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ANALYSIS OF DRUGS IN PHARMACEUTICAL FORMULATIONS
ABSTRACT
T H E S I S SUBMITTED FOR THE AWARD OF THE DEGREE OF
Battor of $t|iIo!Bioptip IN
OHEMISTRY
BY
NADEEM AHMAD KHAISI
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2003
The thesis entitled ''Analysis of Drugs in Pharmaceutical Formulations" is
comprised of five chapters. The first chapter describes a general literature survey of the
subject matter. The very relevant matters include, (i) a brief discussion on analytical
chemistry, its relation with our daily life from old age, role played in the field of
pharmaceutical and biomedical analysis, (ii) various types of analytical techniques and
procedures involving sophisticated instruments which are in use now a days, (iii) the
validation of the developed method and (iv) the statistical treatments adopted during the
data analysis to ensure and enforce the validity of method.
It is well known that human shows briskness in every era of evolutions and
development, which led to the birth of analytical chemistry. It deals with the study of the
intrinsic properties of the materials to discover their valueable applicability or to warn the
concerned one with the possibility of harm from them. Unlike, other areas of studies in
science, there are different properties, which heavily influence the results of the analytical
methodology at each and every step, thus having direct impact on the results. These
important properties have been discussed. By the time, the demands of the biochemical
sciences and the advancement of physical sciences opened the door of instrumentation in
the field of analytical chemistry. The different types of well-established analytical
techniques have been discussed.
Before or during the development of a method, it is necessary to be very careful
of certain important things. These concepts have been discussed briefly. Once the
analytical method is adjusted, it is necessary to decide its suitability for the intended
purpose. This is known as the method validation. Brief discussion of the validation and
TUESI o 2
its components has been presented. When deciding for validation of the method, the role
of statistical analysis can not be ignored. It is the only way to get the most conclusive
results from the mathematical data obtained during the analysis. It also helps to decide
with the progress of the work at each step. A discussion of the statistical analysis has
been given. A brief literature and classification of the concerned pharmaceuticals also
presented.
The second chapter describes kinetic spectrophotometric method for the
determination of silymarin in pure form and in pharmaceutical formulations. The
method is based on the oxidation of the drug with potassium permanganate at pH
7.0±0.2. The reaction is followed spectrophotometrically by measuring the
decrease in the absorbance at 530 nm. Under the optimum experimental
conditions, a calibration curve was constructed by plotting logarithm of initial
rate of reaction (log v) versus logarithm of silymarin concentration (log C),
which was linear over silymarin concentration range of 18-50 | g ml'^ The linear
regression analysis using the method of least square was made to evaluate the
slope, intercept and correlation coefficient. The linear regression equation and
correlation coefficient are log v = 1.024 log C + 3.235 and r = 0.9997 which
indicates an excellent linearity. The confidence limits for the slope and intercept
of the line of regression were calculated and found to be 1.024±3.96x10'" and
3.235±1.67xl0"\ respectively. This indicated the high reproducibility of the
proposed kinetic method. The proposed method was compared with the reference
method.
THESIS
The third chapter deals with the four simple, sensitive and accurate
spectrophotometric methods for the determination of nifedipine in pharmaceutical
formulations. These methods are based on the formation of ion-pair complexes of amino
derivative of the nifedipine with bromocresol green (BCG), bromophenol blue (BPB),
biomothymol blue (BTB) and eriochrome black T (EBT) in acidic medium. The coloured
products are extracted with chloroform and measured spectrophotometrically at 415 nm
(BCG, BPB and BTB) and 520 nm (EBT). Beer's law was obeyed in the concentration
range of 5-32.5, 4-37.5, 6.5-33.0 and 4.5-22.5 \xg ml" with molar absorptivity of 6.41 x
10^ 4.85 X 10^ 5.26 x 10 and 7.69 x 10 L mol" cm" and relative standard deviation of
0.82, 0.72, 0.66 and 0.68 % for BCG, BPB, BTB and EBT methods, respectively. These
methods have been successfully applied for the assay of drug in pharmaceutical
formulations. No interference was observed from common pharmaceutical adjuvants.
Statistical comparison of the results with the reference method shows excellent
agreement and indicates no significant difference in accuracy and precision.
The fourth chapter describes two simple, sensitive and economical
spectrophotometric methods for the determination of amiodarone hydrochloride
in pure form and commercial dosage forms. Method A is based on the charge
transfer complexation reaction of amiodarone base with p-chloranilic acid in
dioxan- dichloromethane. The coloured product is measured at 535 nm. Method
B also utilizes the charge transfer complexation reaction of amiodarone base with
2,3- dichloro 5,6- dicyano 1,4- benzoquinone (DDQ) in acetonitrile medium,
resulting in the formation of DDQ radical anion, which absorbs maximally at 570
THESIS
nm. Beer's law was obeyed in the concentration range of 10 - 360 and 2 - 6 5 |j,g
ml" for methods A and B, respectively. The molar combining ratio, association
constant and optimum assay conditions were studied. The proposed methods
have, been successfully applied to the determination of amiodarone in
commercial dosage forms. The results are validated statistically and compared
with the reference method.
The fifth chapter deals with the two simple and sensitive spectrophotometric
methods for the determination of verapamil hydrochloride in pure and commercial
dosage forms. The first method is based on the oxidation of the drug with potassium
metaperiodate in sulphuric acid medium to give coloured product, which absorbs
maximally at 425 nm. The second procedure involves the formation of coloured
chloroform extractable ion-pair complex of the drug with tropaeolin 000 at pH 4.0
showing absorption peak at 400 nm. Under tlie optimized experimental conditions, Beer's
law is obeyed in the concentration range of 0.00-187.5 and 2-30 |j,g ml"' for methods A
and B, respectively. Both the methods are highly reproducible and have been successfully
applied to the commercial dosage forms. Results of analyses were validated statistically
and through recovery studies. Statistical comparison of the results with the reference
method shows excellent agreement and indicates no significant difference in accuracy
and precision.
ANALYSIS OF DRUGS IN PHARMACEUTICAL FORMULATIONS
l (
T H E S I S \ - ^ \ \ SUBMITTED FOR THE MilKR^ OF THE DEGREE OF
Bottor of $l)ilo£(opt)p
C H E M I S T R Y
BY
NADEEM AHMAD KHAN
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2003
^ s ^
Fed in Compnter
^2ad Libra v.
•/'
/Wtifl-rt. " i ^ ^
0 9 J - i 2005
T5852
v- V/<!k, .
THESIS
DR. NAFISUR RAHMAN Reader
Analytical Research Laboratory Department of Chemistry Aligarh Muslim University Aligarh-202 002 (U.P.) INDIA Tel.:+91-571-2703515 (Office) E-mail: cht17nr@yahoo.co.in
(Date iN&^^mUv 01 tooS
Certificate
T^is is to certify tHat the thesis entitted "JLnaCysis of<Drugs In
(PharmaceuticaC TormuCations" is the originaC wor^ of
Mr. O^adeem JLhmad an, carried out under my supervision and
is suitaBleforsuSmissionforthe award of the degree of<Doctorof
<PhHbsophy in Chemistry.
Na l4h,u^/^^^J^ VlA.-A^
(Ufafisur <l(ahman)
A.CK9^O'WL<E(DGT.9A(E0fT
I Bring all the •praises aitcC commendation to the MmigHty for-providing me
with strength during the xvor^ of my research, which heCped me to overcome the
trouBCes in the wearisome journey.
It wiCf Be hard to find out the words to than^ my sage supervisor,
(Dr. U^afisur <Rfihman, for his excellent guidance, constant support and trust
throughout the course of my research wor^
I am than^Cto the CHairman, department of CHemistry, JlGgarh MusRm
"University, for providing researchfaciCities.
I dufy ac^nowtedge the ^nd cooperation of my friends and coCkagues for
their nd heCp, vaCuaBk suggestions and encouragement.
I eoq^ress my deep sense to my parents whose Blessing have aCways served as a
sheet anchor against the mundane odds arid inspired me to complete my mission.
I egress my deep than^C to my younger Brother and sisters, for the
acQustment they made during my studies.
C^adeemjihmad%lian)
CONTENTS
List of publications I
List of figures H
List of tables V
Chapter 1 General introduction 1
References 50
Chapter 2 Kinetic spectrophotometric method for the determination of 69 silymarin in pharmaceutical formulations using potassium permanganate as oxidant
References 86
Chapter 3 Extractive spectrophotometric methods for the determination 89 of nifedipine in pharmaceutical formulations using bromocresol green, bromophenol blue, bromothymol blue and eriochrome black T
References 120
Chapter 4 Validated Spectrophotometric Methods for the 126 Determination of Amiodarone Hydrochloride in Commercial Dosage Forms using p-chloranilic acid and 2,3 -dichloro 5,6 - dicyano 1,4 - benzoquinone
References jgg
Chapter 5 Quantitative Spectrophotometric Methods for Determination of 159 Verapamil Hydrochloride in Drug Formulations Using Potassium Metaperiodate and Tropaeolin 000
LIST OF PUBLICATIONS
[1]. Kinetic spectrophotometric method for the determination of silymarin in pharmaceutical formulations using potassium permanganate as oxidant; Pharmazie, Govi Verlag GmbH, 2003 (In press).
[2]. Extractive spectrophotometric methods for the determination of nifedipine in pharmaceutical formulations using bromocresol green, bromophenol blue, bromothymol blue and eriochrome black T; IL Farmaco (Elsevier), (Accepted).
[3]. Validated Spectrophotometric Methods for the Determination of Amiodarone Hydrochloride in Commercial Dosage Forms using p-chloranilic acid and 2,3 -dichloro 5,6 - dicyano 1,4 - benzoquinone; Anal. Sci., The Japan Society for Analytical Chemistry, 2003 (Accepted).
[4]. Quantitative Spectrophotometric Methods for Determination of Verapamil Hydrochloride in Drug Formulations Using Potassium Metaperiodate and Tropaeolin 000; AAPS PharmSci, American Association of Pharmaceutical Sciences, 2003 (Communicated).
II
Figures
Fig. 2.1.
Fig. 2.2.
Fig. 2.3.
Fig. 2.4.
Fig. 2.5.
Fig. 3.1.
Fig. 3.2.
Fig. 3.3.
Fig. 3.4.
Fig. 3.5.
Fig. 3.6.
Fig. 3,7.
Fig. 3.8.
Fig. 3.9.
1 Fig. 3.10.
LIST OF FIGURES
Captions
Absorbance-time curves: KMn04, 4.8 xlO" M and silymarin (a) 3.731 X 10- M; (b) 4.146 x 10' M; (c) 6.219 x 10'^ (d) 8.292 X 10- M; (e) 10.365 x 10" M
Absorbance-time curves : silymarin, 10. 365 x 10' M and KMn04 (a) 3.00 x 10"* M ; (b) 2.10 x 10" M; (c) 1.50 x 10" M; (d)9.00xlO-^M.
Calibration curve for the determination of Silymarin.
Error in the determination of the concentration of silymarin using statistical analysis of standard calibration data.
Variation of the confidence limit at (a) p= 0.05 and (b) p= 0.01 level of significance in the form of uncertainty percentage on the concentration of silymarin.
Absorption spectra: (a) Amino derivative of nifedipine-BCG complex; (b) Amino derivative of nifedipine-BPB complex; (c) amino derivative of nifedipine-BTB complex; (d) amino derivative of nifedipine-EBT complex.
Job's plot for drug-dye (1.443 x 10^ M) system: BCG.
Job's plot for drug-dye (1.443 x 10"* M) system: BPB.
Job's plot for drug-dye (1.443 x 10^ M) system: BTB.
Job's plot for drug-dye (1.443 x 10^ M) system: EBT.
Eftect of the concentration of HCI: 31 jig mp' drug + 7 ml of 0.025% BCG.
Effect of the concentration of HCI: 33 |ig ml"' drug + 7ml of 0.025% BPB.
Effect of the concentration of HCI: 33 ug mP' drue + 7 ml of 0.025% BTB. ^
Effect of the concentration of HCI: 22.5 ^g ml"' drug + 7 ml of 0.025% EBT. /mioi
Influence of the volume of 0.025% dve- BCG
Page No.
74
75
78
80
81
93
94
95
96
97
101
102
103
104
106
Ill
Influence of the volume of 0.025% dye: BPB. 107
Influence of the volume of 0.025% dye: BTB. 108
Influence of the volume of 0.025% dye: EBT. 109
Calibration curve for determination of nifedipine with BCG. I l l
Calibration curve for determination of nifedipine with BPB. 112
Calibration curve for determination of nifedipine with BTB. 113
Calibration curve for determination of nifedipine with EBT. 114
Absorption spectra of (a) 2.30 X lO''' M p-chloranilic acid (b) 132 0.36 |ag ml'' amiodarone with 1.15 X 10" M p-chloranilic acid in dioxane- dichloromethane.
Molar ratio plot for amiodarone - p - chloranilic acid complex; 133 amiodarone concentration, 2.933 X 10^ M.
Absorption spectra of (a) 8.81 X 10" M DDQ in acetonitrile 136 (b) 65 tag ml"' amiodarone with 1.06 X 10' M DDQ in acetonitrile.
Fig. 4.4. Molar ratio plot for amiodarone -DDQ complex; amiodarone 137 concentration, 2.933 X 10"* M.
1 \A\\D] 1 140 Fig, 4.5. Plot of versus -'• --"- •'-X—jjr for 1:1
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
3.11.
3.12.
3.13.
3.14
3.15
3.16
Fig.3.17.
Fig.
Fig.
Fig
4.1.
4.2.
4.3.
^x complex of amiodarone - p- chloranilic acid.
1 \A\\D] 1 141
of amiodarone -DDQ.
Fig. 4.7. Influence ofthe volume ofO.08% p-chloranilic acid. 143
Fig. 4.8. Influence ofthe volume of 0.08% DDQ. 145
Fig. 4.9. Calibration curve for determination of amiodarone (method A). 147
Fig. 4.10, Calibration curve for determination ofamiodarone (method B). 148
Fig. 4.11. Plot ofthe variation ofthe confidence limit at 95% confidence 150 level for method A, presented as uncertainty percentage on the concentration ofamiodarone.
IV
Fig. 4.12. Plot of the variation of the confidence limit at 95% confidence 151 level for method B, presented as uncertainty percentage on the concentration of amiodarone.
Fig. 5.1. Absorption spectrum of the reaction product of Verapamil 164 hydrochloride with potassium metaperiodate.
Fig. 5.2. Job's plot for the reaction of verapamil hydrochloride with 165 potassium metaperiodate (each 5. 09 x 10' M).
Fig. 5.3. Absorption spectrum of the reaction product of 168 Verapamil hydrochloride with tropaeolin 000.
Fig. 5.4. Job's plot for the reaction of verapamil hydrochloride with 169 Tropaeolin 000 (each 1.02 x 10" M).
Fig. 5.5. Effect of heating time. 172
Fig. 5.6. Effect of the volume of 0.2% potassium metaperiodate. 173
Fig. 5.7. Effect ofthe volume of 10 moir'H2S04. 174
Fig. 5.8. Effect of the volume of 0.05% tropaeolin 000, 176
Fig. 5.9. Effect of pH: 30 jig ml'' drug + 2.5 ml of 0.05% tropaeolin 000 177 + 5.0 ml buffer of different pH values.
Fig. 5.10. Calibration curve for determination of verapamil hydrochloride 179 (method A).
Fig. 5.11. Calibration curve for determination of verapamil hydrochloride 180 (method B).
Fig. 5.12. Error in the determination of verapamil hydrochloride using 184 statistical analysis of standard calibration data with potassium metaperiodate.
Fig. 5.13. Error in the determination of verapamil hydrochloride using 185 statistical analysis of standard calibration data with tropaeolin 000.
Tables
Table 1.1.
Table 1.2.
Table 1.3
Table 1.4
Table 1.5.
Table 1.6.
Table 2.1.
Table 2.2.
Table 2.3.
Table 3.1.
Table 3.2.
Table 3.3.
Table 3.4.
LIST OF TABLES
Captions
Assay of drugs in drug formulations by Spectrophotometric procedures.
Maximum permitted standard deviation for assay determinations in dependence on the number of repetitions and the specification range
Requirements for different calibration modes with relevant parameters.
Validation characteristics normally evaluated for the different types of test procedures and the minimum number of determination required
Approaches to demonstrate accuracy according to ICH.
Approaches for determining the detection and quantitation limit
Precision test of the proposed kinetic method.
Determination of silymarin in pharmaceutical formulations by standard addition method
Determination of silymarin in dosage forms by the proposed kinetic method and reference method.
Analytical characteristics of the proposed methods.
Determination of nifedipine in pharmaceutical formulations by standard addition method.
Comparison of the proposed methods with existing spectrophotometric methods for the assay of nifedipine in pharmaceutical formulations.
Determination of nifedipine in dosage forms by the proposed methods and reference method.
Page No.
12
27
31
32
33
37
83
84
85
115
116
118
119
VI
Table 4.1.
Table 4.2.
Table 4.3.
Table 4.4.
Table 4.5.
Table 5.1.
Table 5.2.
Table 5.3.
Table 5.4.
Association constant, K; molar absorptivity, z\ from the 142 Ross and Labes plots for the complex and calculated free energy, AG.
Analytical characteristics of the proposed methods. 146
Test of precision and accuracy of the proposed methods. 153
Standard addition method for the determination of 154 amiodarone in dosage forms.
Comparison of the proposed methods with the reference 155 method.
Optical Characteristics and statistical data of the of the proposed methods.
181
Evaluation of the accuracy and precision of the proposed 186 methods.
Standard addition method for the determination of verapamil hydrochloride in dosage forms.
Comparison of the two proposed methods with the reference method.
187
189
CHapter -1 Qenerat Introduction
Medicine is a very ancient art, and drugs have been used in days of antiquity as
far back as history can take us. The early explorers found the South American hidians in
possession of Cinchona bark from which Pelletier and Caventou extracted the
antimalairal alkaloids, quinine. The Roman Naturalist Galenus (131-200 A.D) said that
proper herb mixture could be used for all conceivable health defects. In Galenus
apothecary shop, herbs and some metallic drugs (copper and zinc ores, iron sulfate,
cadmium oxide) were found in abimdance, and a semblance of assaying was maintained
by insistence on "pure" drugs that is the right variety and age of botanical specimens. The
intimation of dosage levels can be seen in his contention that powerfiil narcotics reUeve
pain but may also cause death in excess. In 1646, the French physician Guy Palin (1602-
1672) suggested to a medical student; "Above all flee books on chemistry, in quorum
lectione oleum et Perdes" . This attitude changed as exact laws of chemistry were
formulated as the physiological action of well defined compounds become amenable to
quantitative study and as the active principles of ancient - herbal drugs were extracted,
purified and fitted into stoichiometric laws [1].
hi the mean time, new medicines were discovered and old ones improved.
Withering (1741-1799) introduced digitalis (purple foxglove) as a therapeutic agent for
dropsy, and almost the same material is used in the treatment of cardiac edema today.
Van Swieten (1700-1772) taught the advantages of corrosive sublimate (mercuric
chloride) over older mercurial preparations in "Fevers". Opium was dispensed and
studied by de Quincey (1785-1859) "to tranquilize all irritations of the nervous system, to
stimulate the capacity of enjoyment and to sustain the else drooping animal energies" [2].
Phenacetin, a pain killing drug, was the first pharmaceutical product
developed by the German chemical company, Friedrich Bayer & Co; which, in
1888, licensed May and Baker to manufacture and market it in the United
Kingdom. Higher standards for the preparation of pharmaceutical ingredients had
been set, following the 1858 Medical Act's stipulation that the General Medical
Council (UK) should produce a list of medicines and compounds and manner of
preparing them together with true weights and measures by which they are to be
prepared and mixed [3]. The first British Pharmacopoeia was published in 1864
and thereafter pharmaceutical and fine chemical manufacturers laid greater
emphasis on meeting the standards it set. In 1890, Jesse Boot established an
analytical laboratory and its staff were involved mainly for analyzing proprietory
medicines of competitors in order that Boots should develop new and / or
cheaper formulations [4].
Analj^ical Chemistry is the science and art of determining the
compositions of materials in term of the elements or compounds contained in
them. Historically, the development of analytical methods has followed closely
the introduction of new measuring instruments. Among the many unprecedented
and fundamental changes occurred in the Chemistry during the past century
perhaps the most notable was the increase in the use of sophisticated instruments
and techniques. Researchers in Pharmaceutical Chemistry by the applications of
these techniques are adding a lot in the development of new drugs, which are
improving the life expectancy but also presenting new challenges.
Analytical Chemistry in pharmaceutics is indispensable, without the use of
analytical techniques, it is impossible for the chemists or the researchers to
develop a new approach to solve the problems of quality control and in
ascertaining optimum experimental conditions to obtain a good yield of products.
Development of new pharmaceutical product from naturally occurring
substances undergoes a nimiber of steps from laboratory to semi scale synthesis,
pilot plant scale up, efficacy and toxicology testing, clinical trials and finally full
scale production. In order to safeguard public health, there should be constant
monitoring of impvirities present in the form of inorganic and organic compounds
in the pure drugs and in pharmaceutical formulations. From the last few decades,
Analytical researchers continuing their efforts to produce high quality products
by improving the existing methods of analysis. A very large number of examples
can be cited where analytical chemistry find its applications to drug analysis in
pharmaceutical preparations and biological samples. [5-7].
In pharmaceutical industry, the quality of the manufactured drug and its
formulations must be carefully controlled. Slight changes in composition or in
the purity of drug itself can affect therapeutic values. Therefore, it is necessary to
establish the properties and therapeutic value of a drug before it is approved and
made available in the market. Establishment of the permissible level of dosage of
drug requires the determination of its composition, toxicity and its metabolite at
various stages.
The availability of sub-standard medicines to the general public possesses
many problems, both clinically and economically. It is widely believed that such
sub-standard preparations are readily available in many developing countries [8-
13]. This may be due to poor manufacturing procedures, poor storage conditions
or deliberate counterfeiting of branded or generic products. Therefore, it is
important to recognize that the drugs may contain impurities. These impurities
may result from many sources like raw materials and regents, as reaction by
products, and through degradation during manufacture and storage. Since the
impurities can have safety and efficacy implications, and are therefore the subject
of considerable attention by both manufacturers and regulatory agencies.
Impurities can be classified into three groups, namely: organic, inorganic and
residual solvents. Organic impurities may arise from starting materials,
intermediates and synthetic by-products or from reagents and catalyst or as a
consequence of degradation. Inorganic impurities may result from reagents,
ligands or catalysts as heavy metals or inorganic salts. Residual solvents are
inevitable in drug substance since without solvents, purification and generation
of the desired crystal morphology would be impossible. However, since residual
solvents also arise in excipients and occasionally in the manufacture of drug
products, it was decided to draft a separate guideline to address appropriate
levels. A key component of the guideline, and a fundamental concept is
qualification. Qualification is defined as the process of acquiring and evaluating
data, which establish the biological safety of the individual impurity or a given
impurity profile at the level(s) specified. Thus, the pharmaceutical analyst and
toxicologist must work hand in hand throughout the pre-clinical and clinical
development programme in order to be able to set meaningful specification
requirements. The pharmaceutical analyst must give the usefiil thoughts to the
analytical methods, especially in the development phases. Therefore, it is an
important task for the pharmaceutical analyst to choose an analytical technique
with modem instrimientation, which provides the best solutions to the problems.
The most commonly used analytical techniques in pharmaceutical analysis
are described below. The chromatographic methods include paper
chromatography, thin layer chromatography, column chromatography, capillary
electrochromatography, high performance liquid chromatography, high
performance thin layer chromatography and gas chromatography. The
electrochemical methods mainly used for this purpose are polarography,
volatammetry and amperometry. The radiotracer techniques include mainly the
isotopic dilution and the activation analysis. The spectroscopic techniques used
for this purpose are UV-visible spectrophotometry, derivative
spectrophotometry, difference spectrophotometry, infirared spectroscopy, flame
photometry, fluorometry and nuclear magnetic resonance.
There are number of analysis that could not have been accomplished in
any other ways except the use of chromatographic techniques. High performance
liquid chromatographic methods have been successfully applied in drug analysis
and have now appeared in the dissolution monographs of pharmacopoeias for the
assay of drugs in dissolution fluids [14-16]. It is becoming increasingly popular
because of its sensitivity and specificity in multicomponent analysis and widely
used for the identification of drugs in plasma and drug formulations [17-37].
High performance thin layer chromatographic technique is flexible enough
to analyze different kind of samples. It is an Off - Line technique whose every
stage of analysis can be visualized. It can be used in pharmaceuticals, biomedical
and environmental analysis. A review paper by Renger on contemporary thin
layer chromatography and high performance thin layer chromatography in
pharmaceutical quality control [38,39] discussed development and validation of
methods. Advantages stated include simplicity of handling and performance;
flexibility and short analysis time; the ability to analyze complex or crude
samples with minimimi sample clean up; wide variety of layers; mobile phase;
operational aspects; and detection reagents; no obligation for elution; ability to
evaluate the entire chromatogram stored on the plate with a variety of techniques
and parameters without time constraints; simultaneous but independent
development of multiple samples and standards on each plate, leading to high
sample throughput and increased reliability of results (in-system calibration); and
robustness, allowing easy transfer and adoption of methods. Several drugs have
been investigated by high performance thin layer chromatography in
pharmaceutical preparations [40-49].
Capillary electrochromatography is a method in which a liquid mobile
phase is driven through a stationary phase in a packed capillary column by the
electro-osmotic flow generated by a large difference in potential across the
column [50-53]. A number of papers have appeared that discuss various aspects
of this technique, including one that considers the use of open-tubular capillaries,
packed capillaries and monolithic capillaries [54].
Capillary electrophoresis technique continues to grow in popularity
especially in areas where they can provide complementary informations to the
other more established methodologies or advantages in selectivity and efficiency
[55]. Two of the areas where usage has increased the most are in assessing
enantiomer purity of pharmaceutical via the addition of chiral additives [56,57],
and in determining the physiochemical information about the analyte. Another
important aspect of this technique is that flow volumes are small, and as such,
coupling it to a mass spectrometer is relatively easy [58,59]. A consideration that
is especially important for a technique to gain acceptance in the pharmaceutical
industry is validation [60,61]. The results generated in electrophoresis technique
are comparable to HPLC and GC in terms of accuracy, precision, linearity and
range, specificity and ruggedness.
Flow injection analysis is widely used as analytical technique [62,63] and
has several advantages: (1) reduced reagent consumption, (2) high sampling
frequency and (3) safety in applying toxic reagents because the whole analysis
proceeds in a closed system. An additional advantage observed in flow -
injection analysis is increased selectivity when the analyte is accompanied by
8
more slowly reacting components. Flow injection analysis has been applied in
the analysis of several pharmaceutical compounds [64-73].
Electrochemistry can be used at the early stage of the drug researches for
screening the pharmacological activity of a homologous series of newly
synthesized molecules. There are several examples in the literature showing the
relationship between the electrochemical data and the pharmacological properties
of drug [74-77]. Electrochemical behavior of cefetamet sodium has been studied
using dc, ac, and differential pulse polarography, cyclic voltammetry and
constant potential electrolysis [78]. Likewise, the electrochemical oxidation of
cefotaxime at activated glassy carbon, platinum, and carbon paste electrodes has
been examined [79]. Square wave voltammetry has been used to determine
sulphamethoxypyridazine and trimethoprim simultaneously in mixtures [80].
The antihypertensive drug doxazosin in urine and formulations was
determined by means of both, differential pulse and square wave adsorption
stripping voltammetry at a mercury drop electrode [81]. The potential of electro
analytical techniques for pharmaceutical analysis, covering a broad spectrum of
electrochemical techniques, was reviewed by Kauffmann et al. [82].
Analytical absorption spectroscopy in ultraviolet and visible region of
electromagnetic spectrum is widely used for quantitative analysis in the field of
pharmaceutical and biomedical analysis. The scope of absorption spectroscopy is further
extended by use of colour reactions often with concomitant increase in sensitivity and or
selectivity. Such reactions are used to modify the spectrum of an absorbing molecule, so
that it could be detected in visible region well separated from other interfering
components. Chemical modification is also useful for transforming non-absorbing
molecule to a stable derivative possessing significant absorption spectral behaviour.
A particular advantage of UV Spectrophotometry is that it does not destroy the
sample. After recording the ultra violet spectrum, one can reextract the solution and
recover the absorbing substances for subsequent analytical procedures, which is in sharp
contrast to gas chromatographic and immunological procedures. UV- spectrophotometric
methods are simple, quick, economical and usually do not require elaborate preparation
step prior to assay. However, a UV method is not adequate when two or more drugs with
similar UV spectra are present in the sample or excipients or decomposition product
exhibits UV interference, as in case with dissolution testing of Phenytoin capsules [83].
When a beam of light is passed through material, the incident radiation (IQ) will
always be more intense than the convergent radiation (I). Attenuation of the incident
radiation may be attributed to (1) reflections at the interfaces between air and cell wall
and sample and cell wall, (2) scattering by any suspended particles or dust in the sample
and (3) absorption of radiation by the sample, in normal applications light absorption is
the primary factor reducing the incident radiation.
Fluorescence can often increase the apparent intensity of the emergent radiation,
which may result in inaccurate absorption measurements. Effect of fluorescence can be
reduced by chemically inhibiting fluorescence, locating the sample some distance from
the detector, or using appropriate cutoff filters to block the fluorescence occurring at
longer wavelengths than the incident radiation. When using monochromatic light, the
10
fraction of the radiation absorbed by the substances, ignoring losses due to reflections and
scattering, will be function of the concentration of the substance in the light path and the
thickness of the sample. Mathematically this function can be defined as: A = Log [lo/I] =
ebc, where A is defined as absorbance of the sample, "e" is wavelength dependent
proportionality factor called molar absorptivity, "b" is the light path length through the
sample and "c" is the concentration of the sample in appropriate units. The expression A
= ebc is known as Lambert-Beer's law or Beer's law.
The linearity of Beer's law is subject to some restrictions. Concentrations may not
always be directly proportional to absorbance. Chemical and physical interactions and
instrumental limitations can produce deviations, particularly at high concentrations or
high absorbance. Sample scattering, fluorescence, decomposition, and saturation are
examples of sample-related problems affecting linearity. Hydrogen bonding, ion-pair
formation, solvation and chemical reactions can cause incorrect calculations of sample
concentration as the solvent concentration is varied. Instrument factors that can effect
apparent deviation firom linearity are stray light, resolution and both absorbance and
wavelength calibration. Most of the chemical and instrument effects described above will
result in suppression of absorbance as concentration is increased. To prevent errors.
Beer's Law plots should be constructed for each sample system with at least three points
within the expected concentration range of the unknowns.
Simple colorimetric and UV methods continue to be popular for carrying out
single component assay on a variety of formulated products. Representative examples of
some of the many assays that have been published are given in Table 1.1. Colorimetric
11
methods based on charge transfer reaction of certain n - acceptors have been successfiilly
utilized in pharmaceutical analysis. The u-acceptors such as p-Chloranilic acid,
dichlorophenyl-indophenyl, 2,3-dichloro 5,6-dicyano p-benzoquinone,
tetracyanoethylene, tetrachlorobenzoquinone, p-benzoquinone, p-nitrophenol,
tetracyanoquinodimethane [140-141] have been used to carry out spectrophotometric
assay of certain cardiovascular drugs such as amlodipine besylate [89,90], verapamil
hydrochloride, atenolol [96], acebutalol hydrochloride, carazol, propranolol
hydrochloride [142] and lisinopril [115].
Acid dyes, solochrome dark blue, solochrome black T, bromothymol blue,
bromophenol blue, bromocresol green, bromocresol purple, sunset yellow, tropaeolin 000
and indigo carmine form ion-pair complexes with basic drugs which were quantitatively
extracted into chloroform. Colorimetric methods based on dye pairing continue to be
important and have been used to assay compoimds such as amlodipine besylate [143-
145], verapamil hydrochloride [146], phenothiazine derivative [147] and famotidine
[148].
Derivative Spectrophotometry is an analytical technique of great utility for
extracting both qualitative and quantitative information from spectra composed of
unresolved bands. In recent years, the introduction of electronic differentiation by a
microcomputer interfaced with the spectrophotometer makes possible the plotting of the
first, second or higher order derivatives of a spectrum with respect to wavelength.
12
Table 1.1. Assay of drugs in drug formulations by Spectrophotometric procedures.
Name of Drug
Acetaminophen
Alendronate
Amoxycillin
Amlodipine besylate
Ascorbic acid
Aspirin
Astemizol
Reagents used
p-xylenol
Potassium hexacyanoferrate (HI) and
m-cresol
FeCla
KIO3
Tris- (o-phenanthroline) iron (II)
Ninhydrin
2,3-Dichloro 5,6-dicyano 1,4-
benzoquinone
Ascorbic acid
l-Chloro-2, 4-dinitro benzene
NaOH
p-Chloranilic acid
Suprachen Violet 3B
Tropaeolin 000
Iron (III) and 1,10-Phenanthroline
Amax(nm)
635
260
520
510
590
5S0
530
380
302
540
590
500
515
References
[84]
[85]
[86]
[87]
[88]
[89]
[90]
[90]
[91]
[92]
[93]
[94]
[94]
[94]
13
Acetic acid & N-bromosuccinimide 540
Atenolol
Benidipine
hydrochloride
Bromazepam
Chlorhexidine
Cisapride
HCl & Celestine blue
Iodine
Chloranil
2,3-Dichloro-5, 6-dicyanol, 4-
benzoquinone
Bromanil
Tetracyanoethylene
Ciprofloxacin
p-Chloranilic acid
Methanol
Mohr salt
Alizarin yellow G.
Chromotropic acid
Phloroglucinol
p-Chloranilic acid
540
Folin - Ciocalteau reagent & NaOH 720
Glycine buffer and Azocarmine G 540
365
440
470
450
450
7,7,8,8-Tetracyanoquinodimethane 420
535
238
584
405
530
450
520
[95]
[95]
[95]
[95]
[96]
[96]
[96]
[96]
[96]
[96]
[97]
[98]
[99]
[100]
[101]
[101]
[120]
14
Desipramine
Diltiazem
Hydrochloride
Doxorubicin
hydrochloride
L-Dopa
Etidocaine
Famotidine
Flurazepam
Gentamicin
Imipramine
Levopoda
Lisinopril
Tetracyanoethylene
Anunonium metavandate
Diclofenac Sodium Tris buffer
Sodium metavandate
Bromocresol green
Bromothymol blue
Bromophenol blue
Ferric ammonium sulphate
NaOH
Bromocresol green
Alkaline KMn04
Ninhydrin
HCl
Ninhydrin
Ammonium metavandate
Cerium (IV) nitrate
l,Fluoro-2, 4-dinitro benzene
535
618
284,305
750
415
415
415
[102]
[103]
[104]
[105]
[106]
[106]
[106]
510 [107]
300
625
610
590
230
400
618
510
356
[108]
[109]
[110]
[111]
[112]
[113]
[103]
[114]
[115]
15
Loperamide
hydrochloride
Loratadine
Meclozine
hydrochloride
Methyl dopa
Mefloquine
hydrochloride
Mefenamic acid
Menadione
Metoprolol tartarate
Chloranil
Iodine
Bromothymol Blue
Bromophenol Blue
Naphthol Blue Black
Bromophenol Blue
Chromotrope 2B
Chromotrope 2R
Cerivim (IV) nitrate
HCL
p-Dimethylaminocirmamaldehyde
NaOH
Ethylacetoacetate
3-Methyl 2-benzothiazolinone
hydrazone
Tetracyanoethylene
7,7,8,8-Tetracyanoquinodimethane
346
295
414
415
627
415
540
528
550
222
665
450
550
625
450
420
[116]
[117]
[117]
[117]
[117]
[118]
[119]
[119]
[114]
[120]
[121]
[122]
[123]
[124]
[125]
[125]
16
Nalidixic acid
Nifedipine
Nimisulide
Nitrazepam
Nizatidine
Nortriptyline
Hydrochloride
Oxprenolol
Piroxicam
Salbutamol
Trimethoprim
Thyroxine sodium
Tenoxicam
Verapamil
hydrochloride
Persulphate in alkaline medium
4-(Methyl amino phenol) and
K2Cr207
BCMn04
Iminodibenzyl
3-Aminophenol
Ethylacetoacetate
Alkaline KMn04
3-Methyl 2-benzothiazolinone
hydrazone
Cerium (TV) in H2SO4
Alizarin
2,6-Dichloroquinone chlorimide
7,7,8,8-Tetracyanoquinodimethane
Persulphate in alkaline medium
Nitrous acid
Alizarin
Chloramine-T
320,390
525
530
600
470
482
610
619
480
538
602
842
355
420
538
425
[126]
[127]
[128]
[129]
[129]
[130]
[131]
[132]
[133]
[134]
[135]
[135]
[136]
[137]
[138]
[139]
17
The derivative method has found applications not only in UV-spectrophotometry
but also in infrared [149], atomic absorption, flame spectrophotometry [150,151], and
fluorimetry [152,153]. The use of derivative spectrometry is not restricted to special
cases, but may be of advantage whenever quantitative study of normal spectra is difficult.
Its advantage is that the differentiation degrades the signal-to-nose ratio, so that some
form of smoothing is required in conjunction with differentiation [154].
dA For a single-peak spectrum, the first-derivative is a plot of the gradient — of the
UA
absorption envelope versus wavelength and features maximum and minimum; the vertical
distance between these is the amplitude, which is proportional to the analyte dA
concentration; theoretically, — is zero at max for the band in the normal spectrum. The dX
d^A second-derivative spectrum, —r- versus wavelength, has two maxima with a minimum
dX
between them, at X,max of the normal absorption band [155]. In principle, both peak-
d'^A heights (measured from —^= 0) are proportional to the analyte concentration but the
dX
amplitude can also be measured by the so-called tangent method, in which a tangent is
drawn to the maxima and amplitude is measured vertically from tangent to the minimum
[156]. The differentiation discriminates against broad bands, emphasizing sharper
features to an extent that increases with increasing derivative order, because for bands
(Gaussian or Lorentzian) the amplitude Dn of the nth derivative is related to the n* power
of the inverse of the band width, W, of the normal spectrum [157]:
18
D„a
Thus, for two bands A and B of equal absorbance but different width, the
derivative amplitude of the sharper band (A, for example) is greater than that of the
broader (B) by a factor that increases with increasing derivative order [158,159]:
For the reason, the use of derivative spectra can increase the detection sensitivity
[160-162] of minor spectral features. For quantitative analysis, if Beer's law is obeyed for
normal spectrum, the following equation can be obtained:
= Ic
dX" dA"
Where A = Absorbance, e = molar absorptivity, / = cell path- length and C =
concentration of the analyte and this forms the basis of analytical determinations [163].
Derivative Specti'ophotometry has been applied for quantification of drugs in
pharmaceutical preparations [164-171].
Difference spectrophotometry is a technique, which requires high potentiometric
accuracy and linearity. Small spectral changes in solutions with high initial absorbance
are often measured. This technique has been extensively used in the determination of
drugs by eliminating specific interference from degradation products, coformulated drugs
[172], and also the non-specific irrelevant absorption from the formulation matrix
[173,174]. It provides a sensitive method for detecting small changes in the environment
of chromophores. In general, two solutions at the same concentration are directly
19
compared. One solution is selected as sample, the second as the reference. All coinmon
spectral features to the two solutions cancel out. Only bands, which have been displaced
in one solution relative to the other because of environmental differences, are recorded.
Another emerging trend has been the increasing use of near-infrared (NIR)
spectroscopy in the quality assurance and in process analysis of pharmaceutical products.
Both qualitative and quantitative aspects of NIR spectroscopy as well as uses related to
validation of procedures that employ it are discussed [175-177].
Other important emerging technologies in drug development are proteomics [178]
combinatorial chemistry [179], the application of molecularly imprinted media [180,181],
the use of light scattering particle size analysis for flocculated suspensions [182], and the
profiling of monomeric and dimeric impurities in bulk pharmaceutical batches by
^^FNMR[183].
The number of applications of kinetics to analytical chemistry continues to grow
at a rapid pace. Kinetic methods are good choices for drug analysis as they permit the
sensitive and selective determinations of many drugs within a few seconds with no
sample pretreatment is many cases. Kinetic methods, because of its superiority to the
other colorimetric methods due to the formation of unstable colours in some of these
colorimetric ones, are among the first choices of the researchers in analytical chemistry.
Moreover, the instrumentation required is generally very simple. The principles and
applications of the kinetic methods have been reviewed in papers [184-192] and books
[193,194]. Essentially kinetic methods rely on measurements of concentration changes
(detected via signal changes in a reactant which may be the analyte itself) with time after
20
the sample and reagent have been mixed. The sample and reagent can be mixed manually
or automatically, only slow enough reactions tolerate manual mixing and, even so, they
may be better handled automatically, not only to obtain more rapid and reproducible
results, but also to increase the reaction rate in some cases. Kinetic automatic techniques
are generally based on open system, among the most popular of which is stopped flow
(SF) system and the continuous addition of Reagent (CAR) Technique [195-198]. Several
drugs have been determined by using CAR technique Math photometric [199-200] and
fluorimetric [201] detection. The application of micellar systems to kinetic methods was
the subject of a review by Cai and Co-workers [202]. The discussion included
characteristic of micellar catalysis, micellar modification of properties of system, and
application of micelles in multicomponent determinations. CuUen and Crouch [203], have
described the application of multivariate calibration techniques to multicomponent
kinetic based determinations. These techniques are mandatory for fast reactions but can
also be extended to slow reactions. In both cases, the kinetic curve (variation of the
analytical signal with time) can be recorded immediately. The slope of the initial straight
portion of the kinetic curve gives the reaction rate, which is proportional to the analyte
concentration (initial rate method). The fixed time method [204], also frequently used to
determine the concentration involving the measurement of the signal (the value of which
depends on the analyte concentration) at a preset time.
The determinations can be implemented by using combinations of the SF
technique with (a) fluorescence polarization immunoassay (FPIA), (b) micelle-stabilized
room temperature phosphorimetry and (c) a sensitized energy transfer fluorescence
21
reaction, among other, all are novel strategies which have so far yielded excellent results
in drug analysis. On the other hand, the CAR technique has been extended to
chemiluminescence (CL) reactions as CAR chemiluminescence spectrometry (CARCL),
a new approach that has proved outstanding for the analysis of drugs and other substances
of analytical mterest. Moreover, new kmetic Ghemomettic (kinetometric) approaches
[189] such as the Kalman filter have been developed and applied to the simultaneous
determination of various compounds of pharmaceutical interest.
Over the last few years the term chemometrics has been increasingly used. The
organic chemist S. Wold supposedly coined this term in 1972 when submitting a grant
proposal for the application of statistical methods to chemical data. Together with
American analytical chemist B.R. Kowaiski, Wold formed the international
chemometrics society. Subsequently there have been several major reviews, [205-208]
ACS symposia [209,210], a National Bureau of standards conference, [211] a Nato
School, [212], a text book [213] and a series of monographs [214] devoted to
"Chemometrics". Recently two international journals devoted to Chemometrics have
been established [215,216]. A large variety of clustering methods [217] have been
developed, originally as aid to pharmaceutical and clinical researchers. The ability of
modem analytical instruments helps a lot to acquire large amounts of data rapidly. In a
modem laboratory, measurements are cheap compared with samples: many parameters
(e.g.. Chromatographic and Spectroscopic peak intensities) can be obtained from each
sample. Hence new approaches are needed to interpret what may be described as large
multivariate data matrices, involving specialized statistical and computatioual rricthods,
22
clearly we can say that chemometrics involves the application of statistical methods to
analytical data.
Method Validation
Method validation is an important component in determining the reliability and
reproducibility of analytical method and is a reqiiirement of any regulatory submission
[218], According to the European Union directives in referring to the Good
manufacturing Practice and Quality control of drugs [219-221], validation must be
applied not only in manufacturing process but also in analysis and control methods. The
validation of analytical procedures used for raw materials and a finished product is
gathered in the Pharmaceutical Products and Multistate Register [222-224] also and more
recently it has been reviewed for ICH Harmonized Tripartite Guideline [225,226]. As far
as the validation characteristics are concerned, if the analytical method is described in
Pharmacopoeia, it will be proceeded as follows: (a) with regard to raw materials assay,
the validation will not be necessary, although it must be checked before its routine use,
enclosing information about linearity, sensitivity, precision and selectivity, (b) with
regard to the finished product assay, it is necessary to carry out a brief validation to
demonstrate that the precision, selectivity and accuracy are appropriate.
Validation and Strategy
The overall validation strategy consists of four components, which are the
prevalidation, validation proper, study proper and statistical analysis. These components
23
constitute the platform to evaluate the reliability and ruggedness of an analytical method.
Prevalidation
Prior to initiating the validation proper, a prevalidation is performed by the
primary analyst. This component provides the analyst an opportunity to obtain some
practical experience v^th the methods and helps to identify the optimum conditions. It is
recommended that the following studies be conducted prior to initiating the validation
proper. The appropriate peak response to use in quantitating drug concentrations should
be selected first. The optimum standard curve range and the number of calibrators should
be established. The appropriate regression model which best fits the data is then selected.
The extraction scheme and its recovery should be optimized to give insight into
the limit of quantitation and to help determine if the extraction procedure is reproducible.
If possible, potential compounds that could interfere with the method should be
evaluated. The remaining system suitability parameters are then investigated by
optimizing the other factors.
Validation Proper
The validation proper consists of four analytical runs generated on separate days.
Each validation run emulates the analytical conditions of the study proper and its
expected run time. This strategy helps the analyst develop a daily routine to use during
the study proper and anticipate potential assay problems. Suggestions for additional
samples to be analyzed during each validation run include samples fortified with
24
metabolites and potentially interfering compounds to identify response behaviour and
ensure assay specificity. Stability samples to begin generating information pertinent to
sample processing and storage. From the data generated, specific analytical parameters
are reported, including linearity, accuracy, precision, sensitivity and recovery. From each
standard curve; the slope, intercept, correlation coefficient and variance are monitored.
Statistical analysis are used to determine with in and between-run variance and to
demonstrate how the method can be expected to perform on a daily basis. Based on the
mitial quality control data concentration results, acceptance criteria are established.
Subsequent analytical runs are monitored during the study proper usmg these acceptance
criteria to determine if the data generated are valid.
Acceptance criteria for validation parameters
Using the ICH guidelmes as a basis [227,228], it is the responsibility of the
analyst to select for the given individual test procedure relevant parameters and
appropriate acceptance criteria and to design the experimental studies accordingly. These
acceptance criteria can often be derived from specification limits.
As a general rule, the standard deviation of the analytical procedure should be
lower than 1/6 of the specification range. A detailed approach taking the number of
repeated determinations into account is based on confidence intervals [229,230]. Basic
specification limits (BL) include the variability of the manufacturing process and
represent the final limits (SL) if an error-free analytical procedure is used. Describing the
analytical variability as a normal distribution of the experimental results, confidence
25
intervals can be constructed as a representation of the result probability for a given
number of replicates. The combination of the basic limit and the limit (upper or lower) of
the 95% confidence interval (one sided) then represents the overall specification limit
(Eq. 1).
SL = BL±t„_, 095^^ Eq.(l)
It must be taken into account that this calculation is based on the true standard
deviation whereas the standard deviation obtained in a validation study is only a random
estimate which also displays a variability. A reliable experimental determination of the
true value requires repeated intermediate precision studies, but it can be estimated from
the statistical (z^) distribution of standard deviations. The upper 95% confidence limit of
this distribution (UL) will represent the maximum value for the true standard deviation
(Eq. 2). For six experimental values, it can be approximated by twice the experimental
standard deviation [230].
UL(S,^e) = S,^,x U ^ Eq.(2) V Z "-' J0.95
Rearranging Eqs. (1) and (2) gives the maximum permitted experimental standard
deviation for the given specification limits, i.e. the acceptance limit for the validation. For
drug substances, due to the presence of impurities, these are the lower limits. As the
required standard deviation is dependent on the number of repetitions in the assay,
adjustments are possible (Table 1.2.). Thus, the number of repeated determinations can
also be fine-tuned according to the results of the validation. In case of sufficiently wide
26
specification limits compared to the analytical variability, this allows an efficiency
optimization of the analytical procedure.
While in the case of a drug substance assay the basic limits are defined mainly by
the sum of impurities, in the case of drug products, often only estimation is possible. For
simple dosage forms, the same variability may be expected for analytics and
manufacturing. It should be noted that the estimation of the true standard deviation would
result in a maximum value and, therefore, reduce the required acceptance limit.
Consequently, in critical cases an experimental determination (using at least four to six
series) might be reasonable to obtain an estimate for the true standard deviation. If
specification limits are not yet defined or implied due to safety requirements, Eq. (1) can
be used directly to calculate the lunits. For the true standard deviation, the upper limit of
the experimental determination (Eq. 2) can be calculated or twice this value as an
approximation. Alternatively, a soundly based intermediate precision may be used as
estimation for the true standard deviation.
With respect to impurity detenninations, the ICH reporting threshold of 0.05%
[231] can be regarded as the required quantitation limit for unknown impurities, which
would guarantee a reliable quantitation at the specification limit of 0.1%. If other limits
are required due, for example, to safety considerations, the above-mentioned approach
can be applied. Statistical tests such as the student's Mest or the evaluation of 95%
27
Table 1.2. Maximum permitted standard deviation for assay determinations in dependence on the number of repetitions and the specification range
Drug product (%) Drug product (%) Drug product (%)
Specification range 95-105 95-105 98-102
Basic limit (lower) 97.5 (estimated) 99.0 (estimated) 99.5 (sum of impurities)
Niraiber of repetitions Acceptance limit for experimental standard deviation in validation (n=6)
2 0.28 0.45 0.17
3
4
6
0.74
1.06
1.44
1.19
1.7
2.3
0.45
0.64
0.86
28
confidence intervals should only be carefully (directly) applied as acceptance criteria
because they test for statistical differences. Due to sometimes abnormally small
variabilities in the analytical series, differences are identified as significant which are of
no practical relevance [232]. In addition, when comparing independent methods for the
proof of accuracy, different specificities can be expected which add a systematic bias,
thus increasing the risk of the aforementioned danger.
In addition, the power of these tests increases with the number of determinations.
For practical purposes, a sufficient agreement between two results (two means or a mean
and a nominal value, e.g. recovery) is completely adequate. The acceptance criteria can
be derived from previous experience or calculated based on specification limits and
statistical considerations. For example, assuming an assay procedure with specification
limits from 95 to 105%, a recovery range from 98 to 102% would be acceptable.
The acceptance criteria and limits should be defined before starting the validation
and included in a protocol. After the validation studies, they will serve as a basis for the
evaluation.
Validation characteristics
When performing validation studies, the whole analytical procedure including all
the steps of the sample preparation should be applied, as far as possible. In contrast, the
term 'method' should be restricted to the mode of analytical determination alone (e.g.
capillary electrophoresis, (reversed phase) chromatography and spectrometry). Allowed
exceptions to the written procedure concern the number of repetitions as the number of
29
determinations for the various validation characteristics is described in the ICH guideline
(Table 1.2) and the repetitions in the procedure may be fine-tuned based on the validation
results. Such an adjustment may also be used for the final calibration mode.
As far as possible, the analytical procedure should be independent of the actual
equipment used provided that the equipment has been appropriately qualified. This must
be taken into account for the validation studies.
Specificity
There has been some controversy regarding the technical term for this validation
characteristic, i.e. specificity versus selectivity [233]. In contrast to an isolated test
procedure, in pharmaceutical analysis the sum of various control tests and hence there
combined specificity is used for the overall batch evaluation. A very pragmatic definition
describes selectivity as the (physical) separation of substance mixtures with, for example,
chromatography and electrophoresis, i.e. the determination of the analyte in addition to
other substances. The individual determination of an analyte in the presence of other
substances, (i.e. without significant influence of other substances or classes of
substances) is defined as specific by, for instance, mass spectrometry, NMR, infrared,
fluorescence or UV spectrometry, electrochemical detection and titration [234].
With respect to chromatographic techniques, specificity can be demonstrated by a
sufficient separation of the substances present. For the assay, appropriate separation
means an adequate resolution between the main peak and the impurity and placebo peaks,
which need not to be separated fi-om each other. The required resolution is strongly
30
dependent on the difference in the size of the corresponding peaks as well as on their
elution order [235].
Linearity I range
A linear dependence of the signal and the analyte concentration is certainly the
most convenient case and widely used in pharmaceutical analysis. The requirements and
relevant parameters for the various calibrations are given in Table 1.3.
It should be noted that in most cases only a qualitative statement is needed. For
example, if a single-point calibration (external standard) is aimed at, the requirements are
a linear response flmction and the zero intercept. Care should be taken to remain within
the linear range of the individual detector used.
Accuracy
The ICH guideline recommends the demonstration of accuracy over the whole
working range (Table 1.4). However, if only a narrow range is required (e.g. assay or
impurities with a low specification limit), a six-fold determination at a 100% test
concentration as described for the precision studies may also be used. Several approaches
discussed in the ICH guidelines are given in Table 1.5. If the analytical test to be
validated is compared with another procedure or applied to a reference substance, the
probably different specificities must be taken into account. Therefore, statistical tests
should be performed only if the systematic bias based on these differences can be
quantified and thus corrected or are negligible. Otherwise, the comparison should be
31
Table 1.3 Requirements for different calibration modes with relevant parameters
Quantitation Requirements Relevant parameters
Single-point calibration External standard Linear function
Non-significant Ordinate intercept
Homogeneity of variances"
Multiple-point calibration Linear, unweighted Linear function
Linear, weighted
•Non-linear
100%-method(area normalisation for impurities):
Homogeneity of variances^
Linear function
Continuous function
For main peak: Linear function
Non-significant Ordinate intercept
Homogeneity of Variances"
For impurities Linear Function
Standard error of slope (residual standard deviation), Sensitivities (relative standard deviation, graph), residual analysis, statistical tests (vs. quadratic regression)
Inclusion of zero in confidence interval of the ordinate intercept, magnitude of the intercept (as percent of the signal at 100% test concentration)
F-test of the variances at the lower and upper limit of the range
Standard error of slope (residual standard deviation), sensitivities (relative standard deviation, graph), residual analysis, statistical tests (vs. quadratic regression)
F-test of the variances at the lower and upper limit of the range
Standard error of slope (residual standard deviation), sensitivities (relative standard deviation, graph), residual analysis, statistical tests (vs. quadratic regression)
Appropriate equation
Standard error of slope (residual standard deviation), sensitivities (relative standard deviation, graph), residual analysis, statistical tests (vs. quadratic regression)
Inclusion of zero in confidence interval of the ordinate intercept, magnitude of the intercept (as percent of the signal at 100% test concentration)
F-test of the variances at lower and upper limit of the range
Standard error of slope (residual standard deviation), sensitivities (relative standard deviation, graph), residual analysis, statistical tests (Vs. quadratic regression)
May be presumed for a limited range (factor 10-20).
32
Table 1.4. Validation characteristics normally evaluated for the different types of test procedures and the minimum number of determination required
Validation characheristics
Specificity
Linearity
. Range
Accuracy
Precision
Repeatability '
Intermediate precision reproducibility'^
Detection limit
Quantitation limit
Minimum number
5 concentrations
9 determinations over 3 concentrations levels (e.g. 3x3)
6 determinations at 100% or 9 determinations over 3 concentrations levels (e.g.3x3)
2 series
Test procedure
Identity
Yes
No
No
No
No
No
No
No
Impurities
Quantitative
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Limit
Yes
No
No
No
No
No
«
Yes
No
Assay^
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No Including dissolution content/Potency.
^Lack of specificity of one analytical procedure could be compensated by other supporting analytical procedure. "Intermediate precision sufficient for submission. May be needed in some cases.
33
Table 1.5. Approaches to demonstrate accuracy according to ICH
Drug substance
Drug Product
Impurities (quantitative)
Application of the analytical procedure to a reference
material
Comparison of the results with those of a second, well
characterized procedure.
Application of the analytical procedure to Drug
product synthetic mixtures of drug product
components
Spiking of analyte to drug product
Comparison of the results with those of a second, well
characterized procedure.
Spiking of the impurity to drug substance
or product
Comparison of the results with those of a second well
characterized procedure.
34
performed, as a qualitative verification of plausibility or an acceptable maximum
difference should be defined. Spiking experiments for recovery investigations should be
performed as closely to the authentic conditions as possible so that possible interferences
between the analyte and matrix can be recognized. This ranges, for example, from the
direct preparation of a drug product with various contents of active ingredient to which
the whole analytical procedure is applied to the addition of a drug substance stock
solution to a placebo solution.
For the quantitation of the analyte, the same calibration mode as described in the
final test procedure must be used. Again, statistical tests should be used carefully,
especially with complex matrices and low concentrations of impurities. Alternatively,
acceptable deviations from the theoretical recovery of 100% can be defined based on the
application, experiences or general statistical considerations.
Precision
In addition to the ICH precision levels (Table 1.4), it is advisable to determine the
system precision (injection repeatability) either by repeated determinations of the same
test solution or firom double determinations of each test solution used for repeatability
(Eq. 3).
S''=\~t(\.-\2)' Eq.(3)
Repeatability, also termed as intra-assay precision, refers to the precision obtained
under the same operating conditions over a short interval of time by applying the whole
35
analytical procedure to the sample. Intermediate precision refers to within laboratory
variations. The extent of investigations will depend on the intended use of the analytical
procedure. Repeatability and intermediate precision can be calculated by an analysis of
variances [236,237]. The difference between the precisions levels as well as their
absolute magnitude indicate the robustness of the anal3^ical procedure. For the evaluation
of the suitability (compatibility with specification limits) the intermediate precision can
be regarded as the relevant parameter. From the standard deviation, the repeatability limit
can be calculated (Eq. 4) which represents the maximum permitted difference between
two repeated measurements.
^ = ^«-i,o.95^^^'^*'2.8x5 Eq. (4)
In this way, a straightforward verification is possible if the degree of scattering
imder the actual conditions is comparable to the validation. By this parameter, the
precision obtained during the validation studies using a larger number of data (thus
increasing the reliability) is linked to the routine analyses without requiring a larger
nimiber of experimental data. Reliable estimate of the standard deviation is required to
calculate appropriate repeatability limits.
Most statistical tests and calculations are based on the assumption that the
experimental values are only influenced by random variability (i.e. that they are normally
distributed). Data, which do not fulfill these assumptions (e.g. due to so called 'gross
errors', weighing, dilution, or by problems with the instrument, etc.) will affect the
results. Such values can be identified by statistical outlier tests (e.g. according to Dixon
or Grubbs [236]) in order to eliminate them before performing further calculations.
36
Detection and quantitation limit
Several approaches are given in the ICH guideline to determine the detection and
quantitation limits (Table 1.6). Generally, they are based either on the analysis of blanks
or on the scattering (variability) of the analytical signals in the low concentration range.
Using the blank procedures, the corresponding calculation value is multiplied by the
factors of 3.3 and 10 for the detection and quantitation limits, respectively. The
calculation value may represent the signal of the blank, the standard deviation of the
blank or of the intercept of a calibration line (corresponding to an extrapolated blank). In
the latter tv/o cases, the analytical signal is transformed by the slope of the calibration
line into a concentration [228]. Using the calibration line directly, the aforementioned
factors (3.3 and 10) can be multiplied by the ratio from the residual standard deviation
and the slope (corresponding to the standard error of slope) [228]. Limits calculated or
extrapolated by these procedures should be verified by the analysis of samples in the
corresponding concentration range [228]. This additional verification is already included
in other procedures, which make direct use of the scattering around the calibration line by
means of the 95% prediction interval around the regression line [238,239]. The
quantitation limit can also be obtained directly from precision studies. For this approach.
37
Table 1.6. Approaches for determining the detection and quantitation limit [228]"
Approach Detection limit Quantitation limit
Visual evaluation Minimum level detectable Minimum level quantifiable
Signal-to- noise 3: lor2 :1 10:1
Standard deviation of the 3.3 X a/S lOxa/S
response and the (a) slope (S)
^Verification with suitable number of samples. *" Standard deviation of the blank, residual standard deviation of the calibration line, or standard deviation of the intercept.
38
decreasing analyte concentrations are analyzed repeatedly. The coefficient of variation
(relative standard deviation) is plotted against the corresponding concentration. If a
predefined limit for the coefficient of variation is exceeded (e.g. 10 or 20%), the
corresponding concentration is established as the quantitation limit [240,241].
Study Proper and Statistical Analysis
Daily standard curves are generated to determine the sample concentrations. All
calibrators and quality control samples are analyzed in duplicate. Sample concentrations
are based on single determination. The quality control sample sequence is carefully
monitored for systematic errors. For each standard curve, the slope, intercept, variance,
correlation coefficient and the interpolated calibrated concentration are reported.
Acceptance of the assay results are determined by monitoring the quality control
results. If the interpolated concentrations are v ithin the control charts confidence limits,
established during the method validation, the data are considered valid. Upon completing
a study proper and accepting the analytical runs, the quality control results are
incorporated into their respective data basis to update their confidence limits.
Statistical calculations of the validation parameters and certain others of common
interest
Linear Calibration
Linearity is often tested by using correlation coefficient, r. this quantity, whose
39
full title is "Product-moment Correlation Coefficient", is given by
i:h-^){y.-y)] r = -
i:{^.-^i\L[y.-y)" \ii
Where the points on the graph are {x^,y^), (xz.jVa) {^nV.) (^^'^^J' ^ ^ x
and y are as usual, the mean value of x, and y, respectively. Value of r should be as:
- l < r < + l .
Least square Line
The slope, b, and intercept, a, of the imweighted least square lines are found from.
6 = -YX{^.-x){y,-y)]
/ _ \ 2 E(^'-^)
a = y-bx
Errors and confidence limits
Random errors of the slope and intercept of the regression line involve the
preliminary calculation of Sy/x, which is given by
40
ll/2
Sy/x = Jl{y'-y>f
n-2
In this equation each y, value is measured signal value from the analytical
instruments, while the corresponding y, is the value of y on the fitted straight line of the
same value of x. After the determination of Sy/x, the standard deviation of the slope, Sb,
and the standard deviation of the intercept, Sa can be determined from:
S, 'ylx
S(-,-?)' 1/2
^a - ^y/x
E--
«Z(^.-^)'
1/2
These standard deviations can be used to estimate the confidence limit for the true
slope and intercept values. The confidence limit for the slope are given by b + ts^ , where
the value of "t" is chosen at the desired confidence level and with n-2 degree of freedom.
Similarly, confidence limits for the intercept are given by a±tSi,. Standard
deviation of S^^, can be calculated by using equation:
-11/2
s. = y/n
1 + - + 1 (yo-yf
" b'Zi-,-^f
41
Where XQ and y^ are the value of single concentration and its instrument signal.
In a typical analysis, the value of y^ might be obtained as the mean of m observations of
a test sample, rather than as a single observation.
ylx s„ = b
1 , 1 , jyo-y) — 1 — I m n b^n-.-^r
1/2
Method of standard additions
Matrix effects on the measured signal demand the use of standard additions. The
concentration of the test sample, x^ is given by the intercept on the x-axis, which is
clearly the ratio of the y-axis intercept and the slope of the calibration line as:
a
Limit of detection and sensitivity
The lowest detectable instrument signal, jv , is given by:
y^=ys+KSs
Where y^ and Sg are respectively, the blank signal and its standard deviation. After yj^
has been established, it can readily be converted into a mass or concentration limit of
detection, c , as:
^L=-KS.
42
CLASSIFICATION OF DRUGS
All the drugs according to their chemical nature can be divided into organic and
inorganic compounds. They can be prepared synthetically (from chemicals) or can be
directly obtained or reconstituted from the natural sources/products. All the drugs having
medicinal importance can be broadly divided into two classes.
Chemical classification
Here the drugs are classified according to their chemical structure and properties
without taking the pharmacological actions vmder consideration, hi this class most of the
drugs are having at least an organic substrate, the fiirther classification is done in the
relevant manner.
Pharmacological classification
The drugs are classified according to their action on the organs (Viz., heart, brain,
lymphatic system, respiratory system, endocrine system, central system, nervous system
etc.) hence these drugs are named like antianginal, narcotics, soporifics, analgesics,
diuretics and anaesthetics etc. Further classification of each group is done according to
the therapeutic/pharmacological specificity Vvith the relevant organ.
43
Hepatoprotective Drugs
The liver is subject to acute and potentially lethal injury by a number of
substances, including phalloidm (the toxic constituent of the mushroom amanita
phalloides), carbon tetrachloride, galactosamine, ethanol and other compounds. The
effect of flavonoids on carbon tetrachloride induced toxicity in isolated rat hepatocytes
has been studied by Perrisoud and Testa [242]. The ability to interfere with carbon tetra
chloride-induced release of aspartate aminotransferase was tested on 55 flavonoid
compounds. The more hydrophilic compounds were observed to inhibit the carbon tetra
chloride-induced toxicity, whereas the more lipophilic derivatives actually potentiated the
toxicity. In a number of countries, silybin and other flavonoids are widely utilized in the
treatment of liver diseases [243].
Antianginals
Angina pectoris is a heart ailment characterized by pressing chest pain that often
radiates to the neck area and arms and shoulders (often towards left side). As a clinical
entity William Heberden first characterized it under the name Pectoris Dolor in 1768,
although its symptoms had been noted as early as 1632 [244-246], in the recent medical
terminology it is referred as the symptom of ischaemic heart disease. For nearly a century
following its first description, there was not much that could be done to relieve an angina
patient of his agony: brandy, ether, chloroform, ammonia and other stimulants and
depressants had been tried, but nothing seems to bring comfort. The breakthrough came
in 1867 when T. Lander Bmnton, a British physician, reported his success with amyl
44
nitrite. In the authoritative but lively journal of medicine, the Lancet, he wrote, "On
pouring from five to ten drops of amyl nitrite on a cloth and giving it to the patient to
inhale, the pain completely disappeared and, generally did not return till its wonted time
next night" [247]. And finally the continuous efforts of the medical researchers, they got
succeeded in knowing the different reasons with this life-threatening ailment and its
therapeutic solutions.
Drugs used in angina pectoris are those that reduce cardiac work and myocardial
need by (a) unloading the heart, (b) dilating the capacitance and resistance vessels, (c)
dilating the coronary arteries and (d) blocking P-adrenoceptors. Anginal pain occurs
when the coronary blood flow is insufficient to meet the hearts metabolic requirements.
The drugs that either improve myocardial perfusion or reduce the metabolic demand, or
both can covmteract it. Several antianginal drugs are available for the treatment of angina.
These include (a) organic nitrates, (b) beta-adrenergic blockers, (c) calcium channel
blockers, (d) potassium charmel activators, (e) antiplatelet drugs and (f) certain others like
diazepam, oxyfedrine etc.
This thesis deals with the quantitative analysis of silymarin, nifedipine,
amiodarone hydrochloride and verapamil hydrochloride in pharmaceutical formulations.
SUymarin
Silymarin i.e. 3,5,7-trihydroxy,2-[3-(4-hydroxy-3-methoxy phenyI)-2-
(hydroxy-methyl)-I,4-benzodioxan-6-yl]-4-chromanone(structure I) is a free
radical scavenger and a mixture of flavonolignans from medicinal plant Silybum
45
marianum, officially listed in Martindale [248] is used in supportive treatment of liver
diseases of different etiology due to its hepatoprotective activity,[249-251] which is
considered to involve antioxidative and the membrane stabilizing effects. The liver plays
an important role in regulation of metabolism of plasma lipoproteins and liver injury is
often reflected as a secondary dyslipoproteinaemia, which may lead to the development
of atherosclerosis, particularly when associated with hypercholesterolaemia. [252]
OH 0
0 . ,CH20H
OCH^
Structure I
Nifedipine
Nifedipine, chemically dimethyl-1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)
pyridine-3,5 dicarboxylate, is the prototype compound of dihydro pyridine class of
calcium channel blockers with peripheral and coronary vasodilator properties. It reduces
peripheral resistance, afterload and blood pressure. It is given by mouth in the
management of hypertension and angina pectoris. In human, nifedipine is rapidly
46
metabolized by oxidative mechanism to dehydronifedipine wliich is further metabolized
to more polar compoimds [253-256] and their structures are given below:
HaCOOC^ ,C00CH3
H3C 1 CH3
H
3HCOOC-
H ^ c ^ ^ r ^
^N02
'COOCH3
CH, H
Nifedipine Dehydronifedipine
3HCOOC-
H ^ C - ^ N ^ ,
'COOCH3
CH,
3HC H C2HCOOCV
/ 3HC
HaC I H
-NO2
'COOCH3
^CH,
Nitroso analogue of dehydronifedipine Nisoiidipine
47
Nifedipine is highly sensitive to photo-oxidation, changing in colour from yellow
to brown upon exposure to light [257]. Nifedipine photodegration products have a little or
no pharmacological activity [258,259]. Several studies in the past have been conducted to
determine the photostability of nifedipine in solution and in the solid state, including
photostability of pulverized nifedipine tablet powder [260-262].
Amiodarone hydrochloride
Amiodarone hydrochloride,[2-n-butyl,3-(3,5-diiodo-4-diethylaminoethoxy
benzoyl)-benzofuran] hydrochloride (structure II) is a potent class III
antiarrhythmic drug [263]. It is used to treat ventricular and supraventricular
arrhythmias, especially when they are resistant to other conventional
antiarrhythmic drugs [264,265]. However, its use is sometimes complicated by
serious adverse effects, including occasionally life threatening pulmonary
fibrosis and hepatitis [266]. Therefore, its concentration in patients treated with
therapeutic doses of the drug vary considerably and therapeutic drug monitoring
of amiodarone hydrochloride may assist in individualizing the dosage regimen.
In human, mono-N-desethylamiodarone is known as the major metabolite
[267]. During long-term therapy, the plasma level of mono-N-
desethylamiodarone is comparable with that of amiodarone hydrochloride, and
this metabolite may contribute to the therapeutic effect of amiodarone
hydrochloride.
48
, ^ ^ ^ -O CH2 CH2- -N . /
Ri
\ R2
o
-c—
0112(01^2)2^113
Structure II
Where Ri= R2 = C2 H5 (amiodarone), Ri = Hi, R2 = C2H5 (desythyl amiodarone),
Ri = R2 = H (di-N-desythyl amiodarone).
Verapamil hydrochloride
Verapamil hydrochloride i.e. (±)-5-[(3,4-dimethoxyphenethyl) methyl amino]-2-
(3,4-dimethoxyphenyl)-2-isopropyl valeronitrile monohydrochloride (Structure III) is
clinically a very useful member of the calcium channel blocker. It is used in the treatment
of supraventricular arrhythmias, angina and hypertension [268-270]. The verapamil
shows conduction through the atrioventricular node, and this slows the increased
ventricular response rate that occurs in atrial fibrillation and flutter. A decrease in both
coronary and peripheral vascular resistense together with a sparing effect on myocardial
intracellular oxygen consumption, and the decrease in peripheral vascular resistance may
explain the antihypertensive effect of verapamil [271-273].
49
CH,0 / / ^ CH3
H3C ,CH3 CH
CH2—CH2—N—CH2—CH2- CH2—C—CN
Hsca
OCH,
Structure III OCH3
50
[1]
[2]
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CHapter -2 %inetic spectropHotometric metHocCfor the
determination ofsiCymarin inpharmaceuticaC formulations using potassium permanganate as
oxidant
69
INTRODUCTION
Silymarin i.e. 3,5,7-trihydroxy-2-[3-(4-hydroxy-3-methoxy phenyl)-2-
(hydroxy-methyl)-l,4-benzodioxan-6-yl]-4-chromanone is an important
antihepatotoxic drug wiiich is obtained from the dried fruits of Silybum
marianum and widely used for the treatment of hepatic disorders [1-4]. The
drug is officially listed in Martindale The Extra Pharmacopoeia [5].
The assay procedure listed in the monograph of the Italian
pharmacopoeia describes the UV-Vis spectrophotometric method [6] for
determination of the drug in bulk and pharmaceutical formulations. Several
other methods have been employed to determine silymarin in biological
specimens and/or pharmaceutical formulations such as high performance
liquid chromatography [7-21], thin layer chromatography [22-28], high
performance thin layer chromatography [29], potentiometric titration [30],
diffuse-reflectance fourier transform infrared spectroscopy [31] and UV
spectrophotometry [32,33].
Spectrophotometry as a quantitative analytical tool still belongs to the
most frequently used analytical techniques in pharmaceutical analysis. It
provides practical and significant economic advantages over other methods.
In the literature only few spectrophotometric methods have been reported.
The drug content in pharmaceutical formulations was determined [34], based
on the formation of coloured complex of the drug with 2, 4-dinitrophenyl
hydrazine in the presence of tetramethyl amine hydroxide and quantitatively
measured at 490 nm. Another spectrophotometric method has been developed
70
for the estimation of silymarin based on its reaction with diazotized sulfanilic
acid in alkaline medium to form orange-red coloured chromogen, which
absorbed maximally at 460 nm [35]. Reddy et al. [36] have reported two
spectrophotometric methods for its determination. The first method was based
on the oxidation of the drug by Fe (III) and estimating the reduced Fe (II)
with 1,10-phenanthroline at 510 nm. The other method involved the formation
of a blue complex on treatment with Folin-Ciocalteau reagent in the presence
of NaOH and subsequent determination was done at 740 nm. These
spectrophotometric methods are somewhat tedious and time consuming.
Therefore, there is a need to develop a fast, low cost and selective method for
routine analysis of silymarin in bulk and drug formulations.
The extent of oxidation of organic substrate by potassium
permanganate depends on the pH of the medium. The heptavalent manganese
changes to Mn (VI) in alkaline medium while in neutral and acidic medium,
the permanganate is further reduced forming ultimately Mn (II). This
behaviour 'of permanganate has been exploited to develop a kinetic
spectrophotometric method for assay of nifedipine in drug formulations [37].
In this chapter a fast, simple and sensitive kinetic spectrophotometric
method for the assay of silymarin in bulk and pharmaceutical formulations is
described. The method is based on the oxidation of silymarin by potassium
permanganate at pH 7.0±0.2 and the course of the reaction was followed by
measuring a decrease in absorbance at 530 nm.
71
EXPERIMENTAL
Apparatus
The absorbance was measured on a spectronic 20 D" spectrophotometer
(Milton Roy, USA) with 1 cm glass cells.
Reagents
All chemicals used were of analytical or pharmaceutical grade.
Potassium permanganate, 0.003M was freshly prepared by dissolving 47.41
mg of potassium permanganate in 100 ml of doubly distilled water. The
apparent purity of the potassium permanganate solution was checked
titrimetrically [38].
Test solution
Silymarin (1 mg ml'*) (Micro Labs, Bangalore, India) was prepared in
methanol.
Procedure for silymarin
Kinetic spectropltotometric method for determination of silymarin
Aliquots of 0.18-0.50 ml of silymarin standard test solution were
pipetted into a series of 10 ml standard volumetric flasks. To each standard
flask 1.6 ml of 0.003M potassium permanganate was added and then diluted
to volume with doubly distilled water at 30±1°C. After mixing, reaction
mixture was immediately transferred to spectrophotometric cell and the
72
decrease in absorbance was recorded as a function of time for 20 min at 530
nm. The initial rate of the reaction (v) at different concentrations was
obtained from the slope of the tangent to the absorbance-time curve. The
calibration curve was constructed by plotting the logarithm of the initial rate
of reaction (log v) versus logarithm of the concentration of the silymarin (log
C).
Procedure for determination of silymarin in pharmaceutical formulations
An accurately weighed quantity of the mixed contents of five capsules
or five powdered tablets, equivalent to 50 mg of the drug, was extracted into
50 ml chloroform with shaking and the residue was filtered using Whatman
No. 42 filter paper. The filtrate was evaporated to dryness and the residue was
taken up with methanol and transferred to a 50 ml standard volumetric flask,
diluting to volume. The assay was completed following the recommended
procedure.
RESULTS AND DISCUSSION
The oxidation of organic compounds by permanganate is dependent
upon the pH of the medium. During the course of the reaction the valence of
manganese changes and intermediate ions have been suggested as
participating oxidant. But the species having the main role as potential
oxidant depends on the nature of the substrate and pH of the medium. The
oxidation of silymarin by potassium permanganate at pH 7.0±0.2 was
73
followed spectrophotometrically by measuring the rate of change of
absorbance at 530 nm. The absorbance-time curves are shown in Figs. 2.1.
and 2.2., which are all sigmoid in nature throughout the entire range and
therefore, the initial rates of reaction were determined from the slope of the
initial tangent to the absorbance-time curves. The order with respect to
permanganate was determined by studying the reaction at different initial
concentrations of permanganate with fixed silymarin concentration. Keeping
the silymarin concentration higher than that of the oxidant, the plot of initial
rate [-d[A]/dt] against initial absorbance was linear passing through the origin
suggested that the order of reaction with respect to permanganate at the start
is one. The order with respect to silymarin was evaluated from the
measurement of the rates at several concentrations of silymarin but at fixed
concentration of KMn04 which was also found to be one. The first order
dependence on both permanganate and silymarin in the initial stages leads to
the simple rate expression:
Rate = K [silymarin] [Mn04"]
Silymarin is naturally occurring aryl chromanone (I) related to
flavanone. The flavanones are 2,3-dihydro derivatives of flavones, which are
readily interconvertible. Though the basic skeleton of flavanone and flavone
is the same, the key which distinguishes one structural type from another is
the oxidation level of the various carbons in the heterocyclic ring. The
flavones are in a high state of oxidation where as flavanones are in low state
of oxidation. The oxidation of flavanone with a variety of reagents such as
selenium dioxide [39] or triphenyl methyl carbonium ion [40] has been
74
Time (min)
Fig. 2.1. Absorbance-time curves: KMn04,4.8 xlO" M and silymarin (a) 3.731 X10- M; (b) 4.146 x 10" M; (c) 6.219 x 10" ; (d) 8.292 X 10- M; (e) 10.365 x 10" M
75
Time (min)
Fig. 2.2. Absorbance-time curves: silymarin, 10.365 x 10" M and KMn04 (a) 3.00 x 10"* M; (b) 2.10 x 10"* M; (c) 1.50 x 10"* M; (d)9.00xlO-^M.
76
reported in which flavone is formed. In this study, the same mechanism seems
to be involved. The oxidation of silymarin by permanganate takes place at pH
7.0±02 leading to the formation of chromone (II). Thus, the dehydrogenation
is effected by permanganate and consequently disproportionation between
immediate oxidation states of manganese results in the formation of Mn02.
However no precipitation of MnOa was observed under study time period.
Therefore, the reaction mechanism is presented in scheme 2.1.
Calibration graph and Statistical analysis
Under the optimum experimental conditions, a calibration curve (Fig.
2.3.) was constructed by plotting logarithm of initial rate of reaction (log v)
versus logarithm of silymarin concentration (log C), which showed a linear
relationship over silymarin concentration range of 18-50 \x.g ml''. The linear
regression analysis using the method of least square was made to evaluate the
slope, intercept and correlation coefficient. The linear regression equation and
correlation coefficient are log v = 1.024 log C + 3.235 and r = 0.9997 which
indicates an excellent linearity. The confidence limits for the slope of the line
of regression and intercept were computed using the relation b±tSb and a±tSa
[41], at 95% confidence level and found to be 1.024±3.96xl0"2 and
3.235±I.67x10-', respectively. This indicated the high reproducibility of the
proposed kinetic method. Linearity was also evaluated [42] from the relative
standard deviation of the slope of the calibration line and found to be 1.215.
The limit of detection was established using the equation [43,44]:
77
X o-
o I 5=0 I
u c o E p
u
S
u
u c o c ea
6 a u
78
-4.4 -4.2 -4.1
log of molar concentration
-4.0
Fig. 2.3. Calibration curve for tlie determination of Silymarin.
79
Limit of detection = ^^ x 2 n-2^
where n is the number of standard samples, t is the value of student's t for n-
2 degrees of freedom at 95% confidence level and So = variance. The
variance and detection limit were calculated and found to be 1.100 x 10" ^g
ml"' and 2.823 x 10" ^g ml"' respectively, which confirmed negligible
scattering of experimental data points around the line of regression and good
sensitivity of the method.
The error (Sc) in the determination of a given concentration of
silymarin (C) was established using the equation [45]:
-|l/2
Q _ So
°""b" 1+i^. <>'- >'
n b^(ZC^-nC^)
where y and C are average ordinate and abscissa values, respectively, for n
standard samples. Fig. 2.4. shows the graph of Sc versus the concentration of
silymarin. It is apparent from the graph that the error is reached minimum
when the actual initial rate was equal to the average initial rate in the
calibration graph.
The minimum error of 1.123x10"^ fig ml"' was found in the
determination of about 30 yig ml"' silymarin. The value of Sc also allows
establishing the confidence limit at the selected level of significance for the
determination of unknown concentrations by using the relation, C,±tSc The
results are shown graphically (Fig. 2.5.) in the form of percent uncertaintyfi^. ,oo]
on the concentration [46], at 95% and 99% confidence levels. This is
80
10"" X1.30
I
Concentartion of silymarin (^g ml' )
Fig. 2.4. Error in the determination of tlie concentration of silymarin using statistical analysis of standard calibration data.
81
10-2 X4.5
Concentration of slymarin (ng ml' )
Fig. 2.5. Variation of the confidence limit at (a) p= 0.05 and (b) p = 0.01 level of significance in the form of uncertainty percentage on the concentration of silymarin.
82
a useful way of expressing the confidence limits because the relative
uncertainty can be estimated directly on the concentration over the full range
of the concentration tested.
The reproducibility was established for ten independent analyses of solution
containing 0.40 mg silymarin. The analytical results obtained from the
investigation are summarized in Table 2.1. The relative standard deviation
and mean percent recovery were 0.873% and 100.33%, respectively and
considered to be very satisfactory. The validity of the proposed kinetic
method was checked by applying standard addition method and the results are
reported in Table 2.2.
The applicability of the proposed kinetic method for the determination
of silymarin in dosage forms was examined by analyzing marketed products;
the results were compared to those obtained by the reference method [35] and
are summarized in Table 2.3. The performance of the proposed method was
judged by calculating t- and F-values [47]. At 95% confidence judged level,
the calculated t- and F-values do not exceed the theoretical values indicating
no significant difference between the proposed kinetic method and the
reference method.
83
Table 2.1. Precision test of the proposed kinetic method
Sample No.
1
2
3
4
5
6
7
8
9
10
Mean
Initial rate of reaction (mol r' min'')
11.455x10"^
11.508x10-^
11.376x10"^
11.350x10-^
11.561x10-^
11.534x10-2
11.271x10-2
11.402x10-2
11.324x10-2
11.297x10-2
percent recovery = 100.33 %
Relative standard deviation = •• 0 .833 %
Amount found (Hg per 10ml)
402.97
404.79
400.26
399.37
406.61
405.70
396.68
401.17
398.47
397.57
84
Table 2.2. Determination of silyraarin in pharmaceutical formulations by standard addition method
Preparation Amount taken (Hgml-')
Amount added Total amount found (Hgml-') (Hgml-')
Recovery RSD
Silybin-70 (tablet)
Silvia-70 (tablet)
Limarm-70 (capsule)
Sivylar-70 (capsule)
Levalon-70 (capsule)
10 20
10 20
10 20
10 20
10 20
10 20
10 20
10 20
10 20
10 20
20.03 39.99
20.03 40.02
20.02 40.08
20.03 40.02
20.04 39.99
100.16 99.97
100.18 100.05
100.11 100.20
100.18 100.05
100.20 99.97
0.354 0.343
0.471 0.224
0.585 0.467
0.578 0.224
0.685 0.343
' Three Independent analyses.
o
B
e
•a o
5
u B!
•a
o o< o u
s u .2 Oil
a v> O
C3
S
W
u O
ttH
•"•^
1
^
u
U -P
•s J= 4-*
u 6 u '•5 a c 3
Q ^
041::'
Q tn
i I
•33
c o
C3 Q. U
I u I
0
0
r4
0
en t s
m t ^
-^
0
O N
c<i
0
0 0 CO
o
o 00
85
00 ro CS
( S <o 1-H
00 <N CO
i n ON o
0 0
ON
o\
00 m m o u-i ro
O o o
o o o
o o
en CO
CO o\ o
(S
o o
CO
o
ON ON
o
0 0 ON
o
NO
o o
o NO o
U-I o c3 o
o o CO
o
o o
o
o CO
c5 o
CO
i n o
ON ON O N
O o o o
00 <!i. 00 i ^
ca (in
o
<u
13 3
u
0
r
c _o •3 > u
"3 CO
a u
Cfl
^ CO c a
^-t
a u • 0 a u u •a _c
>
VO
r--ci to
• 3
> 0
g - 0 B 0 U
0 ^ V^ o\ " l - l
, 0 ( M
u 3
A
>
' •5
0 u H
NO en
> u u 0 s u
•0 IS § 0
0 ^ v\ 0
« U, t - i 0
« u 3 d
>
0
86
REFERENCES
[1] S. Cavalieri, Gazz Med. Ital. 133 (1974) 628.
[2] H. A. Salmi, S. Sarna, Scand. J. Gasteroenterol 17 (1982) 517.
[3] P. Ferenci, J. Hepatol. 9 (1989) 105.
[4] K. Hruby, Hum. Toxicol. 2 (1983) 183.
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Society, London, (1996) p. 994.
[6] Farmacopoeia Ufficale Italiana 9th ed., vol. II, tuto Poligrafico e Zecca
cello Stato-Roma, (1985) p. 1673.
[7] E. M. Martinelli, P. Morazzoni, S. Livio, E. Uberti, J. Liq. Chromatogr.
14(1991)1285.
[8] H. Marcher H, C. Klkuta, J. Liq. Chromatogr. 16 (1993) 2777.
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J. Chromatogr. 135 (1977) 499.
[10] G. Tittel, H. Wagner, J. Chromatogr. 135 (1977) 499.
[11] G. Tittel, H. Wagner, J. Chromatogr. 153 (1978) 277.
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Fitoterapia51 (1980) 83.
[13] V. Querela, N. Pierini, U. Valeavi, R. Caponi, S. Innocenti, S. Tederchi,
Anal. Chem. Symp. Ser. 13 (1983) 1.
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[15] K. H. Law, N. P. Das, J. Chromatogr. 388 (1987) 225.
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[16] E. Tomov,M. Simova, T. Pangarova, D. Bocheva, F.E. C.S. Int. Conf.
Chem. Biotechnol. Biol. Act. Nat. Prod. 5 (1985) 534.
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[18] N. Nikolov, A. Bizhev, P. Pashankov, God. Vissb. Khim - Tekhnol. Inst.
30(1990)203.
[19] V. A. Kurkin, Gaertn. fruits. Rastil. Resur. 32 (1996) 80.
[20] P. Wang, R. Cong, J. Wang, L. Zang, Sepu. 16 (1998) 510.
[21] Y. T. Ding, S. Tian, D. Gu, Z. Suu, Z. Zang, Yaowu Fenxi Zazhi 19
(1999)3048.
[22] L. L. Lin, Taiwan Yao. Hsuch. Tsa Chih. 34 (1982) 53.
[23] N. A. Abdel-Salam, M. A. Abdel-Salam, M. A. Elsayed, Pharmazie 37
(1982) 74.
[24] M. Vanhaelen, R. Vaniiaelen - Fastre, J. Chromatogr. 281 (1983) 263.
[25] B. Heinz, W. Funk, A. Voemel, Gaertn. Proc. Int. Symp. lustrum. HPLC
3rd (1985) 313.
[26] Z. Wu, X. Wang, Zhogguo Yaoke Danue Xuebao 20 (1989) 184.
[27] Q. Lin, J. Huang, P. Xie, Guangdong Yaoxue. Yuan. 14 (1998) 250.
[28] R. Szilajtis-Obeiglo, K. Kowalewska, Herba Pol. 30 (1984) 27.
[29] W. Funk, B. Heinz, H. Michel, C. Vonderhied, GIT-Suppl. 4-6 (1987) 9.
[30] J. Koerbl, F. Janick, M. Tkaczyk, K. Havel, (1986) Czech. CS 234387,
IPC GO IN -027126 Appl. 8314077, 06 Jun 1983.
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[32] C. Lan, Z. Kang, S. Zhang, Z. Wu, R. Yu, Nanjung Yaoxueyuan Xuebao
21 (1983)75.
88
[33] V.V Belikov, Rastit. Resur. 21 (1985) 350.
[34] S. S. Zarapkar, N. S. Kanyawar, S. H. Rane, Indian Drugs 37 (2000) 133.
[35] A. Rajasekaran, M. Kumar, G. Krishnamoorthy, B. Jayabar, Indian J.
Pharm. Sci. 59 (1997) 230.
[36] M. N. Reddy, Y. P. N. Reddy, P. J. Reddy, T. K. Murthy, Asian J. Chem.
13 (2001) 1234.
[37] N. Rahman, S. N. H. Azmi, Acta Pharm. 49 (1999) 113-118.
[38] Vogel's Textbook of Quantitative Chemical Analysis 6th ed., Pearson
Education, Singapore, 2002, p. 420.
[39], P. G. Sammel, Comprehensive Organic Chemistry - The synthesis and
Reactions of Organic Compounds, Volume 4, 1st ed., Pergamon Press,
1979, p. 681.
[40] A. Schonberg, G. Schutz, Chem. Ber. 93 (1960) 1466.
[41] J. N. Miller, Analyst 116 (1991) 3.
[42] S. Torrado, S. Torrado, R. Cadorniga, J. Pharm. Biomed. Anal. 12 (1994)
383.
[43] B.Morelli Analyst 108 (1983) 870.
[44] V. V. Nalimov, The Application of Mathematical Statistics to Chemical
Analysis, Pergamon Press, Oxford, 1963, p. 189.
[45] B. Morelli, Anal. Lett. 20 (1987) 141.
[46] R. Cassidy, M. Janoski, LC-GC 10 (1992) 692.
[47] G. D. Christian, Analytical Chemistry, 4th ed., Wiley, New York, 1986,
p. 79.
Cfjcvpter -3 (Ej(tractive spectropHotometric metfiods for the determination of nifedipine in pHarmaceuticaC
formulations using BromocresoCgreen, SromopHenoC SCue, BromotHymoCBCue anderiocHrome 6[ac^^
89
INTRODUCTION
Nifedipine, chemically dimethyl-1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)
pyridine-3,5 dicarboxylate, is an important calcium cliannel blocker with peripheral
and coronary vasodilator activity [1-5]. The drug is frequently used as
antihypertensive and potent arterial vasodilator in the treatment of angina pectoris and
various other cardiovascular disorders [6, 7]. The drug and its formulations are
official in USP [8] and BP [9], which recommended HPLC and non-aqueous titration
for its assay, respectively.
The drug has been determined by a variety of analytical techniques such as
HPLC [10-31], reversed phase HPLC [32-34], HPTLC [35], gas chromatography [36-
53], micellar electrokinetic chromatrography [54], electroanalytical methods [55-60],
flow injection analysis [61], mass spectrometry [62] and UV spectrophotometry [63-
65].
The estimation of nifedipine alone was carried out using second-order
derivative spectra [66] of the compound in 0.1 M HCl whereas first derivative spectra
were utilized for its assay in combined dosage forms [67,68]. The methanolic solution
of the drug reacts with 4-dimethylaminobenzaldehyde in H3PO4 resulting in the
formation of yellow coloured product, which forms a basis for its determination at
380 nm [69]. Two spectrophotometric methods have been recommended, one is based
on the formation of blue coloured complex with Folin Ciocalteau reagent [70] and
second method involves the charge transfer complex formation with chloranil [71]. A
kinetic spectrophotometric method has also been described based on oxidation of drug
with KMn04 at neutral pH [72]. The other two spectrophotometric methods were
developed which involved the reduction of nifedipine with Zn/NHtCl and Zn/HCl to
90
hydoxylamino derivative and primary aromatic amino derivative, respectively
[73,74]. The hydoxylamino derivative was reacted with 4-(methylamino) phenol and
potassium dichromate to give a chromophore, which absorbed maximally at 525 nm.
Thp aromatic amino derivative formed schiffs base with 3,4,5-
trimethoxybenzaldehyde and subsequently determined at 365 nm. So far, no
extractive spectrophotometric method for the assay of nifedipine in pharmaceutical
formulations was reported.
In this chapter, four new extractive spectrophotometric methods for the
determination of nifedipine have been discussed. The methods are based on the
reduction of nitro group of nifedipine by Zn/HCl into primary amino derivative which
forms chloroform extractable ion pair complexes with bromocresol green (BCG),
bromophenol blue (BPB), bromothymol blue (BTB) and eriochrome black T (EBT).
EXPERIMENTAL
Apparatus
A Spectronic 20D* spectrophotometer (Milton Roy, U.S.A.) was used to
measure the absorbance.
An Elico model Li-10 pH meter was used for pH measurements.
Reagents and standards
Preparation of amino derivative of the nifedipine
A 0.05 % solution of reduced nifedipine was prepared by treating a mixture of
12.5 mg of pure nifedipine (J.B. Chemicals and Phannaceuticals Ltd., Mumbai
91
India), dissolved in 3 ml ethanol, 1.8 ml of 5M HCl for BTB (2.5 ml for BCG and
BPB; 3.0 ml for EBT required for ion pair formation) and 3.0 g of Zn dust. The
content was allowed to stand for 20 min at 25 ± 1°C. The solution was then filtered
through a Whatmann No. 42 filter paper, the residue was washed with two 5 ml
portions of doubly distilled water. The filtrate and washings were diluted to volume in
a 25 ml standard volumetric flask with doubly distilled water.
0.025 % solutions of bromocresol green (S.d. fine Chem Limited, India),
bromophenol blue (S.d. fine Chem Limited, India), bromothymol blue (S.d. fine
Chem Limited, India) and eriochrome black T (Fluka Chemie A G, Germany) were
prepared by dissolving 25 mg of each dyestuff in 100 ml standard volumetric flask
and diluting to volume with doubly distilled water.
Procedure of calibration curve
Into a series of 50 ml separating funnel 7 ml of BCG (BPB, BTB or ETB)
followed by an appropriate volume of 0.05 % reduced nifedipine (0.10-0.65 ml for
BCG; 0.08-0.75 ml for BPB; 0.13-0.66 ml for BTB and 0.09-0.45 ml for EBT) were
placed and mixed well. Then 10 ml of chloroform was added to each funnel. The
contents were shaken for 2 min and allowed to separate the two layers. The
absorbance of the organic layer was measured at 415 nm for BCG, BPB, BTB ion-
pair complexes and 520 nm for EBT ion-pair complex against a reagent blank
prepared similarly in each case. The calibration curve was constructed, in each case,
by considering the absorbance measured at seven concentration levels of nifedipine.
The amount of the drug was computed either from calibration curve or from
regression equation. The colour of the complexes were stable for at least 2 hours.
92
Procedure for the assay of drug in dosage forms
An amount of the tablet or capsule equivalent to 12.5 mg of nifedipine was
weighed accurately, and extracted into 25 ml chloroform with shaking. Filtration
through a Whatmann No. 42 filter paper was performed. The filtrate was evaporated
to dryness under vacuum and the residue was dissolved in 3 ml ethanol and converted
into amino derivative following the procedure given under the head "preparation of
amino derivative of the nifedipine" and then subjected to the recommended procedure
for the determination.
RESULTS AND DISCUSSION
Nifedipine contains -NO2 group, which is reduced to amino derivative by zinc
dust and HCl. In the present study, the reduced nifedipine possessing primary
aromatic amino group is protonated in acidic medium, which forms ion-pair
complexes with each of the acid dyes such as BCG, BPB, BTB and EBT. The ion-
associated complexes are quantitatively extracted with chloroform. The absorption
spectra are shown in Fig. 3.1. which revealed that the ion-pair complexes with BCG
(BTB or BPB) and EBT absorbed maximally at 415 and 520 nm, respectively. The
reagent blanks prepared under similar conditions showed no absorption.
Composition and formation constant of ion-pair complexes
The stoichiometry of ion-pair complexes of the reduced drug with each of the
dyestuffs was established following the method of continuous variations [75]. The
results (Figs. 3.2., 3.3., 3.4. and 3.5.) indicated that the molar ratio of the drug to
93
0.7
0.6 -
0.5 -
^ 0.4 ^ a .o 1 . o m < 0.3
0.2
0.1
0.0 -r
350 400 450 500
Wavelength (nm}
550 600
Fig. 3.1. Absorption spectra: (a) Amino derivative of nifedipine-BCG complex; (b) Amino derivative of nifedipine-BPB complex; (c) amino derivative of nifedipiae-BTB complex; (d) amino derivative of nifedipine-EBT complex.
94
0.6
Mole fraction of drug
Fig. 3.2. Job's plot for drug-dye (1.443 x 10" M) system: BCG.
95
0.35
0.2 0.4 0.6
Mole fraction of drug 0.8 1.0
Fig. 3.3. Job's plot for drug-dye (1.443 x 10" M) system: BPB.
96
0.30
0.00 U 0.4
Mole fraction of drug
Fig. 3.4. Job's plot for drug-dye (1.443 x 10" M) system: BTB.
97
o o c n Si
o (0
<
0.2 0.4 0.6
Mole fraction of drug
Fig. 3.5. Job's plot for drug-dye (1.443 x 10 M) system: EBT.
98
dyestuff in each compound is 1:1. The formation constants [76,77] were also
calculated and found to be 2.91 x 10^ 3.32 x 10^ 5.82 x 10 and 5.54 x 10 for
complexes with BCG, BPB, BTB and EBT, respectively.
On the basis of literature background and our experimental findings, the
reaction mechanism is proposed and given in scheme 3.1.
Optimization of the reaction conditions
The optimum conditions for quantitative estimation of the drug were
established via a number of preliminary experiments.
Effect of concentration of hydrochloric acid
The influence of the concentration of HCl on the reduction of the nifedipine
and subsequent ion-pair formation of amino derivative of the drug with various
dyestuffs has been studied. The results are shown in Figs. 3.6., 3.7., 3.8. and 3.9. It is
apparent from figure that the absorbance of ion-pair complexes with BCG (or BPB),
BTB and EBT was found to be constant when the reduction was done in the range of
0.47-0.53 M, 0.34-0.40 M and 0.56-0.64 M HCl, respectively. For the most efficient
extraction of ion-pair with chloroform the optimum value was fixed at 0.50 M, 0.36
M and 0.60 M HCl for ion-pair formation with BCG (or BPB), BTB and EBT,
respectively.
Effect of dye concentration
The effect of dye concentration on the intensity of the colour at the selected
wavelength and constant nifedipine concentration was critically examined using
99
[A]
HjCOOC
HaC^ ^ N ^ ^CH;
HjCOOC
nifedipine
H3C.
COOCH3 H3COOC ^
NH2
amino derivative of nifedipine protonated
Vr' OH HO,
B r ^ ^ * > ^ ^ C C ^ " * * < ^Br Br T ^ \ ^ C H 3
^ A v ^ S O j
bromocresol green (lactoid ring)
(quinoid ring)
HaC^ . N ^ ^CH;
HjCOOC COOCH3 B r - ^ ' ^ * ^ ^ C * ' ' ^ f ^ B r "
r^^ CH3
Br
H3COOC
nifedipine(amino derivative)-bromocresol green complex
Br Br Br
[B]
SO3H
bromoplienol blue (lactoid ring) (quinoid ring)
HaC
H3COOC
Br Br
C ^-^^ "-Br
so7
H3C,
HaCOOC-
Cr nifedipine (amino derivative)-bromophenol blue complex
100
[C]
H3C01
C jH , C3H7
CH3 HO,
COOCH3 Bi
NH3 CH3
H3COOC
C3H7 C3H7
bromothymol blue nifedipine (amino derivative)-bromothynnol blue complex
PI HjCOOC-
nifedipine (amino denvative)-eriochrome black T complex
Scheme 3.1.
101
0.2 0.3 0.4 0.5
Concentration of HCI (M)
0.6 0.7
Fig. 3.6. Effect of the concentration of HCI: 31 ^g ml"' drug + 7 ml of 0.025% BCG.
102
0.3 0.4 0.5
Concentration of HCI (M)
Fig. 3.7. Effect of the concentration of HCi: 33 ^g ml" drug + 7ml of 0.025% BPB.
103
0) u c n Si o tn n <
0.1 0.2 0.3 0.4
Concentration of HCI (M)
0.5
Fig. 3.8. Effect of the concentration of HCI: 33 \xg ml * drug + 7 ml of 0.025% BTB.
104
0.0 0.1 0.2 0.3 0.4 0.5 0.6
Concentration of HCI (M)
0.7 0.8
Fig. 3.9. Effect of the concentration of HCI: 22.5 ^g ml"' drug + 7 ml of 0.025% EBT.
105
different milliliters of the reagent (0.025 %). The results indicated (Figs. 3.10., 3.11.,
3.12. and 3.13) that the maximum absorbance, in each case, was found with 6.0 ml of
the reagent, beyond which absorbance become constant. Therefore, 7.0 ml of each
dyestuff was used for ion-pair formation throughout the experiment.
Choice of organic solvent
A number of organic solvents such as chloroform, carbon tetrachloride,
dichloromethane, benzene and toluene were examined for extraction of the ion-pair
complex in order to provide an applicable extraction procedure. Chloroform was
preferred for its selective extraction of ion-pair complex from the aqueous solution.
Shaking time of 0.5-4.0 min. provided a constant absorbance and hence, 2.0 min was
used as an optimum shaking time throughout the experiment. The ion-pair complexes
were quantitatively recovered in one extraction only and were also stable for at least 2
hours.
Effect of excipients
A systematic study of the effect of excipients was performed, following the
proposed procedures for a 10 ml sample system., by adding a known amount of
excipients to the fixed nifedipine concentration (22.5 \i.% ml"'). The results revealed
the fact that no significant interference was observed from the excipients such as
glucose, fructose, sucrose, lactose and starch commonly present in pharmaceutical
formulations. However, the drug content from the powdered tablets or capsules were
extracted into chloroform which completely eliminates the common excipients found
in drug formulations.
106
o o c n
o lA
<
Volume of BCG (ml)
Fig. 3.10. Influence of the volume of 0.025% dye: BCG.
107
0.50
Volume of BPB (ml)
Fig. 3,11. Influence of the volume of 0.025% dye: BPB.
108
o u c RI a h. o to
<
Volume of BTB (ml)
Fig, 3.12. Influence of the volume of 0.025% dye: BTB.
109
0) u c
o (0
<
Volume of EBT (ml)
Fig. 3.13. Influence of the volume of 0.025% dye: EBT.
110
Analytical data
Calibration graphs (Figs. 3.14., 3.15., 3.16. and 3.17.) were constructed, by
measuring the absorbance at seven concentration levels, which showed linear
response of absorbance in relation to concentration of nifedipine over the range of
5.0-32.5, 4.0-37.5, 6.5-33.0 and 4.5-22.5 |ag ml'' for BCG, BPB, BTB and EBT
methods, respectively. Regression analysis of calibration graphs indicated linear
relationship with negligible intercepts. Table 3.1. summarizes the analytical
parameters, molar absorptivities and the results of statistical analysis of the
experimental data: regression equations computed from calibration graphs along with
standard deviation of slope (Sb) and intercept (Sa), confidence interval of slope (tSb)
and intercept (tSa) on the ordinate. The detection limits [78,79] were found to be 1.06,
0.09, 0.11 and 0.20 [ig ml"' for BCG, BPB, BTB and EBT methods, respectively. The
small value of variance, further, suggested least scatter of experimental data points
around the line of regression.
The repeatability of the proposed procedures was checked by performing ten
replicate determinations of nifedipine at concentration levels of 20 and 30 fig ml''
with BCG (or EBT) and BPB (or BTB), respectively. The relative standard deviations
(RSD %) and recoveries were found to vary over the range of 0.66-0.82 % and 99.8-
100.7 %, respectively.
The accuracy of proposed methods was demonstrated by recovery
experiments, which were carried out by adding a fixed amount of pure drug to the
preanalyzed dosage forms. The analytical results obtained are summarized in Table
3.2.
111
Concentration (ng ml ')
Fig. 3.14. Calibration curve for determination of nifedipine witli BCG.
112
o u c n J3 u O V)
<
Concentration ( g ml )
Fig. 3.15. Calibration curve for determination of nifedipine with BPB.
113
0)
u • c n o in n <
-1 Concentration (^g ml )
Fig. 3.16. Calibration curve for determination of nifedipine witli BTB.
114
Concentration (fxg ml )
Fig. 3.17. Calibration curve for determination of nifedipine with EBT.
u
115
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CHapter -4 VaCidatecfSpectropHotometnc MetHodsfor tde (Determination ofJLtniocCarone HydrocHCoricCe in CommerciaC(Dosage ^orms using p-cHlhraniCic
acicCand2,3 -dicfiCoro 5,6- dicyano 1,4 — Benzaquinone
126
INTRODUCTION
Amiodarone hydrochloride, 2-butyl, 3-benzofliranyl 4-[2-diethyl amino
ethoxy]-3-5-diiodophenyl methanone hydrochloride is an important
antiarrhythmic drug [1-3], used only in the treatment of documented life-
threatening recurrent ventricular dysrhythmias. It undergoes hepatic
metabolism to produce a major metabolite, desythyl amiodarone, which can
approach the concentrations of the parent drug during chronic therapy. The
molecular formulae of amiodarone and its metabolites are given as:
< ^ ^
O
II -c- -O CHz CH, N
/ ' ^
\ R2
^0 CH2(CH2)2CH3 -j
Where Ri= R2= C2 H5 (amiodarone)
Ri = HI , R2 = C2 H5 (desythyl amiodarone)
R] = R2 = H (di- N- desythyl amiodarone)
British Pharmacopoeia [4] describes potentiometric titration and
spectrophotometric method for the assay of amiodarone hydrochloride in pure
form and pharmaceutical formulations, respectively. The different analytical
techniques that have been reported for the determination of cited drug in
biological fluids and pharmaceutical formulations include high performance
liquid chromatography [5-11], high performance thin layer chromatography
[12], liquid chromatography [13-14], infrared spectroscopy [15], fluorimetry
127
[16] and electrochemical methods [17-19]. In the literature only few
spectrophotometric methods have been reported. Sastry et al. [20] have
described two spectrophotometic methods for the determination of
amiodarone hydrochloride based on its reaction with a known excess of iodine
or ammonium molybdate and estimating the unreacted iodine or reduced
ammonium molybdate with metol - sulfanilic acid or potassium thiocyanate.
The reaction [21] between amiodarone and citric acid-acetic anhydride
reagent yielded an intensely blue coloured product and absorbance was
measured at 580 nm. The drug content in pharmaceutical formulations was
also determined spectrophotometrically [22] using the charge transfer
complexation reaction of drug with chloranil and iodine at 500 and 290 nm,
respectively. The extractive spectrophotometric methods [23] have also been
utilized based on extractable ion-pair complexes of the drug with tropaeolin
00, tropaeolin 000 and wool fast blue at 420, 480 and 580 nm, respectively.
A survey of literature reveals that the existing spectrophotometric
methods [20-23] dealing with the estimation of amiodarone in drug
formulations suffer from some disadvantages such as narrow linear dynamic
range requires heating or extraction and long analysis time. Therefore, the
development of fast, simple, sensitive, low-cost and selective methods is the
need of the day especially for a routine quality control analysis of
pharmaceutical products containing amiodarone hydrochloride.
The charge transfer reaction of certain n- acceptors such as p-
chloranilic acid [24-26] and 2,3-dichloro5,6-dicyanol,4-benzoquinone [27-
31 ] has been successfully utilized in pharmaceutical analysis.
128
This chapter describes two rapid and simple visible spectrophotometric
methods for the determination of amiodarone hydrochloride by exploiting its
basic nature and electron donating property. The methods (A and B) are based
on charge transfer complexation reaction of amiodrone base with p-chloranilic
acid and 2,3-dichloro5,6-dicyanol,4-benzoquinone(DDQ) in dioxane-dichloro
methane and acetonitrile media, respectively. The proposed methods were
validated statistically.
EXPERIMENTAL
Apparatus
Spectral runs and absorbances were recorded on a spectronic 20 D"
spectrophotometer (Milton Roy Company, USA) with 1 cm matched glass
cells.
Reagents and Standards
Amiodarone hydrochloride was kindly provided by Troikaa
Pharmaceuticals Ltd. India and was used as received. Commercial dosage
forms of amiodarone hydrochloride such as Aldarone (Zydus Alidac),
Amiodar (Cardicare), Duron (Samarth Pharma) and Eurythmic (Troikaa) were
purchased from local market.
All other reagents used were of analytical grade. Solutions of p-
chloranilic acid (0.08%; Fluka, Switzerland) and DDQ (0.08%; Fluka,
129
Switzerland) were prepared in dioxane and acetonitrile, respectively. An
aqueous solution of sodium carbonate (0.5M, Merck, India) was prepared in
doubly distilled water.
The standard solutions of amiodarone base (2.0 mg ml'' and 0.5 mg
mr') were prepared in dichloromethane.
Recommended Procedures
Preparation of amiodarone base:
To prepare amiodarone base solution, 25.0 ml of 0.2% (2.0 mg ml')
amiodarone hydrochloride and 50.0 ml of 0.5 M NaiCOs solutions were
transferred into a 250 ml separating funnel. The contents of the separating
funnel were mixed well and shaken for 1 min. The two layers were allowed to
separate. The dichloromethane layer was dried over anhydrous sodium sulfate
and diluted to 25 ml with dichloromethane. A working standard solution of
amiodarone base (0.05%) was prepared on further dilution.
Method A:
Aliquots of 0.2%. amiodarone base solution (0.025 - 0.90 ml) were
pipetted into a series of 5.0 ml standard volumetric flasks. 1.5 ml of 0.08% p-
chloranilic acid was added to each flask and diluted to volume with
dichloromethane. The coloured product formed immediately and remained
stable for 1.5 h. The absorbance was measured within the stability period (2-
90 min) after dilution at 535 nm against the reagent blank prepared
simultaneously.
130
Method B:
Aliquots of 0.05% amiodarone base corresponding to 10 - 325 p-g were
pipetted into a series of 5.0 ml standard volumetric flasks. To each flask, 1.5
ml of 0.08% DDQ was added and completed to volume with acetonitrile. The
absorbance was measured at 570 nm within the stability period of 3 - 40 min
against the reagent blank prepared similarly, omitting the drug.
Procedure for determination of commercial dosage forms
Ten tablets were powdered, mixed thoroughly and weighed accurately
to an amount equivalent to 200 mg of amiodarone hydrochloride. It was
stirred well with dichloromethane and filtered through Whatmann No. 42
filter paper. The residue was washed with dichloromethane for complete
recovery of the drug. The filtrate and washings were diluted to volume in a
100 ml standard flask. The amiodrone hydrochloride solution was converted
into amiodrone base following the recommended procedure given under the
head "preparation of amiodarone base solution". It was further diluted
according to the need and then analyzed following the recommended
procedures.
RESULTS AND DISCUSSION
The spectrum of p-chloranilic acid in the dioxan-dichloromethane
mixture exhibits an absorption band at 440 nm. The addition of amiodarone
base to this solution causes an immediate shift with a new characteristic band
131
at 535 nm (Fig. 4.1.). The band may be attributed to the formation of
p-chloranilic acid radical anion. The p-chloranilic acid in dioxane-
dicholoromethane medium exists in unionized form and acts as n -acceptor in
a similar marmer to quinines [32]. Some hydrochloride salts of amines do not
possess a lone pair of electrons and hence, unable to react with a or 71-
acceptors. Similarly, amiodarone hydrochloride does not react with p-
chloranilic acid owing to non-availability of n-electrons. In order to establish
the assay procedure for amiodarone hydrochloride, it is necessary to convert
amiodarone hydrochloride solution into amiodarone base solution. Due to the
same reason, amiodarone hydrochloride was dissolved in dichloromethane
and shaken with a 0.5 M aqueous sodium carbonate solution, resulting in the
formation of amiodarone base in the dichloromethane layer. The addition of
p-chloranilic acid to amiodarone base (n-donor) results in the formation of a
charge transfer complex of the n -T: type. This compound is believed to be an
intermediate molecular association complex, which dissociates in dioxane-
dichloromethane solvent, producing p-chloranilic acid radical anion. The
mole ratio method [33] has been applied to establish the stoichiometric ratio
of amiodarone base to p-chloranilic acid and was found to be 1:1 (Fig. 4.2.).
This indicated the presence of one n-donating center in the amiodarone base
for charge transfer complexation reaction. On the basis of literature
background search and our experimental findings, a reaction mechanism is
proposed (Scheme 4.1.).
DDQ is TT-acceptor, which reacts instantaneously with basic
nitrogenous compounds to form charge transfer complexes of the n-7r type.
132
350 400 450 500 550
Wavelength (nm)
600 650 700
Fig. 4.1. Absorption spectra of (a) 2.30 X 10' M p-ciiloranilic acid (b) 0.36 |ig ml'* amiodarone with 1.15 X 10" M p-chloranilic acid in dioxan- dichloromethane.
133
0.25
0.20 -
0.15 0) u c n o tn
< 0.10
0.05
0.00 0.00 0.25 0.50 0.75 1.00 1.25 1.50
Molar ratio of p-chloranilic acid : drug
1.75 2.00
Fig. 4.2. Molar ratio plot for amiodarone - p - chloranilic acid complex; amiodarone concentration, 2.933 X 10" M.
134
^ ^ ^ ^
O CH2(CH2)2CH3 I
amiodarone
C2H5
0—CH2--CH2—N: +
C2H5
n-n complexation
HO
CI
CI
OH
p-chloranillc acid
<^^^
C2H5
0—CH2—CH2--N I
O CH2(CH2)2CH3 ^1
n - 71 complex
r . ^ ^ ^
in dichloromethane • dioxan solvent
C2H5
0 ^CH2(CH2)2CH3 I
O—CH2—CH2—Nt +
C2H5
Scheme 4.1.
p - chloranilic acid radical anion (coloured species)
135
The absorption spectrum of DDQ in acetonitrile shows a characteristic band
peaking at 350 nm. The addition of amiodarone base to this solution causes an
immediate change in the absorption spectrum with new characteristics bands
peaking at 435, 540 and 570 nm (Fig 4.3.)- These bands may be attributed to
the formation of DDQ radical anions, which probably resulted through the
dissociation of the donor-acceptor complex in a highly polar solvent like
acetonitrile. In order to avoid the maximum interference from the reagent
blank, the absorbance measurements were made at 570 nm. The mole ratio
method (Fig. 4.4.) suggested a donor to acceptor ratio of 1:1, confirming the
presence of one n-donating center in the amiodarone base molecule. The
reaction sequence for the formation of DDQ radical anion is shown in Scheme
4.2.
The molar absorptivities and association constants for amiodarone-p-
chloranilic acid and amiodarone - DDQ reaction products were calculated using
Ross and Labes equation [34].
[A][D] 1 _ j _ 1 ^ 1
[A] + [D] ^ A;,^° KS;, [A] + [D] Ej,
where [A] and [D] are the molar concentrations of the acceptor and donor,
respectively, A / ' ^ and sx are the absorbance and molar absorptivity of the
complex at the specified max- K is the association constant of the complex.
The values of association constant (K) and molar absorptivity (ex) were
obtained from the plot of 1 against [A] [D] i
136
n 1 1 1 I T — ^ — I 1 r
300 340 380 420 460 500 540 580 620 660 700
Wavelenth (nm)
Fig. 4.3. Absorption spectra of (a) 8.81 X10"* M DDQ in acetonitrile (b) 65 xg ral"^ amiodarone with 1.06 X 10" M DDQ in acetonitrile.
137
2.00
0.50 0.75 1.00 1.25
Molar ratio of DDQ : drug
1.50 1.75 2.00
Fig. 4.4. Molar ratio plot for amiodarone -DDQ complex; amiodarone concentration, 2.933 X 10" M,
138
. ^ ^ ^ ^
^O ^CH2(CH2)2CH3
amiodarone
N =
N= '
n-n complexation
CoH 2rl5 N=
0 CH2(CH2)2CH3 ^1
O—CH2—CHz—N:-
2rl5 H='
CI
CI
n-7t complex
in acetonitrite solvent
C2H5
O ^CH2(CH2)2CH3^I
O—CH2—CH,—Nt + 1 C2H5
N =
N =
DDQ radical anion (coloured species)
Scheme 4.2.
139
(Figs. 4.5. and 4.6.). The free energy was also computed. The results are given
in Table 4.1.
Optimization of variables '
The spectrophotometric properties of the coloured species formed with
p-chloranilic acid and DDQ as well as the different parameters affecting the
colour development were extensively studied. The optimum conditions for the
assay procedures (method A and B) have been established by studying the
reactions as a function of the concentration of reagent, nature of the solvent
and the stability of the coloured species.
Effect of the concentration of p-chloranilic acid
For method A, the effect of the volume of 0.08% p-chloranilic acid was
studied over the range of 0.2 -1,5 ml in a solution containing 360 [a,g ml"'
amiodarone. The results (Fig. 4.7) revealed the fact that 1.0 ml of p-
chloranilic acid was required to achieve the maximum intensity of the colour.
Therefore, 1.5 ml was used as an optimum value and maintained throughout
the experiment. The reaction gets stabilized within 2.0 min of mixing at room
temperature and absorbance remained constant for a further 90 min.
Effect of the concentration of DDQ
For method B, the only effective variable is the concentration of DDQ.
To study the influence of the volume of 0.08% DDQ, an aliquot of drug
containing 300 |ag was pipetted into a series of 5.0 ml standard volumetric
140
1 0 ' ^ X 1 .1
1.7 1.8 1.9 2.0 2.1 2.2 2.3 xlO^
lA ] + [Z? ]
Fig. 4.5. Plot of ^ _ ^ _ ^ versus r .-, r J-. x — ^ r for 1:1 complex
of amiodarone - p- chloranilic acid.
AD
141
10"'^x3.9
2.3 x10^
[A]+[D]
Fig. 4.6. Plot of
amiodarone -DDQ.
[A][D] 1 versus , • . • " • , , x
[ ] + [D] , AD for 1:1 complex of
142
Table 4.1. Association constant, K; molar absorptivity, ej. from the Ross and Labes plots for the complex and calculated free energy, AG
Parameter Amiodarone -p-chloranilic acid Amiodarone - DDQ complex complex
3.855 X 10
7.502 X lO'
-15.251
K(lmor')
E X (1 mol'' cm"')
AG (KJ mor')
1.107X10^
1.421 X 10
-17. 953
143
0.2 0.4 0.6 0.8
Volume of p-chloranillic acid (ml)
Fig. 4.7. Influence of the volume of 0.08% p-chloranilic acid.
144
flasks followed by varying volumes of 0.08% DDQ (0.1-1.5 ml) and diluted
to volume with acetonitrile. Fig. 4.8 shows that the highest absorbance was
obtained with 1.0 ml of 0.08% DDQ. Further addition of DDQ caused no
change in the absorbance and hence, 1.5 ml was selected as an optimum
volume for all determinations. The intensity of the colour formed on mixing
the reagent reached maximum within 3 min. and was stable up to 35 min. at
room temperature. Therefore, it is recommended that the absorbance be
measured within this time period.
Analytical data
Table 4.2. summarizes the analytical parameters such as Beer's law
limit, calibration equations obtained by least square treatment of calibration
data, coefficient of correlation, variance, confidence limit for slope and
intercept . The high value of correlation coefficient suggested the excellent
linearity of both the calibration graphs (Figs 4.9. and 4.10.). The values of
confidence limit at 95% confidence level for slope and intercept of the
regression lines pointed towards high reproducibility of the proposed
methods. Test of significance of the intercept of the regression lines showed
that the experimental intercepts, a, for method A and B do not differ
significantly from the theoretical value of zero. For this justification, a
simplified method was used to calculate the quantity from the relation t = a/Sa,
[35] and its comparison with the tabulated data from the t-distribution. The t-
value for method A and B were found to be 0.056 and 0.596, respectively,
which did not exceed the tabulated t- value ( t = 2.365, when v = 7 ) at 95%)
145
o o c a
£1 o in Si
<
Volume of DDQ (ml)
Fig. 4.8. Influence of the volume of 0.08% DDQ.
146
Table 4.2. Analytical characteristics of the proposed methods
Parameters Method A Method B
> ax (nm)
Beer's law limit ((xg ml'')
Molar absorptivity (I mol'' cm"')
Regression equation*
s\ ±ts%
s\ ±ts\ Correlation coefficient (r)
Variance (So'')
Detection limit (ng ml'')
535
10-360
1.421 X 10
A = 3.515X
6.260X10'^
1.480X10'^
2.862 X 10"*
6.769X10-*
09999
1.048X10'*
1.091
A = 3.515 X 10' + 2.076 X 10''C A = 2.734 X 10' + 1.100 X 10' C
570
1-65
7.502 X 10
A = 2.734 X
4.587X10"^
1.085 X 10"'
1.191 X 10"
2.816X10"^
0.9999
6.408 X lO"''
0.161
'With respect to A = a+bC, where a = intercept, b=slope and C= concentration. Standard deviation of the intercept. 'Confidence interval of the intercept. ''standard deviation of the slope. 'Confidence interval of the slope.
147
<u o c ra
o U)
S3 <
100 200 -1
Concentration (fig ml )
300 400
Fig. 4.9. Calibration curve for determination of amiodarone (method A).
148
« o c ni o in n <
-1 Concentration {p.g ml )
Fig. 4.10. Calibration curve for determination of amiodarone (method B).
149
confidence level. It confirmed that intercepts for the proposed methods are not
significantly different from zero. Thus the present methods are fi-ee from
constant errors independent of the concentration of the amiodarone.
The detection limit [36] for method A and B were calculated and found
to be 1.091 and 0.161 ^g ml'', respectively. The small value of the variance
(1.048 X 10" and 6.408 x 10"'' |ig ml'' for method A and B, respectively)
confirmed the small degree of scatter of experimental data points around the
line of regression. Both the detection limit and the slope of the calibration
graphs indicated the good sensitivity.
The error (Sc) in the determination of a given concentration of
amiodarone was calculated by using the following expression [37].
1/2
b
r + _!_+ (A-A) 1
The value of Sc may be used to .establish the confidence limit at the selected
level of significance for the determination of unknown concentrations by
using the relation C, ± tSc. The results are shown in Figs. 4.11. and 4.12. by
plotting percentage uncertainty versus the concentration of amiodarone [38] at
95% confidence level. Hence the confidence limits established for both
methods (A and B) can be used to evaluate the relative uncertainty directly on
the concentration over the full range of the concentration tested.
The reproducibility of the proposed methods were checked by
estimating three different concentration levels within the Beer's law limit in
150
§
120 180 240
Concentration of amiodarone (ng ml" )
360
Fig. 4.11. Plot of the variation of the confidence limit at 95% confidence level for method A, presented as uncertainty percentage on the concentration of amiodarone.
151
o c3
Concentartion of amiodarone (jig ml )
Fig. 4.12. Plot of the variation of the confidence limit at 95% confidence level for method B, presented as uncertainty percentage on the concentration of amiodarone.
152
six replicates. Tlie analytical results obtained from this investigation are
summarized in Table 4.3. The standard deviation, relative standard deviation
and standard analytical error [39] can be considered to be very satisfactory.
The validity of the proposed methods for the determination of drug in
commercial dosage forms was tested by applying standard addition technique.
In this study a known amount of pure amiodarone was added to preanalyzed
commercial dosage forms. The recoveries were obtained in the range of 99.98
- 100.12%. The results are summarized in Table 4.4. The common excipients
present in formulations did not interfere.
The applicability of the proposed methods for the determination of
amiodarone in commercial dosage forms were examined by analyzing
marketed products such as Aldarone (Zydus Alidac), Amiodar (Cardicare),
Duron (Samarth), Eurythmic (Troikaa). The results of the proposed methods
were statistically compared with those obtained by the reference method [21]
and are summarized in Table 4.5. It is evident from the table that the
calculated t and F values [40] are less than the theoretical ones at 95%
confidence level, indicating no significant difference between the methods
compared.
153
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Chem. 14 (2002) 217.
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[22] M. Y. Ebeid, N. M. EI-Kousy, B. A. Mousa, N.G. Mohamed. Egypt. J. Pharm.
Sci. 39 (1998) 31.
[23] T. S. Rao, P. S. N ,H. Ramachandra Rao, A.V.S.S. Prasad, C.S.P. Sastry, Oriental
J. Chem. 17(2001)407.
[24] N. Rahman, S. N. H. Azmi, Anal. Sci. 16 (2000) 1353.
[25] N. A. Zakhari, M. Rizk, F. Ibrahim, M. I. Walsh, Talanta 33(1986) 111.
[26] M. Abdel-Hady Elsayed, S. P. Agarwal, Talanta 29 (1982) 535.
[27] E. A. Taha, S. M. Soliman, H. E. Abdellateef, M. M. Ayad, Microchim. Acta 10
(2002) 175.
[28] H. F. Askal, Talanta 44 (1997) 1749.
[29] L. I. Bebawy, N. El-Kousy, J. K. Suddik, M. Shokry, J. Pharm. Biomed. Anal. 21
(1999)133.
[30] P. Y. Khashaba, S. R. El-Shabouri, K. M. Emara, A. M. Mohamed, J. Pharm.
Biomed. Anal. 22 (2000) 363.
[31] N. Rahman, M. N. Hoda, J. Pharm. Biomed. Anal. 31 (2003) 381.
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(1969) 1479.
158
[33] D. T. Sawyer, W. R. Heinemann, J. M. Beebe, in "Chemistry Experimwents for
Instrumental Metliods", John Wiley and Sons, USA, 1984, p.200.
[34] S. D. Ross, M. M. Labes, J. Am. Chem. Soc. 79 (1957) 76.
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[36] B. Morelli, Analyst 108 (1983) 870.
[37] J. N. Miller, Analyst 116 (1991) 3.
[38] R. Cassidy, M. Janoski, LC-GC 19 (1992) 692.
[39] Vogel's Textbook of Quantitative Chemical Analysis, Sixth edition, Pearson
Education, Singapore, 2002, p. 125.
[40] G. D. Christian, Analytical Chemistry, Fourth edition, John Wiley & Sons, Inc.,
Singapore, 1994, p.35.
CHapter -5 Quantitative SpectropHotometric MetHocCsfor
determination of^erapamidKycfrocHlbncfe in (Drug formulations ZJsing (Potassium Metaperiodate and
TropaeoCin 000
159
INTRODUCTION
Verapamil hydrochloride i.e. (±)-5-[(3,4-dimethoxyphenethyl) methyl
amino]-2-(3,4-dimethoxyphenyl)-2-isopropyI valeronitrile monohydrochloride
is clinically a very useful member of the calcium channel blocker. It is used
in the treatment of supraventricular arrhythmias, angina and hypertension
[1-3].
The drug and its formulations are officially listed in BP [4], which
suggests potentiometric titration method, USP [5] recommends a gas
chromatographic method and IP [6] describes a non-aqueous titration method
for its assay in bulk and dosage forms. The different analytical methods that
are reported for its determination include high performance liquid
chromatography [7-23], high performance thin layer chromatography [24],
liquid chromatography [25,26], gas-liquid chromatography [27,28], capilllary
gas chromatography [29,30], potentiometry [31], potentiometry-
conductometry [32], stripping voltammetry [33], atomic emission
spectrometry [34], mass spectrometry [35] and UV spectrophotometry [36].
Although the above methods have adequate sensitivity to assay lower
concentrations of the drug and hence use of these methods are justified when
the concentration of drug in biological fluids is low. Moreover, these methods
are not easily manageable because of high cost technique. The dru<
concentrations (40-240 mg per tablet) are high enough in pharmaceutical
160
formulations. Therefore, the need for spectrophotometric method is fully
justified.
A review of literature revealed that few spectrophotometric methods
for the assay of verapamil hydrochloride based on charge transfer complexes
with 2,3,5,6,-tetrachloro-p-benzoquinone [37], 2,3-dichloro-5,6-dicyano p-
benzoquinone and 2,5-dichloro-3,6-dihydroxy-p-benzoquinone [38],
chromotrope 2B and chromotrope 2R azodyes [39]; extractable ion-pair
complexes with bromothymol blue, bromophenol blue, bromocresol green,
bromocresol purple and methyl orange [40], solochrome black-T, solochrome
dark blue, fast sulfone FF and solochrome cyanin R [41], erioglaucine and
indigo carmine [42], alizarin red-S [43], picric acid [44] and oxidation of drug
with chloramine T in HCl medium [45].
This chapter describes two simple, sensitive, fast and economical
spectrophotometric methods for the assay of verapamil hydrochloride in pure
and dosage forms. The first method is based on the oxidation of drug with
potassium metaperiodate in sulphuric acid medium. The second method
utilizes the formation of an ion-pair complex of the drug with tropaeolin 000
and subsequent extraction of the yellow colour into chloroform under reaction
conditions used.
E X P E R I M E N T A L
Apparatus
A Milton Roy Spectronic 200"" spectrophotometer (USA) and an Elico
model Ll-10 pH meter were used for absorbance and pH measurements,
161
respectively. A water bath shaker (NSW 133, India) was used to control the
heating temperature for colour development.
Reagents and Standards
All chemicals and reagents used were of analytical grade or
pharmaceutical grade. All solutions were prepared in doubly distilled water.
Aqueous solutions of potassium metaperiodate (0.2%; Otto chemie, India),
tropaeolin 000 (0.05%, Fluka), and 10 M sulphuric acid (Merck, India) were
prepared.
A buffer solution of pH 4 was prepared by mixing 50 ml of 1.0 M
sodium acetate solution with 39.5 ml of 1.0 M HCl solution and diluted to 250
ml with doubly distilled water [46]. The pH of the solution was adjusted to an
appropriate value with the aid of a pH meter.
Verapamil hydrochloride was kindly gifted by Samarth Pharma Pvt.
Limited, India. The standard solutions of verapamil hydrochloride (2.5 mg
ml' and 0.4 mg ml"') were prepared in doubly distilled water.
Recommended Procedures
Method A
Into a series of boiling test tubes different concentrations of verapamil
hydrochloride (0.00 - 0.75 ml; 0.25%) solution were pipetted. To each test
tube 1.2 ml of potassium metaperiodate and 5 ml of 10 M H2SO4 were added,
mixed well and placed on a water bath maintained at 100±1°C for 18 min. The
solutions were cooled to room temperature and transferred to a 10 ml standard
volumetric flask. The contents were diluted to volume with doubly distilled
162
water. The absorbance was measured at 425 nm within the stability period of
2 hr. against the reagent blank treated similarly.
Method B
Into a series of 50 ml separating funnels different volumes of 0.04%
drug solution containing up to 300 \ig were pipetted. 5 ml of buffer solution
of pH 4 and 2 ml of tropaeolin 000 were added to each separating funnel and
mixed well. The funnels were shaken with 10 ml chloroform for 2 min for
complete extraction and then allowed to separate the two layers. The
absorbance of the chloroform layer was measured at 400 nm against a reagent
blank treated similarly. The colour is stable for at least 3 hr.
Analysis of Pharmaceutical Formulations
Twenty tablets were powdered and mixed thoroughly. An amount
equivalent to 250 mg of verapamil hydrochloride was weighed accurately. It
was stirred with chloroform and filtered through Whatmann No. 42 filter
paper. The residue was washed well with chloroform for complete recovery of
the drug. The chloroform was evaporated to dryness and the left drug was
dissolved in doubly distilled water and diluted to 100 ml with doubly distilled
water. It was further diluted according to the need and then analyzed
following the recommended procedures.
163
RESULTS AND DISCUSSION
Potassium metaperiodate is a well known oxidant and has been used for
the oxidation of p-blockers [47-48] containing amino group. The oxidation
products were arylaldhyde or aryloxyacetaldehyde, and secondary or tertiary
butylamine [47]. In all such studies, the presence of a methyl or methylene
group attached to nitrogen is the basic need [49] so that >N—CH2 linkage in
tertiary amines was cleaved preferentially by an oxidant (potassium
metaperiodate or chloramine T or N-bromosuccinimide) resulting in the
formation of yellow coloured product. The oxidation of verapamil
hydrochloride with potassium metaperiodate was carried out in aqueous
sulphuric acid medium as it fulfills the basic need of having tertiary amine
with >N-CH2 linkage. Therefore, >N-CH2 linkage was cleaved preferentially
resulting in the formation of aryloxyacetaldehyde and secondary amine. The
oxidation product absorbs maximally at 425 nm (Fig. 5.1.). In order to
establish stoichiometric ratio between verapamil hydrochloride and potassium
metaperiodate, the method of continuous variations [50] has been applied and
was found to be 1:1 (Fig. 5.2.). On isolation of the two major products, one
produces a blue colour when treated with a benzene solution of chloranil
confirming the presence of amino group and other product reacts with 2,4,-
dinitrophenyl hydrazine to give yellow coloured product indicating the
presence of aryloxy benzene acetaldehyde. It has been confirmed that iodine
was not evolved during this reaction.
164
0.50
400 450
Wavelength (nm)
550
Fig. 5.1. Absorption spectrum of the reaction product of Verapamil hydrochloride with potassium metaperiodate.
165
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Mole fraction of drug (XQ)
Fig. 5.2. Job's plot for the reaction of verapamil hydrochloride with potassium metaperiodate (each 5. 09 x 10" M).
166
Therefore, based on the literature background and our experhiiental
findings, the reaction mechanism was proposed and given in Scheme 5.1.
Verapamil forms coloured chloroform extractable ion-pair complex in
acidic buffer with tropaeolin 000. The absorption spectrum of the ion-pair
complex, which absorbed maximally at 400 nm, is shown in Fig. 5.3. The
reagent blank under similar conditions showed no absorption. The molar ratio
between verapamil and tropaeolin 000 is established by Job's method [51].
This measurement (Fig. 5.4.) showed that 1:1 complex was formed between
drug and tropaeolin 000.
Verapamil hydrochloride contains tertiary amino group which is
protonated in acid buffer, while sulphonic acid group in tropaeolin 000 is the
only group undergoing dissociation in the pH range of 0.5 - 6.9. Finally the
protonated verapamil hydrochloride forms ion-pair with tropaeolin 000, which
is quantitatively extracted into chloroform. The possible reaction mechanism
is proposed and given in Scheme 5.2.
Optimization of Variables
The optimum conditions for the assay of verapamil hydrochloride were
established via a number of preliminary experiments.
Method A
Effect of heating time
To investigate the optimum heating time for color development, 0.75
ml of 0.25% verapamil hydrochloride was mixed with 5 ml of 10 M
167
CHj H3Q ,CH3
'CH
CH2—CH2-N-CH2—CH2-CH2—C—CN
H2SO4 KIO4
OCH3
OCH3
O CH
C H 3 O — / \ - C H 2 - C - H + C H 3 - N - C H 2 - C H 2 - C H 2 — C - C N
H3CO
OCH3
OCH3
3,4-dimethoxy benzene acetaldehyde
5 (N-methyl amino) 2-(3,4-dimethoxy phenyl)-2 isopropyl valeronitile
Scheme 5.1
168
300 350 400 450
Wavelength (nm)
500 550
Fig. 5.3. Absorption spectrum of the reaction product of Verapamil hydrochloride with tropaeolin 000.
169
Mole fraction of drug (XQ)
Fig. 5.4. Job's plot for the reaction of verapamil liydrochloride with Tropaeolin 000 (each 1.02 x 10'^ M).
170
CHj
HjCO'
0 - / / ^
CH3 H3C ^CHj
CH
CH2—CH2—N- CH2—CH2-CH2— C—CN
protonated verapamil "V^ ^OCHs
OCH3
'O3S—C \ - N = N OH
tropaeolin 000
O - / ^ CH3
HjCO'
H3C ^CH3 CH3 CH I I
CH2— CH2—N— CH2—CH2— CH2—C—CN H
O3S—f V-N=N OH
Verapamil -tropaeolin 000 complex
Scheme 5.2
171
H2SO4 and 1.2 ml of 0.2% potassium metaperiodate into a boiling test tube.
The content of the mixture was heated for 20 min. on a water bath at
100±1°C. The intensity of the color (Fig. 5.5.) was reached to maximum after
17 min of heating and remained constant and therefore 20 min of heating time
was used throughout the experiment.
Effect of potassium metaperiodate
To 0.75 ml of 0.25% verapamil hydrochloride, different volumes (0.5 -
1.5 ml) of 0.2% potassium metaperiodate and 5 ml of 10 M H2SO4 were
added. The reaction mixtures were heated for 20 min on a water bath at
100+1°C. The reaction mixture was diluted to 10 ml with doubly distilled
water and the absorbance was measured against a reagent blank at 425 nm.
The results (Fig. 5.6) showed that the highest absorbance was obtained with
1.0 ml, which remained unchanged with higher amount of potassium
metaperiodate. Thus, 1.5 ml of the reagent was added for colour
measurements.
Effect of the concentration of sulphuric acid
The influence of the yolume of 10 M H2SO4 on the formation of yellow
colour was. studied. This was performed by pipetting 0.75 mi of verapamil
hydrochloride (0.25%), 1.5 ml of potassium metaperiodate and different
volumes (1-5 ml) of 10 M H2SO4. It is evident from Fig. 5.7. that the
maximum absorbance was obtained with 4.7 ml of 10 M H2SO4 above this
172
0) o c
o V)
Si
<
Time (min)
Fig. 5.5. Effect of Heating time.
173
0.7
0.6 -I
0.5
• - • - • •
-r
2
Volume (ml)
Fig. 5.6. Effect of the volume of 0.2% potassium metaperiodate.
174
0) u c (0
J3
o m n
Volume (ml)
Fig. 5.7. Effect of the volume of 10 mol I" H2SO4.
175
volume, the absorbance remains constant. Therefore, 5.0 ml of 10 M H2SO4
was used for all the measurements.
Method B
Effect of the concentration of tropaeolin 000
The effect of the volume of 0.05% tropaeolin 000 was studied in a
series of 50-ml separating funnels containing 30 |J.g ml"' of verapamil
hydrochloride, 5.0 ml of buffer solution of pH 4 and different volumes of the
reagent. 10 ml of chloroform was used to extract the ion-pair complex. It is
apparent from Fig. 5.8. that the maximum absorbance was found with 1.9 ml
of tropaeolin 000, beyond which absorbance was constant. Thus, 2.5 ml of
tropaeolin 000 was used for ion-pair formation throughout the experiment.
Effect of the pH
The influence of pH of the buffer solution of sodium acetate HCl on
the formation of extractable ion-pair complex of the drug with tropaeolin 000
was studied. The results are shown in Fig. 5.9. It is evident form the figure
that the absorbance of the complex with tropaeolin 000 was found to be
constant within the pH range 3.5 - 4.5. Thus, all the absorbance measurements
were made at pH 4.
Choice of organic solvent and time of shaking
A variety of organic solvents such as benzene, toluene, hexane,
chloroform, carbon tetrachloride, ethyl acetate and 1,2-dichloromethane were
176
\J.I -
0.6 -
0.5-
g 0.4 -c cs .o o (A
JO
< 0.3 -
0.2-
0.1 -
0.0 i
/
/
^ V
Volume (ml)
Fig. 5.8. Effect of the volume of 0.05% tropaeolin 000.
178
examined for an applicable extraction procedure, but chloroform was
preferred for its selective extraction of the ion-pair complex from the aqueous
solution.
The time of shaking for complete extraction of ion-pair complex was
studied and found that the absorbance of the extract remains constant between
0.5 - 4.0 min. Thus, 2.0 min shaking time was utilized as an optimum value
throughout the experiment. The ion-pair complex was quantitatively
recovered in one extraction only.
Interference studies
The effect of excipients and additives commonly used in drug
formulations, such as starch, lactose, talc, stearic acid, magnesium stearate,
titanium dioxide, tartrazine and lactic acid was evaluated using the proposed
methods. It was found that the common excipients and additives did not
interfere with the analysis.
Analytical data
Under the optimized experimental conditions, linear calibration graphs
(Figs. 5.10. and 5.11.) were obtained over the concentration ranges 0.00 -
187.5 and 2-30 ]xg ml"' of verapamil hydrochloride with molar absorptivities
of 1.18 X 10^ and 9.82 x 10^ 1 mof'cm"' using method A and B, respectively.
Table 5.1. summarizes the analytical parameters such as Beer's law limit,
179
o u c ns Si o (0 £1 <
.1 Concentration (ng ml )
Fig. 5.10. Calibration curve for determination of verapamil hydrochloride (method A).
180
Concentration (ngml )
Fig. 5.11. Calibration curve for determination of verapamil hydrochloride (method B).
181
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182
calibration equations obtained by least square treatment of calibration data,
coefficient of correlation, variance and confidence limit for slope and
intercept. The correlation coefficient was found to be 0.9999 and 0.9998 for
method A and B, respectively, indicating the good linearity of both the
calibration graphs and the intercepts are all close to zero. Test of significance
of the intercept of the regression line showed that the experimental intercept,
a, does not differ significantly from the theoretical value, zero. For this, a
simplified method was used to calculate the quantity from the relation t = a/Sa
[52] and its comparison with the tabulated data from the t-distribution. The t-
value for method A and B are 0.200 and 0.190, respectively which do not
exceed the tabulated t-value (t = 2.365, when v = 7) at 95% confidence level.
It confirmed that intercepts for the proposed methods are not significantly
different from zero. Thus the present methods are free from constant errors
independent of the concentration of the verapamil hydrochloride.
The detection limit [53] and variance were found to be 0.772 |a,g ml"',
7.01 X 10"' |ig ml"' and 0.396 ^g ml"', 1.28 x 10" i g ml"' for method A and B,
respectively. The small value of variance obtained for both the methods
pointed towards negligible scattering of experimental data points around the
line of best fit.
The statistical analysis of the calibration data has been used to
calculate error (Sc) in the determination of a given concentration of verapamil
hydrochloride and also provides a way to establish the confidence limits at the
selected levels of confidence to estimate the relative uncertainty of the
183
concentration over the full range of the concentration tested. These
parameters were established by using the relation [54],
^'~ b 1 1 {A-Af 1 + - + - ^
» b'Yic-cf
1/2
Where A and C are average values of the absorbance and concentration,
respectively, for n standard samples. Figs. 5.12. and 5.13. shows the graphs of
Sc versus concentration of verapamil hydrochloride. The minimum error is
obtained on the determination of about 100 and 16 [ig ml' of verapamil
hydrochloride for method A and method B, respectively.
The accuracy and precision of the proposed methods were evaluated by
the replicate analyses at the three different concentration levels. The results
are summarized in Table 5.2. The relative standard deviation and standard
analytical error were found to be 0.130-1.038% and 0.018-0.102 fag mV\
respectively. The results, therefore, indicated that the precision and accuracy
of the proposed methods were satisfactory.
To check the validity of the proposed methods using standard addition
technique, a fixed amount of pure drug was added to the preanalysed drug
formulations. The results are summarized in Table 5.3. The recovery results
were quite satisfactory.
The applicability of the proposed methods were compared with the
reference method [43] by analyzing authentic commercial products of
verapamil hydrochloride such as Calaptin 80 so (Nicholas Piramal), Verap 80
184
0.42
200 .1
Concentration of verapamil hydrochloride (pg ml )
Fig. 5.12. Error in the determination of verapamil hydrochloride using statistical analysis of standard calibration data with potassium metaperiodate.
185
0.210
0.205
0.200
I
1^
A
1^
6 "1 i i 1 i I i I I I r
10 12 14 16 18 20 22 24 26 28 30 32
Concentration of verapamil hydrochloride ( g ml )
Fig. 5.13. Error in the determination of verapamil Iiydrocliloride using statistical analysis of standard calibration data with tropaeolin 000.
185
u
UJ
0.210
0.205 -
0.200
0.195
0.190
0.185 T i 1 1 1 1 1 1 1 T
12 14 16 18 20 22 24 26 28 30 32
Concentration of verapamil hydrochloride (^g ml" )
Fig. 5.13. Error in the determination of verapamil liydrochloride using statistical analysis of standard calibration data with tropaeolin 000.
186
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s
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187
188
(Mano Pharma) and VPL Amp. 05/2 ml (Samarth Pharma); the results are
summarized in Table 5.4. The results of the proposed methods were statistically
compared with those obtained by the reference method. It is evident from Table
5.4. that the calculated t- and F-values are less than the theoretical ones [55] at
the 95% confidence level, which indicates no significant difference between the
methods compared.
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190
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ORIGINAL ARTICLES
Analytical Chemistty Division, Department of Chemisti-y, Aligarh Muslim University, Aligarh (UP.) India
Kinetic spectropliotometric method for tlie determination of silymarin in pliarmaceutical formulations using potassium permanganate as oxidant
N. RAHMAN, N. A. KHAN, S. N. H. AZMI
Received July 02, 2003, accepted My 22, 2003
Dr. Nafisur Rahman, Analytical Chemistry Division, Department of Chemistry, Aligarh Muslim University, Aligarh - 202002(U.P.) India chtl 7nr@yahoo. co. in
Pharmazie 59: I l l - I l l (2004)
Ug^-
A new simple and sensitive kinetic spectrophotometric method for the detemiinatlon of silymarin in pure form and in pharmaceutical fomnulations Is described. The method Is based on the oxidation of the drug with potassium permanganate at pH 7.0 db 0.2. The reaction is followed spectrophotometri-cally by measuring the decrease in the absorbance at 530 nm. The calibration graph Is linear in the 7ange of 1 8 - 5 ( ^ ml"''. The method has been successfully applied to the determination of silymarin In pharmaceutical formulations. Statistical comparison of the results with the reference method shows excellent agreement and Indicates no significant difference in accuracy and precision.
1. Introduction
Silymarin 3,5,7-trihydroxy-2-[3-(4-hydroxy-3-niethoxy phenyI)-2-(hydroxy-methyI)-l,4-benzodioxan-6-yl]-4-chro-manone, is an important antihepatotoxic drug whicli is obtained from tlie dried fruits of Silybum marianum and widely used for tlic treatment of hepatic disorders (Cava-lieri 1974; Salmi and Sama 1982; Ferenci etaL 1989; Hruby 1983). The drug is officially listed in Martindale The Extra Pharmacopoeia (Martindale 1996). The assay procedure listed in the monograph of the Italian pharmacopoeia describes the UV-\^s spectrophotometric method (Farmacopoeia 1985) for deteimination of the drug . in bulk and pharmaceutical formulations. Several other methods have been employed to determine silymarin in biological specimens and/or pharmaceutical formulations such as high performance liquid chromatography (Martinelli etal. 1991; Marcher and Kikuta 1993; Rickling etal. 1995; Tittel and Wagner 1977; Tittel and Wagner 1978; Quercia etal. 1980; Quercia etal. 1983; Steinigen 1983; Law and Das 1987; Tomov etal. 1985; Pashankov 1988; Nikolov 1990; Kurkin 1996; Wang et al. 1998), thin layer chromatography (Lin 1982; Abdel-Salam 1982; Vanhaelen and Van-haelen-Fastre 1983;Heinz etal. 1985; Wu and Wang 1989; Lin etal. 1998; Szilarjtes-Obeiglo and Kowalesioska 1984), high performance thin layer chromatography (Funk et al. 1987), potentiomeuic tiu-ation (Koerbl et al. 1983), diffuse-reflectance fourier transform infraied specU'oscopy (Martinez et al. 1991) and UV spectrophotometry (Lan et al- 1983^ Belikov 1985). In the literature only few spectrophotometric methods have been reported. The drug content in pharmaceutical formulations was detennined (Zarapkar et al. 2000), based on the formation of a coloured complex of the drug with 2,4-dinitrophenyl hydrazine, in the presence of tetramethyl amincjJiydroxid-\Qualitativc' detemiinations aie done at 490 nm. Another'spectrophotometric method for the csti-
mation of silymarii^based on its reaction with diazotized sulfanilic acid in alkaline medium to form an orange-red coloured chromogen which absorbed maximally at 460 nm (Rajasekaran 1997). Reddy etal. (2001) have reported two spectrophotometric methods for silymarin determination. The first method was based on the oxidation of the drug by Fe(III) and estimating the reduced Fe(II) with 1,10-phenanthroline at 510 nm. The other method involved the formation of a blue complex on treatment with Folin-Cio-calteau reagent in the presence of NaOH and subsequent determination was done at 740 nm. These methods are somewhat tedious and time consuming. Therefore, we develop^ed a fast, low cost and selective method for routine analysis of silymarin in bulk and drug formulations. The extent of oxidation of organic substrate by potassium permanganate depends on the pH of the rnedium. TTie heptavalent manganese changes to Mri(lV).)^n~"aIkaline medium while in neutral and acidic mediurn^the permaii-ganate is further reduced forming ultimately Mn(II). This behaviour of permanganate has been exploited to develop a kinetic spectrophotometric method for assay of nifedipine in drug formulations (Rahman and Azmi 1999). The present communication describes a method for the assay of silymarin in bulk and pharmaceutical formulations. The method is based on the oxidation of silymarin by potassium permanganate at pH 7.0 ± 0.2 and the course of the reaction was followed by measuring a decrease in absorbance at 530 nm.
2. Investigations, results and discussion
The oxidation of organic compounds by permanganate is dependent upon the pH of the medium. During the course of the reaction the valence of manganese changes and in-tennediate ions have been suggested as participating oxidants. But which species have the main role as potential oxidants depends on the nature of the substrate and pH of
•' • o'^ . • C- r/.-'/i
Pharmazie 59 (2004) Ut fi' t
ORIGINAL ARTICLES
1.1-1
1.0-
0.9-
0.8-
0.7-CD
H 0.6-
•e 0.5-o | 0 . 4 -
0.3-
0.2-
0.1-
' 1 1 1 • 1
3 4 8 12
Time(min)
1 16
• a • b
- — - • c
• d
1 20
Rg. 1: Absorbance-time curves: KMn04, 4.8 x 10 *M and silymarin (a) 3.731 X 10-5 M; (j,) 4.146 x 10' 'M; (c) 6.219x10-'; (d) 8.292 x 10-5 M; (e) 10.365 x 10"'M
the medium. The oxidation of silymarin by potassium permanganate at pH 7.0 ± 0.2 was followed spectrophotome-trically at 530 nm. The absorbance-time curves are shown in Figs. 1 and 2, which are all sigmoid in nature throughout the entire range and therefore, the initial rates of reaction were determined from the slope of the initial tangent to the absorbance-time curves. The order with respect to permanganate was determined by studying the reaction at different initial concentrations of permanganate with fixed silymarin concentration. Keeping the silymarin concentration higher than that of the oxidant, the plot of initial rate [-d[A]/dt] against initial absori)ance was linear passing through the origin suggested that the order of reaction with respect to permanganate at the start is one. The order with respect to silymarin was evaluated from the measurement of the rates at several concentrations of silymarin but at fixed concentration of KMn04 which was also found to be one. The first order dependence on both permanganate and silymarin in the initial stages leads to the simple rate expression:
Rate = K[silymarin] [MnO^] (1)
Silymarin is a naturally occurring aryl chromanone(l) related to flavanone. The flavanones are 2,3-dihydro derivatives of flavones, which are readily interconvertible. Though the basic skeleton of flavanone and flavone is the same, the key which distinguishes one structural type from another is the oxidation level of the various carbons in the heterocyclic ring. The flavones are in a high state of oxidation whereas tlavanones are in low state of oxida-
Scheme 1
0.7-1
I
0.6-
0.5-
8 0.4-c
•e 0.3-o
^ 0.2 J
0.1-
0.0-
\
\
\ ^ \ \ , ^ • • N^X"^" • •
^ v ^ ^ " " - • • " • •
' 1 • ' 1 ' 1 •
D 4 8 12
Time (min)
— • — — • — — • — — • —
16
^a • b • c ed
1 20
Fig. 2: Absoibance-lime curves: silymarin, 10. 365 x 10 ' M and KMn04 (a) 3.00 x 10-^ M; (b) 2.10 x 10"^ M; (c) 1.50 x 10"^ M; (d) 9.00 X 10-5 M
tion. The oxidation of flavanone to flavone with a variety of reagents such as selenium dioxide (Sammel 1979) or triphenyl methyl carbonium ion (Schonberg and Schutz 1960) has been reported. In this study, the same mechanism seems to be involved. The oxidation of silymarin by permanganate takes place at pH 7.0 ± 02 leading to the formation of chromone(2). Thus, the dehydrogenation is effected by permanganate and consequently disproportio-nation between inmiediate oxidation states of manganese results in the formation of MnOa. However no precipitation of Mn02 was observed under study time period. The reaction mechanism is presented in the Scheme. Under the optimum experimental conditions, a caUbration curve was constructed by plotting logarithm of initial rate of reaction (log v) versus logarithm of silymarin concentration (log C), which showed a linear relationship over silymarin concentration range of 18-50 ng • ml"'. TTie Un-ear regression analysis using the method of least square was made to evaluate the slope, intercept and correlation coefficienL The linear regression equation and correlation coefficient are log v = 1.024 log C +3.235 and r = 0.9997 which indicates an excellent linearity. The confidence Umits for the slope of the line of regression and intercept were computed using the relation b ± tSb and a ± tSa (Miller 1991), at 95% confidence level and found to be 1.024 ± 3.96 x 10"^ and 3.235 ± 1.67 x 10"', respectively. This indicated the high reproducibility of the kinetic method proposed. Linearity was also evaluated (Torrado etal. 1994) from the relative standard deviation of the slope of the caUbration line and found to be 1.215.
OH 0
,0. ^CHjOH + Mn04
OH O OH I
+ HO-Mn—0
Chromiinone (1) Chronione (2)
Phannazie 59 (2004) I I I
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ORIGINAL ARTICLES / ' / ' , I, /,'L
1.30
128-
126-
124-
O 122-
>< 1.20-
r 118
HI 1.16-
1.14-
112
1.10
Table 1: Precision test of the proposed kinetic method
10 —r—
20 —T— 30 40 60
Concentration of silymarin (PQ -mV)
Rg. 3: Eiror in the detenninalion of the concentration of silymarin using statistical analysis of standard calibration data
The limit of detection was established using the equation (Morelli 1983, NaHmov 1963):
Limit of detection (.2 r^-lV" 7
(2)
where n is the number of standard samples, t is the value of student's t for n—2 degrees of freedom at 95% confidence level and Si = variance. The variance and detection limit were calculated to be 1.100 x 10~* ng • ml~* and 2.823 X 10"^ [ig • ml"' respectively, which confinned negligible scattering of experimental data points around the line of regression and good sensitivity of the method. The error (Sc) in the determination of a given concentration of silymarin (C) was established using the equation (MorelU 1987):
Sc .So • b n b2(2 C2 - nC')
1/2
(3)
Where y and C are average ordinate and abscissa values, respectively, for n standard samples. Fig. 3 shows the
4S-,
4 0
3 5 '
S 3 0 -
o as ^
o <
15-
lo
os-10
— I — 20 30
— 1 — 40
— I 50
Concentration of silymann (ng -mr^)
Fig 4 Variaiion of the confidence limit ai (a) p = 0 05 and (b) p = 0 01 level of significance in ihe form of uncertainly percentage on the conccniralion of iilymann
Piuuma/.ic 59 (2004) III
Sample No Imiial rate of reaciion (mol t"' mm"')
1
2
3
4
5
6
7
8
9
10
11 4x 10-2
11.5 X 10-^
11.3 X 10-2
n.3 X 10-2
11.5 X 10-2
11.5 X 10-2
11.2 X 10-2
11.4 X 10-2
11.3 X 10-2
11.2X 10-2
402
404
400
399
406
405
396
401
398
397
Mean percent recovery = 100% Relative standard dcviauon = 0 8% ' 2 ^ ;< lo
graph of Sc versus the concentration of silymarin. It is apparent from the graph that the error is reached minimum when the actual initial rate was equal to the average initial rate in the calibration, graph. The minimum error of^^^^310^|ig • m r ' was found in the determination of abouTSO ng • ml~^ silymarin. The value of Sc also allows establishing the confidence limit at the selected level of significance for the determination of unknown concentrations by using the relation, C, ± tSc. The results are shown graphically (Fig. 4) in the form of
percent uncertainty ih^ on the concentration at
95% and 99% confidence levels. This is a useful way of expressing the confidence limits because the relative uncertainty can be estimated directly on the concentration over the full range of the concentration tested. The reproducibility was established for ten independent analyses of solution containing 0.40 mg silymarin. The analytical results obtained fi-om the investigation are summarized in Table 1. The relative standard deviation and mean percent recovery were 0.87% and 100.3%, respectively and considered to be very satisfactory. The validity of the proposed kinetic method was checked by applying standard addition method and the results are reported in Table 2. The applicability of the proposed method for the determination of silymarin in dosage forms was examined by analyzing marketed products; the results were compared to those obtained by the reference method (Rajasekaran et al. 1997) and are summarized in Table 3. The performance of the proposed method was judged by calculating t- and F-values (Christian 1986). At 95% confidence level judged, the calculated t- and F-values do not exceed the theoretical values indicating no significant difference between the proposed kinetic method and the reference method.
Table 2: Determination of silymarin in pharmaceutical formulations by the standard addition method
Preparation Amount taken Amount Total amount (Hg ml- ' ) otitied - / found (lie ml'
. f/l^>,fn( I
Recovery RSD ') (%) (%)'
Silybin-70
(tablet) Silvia-70 (tablet) Limann-70 (capsule) Sivylar-70 (capsule) Levalon-70 (capsule)
10 20 10 20 10 20 10 20 10 20
10 20 10 20 )0 20 10 20 10 20
20 0 39 9 20 0 40 0 20 0 40 0 20 0 40 0 20 0 39 9
100 1 99 9
100 1 100 0 100 I 100 2 100 1 100 0 1002 99 9
0 35 0 34 0 47 0 22 0 58 0 46 0 57 0 22 0 68 0 34
Pifi-c intlt.pLnd(.n[ Jtnl\se\
/C
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ORIGINAL ARTICLES
Table 3: Determination of silymarin in dosage fonns by the kinetic method proposed and by the reference metliod
Preparation
Silybin (tablet) Silvia (tablet) Limarin (capsule) Sivylar (capsule) Levalon (capsule)
Labelled amount) (mg/tablct or capsule)
70
70
70
70
70
Proposed 1
Recovery (%)
100 0
100.1
100 0
100.1
99 9
uneuc method
SD
0 13
0 09
0.09
0.12
0 05
RSD
0 33
0.23
0 24
0 30
0.13
Reference method
Recovery (%)
99 8
100.1
99.6
100.0
100.3
SD
0 09
0.06
0.13
0 06
0.10
RSD (%)•
0 23
0 15
0 32
0 15
0.26
0.452
0 205
1 147
0 227
1268
Fcfc
2.010
2 340
1751
3 940
3.830
• Five independent analyses ' Thcorcucal value for t at 95% confidence level is 2 776 = Ihcortacal value for F at 95% confidence level is 639
In conclusion, the proposed method is fast, simple and sensitive for the determination of silymarin in bulk and commercial formulations with good accuracy and precision. The sensitivity is very high and compares favourably with that of other known methods and, can therefore (6^n}success-fully applied as an alternative to the existing nieEods>,
3. Experimental
3.1. Apparatus
The absotbance was measured on a spectronic 20 D"*" spectrophotometer (Milton Roy, USA) with 1 cm glass cells
3.2. Reagents
All chemicals used were of analytical or pharmaceutical grade Potassium permanganate, 0.003M was freshly prepared by dissolving 47.41 mg of potassium permanganate in 100 ml of doubly disalled water. The apparent purity of the potassium permanganate solution was checked titiimetrically (Vogel 2002)
3.3 Test solution
Silymarin (J mg • ml"') (Micro Labs, Bangalore, India) was dissloved in methanol.
3.4. Procedure
341. Stlymarin
Aliquots of 0 18-0 50 ml of silymarin standard test solution were pipetted info a senes of 10 ml sumtiaid volumelnc flasks To each staniiaid flask 1 6 ml of 0 003 M potassium permanganate was added and then diluted to volume with doubly distilled water at 30 ± 1 °C. After mixing, reaction mixture was immediately transfened to a spectrophotometric cell and the decrease in ab-sorbance was recorded as a funcuon of time for 20 rmn at 530 nm The initial rate of the reaction (v) at different concentrations was obtained from the slope of the tangent to the absorbance-time curve The calibration curve was constructed by plotung the loganlhm of the mitral rate of reaction (log v) versus logarthm of the concentration of the silymann (log C)
342 Silymann in pharmaceiilicalformulalions
An accurately weighed quantity of the mixed contents of five capsules or five powdered tablets, equivalent to 50 mg of the drug, was extracted into 50 ml chloroform with shaking and the residue was filtered using Whatman No 42 filter paper The filtrate was evaporated to dryness and the residue was taken up with methanol and transferred to a 50 ml standard volumemc flask, diluting to volume The assay was completed following the recommended procedure
Acknowledgements The authors are grateful to Professor Shahfiullah, Chairman, Department of Chemistry, Ahgarh Muslim University, Aligarh for providing research facilities Financial assistance provided by Council of Scientific and Indusuial Research (CSIR) New Delhi, India to Syed Ndjmul Hejaz Azmi as Research Associate is gratefully acknowledged The authors wish to express their gratitude to M/s Micro Labs Limited, Bangalore India for s.miplc of pure silj mann
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Phaima/,ie 59 (2004) III
20320S8 Die Plinnnazic An.-Nr.P.3105/2 (DISK) (p_21 pzi/pzi04_02/867S/|)7.i867Sii.3d SIGNA Bcnib.: Rcinl
ORIGINAL ARTICLES
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