ANALYSIS OF ANTIHYPERTENSIVE DRUGS IN COMMERCIAL … · Manirul Haque and Suhaib Alam for the care,...
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ANALYSIS OF ANTIHYPERTENSIVE DRUGS IN COMMERCIAL DOSAGE FORMS ABSTRACT THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF Sector of $i|tlo£(op()p IN CHEMISTRY BY HABIBUR RAHMAN DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 2006
Transcript of ANALYSIS OF ANTIHYPERTENSIVE DRUGS IN COMMERCIAL … · Manirul Haque and Suhaib Alam for the care,...
ANALYSIS OF ANTIHYPERTENSIVE DRUGS IN COMMERCIAL DOSAGE FORMS
ABSTRACT
THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF
Sector of $i|tlo£(op()p IN
CHEMISTRY
BY
HABIBUR RAHMAN
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2006
ABSTRACT
The thesis entitled "Analysis of Antihypertensive Drugs in
Commercial Dosage Forms" is comprised of five chapters. The first
chapter describes a general introduction of the subject matter. The
very relevant matters include:
• a brief discussion on drug discovery and drug development.
• the discussion on possible impurities present in active
pharmaceutical ingredients.
• a brief description of various types of analytical techniques used in
the analysis of drugs.
• the discussion on validation strategy. The parameters required for
validation of analytical methods are selectivity / specificity,
linearity, range, accuracy, precision, limits of detection and
quantitation, and robustness / ruggedness. These parameters
have been discussed briefly.
• a description of the classification of drugs based on
pharmacological action on human organs and a brief discussion
mainly on antihypertensive drugs. Finally a brief literature and
chemical structures of the four drugs, i.e., metoprolo! tartrate,
labetalol hydrochloride, ramipril and perindopril erbumine are
presented. An abundant and well-composed list of references is
given at the end of this chapter, comprises 259 citations, taken
from the world's leading scientific journals in the field.
The second chapter describes a simple and sensitive kinetic
spectrophotometric method based on the oxidation of the metoproiol
tartrate with alkaline potassium permanganate. The reaction was
followed spectrophotometrically by measuring the rate of change of
absorbance at 610 nm. The initial rate and fixed time (at 3 min)
methods are utilized for constructing the calibration graphs to
determine the concentration of the drug. The calibration graphs were
linear in the concentration range of 1.0-6.0 ^g ml'^ for both initial
rate and fixed time methods. The regression analysis of calibration
data yielded the regression equations:
log (rate) = 3.634 + 0.999 log C
and A = 6.300 x 10"^ + 6.491 x 10"^ C
for initial-rate and fixed time (at 3 min) methods, respectively. The
detection limits of initial-rate and fixed time methods have been
calculated and found to be 0.04 [ig ml"^ and 0.1 |ag m r \ respectively.
The activation parameters such as Eg, AH*, AS* and AG* were also
evaluated for the reaction and found to be 90.73 KJ nrior\ 88.20 KJ
n)o\'\ 84.54 JK" mol'^ and -63.01 KJ mol'S respectively.
The results were validated statistically and through recovery
studies. The method has been successfully applied to the
determination of metoprolol tartrate in commercial dosage forms.
Statistical comparison of the results with the reference method
showed excellent agreement and indicated no significant difference in
accuracy and precision.
In Chapter three, a validated, selective and sensitive
spectrophotonnetric method has been developed for the determination
of labetalol hydrochloride in commercial dosage forms. The method is
based on the coupling reaction of positive diazonium ion of 4-
aminobenzenesulphonic acid with phenolate ion of labetalol to form a
coloured azo compound which absorbs maximally at 395 nm. Under
the optimized experimental conditions, the colour was stable up to 2
h and Beer's law is obeyed in the concentration range of 0.8 - 17.6 i g
ml'^ with linear regression equation of A = 4.84 xlO'^^^- 7.864 x 10'^
C and coefficient of correlation, r = 0.9999. The molar absorptivity
and Sandell's sensitivity were found to be 2.87 x 10'* I mol'^ cm'^and
0.013 i g cm'^ per 0.001-absorbance unit, respectively. The limits of
detection and quantitation of the proposed method are 0,08 and 0.23
^g m\'^, respectively. The intra day and inter day precision variation
and accuracy of the proposed method was acceptable with low values
of standard analytical error. The recovery results obtained by the
proposed method in drug formulations are acceptable with mean
percent recovery ± RSD of 99.97 ± 0.52% - 100.03 ± 0.63 %. The
results of the proposed method are compared with those of Bilal's
spectrophotometric method which indicated excellent agreement with
acceptable true bias of all samples within ± 2 . 0 % .
The fourth chapter describes a simple and sensitive
spectrophotometric method for the determination of ramipril. The
method is based on the reaction of the drug with l-chloro-2, 4-
dinitrobenzene (CDNB) in dimethylsulphoxide (DI^SO) at 100 ± 1°C.
The absorbance at 420 nm was recorded as a function of time. Fixed-
time (AA) and equilibrium methods were adopted for constructing the
calibration curves. Both the calibration curves were found to be linear
over the concentration range of 20 - 220 |ig m l ' ^ The regression
analysis of calibration data yielded the linear equations; A A = 6.30
X 10'^ + 1.54 X 10"^ C and A = 3.62 xlO"^ + 6.35 x 10'^ C for fixed
time (A A) and equilibrium methods, respectively. The limit of
detection (LOD) for fixed time and equilibrium methods were 1.47
and 1.05 ^g m r \ respectively. The method has been successfully
applied to the determination of ramipril in commercial dosage forms.
Statistical comparison of the results showed that there is no
significant difference between the proposed methods and Abdellatef's
spectrophotometric method.
The last chapter describes two simple, sensitive and accurate
spectrophotometric methods for the determination of perindopril in
pharmaceutical preparations. Method A is based on the formation of
ternary complex between Zn (II), eosin and the perindopril, which is
extractable with chloroform. The absorption spectrum exhibits a band
peaking at 510 nm. Method B is based on the interaction of drug with
iodine in dichioromethane resulting in the formation of charge
transfer complex which absorbs maximally at 365 nm. Beer's law was
obeyed in the concentration range 10-200 pg ml'- and 10-180 pg ml'-
with molar absorptivity of 2.25 xlO^ and 3.71x10^ lit mol'^cm'^ for
methods A and B, respectively. The detection limits for methods A
and B were 0.49 and 0.90 pg mT^ respectively. The proposed
methods have been successfully applied to the determination of
perindopril in commercial dosage forms. The results of the proposed
methods were compared statistically with those obtained by
Abdellatef's spectrophotometric method. The statistical comparision
of these results indicated that there is no significant difference
between the methods compared in terms of accuracy and precision.
ANALYSIS OF ANTIHYPERTENSIVE DRUGS IN COMMERCIAL DOSAGE FORMS
--0S • •S^* - j j , j , . ._^
^ ^THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF
Mottov of ptittofiioplip
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CHEMISTRY
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BY
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HABIBUR RAHMAN
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2006
•• I/: - W l J l
T6415
DEDICATED
MY PARENTS
DR. NAFISUR RAHMAN Reader
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH-202 002 (U.P.) INDIA Tel.: +91-571-2703515 (Office) E-mail: [email protected]
Dated: A ' c ^ w ^ r 2.<^/ 2<}c^
Certificate
This is to certify that the thesis entitled ''Analysis of
antihypertensive drugs in commercial dosage forms" is the
original work of Mr. HABIBUR RAHMAN, carried out under my
supervision and suitable for submission for the award of the
degree of Doctor of Philosophy in Chemistry.
(Dr. Nafisur Rahman)
fmwA m&mmm. ^iivimvWlUs^li/VJjlUfliliJiViit,
First and foremost, I would like to thank the Almighty Allah, The most
merciful and benevolent Allah, whose spiritual inspiration and blessing enabled me
to complete the mission.
I take this opportunity to extend my heartiest gratitude to my respected
supervisor, Dr. Nafisur Rahman for his generous consideration, undaunted help,
unceasing support, valuable suggestions and guidance in the completion of this
work.
I am grateful to Chairman, Department of Chemistry, Aligarh Muslim University,
Aligarh for providing research facilities.
I wish to express my sincere and profound gratitude to Dr. Syed Najmul Hejaz
Azmi, Dr. M. Kashif and Dr. M. Nasrul Hoda for his sympathetic attitude, persistent
interest and consistent encouragement throughout the entire work.
I shall be failing in my duty if I do not acknowledge my colleagues Dr. Yasmin
Ahmad, Dr. Manisha Singh, Mr. Masoom Raza Siddiqui, Ms. Nishat Anwar, Ms. Zehra
Bano and Mr. Rafatullah for providing peaceful and enthusiastic environment
during the progress of this work.
Words fail to express my indebted feelings to my friends Mr. Musawwer, Sk.
Manirul Haque and Suhaib Alam for the care, concern and moral support displayed
at the time of crisis and happiness.
I am extremely grateful to Dr. Abdul Rauf, Professor Mubarak Husain and Dr.
Lutfullah for his unflinching support in enhancing my academic pursuits.
I feel short of words to express my heartful gratitude to my beloved parents,
sisters Habiba and Tayyaba who contributed significantly with their spiritual
inspiration and moral support to complete the work.
(HABIBUR RAHMAN)
na i
CONTENTS List of Publications
List of Tables
List of Figures
CHAPTER 1 General Introduction
References
01
78
CHAPTER 2 Validated kinetic
Spectrophotometric method for the
determination of metoprolol tartrate
in pharmaceutical formulations
96
CHAPTER 3 Determination of labetalol
hydrochloride in drug formulations
by spectrophotometry
124
CHAPTER 4 Kinetic spectrophotometric method
for the determination of ramipril in
commercial dosage forms.
155
CHAPTER 5 Spectrophotometic methods for
the determination of perindopril erbumlne
in pharmaceutical preparations via ternary
complex formation with Zn (II) and eosin
and charge transfer complexation with
iodine.
178
LIST OF PUBLICATIONS
[1] Validated kinetic spectrophotometric method for tine
determination of metoprolol tartrate in plnarmaceuticaj
formulations
Chem. Pharm. Bull 57 (2005) 942 - 948, Chemical Society of Japan
[2] Determination of labetalol hydrochloride in drug
formulations by spectrophotometry
J. Chin. Chem. Soc. (Accepted) Chinese Chemical Society
[3] Kinetic spectrophotometric method for the determination
of ramipril in commercial dosage forms
Canadian Journal of Analytical Sciences and Spectroscopy (CJASS)
(2006) (Communicated)
[4] Spectrophotometric methods for the determination of
perindopril erbumine in pharmaceutical preparations
via ternary complex formation with Zn (II) and eosin
and charge transfer complexation with iodine
Journal of Food and Drug Analysis (2006) (Communicated)
r* LIST OF TABLES
Table 1.1. Chemistry, manufacturing and control testing conducted 05
in the drug development pathway.
Table 1.2. Components of stability testing protocol. 08
Table 1.3. ICH guidelines for identification and qualification 14
of impurities in bulk drug and formulations
Table 1.4. Proportion of various analytical methods prescribed 18
for the assay of bulk drug materials in Ph. Eur. 4 [17]
andUSPXXVlI[18].
Table 1.5. Comparison of HPLC, CE and TLC. 25
Table 1.6. SIA systems for the active ingredient determination. 29
Table 1.7. Voltammetric analysis of drugs. 32
Table 1.8. Quantitative analysis of drugs in pharmaceutical formulations 34
by VU- visible spectrophotometric procedures.
Table 1.9. Analysis of compounds in pharmaceutical formulations. 39
Table 1.10. Validation characteristics normally evaluated for the different 48
types of test procedure and the minimum number of
determinations required ( if applicable).
Table 1.11. Requirements for different calibration modes with 50
relevant parameters.
Table 1.12. Approaches for determining the detection and quantitation [242]. 56
Table 2.1. Sunnmary of data of the initial rate of reaction at different 109
concentration of metaprolol tartrate and KMn04. !
Ill
Table 2.2. Summary of optical characteristics and statistical data for the 111
fixed - time method.
Table 2.3. Test of precision of the proposed methods by intra-day and inter-day 114
assays.
Table 2.4. Summary of data for the determination of metoproloi tartrate in 115
pharmaceutical preparations by standard addition method
Table 2.5. Comparison of the proposed methods using point and Interval 119
hypothesis tests with the reference method at 95% confidence level.
Table 3.1. Optical performance and regression characteristics of the proposed 139
method.
Table 3.2. Intra day and Inter day assays: Test of accuracy and precision of 145
the proposed method in pure form.
Table 3.3. Intra day and Inter day assays: Test of accuracy and precision of the 146
proposed method in pharmaceutical formulations.
Table 3.4. Standard addition method: Evaluation of the validity of the 148
proposed method for the recovery of labetalol hydrochloride.
Table 3.5. Point and interval hypothesis tests: Evaluation of the applicability 149
of the proposed method at 95% confidence level
Table 3.6. Comparison of the proposed method with existing 150
spectrophotometric methods for the determination of labetalol
hydrochloride in drug formulations.
Table 4.1. Calibration equations at different fixed times. 169
Table 4.2. Optical and regression characteristics of the fixed time and 171
equilibrium methods.
IV
Table 4.3. Evaluation of accuracy and precision of the proposed methods 172
Table 4.4. Standard addition method for the determination of ramipril 173
in tablets and capsule
Table 4.5. Comparison of the proposed methods with the reference method 175
Table 5.1. Optical characteristics and statistical data of the regression equations 198
Table 5.2. Evaluation of the accuracy and precision of the two proposed methods 200
Table 5.3. Determination of perindopril in pharmaceutical preparations 201
by standard addition technique
Table 5.4. Determination of perindopril in pharmaceutical preparations 202
by the proposed methods and reference method [12]
LIST OF FIGURES
Figure 1.1. Drug development process. 03
Figure 1.2. ICH decision tree for safety studies. 16
Figure 1.3. The process of development, validation and routine use of an 47
analytical method.
Figure 1.4. Useful range of an analytical method: LOQ = Limit of quantitative 53
measurement and LOL = Limit of linear response.
Figure 1.5. Major pathways of metoprolol metabolism in man. 70
Figure 1.6. Structure of labetalol and proposed pharmacological activity of its 72
4 stereoisomers.
Figure 1.7. Structural formula of (A) ramipril and (B) ramiprilat. 74
Figure 1.8. Simplified representation of the renin-angiotensin system. 75
Figure 2.1. Absorption spectra of (a) I ml of 0.01% metoprolol tartrate 102
(b) 2 ml of 0.015 M potassium permanganate in alkaline medium
(c) coloured product (0.6 ml of metoprolol tartrate (0.01%) +2 ml
of 0.015 M KMn04 + 2.0 ml of 0.6 M NaOH) versus blank diluted
with doubly distilled water in 10 ml standard flask.
Figure 2.2. Limiting logarithmic plot for stoichiometric ratio between 103
metoprolol tartrate and KMn04 (a) log A vs log [drug] and
(b)logAvslog[KMn04].
Figure 2.3. Effect of the molar concentration of NaOH solution on the 105
initial rate of reaction with 60.0 |ag perlO ml metoprolol tartrate
in doubly distilled water.
VI
Figure 2.4. Effect of the molar concentration of KMn04 solution 106
on the initial rate of reaction with 60.0 ^g per 10 ml
metoprolol tartrate in doubly distilled water
Figure 2.5. Absorbance-time curves for the reaction between metoprolol 108
tartrate and KMn04 in aqueous medium: 2.0 ml of 0.015 M KMn04
and metoprolol tartrate: (a) 1.0, (b) 1.5, (c) 2.0, (d) 3.0, (e) 4.0,
(f) 5. 0 and (g) 6.0 ^g ml''. Each set is diluted in 10 mi standard flask
with doubly distilled water.
Figure 2.6. Arrhenius plot: log k versus 1/T for activation energy. 117
Figure 2.7. Eyring plot: log k/T versus 1/T for AH' and AS*. 118
Figure 3.1. Absorption spectra of (a) pure drug solution: 10 )ig ml"'of 132
labetalol hydrochloride in distilled water; (b) 8.67 x lO' M
4-aminobenzene sulphonic acid in distilled water;(c) blank solution:
diazotized solution (2.0 ml of 1.73 x lO'" M 4- aminobenzene
sulphonic acid+1.5 ml of 0.29 NaNOj + 1.8 ml of 0.2 M HCl)
with 4.0 ml of 0.28 M NajCOj in 25 ml distilled water and
(d) Sample solution: 440.0 ^g labetalol hydrochloride and 4.0 ml
of 0.28 M NajCOj with diazotized solution (1.5 ml of 0.029 M
4-aminobenzene sulphonic acid + 1.5 ml of 0.29 M NaN02+
1.8 ml of 0.2M HCl) in 25 ml distilled water.
Figure 3.2. Effect of the molar concentration of reactants: 137
(a) 4-aminobenzenesulphonic acid; (b) NaNOa; (c) HCl; and
(d) Na2C03 with 16.0 ng ml"' labetalol hydrochloride on the
absorbance of coloured product
Vll
Figure 3.3. Plot of error (Sc) versus concentration of labetalol hydrochloride 140
in the determination process.
Figure 3.4. Plot showing variation in the confidence limit at 141
(a) 95% confidence and (b) 99 %confidence level: relative
uncertainty percentage (% AC/C) versus concentration of
labetalol hydrochloride
Figure 4.1. Absorption spectra of (a) 4. 80 x 10"' M ramipril 161
(b) 3.46 X 10"-' M CDNB versus DM.SO (c) complex
(4. 801 X IQ- M ramipril and 1.73 x lO" M CDNB) versus
blank (1.73 x IQ- M CDNB).
Figure 4.2. Effect of heating time on the absorbance of the coloured product 163
(ramipril 200.0 |ig ml"').
Figure 4.3. Effect of the concentration of CDNB on the absorbance of the 164
coloured product (ramipril 200.0 \ig ml'').
Figure 4.4. Limiting logarithmic plots for molar combining ratio between 165
ramipril and CDNB: (a) log Absorbance versus log [Ramipril];
(b) log Absorbance versus log [CDNB].
Figure 4.5. Mole ratio plot for the reaction between ramipril 166
and CDNB in DMSO ([Ramipril] = [CDNB]) = 3.46x10-^ M).
Figure 5.1. Absorption spectra of (a) 4.34x10'^ M eosin; (b) 4.34x10"^ M 184
eosin and 8.35x]0''' M Zn (II); (c) 4.34x10'^ M eosin and 50.0 ^g
ml'' perindopril against doubly distilled water; (d) 200 [ig ml"'
Vlll
perindopril with 6.12 xlO"' M Zn (II) and 5.78 x lO' M eosin extracted
into chloroform against blani< (6.12 xlO" M Zn ^ and 5.78 x 10"''M eosin)
treated similarly.
Figure 5.2. Effect of molar concentration of Zn (II) on the formation of ternary 186
complex.
Figure 5.3. Effect of molar concentration of eosin on the formation of ternary 187
complex.
Figure 5.4. Limitting logarithmic plot for molar combining ratio between 189
perindopril, Zn (II) and eosin: (a) log Absorbance vs log [Perindopril];
(b) log Absorbance vs log [Zn^^]; (c) log Absorbance vs log [eosin].
Figure 5.5. Absorption spectra of (a) 1.5 ml of 2.36 xlO"' M iodine in 193
dichloromethane and (b) 750.0 |j,g perindopril and 2.0 ml of
2.364x10"- M Iodine in dichloromethane. In both cases, the
solutions were diluted to 5 ml.
Figure 5,6, Effect of the molar concentration of iodine on the absorbance of 194
coloured product (perindopril 150.0 figml"').
Figure 5.7. Job's plot for the reaction between Perindopril and iodine in 195
dichlorometane ([Perindopril] = [Iodine] = 1 .OOx lO"'' M).
Figure 5.8, Plot of 1 / A x ([D] [A] / [D] + [A]) Vs. 1 / [D] + [A]. 197
•m
APT GENERAL INTRODUCTION
Discovery and development of a new drug is a long, labour-demanding
process. Recent studies [1] revealed that the average time to discover, develop and
approve a new drug in the United States has steadily increased from 8.1 years in the
1960s, to 14.2 years by the 1990s. The actual time may be even longer as the above
calculation considered the starting point from chemical synthesis, thereby not
including the time for target identification and lead finding/selection process, which
in general takes a minimum of two more years. Typically, the whole process is
fragmented 'Discovery', 'Development' and 'Registration' phases.
The 'Discovery' phase, routinely three to four years, involves identification of
new therapeutic targets, lead finding and prioritization, lead optimization and
nomination of new chemical entities (NCEs). The 'Development' phase, the drug
candidates are subject to preclinical testing in animals (also known as 'Phase I') for
about two years. In addition, preliminary clinical trials in man and the subsequent
full clinical trials (known as Phase II and III) will take a much longer time (-8.6
years). According to DiMasi [1], the 'Registration' phase averaged around 1.8 years
in the late 1990s. Each pharmaceutical firm has their own collection of drug
candidates which are being allocated to different phases of the discovery and
development process, often referred to as the 'pipeline'. Discovery scientists
primarily focus on 'hunting' for drug candidates, whereas in Development one is
trying to 'promote' NCEs as novel and safer commercial medicines. To ensure
scientific and commercial success, it is critical to understand drug development
process, (Fig 1.1) the myriad task and milestones that are vital to a comprehensive
development plan.
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In recent years, the International Conference on Harmonization of technical
requirement for registration of pharmaceuticals for human use (ICH) has adopted
scientific standards for quality control monitoring [2]. These standards are the basis
for most regulatory guidelines, including those published by the Food and Drug
Administration [3]. Key steps on the path include pharmaceutical analysis and
stability studies that are required to determine and assure the identity, potency and
purity of ingredients, as well as those formulated products. Stability testing
facilitates the establishment of recommended storage conditions, determination of
retest periods and definition of acceptable shelf life. These data play a key role in
determining labeling requirements, as well as in the development and monitoring
process.
Stability testing is performed on drug substances and products at various
stages of product development (Tabic 1.1). In early stages, accelerated stability
testing (at relatively high temperatures and /or humidities) is used as a "worst ca.se"
evaluation to determine what types of degradation products may be found after long-
term storage. In preformulation studies, interactions between excipients and the drug
substance are studied under stress conditions to access compatibility.
The design of a complete stability-testing programme for a drug or
nutraceutical product is based upon an understanding of the behaviour, properties
and stability of the drug substance or active ingredient and the experience gained
from preformulation studies and early clinical formulations. Products are analysed at
specific intervals to evaluate a variety of parameters, such as the identity of the
active ingredient, potency, measurement of degradation products, dissolution time,
physical properties and appearance. Samples from production lots of approved
products are retained for stability testing and for comparison testing in the case of
Table 1.1 Chemistry, manufacturing and controls testing conducted in the drug development pathway
Discovery to Phase J • Active pharmaceutical ingredient (API) solubility studies • API identification by spectroscopic analysis • Chromatographic purity • pKa determination • Moisture determination • Partitioning studies • Characterization studies • Development of chromatographic analysis of API • Residual solvents indentification and quantitation • Short-term stability of API • Reference standard certification
Phase I • Validation of analytical methods for API and Phase 1 product • Stability of Phase 1 product • Release of Phase I clinical test materials
Phases II and III • Continued analytical method refinements and validations as
per product and process improvements • Release of Phase II and III clinical test materials • Release of product for long-term toxicology studies • Long-term stability programme • Cleaning validation support • Analysis of manufacturing process validation samples • Container/closure extractable/leachable studies
Post Approval • QC release of product • QC release of API • QC release of excipients • Post approval stability programmes • Scale-up and post approval changes (SUPAC)
product failure. Testing of retained samples alongside returned samples is the key to
ascertaining whether the product failure was manufacturing or storage related.
The objective of analytical testing during preclinical evaluation and Phase I
clinical development is to evaluate the stability of the investigational formulations
used in initial clinical trials, to obtain information needed to develop a final
formulation and to select the most appropriate container and closure (e.g.,
compatibility studies of potential interactive effects between a drug substance and
other components). Information from the experiments listed in Table 1.1 under
discovery to Phase I is summarized in the investigational new drug application
(IND) with the initiation of Phase I clinical trials. When the delivery mechanism of
the drug is an integral part of creating the therapeutic effect and must be used in the
Phase I trials, formulation data, container closure data and corresponding short-term
accelerated stability data should be included in the IND prior to Phase I trials.
Analysis studies on formulations should be underway by the end of Phase II
and the stability protocol for study of both the drug substance and drug product
should be defined. This will help assure that analytical chemistry data generated
during Phase 111 are appropriate for submission. Prior to Phase I, stability of the drug
substance and the formulation to be used must be evaluated. Impurities from the
manufacturing and degradants that form are quantitated and tracked to ensure safety
prior to moving into the Phase I clinical trials and continuity of material used for
laboratory safety testing and clinical trials.
Stability testing done during Phase 111 studies focuses on testing final
formulations in the proposed packaging produced at the manufacturing site. It is
recommended that the stability protocol is defined prior to the initiation of Phase III
studies. In this regard, consideration should be given to establish appropriate linkage
between the non-clinical and clinical batches of the drug substance and drug product
and those of the primary stability batches in support of the proposed expiration-
dating period. Factors to be considered include the source, quality and purity of
various components of the drug product, manufacturing process and production
facility for the drug substance and the drug product, as well as use of same
containers. Data obtained on tests done under controlled conditions replicating
conditions recommended for long-term storage and slightly elevated temperatures
are used to determine a product's shelf life and expiration dates.
Stability testing focuses on the chemical (i.e., integrity, potency,
degradation) and physical properties (e.g., appearance, hardness, particle size,
solubility) of active pharmaceutical ingredients (APIs) and products. In addition,
microbiological testing is done to ensure the substance and products maintain their
resistance to microbial and bacterial growth. Assuring the physical/chemical
properties and effectiveness properties of a pharmaceutical is critical for labeling
and marketing purposes. A wide range of testing is used to evaluate and verify the
identity, potency and availability of the API in the product (Table 1.2). Stability
testing is done at all phases of the development, production and marketing process
for quality control and monitoring purposes. Early in the development of the drug
product, purposeful degradation studies are done as a means to predict possible
degradation pathways of an API. This information is used in the validation of
.stability indicating analytical methods and in pre-formulation studies. Degradation
studies include stress conditions such as heat, oxidative, light, acidic conditions,
basic conditions and heat/humidity. Testing to assure that products meet
specifications for the presence of degradation products and impurities are usually
intensive chromatographic separations with detection down to the 0.01% levels.
Table 1.2 Components of stability testing protocol
Technical grade and manuFacturcr of drug substance and excipients
Type. size, and number of balclies
Type, size, and source of containers and closures
Test parameters
Test methods
Acceptance criteria
Test time points
Test storage conditions
Container storage orientations
Sampling plan
Statistical analysis approaches and evaluations
Data presentation
Retest or expiration dating period
Stability commitment
Typically, impurities and degradation products tliat are 0.1% and above need to be
evaluated for identity and chemical structure. The level of the impurities allowed
depends on the toxicity of the impurity and the daily dose levels of the drug. Identity
information on the stability of a drug substance under defmed storage conditions is
an integral part of the systematic approach to stability evaluation. Stress testing
helps to determine the intrinsic .stability characteristics of a molecule by establishing
degradation pathways to identify the likely degradation products and to validate the
stability indicating power of the analytical procedures used.
Testing is carried out on a single batch of a drug substance and includes the
effects of temperatures in 10°C increments above the accelerated temperature test
condition and humidity, where appropriate (e.g.. 75% or greater). In addition, one
must evaluate oxidation and photolysis on the drug substance, plus its susceptibility
to hydrolysis across a wide range of pll values when in solution or suspension.
Photostability (i.e. light) testing is an integral part of stress testing. Some
degradation pathways can be complex and, under forced conditions, decomposition
products may be observed that are unlikely to be formed under accelerated or loiig-
term testing. This information is useftil in developing and validating suitable
analytical methods, but may not be necessary to examine specifically for all
degradation products if it has been demonstrated that in practice these are not
formed. Information obtained from photostability is the key in choosing appropriate
container/ closure system.
In general, all dosage forms are evaluated for appearance, assay and
degradation products. Additional tests (i.e., potency) are needed for specific dosage
forms. For example, sterility is needed for sterile products but not for tablets or
capsules. In addition, not every test will be performed at each time point.
10
Safety and efficacy are two fundamental issues of importance in drug therapy.
The safety of drug is determined by its pharmacological-toxicological profile as well
as the adverse effects caused by the impurities in bulk and dosage forms.
Impurities in pharmaceuticals are the unwanted chemicals that remain with the
active pharmaceutical ingredients (APIs), or develop during formulation, or upon
aging of both API and formulated APIs to medicines. The presence of these
unwanted chemicals even in small amounts may influence the efficacy and safety of
the pharmaceutical products. Impurity profiling (i.e. the identity as well as the
quantity of impurity in the pharmaceuticals) is now getting important critical
attention from regulatory authorities.
Medicines are the formulated forms of active pharmaceutical ingredients.
There are two types of impurities in medicines:
(1) Impurities associated with active pharmaceutical ingredients and
(2) Impurities that are created during formulation and or with aging or that are
related to the formulated forms.
Impurities associated with APIs
According to ICH guidelines [4], impurities associated with APIs are classified into
the following categories:
• Organic impurities (Process and drug-related)
• Inorganic impurities
• Residual solvents
Organic impurities
Organic impurities may arise during the manufacturing process and/ or storage
of the drug substances. They may be identified or unidentified, volatile or non
volatile, and include the following:
11
Starting materials or intermediates - These are the most common impurities found in
every APIs unless a proper care is taken in every step involved throughout the multi-
step synthesis. Although the end products are always washed with solvents, there are
always chances of having the residual unreacted starting materials may remain
unless the manufacturers are very careful about the impurities.
By-products - In synthetic organic chemistry, getting a single end product with 100%
yield is very rare; there is always a chance of having by-products.
Degradation products - Impurities can also be formed by degradation of the end
product during manufacturing of bulk drugs. However, degradation products
resulting from storage or formulation to different dosage forms or aging are common
impurities in the medicines.
Reagents, ligands, and catalysts - These chemicals are less commonly found in APIs;
however, in some cases they may pose a problem as impurities.
In general, an individual API may contain all of the above- mentioned types of
organic impurities at levels varying from negligible to significant.
Enantiomeric impurities - The single enantiomeric form of a chiral drug is now
considered as an improved chemical entity that may offer a better pharmacological
profile and an increased therapeutic index with more favourable adverse reaction
profile [5]. In any case, cost benefits as well as the patient's compliance need to be
considered in selecting drugs. For the manufacturers of single enantiomeric drug
(eutomer), the undesirable stereoisomers in drug control are considered in the same
manner as other organic impurities.
Inorganic impurities
Inorganic impurities may also derive from the manufacturing processes used
for bulk drugs, They are normally known and identified and include the following:
Reagents, ligands, and catalysts - The chances of having these impurities are rare;
however in some processes, this could create a problem unless the manufactures take
proper care during production.
Heavy metals - The main sources of heavy metals are the water in the processes and
the reactors (if stainless steel reactors are used), where acidification or acid
hydrolysis takes place. These impurities of heavy metals can easily be avoided using
demineralized water and glass- lined reactors.
Other materials (e.g. filter aids, charcoal) - The filters or filtering aids such as
centrifuge bags are routinely used in the bulk drugs manufacturing plants, and, in
many cases, activated carbon is also used. The regular monitoring of fibers and
black particles in the bulk drugs is essential to avoid these contaminafions.
Solvent residues
Residua! solvents are organic volatile chemicals used during manufacturing
process or generated during the production. It is very difficult to remove these
solvents completely by the work- up process; however, efforts should be taken to the
extent possible to meet the safety data. Some solvents that are known to cause
toxicity should be avoided in the production of bulk drugs. Depending on the
possible risk to human health, residual solvents are divided into three classes [6].
Solvents such as benzene (Class I, 2 ppm limit) and carbon tetrachloride (Class I, 4
ppm limit) are to be avoided. On the other hand, the most commonly used solvents
such as methylene chloride (600 ppm), N, N- dimethyl formamide (880 ppm limit)
and acetonitrile (410 ppm limit) are of Class II. Class III solvents (acetic acid,
acetone, isopropyl alcohol, butanol, ethanol and ethyl acetate) have permitted daily
exposures of 50 mg or less per day. In this regard, ICH guidelines [6] for limits
should be strictly followed.
13
Control is more important today than ever. Until the beginning of the 20"
century drug products were produced and sold having no imposed control. Quality
was generally poor. Many products were patent medicines of dubious value. Some
were harmful and addictive. In 1937, elhyicnc glycol was used as vehicle for an
elixir of sulphanilamide, which caused more than 100 deaths [7]. Thereupon the
Food, Drug and Cosmetic act was revised requiring advance proofs of safety and
various other controls for new drugs. The impurities to be considered for new drugs
are listed in regulatory documents of the Food and Drug Administration (FDA) [8],
International Conference on Harmonization of technical requirement for registration
of pharmaceuticals for human use (ICH) [9, 10] and United States Pharmacopoeia
(USP) [11]. Nevertheless, there are many drugs in existence, which have not studied
in such detail. The USP and National Formulary (NF) are the recognized standards
for potency and purity of new drugs. These compendia have become official upon
adoption of first food and drug act. They formulate legal standards of quality, purity
and strength of new drugs. The good manufacturing practices provide minimum
quality standards for production of pharmaceutical as well as their ingredients [12].
The ICH, which took place in Yokohoma, Japan in 1995, has released new
guidelines on impurities in new drug products [13]. These guidelines have number
of advantages., both for industry and regulators. The most critical aspects of the
elaboration of the guidelines were the definition of the levels of the impurities for
identification and qualification (Table 1.3).
Qualification is the process of acquiring and evaluating data for establishing the
biological safety of an individual impurity or a given impurity profile at the levels
specified. The level of any impurity present in a new drug substance that has been
adequately tested in safety and clinical studies is considered qualified. A rationale
14
Table 1.3 ICH guidelines for identification and qualification of impurities in bulk drugs and formulations
Dose
< 1 mg
1- lOing
10- 100 mj
100-2 g
>2g
Threshold for
Identification (%)
1.0
0.5
0.2
0.1
0.1
Qualification (%)
1.0
1.0
0,5
0.2
0,1
15
for selecting impurity limits based on safety consideration has to be provided. The
"Decision tree for safety studies" (Fig. 1,2) describes the considerations for the
qualification of impurities when threshold are exceeded. This has some consequence
for method development.
The monitoring of in-process impurities was an obscure and unidentified about 20
years ago. Now it has become a major factor in modern pharmaceutical industry.
This is mainly the pressure of product quality and the demand for higher standards
of process reliability. Toxicological issues have also brought about a greater
sensitivity to the significance of impurities at trace levels [14]. New attention has
been given to the various classes of toxicants present as impurities in pharmaceutical
products. In view of these changes it has become necessary to pay more attention to
the origins and pathways of a host of impurities within the process. Frequently,
impurities are formed as isomers of the desired reaction products and a critical
impurity can often enter with the feed. Analytical recognition of the problematic
16
Decrease degradation
products level below threshold
Yes Above threshold
No
No Oualified
Yes
Structure elucidated ?
Yes
Yes
Toxicity documented and sufficient?
No
Related to others with known toxicity Yes Acceptable
justification?
No No
Consider patient population and duration of use
Yes
Oualified
Consider need for: 1- Genotoxicity studies (point mutation, chromosomal aberration) 2- General toxicity studies (one species, min.14 days, max. 90 days) 3- Other specific toxicity end point, as appropriate
Adverse effects
Yes,
Consider additional testing or removal /
lowering level of degradation
products
Fig. 1.2. ICH decision tree for safety studies
17
ANALYTICAL TECHNIQUES
From the beginnings of official pharmaceutical analysis, the aim of including
assay methods in compendial monographs has been to characterise the quality of
bulk drug materials by setting limits for their active ingredient content. The
analytical techniques used in drug analysis are given in Table 1.4, based on the
recent editions of European [17] and US [18] Pharmacopoeias.
Titrimetric methods
It is apparent from Table 1.4 that in the majority of cases the titrimetric
methods are still used, especially in European Pharmacopoeia. Advantages of these
methods are saving time and labour, high precision and the fact that there is no need
of using reference standards. Non- aquous titration methods are frequently used in
drug analysis, which can be extended to weak acids and bases. In majority of the
cases, the end point is detected potentiometrically and thus, improving the precision
of the method. In the case of titrimetric methods the lower and upper limhs of assay
are usually 98.0 - 99.0% and 100.5 - 102.0%, respectively (most typically 99.0-
101.0%)). Recently, titrimetric methods have been used for the determination of
captopril [19], gatifloxacin [20], promethazine theodate [21] and nizatidine [22] in
commercial dosage forms.
High performance liquid chromatography
High performance liquid chromatography (HPLC) is widely used both in drug
research and development and also pharmaceutical manufacture. Applications in
research and development include purity control of new drug syntheses, separation
of products during stability testing of drugs and formulations, and pharmacokinetics,
i.e. the determination of drugs and metabolites in biological fluids during
metabolism and clinical investigations. In pharmaceutical manufacture, HPLC is
Table 1.4 Proportion of various analytical methods prescribed for the assay of bulk drug materials in Ph. Eur. 4 [17] and USP XXVII [18]
Method Ph. Eur. 4 USP 27 _!%) (%)
HPLC 15.5% 44%
GC 2 % 2.5 %
Titration 69.5 % 40.5 %
Acid-base 57.5 % 29.5 %
Aqueous mixtures 2 1 % 5.5%
Indicator 6.5 % 4.5 %
Potentiometric 14.5 % 1 %
Non-aqueous 36.5 % 24 %
Indicator 9.5% 14%
Potentiometric 27% 10%
Redox (lodometry, Nitritometry, etc.) 6.5 % 5.5 %
Other (complexometry, argentometry, etc.) 5.5 % 5.5 %
UV-vis spectrophotometry 9.5 % 8.5 %
Microbiological assay (antibiotics) 3 % 2.5 %
Others (IR, NMR, polaremetry, fluorimetry, atomic 0.5%) 2 %
absorption spectroscopy, polarography, gravimetry etc.)
widely used in the quality control of both raw materials and finished dosage forms,
with pharmacopoeial monographs now containing several HPLC assay procedures.
It is evident from Table 1.4; HPLC has become the predominant method in US?
XXVII [18] and- although to a lesser extent - it is one of the most widely used
methods also in European Pharmacopoeia [17].
The problematic points of using specific HPLC methods for the assay of bulk
drug materials were probably taken into consideration by the European
Pharmacopoeia Commission when they summarized their approach regarding this
matter as follows: " Specificity of assays: For elaboration of monographs on
chemical substances, the approach generally preferred by the Commission is to
provide control of impurities via a well designed Test section rather than by
inclusion of an assay that is specific for the active moiety. It is, therefore, the full set
of requirements of monograph that is designed to ensure that the product is of
suitable quality" [17]. Table 1.4 reflects the differences between the approaches of
the European and US Pharmacopoeias: while in USP XXVII the proportion of
specific HPLC (+GC) methods is 46.5%, the same figure in European
Pharmacopoeia is only 17.5%.
Methods based on gradient reversed- phase conditions are found to be very
popular for analyzing cardiovascular and antihypertensive agents. However, the
isocratic conditions for separation of nitrendipine and its impurities of reaction
partners and by-products (methyl-3-amino crotonate, 2-ethyl-2- (nitro-benzylidene)
acetoacetate, l,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic
acid diethyl ester and l,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-
pyridinedicarboxylic acid dimethyl ester) on a Lichrosorb RP-185 [im (25 cm x 4
mm i.d.) methanol- water (70:30) as adjusted to pH 3 with H3PO4 and detection at
20
238 nm were also reported [23]. Ramipril [CAS No.8733-19-5] is one of the several
angiotensin-converting enzyme inhibitors, which is used as a prodrug for regulating
of hypertension. The structure of ramipril is similar to that of a proline containing
natural peptide. Till 1999, methods describing the determination of related
substances of ramipril are not listed in USP or Eur Ph. Quite recently, Hogan et al.
have developed and validated a reversed-phase HPLC method for separation of
related substances of ramipril in Altace capsules using an ion-pairing reagent and a
simple two step gradient in a 40 min run time [24].
The usefulness of HPLC is enhanced when it is coupled with mass
spectrometry: a HPLC / time of flight MS method was developed for screening
anabolic steroids in illegal cocktails [25]. A rapid and sensitive HPLC-MS method
was used for the determination of ramipril and ramiprilat in human serum using
enalapril as an internal standard [26]. The assay linearities of ramipril and ramiprilat
were found over the range 0.10 - 100.0 ng ml"' and 0.25 - 100 ng ml"', respectively.
The hyphenated techniques [27] such as LC-MS and LC-NMR have been employed
to identify drug degradation products in pharmaceutical development. Barret et. al.
[28] have reported a validated HPLC-MS/MS method for simultaneous
determination of simvastatin and simvastatin hydroxy acid in human plasma. HPLC-
MS is an excellent tool for estimating minor components under overlapping peaks,
e.g. the identification and quantitation of as low as 0.01% prednisolone in
hydrocortisone [29]. On- line HPLC-NMR, although only few data are available for
its use in pharmaceutical anaysis, is certainly the method for the future even in this
field. For example, using this technique estradiol and norethisterone acetate was
detected in a galenic emulsion [30]. The structure elucidation of impurities in
21
fluticasone propionate is a good example for the application of on-line HPLC-NMR
[31].
Gas chromatography
Gas chromatography (GC) is based on the volatilized sample transported by
the carrier gas as the moving phase through the stationary phase of the column
where separation takes place by the sorption/desorption process. Samples for gas
chromatographic analysis are normally low molecular weight compounds that are
volatile and stable at high temperature. In this respect, residual solvents in drug
substances and drug products are suitable for gas chromatographic analysis.
Chemical derivatives can also be formed to achieve volatility and thermal stability.
Due to insufficient volatility and thermal stability of the majority of drug
materials, gas chromatography can also be used for their assay in a limited number
of cases only, as reflected by the figures in Table 1.4.
The main application of GC [32] in the determination of impurities in drugs is
the determination of solvent residues [33]. GC is the method of choice for the
determination of volatile, toxic impurities such as N,N- dimethylaniline in
antibiotics [34], 4-chlorophenol in clofibrate [34], and 2,3,7,8- tetrachlorodibenzo-p-
dioxin (down to 2 ppb) in hexachlorophene [34]. GC can be used for the
determination of the synthesis-related impurity dicyclohexylurea in peptides [35]. Of
the limited number of drugs for which GC is prescribed in the pharmacopoeias to
determine their related impurities, biperiden hydrochloride [36], captopril and
griseofulvin [36] clioquinol [37] are mentioned. The case of the purity tests of
desogestrel (13 (3- ethyl-11-methylene-18,19-dinor-17 a-pregen-4-en-20-yn-17-ol)
merits a special mention.
22
In addition to these, a number of papers have been published on the
applications of gas chromatography in pharmaceutical analysis both in dosage forms
and biologicalfluids [38-41].
GC-MS is an ideal tool for screening purposes. The perindopril and its active
free metabolite have been determined after derivatization [42]. GC-MS has been
utilized for simultaneous determination of formic acid and formaldehyde in
pharmaceutical excipients [43]. GC-MS method [44] was also developed for the
determination of 21 different neutral and weakly basic drugs belonging to several
different medicinal classes like antiphlogistics, beta- blockers, P2-
sympathomimetics, lipid regulators, antiepileptic agents, psychiatric drugs and
vasodialators. The possibility to enhance the applicability of GC and GC-MS in
some of the thermally labile compounds is "supersonic GC-MS" using shorter
capillaries with lower film thickness, an increased carrier gas flow rate and a
decreased temperature. This technique was used to determine several steroids [45].
Thin layer chromatography
The invention and rapid spread of thin layer chromatography (TLC) in the
1960s created an entirely new situation in the field of drug analysis. The reasons for
this are as follows;
(1) The method enables the detection, separation, identification and quantitative
determination of organic impurities, which were up to that time not measurable.
(2) The selective chromatographic method was found to be suitable for the reliable
determination of also the main component.
TLC has been used for screening unknown materials in bulk drugs [46]. It
provides a relatively high degree of assurance that all possible components of drug
are separated. However, its application to bulk drugs is limited owing to the
23
problems involved in detection systems. This technique has also been used to
analyse polymyxin B, framycetin, and dexamethasone in an ointment [47] and the
contents of drotaverine and nifuroxazide in capsules [48].
High performance thin layer chromatography
Now a days high performance thin layer chromatography (HPTLC) is
becoming a routine analytical technique due to its advantages of low operating cost,
high sample throughput and need for minimum sample clean up. The major
advantage of HPTLC is that several samples can be run simultaneously using a small
quantity of mobile phase unlike HPLC, thus lowering analysis time and cost per
analysis.
HPTLC has been used for the determination of a number of drugs in
pharmaceutical preparations [49-52]. However, the impurities detected by HPTLC
are limited to 0.1%.
Capillary electrophoresis
The advent of fully automated commercially available capillary
electrophoresis (CE) systems has presented the pharmaceutical analyst with a new
and viable analytical alternative to HPLC and TLC. The modern CE instruments are
capable of the required sensitivity and precision, approaching that of HPLC.
Autosamplers and on-line detection allow simultaneous assay of the main
component and related impurities by CE, which is not a feature of TLC.
As in HPLC, there are distinct operational modes for CE and these are
appropriate to specific drugs and drug classes. For instance free- solution CE
(FSCE) using low pH electrolytes is appropriate for water- soluble basic drugs
whilst FSCE with high pH (pH 7-10) electrolytes is appropriate for water-soluble
acidic compounds. The chromatographic-type CE technique of micellar
24
electrokinetic capillary chromatography (MECC) is applicable to all drug types
including neutral and v/ater- insoluble drugs.
A number of papers have appeared on the use of CE for the determination of
drug impurities due to the advent of highly automated and sensitive capillary
electrophoresis systems. Many pharmaceutical companies are investing heavily in
capillary electrophoresis as this is viewed as a highly complementary technique to
HPLC and the two techniques are often used in combination when assessing drug
purity. The separation mechanisms are widely different in HPLC and CE; being
partitioning and mobility differences, respectively. Therefore, a good agreement
between HPLC and CE results strongly supports a comprehensive evaluation of
sample's purity,
CE can have considerable benefits for analysis of related impurities. Table 1.5
shows a comparison of CE, HPLC and TLC. Specific points from the table are
discussed below.
Combination of HPLC, TLC and CE give widely differing selectivities and
present a good overall assessment of product purity. Preconditioning of CE
capillaries is relatively rapid and is in the order of 2 - 3 min. Often a single capillary
can be used to analyse a range of different compounds, which are separated using
similar electrolytes. Preparation of mobile phase for TLC and HPLC can be a time-
consuming daily task whilst CE electrolyte can be pre-prepared and stored for three
months in many cases. As in HPLC, the use of short columns can considerably
reduce analysis time. The typical CE capillary length is 40 - 60 cm. Reducing this to
20 - 25 cm dramatically reduces analysis time as the field strength (V / cm) is
increased whilst the distance along the capillary to the detector is considerably
shortened. Some resolution is lost [53] but the short capillary gives a good
Table 1.5 Comparison of HPLC, CE and TLC
25
Sensitivity
Precision at low levels
Low UV wavelength detection
Indirect UV detection
Preparative isolation
Running cost
Sample preparation needs
Main peak assay
Automation
Robustness
Analysis time (1-5 samples)
Analysis time (5-50 samples)
Column etc. costs
Experience
Selectivity
HPLC
+++
+++
+
-
++
+
++
+++
+++
++
+
++
+
+++
++
CE
++
++
+++
++
-
+++
++
++
++
++
++
++
+++
+
++
TLC
++
+
+
-
++
+
++
-
+
++
+
++
+
+
++
Key: - poor, + acceptable; ++ good; +++ excellent
26
indication of purity and would be useful as a quality control type analysis or an in-
process check.
The capillary format makes operation robust to the nature of the sample matrix
and composition. The capillary can be rinsed with acid and / or base between
injections to remove unquantified sample components. Strongly retained sample
constituents on HPLC may require use of gradients or extensive analysis times. For
example, ranitidine impurities have been determined [54] in syrup formulations
where the samples are simply diluted with water and analysed at low pH. Under
acidic conditions ranitidine and its impurities are protonated and migrate along the
capillary, the excipients are uncharged and are removed from the capillary by
incorporating a rinse step between injections. Also, operation at low UV
wavelengths allows direct monitoring of compounds and impurities having only
limited chromophores. This possibility can eliminate [55] the time-consuming need
to derivatise samples.
The methods generally employ aqueous buffer systems with daily buffer
requirements of ca. 20 ml. This compares very favourably [56] to the cost of
purchase and disposal of the quantities of organic solvents employed in HPLC.
Capillaries are generally plain fused silica and cost a fraction of that of an HPLC
column. Since the selectivity is independent of the capillary, method transfer is
easier as highlighted by a series of successful inter-company method transfer
exercises [57-59].
A recent survey [60] of a number of pharmaceutical companies in both the UK
and US showed that determination of drug impurities constituted 24% of the
workload of CE within pharmaceutical companies. The survey also indicated [60]
27
that CE method had been successfully included in the marketing submissions to
regulatory authorities.
The majority of attention within the literature has been paid to the purity
testing of batches of drug substances such as domperidone [61], hydrochlorothiazide
[62] and salbutamol [63]. Levels of related impurities have also been established in
pharmaceutical formulations containing various drugs including atenolol [64],
codeine [65], diltiazem [66] and sumatriptan [67].
Analysis of both stored drug substances and formulations constitute a
considerable workload within pharmaceutical analysis laboratories. A number of
stability indicating CE methods have been reported and applied [62, 68] to both
stored drug substance and various formulations.
Capillary electrochromatography
Capillary electrochromatography (CEC), which can be considered to be the
combination of RP-HPLC and CE, has been successfully applied to separation and
quantitation of drugs. The performance of HPLC and CEC was compared using six
steroids (androst-4-ene-3, 17- dione, testosterone, progesterone and the 11 -OH and
20 -OH derivative of the latter). Higher peak efficiencies were obtainable by CEC
[69]. The very promising possibilities of the new technique CEC-MS have been
reviewed with several examples from steroid drugs [70]. The identification of
impurities in fluticasone propionate [71] and the combination of CEC with
nanospray MS with detection limits of 50 fg for corticosteroids merit special
attention [72].
Flow injection analysis
Laboratory automation was initiated in the second half of the XX century.
Steward in the US as well as Ruzicka and Hansen in Denmark, created the flow
28
injection analysis (FIA) technique for the automation of chemical procedures [73,
74].
The flov^ injection analysis (FIA) involves injection of a measured amount of
liquid sample into a moving, nonsegmented stream of reagent. As the injected
sample plus reagent carrier proceeds down a narrow tube, propelled by a constant
flow pump, the sample develops a coloured species, which is detected by a flow-
through photodetector or a spectrophotometer equipped with tlow-through cells. The
signal generated by a coloured species is a sharp peak with a trailing edge. The
height of the peak is proportional to the concentration of analyte.
Following the general application of computers in the routine laboratory, a
second generation of flow analysis was proposed by Ruzicka and Marshall in 1990,
designated sequential injection analysis (SIA) [75]. As with the FIA, this is a non-
segmented continuous flow technique based on the same principles of controlled
dispersion and reproducible manipulation of the FIA concept, but whose mode of
functioning is based on the concept of programmable flow.
The FIA technique has lent an important contribution to the development of
automation in pharmaceutical analysis and its advantages are well documented in
several review articles [76 - 81] as well as in a specialized monograph [82].
The introduction of SIA has awakened the interest of the scientific community
for automation in the pharmaceutical area [83]. Table 1.6 [84 - 97] shows that many
articles dedicated to pharmaceutical analysis have been published, applying
sequential analysis to a wide variety of matrices, such as solid matrices (tablets,
capsules), pastes (ointments, creams), liquids (emulsions, suspensions, solutions)
and covering various active ingredients with different therapeutic activities. By
benefiting from the advantages in the economy of reagents and the elevated
29
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s ( J
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• 4 — •
CJ OJ
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00
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CJ (U
H) i «
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31
sampling rates, the majority of the applications are dedicated to the determination of
active ingredients for quality control in pharmaceutical formulations.
Electrochemical methods
Voltammetry can be carried out using commercially available polarographic
instruments employing the classical polagraphic method (DC polarography) as well
as pusle methods (e.g. DPP). Modern voltammetric instruments with automatic
timing of the individual operations are useful for controlling the individual steps in
adsorptive stripping voltammetry measurements (accumulation time, solution
stirring, rest period, initiation of polarization); a computerized instrument is useful
for this purpose. Square- wave voltammetry (SWV) has become more widely
accessible. Applications of polarography and/or voltammetry in analysis of drugs
have been reviewed in many citations [98 - 102]. In drug analysis, adsorptive
stripping voltammetry (Ad SV) is popular because of the low limit of determination
(reaching few ppb concentration), its accuracy and precision, as well as the low cost
of instrumentation relative to other analytical methods of analysis. Table 1.7 [103-
111] shows the use of voltammetric methods for the determination of drugs.
UV-Visible spectrophotometry
Another group of methods in pharmacopoeias are spectrophotometric methods
based on natural UV-VIS absorption and visual (VIS) spectrophotometric methods
based on chemical reactions such as:
• complex-formation reaction
• oxidation-reduction process
• a catalytic effect
The advantages associated with these methods are low time and labour
consumption as well as good precision. It is worth mentioning that colorimetric
32
Table 1.7 Voltammetric
Drugs
Nifidipine
Isoxsuprine,
Fenoterol
Doxazosin
Lacidipine
Indapamide
Dopamine
Dopamine
Dopamine
Dopamine
: analysis of drugs
Medium
pHl.5
BR, buffer
pH 6.8
BR buffer, pH6.0
pH4.0
-
pH7.4
Physiologi cal buffer
-
Method
LSV, CV
DCP, DPP, ACP
AdSV, SWV
DPV
DPV,ASV
CV, FIA
AdSV
AdSV
LSV
Working electrode
GCE
Hg
MCPE
GCE
MCPE
Carbon-fibre microelectrodes
Grapliite carbon fibre microelectrode
Rotating dislc GCE
AuME
DL
-
0.02,0.01 Mgml"'
0.233 ppm
3.52 ^M
5nM
-
•
Down to 10 nM
-
References
[103]
[104]
[105]
[106]
[107]
[108]
[109]
[110]
[111]
Abbreviations:
MCPE = modified carbon paste electrode,
GCE = glassy carbon electrode,
CV - cyclic voltammetry,
LSV = linear sweep voltammetry,
DCP = direct current polarography,
ACP = alternating current polarography,
DPP = differenfial pulsepolarography,
AdSV= adsorptive stripping voltammetry,
SWV = squarewave voltammetry,
DPV = differential pulse voltammetry,
ASV = anodic stripping voltammetry,
FIA= flow injection analysis,
BR = Britton- Robinson buffer
DL = detection limit.
33
methods based on chemical reactions are still in use for the assay of bulk drug
materials. For example, the blue tetrazolium assay was very popular in the 1950s
and 1960s for the assay of corticosteroid drug formulations, moreover in their
bioassay [112, 113]. However, this method is used for the assay of several bulk
corticosteroids in the recent edition of US Pharmacopoeia (<351> "Assay for
steroids" [18]). Representative examples of UV-visible methods of drug analysis for
some pharmaceuticals that have been published [114-157] are given in Table 1.8.
34
Table 1.8 Quantitative analysis of drugs in pharmaceutical formulations by UV-visible spectrophotonietric procedures
Name of drug Reagents used I, max (nm) References
Acetoaminopheii Amiodarone
hydrchloride
Amlodipine besylate
Amoxycillin &
ampiciilin
Ampiciliin, amoxycillin
& carbenicillin
Ascorbic acid
Benidipine hydrchloride
Diclofenac sodium
Diltiazem hydrchloride
Famotidine
Flunarizine
dihydrochioride
Irbesartan
Lisinopril
m-Cresol p-Chlorani)ic acid
2,3-Dichloro 5,6- dicyano 1,4-
benzoquinone
p-Chloranilic acid
Ninhydrin in DMF medium
2,3-Dichloro 5,6- dicyano 1,4-
benzoquinone
Ascorbic acid
Potassium lodate
Folin ciocalteau phenol
1 -Ch loro-2,4-d in itrobenzene
Methanol
Tris buffer
Sodium metavanadate
Bromothymol blue
Bromophenoi blue
Bromocresol green
KMn04 in alkaline medium
Ninhydrin
Iodine
640 535
575
540
595
580
530
520
750,770
&750
380
238
2S4, 305
750
415
415
415
610
590
295,355
114 115
115
116
117
118
118
119
120
121
122
123
124
125
125
125
126
127
128
Potassium iodate and iodide in
aqueous medium
7,7,8,8-
tetracyanoquinodimethane
p-Chloranilic acid
Ninhydrin
Ascorbic acid
352
743
129
130
525
595
530
130
131
131
35
Labetalol hydrchloride
Losartan potassium
Levodopa
Methyldopa
L-dopa
Menadione
Metoprolol tartrate
Mometasone furcate
Nalidixic acid
Nicorandil
Nifedipine
Norfloxacin
Carbinoxamine
Pantoprazole sodium
Perindopril erbumine
Ramipril
Roxatidine acetate
hydrochloride
Silymarin
Sodium nitroprusside &
hydroxylamine hydrochloride
KMn04 in alkaline medium
Ce(IV) nitrate in H2SO4 medium
Ce(IV) nitrate in H2SO4 medium
NaOH
NaOH in the presence of amine
Ninhydrin
Methanol
Persulfate in alkaline medium
Brucine-sulfanilic acid in H2SO4
medium
3-Methyl-2-benzothiazoline
hydrazone HCl-metol
KMn04 in neutral medium
4-Methyl amino phenol and
K2Cr207
Bromocresol green
Bromophenol blue
Bromothymol blue
Eriochrome Black T
KOH in dimethylsulphoxide
Ammonium molybdate
KMn04 in alkaline medium
Cu (II) & eosin
Potassium ferricyanide and
Ammonium ferric sulfate
l-Chloro-2,4-dinitrobenzene in
dimethyl sulfoxide
Potassium iodate and potassium
iodide in aquous medium
2,3-Dichloro-5,6-dicyano-l ,4-
benzoquinone
p-Chloranilic acid
KMn04 in neutral medium
3-Methyl-2-benzothiazoline
695
/ • ' '"•
603
510
550
300
450
595
248
320,390
410
560
530
525
415
415
415
520
430
830
603
538
725
420
352
530
530
530
430
132
133
134
134
135
136
137
138
139
140
140
141
142
143
143
143
143
144
144
145
146
147
148
149
150
150
151
152
36
Trimethoprim
Tliyroxine
Verapamil HCI
hydrazone HCI
PersLilfate in alkaline medium
Nitrous acid
Chloramine T
N-Bromosuccinimide
Potassium metaperiodate
Tropaeolin 000 No.!
355
420
425
415
425
400
153
154
155
156
157
157
37
Derivative spectrophotometry
Derivative spectropliotometry (DS), which consists in the differentiation of a
normal spectrum, offers a useful means for improving the resolution of mixtures,
because it enhances the detectability of minor spectral features. DS is an analytical
technique of great utility for extracting both qualitative and quantitative information
from spectra composed of unresolved bands by using the first or higher derivatives
of absorbance with respect to wavelength. The derivative spectra are always more
complex than the zero - order spectrum. The first derivative is the rate of change of
absorbance against wavelength. It starts and finished at zero, passes through zero at
the same wavelength as X,max of the absorbance band with first a positive and then a
negative band, with the maximum and minimum at the same wavelengths as the
inflection points in the absorbance band. This bipolar function is characteristic of all
the odd- order derivatives. The most characteristic feature of the second - order
derivative is a negative band with the minimum at the same wavelength as the
maximum on the zero - order band. It also shows two additional positive satellite
bands on either side of the main band. The fourth derivative shows a positive band.
The presence of a strong negative or positive band, with the minimum or maximum
at the same wavelength as Xmax of the absorbance band, is characteristic of the even-
order derivatives.
Compared with conventional spectrophotometric determination, DS has
proved to be a great value in eliminating the interference from excipients and co-
formulated drugs. The DS represents an elegant approach to the problem of
resolving spectral overlap in pharmaceutical analysis. DS permits the simple, rapid,
sensitive and direct determination of mixtures of drugs having closely overlapping
spectra. In pharmaceutical application, DS has led to significant developments in the
38
analysis of drugs in the presence of their degradation products or in multi-
component mixtures.
Derivative UV- spectrophotometry was also successfully applied to the
analysis of steroid hormone formulations. In simple cases (single- component
formulations) only the spectral background caused by excipients should be
eliminated. For this purpose first- derivative spectra are usually sufficient. An
example for this is the determination of triamcinolone acetonide in an ointment
[158]. The same technique using the "zero - crossing" method was found to be
suitable for the simultaneous determination of the components of the dual-
component drug formulations such as creams and tablets containing triamcinolone
acetonide and terbinafine hydrochloride [159] and oral contraceptives containing
ethinylestradiol and levonorgestrel [160,161] as well as ethinylestradiol and
gestodene [162]. A zero- crossing second- derivative method was used for the
determination of fluorometholone and tetrahydrozoline hydrochloride [163] as well
as estradiol and medroxyprogesterone acetate [164] in formulations. Another
possibility to improve the selectivity of the method is the use of "ratio spectrum
(zero - cross) derivative spectroscopic" method. Examples for this are the
determination of spironolactone and hydrochlorothiazide [165], hydrocortisone,
nystatin and oxytetracycline [166], cyproterone acetate and estradiol valerate [167]
in pharmaceutical formulations. DS has wide applications in the field of drug
analysis as represented in Table 1.9 [168 - 174].
Table 1.9 Analysis of compounds in pharmaceutical formulations
39
Compounds Remarks References
Angiotensin-converting enzyme (ACE) inhibitor
Antihypertensive drugs:
alprenolol-hydrochlorothiazide;
hydrochlorothiazide-ramipril;
metoprolol- nifidipine
Amlodipine—its pyridine photodegradation product
Hydrochlorothiazide-irbesarton
Hydrochlorothiazide-losartan potassium
Hydrochlorothiazide-losartan
Hydrochlorothiazide-metoprolol or propranolol
Hydrochlorothiazide-ramipril or spironolactone
Ramipril (third), benazepril (second), enalapril maleate (second), lisinopril (first and second) ,and quinapril (first), in pharmaceutical dosage forms
Use of derivative and derivative compensation techniques to determine a weakly absorbing minor component in the presence of a strongly absorbing major component
Third derivative, the method could usefully be applied to routine quality control of pharmaceutical formulations containing amlodipine
Three methods: (1) compensaUon technique using ratios of the derivative maxima or the derivative minimum. (2) First derivative of the ratio spectra and (3) the absorbance ratio method in the zero- order spectra
Two methods: (1) the amplitude in the first derivative of the ratio spectra at 230.423 and 238.360 nm, respectively and (2) based on compensation technique
Fourth derivative
First and second derivatives, combined formulations
m
Vierordt's method and ratio-zero- crossing derivative
[168]
[169]
[170]
[171]
[172]
[173]
[174]
spectra [165]
40
Fluorlmetry
Fluorimetry finds wide applications in quantitative studies of rates of
degradation, metabolism, and excretion of drugs where other analytical techniques
are not sufficiently sensitive. The native fluorescence of estrogens enables their
direct fluorimetric determination, e.g. the determination without a preliminary
separation of estradiol valerate in an injection formulation in the presence of
hydroxyprogestrone caproate [175]. However, the main application field of this is
their sensitive HPLC detection.
Measurement based on the fluorescence induced by non-stoichiometric
reactions with various strongly acidic reagents [113,176,177] has almost completely
lost their importance in modern pharmaceutical analysis. However, methanol-
sulphuric acid is still an important spray reagent in the thin-layer chromatographic
analysis of steroid drugs. An interesting, highly specific and sensitive flow injection
analysis method for the determination of fluticasone propionate is based on splitting
with sodium hydroxide of its -CO-S-CH2F side chain to form fluoromethylthiol
which is further reacted with glycine and o-phthalaldehyde. The strongly fluorescent
isoindole derivative is formed in the reaction coil of the FIA apparatus [178].
Infrared spectroscopy
Infrared (IR) spectroscopy is the most generally used method for the
identification of bulk drugs in modern pharmacopoeias [17, 34,179]. In addition to
this, IR spectroscopy, especially fourier- transform IR (FTIR) is one of the most
important methods, together with X-ray powder diffractometry, differential scanning
calorimetry (DSC), thermogravimetry (TGA) and thermomicroscopy to characterize
the solvates and polymorphic modifications frequently occurring among steroid
hormone drugs [180], Conventional IR spectrometry using fused KBr discs is not
41
suitable for their differentiation due to the easy transformation of the polymorphs
under pressure. On the basis of diffuse reflectance infrared fourier transform
spectroscopy (DRIFTS) and fourier transform Raman spectroscopic investigations
supported by DSC and TGA measurements [181] four different polymorphic forms
[182] and five solvates [183] were identified and characterized. FT-IR studies
combined with all of the above-mentioned solid-phase techniques revealed the
existence of three polymorphs of fluocinolone acetonide [184] as well as two
polymorphs and a hemihydrate of flunisolide [185].
Near-infrared (NIR) spectroscopy and imaging are fast and nondestructive
analytical techniques that provide chemical and physical information of virtually any
matrix. In combination with multivariate data analysis these two methods open
many interesting perspectives for both qualitative and quantitative analysis. In recent
years, NIR spectroscopy has gained wide acceptance within the pharmaceutical
industry for raw material testing, product quality control and process monitoring.
The growing pharmaceutical interest in NIR spectroscopy is probably a direct resuh
of its major advantages over other analytical techniques, namely, an easy sample
preparation without any pretreatment, the possibility of separating the sample
measurement position and spectrometer by use of fiber optic probes, and the
prediction of chemical and physical sample parameters from one single spectrum.
Actually, the major pharmacopoeias have generally adopted NIR techniques.
The European [186] and United States Pharmacopoeia [187], both contain a general
chapter on near-infrared spectrometry and spectrophotometry, respectively. These
chapters address the suitability of NIR instrumentation for use in pharmaceutical
analysis focusmg mainly on operational qualification and performance verification
comprising scale and repeatibility, response repeatibility, photometric linearity and
42
photometric noise. Only some limited guidance is provided in terms of developing
and validating an application.
Fast and non destructive identification of active ingredients and excipients in
whole tablets, even through the blister packaging, is certainly a domain of NIR
spectroscopy [188 -190]. Generally, the measuring mode is not as critical as with
quantitative applications, except for very thick, highly absorbing tablets and sugar
coated tablets, for which the reflectance mode is recommended to overcome
problems of low analyte signal intensity or even total absorption in transmittance.
Quantitative NIR analysis of active ingredients in tablets has been widely
reported and reviewed in the literature. Within the last 10 years, the number of
publications describing quantitative NIR measurements of active ingredients in
intact tablets has increased tremendously [191 - 205].
Mass spectrometry
In the majority of cases, mass spectrometry is applied in pharmaceutical
analysis associated with various chromatographic techniques. For example, time- of-
flight secondary ion mass spectrometry was successfully applied for the
characterization and imaging of a controlled- released drug delivery system
containing prednisolone sodium metasulphobenzoate. Cross sections of the device
containing a multi- polymer- layer system were proved for the distribution of the
active ingredient [206].
Nuclear magnetic resonance spectroscopy
The process of preclinical drug discovery consists of two steps; finding of
initial hits (binding ligands to a medicinal relevant target, usually a protein) and lead
optimization. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool
that can provide valuable information to every step of drug development. NMR is
43
commonly used for characterizing the structure and molecular dynamics of target or
ligand molecules. During the structure-based lead optimization, NMR provides
insight into the structural and dynamical properties of the target-ligand complex
[207]. Recently NMR spectroscopy has been employed for the rapid screening and
compositional analysis of steroid cocktails and veterinary drug formulations illegally
used to livestock. Stanozolol, fluoxymesterone, methylboldenone, norethandrolone,
clobestol acetate, testosterone and its cypionate and decanoate and other steroid
were identified directly or after HPLC fractionation with the aid of a H-NMR
database [208]. When using interrupted decoupling, solid state '^C-NMR is suitable
not only for detecting low-dose active ingredients (among others prednisolone) in
solid dosage forms, but also for discrimination between the two polymorphic forms
of the latter [209].
Circular dichroism spectropolarimetry
The main application field of circular dichroism (CD) spectroscopy is
structural analysis but this technique can be successfully used also for analytical
purposes. The application of this technique for drug analysis has been reviewed
[210]. The profound difference between the ellipticities of ethisterone and its 5-ene
isomer enables their selective determination (ethisterone at 348 nm at its negative
maximum of the CD spectrum) and the isomer at 296 nm at the positive maximum
of the CD curve. When the simultaneous determination was carried out by HPLC,
the spectropolarimeter could act as a CD-detector [211]. The applicability of the CD
method can be enhanced by the use of preliminary chemical reactions.
Kinetic methods
Kinetic automatic methods are good choices for drug analyses as they permit
the sensitive, selective determination of many drugs within a few seconds with no
44
pretreatment. Moreover, the instrumentation required is generally very simple.
Kinetic methods rely on measurement of concentration changes (detected via signal
changes) in a reactant (which may be the analyte itself) with time after the sample
and the reagents have been mixed. The sample and reagent can be mixed manually
or automatically and the kinetic curve (variation of analytical signal with time) can
be recorded immediately. The slope of the straight initial portion of the kinetic curve
gives the reaction rate, which is proportional to the analyte concentration (initial-rate
method). The fixed-time method also frequently used to derive such a concentration,
involves measuring the signal (the value of which depend on the analyte
concentration) at a pre-set time. The fixed-time and initial-rate methods have been
used more frequently for the determination of drugs in pharmaceutical formulations
and biological fluids [212-214]. Kinetic is relevant to analytical chemistry in at least
four respects:
• it allows the elucidation of the physical, chemical and physico-chemical
mechanisms on which analytical processes are based and hence their rational
optimisation.
• it facilitates the development of new analytical methods and techniques that
are otherwise unattainable if the dynamic aspects are not dealt with.
• it is the foundation of reaction rate method.
• it contributes to the improvement of significant analytical parameters such as
sensifivity, selectivity and precision.
Kinetic automatic techniques are generally based on open systems among the
most popular of which are stopped flow (SF) system [215] and the continuous
addition of reagent (CAR) technique [216-218]. Several drugs have been determined
by using the CAR technique with photometric [219,220] and fluorimetric detection
45
[221]. The use of catalysts to accelerate analytical reactions is feasible with both
reaction rate and equilibrium determinations. In this concern, the use of micellar
media in kinetic method is recently encouraged to enhance the rate of reaction
(through micellar catalysis) and may additionally improves the sensitivity and
selectivity which in turn reduce the analysis time for the analyte [222-224],
Multicomponent kinetic determinations, often called as differential rate methods, are
also receiving popularity in the field of pharmaceutical research [225,226]. Two new
approaches i.e. kinetic wavelength pair method [227] and H-point standard addition
method [228] have been proposed for dealing with overlapping spectra of
components in the binary mixtures.
VALIDATION STRATEGY
Accuracy and reliability of the analytical results is crucial for ensuring
quality, safety and efficacy of pharmaceuticals. Consequently, analytical validation
has been in the focus of regulatory requirements for a long time [229-236].
However, a sensible validation is also essential from a business perspective because
analytical data are the basis of many decisions such as batch release,
establishment/verification of shelf life, etc.
ICH guidelines should be regarded as basis and philosophy of analytical
validation, not as a check list. "// is the responsibility of the applicant to choose the
validation procedure and protocol most suitable for their product" [230]. Suitability
is strongly connected with the requirements and the design of the given analytical
procedure, which obviously varies and must, therefore, be reflected in the analytical
validation. This includes the identification of the performance parameters relevant
for the given procedure, the definition of the appropriate acceptance criteria, and the
appropriate design of the validation studies. In order to achieve this, the analyst must
46
be aware of the fundamental meaning of these performance parameters, calculation
and test and their relationship to his specific application. The process for the
development, validation and use of analytical methods is shown in Fig. 1.3.
Parameters for method validation
To be fit for the intended purpose, the method must meet certain validation
characteristics.Typical validation characteristics (Tablel.lO), which should be
considered are: selectivity/ specificity, linearity, range, accuracy, precision, limits of
detection and quantitation, robustness / ruggedness.
Selectivity / Specificity
Slectivty of a method refers to the extent to which it can determine particular
analyte(s) in a complex mixture without interference from other components in the
mixture. The terms selectivity and specificity have often been used interchangeably.
The term specific generally refers to a method that produces a response for a single
analyte only, v 'hile the term selective refers to a method that provides responses for
a number of chemical entities that may or may not be distinguished from each other.
If the response is distinguished from all other responses, the method is said to be
selective. Since very few analytical methods respond to only one analyte, the use of
the term selectivity is more appropriate than specificity. The International Union of
Pure and Applied Chemistry (lUPAC) has expressed the view that "Specificity is the
ultimate of selectivity". The lUPAC discourages use of the term specificity and
instead encourages the use of the term selectivity.
The selectivity of the analytical method must be demonstrated by providing
data to show the absence of interference with regard to the degradation products,
synthetic impurities and the matrix (excipients present in the formulated product at
their expected levels).
47
Calibration model, range and linearity
Define performance specifications
Devise development experiments
Execute and evaluate results
Plan method validation experiments
Precision and accuracy
Analyte stability
Collate results
Write validation report
Apply validated method
Fig.1.3. The process of development, validation and routine use of an analytical method.
48
Table 1.10 Validation characteristics normally evaluated for the different types of test procedure and the minimum number of determinations required (if applicable).
Validation
characteristics
Specificity''
Linearity
Range
Accuracy
Precision
Repeatability
Intermediate
Precision/ rep
roducibility''
Detection limit
Quantitation
limit
Minimum
number
-
5 concentrations
-
9 determinations
over 3 concen
tration levels
(e.g. 3 X 3)
6 determinations
at 100% or
9 determinations
over 3 concen
tration levels
(e.g. 3 X 3)
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
No
No
^ Including dissolution, content potency Lack of specificity of one analytical procedure could be compensated by other
supporting analytical procedure (s). ' Intermediate precision sufficient for submission. May be needed in some cases.
49
Linearity
The linearity is thie ability of analytical procedure to produce test results which
are proportional to the concentration (amount) of analyte in samples within a given
concentration range, either directly or by means of a well- defined mathematical
transformation. Linearity should be determined by using a minimum of six standards
whose concentration span 80 - 120% of the expected concentration range.
The essential question to be answered is on the suitability of the calibration
mode to be used in the test procedure. The requirements and the relevant parameters
for the various calibrations are given in Tablel.ll.
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 function and the zero intercept. If these
prerequisites are fulfilled, the actual figures for the standard error of slope or the
confidence interval of the intercept, for example, are not used further or referred to.
Therefore, it makes no sense to repeat linearity investigations on other days or with
other operators [237J. However, care should be taken to remain within the linear
range of the individual detector used but this information can be obtained from the
equipment qualification.
The linearity of a method should be established by visual inspection of a plot
of analytical response as a function of analyte concentration. If there is a linear
relationship, test results should be evaluated by appropriate statistical methods, for
example, by calculation of the regression line by the method of least squares. In
some cases, the test data may need to be subjected to a mathematical transformation
prior to regression analysis.
50
Table 1.11 Requirements for different calibration modes with relevant parameters
Quantitation Requirements Relevant parameters
Single-point calibration
External standard
Multiple-point calibration Linear, unweighted
Linear, weighted
Linear function
Non-significant
ordinate intercept
Homogenity of
variances"
Linear function
Homogenity of
variances
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 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,
51
Non-linear
100 % -method (area
normalization for
impurities):
Continuous function
For main peak: linear
function
Non-significant
ordinate intercept
Homogenity of
variances"
For impurities: linear
function
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 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).
May be presumed for a limited range (factor 10-20).
52
Range
The specified range is normally derived from the linearity studies. The range
of an analytical procedure is the interval between the upper and lower concentration
(amount) of analyte in the sample for which it has been demonstrated that the
analytical method has suitable levels of precision, accuracy and linearity. This is
illustrated in Fig. 1.4.
Accuracy
The accuracy of an analytical method is defined as the degree to which the
determined value of analyte in a sample corresponds to the true value. Accuracy
may be measured in different ways and the method should be appropriate to the
matrix. The accuracy of an analytical method may be determined by any of the
following ways:
• Analysing a sample of known concentration and comparing the measured value
to the 'true' value. However, a well characterized sample (e.g. reference
standard) must be used.
• Spiked- placebo (product matrix) recovery method. In the spiked- placebo
recovery method, a known amount of pure active constituent is added to
formulation blank [sample that contains all other ingredients except the active
(s)], the resulting mixture is assayed, and the results obtained are compared with
the expected result.
• Standard addition method: In the standard addition method, a sample is assayed,
a known amount of pure active constituent is added, and the sample is again
assayed. The difference between the results of the two assays is compared with
the expected answer.
53
[y]
Instrumental response (signal)
Signal blank
Dynamic Range < • ,
Concentration [X]
Fig.1.4. Useful range of an analytical method: LOQ = Limit of quantitative measurement and LOL = Limit of linear response
54
• Accuracy may also be determined by comparing test results with those
obtained using another validated test method.
In both method (spiked- placebo recovery and standard addition method),
recovery is defined as the ratio of the observed result to the expected result
expressed as a percentage.
The accuracy of a method may vary across the range of possible assay values
and therefore, must be determined at several different fortification levels. The
accuracy should cover at least three concentrations in the expected range.
Precision
According to ICH, the precision is the closeness of agreement (degree of
scatter) between a series of measurements obtained from multiple sampling of the
same homogeneous sample under the prescribed conditions and may be considered
at three levels:
• repeatability
• intermediate precision
• reproducibility
Repeatability
Repeatability is the precision obtained by independent test results with the
same method on identical test material in the same laboratory by the same operator
using the same equipment within short interval of time. It is also termed as intra-
assay precision [238, 239]. Sometimes it is also termed as within run or within day
precision.
Intermediate precision
Intermediate precision expresses within-laboratories variations: different
days, different analysts, different equipment etc. [240]. The International
55
Organization for Standardization (ISO) definitions used the term "M-factor different
intermediate precision" where the M-factor expresses the number of factors
(reference standard, operator, equipment, laboratory or time) that differ between
successive determinations [241]. Intermediate precision is.sometimes also called
between-run, between-day or inter-assay precision. ^ l-r- /^/^/^Z~~^^^'-
Reproducibility
* • • " ^ , > " '
Reproducibility is the precision obtained within the same mfethod on identical
test material in different laboratories with different operators using different
equipments [242].
Limits of detection and quantitation
Limit of detection (LOD) determines the lowest amount of analyte that can be
detected, as it (the analyte) yields instrumental response greater than a blank, but
cannot be quantified. It is a parameter of "limit test" and expected to produce a
response, which is significantly different fi-om that of a blank. On the other hand
limit of quantitation (LOQ) is a parameter of "determination test" and can be defined
as the lowest concentration of the analyte that can be measured and quantified with
acceptable precision and accuracy.
The most common definition of LOD and LOQ is the analyte concentration
for which the signal exceeds that for a realistic analytical blank by three and ten
times of the standard deviation, respectively. Several approaches have been given in
the ICH guidelines to determine the detection and quantitation limits (Tablel.l2).
Robustness/Ruggedness
ICH [243] defines the "robustness/ruggedness" of an analyfical procedure as
a measure of its capacity to remain unaffected by small, but deliberate variation in
method parameters and provides an indication of its reliability during normal usage.
Table 1.12 Approaches for determining the detection and quantitation [242]
56
Approach Detection limit Quantitation limit
Visual evolution Minimum level detection Minimum level
quantifiable
Signal-to-noise 3:lor2:l 10:
Standard deviation of
the response (So) and -; - x CS Vb
the slope(b) 10.0 X (So)/b
'^Verification with suitable number of samples. ^ Standard deviation of the blank, residual standard deviation of the calibration line, or standard deviation of the intercept.
57
Ruggedness is a measure for the susceptibility of a method to small changes
that might occur during routine analysis like small changes of pH values, mobile
phase composition, temperature etc. Full validation must not necessarily include
ruggedness testing; it can, however, be very helpful during the method development
/ prevalidation phase, as problems that may occur during validation are often
detected in advance. Ruggedness should be tested, if a method is supposed to be
transferred to another laboratory [244].
STATISTICAL ANALYSIS
Recent trend [245-248] in the pharmaceutical analysis emphasizes the use of
statistical analysis and evaluation of the method against statistical parameters like
correlation coefficient, variance, errors and confidence limits, limit of detection and
sensitivity, standard deviation, 7' and 'F' values, standard deviation of slope and
intercept etc. A brief discussion is given here.
Correlation coefficient
When using instrumental methods, it is often necessary to carry out a
calibration process by using a series of samples (standard) each having a known
concentration of analyte. Two statistical procedures should be applied to the
calibration curve:
• test whether the graph is linear or in the form of a curve
• find the best straight line (or curve) through the data points
Linearity is often tested by correlation coefficient, 'r', which can be
calculated for a calibration curve to ascertain the degree of correlation between the
measured instrumental variable and the sample concentration.
58
riLx^y.-Ix^y.^
^5p?F(ioWFfe^
where,
X = mean of all the values of Xj
y - mean of all the values of >'i
n = number of data points.
The maximum value of 'r' is 1. When this occurs there is exact correlation
between the two variables (x and y). When the value of 'r' is zero (xy = 0), there is
complete independence of the variables. The minimum value of 'r' is - 1 , indicates
that the assumed dependence is opposite to what exists. As a general rule,
0.90<r<0.95 indicates a fair curve, 0.95<r<0.99 as a good curve, and r>0.99 includes
excellent linearity.
Linear least squares
The best straight line through a series of experimental points is that line for
which the sum of the squares of the deviation of the points from the line is
minimum. Besides, determining a straight line, uncertainties in the use of calibration
graph for analysis of unknown samples can be specified by this method of least
squares. The equation of the straight line is
A = a + bC
where
A= instrumental response (i.e. absorbance)
b=slope
a=intercept
59
C= concentration of the standards in the spectrophotometric calibration.
To obtain the regression Vine A on C, the sJope 'b' of the line and the
intercept 'a' on the y-axis are given by the following equations
b
a -
Zx,'-[(Zx,)V«]
Y.y, -bY^x.
n
where
X\ = individual value of concentration
_>'i =individual value of absorbance corresponding to Xj
X = mean value of concentration
y - mean value of absorbance
n = number of data points in the calibration line
Errors in the slope and intercept
s _ \ny,~yy n-2
Once the value So has been obtained, both the standard deviation of the
intercept Sa and the slope Sb can be calculated from the following equations:
S a = S , , Ix!
s = 'b a , ^
60
w h e r e ^ — 1 ^ ^ ]
n
Error in the concentration
n l /2 s ' b
i . i . ^y-yy n b'I.ix-xy
vv'here x and y are the average concentration and absorbance values,
respectively, for 'n' standard solutions.
Confidence limit for the slope and intercept
It determines whether the slope and/or intercept of a line differ significantly
from a particular or predicted value. It can be calculated as,
b ± / Sb (for slope)
and/or
a ± ? Sa (for intercept)
where
/= tabulated't' value at desired confidence level for (n-2) degrees of freedom.
Limits of detection and quantitation
Limit of detection (LOD)/ Limit of quantitation (LOQ) are defined as the 3.3
and 10 fold, respectively of the standard deviation of the calibration line. The values
are converted to a concentration by the slope of a corresponding calibration line.
s LOD = 3.3 X —
61
s L O Q = 10 X
Equilance testing
An important property of an analytical method is that it should be free from
the systematic error (bias). Determining bias involves analyzing one or more
standard reference materials whose analyte concentration is known. However,
random errors make it unlikely that, the measured amount will equal to the known
amount even v/hen no systematic errors are present. In order to decide whether the
difference between the observed and standard values can be accounted for by
random variation, a statistical test i.e. a significance test is used for the interpretation
of analytical data.
• Student's /-test: Here comparison is made between two sets of replicate
measurements made by two different methods; one is the test method while
other is accepted (reference method).
where,
X1 = mean from the test method
X2 = mean from the accepted (reference) method
«1 & «2 = number of measurements
Sp=pooled standard deviation of the individual measurements of two sets
is given by
s = I E ( X , -X^f +Z(X2 -X^)'
^ V «, +^2 ~ 2
62
A statistical /-value is calculated and compared with a tabulated value for the
given number of tests at the desired confidence level. If / cai>Aab then there is
significant difference between the results obtained by the two methods at the given
confidence level, but if /cai<' tab then there is no significant difference between the
methods. It is an accuracy-indicating test.
• F-Test: This test indicates whether there is a significant difference between
the two methods (i.e. the new method and the accepted reference method). It
can be represented as,
where
Si and 82= standard deviation of method 1&2.
The calculated value of 'F' is compared with the tabulated standard value at
the selected confidence level and degrees of freedom. If Fcai>Ftab then there is
significant difference between the two methods. It is a precision indicating test.
Interval hypothesis
For pharmaceutical analysis, a bias of ± 2.0 % is acceptable [234] and can be
calculated statistically [249].
For example, a test method (method 2) is considered acceptable if its true mean
value is within that of the reference method (method 1), i.e.
-0.02 ^i< ((12-|ii)< 0.02^1
this can be written as
0.98 <^2 / | i i< 1-02
which can be generalised to
63
0L < \x.2l\i^ < Ou
where 9L and 9u represents the lower and the upper acceptance limits,
respectively when \X2 is expressed as the proportion of the reference mean |ii.
Statistically, OL and 9u can be calculated from the relation
G' c2 .2 A
— 2 p tab X,
V n
20X|X2 +
J
g2,2 ^ — 2 '-^p''tab X2
V n = 0
2 ;
The lower limit (0L) and the upper limit (0u) of confidence interval are obtained
as
0L =
0u =
- b -
- b -
-Vb^ 2a
fVb^
-4ac
-4ac
2a
where, a = X, "^ p *• tab
-2x,x.
c = X2 -2 '-'p'-tab
n-
where x, and Xj estimates of \i\ and )i2 based on ni and n2 measurements
respectively. Sp is the pooled standard deviation and /tab is the tabulated one sided t-
value, with ni+n2-2 degrees of freedom at the specified level of significance.
Testing for outliers
When a series of replicate measurements of same quantity are made, one of
the results will appear too different markedly from the other. There is then a great
temptation to discard this "outliers" before calculating the mean and the standard
64
deviation of tlie data or applying statistical tests to compare the data with other
measurements. The best known method for this purpose is Dixon's Q-test.
_ I Suspected value - Nearest value | [Largest value - Smallest value]
If Qcai Qtab at a given confidence level, then the outliers can be rejected.
65
CLASSIFICATION OF DRUGS
All the drugs according to their chemical nature can be divided into organic
and inorganic compounds. They can be prepared synthetically or reconstituted from
natural sources product. All the drugs having medicinal importance can be broadly
divided into tv/o classes.
Chemical classification
The drugs are classified according to their chemical structure and properties
without taking the pharmacological action. In this class most of the drugs are having
at least an organic substrate; further classification is done in the relevant manner.
Pharmacological classification
In this class the drugs are divided according to their action on the organism's
organ (viz. heart, brain, lymphatic system, respiratory system, endocrine system,
central nervous system etc.) Hence these drugs are called cardiovascular, narcotics,
analgesics, antibiotics, diuretics, and anaesthetics etc. Further classification of each
group is done according to the therapeutic/pharmacological specificity with the
relevant organ. A detailed classification of drugs based on pharmacological action
on human orga.ns has been given in Scheme 1.1.
Antihypertensive drugs
Anti-hypertensive drugs are used to help control blood pressure in people
whose blood pressure is too high. Blood pressure is a measurement of the force with
which blood moves through the body's system of blood vessels. Although everyone's
blood pressure goes up and down in the course of a typical day-getting higher when
they are active and going down when they sleep. Some people have blood pressure
that stays high all the time. This condition is known as hypertension. Hypertension
is not the same as nervous tension. People who have high blood pressure are not
66
1 •
•
•
•
•
•
c o
w c ^ to C <u S
O >^ is c/o C/3
« J
J2 3 03 (U
> o 03 U
>> c =3 o +-' C aj
O
1) c
'C ( • )
T3 C
W
t/i
3 O > fl)
2 m U
c V
U
p: V t/2 K ^
M
s m >-, IT)
<-\ U
t/1
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c m C/3
n o ^
m C O
-*~i
03 t/1
C
(U
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X 03
t/1
0/)
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03
>;
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c 03 -> .2 c
a Q
<
CO > -
O 03
o o S ca ^
« l i£ aj - a DO > ,
o ,^ '5b ® u «
^ -2 -o « .S »J c ob
<! oJ
s
C/5
c
c a;
s -a 3
u
C O W) 03
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o t.
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_o 03 a <u U
t/)
03
<
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5 ^ oD a .
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t/7 <u c
_o "o _c '3 cr o o 3
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67
necessarily tense, high-strung or nervous. They may not even be aware of their
condition.
Being aware of high blood pressure and doing something to control it are
extremely important. However, untreated, high blood pressure can lead to diseases
of the heart and arteries, kidney damage, or stroke, and can shorten life expectancy.
Treatments for high blood pressure depend on the type of hypertension. Most cases
of high blood pressure are called Essential or Primary Hypertension, meaning that
the high blood pressure is not caused by some other medical condition. For most
people with primary hypertension, it is difficult to figure out the exact cause of the
problem. However, such hypertension usually can be controlled by some
combination of anti-hypertensive drugs and changes in daily habits (such as diet,
exercise, and v/eight control).
In people with Secondary hypertension, the high blood pressure may be due to
medical problems such as kidney disease, narrowing of certain arteries, or tumors of
the adrenal glands. Correcting these problems often cures the high blood pressure,
and no further treatment is needed. Controlling primary hypertension, on the other
hand, is usually a life-long commitment. Although people may be able to reduce the
amount of medicine they take as their blood pressure improves, they usually must
continue taking it for the rest of their lives. Many different types of drugs are used,
alone or in combination with other drugs, to treat high blood pressure. The major
categories are:
• Angiotensin-converting enzyme inhibitors (ACE): ACE inhibitors work by
preventing a chemical in the blood, angiotensin 1, from being converted into a
substance that increases salt and water retention in the body. These drugs also
make blood vessels relax, which further reduces blood pressure.
68
• Angiotensin II receptor antagonists: These drugs act at a later step in the
same process that ACE inhibitors affect. Like ACE inhibitors, they lower
blood pressure by relaxing blood vessels.
• Beta blockers: Beta blockers affect the body's response to certain nerve
impulses. This, in turn, decreases the force and rate of the heart's contractions,
which lowers blood pressure.
• Blood vessel dilators (Vasodilators): These drugs lower blood pressure by
relaxing muscles in the blood vessel walls.
• Calcium channel blockers: Drugs in this group slow the movement of
calcium into the cells of blood vessels. This relaxes the blood vessels and
lowers blood pressure.
• Diuretics: These drugs control blood pressure by eliminating excess salt and
water from the body.
• Nerve blockers: These drugs control nerve impulses along certain nerve
pathways. This allows blood vessels to relax and lowers blood pressure
The present thesis deals with the determination of some antihypertensive drugs such
as Metoprolol tartrate; Labetalol hydrochloride; Ramipril and Perindopril erbumine
in pharmaceutical formulations.
69
METOPROLOL TARTRATE ((CisHjsNOa):- C4H6O6)
Metoprolol tartarate, 2-Propanol l-[4-(2-methoxyethyl) phenoxy]-3-[(l-
methyIethyl)amino]-, (±)-, [ R-( R*, R*)]-2,3-dihydroxybutanedioate (2:1) (salt)
[CAS No. 56392-17-7] is a P- selective adrenoceptor antagonist. , It is used in the
treatment of cardiovascular disorders such as hypertension, angina pectoris, cardiac
arrhythmias and myocardial infarction. Metoprolol tartrate is commercially available
as 50 and 100 mg. tablets for oral administration and ampoules for intravenous
administration. Metoprolol tartrate is a white, practically odourless, crystalline
powder with a molecular weight of 684.81. It is very soluble in water, freely soluble
in methylene chloride, chloroform and alcohol; slightly soluble in acetone and
insoluble in ether. The (S)- enantiomer of this agent exhibits 33- times more potency
than (R) - enantiomer in the heart [250]. However, a recemic form of this agent is
used for the treatment of the disease [251]. It is mainly eliminated by hepatic
oxidative metabolism. About 85% of an administered dose is excreted in the urine as
metabolites, with a small amount of the unchanged parent compound [252]. In
human the metabolic pathways of metoprolol are a-hydroxylation, o-dimethylation,
(Fig, 1.5) and oxidative deamination. The a-hydroxylation pathways contributes to
the elimination of only 10% of the dose but it results in the formation of the
pharmacologically a-hydroxy-metoprolol, with 1/10 of the potency of metoprolol in
reducing exercise heart rate [252,253]. The formation of a-hydroxy metoprolol has
the potential to yield diastereomeric products because a new chiral center is formed
upon benzylic hydroxylation [254].
70
H./OH
OCH3
METOPROLOL
H..OH O. N
^
OH
0-DEMETHYLMETOPROLOL
OCH-
a- HYDROXYMETOPROLOL
Fig. I.f5, Major pathways of metoprolol metabolism in man
71
LABETALOL HYDROCHLORIDE (C,9H24N203 • HCI)
Labetalol hydrochloride is chemically known as 2-hydroxy-5-[l-hydroxy-2-
[(l-methyl-3-phenylpropyl) amino] ethylj-benzamide hydrochloride (CAS No.
32780-64-6, M.W. 364.87). It is a white or almost white powder and soluble in
water and alcohol; insoluble in dichloromethane and ether. A 1.0% solution (w/v) in
water is clear and its pH varies between 4.0 and 5.0.Labetalol is unique drug with
complex pharmacological properties exhibiting combined ai and P- adrenoceptor
antagonist properties and partial [ii- agonist activity [255-257]. Labetalol, an
antihypertensive, has been used in the management of pregnancy-induced
hypertension or pre-eclampsia.
Labetalol has two asymmetric centers resulting in four stereoisomers,
designated RR, SS, SR and RS (Fig. L6). The drug is supplied as an approximately
equicomponent mixture of all four isomers, Labetalol is a weak base (pKa= 8.3) and
is administered as the hydrochloride salt. Labetalol exerts reversible and competitive
ai- and ji- blocking activity [258,259] and is approximately one- fourth as potent as
propranolol in blocking y5-receptors and one- tenth as potent as phentolamine in
blocking a\- receptors.
Labetalol is metabolized predominantly in the liver, the metabolites being
excreted in the urine together with only small amounts of unchanged labetalol; its
major metabolite has not been found to have significant a- or ^-adrenoceptor
blocking effects. Excretion also occurs in the feaces via the bile. The elimination
half-life at steady state is reported to be 8 hours. Following intravenous infusion, the
elimination of half- life is about 5.5 hours. Labetalol is not removed by dialysis.
72
H,NOa ^CHjNH CH2CH2-
Labetalol
R" R" R"
RR- isomer: SS- isomer; SR-isomer: RS-isomer:
OH H H OH
H OH OH H
H CHi H
CH3
CH, H
CH^ H
Fig. 1.6, Structure of labetalol and proposed pharmacological activity of its 4 stereoisomers
b- Non- selective beta-adrenoceptor blocking activity
b2- Selective beta2- adrenoceptor agonist activity
aj - Selective betal- adrenoceptor blocking activity
* Denotes chiral center
73
In hypertension labetalol hydrochloride is usually given in an initial dose of
] 00 mg twice daily by mouth with food, gradually increased if necessary according
to the response of the patient and standing blood pressure, to 200 to 400 mg twice
daily; total daily doses of 2.4 g have occasionally been required. An initial dose of
50 mg twice daily has been recommended for elderly patients.
RAMIPRIL(C23H23N2 05)
Ramipril, 2-[N-[(S)-l-ethoxy carbonyl-3-phenyl propyl]-L-alanyl]-(lS, 3S,
5S)-2-azabicyclo [3,3,0]-octane-3-carboxylic acid [CAS No. 87333-19-5, M.W.
416.51] is an angiotensin-converting enzyme inhibitor (ACE).It is a prodrug, which
is rapidly hydrolysed after absorption to form the active metabolite ramiprilat (Fig.
1.7). Ramipril itself is a poor inhibitor of ACE. Ramiprilat exerts all known
haemodynamic effects by inhibiting ACE, thereby decreasing circulating levels of
angiotensin II and leading to a reduction in vasopressor activity and a decrease in
peripheral vascular resistance (Fig. 1.8).
It is used in the treatment of hypertension, heart failure and following
myocardial infarction to improve survival in patients with clinical evidence of heart
failure. It is also used to reduce the risk of cardiovascular events in patients with
certain risk factors. In the treatment of hypertension an initial dose of 1.25 mg once
daily is given by mouth. Since there may be a precipitous fail in blood pressure
when starting therapy with an ACE inhibitor, the first dose should preferably be
given at bed time. The usual maintenance dose is 2.5 to 5.0 mg daily as a single
dose, although upto 10 mg daily may be required. Following myocardial infarction,
treatment with ramipril may be started in hospital 3 to 10 days after the infarction at
a usual initial dose of 2.5 mg twice daily, increased after two days to 5 mg twice
daily. The usual maintenance dose is 2.5 to 5 mg daily. 1 ^
74
..n\\H
C—OH
„a\\H
C—OH
B
Fig. 1.7. Structural formula of (A) Ramipril and (B) Ramiprilat
75
Chymase Trypsin Peptidase
Angiotensin II
AT, AT,
Vasoconstriction . Renal sodium reabsorption
, Cell growth and proliferation (remodelling)
.Vasodilation
. Apoptosis
. Differentiation
. Antiproliferation
Inactive peptides
Fig. 1.8. Simplified representation of the renin-angiotensin-system.
AT 1= angiotensin 11 receptor subtype 1;
AT2= angiotensin II receptor subtype 2;
BK= bradykinin receptor.
76
PERINDOPRIL ERBUMINE (CgHjiNzOs • C4H„N)
Perindopril erbumine is the tert-butylamine salt of perindopril, the ethyl ester
of non-sulphhydryl angiotensin-conerting enzyme (ACE) inhibitor. It is chemically
described as (2S,3aS,7aS)-l-[(S)-N-[(S)-l-Carboxy-butyl]alanyl]hexahydro-2-
indolinecarboxyilc acid, 1-ethyl ester , compound with tert-butylamine(l:-l). Its
molecular formula is Ci9H32N20fi • C4H11N. Its structural formula is:
HOOC.
• H2N-C(CH3)3
Perindopril erbumine is a white, crystalline powder with a molecular weight of
368.47 (free acid) or 441.61 (salt form). It is freely soluble in water (60% w/w),
alcohol and chloroform.
Perindopril, a free acid form of perindopril erbumine, is a pro-drug and
metabolized in vivo by hydrolysis of the ester group to form perindoprilat, the
biologically active metabolite. Perindopril has five asymmetric centers. The drug is
synthesized stereoselectively so that it is a single enantiomer (all S stereochemistry).
Perindopril erbumine is commercially availabe in 2 mg, 4 mg and 8 mg tablets
for oral administration. In addition to perindopril erbumine, each tablet contains the
following inactive ingredients: colloidal silica (hydrphobic), lactose, magnesium
stearate and microcrystalline cellulose. The 4 mg and 8 mg tablets also contain iron
oxide.
Perindopril erbumine is a pro^drug for perindoprilat, which inhibits ACE in
human subjects and animals. The mechanism through which perindoprilat lowers
77
blood pressure is believed to be primarily inhibition of ACE activity. ACE is a
peptidyl dipeptidase that catalyzes conversion of the inactive decapeptide,
angiotensin I, to the vasoconstrictor, angiotensin II. Angiotensin II is a potent
peripheral vasoconstictor, which stimulates aldosterone secretion by the adrenal
cortex, and provides negative feedback on renin secretion.Inhibtion of ACE results
in decreased plasma angiotensin II, leading to decreased vasoconstriction, increased
plasma renin activity and decreased aldosterone secretion. The latter results in
diuretics and natriuresis and may be associated with a small increase of serum
potassium.
Perindopril is extensively metabolized following oral administration, with
only 4 to 12% of the dose recovered unchanged in the urine. Six metabolites
resulting from hydrolysis, glucuronidation and cyclization via dehydration have
been identified. These include the active ACE inhibtor, perindoprilat (hydrolyzed
perindopril) perindopril and perindoprilat glucuronides, dehydrated perindopril and
the diastereoisomers of dehydrated perindoprilat. In human, hepatic esterase appears
to be responsible for the perindopril.
Perindopril erbumine is indicated in patients with stable coronary artery
disease to reduce the risk of cardiovascular mortality or non-fatal myocardial
infarction. It can be used with conventional treatment for management of coronary
artery disease, such as antiplatelet, antihypertensive or lipid-lowering therapy.
Perindopril erbumine is also indicated for the treatment of patients with essential
hypertension. It may be used or given with other classes of antihypertensive,
especially thiazide diuretics.
Pf
78
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96
CHArTER-2 VALIDATED KINETIC
SPECTROPHOTOMETRIC METHOD FOR THE
DETERMINATION OF METOPROLOL
TARTRATE IN PHARMACEUTICAL
FORMULATIONS
97
INTRODUCTION
Metoprolol tartrate is a selective P-adrenergic antagonist, which is used in the
treatment of cardiovascular disorders such as hypertension, angina pectoris, cardiac
arrhythmias and myocardial infarction. The drug is quite sensitive, even a small dose
of the drug gives sufficient blockade. Since the P-blockers are also misused as
doping agents in sports and therefore these drugs have been added to the list of
forbidden drugs by the International Olympic Committee (IOC) [1]. Therefore, the
development of an analytical method for the determination of metoprolol tartrate is
of great significance.
The assay of the drug is listed in the monograph of British Pharmacopoeia,
which describes a potentiometric titration method [2]. Several analytical methods
have been developed for the determination of metoprolol in biological fluids and
pharmaceutical formulations based on high performance liquid chromatography [3-
11], gas chromatography [12,13], capillary electrophoresis [14], thin layer
chromatography [15,16], infrared spectroscopy [17], and electrochemical methods
[18,19]. The above mentioned techniques are sensitive but expensive and require
laborious clean up procedure prior to analysis. Spectrophotometry is the technique
of choice even today due to its inherent simplicity and therefore, frequently used in
the laboratories of the developing countries to overcome versatile analytical
problem. The drug has been determined in the visible region based on ion-pair
formation between drug and reagents like oxidized quercetin [20]; bromophenol
blue, bromocresol purple, bromocresol green [21]; benzyl orange [22]; bromothymol
blue [23]; and carbon disulfide-copper chloride [24]. The charge transfer
complexation reactions of metoprolol tartrate with a and 7c-acceptors [24, 25] have
also been utilized for its quantification in pharmaceutical formulations. Shingbal and
98
Bhangle [26] have reported a spectrophotometric method based on the reaction of
drug with 2, 4-dinitrofluorobenzene in HCl / dioxan medium to form a coloured
chromophore, which absorbs maximally at 380 nm. The quantification of metoprolol
tartrate was done on treatment of the drug with ammonium metavanadate [27], FeCls
[28], N-bromosuccinimide [29], and a mixture of KNO3 and H2SO4 followed by
addition of alkali to get coloured chromophore [30]. The literature is still poor in
analytical procedures based on kinetic spectrophotometry for the determination of
drug in pharmaceutical preparations. There is, therefore, a need for a simple and
sensitive kinetic spectrophotometric method for the determination of metoprolol
tartrate in pharmaceutical formulations. It was found that potassium permanganate
oxidizes the metoprolol in alkaline medium and this reaction has not been used
before to quantify the drug spectrophotometrically.
This chapter describes a simple and sensitive kinetic spectrophotometric method
for the determination of metoprolol tartrate in drug formulations. The method
involves the oxidation of drug with alkaline potassium permanganate at 25 ± 1°C
and subsequent measurement of absorbance at 610 nm. The initial rate and fixed
time methods are adopted for its determination in pharmaceutical formulations.
EXPERIMENTAL
Apparatus
A Shimadzu UV-visible spectrophotometer (model-1601, Japan) with matched
quartz cells was used to measure absorbance.
A water bath shaker (NSW 133, New Delhi, India) was used to control the heating
temperature for colour development.
99
Reagents and standards
All reagents and chemicals used were of analytical or pharmaceutical grade.
Aqueous solutions of 0.6 M sodium hydroxide and 0.015M potassium permanganate
(GR Grade, Merck Limited, Mumbai, India) were prepared in doubly distilled water.
Potassium permanganate (GR Grade, Merck Limited, Mumbai, India) solution
should be freshly prepared and its apparent purity was assayed by titrimetric method
[31].
The standard test solution of metoprolol tartrate (0.01%) was prepared in
doubly distilled water. The formulated dosage forms of metoprolol tartrate such as
betaloc (AstraZeneca Pharma India Ltd., Bangalore, India), metapro (Cardicare,
Bangalore, India) and metolar (Cipla, Mumbai, India) were purchased from the local
market.
Determination procedures for metoprolol tartrate
Initial-rate method: Aliquots of 0.1-0.6 ml of 0.01% metoprolol tartrate were
pipetted into a series of 10 ml standard flasks. To each flask, 2.0 ml of 0.60 M
NaOH was added followed by 2.0 ml of 0.015 M potassium permanganate and then
diluted with doubly distilled water at 25 ± 1°C, The contents of each flask were
mixed well and the increase in absorbance was recorded as a function of time at 610
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 graph was
constructed by plotting the logarithm of the initial rate of reaction (logv) versus the
logarithm of the molar concentration of the metoprolol tartrate (log C). The amount
of the drug was computed either from the calibration graph or the regression
equation.
100
Fixed-time method: The absorbance of each drug sample solution was measured at
610 nm against a reagent blank prepared similarly at a preselected fixed time of 15
min. The calibration curve was constructed by plotting the absorbance against the
final concentration of the drug. The amount of the drug was computed either from
calibration curve or regression equation.
Determination procedure for metoprolol tartrate in pharmaceutical formulations
Five tablets were weighed and powdered. The powder equivalent to 50 mg of
active ingredient was weighed accurately, stirred well with doubly distilled water
and filtered through Whatmann No. 42 filter paper (Whatmann International
Limited, Kent, UK). The residue was washed well with doubly distilled water for
complete recovery of the drug. The content of the drug was then diluted to 250.0 ml
with doubly distilled water. It was further diluted according to the need and
subjected to the determination procedures for metoprolol tartrate. The percent
recovery of the metoprolol tartrate was calculated from the corresponding linear
regression equations or calibration graphs.
Procedure for reference method
Into a series of 10 ml standard volumetric flask, different volumes (0.25-2.5
ml) of 0.01% drug (0.1 mg ml"') solufion were pipetted and diluted to volume with
doubly distilled water. The absorbance was measured against the solvent blank at
224 nm. The amount of the drug in a given sample was computed from the
calibration equation.
RESULTS AND DISCUSSION
Spectral studies
The absorption spectrum of metoprolol tartrate solution in doubly distilled
water shows two absorption bands peaking at 194 and 224 nm while that of
101
potassium permanganate solution in the alkaline medium exhibits an absorption
band peaking at 530 nm. The addition of potassium permanganate to the solution of
pure drug produces a new characteristics band at 610 nm. (Fig. 2.1) This band is
attributed to the formation of manganate ion, which resulted on reduction of
potassium permanganate in alkaline medium. The intensity of the coloured product
increases with time and therefore, a kinetic method was developed for the
determination of metoprolol tartrate in drug formulations. Moreover, potassium
permanganate also oxidizes metoprolol in acid medium resulting in the formation of
a-hydroxy metoprolol and Mn (II). In the presence of acid such as H2SO4, HCl and
H3P04 a-hydroxy metoprolol gives a violet colour which has not been utilized for
quantitative analysis.
Stoichiometry and reaction mechanism
The stoichiometric ratio between metoprolol tartrate and potassium
permanganate was established using limiting logarithmic method [32] by performing
two sets of experiments. In the first set, the concentration of metoprolol tartrate was
varied keeping a constant concentration of KMn04. In the second set of experiment,
concentration of metoprolol tartrate was kept constant while varying the
concentration of KMn04. The logarithm of the absorbance was plotted against the
logarithm of the respective varied concentration of metoprolol tartrate or KMn04
(Fig. 2.2 a and b). It is evident from the slopes of the two straight lines that the
combining molar ratio between metoprolol tartrate and KMn04 is 1:1.
Horai et al. have suggested that the metoprolol tartrate undergoes oxidation [33]
resulting in the formation of a- hydroxy metoprolol. In this study, the potassium
permanganate oxidizes the metoprolol tartrate in alkaline medium producing a-
hydroxy metoprolol and itself reduced loMnO]". The reaction product gives violet
102
1.4
1,2
1.0
0.8
0.6
0.4
200 240 280 320 360 400 440 480 520 560 600 640 680 720 760
Wavelength (nm)
Fig. 2.1. Absorption spectra of (a) 1 ml of 0.01% metoprololol tartrate (b) 2.0
ml of 0.015 M. potassium permanganate in alkaline medium (c) coloured
product (0.6 ml of metoprolol tartrate (0.01%) + 2.0 ml of 0.015 M KMn04 +
2.0 ml of 0.6 M NaOH) versus blank diluted with doubly distilled water in 10
ml standard flask.
lo;
-1.0
-6.0 -5.6 -5.2 -4.8 -4.4 -4.0 -3.6 -3.2 -2.8 -2.4 -2.0
Log [Drug] or [KMnOJ
Fig. 2.2, Limiting logaritlimic plot for stoichiometric ratio between metoproloi
tartrate and ]KMn04 (a) log A vs. log [drug] and (b) log A vs. log [KMn04].
104
colour on treating with formaidchyde-sulphuric acid reagent, which confirmed the
formation of a- hydroxy metoprolol [34].
Optimization of variables
The influence of the concentration of NaOH solution on the rate of reaction
was studied by keeping the constant concentrations of metoprolol tartrate (8.76x10'
M) and KMn04 (3.00x10'^ M) and varying the concentration of NaOH (1.20x10"^-
1.32x10"' M) in a final volume of 10 ml solution. Fig. 2.3 shows that the initial rate
of reaction increased up to 8.4x10'^ M NaOH; beyond this concentration the initial
rate of reaction remained constant. Therefore, a concentration of 1.20x10"' MNaOH
was used throughout the experiment. The effect of the concentration of KMn04
solution on the initial rate of the reaction was studied in the range of 7.50x10"''-
3.30x10"^ M. The inifial rate of reaction (Fig. 2.4) increased with increasing the
concentration of KMn04 and became constant at 2.40x10'"' M. Thus, a concentration
of 3.00x10'^ M KMn04 in the final solution proved to be sufficient for the
maximum concentration of metoprolol tartrate used in the determination process.
The effect of temperature on reaction rate was studied in the range of 298-308 K.
The absorbance-time curves showed the temperature dependence of the reaction
rate. It was observed that metoprolol tartrate reacts faster with potassium
permanganate within the short period of 3-15 min, 3-10 min and 3-7 min. at 298,
303 and 308 K, respectively. At temperatures > 308 K, the decomposition of the
reaction product may take place. To avoid this and for the sake of good results, the
opfimum temperature of 298 K is selected for the determination process.
Analytical data and method validation
Under the optimized experimental conditions, a pseudo-order reaction
condition was worked out by using a large excess of KMn04 and NaOH solution
105
0.040
0.010 0.00 0.02 0.04 0.06 0.08 0.10
Molar concentration of NaOH
0.12 0.14
Fig. 2.3. Effect of the molar concentration of NaOH solution on the initial rate
of reaction with 60.0 i g per 10 ml metoprolol tartrate in doubly distilled
water.
106
0.040
0.035
0.005 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0035
Molar concentration of KMnO,
Fig. 2.4. Effect of the molar concentration of KMn04 solution on the initial rate
of reaction with 60.0 ^g per 10 ml metoprolol tartrate in doubly distilled water.
107
with respect to the initial concentration of metoprolol tartrate. As a result, a pseudo
zero order condition was obtained with respect to the reagents, the overall
concentration change of KMn04 and NaOH during the course of reaction would be
negligible. The initial rates of the reaction were determined from the slopes of the
initial tangent to the absorbance-time curves (Fig. 2.5) and are summarized in Table
2.1. The reaction would obey the following rate equation:
Rate = kt C"
where k^ is the pseudo-order rate constant, C is the concentration of metoprolol
tatrate, n is the order of the reaction. The logarithm form of the above equation is
written as:
Log (rate) = log kn/ + n log C
The linear regression analysis using the method of least square treatment of
calibration data was made to evaluate slope, intercept and correlation coefficient.
Under the working experimental conditions, a calibration graph was constructed by
plotting log of initial rate of reaction (log u) versus log of metoprolol tartrate
concentration (log C), which showed a linear relationship over the concentration
range of 10.0 - 60.0 |.ig per 10 ml. The regression of log rate versus log C gave the
following linear regression equation:
Log (rate) = 3.634 + 0.999 log C
with a correlation coefficient (r) of 0.9999. The value of n in regression equation
confirmed that the reaction is first order with respect to metoprolol tartrate. The
confidence limits for the slope of the line of regression and intercept were computed
108
O c CD
O m
<
Fig. 2.5. Absorbance-time curves for the reaction between metoprolol tartrate
and KMn04 in aqueous medium: 2.0 ml of 0.015 M KMn04 and metoprolol
tartrate: (a) 1.0, (b) 1.5, (c) 2.0, (d) 3.0, (e) 4.0, (f) 5.0 and (g) 6.0 ^g ml ' . Each
set is diluted in 10 ml standard flask with doubly distilled water.
Table 2.1 Summary of data of the Initial rate of reaction at different concentration of metoprolol tartrate and KMn04
109
[Drug] (mol T')
1.460 X lO""
2.190 X IQ-"
2.920 X 10" '
4.380 X lO""
5.840 X 10-'
7.300 X 10"'
8.760 X 10-'
8.760 X 10-"
8.760 X 10-"
8.760 X IQ-"
8.760 X 10""
8.760 X lO""
8.760 X 10-"
[KMn04] (mol P')
3.000 X 10-'
3.000 X 10"
3.000 X 10-'
3.000 X 10-'
3.000 X 10"'
3.000 X 10-'
3.000 X 10-'
7.500 X 10-'
9.000 X 10-
1.200 X 10-'
1.800 X 10-'
2.100 X 10"'
2.400 X 10 '
Initial rate of reaction, u
(mol r' min-')
6.250 X
9.375 X
1.270 X
1.880 X
2.500 X
3 . 1 0 0 ^
3.800 X
8.000 X
1.130X
1.860 X
2.660 X
2.830 X
3.800 X
10-'
10-'
10-
10-
10-
10-
10-
10-'
10-
10-
10-
10-
10-
110
using the relation b ± tSb and a ± tSa [35] at 95% confidence level and found to be
0.999 ± 1.45 X 10" and 3.634 ± 2.60 x 10"', respectively. This indicated the high
reproducibility of the proposed method. The limits of detection (LOD) and
quantitation (LOQ) were evaluated using the following equation:
LOD = 3 .3xSo/b and L O Q = 1 0 x S o / b
where So is the standard deviation of the calibration line and b is the slope and found
to be 1.3x10'^ and 4.0x10'^ [ig ml'', respectively. The variance was calculated
using the equation:
n-2
and found to be 1.576 x 10' f.ig ml"'. The low value of variance indicated negligible
scattering of the experimental data points around the line of regression.
In the fixed-time method, the absorbance of green coloured solution obtained
on interaction of different concentration of metoprolol tartrate with alkaline
potassium permanganate was measured at a preselected fixed time. Calibration plots
of absorbance versus initial concentrations of metoprolol tartrate were established at
a fixed time of 3, 6, 9, 12 and 15 min. The molar absorptivity, regression equations,
coefficient of correlation, limits of detection and quantitation and variance are given
in Table 2.2. It is clear from Table 2.2 that the most acceptable values of molar
absorptivity, limit of detection and quantitation were obtained at a fixed time of 15
min. Therefore, the fixed time of 15 min was adopted as the optimum time for the
determination of metoprolol tartrate in pharmaceutical formulations. The important
analytical parameters of conventional UV spectrophotometric method have been
summarized in Table 2.2. It can be seen that the molar absorptivity of the fixed time
method is higher than that of conventional UV spectrophotometric method
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112
whereas the limits of detection and quantitation of the proposed method are smaller.
The performance of the initial rate method is better since the values of LOD and
LOQ are smaller than that of the fixed time method and analysis can be completed in
a shorter time.
Solution stability and selectivity
The solution stability of metoprolol tartrate was checked by observing UV
spectra of metoprolol tartrate for five days. The aqueous solution of the drug having
two Xynax- 194 and 224 nm, shov/ing no change in the absorption spectra of standard
and sample solutions of drug for at least five days, when the solution were stored
at room temperature. To identify the metoprolol and a-hydroxy metoprolol, thin
layer chromatography was performed. The standard solution, sample solution and
reaction product were applied on TLC plates coated with silica gel and developed in
ethyl acetate-methanol-ammonia (40:5:5 v/v/v) solvent system. The plates were air-
dried and spots were detected in the iodine chamber. In the case of standard and
sample solutions, a single spot was observed with Rf = 0.50 corresponding to
metoprolol, whereas reaction product also showed one spot with Rf = 0.65. This
corresponds to a-hydroxy metoprolol [34]. Thus, the proposed methods are selective
as the major metabolite, a-hydroxy metoprolol does not interfere in the
determination. However, other P-adrenergic antagonists such as propranolol,
atenolol and labetalol react with potassium permanganate in alkaline medium
resulting in the formation of green coloured solution, which absorbs maximally at
610 nm.
Accuracy and precision
The accuracy and precision of the proposed methods was established by measuring
the content of metoprolol tartrate in pure form at three different concentration
113
levels (low, medium and high). The short-term (intra day assay) and the daily
precisions (inter day assay) were performed by measuring five independent analyses
at 1.5, 3.0 and 6.0 |ig ml'' concentration levels within one day and on five
consecutive days, respectively (Table 2.3). The standard deviation, relative standard
deviation and mean percent recoveries obtained by both the initial rate and fixed
time methods can be considered to be very satisfactory.
The validity of the proposed methods was also checked by performing recovery
experiments through standard addition method. For this, a known amount of the pure
drug was added to preanalysed dosage forms and then the total amount of
metoprolol tartrate was determined following the recommended procedures and
reference method. The results are summarized in Table 2.4, which showed
recoveries in the range of 100.02-100.13%, 99.99-100.15 % and 99.94-100.11% for
initial rate, fixed time and reference methods, respectively. No interference from the
common excipients was observed.
Robustness
The conditions are very robust for the application of the proposed methods to
determine the active drug in pharmaceutical formulations. Each operational
parameter was checked and challenged for the robustness of the method. The
operational parameters investigated were:
• volume of0.015MKMnO4(± 0.2 ml)
• volume of 0.60 M NaOH (± 0.2 ml)
Under these conditions a sample solution containing 6.0 \xg ml'' (Metalor 25,
Cipla) of active metoprolol tartrate was assayed five times by the initial rate and
fixed time methods. The values of mean recovery, standard deviafion and relafive
standard deviation represent good reliability of the proposed methods.
114
Table 2.3
Test of precision of the proposed methods by Intra day and Inter day assays
Proposed methods
Initial rate method
Intra day assay
Inter day assay
Fixed time method
Intra day assay
Inter day assay
Amount (Mgml-') Taken
1.5 3,0 6.0
1.5 3.0 6.0
1.5 3.0 6.0
1.5 3.0 6.0
Found iSD"
1.50 ±0.04 3.00 ±0.06 6.01 ±0.06
1.50 ±0.04 2.99 ±0.06 6.00 ±0.06
1.50 ±0.04 2.99 ±0.05 6.01 ±0.05
1.50 ±0.06 2.99 ±0.06 6.00 ±0.07
Recovery (%)
100.03 100.06 100.13
100.14 99.95
100.07
100.21 99.93
100.15
100.01 99.93
100.04
SAE
0.02 0.03 0.03
0.02 0.03 0.03
0.02 0.02 0.02
0.03 0.03 0.03
C.L.
0.05 0.07 0.07
0.05 0.07 0.07
0.06 0.06 0.06
0.07 0.07 0.08
^ Mean for five independent analyses. '' SAE, standard analytical error.
C.L., confidence limit at 95 % confidence level and four degrees of freedom (t = 2.776).
•T3 O
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116
The effect of temperature on reaction rate is well known and important in
understanding the various activation parameters of the reaction products. In order to evaluate
the apparent activation parameters, the reaction rate was studied at 298, 303, and 308 K at
[metoprolol] = 1.46 x 10"''-8.76 x 10' ' M, [KMn04] = 3.00 x 10" M and [NaOH] = 1.20 x 10"
' M.
Arrhenius curve (Fig. 2.6) was constructed by plotting log k versus 1/ T and found to
be linear. Activation energy (Ea) can be calculated from the slope (-Ea / 2.303R) and A from
the intercept of the Arrhenius curve and found to be 90.73 KJmol"' and 4.75 xlO",
respectively. The other activation parameters such as enthalpy, entropy and free energy of
activation of the reaction product were calculated using Eyring equation:
AH* 1 \og- = \log{kJh) + -
2.303/? T T I 2.303 R
The plot of log k'T versus 1/T (Fig 2.7) was linear with correlation coefficient of 0.9999.
A H* was evaluated from the slope (-A H ' / 2.303 R) and AS* from the intercept [log (kb /h) +
AS* / 2.303 R] of the compiled Eyring plot. The values of AH* and AS* were found to be
88.20 KJ mof' and 84.54 JK^'mol"', respectively. The Gibbs free energy of activation was
determined by AG* = AH* - TAS* at 298 K and found to be -63.01 K J mol"'.
Applicability of the proposed methods
The proposed methods (initial rate and fixed time methods) were successfully applied
to the determination of metoprolol tartarte in pharmaceutical formulations. The results of the
proposed methods were statistically compared with those of the reference method using point
hypothesis test. Table 2.5 shows that the calculated t- (paired) and F-values at 95%
confidence level are less than the theoretical ones, confirming no significant differences
between the performance of the proposed methods and the reference method. The previous
17
T 1 ' r
0.00324 0.00326 0.00328 0.00330 0.00332 0.00334 0.00336 0.00338
1/T(K)
Fig. 2.(5. Arrhenius plot: log k versus 1/T for activation energy.
118
en o
-0.7
0,00324 0.00326 0.00328 0,00330 0.00332 0,00334 0.00336 0.00338
1/T(K)
Fig. 2.7. Eyring plot: log k/T versus 1/T for / T for AH^ and AS^
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120
The previous investigations were also checked and confirmed by interval hypothesis tests
[36]. The Canadian Health Protection Branch has recommended that a bias, based on
recovery experiments, of ± 2 % is acceptable [37]. It is evident from Table 2.5 that the true
bias of all samples of drug is smaller than ± 2 %.
121
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[I] C. Ceniceeros, M. I. Maguregui, R. M. Jimenez, R. M. Alonso, J. Chromatogr. B:
Biomed Sci. Appl 705 (1998) 97.
[2] British Pharmacopoeia, I, H. M. Stationery Office, London, (1998) p.889.
[3] B. Mistry, J. Leslie, N. E. Edenton, J. Pharm. Biomed Anal. 16(1998) 1041.
[4] P. Modamio, C. F. Lastra, E. L. Marino,,/. Pharm. Biomed Anal. 17 (1998) 507.
[5] R. Oertel, K. Richter, T. Gramatte, W. Kirch,,/. Chromatogr. A 797 (1998) 203.
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[7] A. G. Gonzalez, M. A. Herrador, A. G. Asuero, Int. J. Pharm., 123 (1995) 149,
[8] S. Svensson, J. Vessman, A. Karlsson,,/. Chromatogr. A 839 (1999) 23.
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[II] Y- .lin Park, D. W. Lee, Won-Yong Lee, Anal. Chim. Acta 47 (2002)51.
[12] G. P. Cartoni, M. A. Ciardi, A. Giarrusso, F. Roast, J. High Resolut. Chromatogr.
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[13] T. A. Ternes, R. Hirsch, R. Mueller, K. Haberer, Fresenius J. Anal. Chem. 362
(1998)329.
[14] J. Sadecka, J. Polonsky, J. Chromatogr. A 735 (1996) 403.
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[16] R. Bhushan, M. Arora, Biomed Chromatgr. 17 (2003) 226.
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S. S. M. Hassan, M. M. Abou-Sekkina, M. A. El-Ries, A. A. Wassel, J. Pharm.
Diomed Anal. 32(2003) 175.
D. Zhou, S. Zhang, C. Me, Y. Gao, X. Wang, Liaoning Shifan Daxue Xuebao, Ziran
Kexueban2\ (1998)43.
H. A, H. Elsherief, S. M. S. Ali, H. F. Askal, I. H. Refaat, Bull. Fac. Sci. 26 (1997)
15.
R. P. G. Somashekhara,H. D. Revanasiddappa, Indian Drugs 38 (2001)97.
Z. Vujic, D. Radulovic, L. Zivanovic, IL Farmaco 50 (1995) 281.
L. Ersoy. S. Kocaman . Arch. Pharm. 324 (1991) 259.
M. A. El-Ries, F. M. Abou Attia, S. A. Ibrahim,,/. Pharm. Biomed. Anal. 24 (2000)
179.
H. ^3\tm,Al-AzharJ. Pharm. Sci. 28 (2001) 319.
D. M. Shingbal, S. R. Bhangle, Indian Drugs 24 (1987) 270.
S. Ahmed, R. D. Sharma, I. C. Shukla, Talanta 34 (1987) 296.
R. B. Patel, A. A. Patel, S. K. Patel, S. B. Patel, S. C. Manakiwala, Indian Drugs 25
(1988)425.
K. V. K. Rao, B. V. V. R. Kumar, M. E. B. Rao, S. S. Rao, Indian J. Pharm. Sci. 65
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N. M. Sanghavi, J. J. Vyas, Indian Drugs 29 (1992) 317.
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123
[34] D. B. Jack, S. Dean, M. J. Kendall, J. Chromatogr. 187 (1980) 277.
[35] J. N. Miller, Analyst 116 (1991) 3.
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Vankeerberghen, D. L. Massart, Anal. Chem. 67(1995)4491.
[37] Canada Health Protection Branch, Drugs Directorate Guidelines: Acceptable methods,
Ministry of National Health and Welfare, Draft, Ottawa, Canada (1992).
124
CHAPTER DETERMINATION OF LABETALOL
HYDROCHLORIDE IN DRUG FORMULATIONS
BY SPECTROPHOTOMETRY
125
INTRODUCTION
Labetalol hydrochloride i.e. Benzamide, 2-hydroxy-5-[l-hydroxy-2-[(l-methyl-
3-phenylpropyl)amino]ethyl]-, monohydrochloride (CAS No. 32780-64-6, M.W.
364.87) is an adrenergic P-receptor blocking agent used in the treatment of
hypertension, which exhibits both a and P-adrenoceptor blocking activity and because
of its use as doping agent in sports, this drug has been added to the list of forbidden
substances issued by the International Olympic Committee [1]. The drug is used to
induce hypotension during surgery as it decreases blood pressure more rapidly than
other beta blockers. The drug is quite sensitive, even a small dose of the drug gives
sufficient blockade, thus indicating that the drug is very much confined with the
cardio protective effects. Hence, it is must to determine the amount of labetalol
hydrochloride in drug formulations. The drug is official in Martindale, The Extra
Pharmacopoeia [2]. The assay procedure is cited in the monographs of British
Pharmacopoeia [3] and United States Pharmacopoeia [4], which describe a
potentiometric titration method and a liquid chromatographic method, respectively.
Several analytical methods such as thin layer chromatography (HPTLC) [5, 6], high
performance liquid chromatography (HPLC) [7, 8], liquid chromatography (LC) [9,
10], liquid chromatography coupled with mass spectrometry (LC-MS) [11,12], gas
chromatography coupled with mass spectrometry (GC-MS) [13] capillary
electrophoresis (CE) [14], electrochemical method [15] and flourimetry [16] are
reported for the estimation of labetalol hydrochloride. The determination in biological
fluids normally requires the use of trace analysis techniques, higher instrumentation
such as HPLC, LC, CE, cyclic voltammetry, flourimetry and hyphenated techniques
like LC-MS, GC-MS, inductively coupled plasma-mass spectrometry which normally
makes the method much more expensive. In addition to this, these methods require
3 26
long and tedious pretreatment of the samples and laborious clean up procedures prior
to analysis. UV-visible spectrophotometry is the technique of choice even today in the
laboratories of research, hospitals and pharmaceutical industries due to its low cost
and inherent simplicity. The analytical procedures based on spectrophotometry [17-
22] were frequently published in reputed journals. Few spectrophotometric methods
have been developed for the determination of labetalol hydrochloride in bulk drug and
pharmaceutical formulations. The quantification of the drug was done based on the
reaction of labetalol hydrochloride with Folin Ciocalteau's reagent, 4-
aminophenazone, 2,6-dichloroquinone chlorimide and ferric alum [23]; benzocaine
and p-nitroaniline in presence of triethylamine and sodium carbonate, respectively
[24]; p-N, N-dimethylphenylenediamine, and 3-methyl 2-benzothiazolinone
hydrazone hydrochloride in the presence of sodium hypochlorite and eerie ammonium
sulphate to form intensely coloured products with Xmax at 685 and 545 nm,
respectively [25]. The estimation of drug was done in the visible region based on ion-
pair formation between drug and reagents like suprachen violet 3 B [25] and wool fast
blue BL [26].
This chapter describes a selective and sensitive spectrophotometric method for
the determination of labetalol in drug formulations. The method is based on the
reaction of 4-aminobenzenesulphonic acid with dilute HCl and NaN02 in a
temperature range of 0 - 3 °C to form an electrophile i.e positive diazonium ion,
which immediately couples at ortho to the hydroxyl group of the benzene ring of
labetalol in Na2C03 medium forming a yellow azo product having Xmax at 395 nm.
The proposed method is carefully optimized and validated as per the International
Conference on Hormonisation (ICH) guidelines [27].
127
EXPERIMENTAL
Apparatus and reagents
The absorbance measurements were made on a Shimadzu UV-visible 1601
spectrophotometer (Kyoto, Tokyo, Japan) with matched quartz cells.
All chemicals and reagents were of analytical grade. Aqueous solutions of 4-
aminobenzenesulphonic acid (0.029 M; Fluka Chemie AG, Switzerland), NaN02
(0.29 M; Otto Chemie, Mumbai, India) and sodium carbonate (0.28 M; GR grade,
Merck Limited, Mumbai, India) were freshly prepared. A 0.2 M HCl was also
prepared by diluting 1.77 ml of pure HCl (35 %, GR grade, Merck Limited, Mumbai,
India) in 100 ml, distilled water.
Drug standard and commercial dosage forms
Labetalol hydrochloride as a reference standard was obtained from Sigma
Chemical Company (St. Louis, MO, USA). Standard solution of Labetalol
hydrochloride (0.04 %) was prepared in distilled water. Commercial dosage forms of
labetalol hydrochloride such as Lobet-100 mg tablet (Samarth Pharma Pvt. Ltd.,
Mumbai, India) and Normadate -50 mg capsule (Glaxo Smithkline, Mumbai, India),
were obtained from local drug store.
Spectrophotometric procedure for the determination of labetalol hydrochloride
Into a series of 25 ml standard volumetric flasks, 1.5 ml of 0.029 M 4-
aminobenzenesulphonic acid, 1.5 ml of 0.29 M NaNOa and 2.0 ml of 0.2 M HCl were
pipetted and kept in an ice-bath (0 - 3 °C) for 10.0 min to complete diazotization
reaction. To each flask, aliquot of 0.04% labetalol hydrochloride reference standard
solution (0.05 -1.1 ml) was added followed by 4.0 ml of 0.28 M sodium carbonate and
then diluted to volume with distilled water at room temperature (25 ± 1 °C). The
contents of each flask were mixed well and the absorbance was measured at 395 nm
128
against the reagent blank prepared similarly except drug. The colour was stable up
to 2 h. The amount of the drug was computed either from the calibration graph or the
regression equation.
Procedure for the determination oflabetalol hydrochloride in drug formulations
The powdered contents of 2 capsules of 50 mg strength (or 1 tablet of 100 mg
strength) were taken in 50 ml distilled water and left for 10 min for complete
dispersion and then filtered through Whatmann No. 42 filter paper (Whatmann
International Limited, Kent, UK) in a 250 ml standard volumetric flask. The residue
was washed well with distilled water for complete recovery of the drug and then
diluted up to the mark with distilled water. The assay was completed following the
recommended spectrophotometric procedure for the determination of labetalol
hydrochloride
Preparation of degraded labetalol
Labetalol hydrochloride was oxidized by treating 100 mg of the drug in 10 ml
distilled water with 0.5 ml saturated sodium carbonate solution and 2.5 ml of 0.3 M
sodium metaperiodate for 10 min at room temperature (25 ± 1°C). The excess of
sodium carbonate was neutralized with the corresponding volume of HCl and then the
products were isolated. The hydrolyzed solution was treated with methyl isobutyl
ketone. The two layers were formed and the organic layer was separated out. The
extract was then evaporated to dryness at room temperature and the residue as
compound 1 (2-hydroxy-5-formylbenzamide) was crystallized from distilled water.
Compound 2 (l-methyl-3-phenylpropylamine), which was in aqueous layer, was
extracted with dichloromethane. The extract was evaporated to dryness and the
residue was crystallized in ether. Thin layer chromatography was applied using silica
gel G plate and a mixture of n -propanol - 1 % ammonia (4:1, v/v) as mobile phase.
129
The bands corresponding to labetalol, compound 1 (2-hydroxy-5-formylbenzamide)
and compound 2 (l-methyl-3-phenylpropylamine) were located under UV lamp at
254 nm and the Rf values were found to be 0.70, 0.80 and 0.35, respectively. The
spots of compound 1 and 2 were too detected by spraying with 2, 4-
dinitrophenylhydrazine in 2M HCl and ninhydrin to give yellow and purple spots,
respectively, thus indicating the presence of carbonyl group in compound 1 and amine
group in compound 2. The literature has confirmed [28] that the two major products
of labetalol were 2-hydroxy-5-formylbenzamide (I, compound 1) and l-methyl-3-
phenylpropylamine (II, compound 2) (Scheme 3.1) thus confirming the above
findings.
Validation
The proposed method has been validated for specificity, linearity, precision,
accuracy, and recovery.
Specificity
The sample solutions of labetalol tablet and capsule were subjected to stress
conditions of light, heat, acid, base and oxidants. All stressed samples were analyzed
for labetalol content and compared to an unstressed time zero reference solution. The
reference assay value for the unstressed product was evaluated and the content of
degradation in the stressed and control samples was calculated relative to this assay
value.
Linearity
The linearity was evaluated by determining labetalol at ten concentration levels:
0.8, 1.6, 3.2, 4.8, 6.4, 8.0, 11.2, 14.4, 16.0 and 17.6 ^g ml''. Each concentration was
analyzed for five times.
130
'V
0 II
H—C
CONH2
, -OH
sodium metaperiodate in Na2C03 medium
li
Scheme 3.1.0xidative degradation of labetalol through N-dealkylation: 1.2-hydroxy-5-formyIbenzamide and II. l-methyI-3-phenylpropylamine.
131
Accuracy and precision
Three concentrations within the Hnearity range were selected: 1.6, 8.0, and 16.0
jig ml' for evaluating accuracy and precision. Five independent analyses at each
concentration level was performed within one day (intra day assay) and repeated for
five consecutive days (inter day assay).
Recovery studies
To study the accuracy of the proposed method and to check the interference
from excipients used in the formulations, recovery experiments were carried out by
standard addition method. For this, 1.2 ml (or 2.0 ml) of sample solution (0.4 mg ml')
was transferred into a 100.0 ml standard volumetric flask followed by 0.8 ml (or 2.0
ml) of reference standard solution (0.4 mg ml'') and volume was completed to the
mark with distilled water. The nominal value was determined by the recommended
procedure.
RESULTS AND DISCUSSION
Spectral studies
The aqueous solutions of pure labetalol and 4-aminobenzenesulphonic acid
(sulphanilic acid) exhibit absorption maxima at 208 and 249 nm, respectively. The
addition of NaN02 and HCl to 4-aminobenzenesuIphonic acid causes diazotization
and the resulting product absorbs maximally at 270 nm. The blank solution is
prepared similarly on addition of sodium carbonate to this diazotized solution, which
causes no shift in the absorption maximum. The blank solution when added to the
drug solution causes a remarkable shift in the absorption maximum due to the
formation of coupling product, which exhibits absorption band peaking at 395 nm
(Fig, 3.1), This reaction is exploited to develop a spectrophotometric method for the
determination of labetalol hydrochloride in drug formulations by measuring the
132
T 1 1 p 1 1 1 1 1 \ 1 1 1 1 r
150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550
Wavelength (nm)
Fig. 3.1. Absorption spectra of (a) pure drug solution: 10.0 jxg ml' of labetalol
hydrochloride in distilled water; (b) 8.67x10" M 4-aminobenzenesulphonic acid
in distilled water; (c) blank solution: diazotized solution (2 ml of l.TSxlO""* M 4-
aminobenzenesulphonic acid + 1.5 ml of 0.29 M NaNO: + 1.8 ml of 0.2 M HCl)
with 4.0 ml of 0.28 M NaiCOj in 25 ml distilled water and (d) sample solution:
440.0 |ig labetalol hydrochloride and 4.0 ml of 0.28 M NajCOs with diazotized
solution (1.5 ml of 0.029 M 4-aminobenzenesulphonic acid + 1.5 ml of 0.29 M
NaN02+ 1.8 ml of 0.2 M HCl) in 25 ml distilled water.
133
concrete absorbance at 395 nm.
Mechanism of the colour reaction
It has been reported that primary aromatic amines such as 4-nitroaniline and 4-
aminobenzenesulphonic acid on treatment with HCl and NaN02 in a temperature
range of 0-3 °C, yield diazonium salt [29]. However, only strongly nucleophilic
electron-rich aromatic or heteroaromatic compounds such as phenol can be coupled
with diazonium salts to yield coloured azo compounds [30]. The diazocoupling
reaction can be considered as a proton eliminating condensation of a positive
diazonium ion with another compound possessing an active hydrogen atom.
The substitution of electrophile usually proceeds para to the active group or ortho if
para position is already substituted. In the similar manner, 4-aminobenzenesulphonic
acid was diazotized to form a positive diazonium ion. Labetalol is a water-soluble
antiadrenergic P-blocker possessing -OH group attached to the benzene ring (phenol)
and forms phenolate ion in sodium carbonate medium [31]. The phenolate ion
undergoes coupling reaction with positive diazonium ion to form a yellow azo
compound, which absorbs maximally at 395 nm. In this case the coupling of
diazonium ion proceeds ortho to the hydroxyl group of the benzene ring of labetalol
because the para position in the drug is already occupied. The reaction sequence is
shown in Scheme 3.2.
Optimization of variables
The different parameters responsible for the formation of diazocoupling
coloured product were extensively studied. The optimum conditions for the
proposed method were established by conducting a series of experiments.
V/^
labetalol
Na2C03 in H2O
OH
phenolate anion
134
HO3S -.r\ NH,
4-aminoben2enesulfonic acid
NaN02 + 2HC1 0-3°C
HO3S- V-N- NCI
+ NaCl + 2H2O
HO3S—< Ch N = N
positive diazonium ion
diazo coupling
V
N=N \ /
SO,H
Azo coupling product (colored)
Scheme 3.2. Azo compound of labetalol (yellow coloured) formed due to union between the positive diazonium ion and a phenolate anion in sodim carbonate medium.
135
Effect of time
To optimize the reaction time for colour development, 1.74 x 10' M 4-
aminobenzenesulphonic acid was treated with 1.74 x 10" M NaN02 and 1.60 x 10"
M HCl in a 25 ml standard volumetric flask. The content of the mixture was
maintained in an ice bath (0 - 3 °C) for varied time of 2 - 14 min and then the flask
was taken out. To each flask, 1.0 ml of 0.04% labetalol hydrochloride and 4.48 x 10'
^ M Na2C03 were added and diluted to volume with distilled water at room
temperature (25 ± TC). The maximum intensity of colour was obtained at 7 min of
cooling and remained constant up to 14 min. Therefore, 10 min of reaction time was
used throughout the determination process.
Effect of 4-aminobenzenesulphonic acid concentration
The influence of the concentration of 4-aminobenzenesulphonic acid on the
development of colour was investigated by treating varied concentration (1.16 x 10'
- 2.32 X lO"-' M) of 4-aminobenzenesulphonic acid with 1.74 x 10' M NaNOa and
1.60 X 10- M HCl in a 25 ml standard volumetric flask and was cooled in an ice
bath for 10 min. After cooling, 400.0 |Lig labetalol hydrochloride and 4.48 x 10' M
NaaCOj were added and diluted to volume with distilled water at room temperature
o
(25 ± 1 C). It was observed that the absorbance measured at 395 nm increased with
increasing concentration of 4-aminoben2enesulphonic acid and became constant at
1.16x10" M, above this concentration absorbance remained constant (Fig. 3.2 a).
Therefore, the concentration of 1.74 x lO'"* M 4-aminobenzenesulphonic acid proved
to be sufficient for the maximum concentration of labetalol hydrochloride.
Effect ofNaN02 concentration
The effect of the concentration of NaNOa on the colour development was
investigated in the range of 1.16 x 10" - 2.32 x IQ" M. The absorbance (Fig. 3.2
136
b) increased with increasing concentration of reagent and became constant at 1.16 x
10" M, above which up to 2.32 x 10" M, absorbance remained constant. Therefore
1,74 X 10" M; NaNOo was chosen as an optimum value for the determination
process.
Effect ofHCl concentration
The influence of the concentration of HCl on the colour development has been
investigated in the range of 1.60 x lO'- M - 1.84 x IQ'- M and the results are shown
in Fig. 3.2 c. The highest absorbance was obtained with 1.04 x 10' M HCl, above
this concentration up to 1.84x10' M, the absorbance remained constant. Therefore,
the concentration of 1.44x10" M HCl was used in all measurements.
Effect ofNa2C03 concentration
The effect of the concentration of NaaCOs was studied in the range of 5.60 x 10' -
5.04 X 10"2 M. As can be seen from Fig. 3.2 d, the absorbance of the coloured
product incretised with increasing concentration of Na2C03 and remained constant at
3.36 X 10" M. Further increase in concentration of the reagent up to 5.04 x 10' M
caused no change in the absorbance. Thus, 4.48 x lO'' M NaiCOs was used as an
optimum value for the determination of iabetalol.
Analytical data and calibration graph
The calibration graph between the absorbance at 395 nm and the Iabetalol
hydrochloride concentration was found to be linear over the concentration range of
0.8 - 17.6 \ig mf' with molar absorptivity and sandeli's sensitivity of 2.874 x IC* 1
mol" cm" and 0.013 jig cm" per 0.001 absorbance unit, respectively. The linear
regression equation of the proposed method has been evaluated by the least square
treatment of the calibration data (n = 10). The linear regression equation and
coiTelation coefficient are A = 4.84 x lO""* + 7.864 x 10" C where A is the
137
o c CD
n L.
o CO
<
.000 0.006 0.012 0.018 0.024 0.030 0.036 0.042 0.048 0.054 0.060
Concentration of labetalol hydrochloride, M
Fig. 3.2. Effect of the molar concentration of reactants: (a) 4-
aminobenzenesulphonic acid; (b) NaNO:; (c) HCI; and (d) Na2C03 with 16.0 jig
ml' labetalol hydrochloride on the absorbance of the coloured product.
138
absorbance and C is the concentration of iabetalol hydrochloride in |j,g ml'' and r =
0.9999, respectively, which indicated an excellent linearity. Table 3.1 summarizes
Beer's law limit; linear regression equation; correlation coefficient; confidence
limits and standard deviations for slope and intercept at 95% confidence level;
variance; detection limit and quantitation limit [32] for the proposed method.
The statistical analysis of the calibration data has made possible to calculate
error (Sc) in the determination of a given concentration of Iabetalol hydrochloride
using the following equation [33];
^•-T
1/2
, 1 (A-A)' 1 + - + n b'Y.(C.-C)'
where A and C are average values of the absorbance and concentration, respectively,
for n standard samples. The plot of Sc versus concentration of Iabetalol
hydrochloride (Fig. 3.3) indicates that the minimum error is obtained when the
analysis is carried out at 8.40 jig ml"'. The value of Sc can be used to establish the
confidence limit at the selected level of significance for the determination of
unknown concentrations of Iabetalol hydrochloride using the relation Cj ± tSc. The
resuhs are presented graphically (Fig. 3.4) by plotting the relative uncertainty
(%AC/C) against the concentration of Iabetalol hydrochloride [34] at 95 and 99 %
confidence levels for n-2 degrees of freedom. Thus, the confidence limit can be
established and the relative uncertainty can be estimated directly on the
concentration over the full range of the concentration tested.
Specificity
The specificity of the proposed method was evaluated by estimating the
amount of Iabetalol in the presence of varying amounts of degraded products of
139
Table 3.1 Optical performance and regression characteristics of the proposed method
Parameters
>max (nm)
Stability (h) Beer's law limit (}j,g ml"') Molar absorptivity (1 mol"' cm"')
Linear regression equation
Sa
±tSa
Sb
±tSb Correlation coefficient (r) Detection limit (fig ml"')
Quantitation limit (|J,g ml"')
Proposed method
395
02 0.8- 17.6
2.87 X 10'
A = 4.84x 10"'+7.86 X 10"^C 1.02 X 10"
2.35 X 10" 9.99 X 10"
2.30 X 10"'
0.9999 0.08
0.23
Sa and Sb are standard deviation of intercept and slope, respectively.
\40
O L_
LU
0.0275
0.0270
0.0265 -
0.0260 -
0.0255
0.0250 -
0.0245
0.0240 — I \ 1 1 1 1 \ — — 1 1 — • — I 1 1 1 1 1 1 1 — ' — I —
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Concentration of labetalol hydrochloride (j g mf^)
Fig. 3.3. Plot of error (Sc) versus concentration of labetalol
hydrochloride in the determination process.
141
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Concentration of labetalol hydrochloride ( ig nril' )
Fig. 3.4. Plot showing variation in the confidence limit at (a) 95% confidence
and (b) 99 %confidence level: relative uncertainty percentage (% AC/C) versus
concentration of labetalol hydrochloride.
142
labetalol such as 2-hydroxy-5-formylbenzamide and l-methyl-3-phenylpropylamine,
which were the major degradants of labetalol. It was found that l-methyl-3-
phenylpropylamine did not interfere with the proposed method whereas 2-hydroxy-
5-formylbezamide interferes seriously.
The various excipients commonly used in dosage forms such as sodium stearyl
fumarate, magnesium stearate, starch, lactose and talc did not interfere with the
proposed method. The samples were stressed by light, acid and base for 2, 2 and 5-
day-time points, respectively at room temperature (25 ± 1°C). It was observed that
stress by such conditions did not cause significant degradation. There was no change
in the absorption spectra of the pure drug and stressed (light, acid and base) sample
solutions. The two reported metabolites of labetalol such as 0-sulphate (I, major)
and 0-glucuronide (11, minor) (Scheme 3.3) were not interfering with the proposed
method because of denature of the phenolic group in these metabolites [35]. The
proposed method is specific and selective as not giving reaction with compounds,
which lack phenol group in the moiety.
Robustness
The robustness of the proposed method relative to each operational parameter
was challenged. The operational parameters investigated were as follows:
• volume of 0.029 M 4-aminobenzenesulphonic acid, 1.5 ml (± 0.5 ml)
• volume of 0.29 M NaN02, 1.5 ml (± 0.5 ml)
• volume of 0.2 M HCl, 2.0 ml (± 0.5 ml)
• volume of 0.28 M Na2C03,4.0 ml (± 1.0 ml)
• cooling time of 10 min (± 3.0 min)
Under these conditions quality control sample solution containing 8.0 fig
ml" (Lobet 100, Samarth Pharma) of labetalol hydrochloride was assayed five times.
143
hepatic metabolism
CONH2
-OSO2O'
Scheme 3.3 Metabolites of labetalol: I by sulphation and II by glucuronidation process.
144
The results obtained by the quality control sample represent good recovery with
acceptable bias of OL >0.98 and BU < 1.02.
Ruggedness
The method ruggedness was evaluated by a second analyst using a different
spectrophotometer (Spectronic 20D" , Milton Roy Company, USA) and freshly
prepared standard sample solutions. The quality control tablet and capsule samples
of labetalol hydrochloride were analyzed five times at one concentration level (8.0
l g ml'') by the ruggedness chemist and developing chemist following the
recommended procedure. The % recovery ± RSD resulted from ruggedness chemist
(tablet: 99.97 ± 0.63%; capsule: 100.06 ± 0.58) and developing chemist (tablet:
99.98 ± 0.52%; capsule; 99.97 ± 0.53%) were compared. The % recovery and RSD
results agreed well within the acceptable limits with permissible bias of all samples
within ± 2.0%).
Accuracy and precision of the proposed methods
The accuracy and precision of the proposed method was established by
determining the content of labetalol in pure and pharmaceutical formulations at three
different concentration levels (1.6, 8.0, and 16.0 j g ml''). The intra day and inter
day precisions of the proposed method in pure form was evaluated by carrying out
five independent analyses at each concentration level within one day and on five
consecutive days (Table 3,2). In the same manner, the intra and inter day precisions
was also performed in drug formulations by measuring the amount of labetalol at
each concentration level (Table 3.3). The results presented in Table 3.2 and 3.3 can
be considered to be very satisfactory.
145
Table 3.2 Intra day and Inter day assays: Test of accuracy and precision of the proposed method in pure form
Intra day Assay
Amount (p.g
Taken
1.6
8.0
16.0
Found ::
1.60 ±
8.00 ±
16.01 ±
ml"')
hSD'
0.04
0.04
0.04
SAE''
0.019
0,019
0.019
C.L'
0.053
0.052
0.053
Inter day Assay
Amount
Taken
1.6
8.0
16.0
(^gml-')
Found ± SD'
1.60 ±0.05
8.01 ±0.05
16.01 ±0.05
SAE''
0.021
0.021
0.022
C.L'
0.059
0.058
0.061
Mean for five independent analyses. '' SAE, standard analytical error. '^ C.L., confidence limit at 95% confidence level and four degrees of freedom (t = 2.776).
146
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147
The validity of the proposed method was evaluated by recovery studies using
the standard addition method. For this purpose, a known amount of reference drug
was added to formulated tablets and capsules at two different concentration levels
and the nominal amount of drug was determined by the proposed method. Each
concentration level was analyzed five times and the results (Table 3.4) show good
recovery with low SD and RSD. No interference from the common excipients was
observed.
The applicability of the proposed method has been tested for the determination
of labetalol in drug formulations. The results of the proposed method were compared
with those obtained by the Bilal's spectrophotometic method [24] using point and
interval hypothesis tests [36]. The student's t and F-values at 95% confidence level
did not exceed the tabulated t- and F-values, confirming no significant difference
between the performance of the proposed method and the reference method (Table
3.5). Once it is ensured that the proposed method is not significantly different from
the reference method, it is necessary to check the bias of each drug sample based on
recovery experiments using the interval hypothesis test. In pharmaceutical analysis,
The Canadian Health Protection Branch has recommended a bias of ± 2 % with
lower limit (BL) of 0.98 and upper limit (0u) of 1.02 [37]. It is evident from Table 3.5
that the true bias of all samples is < ± 2 %. The interval hypothesis test has
confirmed that the accuracy and precision are within the acceptable limits and
indicated no significant difference between the performances of the proposed
method and the reference method at 95% confidence level.
The proposed method for the determination of labetalol hydrochloride in drug
formulations was compared favourably with other existing spectrophotometric
methods (Table 3.6). The molar absorptivity of the proposed method is higher than
148
Table 3.4 Standard addition method: Evaluation of the validity of the proposed method for the recovery of labetaloi hydrochloride
Formulations^
Tablet
Lobet 100 (Samarth Pharma)
Capsule
Normadate 50 (Glaxo Smithline)
Concentration (^gml"') Taken Added
4.8 3.2 8.0 8,0
4.8 3.2 8.0 8.0
Found ± SD''
7.99 ±0.04 16.01 ±0.05
7.99 ±0.04 16.01 ±0.04
Recovery ± RSD^ (%)
99.90 ±0.52 100.09 ±0.27
99.97 ±0.53 100.07 ±0.27
SAE
0.018 0.019
0.019 0.019
C.L.
0.051 0.053
0.053 0.053
^Mean for five independent analyses
149
Table 3.5 Point and interval hypothesis tests: Evaluation of the applicability of the proposed method at 95yo confidence level
Formulations^ Proposed method Reference method t & F- 9L' BU* Recovery RSD' Recovery RSD'' values'' (%) (%) (%) (%)
Tablet
99.97 Lobet 100 (Samarth Pharma)
Capsule
'Normadate 50 100.03 (Glaxo Smithkline)
0.63 99.89 0.8! t = 0.21I 0.991 l.OI F= 1.626
0.52 99.98 0.86 t = 0.148 0.987 1.014 F = 2.722
*Mean for five independent analyses. ''Theoretical t-value (v = 8) and F-value (v = 4, 4) at 95 % confidence level are 2.306 and 6.39, respectively. ' In pharmaceutical analysis, a bias, based on recovery experiments, of ± 2% is acceptable.
150
Table 3.6
Comparison of the proposed method with existing spectrophotometric methods
for the determination of labetalol hydrochloride in drug formulations
Reagents
2,6-dichloroquinone chlorimide
Folin Ciocalteau's reagent
4-aminophenazone
Ferric ammonium sulfate
p-N,N-dimethylphenylene-
diamine hydrochloride & sodium
hypochlorite
'^niiix
(nm)
670
650
510
530
685
Beer's law limit (Mgml-')
5-25
8-40
2,5-15
50-250
-
RSD (%)
-
-
-
-
0.35-0.52
Molar absoptivity
(1 moP' cm"')
1.39x10'
5.88x10'
2.09x10'
1.05 xIO'
References
[23]
[23]
[23]
[23]
[25]
3-methyl 2-benzothiazolinone 545
hydrazone hydrochloride & eerie
ammonium sulfate
0.35-0.52 [25]
Suprachen violet 3B"
Wool fast blue BL'
Diazotized
4-aminobenzenesulfonic acid
565
600
395
-
1-6
0.8-17.6
0.35-0.52
0.64
0.34-0.52 2.87x10'
[25]
[26]
This work
'Extractive method.
151
that of other methods indicating its greater sensitivity. It has the advantage of
determining labetalol as low as 0.8 |.ig ml"'. Moreover, the proposed method is fast
with respect to analysis time and directly applicable to the drug sample without prior
extraction needed in other methods [25, 26]. The method is versatile and useful due
to independence from pH and high tolerance limit from common excipients present
in drug formulations.
152
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2] Martindale, The Extra Pharmacopoeia 33rd edition, UK: Royal Pharmaceutical
Society, London, (2002) p. 918.
3] British Pharmacopoeia, I, H. M. Stationery Office U.K. London (1998) p. 768.
4] The United States Pharmacopoeia, 24th edition Rockville, MD, USA (2000) p.
949.
5] D. R. Jack, S. Dean, M. J. Kandall, S. Laugher, J. Chromatogr. 196 (1980) 189.
6] A. Witek, H. Hopkala, G. Matysik, Chromatographia 50 (1999) 41.
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B: Biomed Sci. Appl. 705 (1998) 97.
8] Y. J. Park, D. W. Lee, W.- Y. Lee,/Ira/. Chim. Acta Al (2002) 51.
9] L Rapado-Martinez. R. M. ViUanueva-Camanas,M. C. Garcia-Alvarez-Coque,
Anal. Chem. 71 (1999)319.
10] S. Cardo-Broach, 1. Rapado-Martinez, J. Esteve-Romera,M. C. Garcia-
Alvarez-Coque J. Chromatogr. Sci. 37(1999) 93.
II] M. Thevis, G, Opfermann, W. Schanzer, Biomed. Chromatogr. 15 (2001) 393.
12] M. Gergov, J. N. Robson, E. Duchoslav, I. Ojanpera,. J. Mass Spectrom. 35
(2000)912.
13] H. Siren, M. Saarinan, S. Hainari, P. Lukkar,M. L. Reikkola, J. Chromatogr.
/( 632 (1993) 215.
14] J. Sadecka, J. ?o\onsky, Pharmazie 50 (1995) 434.
15] A. Voulgaropoulos, M. Sofoniou, E. Kazakou, Electroanalysis, 5 (1993) 525.
16] F. Belal, S. Al-Shaboury, A. S. Al-Tamrah, J. Pharm. Biomed Anal. 30,
(2002)1191.
153
[17] M. I. Walash, M. K. Sharaf-El-Din, M. E. Metwally, M. R. Shabana, J. Chin.
Chem. Soc. 52(2005)71.
[18] A. M. El-Brashy, M. -S. Metwally, A, El-Sepai, J. Chin. Chem. Soc. 52 (2005)
253.
[19] D. R. El-Wasseef, M. Eid, F. Belal,./. Chin. Chem. Soc.52 (2005) 507.
[20] A. Raza, T. M. Ansari, A. Rehman, J. Chin. Chem. Soc. 52 (2005) 1055.
[21] M. M. Hosny,,/. Chin. Chem. Soc. 53 (2006)465.
[22] K. Guclu, K.Sozgen, E. Tutem, M. Ozyurek, R. Apak, Talcmta 65 (2005) 1226.
[23] R. T. Sane, T. G. Chandrashekar, V. G. Nayak, Indian Drugs 23 (1986) 565.
[24] F. Belal, S. Al-Shaboury, A. S. Al-Tamrah,. IL Fdrmaco 58 (2003) 293.
[25] C. S. P. Sastry, D. P. M. Krishna, Mikrochim. Acta 122 (1996) 87.
[26] C. S. P. Sastry, S. G. Rao, K. R. Srinivas, Indian Drugs 35 (1998) 595.
[27] ICH Q2A, Text on Validation of Analytical Procedures, Fed. Regist. 60
(1995)11260.
[28] H. Salomies, L. Luukkanen, R. Knuutila, J. Pharm. Biomed. Anal. 7(1989)
1447.
[29] K. Krohn, M. John, E. I. Demikhov, Russ. Chem. Bull 50 (2001)1248.
[30] H. Zollinger, Colour Chemistry: Synthesis, Properties and Applications of
Organic Dyes and Pigments, VCH, Weinheim, (1987) p. 85.
[31] D. Barton, W. D. Ollis, Alcohols, phenols, ethers and related compounds
Comprehensive Organic Chemistry: The Synthesis and Reactions of Organic
Compounds In: ( edited by J. F. Stoddart)Volume 1, Pergamon Press Ltd.,
Headington Hill Hall, Oxford 0X3 OBW, England (1979) p. 772.
[32] J. Ermer, J. Pharm. Biomed Anal 24 (2001)755.
154
[33] J. Mendham, R. C. Denney, J. D. Barnes, M. Thomas, Vogel's Textbook of
Quantitative Chemical Analysis, Statistics: Introduction to Chemometrics, 6th
edition, Pearson Education, Singapore (2002) p. 137.
[34] R. Cassidy, M. Janoski, LC-GC 10 (1992) 692.
[35] W. O. Foye, Principles of Medicinal Chemistry In: ( edited by D. A. Williams)
New Delhi, India (1995)
[36] C. Hartmann, J. Smeyers-Verbeke, W. Penninckx, Y. V. Heyden, P.
Vankeerberghen, D. L. Massart, Anal. Chem. 67 (1995) 4491.
[37] Canada Health Protection Branch, Drugs Directorate Guidelines: Acceptable
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(1992).
155
CHAPTER-4 KINETIC SPECTROPHOTOMETRIC METHODS
FOR THE DETERMINATION OF RAMIPRIL IN
COMMERCIAL DOSAGE FORMS
156
INTRODUCTION
Ramipril, 2-[N-[(S)-l-ethoxy carbonyl-3-phenyl propyl]-L-alanyl]-(lS, 3S,
5S)-2-azabicyclo [3.3, 0]-octane-3-carboxylic acid [M.W. 416.51] is an angiotensin-
converting enzyme inhibitor (ACE). It is used in the treatment of hypertension, heart
failure and following myocardial infarction. It is also used to reduce the risk of
cardiovascular events in patients with certain risk factors. Ramipril acts as a prodrug
of diacid ramiprilat. Ramipril owes its activity to ramiprilat to which it is converted
after oral administration [1,2]. This drug is officially listed in British Pharmacopoeia
[3] which describes a potentiometric titration procedure for its assay in tablets. In
order to assure the quantity of ramipril in dosage forms, several methods have been
reported which include LC-MS [4], HPLC [5-8], capillary electrophoresis [9],
atomic absorption spectrophotometry [10,11], voltammetry [12], ion selective
electrode potentiometry [13], and radioimmunoassay [14].
The literature revealed that several spectrophotometric methods have been
reported for the quantitative analysis of ramipril in pharmaceutical preparations and
biological fluids. The estimation of ramipril in a binary mixture was carried out by
derivative compensation technique [15] as well as zero crossing derivative technique
[16, 17]. The determination of ramipril was carried out based on ion-pair complex
of the drug with picric acid and bromocresol green [18], formation of ternary
complex of the drug with Cu (II) and eosin [10], and Fe (III) and ammonium
thiocyanate [11]. The charge transfer complexation reaction has also been utilized
for its quantification using 7,7,8,8,tetracyanoquinodimethane, p-chloranilic acid and
2,3-dichloro-5,6-dicyano-p-benzoquinone [19]. Potassium permanganate oxidizes
ramipril in alkaline medium and itself reduces to manganate ion, which was
157
measured at 610 nm [20]. The estimation of ramipril has been done by
spectrophotometry and fluorinietry utilizing the reaction of the drug with 7-fluoro-4-
nitrobenzo-2-oxo-l,3-diazole, which exhibits maximum absorbance at 460 nm, and
maximum fluorescence intensity at 530 nm after excitation at 465 nm [21]. The
reaction of ramipril with a mixture of potassium iodide and potassium iodate has
been utilized for its assay in commercial dosage forms [22].
This chapter describes a simple and sensitive spectrophotmetric method for the
determination of ramipril in bulk and dosage forms. The method is based on the
reaction of ramipril with 1- chloro 2, 4- dinitrobenzene (CDNB) in
dimethylsulphoxide (DMSO) at 100 ± Tc. The absorbance (kmax = 420 nm) of the
coloured product increases as a function of heating time. Therefore, two calibration
procedures i.e. fixed time (AA) and equilibrium methods are adopted for the
determination of ramipril in bulk and commercial dosage forms.
EXPERIMENTAL
Apparatus
Spectral runs were made on a Shimadzu UV-visible 1601 spectrophotometer
(Model no. 1601, Kyoto, Japan). All other spectrophotometric measurements were
made on Spectronic 20 D"*' spectrophotometer (Milton Roy Company, USA). A
water bath shaker (NSW 133, New Delhi, India) was used for heating.
Material and reagents
Ramipril reference standard (Batch No. TR001FO2 ) was kindly provided by
Dr. Reddy's Laboratories Limited (Andhra Pradesh, India). CDNB and DMSO were
purchased from Merck Limited, Mumbai, India. The pharmaceutical preparations of
ramipril such as Ramipres-2.5 (Cipla, Mumbai, India), Ramace-2.5 (AstraZeneca,
158
Mumbai, India) and Hopace-2.5 (Cardicare, Bangalore, India) were obtained from
local market.
Standard solutions:
• 0.7% CDNB (3.46 x 10' M) was prepared in DMSO.
• 0.1% ramipril (2.40 x 10' M) was also prepared in DMSO.
Procedure for determination
Accurately measured aliquots of standard solution of ramipril corresponding
to 0.1 - 1.1 mg were transferred into a series of 5 ml standard volumetric flasks. To
each flask, 2.2 ml of 3.46 x 10' M CDNB was added and diluted to volume with
DMSO. The content of the mixture was kept on water bath at 100 ± 1°C for a
preselected fixed time. The absorbance of the yellow coloured product was
measured at 420 nm against the reagent blank as a function of time. The following
two procedures were adopted for constructing the calibration graphs:
• Fixed-time method (l^A): The change in absorbance (AA) between the times, ti
(3 min) and t2 (6 min) was computed and plotted against the initial
concentration of ramipril.
• Equilibrium method: The equilibrium was attained at 30 min of heating. The
absorbance measured at 32 min of heating was plotted against the initial
concentration of ramipril.
Alternatively, regression equations were also developed for estimation of the
content of ramipril.
Analysis of commercial dosage forms
To determine the amount of ramipril in pharmaceutical preparations (label
claim: 2.5 mg / tablet or capsule), the contents of 20 tablets or capsules were
weighed and finely powdered. A portion of the powder equivalent to 50 mg ramipril
159
was stirred with 20 ml DMSO and let stand for 10 min. The residue was filtered on
Whatmann No, 42 filter paper (Whatmann International Limited, Kent, UK) and
washed with DMSO. The filtrate and washings were diluted to 30 ml with DMSO.
This solution was fiarther diluted as necessary to complete the analysis following the
recommended procedures.
Procedure for reference method [10]
An aliquot of standard solution containing 0.1 - 1.0 mg of ramipril was
transferred into a series of 50.0 ml separating funnels. The volume of each solution
was adjusted to 10.0 ml with distilled water and 3.0 ml of 0.2% Cu (II) sulphate and
1.0 ml of 0.1% eosin were added with swirling. The funnels were shaken vigorously
with 3.0 X 3.0 ml portions of chloroform for 1.0 min, and then allowed to pass the
organic layer through anhydrous sodium sulphate into a 10.0 ml standard volumetric
flask. The volume of chloroform layer was made up to 10.0 ml and the absorbance
was measured at 535 nm against the reagent blank prepared similarly and a
calibration graph was constructed.
Validation Parameters
Specificity
The specificity of the proposed method was evaluated by determining the
ramipril concentration in the presence of varying amounts of degraded product of
ramipril (ramipril diketopiperazine) and common excipients (sodium stearyl
fumarate, magnesium stearate, starch, lactose and talc).
Recovery studies
Recovery experiments were carried out by standard addition method. For this,
4.0 ml (or 8.0 ml) of reference ramipril solution (1.0 mg ml"') was transferred into a
100.0 ml volumetric flask followed by 6.0 ml of sample solution (I.O mg ml'') and
160
volume was completed up to the mark with DMSO. The total amount was
determined by the recommended procedure.
Limits of detection and quantitation
The limits of detection (LOD) and quantitation (LOQ) were calculated using
the following equations [23]:
LOD = 3.3 xSo/bandLOQ=\OxSo/b
where So and b are the standard deviation and slope of the calibration line,
respectively.
RESULTS AND DISCUSSION
A yellov*/ coloured product with maximum absorption at 420 nm was formed
when ramipril was allowed to react with CDNB in DMSO while the reagent, CDNB,
exhibited absorption band at 350.5 nm. (Fig. 4.1) At room temperature, the reaction
did not take place, hence the reaction was carried out at 100 ± 1°C to increase the
rate of reaction and decrease the time for attaining equilibrium. At this temperature,
the intensity of the coloured product increased with time and attained equilibrium in
30 min. The increase in absorbance as a function of time was utilized to develop a
kinetically based spectrophotometric method for the quantitation of ramipril.
Optimization of reaction conditions
The optimum conditions affecting the formation of Meisenheimer complex
were studied and maintained throughout the experiment to determine the quantity of
ramipril in bulk and drug formulations.
Effect of heating time
To optimize the reaction time for colour development, 2.2 ml of 3.46 x 10" M
CDNB and 1.0 ml of 2.40 x lO'' M ramipril were added and kept on water bath at
0
100±1 C for varied time. The maximum intensity of colour was obtained at 30 min
161
280 320 360 400
Wavelength (nm)
520
Fig. 4.1. Absorption Spectra of (a) 4. 80 x lO' M ramipril (b) 3.46 x 10' M
CDNB versus DMSO (c) Complex (4. 80 x 10 M ramipril and 1.73 x 10 M
CDNB) versus Blank (1.73 x 10 M CDNB).
162
of heating and remained constant up to 34 min. Therefore, 32 min of reaction time
was used in equihbrium method (Fig. 4.2).
Effect of the concentration ofCDNB
The effect of the concentration of CDNB on the coloured product was studied
using the equilibrium procedure in the range 1.38 x 10' - 1.66 x 10' M. The
absorbance increased with increase in concentration of CDNB. The maximum
absorbance was obtained with 1.38x10' M; above this concentration absorbance
remained constant (Fig 4.3). Thus, 1.52 x 10' M was used for subsequent studies.
Stoichiometry
The stoichiometry of the reaction was established by limiting logarithmic
method [25]. The slopes of the plots of logarithm of absorbance versus logarithm of
respective varied molar concentration were calculated and found to be 1:1 between
ramipril and CDNB (Fig 4.4). The mole ratio method was also employed to evaluate
the combining ratio. The plot of absorbance versus mole ratio (ramipril/ CDNB) has
also confirmed that one mole of ramipril reacted with 1 mole of CDNB (Fig 4.5).
The apparent formation constant and Gibbs free energy (A G*) were calculated and
found to be 7.20 xlO^ and -56.12 KJ mol"', respectively.
Mechanism of the reaction
A literature survey revealed that brightly coloured products are formed when
polynitroaromatic and halopolynitroaromatic compounds react with bases. The
formation of coloured products is due to various interactions depending upon the
degree to which the base participates through its unshared electron pair with the
nitro compounds [25]. Ramipril possesses -NH group in its structure thus behaves
as a base. The addition of CDNB to ramipril in DMSO yielded the 1-substituted
Meisenheimer complex, which absorbs maximally at 420 nm. On the basis of our
163
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
Time of heating (min)
Fig. 4.2. Effect of heating time on the absorbance of the coloured product
(ramipril 200.0 ^g ml').
164
-^ 1 1 1 1 1 1 r
0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 0.020
Concentration of CDNB (M)
Fig. 4.3. Effect of the concentration of CDNB on the absorbance of the coloured
product (ramipril 200.0 |j,g ml"').
165
-1.6
-2.0 -
-2.4
a, -2S o
TO
o -3.2 m n <
o -3.6
-4.0
-4.4
-4.8
log A = -3.422 ±0.002 (r = 0.99904)
.002 ± 0.003 log [ramipril
log A = -2,063 ± 0.007 + 0.995 ± 0.016 log [CDNB] (r = 0.99999)
-1.0 -0.8 -0.6 -0.4 -0.2
Log [Ramipril] or [CDNB]
0.0 0.2
Fig. 4.4. Limiting logarithmic plots for molar combining ratio between
ramipril and CDNB: (a) log Absorbance versus log [Ramipril]; (b) log
Absorbance versus log [CDNB].
166
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Mole ratio (Ramipril / CDNB)
Fig, 4,5. Mole ratio plot for the reaction between ramipril and CDNB in DMSO
([Ramipril] = [CDNB]) - 3,46x10" M).
167
experimental findings and literature background [26] the reaction mechanism is
proposed and given in Scheme 4.1.
Solution stability
The solution stability was ascertained from UV spectra of reference standard
and quality control samples. The reference standard of the drug showed an
absorption peak at 260 nm. The standard and quality control sample solutions were
kept at room temperature for five days and it was observed that there was no change
in the absorption spectra of these solutions. The solution stability was also
ascertained by applying the standard and quality control samples on TLC plates
coated with silica gel G (Merck Limited, Mumbai, India) and developed in
methanol-chloroform (2:8 v/v) solvent system. The plates were air-dried and spots
were detected in the iodine chamber. In both the cases, a single spot was observed
with Rf = 0.80 corresponding to ramipril.
Analytical performance
Under the optimized experimental conditions mentioned above, the
absorbance of each solution was recorded as a function of time at a regular interval
of 3 min. The change in absorbance (AA) between the times ti (3 min) and t2 (6 or 9,
12, 15 min) was computed and plotted against the initial concentration of ramipril.
The corresponding linear regression equations with coefficient of correlations are
summarized in Table 4.1. As can be seen from the table that the most acceptable
linearity was obtained when the change in absorbance between 3 and 6 min (i.e. AA
= Ae - A3) was plotted against the initial concentration of ramipril. This fixed time
was adopted for the assay procedure.
168
CDNB
Ramipril
DMSO
At 100°C
.„n\\\H
C OH
+ HCl
C OH
Meisenheimer yellow complex
Scheme 4.1
Table 4.1 Calibration equations at different fixed times
169
AA Calibration equation
A6-A3 AA= 6.30X 10""+1.54x 10"- C 0.9999 4.14x10"" 3.14x10""
A9-A3 AA = -1.43 X 10-' + 2.04x 10"^C 0.9998 1.81x10"' 1.37x10"^
A,2-A3 AA = -3.36x 10--' + 2.62x 10"'C 0.9998 2.17x10" ' 1.64x10"'
A,5-A3 AA = -6.90x 10"' + 3.16x 10"'C 0.9997 4.18x10"^ 3.16x10"'
170
The reaction between ramipril and CDNB required 30 min to complete.
Therefore, in equilibrium procedure, the calibration curve was constructed by
plotting absorbance measured after attaining the equilibrium (32 min) against the
initial concentration of ramipril. The linear dynamic range, molar absorptivity,
regression equation, correlation coefficient, confidence limits for slope and
intercept, limits of detection and quantitation, and variance of calibration line for
both the procedures are summarized in Table 4.2. The high values of correlation
coefficient indicated the excellent linearity of the calibration curves. The small
values of confidence limit of slope and intercept pointed towards good
reproducibility of the proposed procedures.
The accuracy and precision of the fixed time and equilibrium methods were
evaluated by performing five successive measurements within one day at three
different concentration levels (40, 120 and 220 |.ig ml"'). The inter day precision was
measured by assaying the bulk sample on five consecutive days. The results are
summarized in Table 4.3. The results showed that the RSD (< 0.22%) and standard
analytical error (< 0.04) found in intra and inter day assays can be considered to be
very satisfactory.
To test the applicability of the proposed methods, recovery experiments were
performed using the standard addition method. For this purpose, a known amount of
pure ramipril was spiked to its formulated preparations and the total amount of the
drug was estimated. The results are summarized in Table 4.4. As can be seen from
the table that the mean recovery ranged from 99.98 - 100.03% with low RSD values.
Moreover, it was also found that the degraded product of ramipril and common
excipients such as sodium stearyl fumarate, magnesium stearate, starch, lactose and
talc did not interfere with quantitation process.
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Table 4.3 Evaluation of accuracy and precision of the proposed methods
Proposed methods
Fixed time (A A)
Intra day assay
Inter day assay
Equilibrium method
Intra day assay
Inter day assay
Amount (^g ml-') Taken
40.0 120.0 220.0
40.0 120.0 220.0
40.0 120.0 220.0
40.0 120.0 220.0
Found ± SD'
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39.97 ±0.08 119.99 ±0.06 219.94 ±0.07
39.99±0.08 119.98 ±0.07 219.98±0.07
40,01 ±0.09 120.01 ±0.08 220.03 ±0.08
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0.22 0.07 0.04
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0.099 0.080 0.092
0.100 0.085 0.089
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174
The proposed methods have been applied for the analysis of ramipril in
commercial dosage forms. The analysis of the same batch of samples was also
completed by Abdellatefs spectrophotometric method [10]. The results of the
proposed method (fixed time or equilibrium method) were compared with those
obtained by the Abdellatefs spectrophotometric method. The student's t- and F-
values at 95% confidence level did not exceed the tabulated t- and F- values. The
interval hypothesis test [27] has also been performed to compare the results at 95%
confidence level. The results presented in Table 4,5 showed that the true bias of all
samples is smaller than ±2% indicating the compliance of the regulatory authorities
[28]. These tests have confirmed that there is no significant difference between the
performances of the methods compared.
175
Table 4.5 Comparison of the proposed methods with the reference method
Formulations
Tablets
Ramipres (Cipla)
Fixed time method Recovery RSD
(%) (%)
100.03 0.05 eL= 0.986 eu= 1.012
Equilibrium method Recovery RSD
(%) (%)
99.99 0.07 eL= 0,986 eu = 1.013
Reference method Recovery RSD
(%) (%)
99.98 0.09
Ramace 100.01 0.06 100.00 0.07 99.98 0.08 (AstraZeneca) eL=0.987 0u= 1.012 0L=O.987 eu= 1.013
t = 0.063 F= 1.889 t =0.038 F= 1.443
Capsule
Hopace 99.95 0.06 99.98 0.09 100.00 0.10 (Cardicare) eL= 0.986 Gu = 1.014 91=0.983 80 = 1.016
t = 0.081 F = 2.819 t = 0.029 F= 1.217
Theoretical t- (v = 8) and F-values (v = 4, 4) at 95 % confidence level are 2.306 and 6.39, respectively. 9L and Gyare within the acceptable limits of ± 2%.
176
REFERENCES
[I] D. N. Franz, ''Cardiovascular Drugs" In: (edited by A. R. Gennaro) 19" edition,
Vol. II, Mack Publishing Company, Pennsylvania (1995) p. 951.
[2] T. G. Warner, and M. C. Perry, Drugs 62 (2002) 1381.
[3] British Pharmacopoeia, II, H. M. Stationery Office, London (2000) p. 1331.
[4] Z. Zhimeng, V. Andre, N. Len, J. Chromatography B. 779 (2002)297.
[5] B. Francesco, M. Patrizia, Q. Fabiasa, IL Farmaco 49 (1994) 457.
[6] U. J. Dhorda, N. B. Shetkar, Indian Drugs 36 (1999) 638.
[7] S. S. Zarapakar, S. H. Rane, Indian Drugs 37 (2000)589.
[8] J. N. Harlikar, A. M. Amlani, Research J. ofChem. & Environ. 7 (2003) 59.
[9] S. Hillaer, K. De Grauwe, W. Van den Bossche, J. Chromatogr. A, 924 (2001)
439.
[10] H. E. Abdellatef, M. M. Ayad, E. A. Taha. J. Pharm. Biomed Anal. 18 (1999)
1021.
[II] M. M. Ayad, A. A. Shalaby, H. E. Abdellatef, M. M. Hosny, J. Pharm.
Biomed Anai 28(2002)311.
[12] J. A. Prieto, R. M. Jimenez, R. M. Alonso, IL Farmaco 58 (2003)343.
[13] H. Y. Aboul-Enein, R. I. Stefen, J. F. van Staden, Anal. Lett. 32 (1999) 623.
[14] H. G. Eckert, G. Muenscher, R. Oekonomopulos, H. Strecker, H. Urbach, H. A,
V^issmsinn, Arzneim.-Forsch. 35 (1985) 1251.
[15] H. H. Abdine, F. A. El-Yazbi, R. A. Shaalan, S. M. Blaih, S. T. P. Pharm Sci.
9(1999)587.
[16] H. Sakm, Chin. Pharm. J. 51(1999)123.
[17] N. Erk, Anal. Lett. 32(1999)1371.
177
fl8] S. M. Blaih, H. H. Abdine, F. A. El-Yazbi, R. A. Shaalan, Spectrosc. Lett. 33
(2000)91.
[19] F. M. Salama, O. I. A. El-Sattar, N. M. El-Aba Sawy, M. M. Fuad, Al Azhar J.
Pharm. 5c/, 27 (2001)121.
[20] A. A. Al-Majed, F. Belal, A. A. Al-Warthan, Spectrosc. Lett. 34(2001) 211.
[21] A. A. Al-Majed, J. Al-Zehouri, IL Fannaco 56(2001) 291.
[22] N. Rahman, Y. Ahmad, S. N. H. Azmi, A. A. P. S. PharmaSciTech. 6 (2005)
543.
[23] J. Ermer, J. Pharm. Biomed. Anal. 24 (2001) 755.
[24] J. Rose, Advanced Physico-Chemical Experiments, Pitman, London (1964) p.
67.
[25] E. Buncell, A. R. Norris, K. E. Russell, Q. Rev. Chem. Soc. 22 (1968) 123.
[26] M. J. Strauss, Chem. Rev. 70 (1970) 667.
[27] C. Hartmann, J. Smeyers-Verbeke, W. Pinninckx, Y. V. Heyden, P.
Vankeerberghen, and D. L. Massart, Anal. Chem. 67 (1995) 4491.
[28] Canada Health Protection Branch, Drugs Directorate Guidelines: Acceptable
methods, Ministry of National Health and Welfare, Draft, Ottawa, Canada
(1992).
178
SPECTROPHOTOMETRIC METHODS FOR
THE DETERMINATION OF PERINDOPRIL
ERBUMINE IN PHARMACEUTICAL
PREPARATIONS VIA TERNARY COMPLEX
FORMATION WITH Zn ( I I ) AND EOSIN AND
CHARGE TRANSFER COMPLEXATION WITH
IODINE
179
INTRODUCTION
Perindopril erbumine is chemically known as, tert-butyl-amine salt of [[2S-1-
(R, R) 2a, 3aP, 7aP]-l-[2-(l-ethoxy carbonyl butyl] amino]-oxopropyl, octa-lH
indole-2 carboxylic acid [CAS No. 107133-36-8, MW. 441.6], It is used for
management of hypertension and congestive heart failure. The effectiveness of
perindopril as an antihypertensive drug has been demonstrated in various clinical
trials [1,2]. The prodrug is transformed in vivo by hydrolysis into pharmacologically
active perindoprilat. It is thought that perindopril reduces blood pressure by
inhibiting the enzyme which catalyses the conversion of angiotensin I to angiotensin
II. Decreased plasma angiotensin II leads to increase plasma rennin activity and a
decrease in aldosterone. Studies in animals and humans suggest that specific and
competitive suppression of the rennin-angiotensin-aldosterone system is the main
mechanism by which blood pressure is reduced.
The drug is officially listed in the monograph of British Pharmacopoeia [3],
which describes a potentiometric titration procedure for its assay in formulations. In
order to assure the quantity of perindopril in pharmaceutical preparations, several
methods have been reported in the literature for the determination of perindopril
such as GC [4], GC-MS [5], HPLC [6], capillary electrophoresis [7], atomic
absorption spectrophotometry [8 ], and radioimmunoassay [9,10].
Few spectrophotometric methods have also been reported for its quantification
in commercial dosage forms using eosin and copper [8], bromothymol blue at pH
5[11], FeCls in the presence of potassium thiocyanate [11]. TT- acceptors [12] such as
2,3- dichloro- 5,6- dicyano-p-benzoquinone, 7,7,8,8- tetracyanoquinodimethane,
tetracyanoethylene, chloranil and p-chloranilic acid as chromogenic reagents.
Kinetic spectrophotometric method was also utilized based on the reaction with 1-
180
chloro 2,4- dinitrobenzene and measuring the rate of change of absorbance at 420
nm [13]. The assay of perindopril was carried out by UV-spectrophotometry using
zero crossing method [14].
This chapter reports two simple, sensitive and accurate spectrophotometric
methods for the determination of perindopril in commercial dosage forms. Method
A is based on the ternary complex formation with Zn (II) and eosin which is
quantitatively extracted into chloroform and measured at 510 nm whereas Method B
is based on the formation of charge transfer complex between perindopril and iodine
in dichloromethane at room temperature.
EXPERIMENTAL
Apparatus
Spectral runs were made on a Shimadzu UV-visible 1601 spectrophotometer
(Kyoto, Tokyo, Japan). All other spectrophotometric measurements were made on
Spectronic 20 D" spectrophotometer (Milton Roy Company, USA).
Materials and reagents
All chemicals and reagents were of analytical or pharmaceutical grade.
Perindopril erbumine was kindly provided by Glenmark Pharmaceuticals Ltd.
Mumbai, India.
Pharmaceutical formulations of perindopril erbumine such as Coversyl (Serdia
Pharmaceuticals Ltd., India) and Perigard (Glenmark Pharmaceuticals Ltd., India)
were purchased from local drug stores.
(I) 1.39x10" M zinc sulphate (Merck limited, Mumbai, India) solution was prepared
in doubly distilled water
(II) 1.45xl0"'M eosin (Fluka Chemie AG, Switzerland) solution was also prepared
in doubly distilled water.
18i
(111) 2.36x10"" M iodine resublimed (Merck limited Munibai, India.) solution was
prepared in dichloromethane (Merck limited, Mumbai, India.).
Standard test solutions
Standard perindopril (Img ml"') (Batch No. K32002003, Glenmark
Pharmaceuticals Ltd, Mumbai) solutions were prepared in doubly distilled water and
dichloromethane for methods A and B, respectively. The solutions were stable for at
least five days if stored at room temperature and dark place.
Proposed procedures
Method A
Appropriate volumes (0.05-1.00 ml) of the standard perindopril solution (1 mg
ml"') were placed into a series of 100 ml separating funnels. To each funnel, 2.2 ml
of zinc sulphate (1.39 xlO"' M) solution was added followed by 2.0 ml of eosin
(1.45 X10"''M) solution and shaken well. The complex was extracted with 2 x 2.5 ml
portions of chloroform and chloroform layer was passed over anhydrous sodium
sulphate. The absorbance of the resulting complex was measured at 510 nm against
blank in which the drug is omitted. The nominal content of the drug was determined
from the calibration graph or corresponding regression equation.
Methods
Appropriate volumes of standard solution of perindopril (1 mg ml"')
corresponding to 50- 900 \xg were transferred into a series of 5 ml volumetric flasks.
To each flask, 2.0 ml of 2.36 xlO"" M iodine was added and diluted to volume with
dichloromethane. The contents of each flask were mixed well and absorbance of the
yellow coloured product was measured at 365 nm against a reagent blank in which
drug is omitted. The absorbance was plotted against the initial concentration to get
the calibration curve. Alternatively, regression equation was derived.
182
Determination procedure for perindopril in pharmaceutical formulations
Twenty five tablets of perindopril (label claim: 2.0 mg/tablet) were powdered
and perindopril was extracted separately by shaking with 20 ml doubly distilled
water and dichloromethane for methods A and B, respectively., followed by another
two extractions, each with 10 ml of distilled water and dichloromethane. The
extracts were filtered through Whatmann No 42 filter paper (Whatmann
International Limited, Kent, UK) into a 50 ml volumetric flask and then diluted to
volume with appropriate solvent. An aliquot of the diluted solution was analysed for
perindopril content following the proposed procedures.
Limits of detection and quantitation
According to International Conference on Harmonisation (ICH) guidelines
[15], the following expressions are used to evaluate LOD and LOQ.
LOD = 3 ,3xSo/bandLOQ=10xSo/b
where So and b are standard deviation and slope of the calibration line, respectively .
Evaluation of bias
The point and interval hypothesis tests [16] have been performed to compare
results of the proposed methods with those of the reference method at 95%
confidence level. The test method is considered acceptable when its true mean is
within ± 2.0 % of that of the reference method. This can be written as
0.98<^2/lii <1.02
which can be generalized to
9L < 1^7/HI <9U
where 9L and 6u are lower and upper acceptance limits, respectively which were
calculated from the following quadratic equation [16].
e'\x',-Syin,]-297,7-,^e\xl~Syin^]=^
183
RESULTS AND DISCUSSION
Method A
Ternary complexes of general formula (LnMxSy) have been widely used in
spectrophotometric analysis [17-23]. In the present study, the ternary complex was
utilized for the determination of perindopril in which main ligand L is perindopril,
the second ligand S is eosin and M is Zn (II). The ternary complex is extractable
with chloroform, whereas the binary system (zinc- drug and zinc - eosin) cannot be
extracted in that way. In the ternary complex, the interaction of Zn (II) (X),
perindopril (Y) and eosin (Z) may be considered as
either XY+Z<^ XZ+Y
or XY +Z ^ XYZ
The absorption spectra have been recorded for each component, separately, and to
their mixture under the experimental conditions discussed above in both aqueous
and organic solvent. The spectra revealed (Fig. 5.1) that aqueous solution of eosin
(Z) absorbed maximally in the visible region at 470 nm and there are no significant
changes in colour and absorption spectrum in the presence of Zn (II) or perindopril,
while neither Zn (II) nor the perindopril shows any absorption in the visible region,
However, when Zn (II) solution is mixed with a mixture containing perindopril and
eosin, the orange - yellow colour changes to red. The absorption spectrum of the
reaction mixture exhibited a new band peaking at 510 nm. The later spectrum is
attributed to the formation of ternary complex.
Optimization of the experimental condition
The different experimental parameters affecting the colour development and
its stability were carefully studied and optimized. Such factors were changed
individually while the others were kept constant.
184
-r 1 1 1 1 1 r
320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
wavelength(nm)
Fig. 5.1. Absorption spectra of (a) 4.34x10''' M eosin; (b) 4.34x10" M eosin and
8.35x10"* M Zn (II); (c) 4.34x10"'' M eosin and 50.0 ng ml' perindopril against
doubly distilled water (d) 200.0 jig ml' perindopril with 6.12xl0''' M Zn (II)
and 5.78 xlO' M eosin extracted into chloroform against blank (6.12x10" M Zn
(II) and 5.78 x l O ^ eosin ) treated similarly.
185
colour changes to red. The absorption spectrum of the reaction mixture exhibited a
new band peaking at 510 nm. The later spectrum is attributed to the formation of
ternary complex.
Optimization of the experimental condition
The different experimental parameters affecting the colour development and
its stability were carefully studied and optimized. Such factors were changed
individually while the others were kept constant.
Effect ofZn (II) concentration
The effect of concentration of Zn (II) on the absorbance of the ternary
complex was studied over the concentration range 2.78xlO'V- 6.68xlO'^M;
keeping the concentrations of the drug (4.53XI0"' 'M) and eosin (5.78X10' ' 'M)
constant. It was observed that increasing the concentration of Zn (II) would result in
a gradual increase in the absorbance of the ternary complex up to5.56x10' M and
remained constant up to 6.68xlO"- M. Thus 6.12xlO'^M of Zn (II) was used as an
optimum concentration (Fig. 5.2).
Effect of eosin concentration
The effect of eosin concentration on the absorbance of the ternary complex
was examined in the concentration range of 2.89xlO"^M- 6.36X10' ' 'M; keeping the
concentration of the drug (4.53xlO"'*M) and Zn (II) (6.12xlO''^M) constant. It was
found that increasing the concentration of eosin resulted in a subsequent increase in
the absorbance value of the ternary complex up to 5.20X10'' 'M and remained
constant up to 6.36 X10"''M. Therefore, a concentration of 5.78X10"''M was used as
the optimum concentration of eosin (Fig. 5.3).
186
-i r
0,000 0,001 0.002 0.003 0.004 0,005 0.006 0.007
2+ Concentration of Zn *M
Fig. 5.2. Effect of molar concentration of Zn (II) on the formation of ternary
complex
187
0,0000 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007
Concentration of eosin, M
Fig. 5.3. Effect of molar concentration of eosin on the formation of ternary
complex
188
Effect of shaking time
In order to examine the effect of shaking time on the complex formation and
the absorbance of the drug- Zn (II) - eosin ternary complex, experiment was
performed for the periods ranging from 1-3 min. A constant absorbance was
obtained from one min. shaking and remained constant up to three min. Therefore,
two min. shaking time was recommended for the determination process.
Stoichiometry of the ternary complex
The stoichiometrty of the reaction was studied adopting the limiting
logarithmic method [24]. For this, three sets of experiments were performed. In the
first set, the concentration of perindopril was varied; keeping excess concentrations
of Zn (II) (6.12 xiO"^M) and eosin (2.89 xlQ-^M). In the second set, the
concentration of Zn (II) was varied while keeping high concentrations of perindopril
(4.53 xlO'^M) and eosin (2.89 xlO"- M). In the last experiment, excess
concentrations of Zn (II) (6.12 x\Q'^M) and perindopril (4.53 xiO'- M) were
employed and the effect on the absorbance was observed on varying the
concentration of eosin. A plot of log absorbance vs. log [perindopril] or log [Zn (II)]
or log [eosin] gave straight lines; the values of the slopes are 0.97, 1.02 and 1.01,
respectively. (Fig. 5.4 a, b and c) Hence, it is concluded that the reaction proceeds
in the ratio 1: 1:1, confirming that one molecule of the drug denses with one
molecule of Zn (II) and one molecule of eosin. Based on the obtained molar
reactivity, and literature background, the possible reaction pathway is proposed and
shown in Scheme 5.1.
189
-3.0
2+1 log[Drug] or [Eosin] or [Zn ]
Fig. 5.4. Limitting logarithmic plot for molar combining ratio between
perindopril, Zn (II) and eosin: (a) log Absorbance vs log [Perindopril]; (b) log .2+
Absorbance vs log [Zn ]; (c) log Absorbance vs log [eosin].
190
CH3
O-V + Br-
• C = 0
OH
^ = ^ ^
COOC2H5
Br + Zn^"
R= CH-CH2CH2CH3
COOC2H5 Extracted in CHCl
1
CH3
/
Zn
•y ^—c
OH
. . ^ ^
Scheme 5.1
Method B
The absorption spectrum of iodine in dichloromethane showed only one peak
with maximum absorption at 500 nm. The colour of iodine changes to yellow upon
reaction with perindopril. This is due to charge transfer complexation reaction
between the n- donor amine and the a- acceptor iodine followed by the formation of
tri- iodide ion pair (Scheme 5.2) that exhibited strong absorption maxima at 290 and
365 nm (Fig. 5.5).
Optimization of the experimental condition
Effect of concentration of iodine
The influence of concentration of iodine on the absorbance of the complex
was investigated by treating varied concentration (4.73 xlO'^M- 1.04 xlO' M) of
iodine with 0.75 ml of 2.26 x]0"^M perindopril into a series of 5 mi flasks. The
contents were diluted with the same solvent at room temperature. The highest
absorbance was achieved with 8.51 xlO''' M iodine. Further addition of iodine
caused no change in the absorbance and therefore, 9.44x10''' M iodine was selected
as an optimum concentration for determination process (Fig. 5.6).
Stoichiometric relationship
Job's Method [25] was applied to determine the molar combining ratio for
charge transfer complex, perindopril- iodine. It is apparent from Fig. 5.7 that the
molar combining ratio between perindopril and iodine is 1:1.
The spectrophotometric data were used to calculate the association constant
and apparent molar absorptivity of the perindopril- iodine charge transfer complex
using the Ross and Labs equation [26]:
MHO) , ± , J , , 1 , ' [A] + ID] A, Ke, [A] + [D] c,
Stepl-
192
H,C O HOOQ
" 1 D ^ H H
N' H 6
H,C HOOC
H,C HOOC
H ^1 0 ^ - /
Step 2-H.C
H.C
HOOC
HX-^ P HOOC
r+i2
Tri- iodide ion pair
Scheme 5.2
193
- I 1 1 i r
200 250 300 350 400 450 500 550 600 650 700
V\^ve length (nm)
-3 Fig. 5.5. Absorption spectra of (a) 1.5 ml of 2.36 x 10" M iodine in
-3 dichloromethane and (b) 750,0 ng perindopril and 2.0 ml of 2.36 x 10 M
iodine in dichloromethane. In both cases, the solutions were diluted to 5 ml.
194
0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 0.0012
Concentration of iodine,M
Fig. 5.6. Effect of the molar concentration of iodine on the absorbance of
coloured product (perindoprii 150.0 |ag ml"')
195
0.0 01 0.2 0.3 0.4 0.5 06 0.7 0.8 0.9 1.0
mole fraction of drug
Fig. 5,7, Job's plot for the reaction between perindopril and iodine in
dichlorometane ([Perindopril] = [Iodine] = 1,00x10' M).
196
where [A] and [D] are the initial molar concentration of iodine and perindopril,
respectively. Aj,^ and f are the absorbance and apparent molar absorptivity of the
charge transfer complex at the wavelength X. K is the association constant of the
complex.
The values of -^—^—— x — were plotted against which yielded a [A] + [D] A, [A] + [D]
•• 1 1 straight line (Fig. 5.8) with the slope of and intercept — . This straight line is
described by the following regression equation:
i i l i ^ x — = 7.62x10^'X ^ + 2.69x10^" (r =0.9999) [A]^[D] A, [A]^-{D]
From the above equation, the association constant and apparent molar
absorptivity of the charge transfer complex were found to be 3.53x10'and 3.71 xlO'
1 mol" cm' , respectively. The standard free energy of complexation, AG , was
calculated using the relation: AG = -RTln K and found to be - 20,25 KJmol" .
Validation parameters
Selectivity
The selectivity of the method was ascertained by analyzing standard
perindopril in presence of excipients such as lactose, magnesium stearate, cellulose
and silicon dioxide. It was observed that both methods are free from interferences.
Analytical data
Standard calibration curves for both the procedures were constructed by
plotting absorbance against concentration of perindopril (^g ml"'). Beer's law range
and molar absorptivity are given in Table 5.1. Regression equations were derived
using the method of least squares. The correlation coefficients, standard
197
0.00041
0.00040 -
0.00039
+
•^ 0.00038 -<
<
0.00037
0.00036
0.00035 1 1 > r 1100 1200 1300 1400 1500 1600 1700 1600
1/[D] + [A]
Fig. 5.8. Plot of 1/A X ([D] [A] / [D] + [A]) Vs. 1/[D] + [A]
198
Table 5.1 Optical characteristics and statistical data of the regression equations
Parameters Method A Method B
Beer's law limit (jLig ml"') 10.0-200.0 10.0- 180.0
Molar absorptivity (I mol"'cm') 2.25x10^ 3.71x10^
Linear regression equation A = 4.15x 10"V 5.10x1 Q-' C A = -4.43x 10"'' +4.50x 1 Q' C
Sa 4.51x10"' 7.58x10"'
tSa 1.07x10'^ 1.79x10"^
Sb 3.71x10"' 6.93X10-'
tSb 8.77x10"' 1.64x10"^
Correlation coefficient (r) 0.9999 0.9999
Variance (So^) 2.25x10"' 1.49x10"'
Detection limit (^g ml"') 0.49 0.90
Quantitation limit (f g ml"') 1.48 2.71
± t Sa, Confidence limit for intercept.
± t Sb, Confidence limit for slope.
199
deviation of slope and intercept, variance of calibration line and detection and
quantitation limits were calculated and listed in Table 5.1. The values of correlation
coefficient and variance clearly indicated the excellent linearity of calibration graph
and negligible scattering of the experimental data points around the calibration line,
respectively.
Accuracy and precision
In order to determine accuracy and precision of the proposed procedures
quality control sample solutions containing three different concentrations (20.0,
100.0 and 180.0 \x% ml'') were prepared and analyzed in five replicates within one
day as well as for five consecutive days. The standard deviations (SD), relative
standard deviations (% RSD), standard analytical error (SAE) and confidence limit
(CL) were calculated and summarized in Table 5.2.
Recovery studies
Recovery experiments were carried out by standard addition method. The
concentration of perindopril was determined in tablets (Coversyl-2.0, Perigard-2.0)
after spiking with 50 and 200% of additional drug. It was observed from Table 5.3
that the methods A and B afforded recoveries in the range of 99.97% - 100.05%.
Application
The applicability of the proposed methods for the assay of perindopril in drug
formulations has been tested on commercially available tablets. The results of the
proposed methods were statistically compared with those of the reference method
[12] using point and interval hypothesis tests. Table 5.4 shows that the calculated t-
(paired) and F-values at 95% confidence level are less than the theoretical ones,
confirming no significant difference between the methods compared. For
pharmaceutical analysis, a bias of ±2.0% is acceptable and the limit of acceptance
200
Table 5.2 Evaluation of the accuracy and precision of the two proposed methods
Proposed methods
Method A
Intra day assay
Inter day assay
Method B
Intra day assay
Inter day assay
Amount
(Mgml-')
Taken
20.0
100.0
180.0
20.0
100.0
180.0
20.0
100.0
180.0
20.0
100.0
180.0
Found ± SD"
19.99 ±0.22
99.96 ±0.16
180.04±0.26
19.99 ±0.33
99.99 ±0.22
179.88±0.29
20.01 ±0.30
100.05 ±0.19
179.97 ±0.34
19.97 ±0.46
100.01 ±0.34
180.05 ±0.43
Recovery ± RSD'
(%)
99.99± 1.12
99.96 ±0.16
100.02±0.15
99.99 ± 1.64
99.99 ±0.22
99.93 ±0.16
100.05 ± 1.49
100.05 ±0.19
99.98 ±0.19
99.83 ±2.3!
100.00 ±0.34
100.03 ±0.24
SAE"
0.10
0.07
0.12
0.15
0.10
0.13
0.13
0.08
0.15
0.21
0.15
0.19
C.L."
0.28
0.20
0.33
0.41
0.28
0.36
0.37
0.23
0.42
0,57
0,42
0.53
^Mean for five independent analyses.
SAE, standard analytical error.
• C.L., confidence limit at 95% confidence level and four degrees of freedom (t = 2.776).
201
I -o e« •o u «s
•o e • * —
?-. £>
a « CS
o. u O. "a _y — s w
I a a u a. o
a
o
O • • - '
w
« i? H ft
o x; • * -»
O
3 o
§
-J
< GO
>
+1
o CO ai
6J) 3 u.
T3 <^-( O t« 1/1 <u u X tu
CJ
< 00
Q u^ c^
D
_^ K) C a o ^-> -a (U • a -a M
<u > o o 1)
a:
t * 3 UH
•a ^+H
o t/1 M
<u o X w
E Q C/5 tf!
(D
a c n) o
• * - '
T3 m •a • a «!
NT
i n
O
O O
o
o +1 o o o
O
00
o o
^
o
o o o
o
o
•^2
o
o
o 4 in o o o
d
ON
o d
r--
d
o d o
o d IT)
oo
d
o o
d
o d
d
o o
d
t n —; d -H o ON ON ON
^ rx) d +1 (N O d o »—
o (N d -H o o d o • — '
ON -^ d -H r--ON ON ON
^ o o o
q fN
o o i n (N
> o o
.2 •5 C/5
o I
u W
*u
0-
e c
O
o t
o
c W
C •a c D D. u •n c u >
w <
E o •o
V 1) u oc <u
•u u 3
<£ T3 C
>
(U o c -o c o o
to ON
^ •? c 03
• a c
o c u
c o o
O
202
Table 5.4 Determination of perindopril in pharmaceutical preparations by the proposed methods and reference method [12]
Formulations Method A Method B
Recovery RSD
(%) (%)
Recovery RSD
(%) (%)
Reference method [ 12]
Recovery RSD
(%) (%)
Coversyl2.0 99.93 0.101 100.09 0.165
(Serdia) eL=0.983 eu=1.016 eL=0.982 00=1.016
99.92
t=0.02 F= 1.387 t=0.27 F=1.08
Perigard2.0 99.80 0.146 100.02 0.093
(Glenmerck) eL=0,983 eu=1.0]9 eL=0.987 eu=1.011
t=0.20 F=1.88 t=0.11 F=1.67
99.94
0.172
0.201
203
interval is within 9L= 0.98 and 0u= 1.02. It is observed from the Table 5.4 that the
true bias is well within the acceptance limit at 95 % confidence level.
204
REFERENCES
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942 Chem. Pluirm. Bull. 53(8) 942—948 (2005) Vol, 53, No. 8
Validated Kinetic Spectrophotometric Metliod for the Determination of Metoprolol Tartrate in Pharmaceutical Formulations
Nafisur RAHMAN,* Habibur RAHMAN, and Syed Najmul Hejaz AZMI
Department of Chemistry, Aligarh Muslim University; Aligarh-202002 (U.P.) India. Received February 28, 2005; accepted May 6, 2005
A kinetic spectrophotometric mettiod lias been described for the determination of metoprolol tartrate in pharmaceutical formulations. The method is based on reaction of the drug with alkaline potassium permanganate at 25±1°C. The reaction is followed spectrophotometrically by measuring the change in absorbance at 610 nm as a function of time. The initial rate and fixed time (at 15.0 min) methods are utilized for constructing the calibration graphs to determine the concentration of the drug. Both the calibration graphs are linear in the concentration range of 1.46x10"'—8.76x10"'vi (10.0—60.0/ig per 10ml). The calibration data resulted In the linear regression equations of log (rate)=3.634+0.999 logC and /l = 6.300xl0"''+6.49lx|0"^ C for initial-rate and fixed time methods, respectively. The limits oi quantitation for initial rate and fixed time methods are 0.04 and O.lO^gmP', respectively. The activation parameters such as £,, 4//*, AS* and 4 0 ' are also evaluated for the reaction and found to be 90.73 kJmor' , 88.20 kJmor' , 84.54 JK~'moP' and 63.01 kJmol"', respectively. The results are validated statistically and through recovery studies. The method has been successfully applied to the determination of metoprolol tartrate in pharmaceutical formulations. Statistical comparison of the results with the reference method shows excellent agreement and indicates no significant difference in accuracy and precision.
Key words spectrophotometry; metoprolol tartrate; validation parameters; pharmaceutical formulation
Metoprolol tartrate is a iBciective /3-adrenergic antagonist, which is used in the treatment of cardiovascular disorders such as hypertension, angina pectoris, cardiac arrhythmias and myocardial infarction. The drug is quite sensitive, even a small dose of the drug gives sufficient blockade. Since the /?-blockers are also misused as doping agents in sports and therefore these drugs have been added to the list of forbidden drugs by the International Olympic Committee (IOC)." Therefore the development of an analytical method for the determination of metoprolol tartrate is of great significance.
The assay of the drug is listed in the monograph of British Pharmacopoeia, which describes a potentiometric titration method. ^ Several analytical methods have been developed for the determination of metoprolol in biological fluids and pharmaceutical formulations based on high performance liquid chromatography,-'"'" gas chromatography,'^''^' capillary electrophoresis,'"*' thin layer chromatography,"'" infrared spectroscopy,'"and electrochemical methods."'" The above mentioned techniques are sensitive but expensive and require laborious clean up procedure prior to analysis. Spectrophotometry is the technique of choice even today due to its inherent simplicity and therefore frequently used in the laboratories of the developing countries to overcome versatile analytical problem. The drug has been determined in the visible region based on ion-pair formation between drug and reagents like oxidized quercetin^"'; bromophenol blue, bromocresol purple, bromocresol green^"; benzyl orange^^'; bromothymol blue^^'; and carbon disulfide-copper chloride.^''' The charge transfer complexation reactions of metoprolol tartrate with a and ;r-acceptors^'*'"' have also been utilized for its quantification in pharmaceutical formulations. Shing-bal and Bhangle^*' have reported a spectrophotometric method based on the reaction of drug with 2,4-dinitrofluo-robenzene in HCl/dioxan medium to form a colored chro-mophore, which absorbs maximally at 380 nm. The quantification of metoprolol tartrate was done on treatment of the
drug with ammonium metavanadate,"' FeClj, **' A/-bromosuc-cinimide,^" and a mixture of KNO3 and H2SO4 followed by addition of alkali to get colored chromophore.-"" The literature is still poor in analytical procedures based on kinetic spectrophotometry for the determination of drug in pharmaceutical preparations. There is, therefore, a need for a simple and sensitive kinetic spectrophotometric method for the determination of metoprolol tartrate in pharmaceutical formulations. It was found that potassium permanganate oxidizes the metoprolol in alkaline medium and this reaction has not been used before to quantify the drug spectrophotometrically.
This paper describes a simple and sensitive kinetic spectrophotometric method for the determination of metoprolol tartrate in drug formulations. The method involves the oxidation of drug with alkaline potassium permanganate at 25±1°C and subsequent measurement of absorbance at 610nm. The initial rate and fixed time methods are adopted for its determination in pharmaceutial formulations.
Experimental Apparatus A Shimadzu UV-visible spectrophotometer (model-1601,
Japan) with matched quartz cells was used to measure absorbance. A water bath shaker (NSW 133, New Delhi, India) was used to control the
heating temperature for color development. Reagents and Standards All reagents and chemicals used were of ana
lytical or pharmaceutical grade. Aqueous solutions of 0.6 M sodium hydroxide and 0.015 M potassium permanganate (GR Grade, Merck Limited, Mum-bai, India) were prepared in doubly distilled water. Potassium permanganate (GR Grade, Merck Limited, Mumbai, India) solution should be freshly prepared and its apparent purity was assayed by titrimetric method."'
The standard test solution of metoprolol tartrate (0.01%) was prepared in doubly distilled water. The formulated dosage forms of metoprolol tartrate such as betaloc (AstraZeneca Pharma India Ltd., Bangalore, India), metapro (Cardicare, Bangalore, India) and metolar (Cipla, Mumbai, India) were purchased from the local market.
Determination Procedures for Metoprolol Tartrate Initial-Rate Method: Aliquots of 0.1—0.6ml of 0.01% metoprolol tartrate were pipetted into a series of 10 ml standard flasks. To each flask 2,0 ml of 0.60 M NaOH followed by 2.0ml of 0.015M potassium permanganate were added and then diluted with doubly distilled water at 25± 1 °C. The contents of each flask
* To whom correspondence shou,ld be addressed, e-mail: [email protected] ) 2005 Pharmaceutical Society of Japan
August 2005 943
were mixed well and the increase in absorbance was recorded as a function of time at 610nm. 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 graph was constructed by plotting the logarithm of the initial rate of reaction (log v) versus the logarithm of the molar concentration of the metoprolol tartrate (logC). The amount of the drag was computed either from the calibration graph or the regression equation.
Fixed-Time Method: The absorbance of each drug sample solution was measured at 610 nm against a reagent blank prepared similarly at a preselected fixed time of 15min. The calibration curve was constructed by plotting the absorbance against the final concentration of the drug. The amount of the drug was computed either from calibration curve or regression equation.
Determination Procedure for Metoprolol Tartrate in Piiarmaceutical Formulations Five tablets were weighed and powdered. The powder equivalent to 50 mg of active ingredient was weighed accurately, stirred well with doubly distilled water and filtered through Whatman No. 42 filter paper (Whatman International Limited, Kent, U.K.). The residue was washed well with doubly distilled water for complete recovery of the drug. The content of the dmg was then diluted to 250.0 ml with doubly distilled water. It was further diluted according to the need and subjected to the determination procedures for metoprolol tartrate. The percent recovery of the metoprolol tartrate was calculated from the corresponding linear regression equations or calibration graphs.
Procedure for Reference Method Into a series of 10 ml standard volumetric flask, different volumes (0.25—2.5 ml) of 0.01% drug (0.1 mgml ') solution were pipetted and diluted to volume with doubly distilled water. The absorbance was measured against the solvent blank at 224 nm. The amount of the drug in a given sample was computed from the calibration equation.
Results and Discussion Spectral Studies The absorption spectrum of metopro
lol tartrate solution in doubly distilled water shows two absorption bands peaking at 194 and 224 nm while that of potassium permanganate solution in the alkaline medium exhibits an absorption band peaking at 530 nm. The addition of potassium permanganate to the solution of pure drug produces a new characteristics band at 610 nm. This band is attributed to the formation of manganate ion, which resulted on reduction of potassium permanganate in alkaline medium. The intensity of the colored product increases with tirne and therefore, a kinetic method was developed for the determination of metoprolol tartrate in drug formulations. Moreover, potassium permanganate also oxidizes metoprolol in acid medium resulting in the formation of a-hydroxy metoprolol and Mn(II). In the presence of acid such as H2SO4, HCl and H3PO4, a-hydroxy metoprolol gives a violet color which has not been utilized for quantitative analysis,
Stoichiometry and Reaction Mechanism The stoichiometric ratio between metoprolol tartrate and potassium permanganate was established using limiting logarithmic method^ ' by performing two sets of experiments. In the first set, the concentration of metoprolol tartrate was varied keeping a constant concentration of KMn04. In the second set of experiment, concentration of metoprolol tartrate was kept constant while varying the concentration of KMn04. The logarithm of the absorbance was plotted against the logarithm of the respective varied concentration of metoprolol tartrate or KMn04 (Figs. 1 a, b). It is evident from the slopes of the two straight lines that the combining molar ratio between metoprolol tartrate and KMn04 is ] : 1.
Horai et al. have suggested that the metoprolol tartrate undergoes oxidation^^' resulting in the formation of a-hydroxy metoprolol. In this study, the potassium permanganate oxidizes the metoprolol tartrate in alkaline medium producing
-4.8 -AA -40 -3.6 -32
Log IDrugl or [KMnO,]
Fig, 1. Limiting Logarithmic Plot for Stoichiometric Ratio between Metoprolol Tartrate and KMn04 (a) log A vs. log[drug] and (b) log/f vs. loglKMnOJ
0 04 0.06 0 08 0 10
Molar concentration of NaOH
Fig. 2. Effect of the Molar Concentration of NaOH Solution on the Initial Rate of Reaction with 60,0/tg per 10 ml Metoprolol Tartrate in Doubly Distilled Water
a-hydroxy metoprolol and itself reduced to Mn04", The reaction product gives violet color on treating with formalde-hyde-sulfuric acid reagent, which confirmed the formation of a-hydroxy metoprolol.^'''
Optimization ofVariables The influence of the concentration of NaOH solution on the rate of reaction was studied by keeping the constant concentrations of metoprolol tartrate (8.76X1 0"^M) and KMn04 (S.OOXIO-^M) and varying the concentration of NaOH (1.20X 10^^—1.32X 10"'M) in a final volume of 10 ml solution. Figure 2 shows that the initial rate of reaction increased up to 8 ,4X10"^M NaOH; beyond this concentration the initial rate of reaction remained con-
944 Vol. 53, No, 8
0.0005 0.0010 0.0015 0.0020 0 0025 0.0030 0 0035
Molar ooncentration of KMnO^
Fig. 3. Effect of the Molar Concentration of KMn04 Solution on the Initial Rate of Reaction with 60.0/ig per 10 ml Metoprolol Tartrate in Doubly Distilled Water
stant. Therefore, a concentration of 1.20X10"' M NaOH was used throughout the experiment. The effect of the concentration of KMn04 solution on the initial rate of the reaction was studied in the range of 7.SOX 10""—3.30x 10'^ M. The initial rate of reaction (Fig. 3) increased with increasing the concentration of KMn04 and became constant at 2 . 4 0 X 1 0 ~ ' M . Thus, a concentration of 3 .00X10~^M KMnO^ in the final solution proved to be sufficient for the maximum concentration of metoprolol tartrate used in the determination process. The effect of temperature on reaction rate was studied in the range of 298—308 K. The absorbance-time curves showed the temperature dependence of the reaction rate. It was observed that metoprolol tartrate reacts faster with potassium permanganate within the short period of 3—15min, 3— lOmin and 3—7min. at 298, 303 and 308 K, respectively. At temperatures >308K, the decomposition of the reaction product may take place. To avoid this and for the sake of good results, the optimum temperature of 298 K is selected for the determination process.
Analytical Data and Method Validation Under the optimized experimental conditions, a pseudo-order reaction condition was worked out by using a large excess of KMn04 and NaOH solution with respect to the initial concentration of metoprolol tartrate. As a result, a pseudo zero order condition was obtained with respect to the reagents, the overall concentration change of KMn04 and NaOH during the course of reaction would be negligible. The initial rates of the reaction were determined from the slopes of the initial tangent to the absorbance-time curves (Fig. 4) and are summarized in Table 1. The reaction would obey the following rate equation:
r a t e = * ^
where k^ is the pseudo-order rate constant, C is the concentration of metoprolol tatrate, n is the order of the reaction. The logarithm form of the above equation is written as:
04 -
0.3 •
02 •
01 -
no 1
- • - a
^^d
I^r - ^ 9
^ ^
Time (min)
Fig. 4. Absorbance-Time Curves for the Reaction between Metoprolol Tartrate and ICMnO^ in Aqueous Medium: 2.0 ml of 0 . 0 1 5 M KMnO, and Metoprolol Tartrate: (a) 1.0, (b) 1.5, (c) 2.0, (d) 3.0, (e) 4.0, (f) 5.0 and (g) 6.0^gml '. Each Set is Diluted in 10ml Standard Flask with Doubly Distilled Water
Table I. Summary of Data of the Initial Rate of Reaction at Different Concentration of Metoprolol Tartrate and K.Mn04
[Drug] (moll ') [KMnOjKmoll Initial rate of reaction, V'(mol 1 ' min ')
1.460X10"'' 2.190X10 ' 2.920X10 ' 4,380X10 ' 5.840X10 ' 7.300X10 ' 8.760X10 ' 8,760X10" 8,760X10 ' 8,760X10 '' 8,760X10 ' 8.760X10" 8.760X10"
3.000X10 ' 3.000X10 ' 3.000X10 ' 3.000X10 •' 3.000X10 ' 3.000X10 ' 3.000X10 ' 7.500X10 " 9.000X10 " 1.200X10 ' 1.800X10 -' 2.100X10^ 2.400X10 '
6.250X10 ' 9.375X10 ^ 1.270X10 ' 1.880X10 ' 2.500X10 • 3.100X10 ' 3.800X10" 8.000X10 ' 1.130X10 ' 1.860X10 ' 2.660X10 ' 2.830X10 ' 3.800X10"^
log(rate)=log^,p+/ilogC
The linear regression analysis using the method of least square treatment of calibration data was made to evaluate slope, intercept and correlation coefficient. Under the working experimental conditions, a calibration graph was constructed by plotting log of initial rate of reaction (log v) versus log of metoprolol tartrate concentration (log Q, which showed a linear relationship over the concentration range of 10.0—60.0/ig per 10 ml. The regression of log rate versus log C gave the following linear regression equation:
log(rate)=3.634+0.999 log C
with a correlation coefficient (r) of 0.9999. The value of n in regression equation confirmed that the reaction is first order with respect to metoprolol tartrate. The confidence limits for
August 2005
Table 2. Summary of Optical Characteristics and Statistical Data for the Fixed-Time Method
945
Parameters
Beer's law limit (/Jg per 10 ml)
Molar absorptivity (1 mol ' cm ') Regression equation
V' Intercept
5.
-IS, Slope
\ i ' \ Correlation coefficient (r)
Variance {S^,)
Detection limit (/igml ')
Quantitation limit (/ig m l ' )
3 min
10.0—60.0
9.133X10' /(=1.0IX10 •' +
1.324X10 'C
5.80X10 '
l.OIXIO '
4.70X10 "
1.208X10 '
1.324X10 ' 1.30x10"
3.342X10 '
0.9997
3.364X10 '
0.15 0.44
6min
10.0—60.0
2.192X10' /(=6.9XI0 ' +
3.178X10 'C
7.80X10 '
6.9X10 *
6.20X10 *
1.594X10 '
3.178X10 '
1.70xi0 '
4.371X10 '
0.9999
6.084X10 '
0.08
0.25
Fixed-lime method
9 min
10.0—60-0
3.151XIO' /4=7.9XI0 •• +
4,580x10 'C
6.30X10 '
7.9X10 '
5.10X10 '
1.311X10 •'
4.580X10"'
1.40X10 '
3.599X10 '
0.9999
3.969X10 '
0.05 0.14
12 min
10.0—60.0 3 973X10' / (= -172X IO '-I
5 831X10 ' C
1.04X10 '
-1.72X10 '
8.31X10 '
1.697X10 '
5.831X10 '
2 30X10 '
5913X10 '
09999 I.082XU) »
0.06
0.18
15 min
10 0—60.0 4.453X10'
/ I=6.3XI0 ' +
6,491X10 ' C
6.60X10 '
6.3X10 '
5.30X10 '
1.363X10 '
6491X10 '
1.50x10 '
3.857X10 '
0.9999
4.356X10 '
0.03
0.10
Reference
method
25.0—250.0 2 028X10'
/(==107X10 ' +
2,940X10 ' C
7,00X10 '
1,07X10 '
5,00X10 '
1,286X10''
2,940X10 •
4,00X10 '
1,03x10 '
0,9999
4,900X10 '
0,08 0,24
a) Calculated t-value, which is less than the theoretical value of ((2.776) for n-l degrees of freedom.
the slope of the line of regression and intercept were computed using the relation b±lS^, and a±tS^^^'' at 95% confidence level and found to be 0.999± 1.45X10"' and 3.634±2.60XI0"^ respectively. This indicated the high reproducibility of the proposed method. The limits of detection (LOD) and quantitation (LOQ) were evaluated using the following equation;
LOD=3.3X5'„/i atid LOQ=10X5(/6
where Sg is the standard deviation of the calibration line and b is the slope and found to be 1.3x10"^ and 4.0X10"'^/igmr', respectively. The variance was calculated using the equation:
X(i°g' • log v„.r
and found to be 1.576X10~'^gmr'. The low value of variance indicated negligible scattering of the experimental data points around the line of regression.
In the fixed-time method, the absorbance of green colored solution obtained on interaction of different concentration of metoprolol tartrate with alkaline potassium permanganate was measured at a preselected fixed time. Calibration plots of absorbance versus initial concentrations of metoprolol tartrate were established at a fixed time of 3, 6, 9, 12 and 15 min. The molar absorptivity, regression equations, coefficient of correlation, limits of detection and quantitation and variance are given in Table 2. It is clear from Table 2 that the most acceptable values of molar absorptivity, limit of detection and quantitation were obtained at a fixed time of 15 min. Therefore, the fixed time of 15 min was adopted as the optimum time for the determination of metoprolol tartrate in pharmaceutical formulations. The important analytical parameters of conventional UV spectrophotometric method have been summarized in Table 2. It can be seen that the molar absorptivity of the fixed time method is higher than that of conventional UV spectrophotometric method whereas the limits of detection and quantitation of the proposed method are smaller. The performance of the initial rate method is better since the values of LOD and LOQ are smaller than that of
the fixed time method and analysis can be completed in a shorter time.
Solution Stability and Selectivity The solution stability of metoprolol tartrate was checked by observing UV spectra of metoprolol tartrate for 5 d. The aqueous solution of the drug having two A, ^ ; 194 and 224 nm, showing no change in the absorption spectra of standard and sample solutions of drug for at least 5 d, when the solutions were stored at room temperature. To identify the metoprolol and a-hydroxy metoprolol, thin layer chromatography was performed. The standard solution, sample solution and reaction product were applied on TLC plates coated with silica gel and developed in ethyl acetate-methanol-ammonia (40: 5 : 5 v/v/v) solvent system. The plates were air-dried and spots were detected in the iodine chamber In the case of standard and sample solutions, a single spot was observed with Rf=0.50 corresponding to metoprolol, whereas reaction product also showed one spot with Rf=0.65. This corresponds to a-hydroxy metoprolol.'"'' Thus, the proposed methods are selective as the major metabolite, a-hydroxy metoprolol does not interfere in the determination. However, other /3-adrenergic antagonists such as propranolol, atenolol and labetalol react with potassium permanganate in alkaline medium resulting in the formation of green colored solution, which absorbs maximally at 610nm.
Accuracy and Precision The accuracy and precision of the proposed methods was established by measuring the content of metoprolol tartrate in pure form at three different concentration levels (low, medium and high). The short-term (intra day assay) and the daily precisions (inter day assay) were performed by measuring five independent analyses at 1.5, 3.0 and 6 .0/ igmr ' concentration levels within 1 d and on 5 consecutive days, respectively (Table 3). The standard deviation, relative standard deviation and mean percent recoveries obtained by both the initial rate and fixed time methods can be considered to be very satisfactory.
The validity of the proposed methods was also checked by performing recovery experiments through standard addition method. For this, a known amount of the pure drug was added to preanalysed dosage forms and then the total amount
946 Vol. 53, No. 8
Table 3. Test of Precision of the Proposed Methods by Intra Day and Inter Day Assays
Proposed methods
Initial rate method Intra day assay
Inter day assay
Fixed time method Intra day assay
Inter day assay
Talcen
1.5 3.0 6.0 1.5 3.0 6.0
1,5 3,0 6.0 1,5 3.0 6,0
Amount ( / jgml ' )
Found ± 5,0,"
1,50+0,04 3.00 6.01 1,50 2,99 6,00
t0.06 t:0.06 t0,04 t0.06 £0,06
1,50+0,04 2.99 + 0.05 6.01+0.05 1,50+0,06 2,99+0,06 6.00+0.07
Recovery
(%)
100,03 100,06 100.13 100.14 99.95
100,07
100,21 99,93
100,15 100,01 99.93
100,04
SAE'"
0,02 0,03 0.03 0.02 0.03 0,03
0,02 0.02 0.02 0.03 0,03 0.03
C.L.'-i
0,05 0,07 0,07 0.05 0.07 0.07
0,06 0.06 0.06 0.07 0.07 0.08
a) Mean for five independent analyses, b) SAE, standard analytical error c) C,L,, confidence limit at 95% confidence level and four degrees of freedom (/ = 2,776),
of metoprolol tartrate was determined following the recommended procedures and reference method. The results are summarized in Table 4, which showed recoveries in the range of 100.02—100.13%, 99.99—100.15% and 99.94—100.11% for initial rate, fixed time and reference methods, respectively. No interference from the common excipients was observed.
Robustness The conditions are very robust for the application of the proposed methods to determine the active drug in pharmaceutical formulations. Each operational parameter was checked and challenged for the robustness of the methods. The operational parameters investigated were:
• volume of 0.015 M KMnO^ (±0,2 ml) • volume of 0 .60 M NaOH (±0.2 ml) Under these conditions a sample solution containing
6.0/ igmr ' (Metalor 25, Cipla) of active metoprolol tartrate was assayed five times by the initial rate and fixed time methods. The values of mean recovery, standard deviation and relative standard deviation represent good reliability of the proposed methods.
The effect of temperature on reaction rate is well known and important in understanding the various activation parameters of the reaction products. In order to evaluate the apparent activation parameters, the reaction rate was studied at 298, 303, and 308K at [metoprolol]=1.46X lO"*"— 8.76XI0""''M, [KMnO4] = 3.00xl0"^M and [NaOH]=l.20x 10''M.
Arrhenius curve (Fig. 5) was constructed by plotting logk versus l/fand found to be linear with coefficient of correlation, r= - 0.9998, Activation energy (£J can be calculated from the slope (-EJ2303R) and A from the intercept of the Arrhenius curve and found to be 90.73 kJinoP' and 4.75X10", respectively. The other activation parameters such as enthalpy, entropy and free energy of activation of the reaction product were calculated using Eyring equation:
AH'-k 4S» log — = \og(kJh) + —
T \ ^^ ^ > 2.303«
0 00324 0.00326 0 00328 0.00330 0.00332 0 00334 0 00336 0 00338
1/T(K)
Fig. 5. Arrhenius Plot: log<r versus l/Tfor Activation Energy
1
2,303/; T
The plot oi\ogklT versus l/7'(Fig, 6) was linear with cor-
0 00324 0,00326 0,00328 0 00330 0.00332 0 00334 0.00336 0 00338
1/T{K)
Fig, 6. Eyring Plot; logA/Z'v'crau.v 1/7" for zJW'and 4 5 '
relation coefficient of -0,9999, 4 / / ' was evaluated from the slope (-zl/-/V2,303/?) and AS^ from the intercept [log(V/') + ^'5''/2,303j^] of the compiled Eyring plot. The values of 4 / / ' and 4 5 ' were found to be 88,20 kJmol"' and 84,54 J K ' ' moP', respectively. The Gibbs free energy of activation was determined by AG^=AH^-TAS^ at 298K and foundtobe-63,01 kJiuoP',
Applicability of the Proposed Methods The proposed methods (initial rate and fixed time methods) were successfully applied to the determination of metoprolol tartarte in pharmaceutical formulations. The results of the proposed methods were statistically compared with those of the reference method using point hypothesis test. Table 5 shows that the calculated t- (paired) and F-values at 95% confidence
August 2005 947
Table 4. Summary of Data for the Determination of Metoprolol Tartrate in I'liarmaceutical Preparations by Standard Addition Method
Preparations"'
Amount ( / rgmr ' )
Initial rate method Fixed time method Reference method
FoundtS.D"' Recovery Found±S.D."' Recovery „ Found±S.D.°' Recovery Taken Added (/Jgml ') (%) ^ ^ ' ^ (/Jgml ') (%) ^^^ ^'^^ (/igml ') (%) ^'^^ ^ ' "
Betaloc-25 3.0 (AstraZeneca) 3.0 Metapro-25 3.0 (Cardicare) 3.0 Metolar-25 3.0 (Cipla) 3.0
1.0 4.00 + 0.06 100.05 0.03 0.07 3.99i0.06 3.0 6.00±0.08 100.02 0.04 0.10 (i.0l±0.05 1.0 4.01+0.05 100.13 0.02 0.06 4.01 ±0.06 3.0 6.00+0.07 100.07 0.03 0.09 6.01 ±0.05 1.0 4.00+0.05 100.06 0.02 0.06 3.99±0.05 3.0 6.00±0.06 100.02 0.03 0.07 6.00+0.05
99.99 0.03 0.07 4.00±0.05 100.09 0.02 0.06 5.99±0.05 100.14 0.03 0.07 3.99±0.09 100.15 0.02 0.06 6.01 ±0.06 99.99 0.02 0.06 4.00±0.05
100.04 0.02 0.06 6.00±0.07
100.11 0.02 0.06 99.94 0.02 0.06 99.94 0.04 0.11
100.17 0.03 0.07 100.11 0.02 0.06 100.05 0.03 0.09
0) Mean for five independent analyses.
Table 5. Comparison of the Proposed Methods Using Point and Interval Hypothesis Tests with the Reference Method at 95% Confidence Level
Initial rate method Fixed time method
Formulations"' Recovery RBD"' /- F-(%) (%) value"' value'"
Recovery RSD°' (- F-(%) (%) value'" value'" "u
Reference method
Recovery R.S.D."' {%) (%)
Betaloc-25 (AstraZeneca) Metapro (Cardicare) Metolar (Cipla)
100.02 0.99 0.23
100.07 1.16 0.23
100.13 0.93 0.18
1.55 0.994 1.008 100.15 1.14 0.55
1.53 0.988 1.010 100.04 0.82 0.37
1.55 0.989 1.012 99.99 0.83 0.13
2.06 0.992 1.012 99.94 0.80
1.32 0.990 1.008 100.17 0.94
1.92 0.986 1.013 100.05 1.15
a) Mean for five independent analyses, h) ThcorcticaW-valuc (v=8) and f-valuc (v=4, 4) ai 95% confidence level arc 2.306 and 6.39, respectively, c) In pharmaceutical analysis, a bias, based on recovery experiments, of ±2% (8^=0.98 and 8^= 1.02) is acceptable.
level are less than the theoretical ones, confirming no significant differences between the performance of the proposed methods and the reference method. The previous investigations were also checked and confirmed by interval hypothesis tests.'^' The Canadian Health Protection Branch has recommended that a bias, based on recovery experiments, of ±2% is acceptable."^ It is evident ft-om Table 6 that the true bias of all samples of drug is smaller than ±2%.
Conclusion Initial rate and fixed time methods are applied for the rou
tine quality control analysis of metoprolol tartrate in pharmaceutical formulations. The proposed method does not require any laborious clean up procedure prior to analysis and therefore, can be fi-equently used in the laboratories of research, hospitals and pharmaceutical industries. It has extremely high sensitivity, selectivity and low limit of detection.
Acknowledgements The authors are gratefiil to The Chairman, Department of Chemistry, Aligarh Muslim University, Aligarh for providing research facilities. Financial assistance provided by Council of Scientific and Industrial Research (CSIR), New Delhi, India to Dr. Syed Najmul Hejaz Azmi (snhazmi(gyahoo.com) as Research Associate (Award No. 9/ 112(329)/2002-EMR-l) is gratefully acknowledged.
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