction with respect to
nalidixic acid. The proposed method can be successfully applied
to the commercial dosage forms.
EXPERIMENTAL
Instrumentation
DU-40 Spectrophotometer (Beckman) was used to record the
spectra and Spectronic-ZOD"" (Milton Roy Co.) was used for
absorbance Vs. time measurements. A temperature controlled
water bath (NSW-133 India) was used to maintain a constant
temperature (50 + 0.2"C).
158
Reagents
All the Chemicals used were of analytical or pharmaceutical
grade. Stock solution of 5.0xlO'^M ferrous ammomium sulphate
was prepared in 1.0X10"' 'M sulphuric acid. Pure nalidixic acid
(Himedia Labs. India) ImgmL"' was prepared in methanol.
Calibration Procedure
In 10ml volumetric flasks, the final concentration of standard
pure solution of nalidixic acid between 0.2-0.8mgmL'' were
placed, flowed by 1.5mL of ferrous ammonium sulphate. The
flasks were immersed in a water bath maintained at 50+0.2°C.
Each flask was taken out at a time interval of five minutes,
cooled at room temperature and diluted upto the mark with
distilled water. The contents were transferred to the
spectrophotometric cell and the absorbance-time curve at 420nm
was recorded against the blank containing all the constituents of
sample except the drug. Calibration graph was prepared by a plot
of logR (Initial rate of reaction) Vs. logC (nalidixic acid
concentration).
Analysis of nalidixic acid in tablets.
The above proposed method was used for the estimation of
nalidixic acid in tablets. About ten tablets were accurately
weighed and grinded into fine powder. An amount of the tablet
power equivalent to lOmg of nalidixic acid was weighed and
treated as described for the calibration procedure. The insoluble
matter was filtered instantaneously during the preparation of
sample solutions.
159
RESULTS AND DISCUSSION
The pure nalidixic acid solution in methanol shows two ?^max. in
the ultravoilet region at 247nm and 338nm.A yellow coloured
chromogen is formed; when ferrous ammonium sulphate is added,
having A,max at 420nm (Fig.5.2),which indicates a bathochromic
shift with respect to A,max. of nalidixic acid.
The reaction product is unstable for forty minutes and
hence the kinetic approach is preffered over conventional
spectrophotometry, where unstability of colour preclude their
efficacy. The kinetics of the formation of reaction product at
maintained temperature (50+0.2°C) is studied at 420nm.The
increase of absorbance is measured as a function of time.
The redox reaction between nalidixic acid and ferrous ammonium
sulphate leads to the formation of a coordination compound. The
reduction of nalidixic acid results in hydrogenation of ethylene
bond in azinone ring. The oxygen atoms, because of lone pair act
as ligands and form coordinate bonds with Iron (III) as shown in
Figure 5.1.
Et . Me. N^ HH
H ^ P J ' " reairangement
•COOH
Fig.5.1 Tentative reaction mechanism of nalidixic acid with
ferrous ammonium sulphate.
To study the rate of reaction with respect to nalidixic acid
(keeping the ferrous ammonium sulphate cone, constant) at
160
50+0.2°C, absorbance versus time observat ions are recorded
(Table 5.1). A plot of absorbance versus time (t) gives straight
lines (Fig 5.3) whose slopes are used as a measure of reaction
rate for solut ions containing different concentrat ion of nalidixic
acid. The kinet ic studies with respect to nal idixic acid gave first
order plots . Job's method of cont inuous variat ion establ ished that
molar s toichiometr ic ratio of reaction between nalidixic acid and
ferrous ammonium sulphate is 1:1 (Fig 5.5). Calibrat ion graphs
constructed by plott ing initial rate of react ion(R) Vs.
concentra t ion (C) or by logR Vs. logC (Fig 5.4(a,b)) show a
linear re la t ionship with respect to the concentrat ion of nalidixic
acid in the range 20-80|j,gmir'. The curve is also used to calculate
order of react ion and found to be equal to +1.0. Validity of the
proposed kinetic method was checked by running different
concentrat ions of nal idixic acid, and the results obtained are
presented in Table 5.2. The optimum reaction temperature was
50+0.2°C, lower temperature showed inaccurate resul ts and the
reaction was incomplete . On the other hand, higher temperature
led to the decomposi t ion of reaction product. Some common
excipients such as lactose, starch, fructose, glucose and sucrose
which are usually present with dosage forms show no
interference in determinat ion and the maximum tolerance amount
was calculated using the procentual change in the absorbance of
nal idixic acid measured value. The results are summarized in
(Table 5.3).
The proposed kinetic method has been successfully applied to the
determinat ion of nal idixic acid in commercial dosage forms. The
results are s tat is t ical ly compared with those obtained with the
161
reference method [17] and are presented in Table 5.4. The
calculated 't' values suggested no significant difference between
the two methods in terms of precision & accuracy. Hence the
recommended method is at par with conventional spectrometric
methods in terms of its reproducibi l i ty and the time required for
the analysis .
a.
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168
10 15 20 25
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Nalidixic acid concentrations are; (a) 8.6x10"^ (b) 1.72x10'' (c) 2.15x10"^
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170
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171
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Pharm. Sci. 60(3) (1998) 140-143.
[15] G.P. Jadhva, H.N. Moore and K.R. Mahadik, Indian Drugs.
35(8) (1998) 475-480.
[16] R. Paliwal, D.K. Jain and P. Trivedi, Indian Drugs, 35(3)
(1998), 165-167.
[17] Shingbal D.M, S. Sarjyotishi, Indian Drugs. 28 (1989) 569.
Reprinted from
JOURNAL OF
PHARMACEUTICAL AND BIOMEDICAL
ANALYSIS
Journal of PliarmatcuUcrfl and Biomedical Aiialyiib 21 (1999) 473-482
Simultaneous spectrophotometric and volumetric determinations of amoxycillin, ampicillin and cloxacillin in
drug formtilations: reaction mechanism in the base catalysed hydrolysis followed by oxidation with iodate in dilute acid
solution
Saidul Zafar Qureshi *, Talat Qayoom, Marud I. Helalet Dcimiiiiiciil of Cliciiinn \ AinihiKdl Rccanli L<ihiii<ii<ir\\ Ahviiili Un\liiii I'niivisin 1lii;,iili-2()2(l()2 (I /') liului
ELSEVIER
Journal of Pharmaceutical and Biomedical Analysis ELSEVIER 21(1999)473-482
lOURNALOr
PHARMACEUTICAL AND BIOMEDICAL
ANALYSIS
www.elsevier.com/locate/jpba
Simultaneous spectrophotometric and volumetric determinations of amoxycillin, ampicillin and cloxacillin in
drug formulations: reaction mechanism in the base catalysed hydrolysis followed by oxidation with iodate in dilute acid
solution
Saidul Zafar Qureshi *, Talat Qayoom, Marud I. Helalet Department of Chemistry, Analytical Research Laboratory, Aligarh Muslim University, Aligarh-202002 (U.P.), India
Received 16 October 1998; received in revised form 29 January 1999; accepted 5 May 1999
Abstract
A method for the analysis of degraded products of amoxycillin, ampicillin and cloxacillin in drug formulations, obtained as a result of their base hydrolysis is described. Simultaneous spectrophotometric and volumetric determinations of the antibiotic is based on the neutralization of the degraded product by dilute hydrochloric acid to get a pH ~ 2 to be conducive for redox titration using potassium iodate as titrant. A red purple colour is developed in carbon tetrachloride at the end point. Spectrophotometry is done after separating the organic layer and measuring the absorbance of red-purple colour at X,„.„ 520 nm. The pathways of different degraded products and their oxidation mechanism is described on IR, TLC and UV spectroscopic studies. © 1999 Elsevier Science B.V. All rights reserved.
Keywords: Amoxycillin; Ampicillin; Cloxacillin; Spcclropholoniclric and volumclric dclcrminalion; Simullancous dclcrmination; Pure and drug Ibrmulations
1. Introduction ^^^ carried out by some oxidants prior to a base or acid catalysed reaction of antibiotics. The de-
A large number of methods for the analysis of S'"'' ' ' hydrolysed products of antibiotics are ti-ampicillin, amoxycillin and cloxacillin and related ^'''^^'^ quantitatively against oxidants such as antibiotics are described [1 -3] . Emphasis has been " § ' ' ["^ ^"'^ '"'^^'^ solutions [5]. It is observed given on spectrophotometric and titrimetric meth- ^^'^^ *^^" hydrolytic conditions are changed from ods, because they are simple and easily manage- moderate concentration of alkali or acid to a
able to the third world. The volumetric titrations ^ . ' , f °' " ' ' "^ '^' f •"'= .?'. P " '^^l^"^. ^^^' ^ diiierent pathway of antibiotics mechanism re-suits. The titrimetric method [4] described by al-
• Corresponding author. Tel; +91-571400515. kaline or enzymic hydrolysis followed by titration
0731-7085/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: 50731-7085(99)00205-8
474 S.Z. Qureshi et at. /J. Pham. Biomed. Anal. 21 (1999) 473-482
with Hg(II) might suffer a setback. The use of nitric acid to neutralize the excess alkali before performing the titration may oxidize thiazolidine ring of the penicillin molecule and therefore could give erroneous results. The authors themselves reported better results with enzymic hydrolysis than alkaline hydrolysis.
In the direct titration of antibiotics [5], with potassium iodate solution, strong acidic conditions are prevailed. The acid plays a dual role, firstly it brings about acid hydrolysis, secondly it makes quantitative oxidation titration feasible. Two procedures are adopted [5]; in the first, the end point is detected by a colour change in the carbon tetrachloride layer from colourless to brown or deep red and in the second one, dye Amaranth is used to detect change in colour from deep red to pale yellow, as the dye is destroyed by an excess of iodate in aqueous medium at the neutralization point.
The present manuscript describes the base catalysed hydrolysis of antibiotics in dilute sodium hydroxide solution. The hydrolysis product of antibiotics which contains sulphur derivatives, eg. -C-S-C groups, -S-H groups are quantitatively oxidized to sulphonic acid by titrating them against potassium iodate solution as oxidant, in dilute hydrochloric acid to prevail conducive atmosphere of pH ~ 2. The chemistry of this reductant system is described which shows the observed stoichiometric value of thiol group to iodate 1:2 which is consistent with theoretical value. The point of difference of our findings to that of previously described by Andrews titration [12] have been highlighted. The volumetric results are compared to spectrophotometric determination. The pathway and the oxidation mechanism of hydrolysis products of antibiotics have been proposed in the light of IR, TLC and spectrophotometric studies.
2. Experimental
2.1. Reagents
All the experiments were performed with analytical reagent grade chemicals using double dis
tilled water. Stock solution of antibiotics, 1 mg ml ~' were prepared by dissolving the pure and standard drugs (Himedia laboratories Bombay India) in water and stored in a well-dark and closed container to avoid direct contact with light. Hydrochloric acid standard solution (1.0 M), potassium iodate and sodium hydroxide of 0.1 M each were prepared in distilled water.
2.2. Apparatus
A Bauch and Lomb Spectonic -200"^ and Spec-tronic-1001 spUt beam spectrophotometer (Milton Roy Co.) with 1 cm path length quartz cells and a controlled water bath (NSW-133 India) were used. IR spectra of the isolated products were recorded on a spectrometer (Shimadzu IR-408, Japan) with KBr disc.
2.3. General procedure
2.3.1. Procedure I: simultaneous determination of penicillins by-volumetric titration against potassium iodate and spectrophotometry of coloured complex in organic layer
Aliquots of 2.5 ml of 0.1 M sodium hydroxide were added into conical flasks followed by suitable volumes of each of the standard penicillin solutions, containing between 0.2-2.0 mg ml""' of amoxycillin and ampiciUin and 0.1-2.5 mg ml~' of cloxacillin. The contents (pH ~ 13) were shaken and placed in a water bath at 80°C for 10-15 min. After completion of the heat treatment, the mixture was cooled to room temperature, followed by 0.5 ml of 1.0 M hydrochloric acid and 5 ml carbon tetrachloride. The mixture contents were titrated against potassium iodate with intermittent shaking. The end point was detected by a colour change from colourless to deep red colour in non-aqueous layer. For spectrophotometric determination, the nonaqueous layer was removed using a 50 ml separating funnel, dried over anhydrous sodium sulphate and measured absorbance at 520 nm against the blank, containing all the species except drug.
S.Z. Qureshi et al. /J. Pharm. Biomed. Anal. 21 (1999) 473-482 475
2.3.2. Procedure II: determination of penicillins by spectrophotometric method
To a known volume of amoxycillin solution ranging between 0.4-3.5 ml, ampicillin 0.4-2.8 ml and cloxacillin 0.1-2.5 ml, 2.5 ml of sodium hydroxide were added in a conical flask. The reaction mixture was kept for 10-15 min on a water bath at 80°C. After cooling to room temperature, 1 ml of potassium iodate, 0.5 ml of hydrochloric acid and 5 ml carbon tetrachloride were added to the reaction mixture. The solution was shaken vigorously till a deep red colour was obtained in the organic layer. It was determined spectrophotometrically by the recommended procedure I.
2.4. Assay of formations
For the determination of penicillins in tablets and capsules, the above methods were used with no modification. About twenty tablets were grinded into finely divided powder. 100 mg of powder was accurately weighed and transferred into a 100 ml standard flask. Similarly the contents of the capsules were weighed and dissolved equivalent to 100 mg in 0.1 1. The solution was well shaken for about 30 min to ensure a homogeneous solution. The residue was removed through Whatman No. 1 filter paper and the washings were taken into a final volume of 0.1 I followed by the recommended procedures I and II.
3. Results and discussion
The mechanism of penicillins in 0.1 M sodium hydroxide resulting in different degraded products can be rationalized in the light of IR, UV spectroscopic studies, TLC and molar stoichiometrics molar ratio of iodate to penicillin titration. The possible degradation pathways are illustrated in Fig. 1.
3.1. TLC and spectrophotometric observations
The presence of la has been suggested on the basis of the results obtained by TLC (Rf= 0.46 in a solvent system made v/v and comprising, 66%
n-butanol, 17% glacial acetic acid, 17% water). This observation is in agreement with previous studies [6], which confirms the existence of 4-hy-droxymethylene oxazolone la, (/?/=0.42 in the same solvent). Our studies of hydrolysis in O.IM NaOH at pH ~ 13 involving a single step reaction using water bath at 80°C, show one X,„„ at 240 nm, at reaction time of 15 min. This attributes the existence of one of the three species viz; lb (i) or lb (ii) or lb (iii). Moreover the formation of penicilloic acid lb (i) from basic hydrolysis of penicillin is well established [7]Tables 1-3. On extending the time interval to 12 h, another X a at 270 nm is obtained, which further confirms the establishment of 1(a). The previous studies show the existence of 1(a) as degraded product of antibiotics in the pH range 13-14 with UV spectroscopic analysis: X ^ x 297.5. The slight difference in "K^.^^ value may be attributed to different experimental conditions of hydrolysis, such as temperature, time of hydrolysis and pH etc.
3.2. IR studies
Three different spectra were taken, (i) pure amoxycillins, spectrum-A (Fig. 2) (ii) degraded in base catalysed hydrolysis, speclrum-B (Fig. 3) (iii) degraded base hydrolysed product treated with 0.1 M hydrochloric acid followed by titration against KI03, spectrum-C (Fig. 4).
The spectrum (B), compared with spectrum (A), revealed that the characteristic bands for different groups at specified frequencies in spectrum (A) are found, (i) missing, (ii) the formation at different frequencies or (iii) remained unchanged. A broad flat portion (3450-2600 cm~') same as it was found in spectrum (A), attributed to primary aliphatic amine N-H (3450-3250 cm" ' ) , car-boxylic (3300-2500 c m " ' ) and S-H (2600-2500 c m " ' ) groupings.
3.3. Missing and formation of new bands
The bands are found missing in the range (1800-1500 c m - ' ) and (1350-1000 cm" ' ) resulting the formation of bands at 860 cm~' , 680cm-' and in the range (1500-1350 cm" ' ) .
476 SZ Qureshi et al /] Pharm Biomed Anal 21 (1999) 473-482
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S.Z. Qureshi et al /J Pharm. Biomed. Anal. 21 (1999) 473-482 479
The identities of thiozolidine and secondary amine groups with different group frequencies lying in the range (1800-1510 cm~') are completely swept out. The second range of missing band (1350-1000 cm"') includes the groups e.g. CHj-S, ester etc. The two missmg ranges are revealed by a descending and ascending portion of spectra-B respectively. The formation of a broad band (1500-1350 cm"') is attributed to the pres
ence of 4-hydroxy-methylene oxazolone (la), which is revealed by the groups frequencies C-0 stretching and 0-H deformation vibration (1440-1395cm~'). The ascending portion (1350 cm~' onwards) of spectrum B further describes the hydrolysis of the rest of the groups present. A strong peak at 860 cm"' may be attributed to S-0 (870-810 cm-') of sulphonic acid. The weak peak at 680 cm " ' suggests to disulphide -S-S, C-S
A Degradation products, Su^e Stage hydrolysis at pH~ 13
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penctllemc acid
Ib(m)
B Oxidized products, Tntnmetncally and
spectrophotometncally using lOo' as Oxidant ^ i I lyiOa'
o II
R—S=0 I OH
SuJphomc acid IVb
O O oxidation 11 11 . . < R - S - S - R °'"'^^°"
Sulphoxide Illb
C-CH-, -C=\ • = \
NH-CH
pencillenic acid (disulphide)
R-S—S-R
lib
- 1 2
Fig 1 The possible degradation pathways of pencilline, followed by oxidation with lodate ions
480 S.Z. Qureshi et at. /J. Pharm. Biomed. Anal. 21 (1999) 473-482
4000 3100 1000 MOO 2000 1*00 l«00 1700 WOO 1S00 HOO 1300 )200 1100 1000 «00 (00 700
WAVENUMBER CM'*
Fig. 2. (i) Pure amoxycillin, spectrum-(A).
4000 3S00 3oS30 It'oO lOOO 1100 1*00 1700 t«00 ttOOUOO 1300 »00 1100 *000 100 ioO 700 WAVENUMBER CM'*
Fig. 3. (ii) Degraded in base catalysed hydrolysis, spectrura-(B).
S.Z Qureshi et al /J. Pharm. Biomed Anal. 21 (1999) 473-482 481
(705-570 cm" ') and sulphoxides ( > S = O, C-S-O at 730-690 cm"') after their partial oxidation to sulphonic acid in a semiquantitative manner as the sulphur compounds especially containing the group S-H, S-S are oxidized by mild oxidant or even atmospheric oxygen [8]. The spectrum -C, which is obtained after completing the titration against iodate in dilute acid conditions, gave the final product corresponding to strong peak at 835 cm"', attributed to the presence of sulphonic acid quantitatively.
The recommended procedure I describes simultaneous volumetric determination of penicillin against potassium iodate as oxidant in dilute acid medium. The deep-violet colour extracted in carbon tetrachloride at the equivalence point is stable for 2 h and has been studied spectrophotometri-cally. The hydrolysis product of penicillins (amoxycillin, ampicillin and cloxacillin) which contained sulphur derivatives, i.e. -C-S-C (lb), -S-H groups are quantitatively oxidized to some intermediate species and finally to sulphonic acid whose identities have already been discussed in IR studies. The hydrolysis by dilute sodium hydrox
ide solution has been carried out slowly at a constant temperature of 80°C with intermittent shaking. The excess of base is neutralized by adding hydrochloric acid (0.5 mmoJes) so that the reaction mixture attains a pH ~ 2. This provides a conducive environment of HjO" to concentration for carrying out quantitative titration of degraded products against potassium iodate. The reaction is summarized as follows;
.6e-[HS-R + 3H20^ RSO3H -H 6H+65] X 2 Step I
[IO3- +C\- -H6H++4e-->ICl-h3H30+]x3
In the first step reaction a quantitative oxidation of thiol to sulphonic acid followed by reduction of lOf to to ICl takes place which may be regarded as the reaction at equivalence point, but without any colour change. This shows that the stoichiometric value calculated theoretically should be of 2 mol of thiol group to 3 mol of iodate (1:1.5) which is inconsistent with experimental value 1:2 [9]. However, the system still involves more iodate ions to make the end point visible due to its reduction to iodine monochlo-
•ooP
90
•0
ij
u (0 z t SO J M
5 401-
}0
20
10 •
4000 ISOO 3000 2S00 2000 ISOO | |00 1700 1(00 ISOO UOO 1300 UOO 1100 tOOO >00 (00 700 WMEMUM&CR CM**
Fig 4. (m) Degraded base hydrolyzed treated with I 0 M hydrochloric acid, followed by titration against KIO3, spectrura-(C)
482 S.Z. Qureshi el a!. /J. Pharm. Bwmed. Anal. 21 (1999) 473-482
ride and finally to free iodine. This is described in step II reaction which is given below:
IO3- + 6H+ + CI- + 4e -"""-^nci + 3H,0 ), —..dissociation-
ici -• r + C1-
I-+H30'^''^''^'^H0I + H +
5HOI°^"^°"4'-''"°"2l2 + IO3- + H- + 2H,0 ofioddte
In the step II, an over consumption of iodate takes place [10], thereby increasing the mole ratio of iodate from 1.5 to 2 mol which is consistent with the experimentally observed stoichiometry of 1 mol thiol to 2 mol iodate. A stable red violet colour of free iodine in organic layer indicates the equivalence point. It is observed that further addition of iodate solution beyond the end point results no decolourization in mixture content. This means that a quantitative oxidation-reduction in dilute hydrochloric acid has taken place in [the system. The titrant iodate is reduced first to iodine monochloride and finally to free iodine to show a visible end point. The E" of iodate in dilute hydrochloric acid (1.19 V) is less than the E" in Andrew titration (1.23 V). The E° 1.19 V is |high enough to oxidize the degraded products of antibiotics containing derivatives. I In Andrev/s titration a strong hydrochloric acid (3-9 M) solution is required for reduction of iodate to iodine monochloride (IClf) complex.
IO3 + 6H++2CI- -h4e--^ICU- +3H,0
E° = 1.23 V
During the course of titration with reducing agent, the presence of free iodine in the system also results from the further reduction of IClj" complex as given below:
2ICl2-+2e-^l2 + 4Cl- E°= 1.06 V
When the reducing agent has been consumed, the free iodine is titrated to form iodine monochloride complex. The end point is marked by the disappearance of the last free iodine. (A point of difference from our observation given in Step II).
2I2 4- IO3- + 6H+ -I- lOCl - -> 5ICI2" + 3H2O
The above conditions prevail if the concentra
tion of the hydrochloric acid at the end point is at least 3 M. The iodate reacts quantitatively with reducing agents such as KI, I2, AS2O3, N2H4 and SO2 to iodine monochloride [11]. In many other cases the concentration of hydrochloric acid is not critical, but for Sb(III), it is 2.5-3.5 M; that is, the optimum acidity for reasonably rapid reaction varies from one reactant to another reactant [12].
The recommended procedure I, which describes the use of dilute hydrochloric acid is applied successfully to the simultaneous quantitative reduction of iodate to free iodine at the equivalence point, especially in a system which contains the degraded products of antibiotics with sulphur derivatives which can be oxidized by very mild oxidizing agents [10]. The half reaction of iodate in dilute acid solution with E° [II] can be proposed as under.
2IO3- + 12H+ -H lOe - -* P -f- 6H2O
E° = 1.19 V
Acknowledgements
The research facilities at the Department of Chemistry, AMU Aligarh are highly acknowledged.
References
[1] A.G. Davidson, J.B. Stenlake, Analyst 99 (1974) 476. [2] J, Talegaonkar, K..S. Boparai, Talanta 29 (1982) 525. [3] U. Forsman, Anal. Chim. Acta. 93 (1977) 153. [4] B. Karibcrg, U. Forsman, Anal. Chim. Acta. 83 (1976)
309. [5] J.K. Grime, B. Tan, Anal. Chim, Acta. 105 (1979) 361. [6] J.L. Longridgc, D. Timms, J. Chcm. Soc. B (1971) 852. [7] M.A. Carroill, J.E. Zarcmbo, J. Antibiol. 30 (1977) 811. [8] D.R. John, M.C. Cascrio, Basic Principles of Organic
Chemistry, 2nd cdn, 1977, p. 745. [9] El-Scbai A. Ibrahim, S.M. Rida, Y.A. Baltagy, M.M.
Abd El-Khalek, J. Drug. Res. (Egypt) 6 (1973) 13. [10] I.M. Kolthoff, R. Belcher, Volumetric Analysis, vol. Ill,
1957, p. 450. [II] I.M. Kolthoff, E.B. Sandell, E.J. Merhan, S. Brucken-
stein. Quantitative Chemical Analysis, 4th edn, 1969, pp. 819,1159.
[12] L.W. Andrews, J. Amer. Chem. Soc. 25 (1903) 756.
Recommended