RP-HPLC METHOD DEVELOPMENT AND VALIDATION STUDIES FOR THE ESTIMATION OF ASPIRIN, CLOPIDOGREL BISULPHATE AND ROSUVASTATIN CALCIUM IN FIXED DOSE COMBINATION CAPSULES

RP-HPLC METHOD DEVELOPMENT AND VALIDATION STUDIES FOR THE ESTIMATION OF ASPIRIN, CLOPIDOGREL BISULPHATE AND ROSUVASTATIN CALCIUM IN FIXED DOSE COMBINATION CAPSULES

R. Singh and T. Khan ^*

Department of Pharmaceutical Chemistry and Quality Assurance SVKM’s Dr. Bhanuben Nanavati College of Pharmacy, Mumbai - 400056, Maharashtra India.

ABSTRACT: The present project was conducted with the objective of developing and validating a RP-HPLC method for the simultaneous estimation of aspirin, clopidogrel bisulphate, and rosuvastatin calcium in fixed-dose combination capsule. The chromatographic separation was carried out on Agilent 1260 series using Waters C18 (250 × 4.6 mm, 5 μ) column as the stationary phase and acetonitrile (ACN): phosphate buffer pH 3, gradient mode at a flow rate of 1.2 ml/min and detection at 230 nm. The validation of the developed method was conducted as per the ICH guidelines Q2 (R1). The retention time of aspirin, rosuvastatin calcium and clopidogrel bisulphate was found to be 3.2 min, 4.7 min, and 12.8 min, respectively, under the optimized chromatographic conditions. The developed method was linear in the concentration range of 6.25-400 μg/ml for aspirin, rosuvastatin calcium, and clopidogrel bisulphate. The developed method was specific, with a mean percent recovery of the three drugs in the range of 99-101%. The relative standard deviation (RSD) was less than 2 in the intraday and inter-day precision studies. A simple, accurate, robust, and precise RP-HPLC method was developed for the simultaneous estimation of aspirin, rosuvastatin calcium, and clopidogrel bisulphate in fixed-dose combination capsule. The developed method was validated for linearity, range, accuracy, precision, robustness, LOD, LOQ, and system suitability. This method can be conveniently used for quantification of the three drugs in fixed-dose combination products.

Aspirin, Rosuvastatin calcium, Clopidogrel bisulphate, RP-HPLC method development, Validation

INTRODUCTION: Cardiovascular diseases such as acute coronary syndrome, myocardial infarction, angina, and stroke are considered as a major cause of death and disability in both developed as well as developing countries.

Various studies have shown that co-administration of aspirin with statins has a synergistic action in the secondary prevention of atherothrombosis and lipophilic statins such as rosuvastatin does not interfere with the antiplatelet effect of clopidogrel in patients with cardiovascular disease.

Fixed-dose combination products containing these drugs are available in the market ^1-2. Aspirin, chemically 2-acetoxybenzoic acid is an odorless, white crystalline powder freely soluble in alcohol, having molecular formula C₉H₈O₄, molecular weight 180.2 g/mol and log P of 1.19.

It is a non-steroidal anti-inflammatory agent and an irreversible COX inhibitor with a prominent antiplatelet effect ^2-3. Rosuvastatin calcium, chemically calcium;(E, 3R, 5S)-7- [4(4-fluorophenyl) -2-[methyl (methylsulfonyl)amino]-6-propan-2-ylpyrimidin-5-yl]-3, 5dihydroxyhept-6-enoate is a white crystalline powder soluble in acetonitrile and slightly soluble in acetone, having molecular formula C₂₂H₂₈FN₃O₆S, molecular weight 481.53 g/mol and log P of 0.13.  It is a statin used to reduce plasma cholesterol levels and prevent cardiovascular disease.  It competitively inhibits hydroxymethylglutaryl-coenzyme an (HMG-CoA) reductase, thereby preventing the conversion of HMG-CoA to mevalonic acid, the rate-limiting step in cholesterol biosynthesis ^{2, 4}. Clopidogrel bisulphate, chemically methyl (2S)-2-(2chlorophenyl)-2-(6,7-dihydro-4H-thieno[3, 2-c]pyridin-5-yl) acetate; sulfuric acid is an off-white powder freely soluble in methanol and practically insoluble in ether, having molecular formula log P of 2.5 Fig. 1. It is an oral, thienopyridine-class antiplatelet agent. It prevents platelet activation by irreversibly blocking one of the three adenosine diphosphate receptors (the P2Y12 receptor) on the platelet surface, thus interfering with platelet activation, degranulation, and aggregation ^{2, 5}.

Fixed-dose combination capsules are available in the market bearing the brand name ROSUMAC GOLD. An exhaustive literature survey indicated that many methods were reported for the estimation of rosuvastatin calcium, aspirin, and clopidogrel bisulphate individually and in combination with other drugs ^6-16. However, no HPLC method for the simultaneous estimation of rosuvastatin calcium, aspirin, and clopidogrel bisulphate has been reported so far. Hence, there exists a need to develop and validate a new accurate, precise HPLC method for the simultaneous estimation of rosuvastatin calcium, aspirin, and clopidogrel bisulphate in fixed-dose combination products as per ICH guidelines Q2(R1) ¹⁷.

FIG. 1: STRUCTURES OF DRUGS ^3-5

MATERIALS AND METHODS:

Chemicals and Reagents: Reference standards of aspirin, rosuvastatin calcium and clopidogrel bisulphate were received as gift samples from Glenmark Generics Ltd., Mumbai, The Andhra Sugars Ltd. Andhra Pradesh, and Watson Pharma Pvt. Ltd, Ambernath, Maharashtra, respectively. Methanol (HPLC grade) was obtained from S. D. Fine Ltd. Mumbai. Rosumac GOLD capsules containing aspirin (75 mg), rosuvastatin calcium (10 mg), and clopidogrelbislphate (75 mg) were purchased from a local pharmacy store in Mumbai.

Instrument: Analytical method development and validation studies were performed on Agilent Technologies 1260 series chromatograph equipped with a quaternary pump and PDA detector.

Method Development Studies: A variety of mobile phases in the isocratic and gradient mode were evaluated in an effort to arrive at the optimum mobile phase composition capable of resolving the three drugs with good resolution and peak shape within a reasonable run time. The optimum chromatographic conditions comprised of Waters C18 (250 × 4.6 mm, 5 μ) column as the stationary phase and acetonitrile (ACN): phosphate buffer pH 3, gradient mode at a flow rate of 1.2 ml/min and detection at 230 nm.

Preparation of Stock Solutions of Reference Standards: 10 mg of aspirin, rosuvastatin calcium and clopidogrel bisulphate each were accurately weighed and transferred to 10 mL volumetric flask each, and the volume was made up to mark with methanol to give 1000 μg/mL solutions of aspirin, rosuvastatin calcium and clopidogrel bisulphate each.

Preparation of Working Solutions of Reference Standards: Working solutions were prepared by taking 1 mL aliquot of standard stock solution of rosuvastatin calcium (1000 μg/mL), aspirin (1000 μg/mL) and clopidogrel bisulphate (1000 μg/mL) individually and transferring to 10 mL volumetric flask each and making up to mark with methanol to give 100 μg/mL solutions of each drug.

Preparation of Stock and Working Sample Solutions: Ten Rosumac GOLD capsule (Label claim: aspirin 75 mg, rosuvastatin calcium 10 mg, and clopidogrel bisulphate 75 mg) were used. These capsules containing pellets were opened, and the pellets triturated, their average weight recorded. These pellets were finely powdered and weight equivalent to aspirin 75 mg, rosuvastatin calcium 10 mg, and clopidogrel bisulphate 75 mg was taken and transferred to 100 mL volumetric flask. 75 mL of methanol was added and sonicated for 15 min, and volume made up to mark with methanol to give a concentration of 750 ppm aspirin, 100 ppm rosuvastatin calcium, and 750 ppm clopidogrel bisulphate. The sample solution was filtered through Whatman filter paper. 1 mL aliquot of filtrate was taken and transferred to 10 mL volumetric flask and volume made up to mark with methanol to give a concentration of 75 ppm aspirin, 10 ppm rosuvastatin calcium and 75 ppm clopidogrel bisulphate.

Validation Studies: The developed analytical method was validated as per the ICH Q2 (R1) guidelines.

Specificity: Specificity was determined by injecting blank and placebo samples. No peaks were observed at the retention times of aspirin, clopidogrel bisulphate, and rosuvastatin calcium.

Linearity and Range: Calibration curves of the three drugs were prepared at a concentration range of 6.25-400 µg/ml (seven concentration levels) versus the peak area. The linearity was determined using the method of least square regression analysis.

Precision: The precision of an analytical method was studied by performing repeatability, intra-day, and inter-day precision as per the ICH guidelines.

Limit of Detection (LOD) and Limit of Quantitation (LOQ): The limit of detection and limit of quantitation were determined based on the standard deviation of the y-intercept and slope of the regression line from the calibration curves of the three drugs in triplicate. The LOD and LOQ were calculated using the formulae given below:

LOD = 3.3 σ / S

LOQ = 10 σ / S

Where, σ is the standard deviation of the response, and S is the slope of the calibration curve.

Robustness: The robustness of the developed method was studied by analyzing the effect of slight variation in the pH of mobile phase (± 0.1 units), change in flow rate (± 0.1 ml/min) and change in mobile phase composition (± 2%) on the retention time, tailing factor, theoretical plates and resolution.

Accuracy: The accuracy of the developed method was determined by calculating the recovery of the three drugs. A fixed concentration of each drug was taken (aspirin-75 µg/ml, rosuvastatin calcium- 10 µg/ml, and clopidogrel bisulphate-75 µg/ml) was taken and the respective reference standard was added at 80%, 100%, and 120% levels. Each level was repeated three times, and the percent recovery and percent relative standard deviation were calculated to estimate the accuracy of the developed method.

System Suitability: The system suitability parameters like retention time, number of USP theoretical plates, USP tailing, peak area, and peak height were evaluated. A standard mixture of aspirin (75 ppm), rosuvastatin calcium (10 ppm), and clopidogrel bisulphate (75 ppm) was injected six times to determine the system suitability of the developed method.

Application of Validated Method for Assay of Fixed-Dose Combination: The assay of the marketed formulation Rosumac GOLD (Label claim: aspirin 75 mg, rosuvastatin calcium 10 mg, and clopidogrel bisulphate 75 mg) was conducted by weighing ten capsules (containing pellets) and determining the average weight. The pellets were finely powdered and weight equivalent to aspirin 75 mg, rosuvastatin calcium 10 mg, and clopidogrel bisulphate 75 mg was transferred to 100 ml volumetric flask. 75 mL of acetonitrile was added and sonicated for 15 min, and volume made up to mark with acetonitrile to give a concentration of 750 ppm aspirin, 100 ppm rosuvastatin calcium and 750 ppm clopidogrel bisulphate.

The sample solution was filtered through Whatman filter paper. 1 mL aliquot of the filtrate was transferred to 10 mL volumetric flask and volume made up to the mark with acetonitrile to give concentration of 75 ppm aspirin, 10 ppm rosuvastatin calcium and 75 ppm clopidogrel bisulphate. 20 μl of this solution was injected, and the chromatogram was recorded and the drug content was calculated from the peak areas.

RESULTS AND DISCUSSION:

Method Development and Optimization: A series of mobile phase compositions were screened in an effort to arrive at an optimum mobile phase that is capable of resolving the three drugs in reasonable run time. The trials were conducted using Waters C18 (250 × 4.6 mm, 5 μ) column as the stationary phase AND various combinations of methanol, ACN, and buffer either alone or combination of two organic solvents with buffer viz. ACN: phosphate buffer pH 3: methanol (50: 20:30 v/v/v) which gave low resolution of aspirin (R_t = 2.7 min) and rosuvastatin (R_t = 3.04 min), ACN: phosphate buffer pH 3 (60: 40 v/v) which gave low resolution of aspirin (R_t = 1.6 min) and rosuvastatin (R_t = 2.4 min) and less retention on the column. A range of wavelengths was explored from 220-248 nm before the selection of the optimum wavelength of 230 nm. A range of flow rates from 0.8 to 1.5 ml/min was evaluated before selecting 1.2 ml/min as the optimum flow rate for this method. The optimum chromatographic conditions are given in Table 1.

Fig. 2 and Fig. 3 are the representative chromatograms of a standard mixture of the three drugs and test sample mixture of the three drugs obtained from the capsules.

TABLE 1: OPTIMISED CHROMATOGRAPHIC CONDITIONS OF THE DEVELOPED METHOD

FIG. 2: CHROMATOGRAM OF STANDARD MIXTURE (ASPIRIN 75 PPM, ROSUVASTATIN CALCIUM 10 PPM AND CLOPIDOGREL BISULPHATE 75 PPM)

Validation Studies:

Specificity: No interferences were observed in the chromatogram at the retention times of the three drugs due to the presence of excipients and blank Fig. 3 indicating that the developed method was specific to the drugs.

FIG. 3: CHROMATOGRAM OF SAMPLE (ASPIRIN 75 PPM, ROSUVASTATIN CALCIUM 10 PPM AND CLOPIDOGREL BISULPHATE 75 PPM

Linearity and Range: A graph of peak area versus concentration (ppm) was plotted for the three drugs at a concentration range between 6.25-400 ppm, and the response was found to be linear in this concentration range. The linear regression equations and correlation coefficients (r²) were calculated for the three drugs, and the correlation coefficients were 0.99 to 1.0 for the three drugs Table 2. The developed method was found to be linear between 6.25-400 ppm for all the drugs. Fig. 4, Fig. 5 and Fig. 6 represent the linearity curve of aspirin, rosuvastatin calcium and clopidogrel bisulphate respectively.

LOD and LOQ: The results of the signal to noise ratio was compared with the response of the three drugs. The LOD and LOQ were estimated from the standard deviation of the y-intercepts and slope of the calibration curves of the three drugs.

The LOD of aspirin, rosuvastatin calcium, and clopidogrel bisulphate was found to be 0.96 ppm, 5.36 ppm, and 1.33 ppm, respectively. The LOQ of aspirin, rosuvastatin calcium, and clopidogrel bisulphate was found to be 2.90 ppm, 16.25 ppm, and 4.03 ppm, respectively.

TABLE 2: LINEARITY AND RANGE STUDIES OF THE DEVELOPED METHOD

*Mean of three determinations

Precision: Precision studies comprising of repeatability, intra-day, and inter-day studies were conducted as per the ICH guidelines. Repeatability of the developed method was assessed using aspirin (75 ppm), rosuvastatin calcium (10 ppm), and clopidogrel bisulphate (75 ppm). The intra-day and inter-day precision studies were conducted at three concentrations of the drugs in triplicate. The % RSD was less than 2 for the three drugs indicating that the developed method was precise in Table 3.

TABLE 3: PRECISION STUDIES OF THE DEVELOPED METHOD

Accuracy:  The % recovery of aspirin, rosuvastatin calcium, and clopidogrel bisulphate was within 98% 102% of the specified limit, indicating that the developed method was accurate Table 4.

TABLE 4: ACCURACY STUDIES OF THE DEVELOPED METHOD

*Mean of three determinations

Robustness: Small, deliberate changes like pH, flow rate, and mobile phase composition were applied, and their impact on parameters like retention time and tailing factor were determined. The results were found to be within the acceptable limits indicating that the developed method was robust in Table 5.

TABLE 5: ROBUSTNESS STUDIES OF THE DEVELOPED METHOD

System Suitability: The developed method showed theoretical plates above 2000 for the three drugs, tailing factor less than 2 for all the three peaks in the chromatogram and the mean resolution more than 2 between all the peaks Table 6.

TABLE 6: SYSTEM SUITABILITY STUDIES OF THE DEVELOPED METHOD

*Mean of six determinations

Application of the Validated Method for the Assay of Fixed-Dose Combination Capsules: The validated method was successfully applied to conduct the assay of marketed fixed-dose combination capsules containing the three drugs. The assay of aspirin, rosuvastatin calcium, and clopidogrel bisulphate was found to be 99.9%, 99.97%, and 99.3%, respectively Table 7. The results of the validation studies of the developed method were satisfactory as per the ICH Q2(R1) guidelines confirming that this method can be applied to fixed-dose combination products of these drugs. The developed method is better than the earlier reported methods (UV and HPLC) of fixed-dose combination products containing any two of the three drugs in terms of range, retention time, and accuracy. In addition, the run time of the developed method is short (15 min) with sharp, symmetrical peaks for the three drugs, which increases the number of analyses that can be done.

TABLE 7:  ASSAY OF FIXED DOSE COMBINATION CAPSULES

CONCLUSION: A simple, accurate, precise, and robust RP-HPLC gradient method was developed for the simultaneous estimation of aspirin, rosuvastatin calcium, and clopidogrel bisulphate in marketed fixed-dose combination capsules. The developed method was validated as per the ICH guidelines Q2 (R1) for parameters, viz. specificity, linearity, range, precision, accuracy, LOD, LOQ, robustness and system suitability. This method can be conveniently applied to quantitative analysis of aspirin, rosuvastatin calcium, and clopidogrelbi sulphate in fixed-dose combination products.

ACKNOWLEDGEMENT: The authors are thankful to Glenmark Generics Ltd., Mumbai, The Andhra Sugars Ltd. Andhra Pradesh and Watson Pharma Pvt. Ltd., Ambernath, for providing gift samples of the active pharmaceutical ingredients.

AUTHOR CONTRIBUTION: Ms. Richa has worked on this project and prepared the manuscript draft. Dr. Tabassum Khan has guided this project and edited the manuscript.

CONFLICTS OF INTEREST: None

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    How to cite this article:

    Singh R and Khan T: RP-HPLC method development and validation studies for the estimation of aspirin, clopidogrel bisulphate and rosuvastatin calcium in fixed dose combination capsules. Int J Pharm Sci & Res 2020; 11(5): 2366-73. doi: 10.13040/IJPSR.0975-8232. 11(5).2366-73.

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