In-Sewer Stability Assessment of Anabolic Steroids and Selective Androgen Receptor Modulators.

Wastewater-based epidemiology is a potential complementary technique for monitoring the use of performance- and image-enhancing drugs (PIEDs), such as anabolic steroids and selective androgen receptor modulators (SARMs), within the general population. Assessing in-sewer transformation and degradation is critical for understanding uncertainties associated with wastewater analysis. An electrospray ionization liquid chromatography mass spectrometry method for the quantification of 59 anabolic agents in wastewater influent was developed. Limits of detection and limits of quantification ranged from 0.004 to 1.56 µg/L and 0.01 to 4.75 µg/L, respectively. Method performance was acceptable for linearity (R2 > 0.995, few exceptions), accuracy (68-119%), and precision (1-21%RSD), and applicability was successfully demonstrated. To assess the stability of the selected biomarkers in wastewater, we used laboratory-scale sewer reactors to subject the anabolic agents to simulated realistic sewer environments for 12 h. Anabolic agents, including parent compounds and metabolites, were spiked into freshly collected wastewater that was then fed into three sewer reactor types: control sewer (no biofilm), gravity sewer (aerobic conditions), and rising main sewer (anaerobic conditions). Our results revealed that while most glucuronide conjugates were completely transformed following 12 h in the sewer reactors, 50% of the investigated biomarkers had half-lives longer than 4 h (mean residence time) under gravity sewer conditions. Most anabolic agents were likely subject to biofilm sorption and desorption. These novel results lay the groundwork for any future wastewater-based epidemiology research involving anabolic steroids and SARMs.

Prevalence of nandrolone preparations with endogenous carbon isotope ratios in Australia.

Nandrolone and its prohormones, including 19-norandrost-4-ene-3,17-dione and 19-norandrost-4-ene-3ß,17ß-diol, are anabolic steroids forbidden at all times in sports according to the World Anti-Doping Code Prohibited List and its metabolite 19-norandrosterone (19NA) is the preferred urinary target compound to identify their abuse. In recent years, an increasing number of 19NA isotope ratio mass spectrometry (IRMS) cases have arisen that, based on the initial testing procedure, were likely to result in an adverse analytical finding but were concluded negative after IRMS analysis. The current study was therefore set up to gain a better insight on the prevalence of nandrolone preparations with endogenous carbon isotope ratio values in Australia. Suitable workplace (non-athlete) urine samples that had previously been reported positive for 19NA were identified and analysed on IRMS. A total of 82% of the samples that were analysed were reported with enriched carbon isotope ratios of 19NA (i.e., 19NA greater than -26‰). This indicates that there is a high prevalence of nandrolone-containing anabolic androgenic steroid preparations in Australia that have 'endogenous' carbon isotope ratios which reduces the ability to identify exogenous nandrolone.

Summer Olympic sports and female athletes: comparison of anti-doping collections and prohibited substances detected in Australia and New Zealand vs. France.

Like any athlete, female athletes may be tempted to use prohibited substances during competition or training to enhance their performance. Anti-doping tests performed on female athletes in summer Olympic sports from two geographical areas: Australia/ New Zealand, and France were compared. First, the distribution of sample collections across different sports disciplines, as well as the distribution of substances was investigated. Then the distribution of collections and substances detected in the five sports categories (Strength/Speed, Endurance, Mixed, Motor Skills with High Energy Expenditure, and Motor Skills with Low Energy Expenditure) were studied with consideration of therapeutic use exemptions obtained by the athlete. Australia/New Zealand and France were similar in their overall number of anti-doping collections performed. Likewise, both regions had the same sports disciplines (athletics, aquatics, cycling) and sport categories (Mixed and Endurance) as having the highest number of sample collections. The Motor Skills with High Energy Expenditure, and Motor Skills with Low Energy Expenditure categories had the lowest number of sample collections. However, the number of substances detected was significantly different (p < 0.05) with a greater number of substances found in the French data. There were a few substances in common between the two geographical areas, namely prednisone/prednisolone, carboxy-THC, terbutaline, vilanterol and methylphenidate, but most were different. In-competition tests were the category where most of the AAFs were found.

Reliability of Drug History to Verify Androgen Abuse in Men.

CONTEXT: Clinical evaluations that require excluding androgen abuse, a secretive, illicit activity, rely on the drug history, but its veracity for androgen abuse has neither been verified nor has any objective corroborating laboratory test been validated. OBJECTIVE: In a high-risk population, to (a) validate the drug history of androgen abuse objectively using state-of-the-art World Anti-Doping Agency-accredited antidoping laboratory urine mass spectrometry tests and (b) to determine what biochemical tests best distinguish androgen abuse from nonuse in this population. METHODS: Urine samples from current (n = 41) and past (n = 31) androgen abusers and nonusers (n = 21) were analyzed by comprehensive mass spectrometry-based detection tests for androgens and related drugs (ARD). RESULTS: No prohibited ARDs were identified among nonusers. Current users had a median of 5 (range 1-13) drugs detected comprising 176 ARDs among 220 drug identifications. Past users had a median of 1 (range 0-9) drugs detected comprising 21 ARDs among 43 drugs. Negative predictive value was high (>0.8) for those denying drug usage while positive predictive value was good (>0.6) for both those reporting currently using (current) and not using (nonusers plus past users) ARD. Serum luteinizing hormone (LH) alone had high, but imperfect, discriminatory power (89%) to distinguish between current and noncurrent androgen use. CONCLUSIONS: We demonstrates that a negative drug history in a high-risk group has high reliability and that even a single suppressed serum LH exhibits high discrimination for objectively detecting androgen abuse.

Profiling Urinary Sulfate Metabolites With Mass Spectrometry.

The study of urinary phase II sulfate metabolites is central to understanding the role and fate of endogenous and exogenous compounds in biological systems. This study describes a new workflow for the untargeted metabolic profiling of sulfated metabolites in a urine matrix. Analysis was performed using ultra-high-performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with data dependent acquisition (DDA) coupled to an automated script-based data processing pipeline and differential metabolite level analysis. Sulfates were identified through k-means clustering analysis of sulfate ester derived MS/MS fragmentation intensities. The utility of the method was highlighted in two applications. Firstly, the urinary metabolome of a thoroughbred horse was examined before and after administration of the anabolic androgenic steroid (AAS) testosterone propionate. The analysis detected elevated levels of ten sulfated steroid metabolites, three of which were identified and confirmed by comparison with synthesised reference materials. This included 5α-androstane-3ß,17α-diol 3-sulfate, a previously unreported equine metabolite of testosterone propionate. Secondly, the hydrolytic activity of four sulfatase enzymes on pooled human urine was examined. This revealed that Pseudomonas aeruginosa arylsulfatases (PaS) enzymes possessed higher selectivity for the hydrolysis of sulfated metabolites than the commercially available Helix pomatia arylsulfatase (HpS). This novel method provides a rapid tool for the systematic, untargeted metabolic profiling of sulfated metabolites in a urinary matrix.

Performance- and image-enhancing drug use in the community: use prevalence, user demographics and the potential role of wastewater-based epidemiology.

Performance- and image-enhancing drug (PIED) misuse is a significant public health issue. Currently, seizure data, surveys, anti-doping testing, and needle service provider data are used to estimate PIED use in populations. These methods are time consuming, single point-in-time measurements that often consist of small sample sizes and do not truly capture PIED prevalence. Wastewater-based epidemiology (WBE) has been used globally to assess and monitor licit and illicit drug consumption within the general community. This method can objectively cover large populations as well as specific subpopulations (gyms, music festivals, prisons), and has potential as a complementary monitoring method for PIED use. Information obtained through WBE could be used to aid public health authorities in developing targeted prevention and education programmes. Research on PIED analysis in wastewater is limited and presents a significant gap in the literature. The focus is on anabolic steroids, and one steroid alternative currently growing in popularity; selective androgenic receptor modulators. This encompasses medical uses, addiction, prevalence, user typology, and associated public health implications. An overview of WBE is described including its benefits, limitations and potential as a monitoring method for PIED use. A summary of previous work in this field is presented. Finally, we summarise gaps in the literature, future perspectives, and recommendations for monitoring PIEDs in wastewater.

Production of certified reference materials for the sports doping control of the REV-ERB agonist SR9009.

Two human metabolites of the REV-ERB agonist SR9009, identified by researchers with an interest in sports doping control, have been synthesized and assessed for purity. The synthesis employed was a modification of published procedures for the parent SR9009, careful attention to the purification of intermediates and the final product ensuring materials of the highest purity were available for certification. For each candidate material impurities of related structure were identified and quantified as a relative mass fraction using high performance liquid chromatography-ultraviolet (HPLC-UV) detection and proton nuclear magnetic resonance (1 H NMR) spectroscopy. The quantification of water, occluded solvent, and inorganic residue was assessed using Karl Fischer, 1 H NMR, and thermogravimetric analysis, thereby completing the assessment of all impurities typically characterized by the mass balance approach. Summation and subtraction from 1000 mg/g afforded the mass fraction of the main component, the associated uncertainty ensuring certified reference material status can be applied to the resulting pure substance calibration standards. The availability of these standards to the sports doping control community will facilitate delivery of metrological traceability to the SI unit for mass (kg) to routine testing results and aid method development for the detection and quantification of SR9009 abuse.

Stable carbon isotope ratio profiling of illicit testosterone preparations--domestic and international seizures.

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.

The synthesis and pharmacological evaluation of adamantane-derived indoles: cannabimimetic drugs of abuse.

Two novel adamantane derivatives, adamantan-1-yl(1-pentyl-1H-indol-3-yl)methanone (AB-001) and N-(adamtan-1-yl)-1-pentyl-1H-indole-3-carboxamide (SDB-001), were recently identified as cannabimimetic indoles of abuse. Conflicting anecdotal reports of the psychoactivity of AB-001 in humans, and a complete dearth of information about the bioactivity of SDB-001, prompted the preparation of AB-001, SDB-001, and several analogues intended to explore preliminary structure-activity relationships within this class. This study sought to elucidate which structural features of AB-001, SDB-001, and their analogues govern the cannabimimetic potency of these chemotypes in vitro and in vivo. All compounds showed similar full agonist profiles at CB1 (EC50 = 16-43 nM) and CB2 (EC50 = 29-216 nM) receptors in vitro using a FLIPR membrane potential assay, with the exception of SDB-002, which demonstrated partial agonist activity at CB2 receptors. The activity of AB-001, AB-002, and SDB-001 in rats was compared to that of Δ(9)-tetrahydrocannabinol (Δ(9)-THC) and cannabimimetic indole JWH-018 using biotelemetry. SDB-001 dose-dependently induced hypothermia and reduced heart rate (maximal dose 10 mg/kg) with potency comparable to that of Δ(9)-tetrahydrocannabinol (Δ(9)-THC, maximal dose 10 mg/kg), and lower than that of JWH-018 (maximal dose 3 mg/kg). Additionally, the changes in body temperature and heart rate affected by SDB-001 are of longer duration than those of Δ(9)-THC or JWH-018, suggesting a different pharmacokinetic profile. In contrast, AB-001, and its homologue, AB-002, did not produce significant hypothermic and bradycardic effects, even at relatively higher doses (up to 30 mg/kg), indicating greatly reduced potency compared to Δ(9)-THC, JWH-018, and SDB-001.

Carbon isotope ratio analysis of endogenous glucocorticoid urinary metabolites after cortisone acetate and adrenosterone administration for doping control.

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 samples. A comprehensive sample 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 11ß-hydroxyetiocholanolone, 11-oxoetiocholanolone and 11ß-hydroxyandrosterone, while pregnanediol functioned as the endogenous reference compound. Urine samples 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 individuals. 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.

Development of a GC/C/IRMS method--confirmation of a novel steroid profiling approach in doping control.

In doping control, an athlete can only be convicted with the misuse with endogenous steroids like testosterone (T), if abnormal values of steroid metabolites and steroid ratios are observed and if the subsequent analysis with isotope ratios mass spectrometry (IRMS) confirms the presence of exogenously administered androgens. In this work, we compare the results of a novel steroid profiling approach with the performance an in-house developed IRMS method. The developed IRMS has the advantage over other methods to be relatively short in time and with target compounds androsterone, etiocholanolone, 5ß-androstane 3α,17ß-diol and 5α-androstane 3α,17ß-diol. Pregnanediol was used as an endogenous reference compound (ERC). Reference limits for the IRMS values were established and applied as decision limits for the evaluation of excretion urine from administration with oral T, T-gel, dihydrotestosterone (DHT) - gel and dehydroepiandrosterone (DHEA). Results indicated the importance of both androstanediols as important IRMS markers where relative values compared to an ERC (Δδ(13)C) yielded better detection accuracy than absolute δ(13)C-values. The detection times of all administered endogenous steroids were evaluated using the proposed thresholds. The results of traditional steroid profiling and a new approach based upon minor steroid metabolites monitoring introduced in a longitudinal framework were evaluated with IRMS. With traditional steroid profiling methods, 95% of the atypical samples could be confirmed whereas an additional 74% of IRMS confirmed was provided by a new biomarkers strategy. These results prove that the other steroid profiling strategies can improve the efficiency in detection of misuse with endogenous steroids.

Development of criteria for the detection of adrenosterone administration by gas chromatography-mass spectrometry and gas chromatography-combustion-isotope ratio mass spectrometry for doping control.

Adrenosterone (androst-4-ene-3,11,17-trione, 11-oxoandrostenedione) is an endogenous steroid hormone that has been promoted as a dietary supplement capable of reducing body fat and increasing muscle mass. It is proposed that adrenosterone may function as an inhibitor of the 11beta-hydroxysteroid dehydrogenase type 1 enzyme (11beta-HSD1), which is primarily responsible for reactivation of cortisol from cortisone. The urinary metabolism of adrenosterone was investigated, after a single oral administration in two male subjects, by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Substantially increased excretion of 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, 11-oxoandrosterone and 11-oxoetiocholanolone was observed. Minor metabolites such as 3alpha,17beta-dihydroxy-5beta-androstan-11-one, 3alpha-hydroxyandrost-4-ene-11,17-dione and 3alpha,11beta-dihydroxyandrost-4-en-17-one were also identified. The exogenous origin of the most abundant adrenosterone metabolites was confirmed by GC-C-IRMS according to World Anti-Doping Agency criteria. Through analysis of a reference population data set obtained from urine samples provided by elite athlete volunteers (n = 85), GC-MS doping control screening criteria are proposed: 11beta-hydroxyandrosterone concentration greater than 10 000 ng/mL (specific gravity adjusted to 1.020) or 11beta-hydroxyandrosterone/11beta-hydroxyetiocholanolone ratio greater than 20.Urine samples fulfilling these screening criteria may be subjected to GC-C-IRMS analysis for confirmation of adrenosterone administration.