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 (d 13 KEYWORDS: endogenous steroids; doping control; carbon isotope ratio (υ 13 C); gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) THE ENDOGENOUS STEROID CHALLENGEDrug use and abuse is an unfortunate feature of sports. The fight against doping in sports is a challenge faced by governments, sports authorities, laboratories, coaches and athletes who wish to contribute to fair, healthy and legal competition. The abuse of synthetic steroid copies is one of the most important issues for doping control. Before 1982, only the use of xenobiotic steroids had been banned, with a positive result requiring unequivocal proof provided by techniques such as GC-MS that the banned substance and/or its metabolites were present in a urine sample. Following the prohibition of testosterone (T; androst-4-ene-17ˇ-ol-3-one) administration by the International Olympic Committee and subsequently by the World Anti-Doping Agency (WADA), 1 new areas of research have been established within doping control laboratories.Methods for the detection of administered synthetic steroids by molecular spectrometry techniques such as GC-MS are limited by their ability to identify and quantify. The GC-MS steroid profiles are, however, an important Ł Correspondence to: Adam T. Cawley, National Measurement Institute, 1 Suakin Street, Pymble NSW 2073, Australia. E-mail: Adam.Cawley@measurement.gov.au means of screening all urine samples entering the laboratory to eliminate those of nonsuspicious nature. The objective for doping control laboratories is, therefore, to maximize the detection of synthetic steroid doping violations while minimizing the additional resources required from stakeholders. This can be achieved with advanced GC-MS steroid profiling strategies based on the analysis of several urinary steroids that can originate from the endocrine system. 2 -7 Control of any endogenous substance requires the establishment of 'normal' reference intervals and subsequent statistical determination of what constitutes an 'abnormal' state. The challenge for doping control is presented by steroid administration altering the human endocrine system in ways that are not yet fully understood, since they are largely dependent on the individual athlete.Synthetic copies of endogenous steroids, namely, dehydroepiandrosterone [(DHEA); androst-5-ene-3ˇ-ol-17-one], androstenedione (ADIONE; androst-4-ene-3,17-dione), 4-androstenediol (4-ADIOL; androst-4-ene-3ˇ,17...

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The proliferation of new psychoactive substances (NPS) in recent years has resulted in the development of numerous analytical methods for the detection and identification of known and unknown NPS derivatives. High-resolution mass spectrometry (HRMS) has been identified as the method of choice for broad screening of NPS in a wide range of analytical contexts due to its ability to measure accurate masses using data-independent acquisition (DIA) techniques. Additionally, it has shown promise for non-targeted screening strategies that have been developed in order to detect and identify novel analogues without the need for certified reference materials (CRMs) or comprehensive mass spectral libraries. This paper reviews the applications of HRMS for the analysis of NPS in forensic drug chemistry, clinical and forensic toxicology. It provides an overview of the sample preparation procedures in addition to data acquisition, instrumental analysis and data processing techniques.Furthermore, it will give an overview of the current state of non-targeted screening strategies with discussion on future directions and perspectives of this technique.3

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 sample 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 samples (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.

Conventional chemical profiling of methylamphetamine 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 methylamphetamine 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 methylamphetamine samples 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 methylamphetamine samples 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.

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Furazadrol ([1',2']isoxazolo[4',5':2,3]-5α-androstan-17β-ol) is a designer anabolic androgenic steroid that is readily available via the internet. It contains an isoxazole fused to the steroid A-ring which offers metabolic stability and noteworthy anabolic activity raising concerns over the potential for abuse of this compound in equine sports. The metabolism of furazadrol was studied by in vivo and in vitro methods for the first time. Urinary furazadrol 17-sulfate and furazadrol 17-glucuronide metabolites were detected in vivo after a controlled administration and compared with synthetically-derived reference materials in order to confirm their identities. They were quantified to establish the excretion profile and a suitable limit of detection. Minor metabolites were also detected, including epifurazadrol, hydroxylated furazadrol, and hydroxylated and oxidised furazadrol, present as the sulfate and glucuronide conjugates. Phase II metabolites were subjected to enzymatic hydrolysis by Escherichia coli β-glucuronidase and Pseudomonas aeruginosa arylsulfatase to further confirm the identity of the corresponding phase I metabolites. The metabolism profile was compared to the products obtained from an in vitro phase I metabolism study, with all but two of the minor in vivo phase I metabolites observed in the in vitro system. These investigations identify the key urinary metabolites of furazadrol following oral administration, which can be incorporated into anti-doping screening and confirmation procedures.

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 sample 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 samples (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.