Comparison of sulfo‐conjugated and gluco‐conjugated urinary metabolites for detection of methenolone misuse in doping control by LC‐HRMS, GC‐MS and GC‐HRMS

Abstract

Methenolone (17β-hydroxy-1-methyl-5α-androst-1-en-3-one) misuse in doping control is commonly detected by monitoring the parent molecule and its metabolite (1-methylene-5α-androstan-3α-ol-17-one) excreted conjugated with glucuronic acid using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for the parent molecule, after hydrolysis with β-glucuronidase. The aim of the present study was the evaluation of the sulfate fraction of methenolone metabolism by LC-high resolution (HR)MS and the estimation of the long-term detectability of its sulfate metabolites analyzed by liquid chromatography tandem mass spectrometry (LC-HRMSMS) compared with the current practice for the detection of methenolone misuse used by the anti-doping laboratories. Methenolone was administered to two healthy male volunteers, and urine samples were collected up to 12 and 26 days, respectively. Ethyl acetate extraction at weak alkaline pH was performed and then the sulfate conjugates were analyzed by LC-HRMS using electrospray ionization in negative mode searching for [M-H](-) ions corresponding to potential sulfate structures (comprising structure alterations such as hydroxylations, oxidations, reductions and combinations of them). Eight sulfate metabolites were finally detected, but four of them were considered important as the most abundant and long term detectable. LC clean up followed by solvolysis and GC/MS analysis of trimethylsilylated (TMS) derivatives reveal that the sulfate analogs of methenolone as well as of 1-methylene-5α-androstan-3α-ol-17-one, 3z-hydroxy-1β-methyl-5α-androstan-17-one and 16β-hydroxy-1-methyl-5α-androst-1-ene-3,17-dione were the major metabolites in the sulfate fraction. The results of the present study also document for the first time the methenolone sulfate as well as the 3z-hydroxy-1β-methyl-5α-androstan-17-one sulfate as metabolites of methenolone in human urine. The time window for the detectability of methenolone sulfate metabolites by LC-HRMS is comparable with that of their hydrolyzed glucuronide analogs analyzed by GC-MS. The results of the study demonstrate the importance of sulfation as a phase II metabolic pathway for methenolone metabolism, proposing four metabolites as significant components of the sulfate fraction.

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

“…[2,[4][5][6] Glucuronoconjugates have been detected after enzymatic hydrolysis with β-glucuronidase and, therefore, only conjugates hydrolyzable in the conditions used have been systematically studied. [8][9][10][11][12][13][14][15] Glucuronoconjugated metabolites of stanozolol and testosterone which are poorly or not hydrolyzed using β-glucuronidase enzymes have been detected using LC-MS/ MS strategies. [7] LC-MS allows for the direct detection of phase II metabolites and its application has resulted in the identification of previously unreported conjugated metabolites, such as glucuronides, sulfates, or cysteinyl conjugates.…”

Doping control container for urine stabilization: a pilot study

“…For the evaluation of endogenous steroid profile, as well as for the screening of prohibited substances of classes S1, S4, S5 and S8, the Agilent GC/MS system of section Incubation experiments with microorganisms was used. For the screening of compounds from S1, S4, S5, S6 and S8, detected as TMS derivatives, the GC/high resolution mass spectrometry (GC/HRMS) analysis was performed on a Waters Micromass Auto Spec Ultima (Manchester, UK) coupled with an Agilent 6890 GC as described in …”

Sulfate metabolites as alternative markers for the detection of 4‐chlorometandienone misuse in doping control

“…Conventional approaches have focused on the detection of the most abundant metabolites after hydrolysis with β-glucuronidase enzymes to release the phase I metabolite and analysis by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). [8][9][10][11][12][13][14][15][16][17][18][19] For some steroids, sulfate metabolites really improved the retrospectivity of the detection. [8][9][10][11][12][13][14][15][16][17][18][19] For some steroids, sulfate metabolites really improved the retrospectivity of the detection.…”