Determination of testosterone and 6 beta-hydroxytestosterone by gas chromatography-selected ion monitoring-mass spectrometry for the characterizationof cytochrome p450 3A activity

A method for the determination of testosterone and its metabolite, 6 beta-h ydroxytestosterone, in liver microsomal incubates employing gas chromatogra phy with selected ion monitoring mass spectrometric detection (GC-SIM-MS) h as been developed. The method is more rapid than previously reported method s. Testosterone and its metabolites are extracted from the incubation mixtu re in a single step with methylene chloride. The method does not require de rivatization and testosterone and its metabolites are separated on a HP-5MS fused-silica capillary column in less than 15 min. The retention times of testosterone (m/z 288), methyltestosterone (m/z 302), and 6 beta-hydroxytes tosterone (m/z 304) are approximately 12.7, 12.8, and 13.4 min, respectivel y. There are no interferences from other known CYP450 metabolites of testos terone. In addition, the selectivity and specificity of the mass spectromet er helps eliminate possible interferences from drugs and new chemical entit ies evaluated using this methodology. Calibration curves for testosterone a nd 6 beta-hydroxytestosterone are Linear from 0.25 to 100 mu M. Extraction recoveries are better than 92% for both analytes and the internal standard, methyltestosterone. Over the course of five separate runs, within-day and inter-day precision (expressed as relative standard deviation) was less tha n 5% for all concentrations of testosterone and 6 beta-hydroxytestosterone. Accuracies ranged from 95.8 to 105.8% for testosterone and 94.6 to 104.2% for 6 beta-hydroxytestosterone. The assay has been used to characterize the CYP3A metabolic activity of multiple preparations of human, rat, and dog l iver microsomes. (C) 1999 Elsevier Science B.V. All rights reserved.