INTRODUCTION:

Atorvastatin calcium (AT) is chemically [R-(R*,R*)]-2-(4-flurophenyl)-β ,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino) carbonyl]-1H-pyrrole-1-heptanoic acid, calcium salt trihydrate.It is an inhibitor of 3-hydroxy-3methyl glut aryl coenzyme A (HMG-Co A) reductase. This enzyme catalyses the conversion of HMG-Co A to mevalonate, an early and rate-limiting step in cholesterol biosynthesis.^1-4 Bio analytical, HPLC, HPTLC and UV-visible spectrophotometry, methods have been reported for its individual determination of atorvastatin calcium and in combination with other drugs.^5-12 While for estimation of atorvastatin calcium and aspirin combination, RP-HPLC¹³ method has been reported. Aspirin, 2-acetoxy benzoic acid is cyclo oxygenase inhibitor. It is used as an analgesic, antipyretic, anti-inflammatory and anti thrombic agent. Aspirin is official in IP¹⁴, BP¹⁵ and USP.¹⁶ Severel spectrophotometric method¹⁷, Spectrofluorimetric¹⁸ HPTLC¹⁹have been reported for estimation of aspirin. Clopidogrel bisulphate is methyl (s)-2-chlorophenyl (4, 5, 6, 7-tetrahydrothieno- [3, 2-C] pyridin-5-yl) acetate bisulphate, an ADP antagonist.

Several spectrophotometric methods, HPLC methods, Capillary electrophoresis GC method have been reported for estimation of Clopidogrel bi sulphate^20-24 While for estimation of Clopidogrel bi sulpate and aspirin combination, several RP-HPLC^25-26method has been reported.

A capsule formulation containing 150 mg of aspirin 10mg of Atorvastatin and 150 mg of clopidogrel bisulphate is available (Noklot-CV, Zydus Cadila Health care limited). The combination of Asprin Atorvastatin and Clopidogrel is not official in any Pharmacopeia. So far, no HPLC method is reported for this combination. The present manuscript describes a novel LC method which is simple, rapid, precise, sensitive, selective and accurate isocratic reverse phase HPLC-UV method for simultaneous determination of asprin ,atorvastatin and Clopidogrel in capsule dosage form. The method proved to be simple model since it does not contain any buffer system. During present study efforts were directed use of mobile phase without salt to increase column life.

EXPERIMENTAL:

Reagents and chemicals:

Acetonitrile HPLC grade was procured from E. Merck Ltd, Mumbai. Glacial acetic acid and Methanol AR grade were procured from Qualigens Fine Chemicals, Mumbai. Water HPLC grade was obtained from a Milli-QRO water purification system. Reference standards Atorvastatin calcium and Clopidogrel bi sulphate were procured from Madras pharmaceuticals, Chennai and Asprin was procured from Aarthi Drugs, Mumbai.

Apparatus and Chromatographic conditions:

The HPLC system consisted of a separation module (Water Alliance 2695) and Photo Diode Array (PDA) detector all from waters. (Water’s Corporation, (USA). An isocratic elution was performed on a Hypersil BDS C₁₈ column (250 mm x 4.6 mm, 5µ)

The mobile phase was degassed and filtered (0.45 µ, Millipore) mixture of acetonitrile, water (p^H3)and methanol (50:40:10 v/v). Injection volume was 20µl and run time was 10min and flow rate was 1.5ml ml/min. The column was maintained ambient temperature and the eluents were detected at 248nm.Quantification was achieved by peak-area-ratio method with reference to the standards.

Preparation of stock and standard solutions Atorvastatin calcium, Asprin and Clopidogril bisulphate

Standard stock solution (1000µg/ml) of Atorvastatin calcium, Asprin and Clopidogril bi sulphate were prepared separately in methanol.The working standard solutions were prepared and furthur diluted in mobile phase to contain a mixture of asprin ,atorvastatin calcium, and Clopidogril in over the linearity range from 120-480µg/ml ,8-32µg/ml and 60-240µg/ml respectively.

Assay in formulations

Twenty capsules, Noklet-CV,(Zydus cadila Health care limited ),each containing 10mg of AT, 150 mg of AS and 75mg of CL were weighed and capsule contents are finely powdered. A quantity of powder equivalent to10mg of AT,75 mg of CL and 150mg of AS were weighed and transferred in to 100ml of standard volumetric flask and added 50 ml of methanol. The sample was kept in an ultrasonic bath for 20 min and furthur diluted to 100ml by using mobile phase. Then it is filtered through 0.22µ membrane filter paper. Two ml of this solution further diluted to 10ml to get a concentration of 20 µg/ml of AT, 150µg/ml of CL and 300µg/ml of AS.20µl of this solution was injected in to HPLC system and chromatograms were recorded. A duplicate injection of the standard solution was also injected into the HPLC system and the chromatograms were recorded. The amount of Atorvastatin calcium, Clopidogrel bisulphate ,and Asprin present in each capsule was calculated by comparing the peak area of the standard solution and sample. The amount of the drugs were calculated and tabulated in [Table 1].

RESULTS AND DISCUSSION:

The HPLC procedure was optimized with a view to develop precise and accurate assay method. Various mobile phase systems were prepared and used to provide an appropriate chromatographic separation, but the proposed mobile phase comprising of acetonitrile, water (p^H3) and methanol (50:40:10 v/v) gave a better resolution. The detection was carried out by using UV-visible PDA detector at 248nm. Amongest the several flow rates tested (0.5 - 2.0 ml), the flow rate of 1.50 ml/min was the best for all the drugs with respect to location and resolution of peaks. The retention time of Asprin, Atorvastatin calcium, Clopidogrel bisulphate was found to be 2.552, 7.472 and 9.195 min respectively. The chromatograms of standard and sample solution of AS, AT and CL were shown in [Figure 1] and [ Figure 2] .The asymmetry factor of Asprin, Atorvastatin calcium and Clopidogrel bi sulphate was found to be 0.5612, 0.497 and 0.6944 respectively, which indicates symmetrical nature of the peak. The percentage label claim of individual drugs found in formulations were calculated and presented in [Table 1] .The results of analysis shows that the amounts of drugs estimated were in good agreement with the label claim of the formulations.

Figure 1: Chromatogram of standard

Table 2: Accuracy of the method

Amount(%) of drug added	Theoretical content(µg/ml)	Conc. found (µg/ml)±SD*	Recovery (%)	SEM	RE (%)	RSD (%)
ASPRIN
0	300	301.12±0.382	100.36	0.215	0.72	1.24
75	525	524.16±0.482	99.84	0.354	0.86	1.34
100	600	601.21±0.261	100.16	0.165	0.88	0.97
125	675	748.19±0.167	99.75	0.148	1.21	0.46
ATORVASTATIN
0	20	20.39±0.216	101.95	0.128	0.45	0.96
75	35	35.42±0.314	101.21	0.259	0.69	0.46
100	40	39.51±0.169	98.77	0.368	1.24	0.62
125	45	45.18±0.145	100.41	0.452	1.12	0.37
CLOPIDOGREL
0	150	149.23±0.231	99.48	0.275	1.26	0.51
75	262.5	261.36±0.452	99.75	0.429	1.52	0.86
100	300	301.14±0.156	100.36	0.398	0.93	0.42
125	337.5	336.25±0.264	99.78	0.514	0.65	0.76

*SD= standard deviation(n=3),SEM= Standard Error of Mean,*RSD=SD/Mean×100,

RE(%)=%Relative Error =(Mean assayed concentration-Added Conentration/ Added Conentration×100)

Figure 2: Chromatogram of sample

Method Validation:

The developed method is validated according to ICH guidelines.²⁷

Accuracy and Precision:

The accuracy of the method was determined by recovery experiments. It was confirmed by studying the recovery at three different concentrations, 75%, 100%, and 125 % of those expected by spiking a previously analyzed test solution with additional drug standard solutions, the analysis being done in replicate. The %RSD and % relative error in all cases were within the acceptable limit ( ≤ 2%). It is evident from the results of accuracy study, reported in [Table 2], that the proposed method enables very accurate quantitative simultaneous estimation of AS, AT, and CL.

The Precision of the method was demonstrated by system precision and method precision studies. In the system precision studies, six replicate injections of the working standard solution prepared as per the proposed method and chromatograms were recorded. Standard deviation and relative standard deviation for the area was calculated and presented in [Table 3]. In the method precision studies, six replicate injections of the analyte solution prepared as per the proposed method and chromatograms were recorded. Standard deviation and relative standard deviation for the area was calculated and presented in [Table 4]. From the data obtained, the developed RP-HPLC method was found to be precise.

Table3: System precision Report

Parameters	Area of Asprin	Area of Atorvastatin	Area of Clopidogrel
Trail1	42457998	1813556	7134569
Trail2	42458699	1815331	7135698
Trail3	42456586	1814229	7136895
Trail4	42457569	1815226	7135421
Trail5	42456987	1814665	7135215
Trail6	42456523	1815496	7136842
Average	42457379	1814751	7135773
Standard deviation	859.3937	751.6596	926.5153
% Relative standard deviation	0.0020	0.0414	0.0129

Table 4: Method precision Report

Parameters	Area of Asprin	Area of Atorvastatin	Area of Clopidogrel
Trail1	42456775	1813123	7135125
Trail2	42458281	1814256	7134456
Trail3	42454558	1815469	7135745
Trail4	42457569	1816542	7136546
Trail5	42456561	1816542	7134453
Trail6	42454123	1814256	7135856
Average	42456311	1815031	7135364
Standard deviation	1649.3412	1385.626	836.1643
% Relative standard deviation	0.0038	0.076	0.0117

Linearity and Range:

A linear relationship was observed between the absorbance and concentration over the range from 120-480µg/ml for asprin, 8-32 µg/ml for atorvastatin and 60-240µg/ml for clopidogrel [Table 5]. The linearity was expressed as R², which was 0.999 for Asprin, 0.999 for atorvastatin and 0.998 for clopidogrel. Values of R²y-intercept, and slope of the regression line are shown in [Figures 3, 4 and 5].

Table5.Linearity Report

Parameter	Results
Parameter	Asprin	Atorvastatin	Clopidogrel
Linearity(R²)	0.999	0.999	0.998
RSD,%	≤ 2	≤ 2	≤ 2
Mean Recovery,%	100.16	99.24	101.27
LOD,µg/ml	0.4	0.05	0.06
LOQ,µg/ml	1.2	0.15	0.2
Range, µg/ml	120-480	8-32	60-240

Figure 3: Linearity curve for Asprin

Figure 4: Lineatity curve for Atorvastatin

Figure 5:Linearity curve for Clopidogrel

Limit of Detection (LOD) and Limit of Quantification (LOQ):

Limit of detection (LOD) and limit of quantification (LOQ) were calculated as 3.3 ∂/S and 10 ∂/S, respectively as per ICH guidelines, where ∂ is the standard deviation of the response (y-intercept) and S is the slope of the calibration plot. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3).The LOD for AS, AT and CL was found to be 0.4 µg/ml, 0.05 µg/ml and 0.06µg/ml, respectively. The LOQ is the smallest concentration of the analyte which gives response that can be accurately quantified (signal to noise ratio of 10).The LOQ of AS, AT and CL was found to be 1.2 µg/ml, 0.15 µg/ml and 0.2 µg/ml respectively. [Table 5].

Ruggedness and Robustness:

The ruggedness of the method was determined by carrying out the experiment on different instruments like Shimadzu HPLC (LC 10 AT), Water Alliance 2695 by different operators using different columns. Robustness of the method was determined by making slight changes in the chromatographic conditions. It was observed that there were no marked changes in the chromatograms, which demonstrated that the RP- HPLC method developed is rugged and robust.

Solution Stability:

In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 6 h at room temperature. The results show that for both solutions, the retention time and peak area of Asprin, Atorvastatin and Clopidogrel remained unchanged (percentage RSD less than 2.0), thus indicated that both solutions were stable for atleast 6 h, which was sufficient to complete the whole analytical process.

System suitability studies:

The column efficiency, resolution and peak asymmetry were calculated for the standard solutions (Table No.6). The values obtained, demonstrated the suitability of the system for the analysis of this drug combinations.

CONCLUSION:

The proposed RP-HPLC method is precise, rugged, robust, simple and rapid. Hence the present RP-HPLC method is suitable for the simultaneous estimation of Asprin, Atorvastatin calcium and Clopidogrel bi sulphate in capsule dosage form.

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Received on 01.03.2011 Modified on 16.03.2011

Asian J. Research Chem. 4(5): May, 2011; Page 795-799

S. No	Capsule sample	Label claim (mg/capsule)	Peak Area	^*Amount Present ( mg/capsule)	^*Percentage Label claim (%w/w)
1	Asprin	150	42457921	148.98±0.02	99.32±0.05
2	Atorvastatin	10	1813578	10.11±0.04	101.11±0.08
3	Clopidogrel	75	7134522	74.53±0.05	99.37±0.12