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Cawley A.T.,Australian Racing Forensic Laboratory | Blakey K.,Queensland Government | Waller C.C.,Australian National University | McLeod M.D.,Australian National University | And 6 more authors.
Drug Testing and Analysis | Year: 2016

In 2012, seized capsules containing white powder were analyzed to show the presence of unknown steroid-related compounds. Subsequent gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) investigations identified a mixture of 3α- and 3β- isomers of the novel compound; 3-chloro-17α-methyl-5α-androstan-17β-ol. Synthesis of authentic reference materials followed by comparison of NMR, GC-MS and gas chromatography-tandem mass spectrometry (GC-MS/MS) data confirmed the finding of a new ‘designer’ steroid. Furthermore, in vitro androgen bioassays showed potent activity highlighting the potential for doping using this steroid. Due to the potential toxicity of the halogenated steroid, in vitro metabolic investigations of 3α-chloro-17α-methyl-5α-androstan-17β-ol using equine and human S9 liver fractions were performed. For equine, GC-MS/MS analysis identified the diagnostic 3α-chloro-17α-methyl-5α-androstane-16α,17β-diol metabolite. For human, the 17α-methyl-5α-androstane-3α,17β-diol metabolite was found. Results from these studies were used to verify the ability of GC-MS/MS precursor-ion scanning techniques to support untargeted detection strategies for designer steroids in anti-doping analyses. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Cawley A.T.,Australian Racing Forensic Laboratory | Cawley A.T.,University of Sydney | George A.V.,University of Sydney
Drug Testing and Analysis | Year: 2012

The detection of steroids originating from synthetic precursors against a background of their chemically identical natural analogues has proven to be a significant challenge for doping control laboratories accredited by the World Anti-Doping Agency (WADA). The complementary application of gas chromatography-mass spectrometry (GC-MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) has been demonstrated to provide specific detection of endogenous steroid misuse for improved anti-doping analysis. Markers of synthetically derived steroids are reviewed on the basis of abnormal urinary excretions and low 13C content. A combinatorial approach is presented for the interpretation of GC-MS and GC-C-IRMS data in the anti-doping context. This methodology can allow all relevant information concerning an individual's metabolism to be assessed in order to make an informed decision with respect to a doping violation. © 2012 John Wiley & Sons, Ltd.

Schneiders F.I.,Charles Sturt University | Noble G.K.,Charles Sturt University | Boston R.C.,University of Pennsylvania | Dunstan A.J.,Australian Racing Forensic Laboratory | And 2 more authors.
Veterinary Journal | Year: 2012

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 30. mg of ACP, was given IV to 12 racing-bred geldings. Blood and urine were collected for 7. days 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, r2=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. © 2012 Elsevier Ltd.

Mcgree J.M.,Queensland University of Technology | Noble G.,Charles Sturt University | Schneiders F.,Charles Sturt University | Dunstan A.,Australian Racing Forensic Laboratory | And 3 more authors.
Journal of Veterinary Pharmacology and Therapeutics | Year: 2012

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 (30mg) 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 769L/h and 6874L, respectively. For a typical horse in the study, after receiving 30mg of ACP, the upper limit of the detection time was 35h in plasma and 100h in urine, assuming an arbitrary limit of detection of 1μg/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. © 2012 Blackwell Publishing Ltd.

Richards S.L.,Australian Racing Forensic Laboratory | Richards S.L.,University of Sydney | Cawley A.T.,Australian Racing Forensic Laboratory | Raftery M.J.,University of Sydney
Bioanalysis | Year: 2013

Background: Dermorphin, a hepta-peptide with potent analgesic properties, is classified as a doping agent in equine racing. Since its discovery, a number of biologically active structural analogs have been synthesized and made commercially available so there is a need for reliable methods of detection. Methodology/Results: A sensitive detection method was developed for dermorphin and six analogs in equine urine. Peptide enrichment was achieved using weak cation exchange with subsequent separation and detection by nano-UHPLC-MS/MS. Method validation parameters included: specificity, linearity (5-10000 pg/ml), recovery (58-93%), intra and inter-assay repeatability, LOD (5-50 pg/ml) and matrix effects. Conclusion: The presented method will facilitate the control of the abuse of dermorphin and selected analogs in equine sports. © 2013 Future Science Ltd.

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