Development and Validation of a Simultaneous HPLC Method for Estimation of Bisoprolol Fumarate and Amlodipine Besylate from Tablets

Abstract

A fast, robust and stability indicating RP-HPLC method was developed for simultaneous determination of bisoprolol fumarate and amlodipine besylate in tablets. The mobile phase was mixture of 25 mM ammonium acetate adjusted to pH 5.0 and methanol (65: 35) at 0.8 ml/min. The stationary phase was Luna C18-2 column (3 mu, 50x4.6 mm ID). UV detection was performed at 230 nm. Retention time was 1.45 min and 3.91 min for bisoprolol and amlodipine, respectively. Linearity was established in the range of 8-33 mug/ml. Mean recovery was 99.1% and 98.6% for bisoprolol fumarate and amlodipine besylate, respectively.

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Indian Journal of Pharmaceutical Sciences542 July - August 2008
occlusive, protective skin barrier and actively
replenishes moisture for better skin protection
thereby leaving the skin silky, smooth and hydrated.
The repairing and protecting properties open up
possibilities to formulate caring products for sensitive
skin. Improving the product features and medicinal
functionality further validate mango butter as a
specialty excipient in development of cosmeceuticals
and has an immense value for its commercialization.
ACKNOWLEDGEMENTS
Authors wish to express their sincere gratitude to Charbhuja
Trading Agencies and Pvt. Ltd., Mumbai for gift samples
of mango butter and the refined fractions. We are also
thankful to Mr. Devang Shah for his kind assistance in the
in vivo studies.
TABLE 1: COMPARATIVE EVALUATION OF THE PARAMETERS IN IN VIVO EXCISION AND INCISION WOUND MODELS
Group Excision Incision
Percentage Wound Contraction Time of Scar Area Tensile
d 4 d 8 d 16 Re-epithelization (mm2) Strength (g)
(d)
Control 13.2±3.6 50.9±4.9 78.1±1.6 24.0±0.7 1.38±0.05 160.0±8.6
Foot Care Cream 14.4±3.1 41.9±7.1 91.1±1.2# 16.8±0.6# 1.05±0.23* 341.7±23.1#
Marketed Cream 17.3±3.5 51.1±4.4 85.5±0.7* 19.2±0.5# 1.08±0.04* 215.0±6.2*
All values are given in mean±SE; *p<0.05 = signiÞ cant Vs. Control.
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Accepted 23 August 2008
Revised 04 February 2008
Received 04 July 2007
Indian J. Pharm. Sci., 2008, 70 (4): 539-542
Development and Validation of a Simultaneous HPLC
Method for Estimation of Bisoprolol Fumarate and
Amlodipine Besylate from Tablets
D. N. VORA* AND A. A. KADAV
Chemistry Dept, Mithibai college of Arts, Chauhan Institute of Science and A. J. College of Commerce and Economics,
Vile Parle (W), Mumbai–400 056, India
Vora, et al.: Simultaneous HPLC Method for Bisoprolol Fumarate and Amlodipine Besylate
A fast, robust and stability indicating RP-HPLC method was developed for simultaneous determination of bisoprolol
fumarate and amlodipine besylate in tablets. The mobile phase was mixture of 25 mM ammonium acetate adjusted
to pH 5.0 and methanol (65: 35) at 0.8 ml/min. The stationary phase was Luna C18-2 column (3 µ, 50×4.6
mm ID). UV detection was performed at 230 nm. Retention time was 1.45 min and 3.91 min for bisoprolol and
amlodipine, respectively. Linearity was established in the range of 8–33 µg/ml. Mean recovery was 99.1% and
98.6% for bisoprolol fumarate and amlodipine besylate, respectively.
Key words: Bisoprolol fumarate, amlodipine besylate, RP-HPLC
*For correspondence
E-mail: dn.vora@rediffmail.com
Bisoprolol fumarate is a synthetic beta1-selective
cardioselective adrenoceptor blocking agent. The

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Indian Journal of Pharmaceutical Sciences 543July - August 2008
chemical name for bisoprolol fumarate is (±)-1-[4-
[[2-(1-methylethoxy) ethoxy] methyl]phenoxyl-3-
[(1-methylethyl)amino]-2-propanol(E)-2-butenedioate
(2:1). It is a white crystalline powder, which is readily
soluble in water, methanol, ethanol, and chloroform1.
It is ofÞ cial in USP2.
Amlodipine besylate, a long-acting calcium channel
blocker, is chemically described as 3-ethyl-5-methyl(±)-
2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-
1,4-dihydro-6-methyl-3,5-pyridine dicarboxylate,
monobenzenesulphonate. Amlodipine besylate is a
white crystalline powder. It is slightly soluble in
water and sparingly soluble in ethanol3. It is ofÞ cial
in BP4. Beta blocker plus calcium channel blocker
combinations have utility in certain cardiovascular
diseases like angina pectoris, myocardial infarction
and hypertension. A tablet formulation containing
bisoprolol fumarate and amlodipine besylate has been
recently introduced on the market.
Various methods for determination of bisoprolol
by fluorimetry5,6, HPLC7-9 and densitometry10-12 are
reported in literature. Also HPTLC13-16, HPLC17-22,
spectrophotometry23-28 methods are reported for
determination of amlodipine alone or in combination
with other drugs. But, literature survey did not
reveal any method for simultaneous determination
of bisoprolol and amlodipine. The aim of this study
was to develop a fast, precise, accurate, rugged and
robust HPLC method for simultaneous determination
of bisoprolol and amlodipine in tablets. Criteria
employed for assessing suitability of proposed method
was cost effectiveness and speed of analysis.
A liquid chromatographic system comprising of
Waters 2695 separation module and Waters 2996
PDA detector (Waters Corporation, Milford, USA)
connected to Empower chromatography software for
processing the data generated were used. Reference
standard of bisoprolol fumarate and amlodipine
besylate was kindly supplied by Indoco Remedies
along with certificate of analysis, and used as
received. HPLC grade acetonitrile was purchased
from J. T. Baker, NJ, USA, GR grade ammonium
acetate was obtained from Merck, Mumbai, India and
ExcelaR grade glacial acetic acid was supplied by
Qualigens Fine Chemicals, Mumbai, India. The Þ lter
used in sample preparation was mdi SY25NN which
was manufactured by Advanced Microdevices (P) Ltd,
Ambala, India. The combination tablets containing
bisoprolol fumarate and amlodipine besylate (Concor
AM, Merck) were procured from the market.
A buffer solution was prepared by adjusting the pH of
25 mM ammonium acetate solution to 5.0 with acetic
acid. The mobile phase was filtered and degassed
mixture of buffer pH 5.0 and acetonitrile (65:35, v/v).
Luna C18-2 column, (3 µ, 50×4.6 mm) was used as
stationary phase. A constant ß ow of 0.8 ml/min was
maintained throughout the analysis. Detection was
carried out using PDA detector at 230 nm.
A combined standard stock solution of bisoprolol
fumarate and amlodipine besylate was prepared in
methanol (200 µg/ml). Five ml of standard stock
solution was diluted to 50 ml with mobile phase
to obtain a 20 µg/ml solution of bisoprolol and
amlodipine and used as working standard for assay
analysis. Twenty tablets were weighed and crushed
to fine powder. An accurately weighed portion
of the powder equivalent to 10 mg of bisoprolol
fumarate and 10 mg of amlodipine was taken in 50
ml volumetric flask, about 30 ml of methanol was
added to it and ß ask was kept in an ultrasonic bath
for 2 min with intermittent swirling. This solution
was then diluted to the mark with methanol and
centrifuged. Five ml of the supernatant solution was
diluted to 50 ml with mobile phase and mixed. This
solution was Þ ltered through mdi SY25NN Þ lter and
used for assay analysis. Five µl of each of working
standard and sample solution were injected into the
chromatograph and the peak areas were recorded.
The amount of each active was computed by external
standard quantiÞ cation.
In order to optimize the LC separation of bisoprolol
and amlodipine, initially, mobile phases of buffer
and acetonitrile were used. The retention behavior
of both the drugs was studied with respect to pH of
buffer solution in the range of 3.0–6.8, and aqueous
composition of mobile phase. Retention of both the
drugs was found slightly dependant on pH of buffer
(slight increase in retention with increase in pH).
Bisoprolol was found relatively less sensitive to
aqueous composition as against amlodipine, which
was found more sensitive to aqueous composition.
A ten percent increase in aqueous composition
resulted in 1.6 and 3.3 times increase in retention for
bisoprolol and amlodipine respectively. The buffer
solution of pH 5.0 and mobile phase composition of
buffer:acetonitrile (65:35) was found most appropriate

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Indian Journal of Pharmaceutical Sciences544 July - August 2008
for separation of bisoprolol and amlodipine on Luna
C18-2 column. Flow rate was optimized based on
capacity factor and column efÞ ciency. Bisoprolol and
amlodipine were well resolved in reasonable time of
about 5 minutes. The retention times were 1.45 min
and 3.91 min, respectively. The resolution between
bisoprolol and amlodipine was 14.2. The Þ nal dilution
of analytes with mobile phase helped to minimize the
interference due to blank peaks. The wavelength of
230 nm was selected for the UV detection because
at this wavelength there was maximum overlap of
the spectra of bisoprolol and amlodipine. The peak
purity of the peak due to bisoprolol and amlodipine
was tested using PDA detector and were found to be
pure.
To ascertain effectiveness of system suitability test,
Þ ve replicate injections of freshly prepared working
standard solution were injected into the chromatograph
and relative standard deviation (RSD) of peak areas
was calculated. The data is presented in Table 1.
System suitability parameters such as tailing factor,
resolution factor, capacity factor and theoretical plates
of a typical chromatogram are tabulated in Table 2.
Linearity (described by equation and corresponding
corre lation coefficient) was determined using five
calibration levels for both the compounds (at 50-150%
levels). The concentrations of calibration solutions of
both the drugs were from 8 to 33 µg/ ml. The method
of linear regression was used for data evaluation.
Peak area of standard compounds was plot ted against
respective concentrations. The content of actives found
in the commercial brand of tablets (Concor AM,
Merck) by proposed method is shown in Table 1. The
low values of RSD, indicates that method is precise.
Intermediate precision was studied using different
column, HPLC instrument and performing the analysis
on different day. The results are presented in Table 1,
along with repeatability data. Sample solution injected
after 24 h of preparation did not show any appreciable
change in assay value. To conÞ rm the accuracy of the
proposed method, recovery experiments were carried
out by standard addition technique. Three different
levels of standards were added to pre-analyzed tablet
samples in triplicate. The mean percentage recoveries
of bisoprolol and amlodipine were 99.1% and 98.6%,
respectively. The results are shown in Table 1, which
indicates that the method is accurate and precise and
also there is no interference due to excipients present
in the tablets. To ascertain the suitability of Þ lter used
TABLE 1: METHOD VALIDATION RESULTS FOR
INDIVIDUAL COMPOUND
Parameter Bisoprolol Amlodipine
System precisiona (% RSD) 0.5 0.8
Repeatabilityb (% assay) 98.5 99.9
Repeatabilityc (% RSD) 0.3 0.4
Intermediate precisionb (% assay) 99.5 99.9
Intermediate precisionc (% RSD) 0.5 0.4
Linearityd (correlation coefÞcient) 0.99999 0.99999
Linearityd (equation) y = 8922.1x y = 14126.9x
- 1466.6 - 5056.6
Accuracye (% RSD) 0.8 0.4
Accuracye (% recovery) 99.1 98.6
Selectivityf No No
interference interference
Stability—ambient[%]g 99.5 98.7
Filter recoveryh 99.8 99.6
aDetermined on five replicate injections of working standard solution.
bDetermined on six real samples of Concor AM tablets and average is reported.
cPercent RSD of six values of% assay of Concor AM tablets.dDetermined at Þ ve
levels, from 50–150% of working standard concentration.eDetermined at three
levels with triplicate determination at each level. Mean of 9 values and% RSD
is reported. fDemonstrated by forced degradation and peak purity of main
peaks in degraded samples. gPercent correlation of assay after 24 h of sample
preparation against freshly prepared sample. hPercent correlation of assay of
Þ ltered sample against assay of centrifuged sample.
TABLE 2: SYSTEM SUITABILITY PARAMETERS AND ROBUSTNESS
Component Robustness parameter k’a Tb Rc Nd %Assay
Bisoprolol No change (repeatability) 1.08 1.48 - 2596 98.5
Organic in mobile phase (+3%) 1.20 1.38 - 1522 98.4
Organic in mobile phase (-3%) 1.31 1.39 - 1928 98.8
pH of buffer (+0.2 units) 1.18 1.50 - 2524 98.5
pH of buffer (-0.2 units) 1.14 1.41 - 2770 98.5
Column temperature 35° 1.31 1.39 - 3217 98.3
Flow (+0.1 mL) 1.05 1.52 - 2733 98.1
Flow (-0.1 mL) 1.20 1.43 - 2628 98.4
Amlodipine No change (repeatability) 4.58 1.53 14.23 4985 99.9
Organic in mobile phase (+3%) 3.79 1.54 9.23 3545 100.3
Organic in mobile phase (-3%) 5.25 1.52 13.26 4618 99.7
pH of buffer (+ 0.2 units) 4.53 1.52 13.24 4835 99.8
pH of buffer (- 0.2 units) 4.42 1.50 13.71 5062 99.8
Column temperature 35° 4.96 1.51 14.61 5614 99.4
Flow (+ 0.1 mL) 3.92 1.50 12.16 4738 99.1
Flow (- 0.1 mL) 4.29 1.55 12.65 4933 99.6
aCapacity factor determined for individual peak. bTailing factor determined for individual peak. cResolution factor determined between bisoprolol and amlodipine
peaks. dColumn efÞ ciency expressed as number of theoretical plates for bisoprolol and amlodipine peaks.

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Indian Journal of Pharmaceutical Sciences 545July - August 2008
for Þ ltering sample preparation, the aliquot of sample
solution was centrifuged and another aliquot of same
sample solution was Þ ltered through SY25NN Þ lter.
The percentage assay result of Þ ltered sample was in
close agreement with result of centrifuged sample,
indicating that there was no adsorption of analytes on
the Þ lter. In order to evaluate speciÞ city and stability
indicating capability of the proposed method forced
degradation studies were performed. The powdered
samples of tablets were exposed to acidic, alkaline,
strong oxidizing, heat and UV light conditions.
Also, standard of bisoprolol and amlodipine were
exposed to the above stress conditions, individually
and in combination with each other to identify
source of degradation peaks, if any. All the exposed
standards and tablet samples were then analyzed by
the proposed method. The results are given in Table
3. The assay values found lowered for bisoprolol in
all the degradation conditions, however in case of
amlodipine the assay values found lowered in all
conditions except light. The assay values of both
the ingredients were found decreased signiÞ cantly in
case of samples exposed to strong heat. Peaks due
to bisoprolol and amlodipine in the chromatogram
of all exposed samples were investigated using
PDA detector and were found spectrally pure. The
proposed method was subjected to robustness studies
with respect to change in pH of buffer (±0.2 units),
change in mobile phase composition (±3%), change
in column temperature (35°) and change in ß ow rate
(±0.1 ml). The results are presented in Table 2. The
method was found robust with respect to variability
in above conditions.
The proposed method is fast, precise, accurate,
rugged and robust for the simultaneous determination
of bisoprolol and amlodipine from tablets. Hence
it can be easily and conveniently adopted for the
routine quality control analysis for assay as well as
dissolution and content uniformity testing.
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TABLE 3: FORCED DEGRADATION DATA
Degradation condition Bisoprolol Amlodipine
% Assay Purity Purity % Assay Purity Purity
anglea threshold anglea threshold
No degradation (Control) 98.5 1.177 1.598 99.9 0.480 1.278
Acid hydrolysis (1N HCl, 80°, 20 min) 95.3 1.191 1.357 94.1 0.380 0.981
Alkali hydrolysis (1N NaOH, 80°, 10 min) 95.4 0.725 1.421 93.9 0.406 0.942
Oxidation (30% H2O2, 80°, 30 min) 91.5 1.327 1.478 91.6 0.417 0.974
Thermal (105°, 1 d) 79.7 3.501 26.787 84.6 1.126 18.860
Photolytic (UV@254 nm, 1 d) 93.9 1.791 29.484 98.5 0.845 20.924
aFor the peak to be pure, purity angle is required to be less than purity threshold.

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Indian Journal of Pharmaceutical Sciences546 July - August 2008
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Accepted 25 August 2008
Revised 04 February 2008
Received 03 April 2007
Indian J. Pharm. Sci., 2008, 70 (4): 542-546