Bio-Analytical Method Development and Validation of RP-HPLC Method For the Simultaneous Estimation of Drotavarine Hydrochloride and Mefenatic Acid in Human Plasma by Applying Design of Experiment Approach

Abstract

Proposed work describes development and validation of simple, accurate and precise Bioanalytical RP- HPLC method for the estimation of MFA and DRT simultaneously in the human plasma by applying DoE approach. DoE was successfully applied by selecting critical parameters. Optimization of conditions of HPLC method was done by taking trials at different PH and mobile phase ratio. C8 KROMASIL 100 (5 um x 15 cm x 4.6 um) column was selected for as a stationary phase. Wavelength selected for the estimation was 250nm for both the drugs. Mobile phase consisting of ACN: Ammonium acetate buffer (55:45, PH-3.5) solution and a flow rate of 1ml/min. The proposed method includes all type of extraction methods like Protein precipitation, Solid phase extraction, Liquid-liquid extraction. Comparison of all three methods was done and among them LLE gives highest recovery so it was selected for extraction and estimation of MFA and DRT. Linearity range observed for DRT and MFA was 50-2000ng/ml and 156.25-6250ng/ml with the correlation co-efficient values of 0.996 and 0.995 respectively. Average recovery of the extracted sample from the plasma was found to be 93.04 and 82.73 for DRT and MFA respectively. LOD and LOQ were found to be 40 and 120ng/ml for DRT and 150 and 468.75ng/ml for MFA.RSD values for DRT and MFA was found to be less than 5% and less than 10% respectively. Stability study was performed by carrying out Freeze-thaw cycle which showed good stability of the drugs in plasma. Developed Bio-analytical method was validated according to US-FDA guidelines and can be applicable for the pharmacokinetic profiling as of these drugs in the human plasma.