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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.


PubMed | Australian Racing Forensic Laboratory, University of Technology, Sydney, Australian National University, Queensland Government and 2 more.
Type: Journal Article | Journal: 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 3- and 3- isomers of the novel compound; 3-chloro-17-methyl-5-androstan-17-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 3-chloro-17-methyl-5-androstan-17-ol using equine and human S9 liver fractions were performed. For equine, GC-MS/MS analysis identified the diagnostic 3-chloro-17-methyl-5-androstane-16,17-diol metabolite. For human, the 17-methyl-5-androstane-3,17-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.


PubMed | Forensic Toxicology Laboratory, Australian Racing Forensic Laboratory and University of Technology, Sydney
Type: | Journal: Drug testing and analysis | Year: 2017

Hallucinogenic phenethylamines such as 2,5-dimethoxyphenethylamines (2C-X) and their N-(2-methoxybenzyl) derivatives (25X-NBOMe) has seen an increase in novel analogues in recent years. These rapidly changing analogues make it difficult for laboratories to rely on traditional targeted screening methods to detect unknown new psychoactive substances (NPS). In this study, twelve 2C-X, six 2,5-dimethoxyamphetamines (DOX) and fourteen 25X-NBOMe derivatives, including two deuterated derivatives (2C-B-d


McKinney A.R.,Australian Racing Forensic Laboratory | Cawley A.T.,Australian Racing Forensic Laboratory | Young E.B.,Queensland Government | Kerwick C.M.,Queensland Government | And 5 more authors.
Bioanalysis | Year: 2013

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,5- or 3,5α-stereochemistry, the oxidation of secondary 17-hydroxyl groups and 16α-hydroxylation. The Phase II -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. © 2013 Future Science Ltd.


PubMed | Racing NSW, Australian Racing Forensic Laboratory and University of New South Wales
Type: | Journal: Talanta | Year: 2016

Over recent years threats to racing have expanded to include naturally occurring biological molecules, such as peptides and proteins, and their synthetic analogues. Traditionally, antibodies have been used to enable detection of these compounds as they allow purification and concentration of the analyte of interest. The rapid expansion of peptide-based therapeutics necessitates a similarly rapid development of suitable antibodies or other means of enrichment. Potential alternative enrichment strategies include the use of aptamers, which offer the significant advantage of chemical synthesis once the nucleic acid sequence is known. A method was developed for the enrichment, detection and quantitation of gonadotropin-releasing hormone (GnRH) in equine urine using aptamer-based enrichment and LC-MS/MS. The method achieved comparable limits of detection (1 pg/mL) and quantification (2.5 pg/mL) to previously published antibody-based enrichment methods. The intra- and inter-assay precision achieved was less than 10% at both 5 and 20 pg/mL, and displayed a working dynamic range of 2.5-100 pg/mL. Significant matrix enhancement (170 8%) and low analytical recovery (29 15%) was observed, although the use of an isotopically heavy labelled GnRH peptide, GnRH (Pro(13)C5,(15)N), as the internal standard provides compensation for these parameters. Within the current limits of detection GnRH was detectable up to 1h post administration in urine and identification of a urinary catabolite extended this detection window to 4h. Based on the results of this preliminary investigation we propose the use of aptamers as a viable alternative to antibodies in the enrichment of peptide targets from equine urine.


Sutton G.J.,University of New South Wales | Cawley A.,Australian Racing Forensic Laboratory | Murphy C.,Australian Racing Forensic Laboratory | Lau M.L.,Australian Racing Forensic Laboratory | Hibbert D.B.,University of New South Wales
Drug Testing and Analysis | Year: 2014

Horse racing authorities impose a limit on the concentration of plasma 'total carbon dioxide' (TCO2), typically 36mM with action taken above 37mM, as measured by an electrochemical gas analyzer. It is of interest to understand the distribution of TCO2 in a 'normal' population of racehorses and determine probabilities of members of this population exceeding these current regulatory and action limits. TCO2 levels in equine plasma samples have been modelled for 12 months (2011-2012) of thoroughbred (3076 measurements) and standardbred (3788 measurements) data in Australia. The two populations have a common seasonal pattern, while the non-seasonal distributions differ. A single Gaussian distribution about the seasonal pattern explains the thoroughbred data, but there is evidence for a second Gaussian component for the standardbred horses. A Gaussian mixture model for standardbred horses gave a main component that matched the thoroughbred distribution, which was centred about 30.2mM, and a smaller (about 20 % of the total density) Gaussian centred at 32.3mM. The existence of a second, higher-meaned population of standardbred horses points to increased use of alkalinizing salts among a minority of trainers, whom still, however, maintain mostly legal levels of TCO2. Identification of this group can be used to direct intelligence-based testing with a view to limiting use of these products. Probabilities of exceeding limits are affected by seasonality, but the current rules remain conservative. © 2014 John Wiley & Sons, Ltd.


Richards S.L.,Australian Racing Forensic Laboratory | Richards S.L.,University of New South Wales | Cawley A.T.,Australian Racing Forensic Laboratory | Cawley A.T.,University of New South Wales | And 4 more authors.
Talanta | Year: 2016

Over recent years threats to racing have expanded to include naturally occurring biological molecules, such as peptides and proteins, and their synthetic analogues. Traditionally, antibodies have been used to enable detection of these compounds as they allow purification and concentration of the analyte of interest. The rapid expansion of peptide-based therapeutics necessitates a similarly rapid development of suitable antibodies or other means of enrichment. Potential alternative enrichment strategies include the use of aptamers, which offer the significant advantage of chemical synthesis once the nucleic acid sequence is known. A method was developed for the enrichment, detection and quantitation of gonadotropin-releasing hormone (GnRH) in equine urine using aptamer-based enrichment and LC-MS/MS. The method achieved comparable limits of detection (1 pg/mL) and quantification (2.5 pg/mL) to previously published antibody-based enrichment methods. The intra- and inter-assay precision achieved was less than 10% at both 5 and 20 pg/mL, and displayed a working dynamic range of 2.5-100 pg/mL. Significant matrix enhancement (170±8%) and low analytical recovery (29±15%) was observed, although the use of an isotopically heavy labelled GnRH peptide, GnRH (Pro13C5,15N), as the internal standard provides compensation for these parameters. Within the current limits of detection GnRH was detectable up to 1 h post administration in urine and identification of a urinary catabolite extended this detection window to 4 h. Based on the results of this preliminary investigation we propose the use of aptamers as a viable alternative to antibodies in the enrichment of peptide targets from equine urine. © 2016 Elsevier B.V. All rights reserved.


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.


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.


PubMed | Australian Racing Forensic Laboratory
Type: Journal Article | Journal: Bioanalysis | Year: 2013

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.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.The presented method will facilitate the control of the abuse of dermorphin and selected analogs in equine sports.

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