Andrew R. McKinney

Metabolism of methandrostenolone in the horse: a gas chromatographic-mass spectrometric investigation of phase I and phase II metabolism

The phase I and phase II metabolism of the anabolic steroid methandrostenolone was investigated following oral administration to a standardbred gelding. In the phase I study, metabolites were isolated from the urine by solid-phase extraction, deconjugated by acid catalysed methanolysis and converted to their O-methyloxime trimethylsilyl derivatives. GC-MS analysis indicated the major metabolic processes to be sequential reduction of the A-ring and hydroxylation at C6 and C16. In the phase II study, unconjugated, beta-glucuronidated and sulfated metabolites were fractionated and deconjugated using a combination of liquid-liquid extraction, enzyme hydrolysis, solid-phase extraction and acid catalysed methanolysis. Derivatization followed by GC-MS analysis revealed extensive conjugation to both glucuronic and sulfuric acids, with only a small proportion of metabolites occurring in unconjugated form.

Equine Metabolites of Norethandrolone: Synthesis of a Series of 19-Nor-17α-pregnanediols and 19-Nor-17α-pregnanetriols

A range of 19-nor-17α-pregnanediols and 19-nor-17α-pregnanetriols have been synthesized and used to confirm the structures of major equine urinary metabolites of the synthetic anabolic steroid norethandrolone (1). 19-Nor-5α,17α-pregnane-3α,17β-diol (2), 19-nor-5α,17α-pregnane-3β,17β-diol (4), 19-nor-5β,17α-pregnane-3α,17β-diol (6), and 19-nor-5β,17α-pregnane-3β,17β-diol (7) were prepared by stereoselective reduction of the 3-ene-4-one of norethandrolone. The 19-nor-5α,17α-pregnane-3β,16α,17β-triol (8) and 19-nor-5α,17α-pregnane-3β,16β,17β-triol (9) were prepared from 19-nortestosterone (11) by multistep processes in which the critical step involved Grignard additions to 16-acetoxy-17-ones. The triols (20R)-19-nor-5α,17α-pregnane-3β,17β,20-triol (22) and (20S)-19-nor-5α,17α-pregnane-3β,17β,20-triol (23) were prepared from norethindrone (24) by initial selective A-ring reduction, then subsequent modification of the 17-ethynyl group. By comparison of these compounds with post-administration equine urine samples it was possible to establish A-ring reduction with 3β,5α stereochemistry as well as non-stereospecific 16-hydroxylation and 20-hydroxylation as significant metabolic pathways affecting norethandrolone in the horse.

The metabolism of anabolic-androgenic steroids in the greyhound.

BACKGROUND Effective control of the use of anabolic-androgenic steroids (AASs) in animal sports is essential in order to ensure both animal welfare and integrity. In order to better police their use in Australian and New Zealand greyhound racing, thorough metabolic studies have been carried out on a range of registered human and veterinary AASs available in the region. RESULTS Canine metabolic data are presented for the AASs boldenone, danazol, ethylestrenol, mesterolone, methandriol, nandrolone and norethandrolone. The principal Phase I metabolic processes observed were the reduction of A-ring unsaturations and/or 3-ketones with either 3α,5β- or 3β,5α-stereochemistry, the oxidation of secondary 17β-hydroxyl groups and 16α-hydroxylation. The Phase II β-glucuronylation of sterol metabolites was extensive. CONCLUSION The presented data have enabled the effective analysis of AASs and their metabolites in competition greyhound urine samples.

Acepromazine pharmacokinetics: a forensic perspective.

Acepromazine (ACP) is a useful therapeutic drug, but is a prohibited substance in competition horses. The illicit use of ACP is difficult to detect due to its rapid metabolism, so this study investigated the ACP metabolite 2-(1-hydroxyethyl)promazine sulphoxide (HEPS) as a potential forensic marker. Acepromazine maleate, equivalent to 30mg of ACP, was given IV to 12 racing-bred geldings. Blood and urine were collected for 7days post-administration and analysed for ACP and HEPS by liquid chromatography-mass spectrometry (LC-MS). Acepromazine was quantifiable in plasma for up to 3h with little reaching the urine unmodified. Similar to previous studies, there was wide variation in the distribution and metabolism of ACP. The metabolite HEPS was quantifiable for up to 24h in plasma and 144h in urine. The metabolism of ACP to HEPS was fast and erratic, so the early phase of the HEPS emergence could not be modelled directly, but was assumed to be similar to the rate of disappearance of ACP. However, the relationship between peak plasma HEPS and the y-intercept of the kinetic model was strong (P=0.001, r(2)=0.72), allowing accurate determination of the formation pharmacokinetics of HEPS. Due to its rapid metabolism, testing of forensic samples for the parent drug is redundant with IV administration. The relatively long half-life of HEPS and its stable behaviour beyond the initial phase make it a valuable indicator of ACP use, and by determining the urine-to-plasma concentration ratios for HEPS, the approximate dose of ACP administration may be estimated.

Metabolism of stanozolol: chemical synthesis and identification of a major canine urinary metabolite by liquid chromatography-electrospray ionisation ion trap mass spectrometry.

The canine phase I and phase II metabolism of the synthetic anabolic-androgenic steroid stanozolol was investigated following intramuscular injection into a male greyhound. The major phase I biotransformation was hydroxylation to give 6alpha-hydroxystanozolol which was excreted as a glucuronide conjugate and was identified by comparison with synthetically derived reference materials. An analytical procedure was developed for the detection of this stanozolol metabolite in canine urine using solid phase extraction, enzyme hydrolysis of glucuronide conjugates and analysis by positive ion electrospray ionisation ion trap LC-MS.

Synthesis of Equine Metabolites of Anabolic Steroids: Reformatsky Reactions on Estran-17-ones

Reformatsky reactions involving ethyl bromoacetate/zinc are reported for 19-norandrosterone acetate and 19-norepiandrosterone acetate. In each case the major product was the 17β-alcohol from α-attack, although a significant amount of the 17α-alcohol from β-attack was also isolated. The ethyl 3-acetoxy-17β-hydroxy-19-nor-5α,17α-pregnan-21-oates were then hydrolysed to 3,17β-dihydroxy-19-nor-5α,17α-pregnan-21-oic acids or reduced to 19-nor-5α,17α-pregnane-3,17β,21-triols. Comparison of the synthetic products with compounds previously reported as metabolites of norethandrolone in the horse provided valuable information on the regio- and stereo-chemistry of equine steroid metabolism.

A Bayesian approach for estimating detection times in horses: exploring the pharmacokinetics of a urinary acepromazine metabolite

We describe the population pharmacokinetics of an acepromazine (ACP) metabolite (2-(1-hydroxyethyl)promazine) (HEPS) in horses for the estimation of likely detection times in plasma and urine. ACP (30 mg) was administered to 12 horses, and blood and urine samples were taken at frequent intervals for chemical analysis. A bayesian hierarchical model was fitted to describe concentration-time data and cumulative urine amounts for HEPS. The metabolite HEPS was modelled separately from the parent ACP as the half-life of the parent was considerably less than that of the metabolite. The clearance (Cl/F(PM)) and volume of distribution (V/F(PM)), scaled by the fraction of parent converted to metabolite, were estimated as 769 L/h and 6874 L, respectively. For a typical horse in the study, after receiving 30 mg of ACP, the upper limit of the detection time was 35 h in plasma and 100 h in urine, assuming an arbitrary limit of detection of 1 lg/L and a small (≈0.01) probability of detection. The model derived allowed the probability of detection to be estimated at the population level. This analysis was conducted on data collected from only 12 horses, but we assume that this is representative of the wider population.