ISOCRATIC HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR THE SEPARATION OF TESTOSTERONE METABOLITES

An isocratic reversed-phase high-performance liquid chromatographic (H PLC) method using an Ultrasphere IP column has been developed for the determination of testosterone and its metabolites after incubation of 4-C-14-labelled or unlabelled testosterone with rat liver microsomes. Compounds were eluted with methanol-water-tetrahydrofuran (35:55:10, v /v, pH 4.0) and detected by ultraviolet (UV) absorption at 245 nm. UV or on-line radioactivity detection can be used although, due to differ ences in detector cell volumes, peak resolution is slightly better wit h UV detection. Selectivity was validated by collecting HPLC peaks and verifying their identity by gas chromatography-mass spectrometry afte r derivatization by N,O-bis(trimethylsilyl)trifluoroacetamide chlorosi lane. A three-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method, using cort icosterone as internal standard. The method is applicable to the measu rement of cytochrome P-450 isoenzyme activities in rat liver.