Metabolism of methylstenbolone studied with human liver microsomes and the uPA^+/+‐SCID chimeric mouse model

Abstract

Anti-doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono-hydroxylated derivatives (U1-U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di-hydroxylated metabolites (U11-U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC-MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method.

New Potential Biomarker for Methasterone Misuse in Human Urine by Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry

“…Human liver microsomes and an uPA +/+ -SCID chimeric mouse model were employed to produce its different metabolites, and the reduction in C3 together with hydroxylation in C2, C12 (minor), C16 and C20 constitute the main metabolism pathways [10,11,12]. Methasterone and its hydroxylated metabolites were also excreted as glucuronidated compounds, while no sulfated metabolites could be detected in phase II metabolism [13].…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…Exploiting human liver microsomal in vitro approaches and a mouse model exhibiting humanized liver properties, the metabolic fate of methylstenbolone was elucidated using LC‐MS/MS and GC‐MS/MS . Most of the 13 observed metabolites represented monohydroxylated derivatives and resulted only from in vitro incubations.…”

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

“…These results, however, did not reflect recent findings in doping control samples in which MSTEN glucuronide but not the reported hydroxylated metabolites were detected. 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 .…”