Novel Bio-analytical RP-HPLC Method for Simultaneous Estimation of Aceclofenac and Drotavarine Hydrochloride in Human Plasma by DOE Approach

Abstract

A simple, accurate, precise, specific and robust RP-HPLC method is developed for simultaneous estimation of aceclofenac and drotaverine hydrochloride in human plasma by applying DoE approach. Liquid chromatography was performed on a Kromasil C8 column (150 × 4.6 mm, 5μm) and the mobile phase consisted of Acetonitrile: Ammonium acetate buffer pH 3.5 (53:47 v/v) with 1ml/min of flow rate. The effluent was monitored on a UV detector at 230 nm. Out of three types of extraction procedures viz. protein-precipitation, liquid-liquid extraction and solid-phase extraction applied; good recovery was obtained using solid-phase extraction and hence was selected for further extraction. Average recovery of extracted sample from plasma was found to be 71 % and 98 % for aceclofenac and drotaverine hydrochloride respectively. There was a good linear relationship of peak area of both the drugs with the concentration which ranges from 30 – 9000 ng/ml and 50 – 180 ng/ml for aceclofenac and drotaverine hydrochloride respectively. Bio-analytical samples were found to be stable over three freeze-thaw cycles showing good stability of drugs in plasma. The bio-analytical method was validated for selectivity, linearity, accuracy, precision and stability according to USFDA guidelines and can be further applied for therapeutic monitoring of these drugs in patients