Simultaneous Determination of Tolbutamide and Its Metabolite 4-hydroxytolbutamide,Chlorzoxazone in Rat Plasma by LC-MS-MS and Application to Pharmacokinetic Study

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.