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Endogenous steroid use can increase urinary testosterone/epitestosterone (T/E) values. In addition, ethanol in amounts >0.5 g per kg of body weight (g/kg) can also increase T/E values. However, the effect of smaller doses of ethanol on T/E values is unknown. The influence of 0.2 and 0.4 g/kg of ethanol on baseline T/E values in 20 men and 20 women with low and high baseline T/E values was investigated and correlated with ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentrations. T/E values for 7 of the women were excluded from the study because of undetectable T concentrations or for other reasons. One man and 1 woman with a high T/E baseline value had a significant increase in their T/E value after ingestion of 0.2 g/kg of ethanol. One man and 2 women with a high T/E baseline, and 1 woman with a low T/E baseline had significantly increased T/E values after ingestion of 0.4 g/kg of ethanol. There was wide variability in peak EtG concentrations and a lack of correlation between ethanol dose and EtG concentrations. Interestingly, 1 man and 2 women with increased T/E values following ethanol ingestion had EtG concentrations below the World Anti-Doping Agency (WADA) cut-off of 5000 ng/mL. These findings demonstrate that small amounts of ethanol can elevate T/E values, with women being more susceptible. In addition, consideration should be given to the lowering of the WADA EtG cut-off to detect samples with elevated T/E values from ingestion of low doses of ethanol.

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Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and can normalize suppressed testosterone concentrations in males following prolonged anabolic steroid use. Because of the potential for abuse by males, hCG is on the World Anti-Doping Agency (WADA) list of prohibited substances. The majority of WADA-accredited laboratories measure urinary hCG using an automated immunoassay. Only immunoassays that recognize the intact alpha and beta heterodimer of hCG (intact hCG) should be used to measure urinary hCG for doping control purposes since intact hCG is the only biologically active molecule. WADA further requires that confirmation testing is performed using an intact hCG immunoassay that is different from the one used in the initial testing procedure or by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study we measured the concentration of intact hCG, free ß-subunit (hCGß) and ß-subunit core fragment (hCGßcf) in 570, 275, and 256 male urine samples, respectively, by an immunoextraction LC-MS/MS method. Mean concentrations of intact hCG, hCGß and hCGßcf were 0.04 IU/L, 0.47 pmol/L and 0.16 pmol/L, respectively. The upper reference limits (97.5th percentile) for intact hCG, hCGß and hCGßcf were 0.21 IU/L, 0.40 pmol/L, and 1.86 pmol/L, respectively. Based on these data, we recommend a threshold of 1.0 IU/L for intact hCG (false positive rate of <1 in 10 000) for detecting male athletes that dope with hCG.

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Sports drug testing laboratories are required to detect several classes of compounds that are prohibited at all times, which include anabolic agents, peptide hormones, growth factors, beta-2 agonists, hormones and metabolic modulators, and diuretics/masking agents. Other classes of compounds such as stimulants, narcotics, cannabinoids, and glucocorticoids are also prohibited, but only when an athlete is in competition. A single class of compounds can contain a large number of prohibited substances and all of the compounds should be detected by the testing procedure. Since there are almost 70 stimulants on the prohibited list it can be a challenge to develop a single screening method that will optimally detect all the compounds. We describe a combined liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) testing method for detection of all the stimulants and narcotics on the World Anti-Doping Agency prohibited list. Urine for LC-MS/MS testing does not require sample pretreatment and is a direct dilute and shoot method. Urine samples for the GC-MS method require a liquid-liquid extraction followed by derivatization with trifluoroacetic anhydride.

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Isotope ratio mass spectrometry (IRMS) testing is performed to determine if an atypical steroid profile is due to administration of an endogenous steroid. Androsterone (Andro) and etiocholanolone (Etio), and/or the androstanediols (5α- and 5ß-androstane-3α,17ß-diol) are typically analyzed by IRMS to determine the (13) C/(12) C ratio. The ratios of these target compounds are compared to the (13) C/(12) C ratio of an endogenous reference compound (ERC) such as 5ß-pregnane-3α,20α-diol (Pdiol). Concentrations of Andro and Etio are high so (13) C/(12) C ratios can easily be measured in most urine samples. Despite the potentially improved sensitivity of the androstanediols for detecting the use of some testosterone formulations, additional processing steps are often required that increase labour costs and turnaround times. Since this can be problematic when performing large numbers of IRMS measurements, we established thresholds for Andro and Etio that can be used to determine the need for additional androstanediol testing. Using these criteria, 105 out of 2639 urine samples exceeded the Andro and/or Etio thresholds, with 52 of these samples being positive based on Andro and Etio IRMS testing alone. The remaining 53 urine samples had androstanediol IRMS testing performed and 3 samples were positive based on the androstanediol results. A similar strategy was used to establish a threshold for Pdiol to identify athletes with relatively (13) C-depleted values so that an alternative ERC can be used to confirm or establish a true endogenous reference value. Adoption of a similar strategy by other laboratories can significantly reduce IRMS sample processing and analysis times, thereby increasing testing capacity.

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Drug testing for sports doping control programs is extensive and includes numerous classes of banned compounds including anabolic androgenic steroids, ß2-agonists, hormone antagonists and modulators, diuretics, various peptide hormones, and growth factors. During competition, additional compounds may also be prohibited such as stimulants, narcotics, cannabinoids, glucocorticosteroids, and beta-blockers depending both on the sport and level of competition. Each of these classes of compounds can contain many prohibited substances that must be identified during the testing procedure. Various methods that have been designed to detect a large number of compounds in different drug classes are highly desirable as initial screening tools. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is widely used by anti-doping testing laboratories for this purpose and several rapid methods have been described to simultaneously detect different classes of compounds. Here, we describe a simple urine sample cleanup procedure that can be used to detect numerous anabolic androgenic steroids, ß2-agonists, hormone antagonists and modulators, glucocorticosteroids, and beta-blockers by LC-MS/MS.

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Madol (17alpha-methyl-5alpha-androst-2-en-17beta-ol) was identified in an oily product received by our laboratory in the context of our investigations of designer steroids. The product allegedly contained an anabolic steroid not screened for in routine sport doping control urine tests. Madol was synthesized by Grignard methylation of 5alpha-androst-2-en-17-one and characterized by mass spectrometry and NMR spectroscopy. We developed a method for rapid screening of urine samples by gas chromatography/mass spectrometry (GC/MS) of trimethylsilylated madol (monitoring m/z 143, 270, and 345). A baboon administration study showed that madol and a metabolite are excreted in urine. In vitro incubation with human liver microsomes yielded the same metabolite. Madol is only the third steroid never commercially marketed to be found in the context of performance-enhancing drugs in sports.

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Tetrahydrogestrinone (18a-homo-pregna-4,9,11-trien-17beta-ol-3-one or THG) was identified in the residue of a spent syringe that had allegedly contained an anabolic steroid undetectable by sport doping control urine tests. THG was synthesized by hydrogenation of gestrinone and characterized by mass spectrometry and NMR spectroscopy. We developed and evaluated sensitive and specific methods for rapid screening of urine samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS) of underivatized THG (using transitions m/z 313 to 241 and 313 to 159) and gas chromatography/high-resolution mass spectrometry (GC/HRMS) analysis of the combination trimethylsilyl ether-oxime derivative of THG (using fragments m/z 240.14, 254.15, 267.16, and 294.19). A baboon administration study showed that THG is excreted in urine.

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Norbolethone (13-ethyl-17-hydroxy-18,19-dinor-17alpha-pregn-4-en-3-one) is a 19-nor anabolic steroid first synthesized in 1966. During the 1960s it was administered to humans in efficacy studies concerned with short stature and underweight conditions. It has never been reported by doping control laboratories. Norbolethone was identified in two urine samples from one athlete by matching the mass spectra and chromatographic retention times with those of a reference standard. The samples also contained at least one likely metabolite. The samples were also unusual because the concentrations of endogenous steroids were exceptionally low. Since norbolethone is not known to be marketed by any pharmaceutical company, a clandestine source of norbolethone may exist.

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Androstenedione is a steroid hormone sold over-the-counter to individuals who expect that it will enhance strength and athletic performance. Endogenous androstenedione is the immediate precursor of testosterone. To evaluate the metabolism of oral androstenedione, we randomly assigned 37 healthy men to receive 0 (group 1), 100 mg (group 2), or 300 mg (group 3) of androstenedione in a single daily dose for 7 days. Eight-hour urines were collected 1 day before the start of androstenedione, and on days 1 and 7. Using gas chromatography-mass spectrometry, we measured excretion rates of glucuronide-conjugated epitestosterone, its putative precursor (E-precursor), and metabolites (EM-1 and EM-2), and we evaluated possible markers of androstenedione administration. Day 1 and 7 rates were not different: the means were averaged. The means (microg/h) for groups 1, 2, and 3, respectively were, for epitestosterone 2.27, 7.74, and 18.0; for E-precursor, 2.9, 2.0, and 1.5; for EM-1/E-precursor 0.31, 1.25, and 2.88; for EM-2/E-precursor 0.14, 0.15, and 1.15; for testosterone/epitestosterone (T/E) 1.1, 3.5, and 3.2. Epitestosterone, EM-1, and EM-2 excretion was greater in groups 2 and 3 versus group 1 (0.0001 < P < 0.03), as were EM-1/E-precursor, EM-2/E-precursor, and T/E. E-precursor excretion was lower in groups 2 (P = 0.08) and 3 (P = 0.047) versus group 1. Androstenedione increases excretion of epitestosterone and its two metabolites, while decreasing that of its precursor. Elevated ratios of EM-1- and EM-2/E-precursor, and the presence of 6alpha-hydroxyandrostenedione are androstenedione administration markers.

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