In vivo biotransformation of 17α‐methyltestosterone in the horse revisited: identification of 17‐hydroxymethyl metabolites in equine urine by capillary gas chromatography/mass spectrometry

Abstract

The in vivo phase I biotransformation of 17α-methyltestosterone in the horse leads to the formation of a complex mixture of regio- and stereoisomeric C₂₀O₂, C₂₀O₃ and C₂₀O₄ metabolites, excreted in urine as glucuronide and sulphate phase II conjugates. The major pathways of in vivo metabolism are the reduction of the A-ring (di- and tetrahydro), epimerisation at C-17 and oxidations mainly at C-6 and C-16. Some phase I metabolites have been identified previously by positive ion electron ionisation capillary gas chromatography/mass spectrometry (GC/EI + MS) mainly from the characteristic fragmentation patterns of their methyloxime-trimethylsilyl ether (MO-TMS), enol-TMS or TMS ether derivatives. Following oral administration of 17α-methyltestosterone to two castrated thoroughbred male horses, the glucuronic acid conjugates excreted in post-administration urine samples were selectively hydrolysed by E. coli β-glucuronidase enzymes. Unconjugated metabolites and the steroid aglycones obtained after enzymatic deconjugation were isolated from urine by solid-phase extraction, derivatised as MO-TMS ethers and analysed by GC/EI + MS. In addition to some of the known metabolites previously identified from the characteristic mass spectral fragmentation patterns of 17α-methyl steroids, some isobaric compounds exhibiting a diagnostic loss of 103 mass units from the molecular ions with subsequent losses of trimethylsilanol or methoxy groups and an absence of the classical D-ring fragment ion were detected. From an interpretation of their mass spectra, these compounds were identified as 17-hydroxymethyl metabolites, formed in vivo in the horse by oxidation of the 17-methyl moiety of 17α-methyltestosterone. This study reports on the GC/EI + MS identification of these novel 17-hydroxymethyl C₂₀O₃ and C₂₀O₄ metabolites of 17α-methyltestosterone excreted in thoroughbred horse urine. Copyright © 2003 John Wiley & Sons, Ltd.