Determination of Metformin in Human Plasma and Urine by High-Performance Liquid Chromatography Using Small Sample Volume and Conventional Octadecyl Silane Column

Purpose. To develop a selective and sensitive high-performance liquid chromatographic method for the determination of metformin in human plasma and urine, using a conventional reverse phase column and low specimen volume. Methods. Extraction of metformin and ranitidine (as internal standard) from plasma and urine samples (100 mu L) was performed with a 1-butanol-hexane (50: 50, v/v) mixture under alkaline conditions followed by back-extraction into diluted acetic acid. Chromatography was carried out using a C18 column (250 mmx4.6 mm, 5 mu m). A mobile phase consisting of acetonitrile and KH2PO4 (34: 66, v/v) and sodium dodecyl sulphate (3 mM) was pumped at an isocratic flow rate of 0.7 mL/min. Results. The calibration curves were linear (>0.995) in the concentration ranges of 10-5000 ng/mL and 2-2000 mu g/mL for metformin HCl equivalents in plasma and urine respectively. The mean absolute recoveries for 100 and 1000 ng/mL metformin HCl in plasma using the present extraction procedure were 93.7 and 88.5%, respectively. The intra- and inter-day coefficients of variation in plasma and urine were <20% at the lowest, and <16% at other concentrations. The percent error values were less than 2% in plasma while it reached similar to 9% in urine. The lower limits of quantification were 7.8 ng/mL and 1.6 mu g/mL of metformin base in plasma and urine respectively. Conclusions. The method showed high calibers of sensitivity and selectivity for monitoring therapeutic concentrations of metformin in both plasma and urine based on a 0.1 ml sample size.