Objective: To set up an LC-MS-MS method for the simultaneous determination of Cytochrome P450 probe substrates

including tolbutamide and its metabolite 4-hydroxytolbutamide for CYP2C9

chlorzoxazone for CYP2E1. Method: Probe drugs with the IS gliclazide were extracted using ethyl acetate. Gradient elution was performed on an Agelient Eclipse Plus-C18 column (2.1 mm×50 mm

3.5 μm). The mobile phase consisted of 0.01% formic acid (1 mmol·L-1 ammonium formate) and acetonitrile. The flow-rate was 0.3 mL·min-1

and the injection volume was 10 μL. The probe drugs were analyzed by ESI MS in MRM mode. The MS-MS reaction selected ions 269.1/170.0 m/z for tolbutamide

285.1/186.0 m/z for 4-hydroxytolbutamide

168.1/132.1 m/z for chlorzoxazone and m/z 322.3/170.2 ions for gliclazide. Result: The tolbutamide

4-hydroxytolbutamide and chlorzoxazone had good linear relationship within the range of 0.98-4 000

0.25-125

0.98-2 000 μg·L-1

respectively. The extraction recoveries for all probe dings were more than 70%.The stability RSD were less than 11% and matrix effects in plasma on the ionization of probe drugs were negligible. Conclusion: The method described in this report has accuracy

sensitivity and reproducibility. The established LC-MS-MS method was suitable for pharmacokinetic study of tolbutamide and chlorzoxazone as a cocktail probe group and could be applied to hepatic microsomal enzyme study.