ORCID Profile
0000-0002-3442-8617
Current Organisation
Racing New South Wales
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In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Analytical Chemistry | Instrumental Methods (excl. Immunological and Bioassay Methods) | Organic Chemical Synthesis | Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Analytical Spectrometry | Immunological and Bioassay Methods
Expanding Knowledge in the Chemical Sciences | Crime Prevention | Animal Welfare | Horses | Expanding Knowledge in the Biological Sciences | Professional, Scientific and Technical Services |
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6AY00156D
Abstract: The use of LC-HRAM spectrometry to identify ‘unknown’ compounds by non-targeted screening provides a potential advantage for forensic toxicology.
Publisher: Wiley
Date: 18-07-2018
DOI: 10.1002/DTA.2224
Abstract: In vitro technologies provide the capacity to study drug metabolism where in vivo studies are precluded due to ethical or financial constraints. The metabolites generated by in vitro studies can assist anti-doping laboratories to develop protocols for the detection of novel substances that would otherwise evade routine screening efforts. In addition, professional bodies such as the Association of Official Racing Chemists (AORC) currently permit the use of in-vitro-derived reference materials for confirmation purposes providing additional impetus for the development of cost effective in vitro metabolism platforms. In this work, alternative conditions for in vitro phase II sulfation using human, equine or canine liver S9 fraction were developed, with adenosine triphosphate (ATP) and sodium sulfate in place of the expensive and unstable co-factor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), and employed for the generation of six representative steroidal sulfates. Using these conditions, the equine in vitro phase II metabolism of the synthetic or so-called designer steroid furazadrol ([1',2']isoxazolo[4',5':2,3]-5α-androstan-17β-ol) was investigated, with ATP and Na
Publisher: Wiley
Date: 13-07-2012
DOI: 10.1002/DTA.1378
Abstract: 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 (13)C 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 in idual's metabolism to be assessed in order to make an informed decision with respect to a doping violation.
Publisher: Wiley
Date: 24-11-2008
DOI: 10.1002/RCM.3826
Abstract: Studies have shown that the administration of androstenedione (ADIONE) significantly increases the urinary ratio of testosterone glucuronide to epitestosterone glucuronide (T/E) - measured by gas chromatography/mass spectrometry (GC/MS) - in subjects with a normal ( approximately 1) or naturally high (>1) initial values. However, the urinary T/E ratio has been shown not to increase in subjects with naturally low (<1) initial values. Such cases then rely on the detection of C(6)-hydroxylated metabolites shown to be indicative of ADIONE administration. While these markers may be measured in the routine GC/MS steroid profile, their relatively low urinary excretion limits the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to specifically confirm ADIONE administration based on depleted (13)C content. A mass spectrometry strategy was used in this study to identify metabolites of ADIONE with the potential to provide compound-specific detection. C(4)-hydroxylation was subsequently shown to be a major metabolic pathway following ADIONE administration, thereby resulting in urinary excretion of 4-hydroxyandrostenedione (4OH-ADIONE). Complementary analysis of 4OH-ADIONE by GC/MS and GC/C/IRMS was used to confirm ADIONE administration.
Publisher: Future Science Ltd
Date: 04-2013
DOI: 10.4155/BIO.13.40
Abstract: 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 s les.
Publisher: Wiley
Date: 20-03-2022
DOI: 10.1002/DTA.3250
Abstract: The concept of biomarker measurements in the form of a ratio has not been explored in detail. This is surprising considering the current and future potential for biomarkers incorporating endogenous reference compounds (ERCs) in a range of fields. A selection of these relating to clinical and forensic applications, human antidoping, equine antidoping and veterinary residues are discussed.
Publisher: Wiley
Date: 03-06-2008
DOI: 10.1002/JMS.1437
Abstract: The administration of synthetic steroid copies is one of the most important issues facing sports. Doping control laboratories accredited by the World Anti-Doping Agency (WADA) require methods of analysis that allow endogenous steroids to be distinguished from their synthetic analogs in urine. The ability to measure isotope distribution at natural abundance with high accuracy and precision has increased the application of Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS) to doping control in recent years. GC-C-IRMS is capable of measuring the carbon isotope ratio (delta(13)C) of urinary steroids and confirm their synthetic origin based on the abnormal (13)C content. This tutorial describes some of the complexities encountered by obtaining valid delta(13)C measurements from GC-C-IRMS and the need for careful interpretation of all relevant information concerning an in idual's metabolism in order to make an informed decision with respect to a doping violation.
Publisher: Wiley
Date: 17-04-2017
DOI: 10.1002/DTA.2180
Abstract: The move towards personalized medicine derived from in idually focused clinical chemistry measurements has been translated by the human anti-doping movement over the past decade into developing the athlete biological passport. There is considerable potential for animal sports to adapt this model to facilitate an intelligence-based anti-doping system. Copyright © 2017 John Wiley & Sons, Ltd.
Publisher: Elsevier BV
Date: 10-2005
DOI: 10.1016/J.JCHROMB.2005.01.042
Abstract: The need for laboratories accredited by the World Anti-Doping Agency (WADA) to develop methods of analysis for steroids excreted primarily as their sulfate conjugates has faced significant analytical challenges. One of the issues relates to the extraction of these metabolites from urine in a relatively pure state. The use of (-)-N,N-dimethylephedrinium bromide as an ion pairing reagent was optimised to produce a method that is selective for the extraction of steroid sulfates prior to GC-MS or LC-MS analysis, with minimal contributions from the urine matrix. The recovery of androsterone from its sulfate conjugate was determined to be 67% with a relative quantitative uncertainty of +/-14% (k = 2).
Publisher: Wiley
Date: 07-09-2012
DOI: 10.1002/DTA.321
Abstract: Conventional chemical profiling of methyl hetamine has long been employed by national forensic laboratories to determine the synthetic route and where possible the precursor chemicals used in its manufacture. This laboratory has been studying the use of stable isotope ratio mass spectrometry (IRMS) analysis as a complementary technique to conventional chemical profiling of fully synthetic illicit drugs such as methyl hetamine. As part of these investigations the stable carbon (δ(13) C), nitrogen (δ(15) N), and hydrogen (δ(2) H) isotope values in the precursor chemicals of ephedrine and pseudoephedrine and the resulting methyl hetamine end-products have been measured to determine the synthetic origins of methyl hetamine. In this study, results are presented for δ(13) C, δ(15) N, and δ(2) H values in methyl hetamine synthesized from ephedrine and pseudoephedrine by two synthetic routes with varying experimental parameters. It was demonstrated that varying parameters, such as stoichiometry, reaction temperature, reaction time, and reaction pressure, had no effect on the δ(13) C, δ(15) N, and δ(2) H isotope values of the final methyl hetamine product, within measurement uncertainty. Therefore the value of the IRMS technique in identifying the synthetic origin of precursors, such as ephedrine and pseudoephedrine, is not compromised by the potential variation in synthetic method that is expected from one batch to the next, especially in clandestine laboratories where manufacture can occur without stringent quality control of reactions.
Publisher: Elsevier BV
Date: 05-2011
DOI: 10.1016/J.FORSCIINT.2010.11.016
Abstract: Doping control laboratories accredited by the World Anti-Doping Agency (WADA) require criteria that allow endogenous steroids to be distinguished from their synthetic analogues in urine. Methodology based on "looking outside the metabolic box" was used in this study to identify diagnostic urinary markers of 4-androstenediol (4-ADIOL) administration. Androst-2,4-diene-17-one and androst-3,5-diene-17-one are proposed to be formed in urine from acid-catalyzed hydrolysis of 4-ADIOL sulfoconjugate, a major phase II metabolic product of 4-ADIOL. The presence of these markers in the routine gas chromatography-mass spectrometry (GC-MS) steroid screen was suitable to identify s les requiring confirmation by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) - to measure the carbon isotope ratio (δ(13)C) of the androstdiene markers and confirm their likely synthetic origin based on depleted (13)C content.
Publisher: Wiley
Date: 24-02-2014
DOI: 10.1002/DTA.1600
Abstract: Horse racing authorities impose a limit on the concentration of plasma 'total carbon dioxide' (TCO2), typically 36 mM with action taken above 37 mM, 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 s les 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 30.2 mM, and a smaller (about 20 % of the total density) Gaussian centred at 32.3 mM. 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.
Publisher: Wiley
Date: 17-09-2012
DOI: 10.1002/DTA.1403
Abstract: Glucocorticoids are listed on the World Anti-Doping Agency (WADA) Prohibited List of substances. The detection of the administration of hydrocortisone and cortisone is complicated by the fact that the human body also produces these steroids naturally. Gas chromatography-combustion-isotope ratio mass spectrometry can be utilized to determine the use of endogenous glucocorticoids by measuring the carbon isotope ratio (CIR) of their resulting metabolites in human urine s les. A comprehensive s le preparation protocol for the analysis of endogenous glucocorticoid urinary metabolites was developed and validated, incorporating the use of high performance liquid chromatography (HPLC) for purification and chemical oxidation for derivatisation. Target compounds were tetrahydrocortisol and tetrahydrocortisone, and 11β-hydroxyetiocholanolone, 11-oxoetiocholanolone and 11β-hydroxyandrosterone, while pregnanediol functioned as the endogenous reference compound. Urine s les from a population of 50 volunteers were analyzed to determine CIR reference limits. Excretion studies of the endogenous glucocorticoid preparation cortisone acetate (25 mg oral) and the dietary supplement adrenosterone (75 mg oral) were conducted with six male in iduals. Variable changes in steroid metabolite isotopic composition were found across subjects after administration. The study also revealed that CIR analysis of the major glucocorticoid metabolites tetrahydrocortisol and tetrahydrocortisone is necessary to unambiguously distinguish administration of cortisone and adrenosterone, the former officially restricted to out-of-competition use by athletes, the latter not being restricted at the current time. Moreover, this study reaffirms that CIR methods for the doping control of endogenous steroids should not rely upon a single ERC, as the administration of an appropriate precursor to that ERC could cause complications during analysis.
Publisher: Wiley
Date: 09-12-2021
DOI: 10.1002/DTA.2976
Abstract: The constant evolution of the illicit drug market makes the identification of unknown compounds problematic. Obtaining certified reference materials for a broad array of new analogues can be difficult and cost prohibitive. Machine learning provides a promising avenue to putatively identify a compound before confirmation against a standard. In this study, machine learning approaches were used to develop class prediction and retention time prediction models. The developed class prediction model used a naïve Bayes architecture to classify opioids as belonging to either the fentanyl analogues, AH series or U series, with an accuracy of 89.5%. The model was most accurate for the fentanyl analogues, most likely due to their greater number in the training data. This classification model can provide guidance to an analyst when determining a suspected structure. A retention time prediction model was also trained for a wide array of synthetic opioids. This model utilised Gaussian process regression to predict the retention time of analytes based on multiple generated molecular features with 79.7% of the s les predicted within ±0.1 min of their experimental retention time. Once the suspected structure of an unknown compound is determined, molecular features can be generated and input for the prediction model to compare with experimental retention time. The incorporation of machine learning prediction models into a compound identification workflow can assist putative identifications with greater confidence and ultimately save time and money in the purchase and/or production of superfluous certified reference materials.
Publisher: Wiley
Date: 05-03-2015
DOI: 10.1002/DTA.1782
Abstract: The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected ex les. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3β,17β-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical s le preparation.
Publisher: Wiley
Date: 15-07-2021
DOI: 10.1002/DTA.2893
Abstract: Synthetic opioids are a class of compounds that are of particular concern due to their high potency and potential health impacts. With the relentless emergence of new synthetic opioid derivatives, non-targeted screening strategies are required that do not rely on the use of library spectra or reference materials. In this study, product ion searching, and Kendrick mass defect analysis were investigated for non-targeted screening of synthetic opioids. The estimated screening cut-offs for these techniques ranged between 0.05 and 0.1 ng/mL. These techniques were designed to not be reliant on a particular vendor's software, meaning that they can be applied to existing drug screening protocols, without requiring the development and validation of new analytical procedures. The efficacy of the developed techniques was tested through blind trials, with spiked s les inserted amongst authentic plasma s les, which demonstrated the usefulness of these methods for high-throughput screening. The use of a non-targeted screening workflow that contains complementary techniques can increase the likelihood of detecting compounds of interest within a s le, as well as the confidence in detections that are made.
Publisher: Wiley
Date: 08-03-2022
DOI: 10.1002/DTA.3245
Abstract: Metabolomics is a multidisciplinary field providing workflows for complementary approaches to conventional analytical determinations. It allows for the study of metabolically related groups of compounds or even the study of novel pathways within the biological system. The procedural stages of metabolomics experimental design, s le preparation, analytical determinations, data processing and statistical analysis, compound identification and validation strategies are explored in this review. The selected approach will depend on the type of study being conducted. Experimental design influences the whole metabolomics workflow and thus needs to be properly assessed to ensure sufficient s le size, minimal introduced and biological variation and appropriate statistical power. S le preparation needs to be simple, yet potentially global in order to detect as many compounds as possible. Analytical determinations need to be optimised either for the list of targeted compounds or a universal approach. Data processing and statistical analysis approaches vary widely and need to be better harmonised for review and interpretation. This includes validation strategies that are currently deficient in many presented workflows. Common compound identification approaches have been explored in this review. Metabolomics applications are discussed for clinical and forensic toxicology, human and equine sports anti-doping and veterinary residues.
Publisher: Wiley
Date: 21-06-2018
DOI: 10.1002/DTA.2411
Abstract: The use of testosterone and its pro-drugs, such as dehydroepiandrosterone (DHEA), is currently regulated in horseracing by the application of international testosterone thresholds. However, additional steroidomic approaches, such as steroid ratios, to distinguish overall adrenal stimulation from drug administrations and an equine biological passport for longitudinal steroid profiling of in idual animals could be advantageous in equine doping testing. Thus, DHEA concentrations and related ratios (testosterone [T] to DHEA and DHEA to epitestosterone [E]) were assessed in the reference population by quantitative analysis of 200 post-race gelding urine s les using liquid chromatography-tandem mass spectrometry. DHEA concentrations ranged between 0.9 and 136.6 ng/mL (mean 12.8 ng/mL), T:DHEA ratios between 0.06 and 1.85 (mean 0.43), and DHEA:E ratios between 0.21 and 13.56 (mean 2.20). Based on the reference population statistical upper limits of 5.4 for T:DHEA ratio and 48.1 for DHEA:E ratio are proposed with a risk of 1 in 10 000 for a normal outlier exceeding the value. Analysis of post-administration urine s les collected following administrations of DHEA, Equi-Bolic® (a mix of DHEA and pregnenolone) and testosterone propionate to geldings showed that the upper limit for T:DHEA ratio was exceeded following testosterone propionate administration and DHEA:E ratio following DHEA administrations and thus these ratios could be used as additional biomarkers when determining the cause of an atypical testosterone concentration. Additionally, DHEA concentrations and ratios can be used as a starting point to establish reference ranges for an equine biological passport.
Publisher: Wiley
Date: 05-2021
DOI: 10.1002/DTA.3049
Abstract: Androgens, both steroidal and nonsteroidal in nature, are among the most commonly misused substances in competitive sports. Their recognized anabolic and performance enhancing effects through short- and long-term physiological adaptations make them popular. Androgens exist as natural steroids, or are chemically synthesized as anabolic androgenic steroids (AAS) or selective androgen receptor modulators (SARMs). In order to effectively detect misuse of androgens, targeted strategies are used. These targeted strategies rely heavily on mass spectrometry, and detection requires prior knowledge of the targeted structure and its metabolites. Although exquisitely sensitive, such approaches may fail to detect novel structures that are developed and marketed. A nontargeted approach to androgen detection involves the use of cell-based in vitro bioassays. Both yeast and mammalian cell androgen bioassays demonstrate a clear ability to detect AAS and SARMS, and if paired with high resolution mass spectrometry can putatively identify novel structures. In vitro cell bioassays are successfully used to characterize designer molecules and to detect exogenous androgens in biological s les. It is important to continue to develop new and effective detection approaches to prevent misuse of designer androgens, and in vitro bioassays represent a potential solution to nontargeted detection strategies.
Publisher: Wiley
Date: 05-04-2022
DOI: 10.1002/DTA.3264
Abstract: Equine urine analysis has evolved over time to detect thousands of urinary compounds for doping control in the horse racing industry. The longitudinal assessment of 3‐methoxytyramine to tyramine ratio (3‐MT/T) values in equine urine by GC–MS profiling was investigated to support the Racing NSW Equine Biological Passport (EBP) for detection of dopaminergic manipulation in racehorses. This involved comparison of routine urine s les to administration studies of Sinemet , a common Parkinson's disease medication containing levodopa. Using an endogenous reference compound (ERC) in a urinary ratio enabled greater confidence to provide intelligence of pharmaceutical manipulation as distinct from physiological variation. Population reference limits (PRLs) of 776 ng/ml for urinary 3‐MT and 5.3 for 3‐MT/T, together with the use of in idual reference limits (IRLs), are proposed.
Publisher: Wiley
Date: 03-03-2021
DOI: 10.1002/DTA.3021
Abstract: The emergence of novel doping agents is a continuous issue for analysts who aim to maintain the integrity of horseracing together with the well-being and safety of the animals and riders involved. Untargeted mass spectrometric analysis presents a potential improvement for antidoping as it enables the detection of compounds being indirectly affected by an administered drug. In this study, liquid chromatography-high-resolution mass spectrometry was used to investigate a 12-horse administration study of the synthetic opioid, butorphanol. A mass spectrometric workflow capable of detecting metabolic differences for an extended period of time was successfully developed. This proof-of-concept study demonstrates the potential of untargeted workflows to provide a list of biomarkers of exposure and effect that are indicative of drug administration which may be implemented into routine testing for improved doping control.
Publisher: Wiley
Date: 04-03-2022
DOI: 10.1002/DTA.3244
Abstract: The conventional detection of exogenous drugs in equine doping s les has been used for confirmation and subsequent prosecution of participants responsible. In recent years, alternative methods using indirect detection have been investigated due to the expanding number of pharmaceutical agents available with the potential of misuse. The monitoring of endogenous biomarkers such as hydrocortisone (HC) has been studied in equine urine with an international threshold of 1 μg/ml established however, there is no current threshold for equine plasma. The aim of this research was to investigate plasma concentrations of HC and cortisone (C) in race day s les compared to an administration of Triamcinolone Acetonide (TACA). The reference population ( n = 1150) provided HC (6 to 145 ng/ml) and C (0.7 to 13 ng/ml) levels to derive the HC to C ratio (HC/C). Population reference limits (PRLs) were proposed for HC/C values at 0.2 (lower) and 61 (upper). Administration of TACA resulted in down‐regulation of HC/C values below the estimated PRLs for up to 96 h post‐administration. This indirect detection period was longer than the detection of TACA for 72 h. The use of in idual reference limits (IRLs) for HC/C values was investigated to support the Equine Biological Passport (EBP), an intelligence model developed by Racing NSW for longitudinal monitoring of biomarkers.
Publisher: Wiley
Date: 08-03-2017
DOI: 10.1002/DTA.2171
Abstract: Hallucinogenic phenethylamines such as 2,5-dimethoxyphenethylamines (2C-X) and their N-(2-methoxybenzyl) derivatives (25X-NBOMe) have 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-dimethoxy hetamines (DOX), and fourteen 25X-NBOMe derivatives, including two deuterated derivatives (2C-B-d
Publisher: Wiley
Date: 06-2009
DOI: 10.1002/RCM.4109
Abstract: Conventional chemical profiling of methyl hetamine has been used for many years to determine the synthetic route employed and where possible to identify the precursor chemicals used. In this study stable isotope ratio analysis was investigated as a means of determining the origin of the methyl hetamine precursors, ephedrine and pseudoephedrine. Ephedrine and pseudoephedrine may be prepared industrially by several routes. Results are presented for the stable isotope ratios of carbon (delta(13)C), nitrogen (delta(15)N) and hydrogen (delta(2)H) measured in methyl hetamine s les synthesized from ephedrine and pseudoephedrine of known provenance. It is clear from the results that measurement of the delta(13)C, delta(15)N and delta(2)H stable isotope ratios by elemental analyzer/thermal conversion isotope ratio mass spectrometry (EA/TC-IRMS) in high-purity methyl hetamine s les will allow determination of the synthetic source of the ephedrine or pseudoephedrine precursor as being either of a natural, semi-synthetic, or fully synthetic origin.
Publisher: Elsevier BV
Date: 03-2009
DOI: 10.1016/J.STEROIDS.2008.11.004
Abstract: The detection of steroids originating from synthetic precursors in relation to their chemically identical natural analogues has proven to be a significant challenge for doping control laboratories accredited by the World Anti-Doping Agency (WADA). Endogenous steroid abuse may be confirmed by utilising the atomic specificity of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) that enables the precise measurement of differences in stable isotope ratios that arise as a result of fractionation patterns inherent in the source of steroids. A comprehensive carbon isotope ratio (delta(13)C) profiling study (n=1262) of urinary ketosteroids is reported that demonstrates the inter-in idual variation that can be expected from factors such as diet, ethnicity, gender and age within and between different populations (13 countries). This delta(13)C distribution is shown by principal component analysis (PCA) to provide a statistical comparison to delta(13)C values observed following administration of testosterone enanthate. A limited collection of steroid diol data (n=100 consisting of three countries) is also presented with comparison to delta(13)C values of excreted testosterone to validate criteria for WADA accredited laboratories to prove doping offences.
Publisher: Wiley
Date: 10-05-2010
DOI: 10.1002/RCM.4563
Abstract: Previous work in these laboratories and by Butzenlechner et al. and Culp et al. has demonstrated that the delta(2)H isotope value of industrial benzaldehyde produced by the catalytic oxidation of toluene is profoundly positive, usually in the range +300 per thousand to +500 per thousand. Synthetic routes leading to hetamine, methyl hetamine or their precursors and commencing with such benzaldehyde may be expected to exhibit unusually positive delta(2)H values. Results are presented for delta(13)C and delta(2)H isotope values of 1-phenyl-2-nitropropene synthesized from an industrial source of benzaldehyde, having a positive delta(2)H isotope value, by a Knoevenagel condensation with nitroethane. Results are also presented for delta(13)C and delta(2)H isotope values for hetamine prepared from the resulting 1-phenyl-2-nitropropene. The values obtained were compared with delta(13)C and delta(2)H isotope values obtained for an hetamine s le prepared using a synthetic route that did not involve benzaldehyde. Finally, results are presented for s les of benzaldehyde, 1-phenyl-2-nitropropene and hetamine that had been seized at a clandestine hetamine laboratory.
Publisher: Elsevier BV
Date: 07-2004
Publisher: Elsevier BV
Date: 02-0001
Publisher: Wiley
Date: 22-10-2010
DOI: 10.1002/DTA.175
Abstract: Gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS) is the preferred method of confirming the administration of exogenous testosterone by athletes. This relies on synthetic testosterone preparations being depleted in 13 C compared to natural testosterone. There is concern, however, about the existence of synthetic testosterone products that are unexpectedly 13 C‐enriched and which may allow athletes to circumvent the current GC‐C‐IRMS test. Further to the reported studies of legitimate pharmaceutical‐grade testosterone products, a detailed analysis of seized materials from border‐level seizures was required to obtain intelligence concerning trends in ‘black market’ testosterone manufacture and distribution. The s le set collected for this study between 2006 and 2009 inclusive provided a δ 13 C range (n = 266) of − 22.9‰ to − 32.6‰ with mean and median values of − 28.4‰ and − 28.6‰, respectively. Within this distribution there were 24 s les (9%) confirmed to have δ 13 C values in the range reported for endogenous urinary steroid metabolites (≥− 25.8‰). The benefit of δ 13 C profiling for testosterone preparations was demonstrated by the ability to identify specific seized products that can be target tested for future intelligence purposes. In addition, the potential of stable hydrogen isotope ratio ( 2 H/ 1 H δ 2 H) discrimination to complement δ 13 C analysis was investigated. Methodologies for the determination of δ 2 H values by gas chromatography‐thermal conversion‐isotope ratio mass spectrometry (GC‐TC‐IRMS) were developed to provide a δ 2 H range (n = 173) of − 177‰ to − 268‰ with mean and median values of − 231‰ and − 234‰, respectively. Copyright © 2010 John Wiley & Sons, Ltd.
Publisher: Wiley
Date: 04-02-2020
DOI: 10.1002/DTA.2769
Abstract: Hemapolin (2α,3α-epithio-17α-methyl-5α-androstan-17β-ol) is a designer steroid that is an ingredient in several "dietary" and "nutritional" supplements available online. As an unusual chemical modification to the steroid A-ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC-MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α-methyl-5α-androst-2-en-17β-ol), reduced and dihydroxylated madol (17α-methyl-5α-androstane-2β,3α,17β-triol), and the isomeric enone metabolites 17β-hydroxy-17α-methyl-5α-androst-3-en-2-one and 17β-hydroxy-17α-methyl-5α-androst-2-en-4-one, were detected and confirmed in equine urine extracts by comparison with a library of synthetically derived reference materials. A number of additional madol derivatives derived from hydroxylation, dihydroxylation, and trihydroxylation were also detected but not fully identified by this approach. A yeast cell-based androgen receptor bioassay of available reference materials showed that hemapolin and many of the metabolites identified by this study were potent activators of the equine androgen receptor. This study reveals the metabolites resulting from the equine administration of the androgen hemapolin that can be incorporated into routine GC-MS antidoping screening and confirmation protocols to detect the illicit use of this agent in equine sports.
Publisher: Wiley
Date: 12-2012
DOI: 10.1002/DTA.1439
Publisher: Wiley
Date: 19-08-2014
DOI: 10.1002/DTA.1533
Abstract: Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is now established as a robust and mature analytical technique for the doping control of endogenous anabolic androgenic steroids in human sport. It relies on the assumption that the carbon isotope ratios of naturally produced steroids are significantly different to synthetically manufactured testosterone or testosterone prohormones used in commercial medical or dietary supplement products. Recent publications in this journal have highlighted the existence of black market testosterone preparations with carbon isotope ratios within the range reported for endogenous steroids (i.e. δ(13) C ≥ -25.8 ‰). In this study, we set out to profile domestic and international law enforcement seizures of illicit testosterone products to monitor the prevalence of 'enriched' substrates--which if administered to human subjects would be considered problematic for the use of current GC-C-IRMS methodologies for the doping control of testosterone in sport. The distribution of δ(13) C values for this illicit testosterone s le population (n = 283) ranged from -23.4 ‰ to -32.9 ‰ with mean and median of -28.6 ‰--comparable to previous work. However, only 13 out of 283 testosterone s les (4.6 %) were found to display δ(13) C values ≥ -25.8 ‰, confirming that in the vast majority of cases of illicit testosterone administration, current GC-C-IRMS doping control procedures would be capable of confirming misuse.
Publisher: Wiley
Date: 31-05-2017
DOI: 10.1002/DTA.2205
Abstract: Acadesine, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, commonly known as AICAR, is a naturally occurring adenosine monophosphate-activated protein kinase (AMPK) activator in many mammals, including humans and horses. AICAR has attracted considerable attention recently in the field of doping control because of a study showing the enhancement of endurance performance in unexercised or untrained mice, resulting in the term 'exercise pill'. Its use has been classified as gene doping by the World Anti-Doping Agency (WADA), and since it is endogenous, it may only be possible to control deliberate administration of AICAR to racehorses after establishment of an appropriate threshold. Herein we report our studies of AICAR in post-race equine urine and plasma s les including statistical studies of AICAR concentrations determined from 1,470 urine s les collected from thoroughbreds and standardbreds and analyzed in Australia, France, and Hong Kong. Quantification methods in equine urine and plasma using liquid chromatography-mass spectrometry were developed by the laboratories in each country. An exchange of spiked urine and plasma s les between the three countries was conducted, confirming no significant differences in the methods. However, the concentration of AICAR in plasma was found to increase upon haemolysis of whole blood s les, impeding the establishment of a suitable threshold in equine plasma. A possible urine screening cut-off at 600 ng/mL for the control of AICAR in racehorses could be considered for adoption. Application of the proposed screening cut-off to urine s les collected after intravenous administration of a small dose (2 g) of AICAR to a mare yielded a short detection time of approximately 4.5 h. Copyright © 2017 John Wiley & Sons, Ltd.
Publisher: Future Science Ltd
Date: 12-2013
DOI: 10.4155/BIO.13.281
Abstract: 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.
Publisher: Wiley
Date: 23-06-2016
DOI: 10.1002/DTA.1832
Abstract: 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.
Publisher: Elsevier BV
Date: 08-2011
DOI: 10.1016/J.CHROMA.2011.06.014
Abstract: An alternative calibration procedure for the gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) measurements of the World Antidoping Agency (WADA) Accredited Laboratories is presented. To alleviate the need for externally calibrated CO₂ gas for GC-C-IRMS analysis of urinary steroid metabolites, calibration using an external standard mixture solution of steroids with certified isotopic composition was investigated. The reference steroids of the calibration mixture and routine s les underwent identical instrumental processes. The calibration standards bracketed the entire range of the relevant δ¹³C values for the endogenous and exogenous steroids as well as their chromatographic retention times. The certified δ¹³C values of the reference calibrators were plotted in relation to measured m/z ¹³CO₂/¹²CO₂ (i.e. R(45/44)) mass spectrometric signals of each calibrator. δ¹³C values of the s le steroids were calculated from the least squares fit through the calibration curve. The effect of the external calibration on δ¹³C values, using the same calibration standards and set of urine s les but different brands of GC-C-IRMS instruments, was assessed by an interlaboratory study in the WADA Accredited Laboratories of Sydney, Australia and Athens, Greece. Relative correspondence between the laboratories for determination of androsterone, etiocholanolone, 5β-androstane-3α,17β-diacetate, and pregnanediacetate means were SD(δ¹³C)=0.12‰, 0.58‰, -0.34‰, and -0.40‰, respectively. These data demonstrate that accurate intralaboratory external calibration with certified steroids provided by United States Antidoping Agency (USADA) and without external CO₂ calibration is feasible and directly applicable to the WADA Accredited Laboratories for the harmonization of the GC-C-IRMS measurements.
Start Date: 09-2019
End Date: 12-2023
Amount: $313,659.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2013
End Date: 11-2017
Amount: $180,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 03-2013
End Date: 12-2016
Amount: $195,000.00
Funder: Australian Research Council
View Funded Activity