Chlorzoxazone,tolbutamide and midazolam,the probe drugs of three cytochrome P450,can be determined in plasma of rats by HPLC in a single run

Objective To establish an efficient,rapid,selective HPLC method for determining three CYP450 s probe drugs,chlorzoxazone,tolbutamide and midazolam,in single run in rat plasma. Methods The three specific probe substrates of chlorzoxazone,tolbutamide,and midazolam,together with the internal standard diazepam,could be extracted using liquid-liquid extraction in rat plasma,followed by high-performance liquid chromatography( HPLC) using a C18column( 250 mm × 4. 6 mm,5 μm). The mobile phase was constituted of a methanol and 0. 05 mol·L- 1phosphate buffer( 61 ∶ 39,V / V). Results All analytes were separated simultaneously in a single run. The detection limits ranged from 0. 1 ~ 50 mg·L- 1for chlorzoxazone,tolbutamide and midazolam. The precisions for three probe substrates were 4. 78% ~ 9. 78%,the accuracy of three probe substrates ranged from 92. 71% ~ 109. 79%. The limit of quantification( LOQ) was 0. 1 mg·L- 1for chlorzoxazone,tolbutamide and midazolam. Conclusions The present method provides a robust,fast analytical tool for the three-probe drug cocktail. Finally,the method was suitable for determining the plasma concentration of these compounds and evaluating the CYP2E1,CYP2C9 and CYP3A4 activities in the drug-drug interaction studies.