Biotransformation of 1-dehydrotestosterone in the equine male castrate: Identification of the neutral unconjugated and glucuronic acid conjugated metabolites in horse urine

Abstract

The in vivo biotransformation of (1,2(n)-3H)1-dehydrotestosterone was studied in three equine male castrates and a number of neutral metabolites were identified in the urinary unconjugated and glucuronic acid conjugate fractions by gas chromatography/mass spectrometry. The metabolites were extracted from aliquots of the 0-24 h urine samples by Amberlite XAD-2 and separated into combined unconjugated plus glucuronic acid conjugated and sulphoconjugated fractions by Sephadex LH-20 column chromatography. After enzymatic hydrolysis of the glucuronides, the crude neutral unconjugated steroids plus the aglycones were partially purified by Kieselgel H chromatography and identified as their methyloxime trimethylsilyl derivatives. In the unconjugated fraction, the major metabolites were isomers of androsta-1,4-diene-6,16,17-triol-3-one. In the aglycone fraction a small amount of the parent steroid was present but the major metabolite was the 17 alpha isomer androsta-1,4-dien-17 alpha-ol-3-one. Other metabolites containing the 1,4-dien-3-one group were isomers of androsta-1,4-diene-16,17-diol-3-one and androsta-1,4-diene-6,16-diol-3-one. Reduction of the 4-ene functionality leading to the formation of 5-androst-1-en-16-ol-3,17-dione, 5-androst-1-ene-16,17-diol-3-one and of the 1-ene functionality leading to the formation of testosterone and its further reduction leading to the formation of C19O2 and C19O3 androstane metabolites was observed. Some interesting features on the electron impact fragmentations of the methyloxime trimethylsilyl derivatives of steroids containing a 1,4-dien-3-one group were also observed.

Identification and characterization of novel long‐term metabolites of oxymesterone and mesterolone in human urine by application of selected reaction monitoring GC‐CI‐MS/MS

“…Previous research pointed this out for 19‐nortestosterone sulfate . Similar results were found for testosterone sulfate and boldenone sulfate . This means that if larger amounts of mestM22 can be hydrolysed to its free form, the detection time can be drastically increased.…”

Confirmatory analysis of 17β-boldenone, 17α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography–electrospray ionisation tandem mass spectrometry

“…Since then ␣-Bol is considered as a naturally occurring steroid in cattle and findings of only the ␣-Bol metabolite do not provide sufficient evidence for illegal treatment. The excretion of boldenone metabolites has been studied in human-and equine urine, and also in bovine urine and faeces [5][6][7]. In the latter study microsomes and hepatocytes from liver tissue were used to investigate metabolic pathways of boldenone in cattle in vitro and, combined with data from in-vivo findings, used in order to identify new metabolites in urine and faeces [7].…”

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

“…The in vivo biotransformation and detection of anabolic steroids in the horse have been the subject of a number of studies from this laboratory1–11and from other laboratories 12–15. Following enzymatic hydrolysis of the glucuronides and chemical solvolysis of the sulfoconjugates,1,4,8 the phase I aglycones are isolated from urine by liquid‐liquid or solid‐phase extraction (SPE). The complex mixtures are then purified and derivatised mainly as MO‐TMS or TMS ethers and the metabolites identified by GC/EI + MS on the basis of the molecular weights and characteristic fragmentation patterns of the derivatised steroids.…”

“…In the horse, the main phase I biotransformation pathways of the C 18 O 2 and C 19 O 2 injectable anabolic steroids are sequential reduction of the A‐ring, oxidation at C‐6 and C‐16 and epimerisation of the 17β‐ to the 17α‐hydroxyl group, resulting in the formation of a number of isomeric C‐18 and C‐19 O 2 , O 3 and O 4 metabolites. Metabolites retaining the original 17β‐hydroxy stereochemistry are excreted in equine urine mainly as sulphate conjugates while their 17α‐hydroxy isomers are primarily excreted as glucuronides 1,2,4,8. The stereochemistry of the isomers of the reduced and oxidised phase I metabolites can only be fully characterised by comparison with authentic compounds where available.…”