INTRODUCTION:

Amlodipine besylate and Atorvastatin calcium tablets combine the long-acting calcium channel blocker amlodipine besylate with the synthetic lipid-lowering agent atorvastatin calcium.

The amlodipine besylate is chemically described as 3-Ethyl-5 methyl (±)-2-[(2-aminoethoxy) methyl]-4-(o-chlorophenyl)-1, 4-dihydro-6-methyl-3, 5 pyridinedicarboxylate, monobenzenesulphonate. Its empirical formula is C₂₀H₂₅ClN₂O₅•C₆H₆O₃S.

Amlodipine besylate is a white crystalline powder with a molecular weight of 567.1.It is slightly soluble in water and sparingly soluble in alcohol.

Atorvastatin calcium component is chemically described as [R-(R*, R*)]-2-(4-fluorophenyl)-β, δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenyl amino) carbonyl]-1Hpyrrole-1-heptanoic acid, calcium salt (2:1) trihydrate. The empherical formula of atorvastatin calcium is (C₃₃H₃₄ FN₂O₅)2Ca•3H₂O and its molecular weight is 1209.42. Its structural formula is:

Atorvastatin calcium is a white to off-white crystalline powder that is insoluble in aqueous solutions of pH 4 and below. Atorvastatin calcium is very slightly soluble in distilled water, pH 7.4 phosphate buffer, and acetonitrile; slightly soluble in ethanol; and freely soluble in methanol.

Several RP-HPLC^1-7, Spectrophotometric^8-11, TLC¹², HPTLC¹³, LC-MS/MS^14-15 and determination in human plasma have been reported for the estimation of Amlodipine and Atorvastatin in combination.

EXPERIMENTAL:

Instrumentation:

An Automated HPLC (Agilent 1200) with quaternary pump, Auto sampler, PDA detector and Hypersil BDS C₁₈, particle size 5µ column was used. The Agilent system was equipped with Chemstation software for data processing.

Chemicals and Reagents:

All solvents were HPLC grade, purchased from RFCL Ltd and AR grade salts and acids were obtained from Merck chemicals. Standard and samples of Amlodipine were kindly gifted by BioPlus Life Sciences Pvt. Ltd., Bangalore.

Chromatographic Conditions:

Method was developed by using a Hypersil BDS C₁₈ (250x4.6 mm) 5µm column. Fixed mobile phase composition for isocratic elution was 0.02M ammonium acetate buffer solution pH 4.0: Acetonitrile (50:50v/v). Flow rate was employed as 1.0mL/min. Detection was carried out at 238nm. pH 6.8 Phosphate buffer was used as diluent.

Standard Preparation:

For Amlodipine : Weighed accurately about 56 mg of Amlodipine working standard into a 100 mL clean and dry volumetric flask, with this about 50mL of methanol was added and sonicated to dissolve and made up to the volume with methanol. Further dilution, 5mL of the above solution was taken in a 100mL volumetric flask and the volume was made up to the mark with the diluent, the final concentration of the above solution was 0.02mg/mL solution. The solution was filtered through 0.45µ membrane nylon filter.

For Atorvastatin : Weighed accurately about 88 mg of Atorvastatin working standard into a 50 mL clean and dry volumetric flask, with this about 20mL of methanol was added and sonicated to dissolve and made up to the volume with methanol. Further dilution, 10mL of the above solution was taken in a 50mL volumetric flask and the volume was made up to the mark with the diluent, the final concentration of the above solution was 0.08mg/mL solution. The solution was filtered through 0.45µ membrane nylon filter.

Sample Preparation:

Twenty tablets were weighed and crushed into fine powder. The tablet powder, equivalent to 80mg of Atorvastatin was weighed and transferred to a 100mL volumetric flask and 60mL of methanol was added to disperse the blend completely and kept it for ultrasonication for about 20minutes. Finally the volume was made up to the mark with the methanol. The solution was centrifuged and diluted 10mL of the supernant solution to 50mL with the diluent and mixed. Filtered through 0.45µ membrane nylon filter paper. A 50µL of standard was injected into the system for six times under the chromatographic conditions as described above to get the system suitability results. The 50µL of sample solution also injected in duplicate for six times into the system for assay results of Amlodipine and Atorvastatin. Area of each peak was measured at 238nm. The amount of Amlodipine and Atorvastatin present in the tablets was calculated with the standard areas and the standard deviation of standard and relative standard deviation and USP plate counts and tailing factor also calculated. The system suitability results are presented in table 1 and .Typical chromatogram of Amlodipine present in the tablet is given in the figure no 1.

A successful attempt was made to estimate the Amlodipine and Atorvastatin. Therefore it was thought worthwhile to develop an accurate and rapid RP-HPLC method for the estimation of Amlodipine and Atorvastatin from tablet formulations and it belongs to the class of calcium channel blocker¹⁶. Amlodipine and Atorvastatin tablets are formulated for oral administration in following strengths.

Fig 1: Tipical chromatogram of Amlodipine and Atorvastatin Sample RT 2.6 min and 4.995min respectively

Table1: System suitability results

Parameter	USP Tailing	USP Plate count	SD	%RSD
Amlodipine	1.3	6561	1.9987	0.53%
Atorvastatin	1.0	10067	1.6803	0.04%

Validation of HPLC Method:

The proposed RP-HPLC method was validated as per ICH guidelines.

Specificity:

The specificity of the RP-HPLC method was determined by comparison of chromatograms of diluent, placebo (mixture of excipients with coating materials) (Fig. 2) and with respect to impurities. It is found that there is no co-elution of impurities with the main analyte and all impurities with main analyte are passing the peak purity test.

Fig 2: Placebo chromatogram

Precision:

System Precision:

As a part of system precision prepared the standard solution and injected into the injector six times and found the USP plate count, USP tailing, Standard deviation and percentage standard deviation. All results found to be satisfactory.

Method Precision:

Precision study was performed to find out the assay percentage in six sample preparations with six individual weights of Amlodipine Besilate and Atorvastatin Calcium Tablets. The results are summarized below in the table 2 and 3.

S. No	mg/Tablet	% Assay	Average	%RSD
Sample 1	9.8	98.9	99.7	0.74
Sample 2	9.9	99.2
Sample 3	10.1	101.0
Sample 4	9.9	99.6
Sample 5	9.9	99.5
Sample 6	10.0	100.0

Table 2.Assay results for Amlodipine

S. No	mg/Tablet	% Assay	Average	%RSD
Sample 1	80.0	100.0	100.3	0.61
Sample 2	79.9	99.9
Sample 3	80.1	101.1
Sample 4	80.1	101.1
Sample 5	79.8	99.8
Sample 6	80.0	100.0

Table 3.Assay results for Atorvastatin

Accuracy (Recovery Studies):

Recovery studies were performed by individual addition of active pharmaceutical ingredient into the placebo at four levels i.e., 80%, 100%, 120%, and 150%. Known amounts of standard (API) and placebo were added for preparation of recovery sample. Results of recovery studies are shown in table 4.

Table4: Recovery results.

%Recovery (Accuracy) results of Amlodipine
% of drug added		80%	100%	120%	150%
% recovered	AML	100.2	100.2	100.0	100.1
% recovered	ATVR	100.1	100.0	99.9	99.8

Linearity Studies:

Linearity studies were performed by preparing standard stock solution in the range of 20%-120% of sample concentration. The linearity graph is presented in the below fig no. 3 and 4

Fig no 3: Linearity graph for Amlodipine

Fig no 4: Linearity graph for Atorvastatin

Robustness:

The robustness study was done by making small changes in the optimized method parameters like ±0.2 change in the pH, ±5% change in the mobile phase composition, ±5°C in the column temperature and ±0.2mL change in the flow. There was no significant impact on the retention time, USP tailing,USP plate count and %RSD.

Ruggedness:

Ruggedness study was done by the two analysts in different day with different system and with different column. The % RSD for analyst-I was 0.16% and for analyst-II was 0.18% for Amlodipine and Atorvastatin respectively.

RESULTS AND DISCUSSION:

The present work describes RP-HPLC method for estimation of Amlodipine and Atorvastatin in Amlodipine Besilate and Atorvastatin Calcium Tablets. The drug was resolved on Hypersil BDS C₁₈ (250x4.6) mm 5µm column using 0.02M ammonium acetate buffer solution pH 4.0 and Acetonitrile in the ratio of 50:50v/v with a flow rate of 1.0mL/min. UV detection was performed at 238nm. Linearity range was performed in the concentration range of 0.004mg/mL to 0.96mg/mL for Amlodipine and 0.03mg/mL to 0.2mg/mL for Atorvastatin. The correlation co-efficient (r²value) for Amlodipine and Atorvastatin was 1. The %RSD for the tablet analysis and recovery studies was less than 2% indicating high degree of accuracy. The %RSD for intraday precision and inter day precision was less than 2% indicating high degree of precision. The results of robustness study also indicated that the method is robust and is unaffected by small variations in the chromatographic conditions. The results of ruggedness study were found to be satisfactory. Hence it can be concluded that the developed RP-HPLC method is accurate, precise and selective and can be employed successfully for the estimation of Amlodipine and Atorvastatin in the formulations.

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Received on 09.05.2010 Modified on 18.05.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 763-766