RESEARCH ARTICLE RP-HPLC Method for Simultaneous Estimation of Atorvastatin Calcium and Ramipril from Capsule Dosage Form. Jadhav S. D.1*, Thamake S. L.2 Pishawikar S. A.2 Abstract: A reverse phase high performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of atorvastatin and ramipril in marketed formulation is developed. The determination was carried out on a HIQ SII C 18 column -10 (4.5 mm x 250 mm).column using a mobile phase of acetonitrile: 0.02 M Potassium dihydrogen phosphate (pH 3.2) [75: 25%v/v]. The flow rate was 1 ml/min with detection at 217 nm. The Valsartan is used as internal standard in this method. The retention time for atorvastatin was 6.1 min, for ramipril 3.43 min. Atorvastatin and ramipril showed a linear response in the concentration range of 20-180 μg/ml and 10- μg/ml respectively. The correlation co-efficient r value for atorvastatin and ramipril was 0.9999 for both drugs. The results of analysis have been validated statistically and by recovery studies. The percentage recoveries obtained for atorvastatin and ramipril ranges from 99.04 to 101.15 %. Keywords: Atorvastatin, Ramipril, RP-HPLC. INTRODUCTION Atorvastatin [R-(R*, R*)]-2-(4-fluorophenyl),∂ dihydroxy-5-(1-methylethyl)-3-phenyl-4[(phenyl amino) carbonyl]-1H-pyrrole-1heptanoic acid calcium salt for trihydrate] is Anti-Lipidemic agent act by inhibiting HMG CoA reductase enzyme.[1] Ramipril [2S[ [R* R* ], , , ]]-1[2[[1(Ethoxycarbonyl)-3-phenylpropyl] amino]-1-oxon propyl] octahydro cyclopenta [b] pyrrole-2-carboxylic acid.] is ACE inhibitor.[2-3] The combination of both drugs is used in treatment of Hypertension. Several HPLC[4-6] , HPLC[7], Liquid Chromatography– electro Spray Ionization Tandem Mass Spectrometry[8] methods have been reported for atorvastatin alone or in combination with other drugs.[9-11] Only one UV spectroscopy i.e. derivative spectroscopy method has been reported for simultaneous estimation of atorvastatin (ATR) and ramipril (RAM).[12] The present method (Internal Standard RP-HPLC method) quantitates both drugs simultaneously without exipients interference in analysis from capsule dosage form [13] MATERIALS AND METHODS: Instrumentation: The separation of ATR and RAM was carried out on an isocratic JASCO RP-HPLC system using C18 column (150x4.6mm internal diameter, particle size 5µm).The pump used in this HPLC system was PU 2080 pump (Dual piston with gear driven pump). The 20 µl Department of Pharmaceutical Chemistry, Tatyasaheb Kore College of Pharmacy, Warananagar-416113(M.S.) India. 1 swapnil.mpharm@gmail.com *(For correspondence) Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College Of Pharmacy, Kolhapur-416013(M.S.) India. 2 Inventi Impact: Pharm Ana & Qual Assur Vol. 1, Issue 1 www.inventi.in www sample solutions were injected to chromatographic system using Rheodyne Injector. The UV detector used in this HPLC system was Czerny turners mount monochromater with deuterium lamp as light source. The chromatographic and the integrated data were recorded using Hercule 2000 (interface) computer system. Data processing was carried out using Borwin® Version 1.5software. Reagents and Chemicals: The solvents used were of HPLC/AR grade. Pure drug samples of ATR, RAM and Valsartan (VAL) are obtained as gift samples from Cipla India Pvt. Ltd. Kurkumbh, Mumbai. Marketed formulation ATOCORR(Company Name) was procured from the local market. Chromatographic Conditions: The analysis was carried out by HPLC using Acetonitrile: 0.02 M Potassium dihydrogen phosphate (pH 3.2) [75:25 %V/V] as a mobile phase and HIQ SII C18 column -10 (4.5 mm x 250 mm) as a stationary phase at a flow rate of 1ml /minute in an isocratic elution mode. Before delivering the mobile phase in to the system, it was degassed and filtered through 0.20µm syringe filter. The injection volume was 20 µl and the detection was performed at 217 nm. Standard Stock Solution: About 20 mg of each of reference standard of ATR, RAM and VAL (Internal Standard) was weighed accurately and transferred to three separate 100 ml volumetric flasks. Both drugs were dissolved in 50 ml of mobile phase with shaking and volume was made up to the mark with mobile phase to get μg/ml of standard stock solution of each drug. These stock solutions were then sonicated and filtered through . μ syringe filter. Calibration Curves: For each drug, appropriate aliquots were pipetted out from each standard stock solution into a series of 10 ml volumetric flasks. The volume was made up to mark with mobile phase to get set of solutions having concentrations 10μg/ml , μg/ml and μg/ml in each flask for RAM, ATR and VAL respectively. Triplicate dilutions of each concentration of each drug were prepared separately. From these triplicate solutions, 20 μl injections of each concentration of each drug were injected into the RP-HPLC system separately and chromatographed under the conditions as described above. Evaluation of both drugs was performed with UV detector at 217 nm. Peak areas were recorded for all the peaks. Retention factors were calculated and plotted against the concentrations to obtain the standard calibration curves (Figure 1). Analysis of capsule Formulation: Twenty capsules of ATOCORR were weighed and powder separated from capsule shells. From the content of 20 capsules, an amount equivalent to 20 mg of ATR and 10 mg of RAM was weighed and dissolved in 50 ml of mobile phase and sonicated for 20 minutes. The solution was filtered through . μm Nylon , 6(N66) membrane filter and then final volume of the solution was made up to 100 ml with acetonitrile to get stock solution containing μg ml -1 of ATR and RAM μg ml -1 in 100 ml volumetric flask. After appropriate dilutions, the solutions were run on HPLC system and the concentration of each analyte was determined using calibration curve data.[14] The statistical data obtained after replicate determinations (n = 6) is shown in Table 1 and chromatogram is shown in Figure 2. Validation of HPLC Method: The proposed RP-HPLC method was validated as per ICH guidelines.[15] Specificity The specificity of the RP-HPLC method was determined by comparison of the chromatogram of mixed standards and sample solutions. The parameters like retention time, resolution and tailing factor were calculated. Good correlation was found between the results of mixed standards and sample solutions. Precision Precision study was performed to find out intra-day and inter-day variations. The %relative standard deviation (RSD) for intraday precision was 0.993 % for ATR and 0.877 % for RAM and for inter-day precision was 1.025% for ATR and 0.743% for RAM respectively which is less than 2% indicating high degree of precision. Accuracy (Recovery studies) Recovery studies were performed by standard addition method at three levels i.e., 80%, 100% and 120%. Known amounts of standard ATR and RAM were added to preanalyzed samples and they were subjected to proposed 2010paqa009, CCC: $10 © Inventi Journals (P) Ltd Published on Web 15/07/2010 RESEARCH ARTICLE Table 1: Results of Marketed Formulation Analysis. Drug Label claim (mg/capsule) Estimated % of Label claim ±S.D.* % Recovery ± S.D.* 80 100 120 ATR 10 99.73±0.568 100.13±0.853 99.47±0.752 100.90±0.905 RAM 5 99.97±1.069 99.13±1.053 100.07±0.619 99.23±1.072 *Average of six determinations; S.D., standard deviation. Table 2: System Suitability Parameters. Sr. No. Parameters ATR RAM 1. Theoretical Plates 4813 4781 2. Tailing Factor 1.21 1.17 3 Separation Factor 1.78 0.00 4. Resolution 10.90 0.00 5. Limit of Detection (µg ml -1) 0.1712 0.2423 6. Limit of Quantitation (µg ml -1) 0.5189 0.7342 Figure1: Overlain Chromatogram of Mixed Standards. Inventi Impact: Pharm Ana & Qual Assur Vol. 1, Issue 1 www.inventi.in www Figure 2: Chromatogram of Marketed Formulation Analysis 2010paqa009, CCC: $10 © Inventi Journals (P) Ltd Published on Web 15/07/2010 RESEARCH ARTICLE HPLC. Results of recovery studies are shown in Table 1. Limit of detection (LOD) and limit of quantitation (LOQ) The LOD and LOQ were separately determined based on the calibration curves. The standard deviation of the y-intercepts and slope of the regression lines were used. Results of LOD and LOQ are given in Table 2. Robustness The robustness study was done by making small changes in the optimized method parameters like ±0.1 change in pH ±1% change in mobile phase ratio and column temperature. There was no significant impact on the retention time and tailing factor. Ruggedness The ruggedness study was done by the two analysts. The %RSD for analyst-I was 0.8526% for ATR and 0.6390% for RAM and for analystII was 0.5941% for ATR and 0.6829 % for RAM respectively. System Suitability Parameters System suitability parameters were analyzed on freshly prepared standard stock solutions of ATR and RAM. Both the drugs were injected into the chromatographic system under the optimized chromatographic conditions. Parameters that were studied to evaluate the suitability of the system were number of theoretical plates, tailing factor, resolution, separation factor etc.[16] RESULT AND DISCUSSION The proposed method describes a RP-HPLC procedure employing a C18 column and a mobile phase water and acetonitrile in the ratio 25:75 (pH 3.2). The two drug solutions having concentration of 10 µg ml -1 were scanned in range of 200 nm to 400 nm on a UV-Visible spectrophotometer for selection of sampling wavelength. After recording the spectra of the two drugs, 217 nm was selected as suitable wavelength for estimation. The best resolution of the two drugs and internal standard eluting within 7.5 minutes was achieved with a flow rate of 1 ml min -1. The linearity response of the HPLC system for ATR and RAM were obtained over the range of 20-180 µg ml-1 and 10-90 µg ml-1 Inventi Impact: Pharm Ana & Qual Assur Vol. 1, Issue 1 www.inventi.in www respectively. Accuracy of the method was checked by adding known amounts of pure drug to each known concentration of the capsule formulations. The %RSD for the tablet analysis and recovery studies was less than 2% indicating high degree of accuracy. The other validation parameters have proved accuracy, precision, sensitivity and selectivity of the proposed method as number of theoretical plates, tailing factor, resolution, separation factor, results of analysis and statistical parameters were found be within limits. CONCLUSION The statistical data have proven that developed HPLC method for simultaneous estimation of ATR and RAM was found to be more accurate, precise, sensitive and selective. Therefore the proposed method could be applied for routine analysis in quality control laboratories for both in bulk and multicomponent formulations. 8. 9. 10. 11. 12. REFERENCES AND NOTES 1. 2. 3. 4. 5. 6. 7. Budawari S, The Merck Index, 23rd Edn, Merck and Co. Inc.; Whitehouse Station, NJ, 1996, 893; 8280. Ministry of Health and Family Welfare; Government of India, Indian Pharmacopoeia, 5th Edn, Vol. II, The Controller of Publication, New Delhi, 2007, 749. Florey K, Analytical Profiles of Drug Substances, Vol-10, Elsevier India Private Limited, New Delhi, 2005, 405-461. Mishra P, Gupta A and Shah K, Simultaneous Estimation of Atorvastatin Calcium and Amlodipine Besylate from Tablets, Indian Journal of Pharmaceutical Sciences, 69(6), 2007,831-833. Jain N, Raghuwanshi R and Jain D, Development and Validation of RP-HPLC Method for Simultaneous Estimation of Atorvastatin Calcium and Fenofibrate in Tablet Dosage Forms, Indian Journal of Pharmaceutical Sciences, 70(2), 2008, 263-265. Maheshwari K R, Sankar G G and Rao A L, HPLC Method for the Simultaneous Determination of Atorvastatin and Amlodipine in Tablet Dosage Forms, Indian Journal of Pharmaceutical Sciences, 68(2), 2006, 275-277. Patel N M, et al., Development and Validation of HPTLC Method for the Simultaneous Estimation of Atorvastatin Calcium and Ezetimibe, Indian 13. 14. 15. 16. Journal of Pharmaceutical Sciences, 68, 2006, 793-796. Miao X S and Metcalfe C D, Determination of Cholesterol-lowering Statin Drugs in Aqueous Samples Using Liquid Chromatography–electro Spray Ionization Tandem Mass Spectrometry, Journal of Chromatography A, 998, 2003, 133– 141. Bonazzi D and Cavrini V, Analysis of ACE inhibitors in Pharmaceutical Dosage Forms by Derivative UV Spectroscopy and Liquid Chromatography (HPLC), Journal of Pharmaceutical and Biomedical Analysis, 16, 1997, 431-438. Mohammadia A and Rezanour N, A stabilityindicating high performance liquid chromatographic (HPLC) assay for the simultaneous determination of atorvastatin and amlodipine in commercial tablets, Journal of Chromatography B, 846, 2007, 215–221. Zaheer Z and Farooqui M N, Stability-indicating High Performance Liquid Chromatographic Determination of Atorvastatin Calcium in Pharmaceutical Dosage Form, African Journal of Pharmacy and Pharmacology, 2, 2008, 204-210. Thamake S L, Jadhav S D and Pishawikar S A, Development and Validation of Method for Simultaneous Estimation of Atorvastatin Calcium and Ramipril from Capsule Dosage form by First Order Derivative Spectroscopy Asian Journal of Research in Chemistry, 2 (1), 2009, 52-53. Chitlange S S, Bagri K, Stability Indicating RPHPLC Method for Simultaneous Estimation of Valsartan and Amlodipine in Capsule Formulation, Asian Journal of Research in Chemistry, 1, 2008, 15-18. Shethi P D, Quantitative Analysis of Drugs in Pharmaceutical Formulations, 3rd Edn, CBS Publishers and Distributors, New Delhi, 1997, 17. ICH Q2B, Text on Validation of Analytical Procedures, Geneva, 1994, 1–5. Snyder L R, Kirkland J J and Glajch J L, Practical HPLC method Development, 2nd Edn, A WileyInterscience Publication, 1997, 250-747. ACKNOWLEDGEMENT: The authors are very thankful to Cipla India Pvt. Ltd. for providing gift sample of Atorvastatin Calcium and Ramipril. The authors are also thankful to Tatyasaheb Kore College of Pharmacy, Warananagar and Bharati Vidyapeeth College of Pharmacy, Morewadi, Kolhapur for provision of facilities for this research work. 2010paqa009, CCC: $10 © Inventi Journals (P) Ltd Published on Web 15/07/2010