Development and Validation of Liquid Chromatographic Methods for Anti- Hyperlipidemic Drugs in Binary Combinations

Abstract

In the present dissertation, stress was applied to determine anti-hyperlipidemic drugs in combination form especially in binary combinations using simple, sensitive and economic HPLC methods. Seven HPLC methods have been developed for Atorvastatin- Ezetimibe, Ezetimibe-Simvastatin, Gemfibrozil-Simvastatin, Ezetimibe-Fenofibrate, Ezetimibe-Lovastatin, Atorvastatin-Gemfibrozil and Rosuvastatin-Ezetimibe dual formulations. The first HPLC method was developed for the simultaneous determination of atorvastatin and ezetimibe in tablet formulations. Chromatographic separation was achieved on a 250 × 4.6 mm, 5μ Hypersil phenyl-2 column at 242 nm using a mixture of 0.1 M ammonium acetate (pH 6.5) and acetonitrile in the ratio of 28:72 (v/v) as a mobile phase. The method was linear in the concentration range of 12-52 μg/ml for both atorvastatin and ezetimibe with correlation coefficient between 0.9966 and 0.9993. The total run time was less than 5 min. The second method which was developed was for the simultaneous determination of ezetimibe and simvastatin in pharmaceutical formulations. Chromatographic separation was performed on a Merck C18 column at a wavelength of 240 nm using a mixture of 0.1M ammonium acetate buffer pH 5.0 and acetonitrile in the ratio of (30:70, v/v). The method results in excellent separation with good resolution between the two analytes. The within day variation was between 0.28 and 1.10 % and between day variation was between 0.56 and 1.32 %. The recovery was greater than 99.12 % with RSD less than 1.38 %. In the third method, conditions were optimized to develop a simple, sensitive and validated HPLC method to determine gemfibrozil and simvastatin simultaneously in synthetic mixture form. Chromatographic separation was achieved on a C-18 column using a mixture of 0.1 M ammonium acetate pH 5.0 and acetonitrile in the ratio of 15:85 (v/v) at a wavelength of 237 nm. Linearity of the method was found to be in the concentration range of 60-420 μg/ml for gemfibrozil and 1-7 μg/ml for simvastatin with correlation coefficient greater than 0.9999. The fourth method developed for available binary combination was the simultaneous iABSTRACT determination of ezetimibe and fenofibrate in tablets. Isocratic chromatography was performed on a Merck C-18 column using a mixture of 0.1 M ammonium acetate pH 5.0 and acetonitrile in the ratio of (25:75, v/v) at a flow rate of 1.5 ml/min. The detection was carried out at a wavelength of 240 nm using a photodiode array detector. The method was linear in the concentration range of 0.8-40 μg/ml for ezetimibe and 12.8-640 μg/ml for fenofibrate. The fifth method developed was for the simultaneous determination of ezetimibe and lovastatin in synthetic mixture form. Chromatographic separation was performed on a C- 18 column using a mixture of 0.1M ammonium acetate buffer pH 5.0 and acetonitrile in the ratio of (28:72, v/v). The detection was carried out at a wavelength of 240 nm using a photodiode array detector. The method was linear in the concentration range of 0.2-100 μg/ml for ezetimibe and 0.4-200 μg/ml for lovastatin. The within day variation was between 0.32 and 1.22 % and between day variation was between 0.98 and 1.63 %. The recovery was greater than 102 % with RSD less than 1.5 %. Later the method was also applied for the determination of these two drugs in spiked human plasma. No plasma peaks interfered with the peaks of active anaytes, which means it can also be used for the determination in human plasma. The separation procedure for the simultaneous determination of atorvastatin and gemfibrozil in synthetic mixture form was also developed. Chromatographic separation was achieved on a C-18 column using a mixture of 0.1 M ammonium acetate pH 5.0 and acetonitrile in the ratio of 45:55 (v/v) at a wavelength of 240 nm. Linearity of the method was found to be in the concentration range of 0.1-20 μg/ml for atorvastatin and 6-1200 μg/ml for gemfibrozil with correlation coefficient 0.9997 for atorvastatin and 0.9976 for gemfibrozil. The elution time for the two components was less than twelve minutes. Forced degradation study was also applied to both the drugs individually and in combination form. During the forced degradation study under oxidative stress, a novel degradation product was also isolated in crystalline form. Later the developed method under the same chromatographic conditions was also applied for the determination of these two drugs in spiked human plasma. No plasma peaks interfered with the peaks of active anaytes, which means it can also be used for the determination in human plasma.