Simultaneous quantification of metformin and glipizide in human plasma by high-performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study.

A selective, sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to determine metformin and glipizide simultaneously in human plasma using phenacetin as internal standard (IS). After one-step protein precipitation of 200 μL plasma with methanol, metformin, glipizide and IS were separated on a Kromasil Phenyl column (4.6 × 150 mm, 5 µm) at 40°C with an isocratic mobile phase consisting of methanol-10 mmol/L ammonium acetate (75:25, v/v) at a flow rate of 0.35 mL/min. Electrospray ionization source was applied and operated in the positive mode. Multiple reaction monitoring using the precursor → product ion combinations of m/z 130 → m/z 71, m/z 446 → m/z 321 and m/z 180 → m/z 110 were used to quantify metformin, glipizide and IS, respectively. The linear calibration curves were obtained over the concentration ranges 4.10-656 ng/mL for metformin and 2.55-408 ng/mL for glipizide. The relative standard deviation of intra-day and inter-day precision was below 10% and the relative error of accuracy was between -7.0 and 4.6%. The presented HPLC-MS/MS method was proved to be suitable for the pharmacokinetic study of metformin hydrochloride and glipizide tablets in healthy volunteers after oral administration.