Liquid chromatographic–mass spectrometric method to assess cytochrome P450-mediated metabolism of testosterone by rat everted gut sacs

A rapid, sensitive and specific method was developed for the simultaneous assay of testosterone, androstenedione and 6β-hydroxytestosterone (6β-OHT) in the TC199 tissue culture medium used in intestinal drug metabolism studies with the rat everted gut sac model. An electrospray LC–MS method was validated in the concentration range of 0.025–9.5 μM (7.2 ng–2.7 μg/mL) for testosterone and androstenedione and 0.01–4 μM (3 ng–1.2 μg/mL) for 6β-hydroxytestosterone. The limits of quantification (LOQ) with an injection volume of 10 μL were 0.0005 μM (4.9 fmol, 1.4 pg injected), 0.004 μM (0.04 pmol, 11.4 pg injected) and 0.03 μM (0.3 pmol, 91 pg injected), respectively. The method also detected the other testosterone metabolites, the 16α-, 16β-, 2β- and 2α-hydroxytestosterones and was then used to study the metabolism of testosterone during its absorption by rat intestine in vitro, using everted gut sacs.