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In vivo biotransformation of 17α‐methyltestosterone in the horse revisited: identification of 17‐hydroxymethyl metabolites in equine urine by capillary gas chromatography/mass spectrometry
Author(s) -
Dumasia M. C.
Publication year - 2003
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.909
Subject(s) - chemistry , chromatography , glucuronic acid , glucuronide , mass spectrometry , gas chromatography–mass spectrometry , in vivo , methyltestosterone , urine , gas chromatography , biotransformation , ether , tandem mass spectrometry , zeranol , enzyme , biochemistry , organic chemistry , polysaccharide , medicine , microbiology and biotechnology , biology
The in vivo phase I biotransformation of 17α‐methyltestosterone in the horse leads to the formation of a complex mixture of regio‐ and stereoisomeric C 20 O 2 , C 20 O 3 and C 20 O 4 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 C 20 O 3 and C 20 O 4 metabolites of 17α‐methyltestosterone excreted in thoroughbred horse urine. Copyright © 2003 John Wiley & Sons, Ltd.