New potential biomarkers for mesterolone misuse in human urine by liquid chromatography quadrupole time-of-flight mass spectrometry

Abstract

In this paper, mesterolone metabolic profiles were investigated carefully. Mesterolone was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry (LC-QTOFMS) for the first time. Liquid-liquid extraction was applied to processing urine samples, and dilute-shoot analyses of intact metabolites were also presented. In LC-QTOFMS analysis, chromatographic peaks for potential metabolites were hunt down by using the theoretical [M-H](-) as target ions in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Ten metabolites including seven new sulfate and three glucuronide conjugates were found for mesterolone. Because of no useful fragment ion for structural elucidation, gas chromatography-mass spectrometry instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after solvolysis. Thus, their potential structures were proposed particularly by a combined MS approach. All the metabolites were also evaluated in terms of how long they could be detected, and S1 (1α-methyl-5α-androst-3-one-17β-sulfate) together with S2 (1α-methyl-5α-androst-17-one-3β-sulfate) was detected up to 9 days after oral administration, which could be the new potential biomarkers for mesterolone misuse.

Markers of mesterolone abuse in sulfate fraction for doping control in human urine

“…According to the data of the Table it can be assumed that M1 is the 3β‐epimer of M2, namely 1α‐methyl‐5α androstan‐3β ol‐17one. This metabolite has also been identified by Lu et al . Further evidence for this structural assignment can be drawn by elution order in LC and GC analysis which gives structural evidence as described by Schanzer et al .…”

“…a) is similar to that of 1α‐methyl‐5α‐androstan‐3α‐ol‐17one diTMS derivative and differs significantly from that of TMS,enolTMS of parent mesterolone (Fig. c) that has been proposed as candidate structure for this metabolite . 17 Epimer of mesterolone is expected to show similar fragmentation pattern and hence can also be excluded as possible structure for this metabolite.…”

Development of a multi‐functional concurrent assay using weak cation‐exchange solid‐phase extraction (WCX‐SPE) and reconstitution with a diluted sample aliquot for anti‐doping analysis

“…Meldonium is known to reduce lactic acid levels, thus enhancing both the energy storage and the endurance of elite athletes . Furthermore, to evaluate the exact concentration of drugs in urine, the conjugated metabolites, such as glucuronides, sulfates, or cysteinyl conjugates, have to be de‐conjugated in order to use the multiclass doping analysis method …”

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.…”