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 17 alpha-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 17 alpha-methyltestosterone to two castrated thoroughbred male horses, the glucuronic acid conjugates excreted in post-administration urine samples were selectively hydrolysed by E. coli beta-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 17 alpha-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 17 alpha-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 17 alpha-methyltestosterone excreted in thoroughbred horse urine.

Detection and Characterization of a New Metabolite of 17α-Methyltestosterone

“…It represented less than 0.2% of the total excretion for this steroid. In addition, this epimerization has also been found in the horse (Dumasia, 2003). Abundances observed in the spectrum of A4 are in agreement with those reported for 17-epimethyltestosterone (Schänzer et al, 1992).…”

“…Two main metabolites were identified in the metabolism of 17␣-methyltestosterone in human: 17␣-methyl-5␣-androstane-3␣,17␤-diol (A2) and 17␣-methyl-5␤-androstane-3␣,17␤-diol (A3) (Schänzer and Donike, 1993). Hydroxylated metabolites have been described in several animals including the horse (Dumasia, 2003;McKinney et al, 2007;Yamada et al, 2007) and the heifer (Blokland et al, 2005).In doping control analysis, the detection of steroid metabolites is normally performed by gas chromatography-mass spectrometry (GC-MS) after trimethylsilylation (Schänzer and Donike, 1993;Ayotte et al, 1996;Saugy et al, 2000), achieving the required sensitivity of 2 ng/ml (WADA Technical Document TD2004MRPL version 1.0, 2007, http://www.wada-ama.org/rtecontent/document/perf_limits_2. pdf).…”

“…Two main metabolites were identified in the metabolism of 17␣-methyltestosterone in human: 17␣-methyl-5␣-androstane-3␣,17␤-diol (A2) and 17␣-methyl-5␤-androstane-3␣,17␤-diol (A3) (Schänzer and Donike, 1993). Hydroxylated metabolites have been described in several animals including the horse (Dumasia, 2003;McKinney et al, 2007;Yamada et al, 2007) and the heifer (Blokland et al, 2005).…”

Identification and quantification of metabolites common to 17α-methyltestosterone and mestanolone in horse urine

“…Studies have shown that structurally related MTS and MSL yield metabolites in common in human [1,2]. There are also reports of the urinary metabolites of MTS in horse; Stanley et al [3] and Dumasia [4] used GC/MS to identify 17-hydroxymethyl metabolites, and McKinney et al examined the stereochemistry of urinary metabolites by comparing with authentic reference standards [5]. The main steps in the metabolism of MTS in horses are as follows: reduction of A-ring 3-oxo and 4-ene groups; oxidation at C-6, C-15, and C-16; and epimerization at C-17 during phase I biotransformation, followed by sulfate and glucuronide conjugation during phase II biotransformation [3,4].…”

In vitro and in vivo studies of androst‐4‐ene‐3,6,17‐trione in horses by gas chromatography–mass spectrometry

“…There was little information on the biotransformation of oral anabolic steroids in horses, although this class of steroids is known as potential drugs of abuse in human sports due to their ease of administration and normally shorter excretion times. It was not until the late 1990s that racing chemists started to direct more eff ort to studies of these substances, including ethylestrenol (Kim et al, 1996), oxymetholone and mestanolone (Tang et al, 2000), danazol (Tang et al, 2001), methandrostenolone (McKinney et al, 2001a), norethandrolone (McKinney et al, 2001b), normethandrone (Fox et al, 2001), 17a-methyltestosterone (Dumasia, 2003), 1-testosterone (Kwok et al, 2004), clostebol acetate and mesterolone , 7-keto-dehydroepiandrosterone acetate , methenolone acetate and turinabol (Ho et al, 2007).…”