GC-MS-Based Quantitative Signatures of Cytochrome P450-Mediated Steroid Oxidation Induced by Rifampicin

Background: Drug-induced cytochrome P450 (CYP) activity affects endocrine function and drug clearance rates, leading to the development of unpredictable pathologic and toxicologic risks.Methods: Urinary steroid profiling based on gas chromatography-mass spectrometry (GC-MS) was used for simultaneous quantification of CYP-mediated regioselective hydroxysteroids and their substrates, including 26 androgens, 9 estrogens, 5 progestins, and 7 corticoids. The quantitative data were visualized using a hierarchically clustered heat map to allow identification of CYP-mediated steroid signatures. Twelve healthy subjects were orally administered 600 mg of rifampicin a day for 7 days, and their CYP enzyme activity was evaluated.Results: Using GC-MS, all 47 steroids were well separated with good peak shapes. This assay had good linearity (r(2) > 0.994) in a dynamic range, and the interassay imprecision (% CV) and inaccuracy (% bias) were 3.0%-15.6% and 98.0%-109.2%, respectively. Administration of the CYP3A4 inducer rifampicin produced distinct differences in CYP3A4 and CYP11B1, CYP19A1, HSD11B, and HSD17B, which were indicated by their heat map-visualized steroid signatures.Conclusions: This CYP-mediated steroid signature profile allows simultaneous assessment of CYP1A, CYP1B, CYP2C, CYP3A, CYP11B, CYP17A, CYP19A, and CYP21A in urine samples. This method could therefore be a useful tool for assessing drug efficacy.