Biotransformation of 17-alkyl steroids in the equine: high-performance liquid chromatography–mass spectrometric and gas chromatography–mass spectrometric analysis of fluoxymesterone metabolites in urine samples

Detection of stanozolol and its metabolites in equine urine by liquid chromatography?electrospray ionization ion trap mass spectrometry

“…If it is allowed that the hydroxylation site is C16, there are four possible stereochemistries based on variation at C16 and C17. 16␣-Hydroxylation and 17-epimerization have both been reported for stanozolol in humans [1,3], while in the horse they have been variously reported for the analogous anabolic steroids methandrostenolone [15][16][17], 17␣-methyltestosterone [18], fluoxymesterone [19] and norethandrolone [20]. However, given that the accepted method of formation of 17-epimers from 17␣-alkyl-17␤-hydroxysteroids is via spontaneous hydrolysis of the tertiary 17␤-sulfate conjugate in the urine [15], their formation is obviously critically dependant on the phase II metabolism.…”

Modern Techniques for the Determination of Anabolic–Androgenic Steroid Doping in the Horse

“…In equine competition, this may include performance inhibitors as well as enhancers future science group testosterone [8,9], boldenone [10], methandienone [11][12][13], stanozolol [14,15], 17a-methyltestosterone [16][17][18][19], fluoxymesterone [20,21], norethandrolone [22], danazol [23], methenolone [24], turinabol [25], mesterolone [26], methandriol [27] and tetrahydrogestrinone [28]), and the various international AAS regulatory thresholds are also targeted at this matrix. The sample preparation required for the MS based ana lysis of AASs in equine urine is necessarily complex (FiguRe 2).…”

“…Common derivatization procedures include O-methyloximination followed by trimethylsilylation [6-10, 13,[16][17][18]20,22,23], enol-trimethylsilylation [17,21,[24][25][26][27]45,48,[52][53][54][55], oximination followed by tert-butyldimethylsilylation [29] and perfluoroacylation [30,34,41,47]. The oximination/tert-butyldimethylsilylation approach was the first to be adapted for general equine AAS screening and makes use of the strong [M-57] + fragments exhibited by steroidal tert-butyldimethylsilyl ethers under electron ionization (EI) conditions, corresponding to the loss of a single tert-butyl radical.…”

Characterization and Quantification of Fluoxymesterone Metabolite in Horse Urine by Gas Chromatography/Mass Spectrometry

“…Based on these results, the metabolites were presumed to be a C-16 hydroxylated isomer; a 3-keto or 4-ene reduced and C-16 hydroxylated isomer; a 3-keto and 4-ene reduced and C-16 hydroxylated isomer; a C-6,16 dihydroxylated isomer; a 3-keto or 4-ene reduced and C-6,16 dihydroxylated isomer; and a 3-keto and 4-ene reduced and C-6,16 dihydroxylated isomer, and they have been reported as FOM metabolites in horse or humans. [3][4][5][6][7] However, their peaks in the total ion chromatograms were very small compared with those of the metabolites of the unconjugated fraction, particularly F1.…”

“…[3][4][5][6] On the other hand, Stanley et al reported on FOM metabolites in horse urine; metabolites originating from C-6 or C-6, C-16 hydroxylation, and the 17-epimer were confirmed by LC/MS and GC/MS analyses. 7 To develop a suitable doping analysis of FOM in our laboratory, we investigated FOM metabolites in horse urine, and detected metabolites that have not been reported to date. In this study, based on GC/MS data of the metabolites excreted into horse urine after FOM administration, we synthesized the main metabolite and used it as a reference for identification and quantification.…”