Determination of metformin in human plasma by HPLC-MS/MS method

Objective: To establish a HPLC-MS/MS method for determining the concentration of metformin in human plasma. Methods: Metformin and phenformin (internal standard) were separated from plasma by protein precipitation with acetonitrile, and determined by HPLC-MS/MS method with the mobile phase consisting of methanol-acetonitrile-10 mmol/L ammonium acetate (20: 20: 60), at a flow rate of 0.2 mL/min. Atmospheric pressure chemical ionization(APCI) source was applied and operated in the positive ion mode in multiple reaction monitoring (MRM) mode. The fragmentation transitions from precursor to production were m/z 130.2 → 71.0 for metformin and m/z 206.1 → 59.9 for phenformin,respectively. Results: The linear range of metformin was 2.0-2 000 μg/L (n=7,r=0.999 6). The lowest limit of quantitation of metformin was 2.0 μg/L. At low,middle and high concentrations (10,500,1 500 μg/L),the intra-day RSDs were 4.65%,2.84% and 3.41%(n=5), the inter-day RSDs were 7.96%,3.98% and 4.77%(n=5),and the accuracy rates were 95.9%,97.4% and 89.9%(n=5). Conclusion: The method is sensitive,specific,reproducible and accurate for the determination of metformin in human plasma.