IL FARMACO 59 (2004) 913919

http://france.elsevier.com/direct/FARMAC/

Application of oxidants to the spectrophotometric determination

of amlodipine besylate in pharmaceutical formulations
Nafisur Rahman *, Manisha Singh, Md. Nasrul Hoda
Analytical Research Division, Department of Chemistry, Aligarh Muslim University, Aligarh, 202002 UP, India
Received 21 January 2004; received in revised form 26 May 2004; accepted 17 July 2004

Available online 16 September 2004

Abstract
Three new spectrophotometric methods for the determination of amlodipine besylate have been proposed. The first two methods, i.e. A and
B, are based on the oxidation of the drug with Fe(III) and the estimation of Fe(II) produced after chelation with either 1,10-phenanthroline or
2,2-bipyridyl at 500 and 515 nm, respectively. The Beers law was obeyed in the concentration ranges of 210 and 414 g ml1 with molar
absorptivity of 2.9 104 and 2.7 104 l mol1 cm1 for methods A and B, respectively. The third procedure depends on the interaction of
amlodipine besylate with ammonium heptamolybdate tetrahydrate, which resulted in the formation of molybdenum blue (kmax 825 nm). The
linear dynamic range and the molar absorptivity values were found to be 1559 g ml1 and 1.8 104 l mol1 cm1, respectively. The results
of the proposed procedures were validated statistically and compared with those obtained by the reference method. The proposed methods
were applied successfully to the determination of amlodipine besylate in commercial tablets.
2004 Elsevier SAS. All rights reserved.
Keywords: Amlodipine besylate; Visible spectrophotometry; Oxidation; Pharmaceutical analysis

1. Introduction

Amlodipine besylate (I) is a dihydropyridine type long

acting calcium channel blocker with slow onset of vasodia-
latory action [1,2]. The use of this important life saving drug
is approved for the treatment of variant and stable angina and
hypertension, too [3,4]. As an additional property, amlodi-
pine inhibits vascular smooth muscle cell growth through
interactions with targets other than L-type calcium channels
quid chromatography with tandem mass spectrometry [23]
[5] and is more selective for arterial vascular smooth muscle
and fluorimetry [24].
than cardiac tissues. It has been investigated that amlodipine
As far as sensitive and economical methods of assay are
exhibits ameliorating effects on plasma and myocardial ca-
concerned, few spectrophotometric methods have been repor-
techolamines with a significant reduction of calcium deposi-
ted for the quantification of amlodipine besylate based on
tion and may be useful in dilated cardiomyopathy [6,7].
extractable ion pair complexes [2529], oxidative coupling
Various analytical methods have been reported for the
with 3-methyl 2-benzothiazolinone hydrazone hydrochlo-
assay of amlodipine besylate in pure form as well as in
ride [30], reactions with sodium hydroxide, ninhydrin and
pharmaceutical formulations. They include high perfor-
ascorbic acid in N, N-dimethylformamide medium [3133],
mance liquid chromatography [813], reversed phase high
derivative spectroscopy [34,35], simultaneous multicompo-
performance liquid chromatography [4,1416], high perfor-
nent mode of analysis and difference spectrophotometry
mance thin layer chromatography [1720], gas chromatogra-
[3638], charge transfer complexation [33,3941], and reduc-
phy [21], gas chromatographymass spectrometry [22], li-
tion of Fe (III) and subsequent interaction of Fe (II) with
ferricyanide [42]. The inorganic oxidants such as Fe(III) [43],
* Corresponding author. vanadate [44], molybdate [45] and permanganate [46] have
E-mail address: cht17nr_amu@yahoo.com (N. Rahman). been used to determine pharmaceuticals in dosage forms.
0014-827X/$ - see front matter 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.07.009
914 N. Rahman et al. / IL FARMACO 59 (2004) 913919
Fig. 1. Effect of time on the oxidation of amlodipine besylate(a) methods
A and B, and (b) method C.
In this communication, three spectrophotometric methods
for the determination of amlodipine besylate have been pro-
posed. The first two methods (method A and B) are based on
the reduction of Fe(III) by amlodipine besylate and conse-
quently Fe(II) produced is estimated by chelation with 1,10-
phenanthroline (method A) or 2,2-bipyridyl (method B). In a
third procedure (method C), molybdate is reduced to molyb-
denum blue which is determined at 825 nm. All the three
methods were found to be more sensitive in comparison to
the existing spectrophotometric methods.
2. Experimental
2.1. Instruments
Spectronic 20D+ spectrophotometer (Milton Roy, USA)
and pH-meter model L1-10T (Elico, India) were used to
Fig. 2. Effect of 2 ml of varying concentration of ferric ammonium sulphate
on the oxidation of amlodipine besylate.
Fig. 3. Effect of 0.5 ml of varying concentration of 1,10-phenanthroline
(method A) and 1.0 ml of varying concentration of 2,2-bipyridyl (method
B).
measure the absorbance and pH, respectively. A temperature
controlled water bath (NSW, New Delhi) was used to control
the temperature.
2.2. Reagents and materials
(i) A 0.1% amlodipine besylate stock solution was prepared
by dissolving 100 mg of pure drug (M/s. Wockhardt Ltd.,
India) in minimum volume of glacial acetic acid for
methods A and B, and in minimum volume of methanol
for method C. The whole contents were diluted to 100 ml
with doubly distilled water. Further dilutions were made
with water according to the need.
 N. Rahman et al. / IL FARMACO 59 (2004) 913919 915

2.3. Recommended procedures

2.3.1. Method A
Aliquots corresponding to 210 g ml1 of amlodipine
besylate were pipetted into a series of boiling tubes. Then
2.0 ml of 0.25% ferric ammonium sulphate, 2.5 ml of sodium
acetateacetic acid buffer of pH 4.6 and 0.5 ml of 0.6%
1,10-phenanthroline were added successively in each tube.
The contents were mixed well and placed in a water bath for
20 min to develop the colour. After heating, the tubes were
cooled to room temperature and the contents were finally
transferred to 10 ml volumetric flasks and completed to
volume with doubly distilled water.

2.3.2. Method B
Into a series of boiling tubes, amlodipine besylate solution
in the concentration range of 414 g ml1 were transferred,
followed by 2.0 ml of 0.25% ferric ammonium sulphate
solution. The contents of each tube were heated on a water
bath for 20 min. A 2.5 ml of sodium acetateacetic acid
Fig. 4. Effect of volume of 2.5 102 M ammonium heptamolybdate
buffer of pH 4.6 and 1.0 ml of 1.0% 2,2-bipyridyl were
tetrahydrate solution on the oxidation of amlodipine besylate. added in each tube after taking them out from the water bath.
The contents were transferred to 10 ml volumetric flasks and
(ii) A 0.25% ferric ammonium sulphate solution was prepa- made upto the mark with doubly distilled water.
red in 0.08 M sulphuric acid.
(iii) A 2.5 102 M ammonium heptamolybdate tetrahydrate 2.3.3. Method C
solution was prepared in 4 M sulphuric acid. Aliqouts of amlodipine besylate solution corresponding to
(iv) A 0.6% 1,10-phenanthroline and 1.0% 2,2-bipyridyl 1559 g ml1 were transferred into a series of boiling tubes.
solutions were prepared in ethanol. To each tube 2.5 ml of 2.5 102 M ammonium heptamoly-
(v) Buffer solutions: sodium acetateacetic acid buffer of bdate tetrahydrate and 5 ml of disodium hydrogen phospha-
pH 4.6 was prepared by mixing equal volumes of 0.2 M tecitric acid buffer solution of pH 4.0 were added and the
sodium acetate and acetic acid. The disodium hydrogen contents were heated on a water bath for 25 min. It was
phosphatecitric acid buffer of varying pH values were transferred to 10 ml volumetric flasks after cooling and
prepared by mixing appropriate volume of 0.2 M diso- diluted to the mark with doubly distilled water.
dium hydrogen phosphate and 0.1 M citric acid. The pH The absorbances were recorded against their correspon-
was adjusted with the aid of pH-meter. All other chemi- ding reagent blanks prepared simultaneously at 500, 515 and
cals used were of analytical reagent grade. 825 nm for methods A, B and C, respectively.
Table 1
Spectrophotometric characteristics and statistical data of the regression equations
Parameters Methods
A B C
Beers range (g ml1) 210 414 1559
Absorption maxima (kmax nm) 500 515 825
Molar absorptivity (l mol1 cm1) 2.9 104 2.7 104 1.8 104
Sandells sensitivity (g cm2 per 0.001 AU) 1.95 102 2.10 102 3.15 102
Limit of detection (g ml1) 0.08 0.12 0.35
Limit of quantitation (g ml1) 0.83 1.2 3.5
Regression data
Intercept (a) 0.8 103 4.9 103 2.2 103
S.D. of intercept (Sa) 2.2 103 3.2 103 1.1 102
Slope (b) 4.7 103 6.5 103 2.2 102
S.D. of slope (Sb) 5.1 102 4.6 102 3.1 102
tSa a 3.3 104 3.4 104 2.6 104
tSb a 7.0 104 6.8 104 5.2 102
Correlation coefficient (r) 0.9998 0.9996 0.9995
2
Variance (So ) 4.3 106 8.2 106 6.6 105
Standard error of estimate (Se) 2.1 103 2.8 103 9.9 103
Linearity (Sb rel)% 0.65 0.73 0.83
a
Confidence interval of intercept and slope at 95% confidence level [48], t-values as in reference [51].
916 N. Rahman et al. / IL FARMACO 59 (2004) 913919

Table 2
Comparison of the proposed methods with the existing spectrophotometric methods for the determination of amlodipine besylate
Reagents kmax (nm) Beers range (g ml1) Molar absorptivity (l mol1 cm1) %RSD References
Eriochrome black-T a 495.0 550 6.54 103 0.88 [25]
Indigocarmine a 590.0 25150 1.96 103 0.87 [25]
Bromocresol green a 409.0 080 [26]
Bromophenol blue a 409.0 080 [26]
Methylene blue a 668.2 080 [26]
Rhodizonic acid a 450.0 1001500 [27]
MBTH b 630.0 540 9.07 103 0.87 [30]
Sodium hydroxide 456.0 20100 1.90 [31]
Ninhydrin 595.0 1060 6.52 103 0.66 [32]
DDQ c 580.0 1125 0.6 103 0.321.53 [33]
Ascorbic acid 530.0 10140 0.32 103 0.361.05 [33]
p-chloranilic acid 540.0 100600 0.91 103 0.52 [38]
Fe(III)-ferricyanide 760.0 5-15 1.76 104 0.35-4.90 [42]
Fe(III)-PTL d 500.0 210 2.9 104 0.390.52 This work
Fe(III)-BPL e 515.0 414 2.7 104 0.630.78 This work
Ammonium heptamolybdate 825.0 1559 1.8 104 0.300.35 This work
a
Extractive methods.
b
3-Methyl 2-benzothiazolinone hydrazone hydrochloride.
c
2,3-Dichloro 5,6-dicyano 1,4-benzoquinone.
d
1,10-Phenanthroline.
e
2,2-Bipyridyl.

2.3.4. Analysis of pharmaceutical formulations
Ten tablets of different brands available in the local market
equivalent to 100 mg of amlodipine besylate were crushed
and finely grounded to powder and extracted into sufficient
volume of chloroform with shaking. It was filtered through
sodium sulphate kept on a glass wool bed. The filtrate was
evaporated to dryness. Dry residue was dissolved with gla-
cial acetic acid for methods A and B, and in methanol for
method C, and made upto the mark with doubly distilled
water. The assay was completed following the recommended
procedures. The amount of drug was calculated either from
the calibration graph or the regression equation.
3. Results and discussion
The amlodipine besylate contains amino group and hence
able to reduce Fe (III) to Fe (II) and Mo (VI) to Mo (V). The
reduced Fe (II) reacts with 1,10-phenanthroline (method A)
or 2,2-bipyridyl (method B) to form red coloured complexes
which absorbed maximally at 500 and 515 nm, respectively.
In method C, the intense blue colour owing to the formation
of molybdenum blue having an absorption maximum at
825 nm is utilized for quantification of the cited drug.
530 min. Maximum and constant absorbance was obtained
after 18 min for the methods A and B whereas 22 min for the
method C. The results are shown in Fig. 1a and b.
The effects of other variables (methods A and B) such as
concentration of Fe(III) and 1,10-phenanthroline or 2,2-
bipyridyl have been investigated to develop maximum colour
by adding to 10 g ml1 amlodipine besylate. The results are
shown in Figs. 2 and 3. Therefore, 2.0 ml of 0.25% Fe(III)
and 0.5 ml of 0.6% 1,10-phenanthroline or 1.0 ml of 1%
2,2-bipyridyl were used throughout the experiment.
The effect of reagent concentration for method C was
studied by adding different volumes of 2.5 102 M ammo-
nium heptamolybdate tetrahydrate solution to a constant
amount of amlodipine besylate (250 g). It was observed
(Fig. 4) that the maximum colour intensity was obtained with
1.1 ml of the reagent, after which further increase in volume
resulted in no change in absorbance. Thus, 2 ml of the
reagent was sufficient to reach with the maximum drug
concentration in the Beers range.
The second variable in this method was the pH of the
medium. An investigation of the pH of the medium showed
that the pH in the range of 3.05.0 was sufficient to develop
the colour. Therefore, disodium hydrogen phosphatecitric
acid buffer of pH 4.0 was used throughout the experiment.

3.1. Optimization of reaction conditions 3.2. Analytical data

The optimum conditions for the formation of maximum
colour were studied and maintained throughout the experi-
ment. In order to obtain the highest and most stable absor-
bance the effect of the reaction time on the absorbance of the
product was studied. The reactions were performed on a
water bath at 100 1 C for the periods ranging from
Under the optimized experimental conditions, the optical
characteristics and the statistical parameters for the proposed
methods are summarized in Table 1. An investigation of the
intercept, standard deviation of the intercept and slope [47]
variance [48], standard error of estimate, correlation coeffi-
cient and linearity [49] suggest the excellent linearity of the
 N. Rahman et al. / IL FARMACO 59 (2004) 913919 917

indicated good sensitivity of the proposed methods which

again follow the order A > B > C. A comparison of all these
methods with the existing spectrophotometric methods
(Table 2) shows that the proposed methods are more sensitive
with lower RSD. However, the linear dynamic ranges of the
developed methods are narrow.
The uncertainty (% DC/C) in the determination of amlo-
dipine besylate throughout the range of calibration of each
method was calculated at 95% and 99% confidence levels
[51] and plotted against the concentrations (Fig. 5a and b). It
is apparent from the figures that the uncertainties are higher
at very low concentrations. Therefore, it is suggested that the
methods be operated above 4 and 6 g ml1 (method A),
6 and 8 g ml1 (method B) and 25 and 35 g ml1 (method
C) concentrations at 95% and 99% confidence levels, respec-
tively, to avoid higher uncertainty on the results of the analy-
ses.
The precision studies of the proposed methods were done
by carrying out 10 independent determinations at two
concentration levels (Table 3). The relative standard devia-
tions (RSD) were found in the range of 0.390.52%, 0.63
0.78% and 0.300.35% for the methods A, B and C, respec-
tively. The results are presented in Table 3.
For additional confirmation of the accuracy and precision
of the proposed methods, standard addition method was
performed by adding a known amount of pure drug to the
preanalyzed dosage forms. The results are summarized in
Table 4 and considered to be very satisfactory.
Table 5 shows the results obtained for the determination of
amlodipine besylate in pharmaceutical formulations by the
proposed methods and the reference method [30]. The t- and
F- values calculated from the experimental data were found
to be smaller than the theoretical ones at 95% confidence
level [52]. Thus, there were no significant differences
between the two sets of the results.

Acknowledgements
Fig. 5. Variation of confidence limit at 95% and 99% confidence levels in
the determination of amlodipine besylate(a) methods A and B, and (b)
The authors are grateful to Professor Kabiruddin, Chair-
method C.
man, Department of Chemistry, Aligarh Muslim University,
calibration graph of method A followed by method B and C, Aligarh, for providing the research facilities. One of the
respectively. The comparative study of the molar absorpti- authors (M.N. Hoda) is thankful to C.S.I.R., India for the
vity, Sandells sensitivity [50], detection limit [48] and slope award of Senior Research Fellowship to carry out this work.
Table 3
Evaluation of the accuracy and precision of the proposed procedures
Proposed methods Amount (g ml1) %RSD a SAE b Confidence limit c
a
Taken Found SD
A 5 4.97 0.03 0.52 0.01 0.0174
10 10.01 0.04 0.39 0.013 0.0232
B 8 8.06 0.06 0.78 0.019 0.0348
12 12.05 0.08 0.63 0.025 0.0464
C 25 25.01 0.09 0.35 0.029 0.0522
40 39.88 0.30 0.3 0.038 0.0696
a
Mean S.D. for 10 determinations.
b
Standard analytical error.
c
Confidence limit at 95% confidence level and 9 of freedom with t-values as in reference [51].
918 N. Rahman et al. / IL FARMACO 59 (2004) 913919

Table 4
Standard addition method for the determination of amlodipine besylate in dosage forms
Formulation Method A Method B Method C
names Amount (g ml1) %Recovery Amount (g ml1) %Recovery Amount (g ml1) %Recovery
Taken + Added Found a %RSD a Taken + Added Found a %RSD a Taken + Added Found a %RSD a
Amdepin-10 4+4 7.99 99.90 0.34 5+5 10.02 100.17 0.26 12.5 + 12.5 24.94 99.74 0.35
Amlogard-10 4+4 8.03 100.40 0.53 5+5 10.05 100.50 0.72 12.5 + 12.5 24.98 99.91 0.62
Amlopres-10 4+4 8.01 100.17 0.51 5+5 9.98 99.80 0.29 12.5 + 12.5 24.95 99.80 0.53
Myodura 4+4 8.02 100.25 0.76 5+5 10.03 100.35 0.50 12.5 + 12.5 25.08 100.34 0.42
a
Average of six independent analyses.

Table 5
Comparison of the three proposed methods with the reference method [30]
Formulation Methods
names A B C Reference [30]
Recovery (%) a,b %RSD a,b Recovery (%) a,b %RSD a,b Recovery (%) a,b %RSD a,b Recovery (%) c % RSD c
Amdepin-10 99.9 0.34 100 0.41 100.35 0.57 100.00, 100.24 0.41, 0.57
(1.343, 2.21) (0.879, 1.53) (0.434, 3.24)
Amlogard-10 100.07 0.2 100.27 0.34 100.59 0.57 100.17, 100.39 0.39, 0.36
(0.971, 3.64) (1.041, 1.29) (1.147, 4.34)
Amlopres-10 100.1 0.14 100.06 0.17 100.1 0.44 99.91, 100.19 0.16, 0.28
(1.823, 1.29) (1.478, 1.13) (0.403, 2.60)
Myodura-10 100.07 0.19 100.1 0.21 100.26 0.45 100.12, 100.14 0.12, 0.25
(0.511, 2.37) (0.163, 2.98) (0.553, 3.65)
a
Average of six independent analyses.
b
Values in the parentheses shows (t, F) values, for which theoretical values at 95% confidence level are 2.132 and 5.05, respectively [51].
c
The first value is from the batch of tablets used for methods A and B while second value is from those used for the method C.

Thanks are also extended to M/s. Wockhardt Ltd., India for
providing the gift sample of pure amlodipine besylate and to
Dr. S.M. Jaweed Mukarram (Associate Director, Wockhardt
Ltd.) for such a generous help and cooperation to streamline
this project.
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