Development and validation of a reverse phase high-performance liquid chromatographic (RP-HPLC) method for the determination of metformin in human plasma

Abstract

A simple, rapid, efficient, and precise reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the determination of metformin levels in human plasma. Plasma samples were acidified with 0.5ml of HCl, deproteinized with acetonitrile and centrifuged. The supernatant was washed with dichloromethane and injected into the HPLC system. Separation was achieved within 10 min on a Poroshell 120 EC-C18 (4.6 mmx 50 mm 2.7 Microns)column with an isocratic mobile phase consisting of acetonitrile and methanol (10:90) containing 0.03Mdi basic  ammoniumphosphate (pH 7) isocratic elution mode, an injection volume of 2 µl , flow rate of 0.8 ml min -1 at a detection wave length of 236 nm, and at  ambient temperature. Calibration curve (0.05-5 µgml ^-1) was constructed by plotting the peak arearatiosagainst their corresponding concentrations. The  method was validated according to ICH guidelines. Metformin andphenytoineluted at 2.23 and 4.41 minutes respectively. The method was precise (RSD ±  SD 3.647 ± 4.36) and accurate (%ER3.43and % recovery 96.52%), with correlation coefficient (R² = 0.995. Limit of detection (LOD) and Limit of  quantification(LOQ) of the developed method were 0.05 and 0.02 µgml ^-1 respectively. All the parameters are within the acceptable limits of ICH  guidelines. The developed method was found to be simple, rapid, efficient, precise and accurate for the quantification of metformin in human plasma