Determination of metformin hydrochloride and glibenclamide using HPLC and LC-MS / 第三军医大学学报

ABSTRACT

Objective To establish an HPLC method for the determination of plasma metformin hydrochloride and an HPLC/ESI-MS method for the determination of plasma glibenclamide in human. Methods The metformin plasma samples were added with acetonitrile to denature proteins and the supernatant was injected into the system directly by using EC 250/4.6 NUCLEOSIL 100-5 analytical column (4.6 mm?250 mm, 5 ?m) with a mobile phase of 0.03 mol/L (NH 4) H 2PO 4∶acetonitrile (75∶25, pH 7.0) at the flow rate of 1.0 ml/min and the detection wavelength of 240 nm. Using glipizide as a internal standard, the glibenclamide plasma samples were extracted with ethyl acetate and determined by LC-MS using a chromatographic column ORBAX SB300C 18 (2.1 mm?150 mm, 5 ?m) with a mobile phase of acetonitrile-0.1% ammonium acetate (55∶45, V/V) at the flow of 0.2 ml/min. Results The glibenclamide concentration had a good linear correlation in the range of 2-200 ng/ml (r=0.999 8) while the metformin concentration in the range of 6.25-4 000 ng/ml (r= 0.999 9 ). Both the inter-day and intra-day RSD of metformin and glibenclamide were lower than 5%. Conclusion The assays have been proved to be sensitive, accurate and convenient and can be used in the study of human pharmacokinetics of glibenclamide and metformin hydrochloride.