INTRODUCTION:
Atorvastatin calcium is a hypolipidemic agent which
inhibits HMG-CoA reductase enzyme involved in lipid synthesis. Amlodipine
besylate is a Calcium channel blocker used in the treatment of hypertension.
The combination is used to treat high blood pressure (hypertension), chest pain
(angina), and to lower the risk of stroke, heart attack and other heart complications
in people with type 2 diabetes, coronary heart disease or other risk factors1.
Literature
survey reveals that analysis of Atorvastatin calcium and Amlodipine besylate
either alone or in combination has been attempted previously using UV
Spectroscopy2-4, Fluorimetry5 and RP-HPLC methods6-21.
However, most of the HPLC methods employ mobile phases having extreme pH
values or a high buffer concentration. The retention time of the last eluting
drug ranged from 3.71 to 8.6 mins. Efficiency in analysis could be achieved if
retention time could be reduced and inexpensive columns were employed, which
would thereby decrease the time of analysis and bring about savings in cost.
Further, the use of simpler solvents, moderate pH of mobile phase and lower
buffer concentration would help to prolong column life. Hence, the aim of the
present work was to develop and validate a simple and rapid RP-HPLC method for
the simultaneous estimation of Amlodipine and Atorvastatin in tablet dosage
form. Further an attempt was made to adopt a tablet drop method in place of the
conventional tablet crush method for the assay of tablet formulation.
MATERIALS
AND METHODS:
Chemicals
and Reagents:
Atorvastatin
calcium and Amlodipine besylate were procured from Vergo Pharma research
laboratory, Verna-Goa and Cipla Pvt. Ltd. Verna-Goa respectively as gift
samples. The tablet formulation ‘Zivast AM’ was purchased from a local retail
pharmacy. All chemicals and reagents were of HPLC / AR grade.
Instrumentation:
An
Agilent HPLC of 1120 Compact LC series with Open lab software having VW
detector was used for the analysis.
Preparation
of Mobile phase:
Mobile
phase consisting of pH 5.8 Potassium di-hydrogen phosphate buffer (20mM) and
Acetonitrile in a ratio of 55:45, v/v was employed.
Preparation
of Diluent:
A solution of 45% Acetonitrile in water was used as
Diluent / Blank.
Preparation
of Test sample:
Five
tablets were weighed and transferred directly to 100mL volumetric flask. To
this 70mL of diluent was added, vortexed and sonicated for 10 minutes. Volume
was made up to the mark with diluent and mixed well. This was centrifuged for
10 minutes at 5000 rpm. From the supernatant 2mL was taken and transferred to a
100mL volumetric flask and volume was made up to the mark with diluent and
injected in the HPLC system.
Preparation
of placebo sample:
The
amount of placebo powder equivalent to 5 tablets was weighed and transferred to
100mL volumetric flask and procedure was adopted as for test preparation.
Chromatographic
conditions:
Column: Waters Symmetry C-18 (150mm×3.9mm×5µ)
Column temperature: 30°C
Mobile phase: Acetonitrile: 20mM Phosphate buffer pH
5.8 (45:55, v/v)
Flow rate: 1.0 mL/min
Injection volume: 20 µL
Run time: 5 minutes
Detection wavelength: 254nm
Validation:
The
mixed drug standard solution was injected six times to establish the system
suitability. The developed method was validated for specificity, linearity,
range, accuracy, precision, and robustness as per ICH guidelines22.
RESULTS AND DISCUSSION:
Initial
trials were conducted for the simultaneous estimation of Amlodipine and Atorvastatin
using mobile phase consisting of Phosphate buffer and Acetonitrile in different
proportions. Mobile phase consisting of Phosphate buffer pH 5.8
(20mM): Acetonitrile (55:45, v/v) with flow rate of 1 mL/min and a column
temperature of 30°C was found to be optimal for obtaining well defined and
resolved peaks.
Accuracy
was determined by performing % recovery in triplicate at 80%, 100% and 120% of
the test concentration. The results are given in Table 3.
Precision
of the method was determined by performing repeatability studies on six
injections of standard solution. Resulting chromatograms were recorded and %
RSD for the peak area was calculated. The results of precision analysis are
given in Table 4.
Robustness
of the method was established by making small deliberate changes in the flow
rate, column oven temperature, composition of Mobile Phase and pH of the
buffer. The system suitability parameters were measured and it was found that
small, deliberate changes in chromatographic conditions did not alter the
results of analysis significantly.
The
assay of the formulation was performed employing the tablet drop technique. The
mean % assay for Amlodipine was found to be 91.90% and for Atorvastatin it was
found to be 107.91% which was within the acceptance limits of 90-110%.
CONCLUSION:
A
simple, rapid, specific, accurate, precise and robust reversed phase high
performance liquid chromatographic method has been developed for the
simultaneous estimation of Amlodipine and Atorvastatin in tablet dosage form.
The developed method imparted efficiency in analysis as with reduced retention
time there was decrease in the time of analysis which resulted in savings in
cost. Further, the use of simpler solvents, moderate pH of mobile phase and lower
buffer concentration would help to prolong column life. Also, by adopting a
tablet drop method in place of the conventional tablet crush method for the
assay of tablet formulation convenience in analysis along with minimization in
error while transfer of weighed tablet powder was achieved. Thus, the
developed method can be used as a powerful quality control tool for the
analysis of Amlodipine and Atorvastatin in bulk or in their combined tablet
formulation.
ACKNOWLEDGEMENT:
The
authors are thankful to Cipla Pvt. Ltd. Verna-Goa for gift sample of Amlodipine
besylate and to Vergo Pharma Research Laboratory, Verna-Goa for gift sample of
Atorvastatin calcium.
CERTIFICATE
OF CONFLICT OF INTEREST:
The
authors declare no conflict of interest.
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