Synthesis and characterization of 6β-hydroxyandrosterone and 6β-hydroxyetiocholanolone conjugated with glucuronic acid

© 2014 John Wiley & Sons, Ltd. The detection of testosterone (T) misuse by doping control laboratories is mainly based on monitoring urinary T phase I metabolites released after enzymatic hydrolysis of the corresponding phase II glucuronide metabolites by gas chromatography (tandem) mass spectrometry (GC-MS(/MS)) methods. However, this strategy fails to properly determine two recently reported phase II metabolites of T conjugated with glucuronic acid that remained mostly conjugated after the hydrolysis step. These metabolites were identified as glucuronides of 6β-hydroxyandrosterone (6β-OH-And) and 6β-hydroxyetiocholanolone (6β-OH-Etio) but their exact conjugation site remained undetermined. In this study, the four possible glucuronides of 6β-OH-And and 6β-OH-Etio were synthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy. Moreover, their chromatographic properties and MS spectra were compared to those obtained for the urine samples collected after administration of T. Results confirmed that the recently reported metabolites were the 3α-glucuronides of 6β-OH-And and 6β-OH-Etio. The synthesis and the elucidation of the exact structure of the metabolites presented in this study are crucial steps for the development of analytical methods in order to explore their role in T metabolism and their potential usefulness as biomarkers of T misuse.