Asian J. Research Chem. 1(1): July-Sept.

2008,
,

www.ajrconline.org

ISSN 0974-4169

RESEARCH ARTICLE

Stability Indicating RP- HPLC Method for Simultaneous Estimation of Valsartan

and Amlodipine in Capsule Formulation.
1

SS Chitlange1*, Kiran Bagri1 and DM Sakarkar2.

Dr. D.Y.Patil Institute of Pharmaceutical Science and Research, Pimpri, Pune- 411018
2
S. N. Institute of Pharmacy, Pusad
*Corresponding Author E-mail: sohanchitlange@rediffmail.com

ABSTRACT
Present work describes a precise, accurate and reproducible Reverse phase High Performance Liquid Chromatographic
(RP-HPLC) method for simultaneous estimation of Amlodipine besylate (AMLB) and Valsartan (VAT) on RP C-18
Column (Kromasil, 250 x 4.6 mm) using acetonitrile: phosphate buffer (0.02M, pH 3.0), (56:44 v/v) as mobile phase at
a flow rate of 1.0 ml/min and the detection wavelength was 234 nm. The retention time for AMLB and VAT was found
to be 3.07 and 6.20 min, respectively. The method was also applied for the determination of AMLB and VAT in the
presence of their degradation products formed under variety of stress conditions. Proposed method was validated for
precision, accuracy, linearity range, robustness and ruggedness.

KEY WORDS

Amlodipine besylate, Valsartan, Reverse phase High Performance Liquid Chromatography,

Stability indicating method.

INTRODUCTION:

MATERIAL AND METHOD:

Amlodipine besylate (AMLB), 2-[(2-amino ethoxy)methyl]-4-(2-cholophenyl)-1, 4-dihydro-6-methyl-3, 5pyridine dicarboxylic acid 3-ethyl-5-methyl ester,
benzosulfonate, is a potent dihydro calcium channel
blocker1.

Chemicals and Reagents

AMLB and VAT were obtained as gift samples from
Glenmark Pharmaceuticals Limited, Nashik and Lupin
Laboratories Ltd, Pune respectively. Acetonitrile (HPLC
grade), potassium phosphate and orthophosphoric acid
were of reagent grade. Double distilled water was used in
preparation of mobile phase. The commercial formulation
of AMLB and VAT is available in ratio of 1:32{Valzaarsm (2.5/80 mg)} as capsules.

Valsartan (VAT), N-(1-Oxopentyl)-N-[[2'-(1H-tetrazol5-yl) [1, 1'-biphenyl]-4-yl] methyl]-L-valine, is a potent

angiotensin receptor blocker 1. Literature survey
revealed HPLC2-4, RP-HPLC5,6, HPTLC7,8, LCMS/MS9,
LC-MS10
and
simultaneous
UVspectrophotometric methods11,12 are reported for the
estimation of AMLB alone or in combination with other
anti-hypertensive agents. Methods such as HPLC 13-15,
LC-MS 16-18, Protein precipitation19, Capillary
electrophoresis20
and
simultaneous
UVspectrophotometric methods21,22 are reported for
estimation of VAT alone or in combination with other
agents. As no method is reported for AMLB and VAT
in combination, the aim of the present study was to
develop accurate, precise and selective reverse phase
HPLC assay procedure for the analysis of AMLB and
VAT in bulk drug samples and in combined dosage
formulation.
Received on 20.08.2008
Accepted on 14.09.2008

Modified on 30.08.2008
AJRC All right reserved

Asian J. Research Chem. 1(1): July-Sept. 2008;Page 15-18

Instrumentation
A Gradient HPLC (Merck Hitachi) with L-7100 double
reciprocating pump, L-7400 UV detector and RP-C18
column was used. A Rheodyne injector with a 20 l loop
was used for the injection of sample. The HPLC system
was equipped with Winchrom software for data
processing.
Chromatographic Condition
The mobile phase containing acetonitrile: potassium
dihydrogen phosphate buffer (0.02M, pH 3.0) (56:44v/v)
was found to resolve AMLB and VAT. Orthophosphoric
acid was used for pH adjustment of buffer. The mobile
phase was filtered on a 0.45 micron membrane filter and
then ultrasonicated for 30 min. The flow rate was set to1.0
ml/min. Both drugs shows good absorbance at 234 nm,
which was selected as wavelength for further analysis. All
determinations were performed at constant column
temperature (250C).

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Asian J. Research Chem. 1(1): July-Sept. 2008,

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Preparation of Stock Solutions

The standard stock solution of AMLB and VAT was
prepared by dissolving 10 mg of each drug separately in
100.0 ml mobile phase.
Calibration curve
Calibration curves were prepared by taking appropriate
aliquots of standard AMLB and VAT stock solutions in
different 10 ml volumetric flask and diluted up to the
mark with mobile phase to obtain final concentrations
of 1.0,2.5, 5.0, 10, 15, 20, 25, 30, 40 g/ml of AMLB
and 10, 20, 30, 40, 50, 60, 70 ,80 g/ml of VAT.
Standard solutions (n=6) were injected through 20 l
loop system and chromatograms were obtained using
1.0 ml/mim flow rate. The effluent was monitored at
234 nm. Calibration curve was constructed by plotting
average peak area against concentration and regression
equation was computed.
Table no. 1. Result of AMLB and VAT in marketed
formulation (n=6).
Marketed
Drug
% Amount
%
Formulation
found SD
RSD
Valzaar-sm
AMLB
99.590.462
0.464
(Torrent
VAT
99.820.646
0.647
Pharma. ltd.)
S.D: Standard deviation, RSD: Relative standard deviation

Validation of the method

The developed method was validated in terms of
linearity, accuracy, specificity, limit of detection, limit
of quantification, intra-day and inter-day precision and
repeatability of measurement.

FIG.-1 Typical chromatogram of AMLB (RT=3.06 min)

and VAT (RT= 6.22 min).

Sample Preparation
A total of 20 capsules were uncapped and the contents
of the capsules were accurately weighed. An amount
equivalent to one capsule (containing 2.5 mg of AMLB
and 80 mg of VAT) was transferred to a 100ml
volumetric flask; 50 ml of mobile phase was added and
the flask was kept in an ultrasonic bath for 10 min. The
volume was made upto mark and the solution was
filtered through 0.2 micron nylon membrane filter.

To 1ml of the filtered solution 15.5 ml of standard stock

solution of AMLB (100 g/ml) was added in a 50 ml
volumetric flask and final volume was made up with mobile
phase to yield a solution containing 16 g/ml of each drug.
The diluted solution was analysed under optimized
chromatographic conditions and chromatogram is depicted
in fig. No.1.
Degradation Studies
Stress testing of the drug substance can help identify the
likely degradation products, the stability and specificity of
the analytical procedure.
Acidic degradation studies
A 1 ml of 0.1 M hydrochloric acid was added to 9 ml of drug
solution to get final concentration of 16 mcg/ml of each
drug. This solution was allowed to stand for 24 hrs.
Alkali degradation studies
A 1 ml of 0.1 M sodium hydroxide was added to 9 ml of
drug solution to get final concentration of 16 mcg/ml of each
drug. This solution was allowed to stand for 24 hrs.
Oxidative studies
A 1 ml of 3% hydrogen peroxide was added to 9 ml of drug
solution to get final concentration of 16 mcg/ml of each
drug. This solution was allowed to stand for 24 hrs.
Temperature stress studies
A drug solution containing 16 mcg/ml of each drug was
maintained at 500C for 24 hrs.
After the stress assays, the sample were analyzed in the
above reported chromatographic conditions. Fig. no.2-5
shows the degradation behavoiur of combination.

FIG.-2 Chromatogram showing degradation in 0.1 M HCl.

RESULT AND DISCUSSION:

To develop a precise, accurate and suitable HPLC method
for the simultaneous estimation of AMLB and VAT,
different mobile phases were tried and the proposed
chromatographic conditions were found to be appropriate
for the quantitative determination. The results obtained by
the assay of marketed formulation are summarized in
Table.1. System suitability tests were carried out as per
USP XXIV and parameters are summarized in Table.2.

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Degradation Behavior- The results of the stress

studies indicated the specificity of the method that
has been developed. VAT was degraded only in 3%
H2O2 and in temperature stress conditions whereas
AMLB was degraded in all conditions. The degraded
products appeared at retention time (RTs) 2.18 and
2.48 in 0.1 M HCl, 2.16 and 2.48 in 0.1 M NaOH,
2.57 and 5.17 in 3% H2O2 and 2.15, 2.45 and 5.08 in
temperature degradation studies. The result of forced
degradation studies are given in table no. 4.

FIG.-3 Chromatogram showing degradation in 0.1 M

NaOH

FIG.-4 Chromatogram showing degradation in 3% H2O2

Method Validation23 - The proposed HPLC method

was validated as per ICH guidelines.
Specificity
The peak purity of AMLB and VAT were assessed
by comparing the retention time (TR) of standard
AMLB and VAT. Good correlation was obtained
between the retention time of standard and sample of
AMLB and VAT.
Linearity
Linearity was studied by preparing standard
solutions at different concentration levels. The
linearity range for AMLB and VAT were found to be
1- 40 g/ml and 10-80 g/ml, respectively. The
regression equation for AMLB and VAT were found
to be y = 413580x 78268 and y = 546553x 62276
with coefficient of correlation, (r) 0.9996 and 0.9995,
respectively.
Precision
Precision was evaluated by carrying out six
indepndent sample preparation of a single lot of

formulation. The sample solution was prepared in the

same manner as described in sample preparation.
Percentage relative standard deviation (%RSD) was found
to be less than 2% for within a day and day to day
variations, which proves that method is precise. Results
are shown in Table 3.
Table no. 2. System Suitability Parameters
Parameter
AMLB
Linearity range
1-40 g/ml
Correlation coefficient*
0.9996
Slope*
413580
Limit of detection
(g/ml)
0.03
Limit of quantitation
(g/ml)
0.089
Retention time*
3.07
Resolutionfactor*
2.43
Tailing factor*
0.90
Accuracy* 80%
99.750.492
(Recovery 100%
100.400.604
studies )
120%
101.560.810
*Average of six readings Standard deviation

VAT
10-80 g/ml
0.9995
546553
0.018
0.054
6.20
0.83
100.810.63
99.090.112
101.790.36

Accuracy (Recovery studies)

To check the degree of accuracy of the method, recovery
studies were performed in triplicate by standard addition
method at 80%, 100% and 120%. Known amounts of
standard AMLB and VAT were added to pre-analyzed
samples and were subjected to the proposed HPLC method.
Results of recovery studies are shown in Table 2.
Robustness of method
To evaluate the robustness of the developed RP-HPLC
method, small deliberate variations in the optimized method
parameters were done. The effect of change in flow rate,
mobile phase ratio and column temperature on the retention
time and tailing factor were studied. The method was found
to be unaffected by small changes like 0.1 change in pH,
0.1 change in flow rate and 1 change in mobile phase.
Ruggedness of method
Ruggedness of the proposed RP-HPLC method was
evaluated by comparing the results obtained by two different
analysts. The standard deviation for AMLB and VAT for
analyst I was 0.4039, 0.5742 and for analyst II was 0.2201,
0.4130 respectively.
Table No.3: Statistical Evaluation of Precision of developed
method.
Drug

Repeatability

AMLB
99.870.625
VAT
100.760.542
S.D: Standard deviation

Intraday

Interday

% Mean SD
99.980.751
101.310.623
99.210.757
101.980.953

CONCLUSION:
The proposed method is simple, sensitive and reproducible
and hence can be used in routine for simultaneous
determination of AMLB and VAT in bulk as well as in
pharmaceutical preparations. Statistical analysis of the
results has been carried out revealing high accuracy and
good precision

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Asian J. Research Chem. 1(1): July-Sept. 2008,

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Table No. 4: Results of Forced Degradation studies.

Stress condition
Time (hours) % Assay of active substance

Acid hydrolysis (0.1 M HCl)

Base hydrolysis (0.1 NaOH)
Oxidation (3% H2O2)
Thermal degradation (500C)

24
24
24
24

AMLB
82.6
85.72
86.81
84.2

FIG.- 5Chromatogram showing thermal degradation

ACKNOWLEDGEMENTS:
The authors are thankful to Glenmark Pharmaceuticals
Limited, Nashik and Lupin Laboratories Ltd., Pune for
providing gift samples of drugs AMLB and VAT.

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