The in vivo metabolism of Furazadrol in greyhounds

Abstract

Samples of the ‘dietary supplement’ Furazadrol sourced through the internet have been reported to contain the designer anabolic androgenic steroids [1′,2′]isoxazolo[4′,5′:2,3]‐5α‐androstan‐17β‐ol (furazadrol F) and [1′,2′]isoxazolo[4′,3′:2,3]‐5α‐androstan‐17β‐ol (isofurazadrol IF). These steroids contain an isoxazole fused to the A‐ring and were designed to offer anabolic activity while evading detection, raising concerns over the potential for abuse of this preparation in sports. The metabolism of Furazadrol (F:IF, 10:1) was studied by in vivo methods in greyhounds. Urinary phase II Furazadrol metabolites were detected as glucuronides after a controlled administration. These phase II metabolites were subjected to enzymatic hydrolysis by Escherichia coli β‐glucuronidase to afford the corresponding phase I metabolites. Using a library of synthetically derived reference materials, the identities of seven urinary Furazadrol metabolites were confirmed. Major confirmed metabolites were isofurazadrol IF, 4α‐hydroxyfurazadrol 4α‐HF and 16α‐hydroxy oxidised furazadrol 16α‐HOF, whereas the minor confirmed metabolites were furazadrol F, 4β‐hydroxyfurazadrol 4β‐HF, 16β‐hydroxyfurazadrol 16β‐HF and 16β‐hydroxy oxidised furazadrol 16β‐HOF. One major hydroxyfurazadrol and two dihydroxyfurazadrol metabolites remained unidentified. Qualitative excretion profiles, limits of detection and extraction recoveries were established for furazadrol F and major confirmed metabolites. These investigations identify the key urinary metabolites of Furazadrol following oral administration, which can be incorporated into routine screening by anti‐doping laboratories to aid the regulation of greyhound racing.

Sample Preparation Techniques for Growth-Promoting Agents in Various Mammalian Specimen Preceding MS-Analytics

“…Pranata et al [ 56 ] analyzed unfractioned dog urine by mixing it with phosphate buffer (pH 7.4) and extracting the mixture using mixed-mode weak anion exchange (WAX) cartridges. The eluate was dried and reconstituted in aqueous FA (0.1%)/MeOH (95/5, v/v ).…”

“…The eluate was dried, reconstructed, and subjected to LC-MS analysis. The LODs were determined for the routine analysis [ 56 ]. Similarly, Jungle Warfare (Δ6-methyltestosterone) was extracted using three different protocols.…”