VALIDATED METHOD DEVELOPMENT FOR ESTIMATION OF
ATORVASTATIN
AND
AMLODIPINE
IN
SOLID
DOSAGE
REGIMENS
Kapil Sharma*1, Yogesh Sharma 2, Priyanka Sharma1
1
Yaresun Pharmaceutical Pvt. Ltd.,India.
2
Yaresun labs jaipur-302006,rajasthan,india.
1
pharma_kapil@rediffmail.com
1
ABSTRACT
A method for simultaneous estimation of Atorvastatin Calcium (AVS) and Amlodipine
Besilate (AML) in combined tablet dosage form has been developed. The method
employs the application of multicomponent mode of analysis. This method utilize
Phosphate buffer (pH6.8). AVS show maximum absorbance at a wavelength of 240 nm
and AML at 369 nm. The method is fast,economical and very cheap as compared to other
simultaneous spectrophotometric method using multi-component mode of analysis for
estimation of Atorvastatin Calcium and Amlodipine Besilate due to using phosphate
buffer instead of costly solvent.Where the linearity ranges for AVS and AML were 525µg/ml and 10-50 µg/ml respectively. The procedure was successfully applied for the
simultaneous determination of both drugs in laboratory prepared mixture and in market
available tablet dosage form. The accuracy of the method was assessed by recovery
studies and was found to be 99.41±0.83 and 98.65±0.54 for AVS and AML respectively.
Results of the analysis were validated statistically so that it can be used for routine
analysis of AVS and AML in combined tablet dosage form.1,6-9
Keywords: UV-spectroscopy-multicomponent mode, validation, Atorvastatin Calcium,
Amlodipine Besilate,combined tablet dosage form.
2
INTRODUCTION
Atorvastatin calcium is chemically [R-(R, R*)]-2-(4-fluorophenyl)-β-δ-dihydroxy-5 (1methylethyl)-3-phenyl-4 [(phenylamino) carbonyl]-1H-pyrrole-1-heptanoic acid, calcium
salt (2:1) trihydrate. It is inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase. This enzyme catalyzes the conversion of HMG-CoA to mevalonate, an
early and rate limiting step in cholesterol biosynthesis. Amlodipine Besilate is
chemically 3-Ethyl 5-methyl (4RS)-2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-6methyl-1, 4- dihydropyridine-3, 5-dicarboxylate benzenesulphonate. Amlodipine is a
calcium ion antagonist or slow-channel blocker that inhibits the influx of calcium ions
into vascular smooth muscle and cardiac muscle. 2 It is official in British Pharmacopoeia1
but not in I.P and USP. Review of literature revels that there is not any method described
for simultaneous estimation of Atorvastatin calcium and Amlodipine Besilate using
multi-component mode of analysis6-9. The present paper describes simple, reproducible
and sensitive UV spectroscopy method for the determination of Atorvastatin Calcium
and Amlodipine Besilate combined dosage form, using multi-component mode of
analysis. The aim of this paper was to develop simultaneous spectrophotometric method
for quantitative determination of AVS and AML in combined tablet dosage form by
using multicomponent mode of analysis. The results of analysis using the
spectrophotometric method for estimation was found to be satisfactory such that the
developed method can be used for routine analysis of drugs from tablet dosage form.
MATERIAL AND METHOD
3
Instrument, reagents and chemicals
A dual-beam shimadzu UV-visible spectrophotometer 1601 was used. Freshly prepared
Phosphate buffer (pH6.8) (LR, Merck, Mumbai) was used as solvent. Gift sample of
AVS and AML was procured from Yaresun Pharmaceuticals p.ltd.rajasthan. Tablets of
10 mg AVS and 5 mg AML strength were procured from Yaresun Pharmaceuticals
p.ltd.rajasthan.
Preparation of stock solution of Amlodipine Besilate
Stock solution of containing 100 µg/ml of Amlodipine Besilate was prepared by
dissolving equivalent to about 25 mg of Amlodipine in 250 ml Phosphate buffer
(pH6.8).
Preparation of stock solution of Atorvastatin calcium
Stock solution containing 200 µg/ml of Atorvastatin calcium was prepared by
dissolving equivalent to about 20 mg of Atorvastatin in 100 ml Phosphate buffer
(pH6.8).
Preparation of laboratory synthetic mixture of Atorvastatin and Amlodipine
Laboratory synthetic mixture of AVS and AML were prepared in the ratio of 2:1
respectively. This decision is based on the combination of AVS and AML available in
the combined tablet dosage form. Laboratory synthetic mixture of AVS and AML were
prepared in the ratio of 20:10 respectively was prepared by dissolving 10 ml of AVS
stock solution and 10 ml AML stock solution in to 100 ml Phosphate buffer (pH6.8).
Maximum absorbance was found to be at 240 nm and 369 nm for AVS and AML
4
respectively. The spectrums are shown in figure 1 and 2. Overlay spectrum is shown in
figure 3.
Linearity and accuracy
Linearity and accuracy in the concentration range of 5-26 g/ml for AVS and 10-50
g/ml for AML were examined employing intraday and interday studies for the
determination of AVS and AML. The results for intraday and interday experiments with
a good correlation were obtained and evaluated statistically as demonstrated in table 1
and 2. Calibration curve is shown in figure 4 and 5. Optical parameters and Regression
characteristics are shown in table 3.
Application of the proposed procedure for the estimation of Atorvastatin and
Amlodipine in combined tablet dosage form
Twenty tablets were taken, and accurately weighed. The tablets were crushed to a fine
powder. The powder sample equivalent to 20 mg of Atorvastatin and equivalent 10 mg
of Amlodipine was transferred to a 100 ml volumetric flask and about 80 ml of
Phosphate buffer (pH6.8) was added and sonicated to dissolve. The volume was made
up to the mark with Phosphate buffer (pH6.8). This solution was filtered through
whatman filter paper 42. This solution (10 ml) was diluted to 100 Phosphate buffer
(pH6.8). The solutions were analyzed by multicomponent mode of analysis. As blank
Phosphate buffer (pH6.8) was used.
5
RESULTS AND DISCUSSIONS3-5
A UV-spectrophotometric (multicomponent mode of analysis) method was developed
for the estimation of Atorvastatin and Amlodipine in combined tablet dosage form.
Solvent used was Phosphate buffer (pH6.8). Measurement was done at 240 nm and 369
nm for Atorvastatin and Amlodipine respectively. Beer’s law was obeyed in the
concentration range of 5-25 g/ml for Atorvastatin and 10-50 g/ml for Amlodipine.
The value of standard deviation was satisfactory and the recovery studies were 99.41%±
0.83 for AVS and 98.65 %±0.54 for AVL. The value of relative standard deviation in
repeatability study was found 1.654 % for Atorvastatin and 0.894 % for Amlodipine. In
intra-day precision value of relative standard deviation was found 0.841 % for
Atorvastatin and 1.113 % for Amlodipine. The value of relative standard deviation for
inter-day precision was found 0.754 % for Atorvastatin and 0.458 % for Amlodipine.
Hence the newly developed method can be used for routine analysis for the estimation
of Atorvastatin and Amlodipine in combined tablet dosage form.
6
ACKNOWLEDGEMENTS
The authors are to Yaresun pharmaceutical Pvt. ltd, for providing the facilities to carry
out the work. The authors are also thankful to yaresun labs rajasthan for providing
working standard of Atorvastatin and Amlodipine.
7
REFERENCES
1. British Pharmacopoeia, department of health, Great Britain, Vol-1, 2003; 65,
124.
2. Rang H.P., Dale M.M., Ritter J.M., and Moore P. K., Pharmacology, 5th Edition,
2003, Churchill Living Stone, 345.
3. Robert A. Nash, Alfred H. Wachter, Pharmaceutical process validation, Marcel
Dekker, Inc., 2003, 507-22
4. Text on Validation of Analytical Procedures Q2A in; I.C.H. Harmonised
Tripartite Guidelines; Oct. 1994.
5. Guidelines for the validation of analytical methods for active constituent,
agriculture and veterinary chemical products, Australian Pesticides & Veterinary
Medicines Authority, October 2004.
6. Malesuik MD, Cardoso SG, Bajerski L, Lanzanova FA. Determination of
amlodipine in pharmaceutical dosage forms by liquid chromatography and
ultraviolet spectrophotometry, Journal of AOAC International, 2006 Mar-Apr;
89(2): 359-64.
7. Rajeswari K Raja, et. al., RP-HPLC method for the simultaneous determination
of Atorvastatin and Amlodipine in tablet dosage form, Indian Journal of
Pharmaceutical Sciences, year 2006, volume 68, issue 2, page 275-277.
8. Manoj K, Shanmugapandiyan P, Anbazhagan S. RP HPLC method for
simultaneous estimation of Atorvastatin and aspirin from capsule formulation.
Indian Drugs, 2004; 41(5): 284-289.
8
9. Erturk S, Sevinc Aktas E., et. al, An HPLC method for the determination of
Atorvastatin and its impurities in bulk drug and tablets, J Pharm Biomed Anal.,
2003 Dec 4; 33(5):1017-23.
9
Table 1: Recovery study of Atorvastatin and Amlodipine
S.No.
Drug
Conc.
before
Reference Std. Conc. after
Percent
Mean
Recovery
percent
added
spiking
(g/ml)
(g/ml)
19.620
15.746
35.312
99.85
19.620
19.723
39.317
99.93
spiking
Recovery
(g/ml)
1
Atorvastatin
99.41
±
0.83
2
Amlodipine
19.620
23.855
43.251
98.45
9.907
7.936
17.585
98.55
9.907
9.822
19.566
98.17
98.65
±
0.54
9.907
11.854
21.594
99.23
10
Table 2: Summary of Validation Parameters Atorvastatin and Amlodipine
OBSERVATION
PARAMETER
Atorvastatin
Amlodipine
0.999
0.999
99.41%
98.65%
±
±
0.83
0.54
Repeatability (n= 6)
1.654
0.894
Intra-day (n=3)
0.841
1.113
0.754
0.458
Linearity
(Correlation coefficient r2)
Accuracy
(% Recovery)
Precision RSD
Inter-day (days=3)
11
Table 3: Optical parameters and Regression characteristics of Atorvastatin and Amlodipine
Atorvastatin
Amlodipine
240 nm
369 nm
0-25
0-50
5.0014 x 104
1.0024 x 104
2.214 x 10-2
5.657 x 10-2
slope (b)
0.99
0.98
intercept (a)
0.11
0.13
Correlation coefficient (r)
0.999
0.999
Parameters
Beer’s law limit
(g/ml)
Molar absorptivity
(l mole-1cm-1)
Sandell's sensitivity
(mg/cm2/0.001absorbance unit)
Regression equation
(y= a + bc)
12
Figure 1: Absorbance of Atorvastatin
13
Figure 2: Absorbance of Amlodipine
14
Figure 3: Overlay absorbance Atorvastatin and Amlodipine
15
Figure 4: Clibration curve of Atorvastatin
16
Figure 5: Calibration curve of Amlodipine
17