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

Abstract

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.

Screening for anabolic steroids in sports: Analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem mass spectrometry

“…The synthesis of the glucuronides was based on a previously described method [32,33]. Briefly, in 0.5 mL of toluene, 100 g (0.36 mol, 1 equivalent) of either 17␣-methyl-5␣-androstan-3␣,17␤-diol or 17␣-methyl-5␤-androstan-3␣,17␤-diol were added followed by 142 g (0.36 mol, 1 equivalent) of acetobromo-␣-d-glucuronic acid methyl ester and 500 g of Ag 2 CO 3 .…”

LC‐MS/MS detection of unaltered glucuronoconjugated metabolites of metandienone

“…[27] Briefly, 40 μg of epimetendiol (0.13 μmol, 1 equivalent) or 40 μg of 18-nor-dimethyl (0.14 μmol, 1 equivalent) were dissolved in 0.5 mL of toluene. [27] Briefly, 40 μg of epimetendiol (0.13 μmol, 1 equivalent) or 40 μg of 18-nor-dimethyl (0.14 μmol, 1 equivalent) were dissolved in 0.5 mL of toluene.…”

Ultra high performance liquid chromatography tandem mass spectrometric detection of glucuronides resistant to enzymatic hydrolysis: Implications to doping control analysis

“…3a-glucuronide-6b-hydroxyandrosterone (6OH-Andros-3-Gluc), 3a-glucuronide-6b-hydroxyetiocholanolone (6OH-Etio-3-Gluc) and 6b-glucuronide-3a-hydroxyandrosterone (6OH-Andros-6-Gluc, used as internal standard, ISTD) were synthesized as previously described [11]. T, E, Andros, Etio, T-Gluc, E-Gluc, 5a-androstan-3a,17b-diol-3a-glucuronide, 5b-androstan-3a,17b-diol3a-glucuronide, 5a-androstan-3a,17b-diol-17a-glucuronide, 5b-androstan-3a,17b-diol-17a-glucuronide, d 3 -testosterone (d 3 -T), d 3 -epitestosterone (d 3 -E), d 5 -etiocholanolone (d 5 -Etio), d 3 -testosterone glucuronide (d 3 -T-Gluc) and d 3 -epitestosterone glucuronide (d 3 -E-Gluc) were purchased from the Australian National Measurement Institute (Pymble, Australia).…”

“…Recently, the application of a UHPLCeMS/MS method revealed the presence of two glucuronides, 3a-glucuronide-6b-hydroxyandrosterone (6OH-Andros-3-Gluc) and the 3a-glucuronide-6b-hydroxyetiocholanolone (6OH-Etio-3-Gluc), which remain conjugated after enzymatic hydrolysis [10,11]. After a qualitative determination, the urinary levels of these compounds were shown to increase their concentrations around 300 fold after an oral administration of T undecanoate.…”