Metabolic study of methylstenbolone in horses using liquid chromatography-high resolution mass spectrometry and gas chromatography-mass spectrometry

Studies on the in vivo metabolism of methylstenbolone and detection of novel long term metabolites for doping control analysis

“…Comparing the order of elution and mass spectra, the herein observed metabolites 14 and 18 were assigned to urinary metabolites of methylstenbolone reported by Calvacanti et al Unfortunately, applying a similar approach concerning the numerous metabolites identified in the horse proved considerably more complex as the gas chromatographic conditions differed substantially and the majority of the metabolites was discussed based on liquid chromatography‐mass spectrometry‐derived data . With a distinct loss of 103 Da under EI‐MS conditions, one metabolite (number 5 in Table ) showed similar features to the metabolite M1c tentatively characterized as 20‐hydroxymethylstenbolone by Choi et al After close scrutiny of the corresponding mass spectrum of metabolite 5, however, the structure of 20‐hydroxymethylstenbolone was not confirmed in the present study as depicted in Figure . For both the deuterated and the non‐deuterated metabolite, the characteristic fragment at m/z 143.0886 representing a diagnostic D‐ring fragment composed of carbons C‐15‐17 and C‐20 plus the O‐TMS moiety is still present, which precludes an additional hydroxyl group at C‐20 .…”

“…The accurate mass of m/z 464.3457 corresponds to the elemental composition of C 27 H 48 2 H 2 O 2 Si 2 (mass error = 2.7 ppm) and besides the consecutive losses of a methyl radical (15 u) and the TMSOH (90 u), a characteristic loss of C 3 H 6 2 H (44 u) is observed. In the publication of Choi et al, methasterone is described as one metabolite of methylstenbolone in the equine . An initial comparison of the mass spectra obtained for methasterone and the new long‐term metabolites yielded similar fragments, but the comparison of the methasterone certified reference material and both the metabolites resulted in differing retention times under the described chromatographic conditions.…”

“…In agreement with literature data on human and especially horse metabolism, the majority of the methylstenbolone metabolites was found to be hydroxylated (24 out of 40 metabolites. 3,5 Regarding phase-II-metabolism, 25 metabolites were excreted glucuronidated and only six metabolites were found simultaneously as both glucurono-and sulfo-conjugated analytes.…”

“…Also, additional hydroxylated metabolites identified in in vitro and humanized mouse metabolism studies on methylstenbolone are not directly applicable to sports drug testing . In a recent investigation focusing on the metabolism of methylstenbolone in the horse, several metabolites were described that were detectable for a longer time period than methylstenbolone itself . Based on these results, revisiting the metabolism of methylstenbolone in humans seemed advisable in order to elucidate the discrepancies found between the described and observed urinary metabolites and to facilitate the improved detection of this banned steroid in doping control analysis.…”

Mass Spectrometric Fragmentation of Trimethylsilyl and Related Alkylsilyl Derivatives

“…a) contains a group of ions at m/z 179, 181, 194 (base peak), 195 and 206–208. Their relative abundances vary in corresponding 3‐TMSO‐Δ 1,3 ‐steroids depending on the substituent at C17 (enolized 17‐oxo (Parr et al, ; Schänzer & Donike, ) and in the spectra of various anabolic steroids with methyl groups at C2 or C17 (Choi et al, ) suggesting multiple fragmentation pathways. D 9 ‐TMS labeling showed the presence of an intact TMS group in these ions (Massé et al, ).…”