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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407

Research Article

SIMULTANEOUS ESTIMATION OF ROSUVASTATIN CALCIUM AND CLOPIDOGREL BISULPHATE FROM

BULK AND COMMERCIAL PRODUCTS USING A VALIDATED REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC TECHNIQUE

Avani Sheth1*, K N Patel1, Badmanaban Ramlingam2, Nehal Shah3 1SAL Institute of Pharmacy, Opp Science City, Ahmedabad, Gujarat, India

2Department of Quality Assurance, Shri Sarvajanik Pharmacy College, Mehsana, Gujarat, India 3Department of Quality Assurance, Dharmaj Degree of Pharmacy, Dharmaj, Anand, Gujarat, India

Article Received on: 15/09/12 Revised on: 23/10/12 Approved for publication: 13/11/12

*Email: avani_sheth12@yahoo.co.in ABSTRACT A simple, rapid, accurate, precise and economical reverse phase high performance liquid chromatographic method is developed for simultaneous separation and quantification of two cardiovascular drugs, viz., Rosuvastatin Calcium and Clopidogrel Bisulphate. The separation of both the drugs was achieved on Novapak C18 column (100 x 3.9 mm, 4 µm) column using a mobile phase of Sodium Perchlorate buffer solution (at pH 2.5): Acetonitrile (65:35 v/v). The flow rate was 1 ml/min and detection was done at 242 nm. The retention time for Rosuvastatin Calcium was 4.4 min and Clopidogrel Bisulphate was 7.6 min. Rosuvastatin Calcium and Clopidogrel Bisulphate showed a linear response in the concentration range of 10-60 µg/ml and 18.75 - 112.5 µg/ml respectively. The correlation co-efficients for Rosuvastatin Calcium and Clopidogrel Bisulphate were 0.9999 and 0.9999 respectively. The percentage recoveries obtained for Rosuvastatin Calcium and Clopidogrel Bisulphate ranges from 99.1% to 100.5% and 99.1% to 99.6% respectively. The results of analysis have been validated as per ICH guidelines. Validation results indicated that method shows satisfactory linearity, accuracy, precision and ruggedness. The extremely low flow rate, simple mobile phase composition makes this method cost effective, rapid, non-tedious and can also be successfully employed for simultaneous estimation of both drugs in commercial products. Keywords: Rosuvastatin Calcium, Clopidogrel Bisulphate, HPLC INTRODUCTION Rosuvastatin Calcium (Figure 1) is chemically, bis[(E)-7-[4-(4-fluorophenyl)-6-isopropyl-2-[methyl (methylsulfonyl) amino] pyrimidin-5-yl](3R,5S)-3,5-dihydroxyhept-6-enoic acid] calcium salt . Rosuvastatin Calcium is a statin drug used as HMG Co A reductase inhibitor for controlling hyperlipidemic conditions. The literature survey reveals that Rosuvastatin Calcium was analyzed by the Spectrophotometric, HPTLC and Reverse phase High Performance Liquid Chromatography (RP-HPLC) methods1-

8.Clopidogrel Bisulphate (Figure 2) is an orally administered Antiplatelet agent which is used to inhibit clot in blood vessels. Clopidogrel Bisulphate is chemically methyl (+)-(S)-α-(2-chlorophenyl)-6, 7-dihydrothieno [3, 2-c] pyridine-5(4H) acetate sulphate. Literature survey reveals bio-analytical methods by LCMS for detection of Clopidogrel Bisulphate in human serum and blood, few spectrophotometric methods and HPLC methods for the quantitative estimation of Clopidogrel Bisulphate in bulk and pharmaceutical formulations9-13.Currently most commonly prescribed medicines for cardiovascular diseases are Statins and Antiplatelet agents. Monotherapy with oral cardiovascular agents is not sufficient to control heart diseases and henceforth a combination tablet formulation is beneficial in terms of its convenience and patient compliance. The present drug combination has promising effect to control heart diseases. Literature review suggests no analytical methods reported for simultaneous analysis of both the drugs together and henceforth the following experiment was performed.

MATERIALS AND METHODS Pure Samples Rosuvastatin Calcium and Clopidogrel Bisulphate were received from Cadila Pharmaceuticals Ltd., Ahmedabad, Gujarat. Marketed Preparation Rosuva-CL Tablets was obtained from Zydus Cadila Healthcare Ltd., Moraiya,

Ahmedabad. Dose of Rosuvastatin Calcium was 40 mg and Clopidogrel Bisulphate was 75 mg in Rosuva-CL Tablets. Acetonitrile (HPLC Grade) used was purchased from Merck (India) Ltd., Mumbai. Orthophosphoric acid and Sodium Perchlorate were purchased from Spectrochem, India. RP-HPLC was performed using Shimadzu HPLC system (LC 2010CHT, Shimadzu Corporation, Japan) equipped with quaternary pump, auto injector, column oven and PDA detector. Chromatographic condition Column : Novapak C18 3.9 x 100 mm 4µm Detector : 242 nm Injection Volume : 10 µl Flow Rate : 1.0 mL min-1

Temperature : 40º C Run Time : 10 minutes Mobile Phase : Sodium perchlorate Buffer (at pH 2.5): Acetonitrile (65:35) Diluent : Methanol Experimental work Buffer preparation Accurately weighed 14 g Sodium Perchlorate was dissolved in to 1000 mL Milli-Q water and 1ml Triethylamine was added to this buffer Solution, than pH was adjusted to 2.5 with ortho-phosphoric acid. Preparation of standard solution The standard stock solution Rosuvastatin Calcium (400 ppm) and Clopidogrel Bisulphate (750 ppm) were prepared by weighing Rosuvastatin Calcium 80 mg and Clopidogrel Bisulphate 150 mg in 200ml volumetric flask respectively. Add 80ml diluent to it and sonicated well for 10 minutes and making volume up to mark with diluent. Then 10 ml of standard stock solution was diluted to 100 ml with diluent to make final standard concentration of Rosuvastatin Calcium (40 ppm) and Clopidogrel Bisulphate (75 ppm) respectively.

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Preparation of test solution Accurately 20 intact tablets were weighed and average weight of tablet was calculated. Then tablets were finely crushed, powdered and sample powder about 640 mg (Tablet powder Equivalent to 80 mg Rosuvastatin Calcium and 150 mg Clopidogrel Bisulphate) was transferred into 200 ml volumetric flask. Then add about 100.0 ml diluent was added and sonicate for 40 minutes with intermittent shaking. Then volume was made up to mark with diluent. Then 10 ml of standard stock solution was diluted to 100 ml with diluent to make final standard concentration of Rosuvastatin Calcium (40 ppm) and Clopidogrel Bisulphate (75 ppm) respectively. The Test solution was filtered through 0.45µ (PVDF Millipore Filter) and analyzed by using HPLC Reason for selecting this experimental work Literature review reveals only individual methods for estimation of Rosuvastatin Calcium and Clopidogrel Bisulphate individually but no methods were reported for simultaneous estimation of Rosuvastatin Calcium and Clopidogrel Bisulphate was reported till date. So method was developed method more superior to previously published methods of individual estimation of all drugs. The detection wavelength of 242 nm was chosen in order to achieve a good sensitivity for quantitative determination of Rosuvastatin Calcium and Clopidogrel Bisulphate in solid dosage form. The retention time for Rosuvastatin Calcium was 4.4 min, and for Clopidogrel Bisulphate was 7.6 min. The isocratic program throughout HPLC method was adopted to analyze both components in a single run as shown in (Figure 3). Method Validation14 Validation was carried out with respect to various parameters, as required under ICH guideline Q2 (R1). The developed method validated with respect to parameters such as linearity, precision, accuracy, specificity, ruggedness, robustness and solution stability. System suitability and System Precision The results of system suitability and system precision are presented in Table 1. Linearity To achieve linearity and range, stock solution containing Rosuvastatin Calcium (400 ppm) and Clopidogrel Bisulphate

(750 ppm) were prepared. Rosuvastatin Calcium and Clopidogrel Bisulphate Stock solutions were diluted to yield solutions in the concentration range of 10-60 µg mL-1 and 18.75-112.5 µg mL-1 respectively. The solutions were analyzed by using HPLC. Calibration curve for both the drugs are shown in (Figure 4) and (Figure 5). The results of linearity are presented in Table 2. Precision The method precision was done by preparing six different sample preparations by one analyst. The results are presented in Table 3. The results obtained were within 2% RSD. Ruggedness Ruggedness test was determined between different analyst, instrument and Column. The value of percentage RSD was below 2.0%, showed ruggedness of developed analytical method. The results are presented in Table 3. Accuracy The difference between theoretical added sample amount to the placebo and practically achieved sample amount from placebo (after HPLC analysis) is called accuracy of analytical method. Accuracy was determined at three different level 50%, 100% and 150% of the target concentration in triplicate. The results are presented in Table 4. Solution stability

The standard and sample solutions were found stable up to 24 hours at room temperature. Robustness

Robustness of the method was carried out by deliberately made small changes in the flow rate, pH, organic phase ratio and column oven temperature. Results are presented in Table 5 LOD (Limit of Detection and LOQ (Limit of Quantitation) In order to estimate the limit of detection (LOD) and limit of quantitation (LOQ) values, the blank sample was injected six times and the peak area of this blank was calculated as noise level. The LOD was calculated as three times the noise level while ten times the noise value gave the LOQ. The results of LOD and LOQ are mentioned in Table 6.

Table 1: General Parameters found after RP HPLC Analysis

Compound Retention Time Theoretical Plates Asymmetry Rosuvastatin Calcium 4.4 ± 0.0031 9819 1.12

Clopidogrel Bisulphate 7.6 ± 0.0022 9351 1.09

Table 2: Linearity Data of Rosuvastatin Calcium and Clopidogrel Bisulphate Linearity Data for Rosuvastatin Calcium & Clopidogrel Bisulphate.

Linearity Range

Stock Solution used for Linearity

Diluted to volume ml

Final Conc Rosuvastatin (ppm)

Rosuvastatin Area

Final Conc Clopidogrel (ppm)

Clopidogrel Area

25% 2.50ml 100 ml 10 342118 18.75 174181 50% 5.00ml 100 ml 20 510813 37.50 264298 75% 7.50ml 100 ml 30 686958 56.25 355169

100% 10.0 ml 100 ml 40 859291 75.00 442878 125% 12.5 ml 100 ml 50 1030113 93.75 531285 150% 15.0 ml 100 ml 60 1210101 112.50 624321

Table 3: Precision and Ruggedness Data for Rosuvastatin Calcium and Clopidogrel Bisulphate

Parameters Rosuvastatin Calcium Clopidogrel Bisulphate %Assay

Mean (n=6) % RSD %Assay

Mean (n=6) % RSD

Method Precision 100.4 0.4 100.2 0.7 Ruggedness 99.8 1.0 99.4 0.9

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Table 4: Accuracy Data of Rosuvastatin Calcium and Clopidogrel bisulphate Levels Rosuvastatin Calcium Clopidogrel Bisulphate

%Assay Mean (n=3)

% RSD %Assay Mean (n=3)

% RSD

50% 99.8 0.6 99.6 0.4 100% 99.1 0.7 99.4 1.2 150% 100.5 0.2 99.1 0.7

Table 5: Robustness Data for Rosuvastatin Calcium and Clopidogrel Bisulphate

Rosuvastatin Calcium Robustness Study Sys. Suit.

Temp. -5°C Temp.

+5°C Flow -

10% Flow +10%

Org. -2%

Org. +2% pH = 2.3 pH = 2.7 Mean % RSD

0.6 0.4 0.3 0.8 0.1 0.4 0.3 0.6 0.9 0.4% Clopidogrel Bisulphate Robustness Study

Sys. Suit. Temp. -5°C

Temp. +5°C Flow -10%

Flow +10%

Org. -2%

Org. +2% pH = 3.2 pH = 2.8 Mean % RSD

0.8 0.6 0.5 0.9 0.7 0.3 0.1 0.3 0.2 0.5%

Table 6: Summary of Validation Parameters of RP HPLC Method for Simultaneous estimation of Rosuvastatin Calcium and Clopidogrel Bisulphate

Parameters of Validation Acceptance Criteria Rosuvastatin Calcium Clopidogrel Bisulphate Range of Linearity Follows Beer Lambert’s law 10 – 60 mg/ml 18.75-112.5 mg/ ml

Correlation Coefficient Correlation coefficient r2 > 0.999 or 0.995 0.9999 0.9999 LOD S/N > 2 or 3 0.2 mg/ml 0.375 mg/ml LOQ S/N > 10 0.6 mg/ml 1.13 mg/ml

Precision RSD < 2% 0.4% 0.7% Ruggedness RSD < 2% 1.0% 0.9%

Accuracy Recovery 98- 102% 99.1% to 100.5% 99.1% to 99.6% Specificity No interference Complies Complies

Solution Stability > 12 hour Stable for 24 hr %RSD = 0.7%

Stable for 24 hr %RSD =0.9%

Robustness RSD NMT 2% in given condition Complies Complies

Figure 1: Structure of Rosuvastatin Calcium

Figure 2: Structure of Clopidogrel Bisulphate

Figure 3: HPLC Chromatogram of Rosuvastatin Calcium and Clopidogrel Bisulphate

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Figure 4: Calibration Curve of Rosuvastatin Calcium

Figure 5: Calibration Curve of Clopidogrel Bisulphate

RESULT AND DISCUSSIONS The proposed method was found to be simple, accurate, precise and rapid for simultaneous estimation of Rosuvastatin Calcium and Clopidogrel Bisulphate in all dosage forms. The values of relative standard deviation are satisfactorily low and recovery was close to 100% which indicated accuracy and reproducibility of methods. CONCLUSION Thus proposed method was found to be simple, accurate, precise selective and economical for simultaneous routine analysis of Rosuvastatin Calcium and Clopidogrel Bisulphate in bulk and commercial dosage form. REFERENCES

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