Searching for new long‐term urinary metabolites of metenolone and drostanolone using gas chromatography–mass spectrometry with a focus on non‐hydrolysed sulfates

Abstract

Sulfated metabolites have been shown to have potential as long‐term markers of anabolic–androgenic steroid (AAS) abuse. In 2019, the compatibility of gas chromatography–mass spectrometry (GC–MS) with non‐hydrolysed sulfated steroids was demonstrated, and this approach allowed the incorporation of these compounds in a broad GC–MS initial testing procedure at a later stage. However, research is needed to identify which are beneficial. In this study, a search for new long‐term metabolites of two popular AAS, metenolone and drostanolone, was undertaken through two excretion studies each. The excretion samples were analysed using GC–chemical ionization–triple quadrupole MS (GC–CI–MS/MS) after the application of three separate sample preparation methodologies (i.e. hydrolysis with Escherichia coli–derived β‐glucuronidase, Helix pomatia–derived β‐glucuronidase/arylsulfatase and non‐hydrolysed sulfated steroids). For metenolone, a non‐hydrolysed sulfated metabolite, 1β‐methyl‐5α‐androstan‐17‐one‐3ζ‐sulfate, was documented for the first time to provide the longest detection time of up to 17 days. This metabolite increased the detection time by nearly a factor of 2 in comparison with the currently monitored markers for metenolone in a routine doping control initial testing procedure. In the second excretion study, it prolonged the detection window by 25%. In the case of drostanolone, the non‐hydrolysed sulfated metabolite with the longest detection time was the sulfated analogue of the main drostanolone metabolite (3α‐hydroxy‐2α‐methyl‐5α‐androstan‐17‐one) with a detection time of up to 24 days. However, the currently monitored main drostanolone metabolite in routine doping control, after hydrolysis of the glucuronide with E.coli, remained superior in detection time (i.e. up to 29 days).

Detection of testosterone microdosing in healthy females

“…Androgen doping originally involved using marketed synthetic androgens that are now readily detectable by highly sensitive urine mass spectrometry methods to identify distinctive xenobiotic chemical signatures 4,5 as well as long‐term metabolites such as sulfate conjugates with longer detection windows 6–9 . Consequently, deterrence from using marketed synthetic androgens has led to alternative approaches aiming to continue exploiting the ergogenic advantages of androgen doping surreptitiously.…”

Olympic Anti-Doping Laboratory: The Analytical Technological Road From 2016 Rio De Janeiro to 2021 Tokyo

“…The doping control laboratory in Ghent analyzed steroids' nonhydrolyzed sulfate metabolites using GC-MS/MS instead of LC-MS/MS to detect the metabolites' artifacts [153]. Another study by this laboratory [154] developed a method for identification of new sulfo-conjugated metabolites for methenolone and drostanolone using GC-MS/MS. The results showed the identification of nonhydrolyzed sulfate metabolite of methenolone detected with longer detection window up to 17 days compared with hydrolyzed metabolites.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…By means of an elimination study with 25 mg of orally administered mesterolone, it was shown that the intake of a single dose was detected up to 22 days in urine samples 63 . Similarly, Albertsdottir et al assessed the potential of targeting intact sulfo‐conjugates of AAS metabolites of metenolone and drostanolone by GC‐MS/MS in routine doping controls, 64 which led to the identification of five sulfo‐conjugated metabolites of each of the two AAS. 1β‐Methyl‐5α‐androstan‐17‐one‐3‐sulfate offered a substantially longer detection window than previously reported and employed ITPs as demonstrated with elimination study urine samples collected after the oral administration of 50 mg of metenolone acetate.…”