Abstract

A NOVEL ENANTIO-SELECTIVE REVERSE PHASE HPLC METHOD FOR THE DETERMINATION OF EZETIMIBE AND IT’S ENANTIOMER

Pavani Jagu*, Vijay Kumar Baksam, B.M. Rao, Someswara Rao Nittala

Abstract

An accurate and reproducible High Performance Liquid Chromatographic (HPLC) method has been developed and validated for the direct separation of individual enantiomers of Ezetimibe, a selective inhibitor of intestinal cholesterol. The enantiomers were resolved by reversed phase chromatography, on a Chiralpak AS-RH column by using a mobile phase with gradient composition of Water- Acetonitrile-Formic acid. Formic acid, the mobile phase additive played a very significant role in improving the chromatographic resolution and also in enhancing the chromatographic efficiency. Baseline separation of the enantiomers of Ezetimibe was obtained with a pronounced resolution of about 2.0. The developed method was extensively validated and proved its suitability and robustness. The standard curves for the two enantiomers were linear (r > 0.999) in the concentration range from 0.47 μg/mL (LOQ) to 4.7 μg/mL for an analyte concentration of 0.5 mg/mL for each enantiomer. The percentage recovery of (R)-Ezetimibe was ranged from 93.3 to 98.9 in bulk drug samples of Ezetimibe. The limit of detection and limit of quantitation for (R)-Ezetimibe was 0.14 μg/mL and 0.47 μg/mL respectively. The intra-day precision (%RSD) results were 0.89, while the inter-day precision results were 0.79 calculated for content of (R) - Ezetimibe. Ezetimibe sample solution stability and mobile-phase stability were studied for 48 h and found to be stable during the period. The validated method yielded good results of selectivity, linearity, precision, accuracy and robustness. The method was also able to resolve the enantiomers from the degradation impurities of Ezetimibe.

Keywords: Ezetimibe, Chiral HPLC, Reverse Phase, Chiralpak AS-RH, Method validation.