Determination of metformin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A simple, selective, sensitive and precise high-performance liquid chromatographic plasma assay for the hypoglycemic agent metformin is described. Acidified samples of plasma were deproteinated with acetonitrile, washed with dichloromethane and the resulting supernatant injected. Chromatography was performed at 40°C by pumping a mobile phase of acetonitrile (250 ml) in pH 7, 0.03 M diammonium hydrogen phosphate buffer (750 ml) at a flow-rate of 1 ml/min through a silica column. Metformin and the internal standard (atenolol) were detected at 240 nm and were eluted 7.8 and 6.8 min, respectively, after injection. No endogenous substances were found to interfere. Calibration curves were linear (r>0.999) from 10 to 2000 ng/ml. The absolute recovery of both metformin and atenolol was greater than 76%. The detection limit and limit of quantitation were 2.5 and 10 ng/ml, respectively. The intra- and inter-day precision (C.V.) was 12%, or less, and the accuracy was within 6.2% of the nominal concentration. This method is suitable for clinical investigation and monitoring metformin concentration.