Ion-pair HPLC Method for the Quantification of Metformin in Human Plasma and Its Application

Abstract

The aim of this study is to develop and validate a rapid, selective and sensitive ion-pairing HPLC-UV method for the determination of metformin in human plasma, using a conventional reverse phase column. This Experimental study was conducted at the Department of Biomedical Sciences, Faculty of Medicine, University of Medicine, “Mother Tereza” hospital center, between November 2014 and February 2015. In this study, Ion-pair separation followed by UV detection performed on deproteinised and dichloromethane washed plasma samples was chosen for the determination of metformin. The separation was carried out on an analytical LiChrocart ® 100 RP 18 (125 x 4.0mm i.d., 5 µm particle size) C18 column. A mobile phase of acetonitrile and 10 mM sodium phosphate buffer (pH=6.0; 32.5:67.5, v/v) and sodium dodecyl sulphate (0.3%) was pumped at an isocratic flow rate of 1.25 mL/minute, and quantification was performed at 236 nm using a UV/Vis DAD.  The calibration curves were linear (r > 0.9998) in the concentration ranges of 50-1600 ng/mL for metformin in plasma. The assay enables the measurement of metformin for therapeutic drug monitoring with a minimum detectable limit of 18 ng/ml. The coefficients of variance for the inter-day and intra-day assays were within clinically relevant ranges. Absolute recovery was reported to be greater than 90% for all three plasma quality control concentrations examined. The proposed approach for quantifying metformin in human plasma was found to be fast, precise, and accurate.This strategy was successfully utilised to a pharmacokinetic investigation in people by oral dosing.