Detection and quantification of glucuro- and sulfoconjugated metabolites in human urine following oral administration of xenobiotic 19-norsteroids

Recently, the endogenous origin of nandrolone (19-nortestosterone) and other 19-norsteroids has been a focus of research in the field of drug testing in sport. In the present study, we investigated metabolites conjugated to a glucuronic acid and to a sulfuric acid in urine following administration of four xenobiotic 19-norsteroids. Adult male volunteers administered a single oral dose (10 mg) of each of four 19-norsteroids. Urinary samples collected from 0 to 120 h were subjected to methanolysis and β-glucuronidase hydrolysis and were derivatized by N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) before gas chromatography–mass spectrometry analysis. We confirmed that 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) were present in both glucuronide (g) and sulfate (s) conjugates and 19-norepiandrosterone (19-NEA) was excreted exclusively as a sulfate fraction in urine of all 19-norsteroids tested. The overall levels of the three metabolites can be ranked as follows: 19-NAg+s > 19-NEg+s > 19-NEAs. The concentration profiles of these three metabolites in urine peaked between 2 to 12 h post-administration and declined thereafter until approximately 72–96 h. 19-NA was most prominent throughout the first 24 h post-administration, except for a case in which an inverse relationship was found after 6 h post-administration of nandrolone. Furthermore, we found that sulfate conjugates were present in both 19-NA and 19-NE metabolites in urine of all 19-norsteroids tested. The averaged total amounts of metabolites (i.e. 19-NAs+g + 19-NEs+g + 19-NEAs) excreted in urine were 38.6, 42.9, 48.3 and 21.6% for nandrolone, 19-nor-4-androsten-3,17-dione, 19-nor-4-androsten-3β,17β-diol and 19-nor-5-androstene-3β,17β-diol, respectively. Results from the excretion studies demonstrate significance of sulfate-conjugated metabolites on interpretation of misuse of the 19-norsteroids.