Bio-Analytical Method Development and Validation for Simultaneous Estimation of Aceclofenac and Drotaverine Hydrochloride in Human Plasma by RP-HPLC

Abstract

The proposed work describes a simple, accurate, precise and robust RP-HPLC method developed and validated for the simultaneous estimation of Aceclofenac and Drotaverine Hydrochloride in human plasma by DoE approach. By selecting the critical quality attributes, DoE was successfully applied to the analytical method development. The optimization of conditions for HPLC method was done by taking trials at different mobile phase ratio and pH. KROMASIL C8 (150*4.6 mm, 5μm) column was selected as the stationary phase. For the estimation of both the drugs, UV detection was done at 230 nm wavelength using mobile phase of ACN: Ammonium acetate buffer pH 3.5 (53:47 v/v) with flow rate of 1 ml/min. The proposed method includes three types of extraction methods; Protein precipitation, Liquid-liquid extraction and Solid-phase extraction. All these extraction methods were compared and among them, SPE gave better recovery. For extraction and estimation of Aceclofenac and Drotaverine Hydrochloride, SPE was selected. Average recovery of the extracted sample from the plasma was found to be 71 % and 98 % for Aceclofenac and Drotaverine Hydrochloride, respectively. Linearity over the concentration ranges of 30 – 9000 ng/ml and 50 – 180 ng/ml with correlation coefficient values 0.9952 and 0.9926 was obtained for Aceclofenac and Drotaverine Hydrochloride, respectively. The % CV for Aceclofenac and Drotaverine Hydrochloride was found to be less than 10% and less than 5%, respectively. Bioanalytical samples found to be stable over the three freeze-thaw cycles, which showed good stability of drugs in plasma. The bioanalytical method was validated for selectivity, linearity, accuracy, precision, stability according to USFDA guidelines and can be applicable for the pharmacokinetic profiling of these drugs in human plasma.