Rapid Determination of CYP2 C9 Activity by UPLC-MS/MS / 中国药师

ABSTRACT

Objective:To establish an ultra performance liquid chromatography-tandem quadruple mass spectrometry ( UPLC-MS/MS) method to determine CYP2C9 activity in vitro. Methods:An ACQUITY UPLC? BEH C18 (100 mm × 2. 1 mm, 1. 7 μm) column was used as the stationary phase at 30℃. The mobile phase consisted of acetonitrile-water ( containing 0. 1% formic acid and 0. 5%ammonia water) (40∶60, v/v). The flow rate was 0. 2 ml·min-1. Chlorpropamide was used as the internal standard. The MS condi-tions were as followsESI with positive ion detection mode. Self-prepared CYP2C9?1, ?2, ?3 and ?13 protein were incubated with tolbutamide at 37℃ and 800μl ethyl acetate was added to stop the reaction. After centrifuged at 10 000g, the organic layer was then dried using nitrogen, the residue was re-dissolved in 200μl mobile phase and determined by UPLC-MS/MS. Results: The reten-tion time of 4-hydroxytolbutamide was 1. 21 min. An excellent linear calibration curve of 4-hydroxytolbutamide was obtained within the concentration range of 0. 05-5 ng·μl-1(r=0. 999 8). The lower limit of quantification of 4-hydroxytolbutamide was 0. 01 ng·μl-1 with the average recovery of 99. 3%-100. 3%. The intra- and inter-day RSDs were all less than 5%. There was no interference from the endogenous substances existing in the incubation system. The catalytic activity of the variants CYP2C9?2,?3 and?13 after tol-butamide was incubated with CYP2C9?1,?2,?3 and?13 was 47. 3%, 11% and 0. 3% of wild type CYP2C9?1. Conclusion:The method is simple and stable, and suitable for the fast evaluation of cytochrome CYP2C9 activity in vitro and relevant studies on the inhibitors.