In vivo and in vitro metabolism of the designer anabolic steroid furazadrol in thoroughbred racehorses

•In vivo and in vitro equine metabolic profile of the designer steroid furazadrol.•Phase I and phase II in vivo urinary metabolites characterised and reported.•Major metabolites quantified to establish excretion profile.•Translation to locally implemented routine screening and confirmation protocols.•Major metabolites matched against characterised reference materials.

Furazadrol ([1′,2′]isoxazolo[4′,5′:2,3]-5α-androstan-17β-ol) is a designer anabolic androgenic steroid that is readily available via the internet. It contains an isoxazole fused to the steroid A-ring which offers metabolic stability and noteworthy anabolic activity raising concerns over the potential for abuse of this compound in equine sports. The metabolism of furazadrol was studied by in vivo and in vitro methods for the first time. Urinary furazadrol 17-sulfate and furazadrol 17-glucuronide metabolites were detected in vivo after a controlled administration and compared with synthetically-derived reference materials in order to confirm their identities. They were quantified to establish the excretion profile and a suitable limit of detection. Minor metabolites were also detected, including epifurazadrol, hydroxylated furazadrol, and hydroxylated and oxidised furazadrol, present as the sulfate and glucuronide conjugates. Phase II metabolites were subjected to enzymatic hydrolysis by Escherichia coli β-glucuronidase and Pseudomonas aeruginosa arylsulfatase to further confirm the identity of the corresponding phase I metabolites. The metabolism profile was compared to the products obtained from an in vitro phase I metabolism study, with all but two of the minor in vivo phase I metabolites observed in the in vitro system. These investigations identify the key urinary metabolites of furazadrol following oral administration, which can be incorporated into anti-doping screening and confirmation procedures.

Graphical abstractThe in vivo metabolism of the designer steroid furazadrol identifies the key urinary metabolites following oral administration, which can be incorporated into anti-doping screening and confirmation procedures.Figure optionsDownload full-size imageDownload as PowerPoint slide