96 research outputs found
Recent developments in the use of isotope ratio mass spectrometry in sports drug testing
According to the annual report of the World Anti-Doping Agency, steroids are the most frequently detected class of doping agents. Detecting the misuse of endogenously occurring steroids, i.e. steroids such as testosterone that are produced naturally by humans, is one of the most challenging issues in doping control analysis. The established thresholds for urinary concentrations or concentration ratios such as the testosterone/epitestosterone quotient are sometimes inconclusive owing to the large biological variation in these parameters. For more than 15years, doping control laboratories focused on the carbon isotope ratios of endogenous steroids to distinguish between naturally elevated steroid profile parameters and illicit administration of steroids. A variety of different methods has been developed throughout the last decade and the number of different steroids under investigation by isotope ratio mass spectrometry has recently grown considerably. Besides norandrosterone, boldenone was found to occur endogenously in rare cases and the misuse of corticosteroids or epitestosterone can now be detected with the aid of carbon isotope ratios as well. In addition, steroids excreted as sulfoconjugates were investigated, and the first results regarding hydrogen isotope ratios recently became available. All of these will be presented in detail within this review together with some considerations on validation issues and on identification of parameters influencing steroidal isotope ratios in urin
Urinary Analysis of Four Testosterone Metabolites and Pregnanediol by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry after Oral Administrations of Testosterone
The most frequently used method to demonstrate testosterone abuse is the determination of the testosterone and epitestosterone concentration ratio (T/E ratio) in urine. Nevertheless, it is known that factors other than testosterone administration may increase the T/E ratio. In the last years, the determination of the carbon isotope ratio has proven to be the most promising method to help discriminate between naturally elevated T/E ratios and those reflecting T use. In this paper, an excretion study following oral administration of 40 mg testosterone undecanoate initially and 13 h later is presented. Four testosterone metabolites (androsterone, etiocholanolone, 5α-androstanediol, and 5β-androstanediol) together with an endogenous reference (5β-pregnanediol) were extracted from the urines and the δ13C/12C ratio of each compound was analyzed by gas chromatography-combustion-isotope ratio mass spectrometry. The results show similar maximum δ13C-value variations (parts per thousand difference of δ13C/12C ratio from the isotope ratio standard) for the T metabolites and concomitant changes of the T/E ratios after administration of the first and the second dose of T. Whereas the T/E ratios as well as the androsterone, etiocholanolone and 5α-androstanediol δ13C-values returned to the baseline 15 h after the second T administration, a decrease of the 5β-androstanediol δ-values could be detected for over 40 h. This suggests that measurements of 5β-androstanediol δ-values allow the detection of a testosterone ingestion over a longer post-administration period than other T metabolites δ13C-values or than the usual T/E ratio approac
Detection of Exogenous GHB in Blood by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry: Implications in Postmortem Toxicology
Because GHB (γ-hydroxybutyrate) is present in both blood and urine of the general population, toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. In this paper, we propose a procedure for the detection of exogenous GHB in blood by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Following liquid-liquid and solid-phase extractions, GHB is derivatized to GHB di-TMS before analysis by GC-C-IRMS. Significant differences in the carbon isotopic ratio (Δδ13C-values > 13.5‰) were found between endogenous and synthetic GHB. Indeed, for postmortem blood samples with different GHB concentrations (range: 13.8-86.3 mg/L), we have obtained GHB δ13C-values ranging from −20.6 to −24.7‰, whereas δ13C-values for the GHB from police seizure were in the range −38.2 to −50.2‰. In contrast to the use of cut-off concentrations for positive postmortem blood GHB concentrations, this method should provide an unambiguous indication of the drug origi
Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing
Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5β-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 3β-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5β-androstane-3α,17β-diol (5aDIOL and 5bDIOL), 17β-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3β-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3β,17β-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n = 67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroi
Source inference of exogenous gamma-hydroxybutyric acid (GHB) administered to humans by means of carbon isotopic ratio analysis: novel perspectives regarding forensic investigation and intelligence issues
γ-Hydroxybutyric acid (GHB) is an endogenous short-chain fatty acid popular as a recreational drug due to sedative and euphoric effects, but also often implicated in drug-facilitated sexual assaults owing to disinhibition and amnesic properties. Whilst discrimination between endogenous and exogenous GHB as required in intoxication cases may be achieved by the determination of the carbon isotope content, such information has not yet been exploited to answer source inference questions of forensic investigation and intelligence interests. However, potential isotopic fractionation effects occurring through the whole metabolism of GHB may be a major concern in this regard. Thus, urine specimens from six healthy male volunteers who ingested prescription GHB sodium salt, marketed as Xyrem®, were analysed by means of gas chromatography/combustion/isotope ratio mass spectrometry to assess this particular topic. A very narrow range of δ13C values, spreading from −24.81‰ to −25.06‰, was observed, whilst mean δ13C value of Xyrem® corresponded to −24.99‰. Since urine samples and prescription drug could not be distinguished by means of statistical analysis, carbon isotopic effects and subsequent influence on δ13C values through GHB metabolism as a whole could be ruled out. Thus, a link between GHB as a raw matrix and found in a biological fluid may be established, bringing relevant information regarding source inference evaluation. Therefore, this study supports a diversified scope of exploitation for stable isotopes characterized in biological matrices from investigations on intoxication cases to drug intelligence programme
Plasma and urine profiles of Δ9-tetrahydrocannabinol and its metabolites 11-hydroxy-Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol after cannabis smoking by male volunteers to estimate recent consumption by athletes
Since 2004, cannabis has been prohibited by the World Anti-Doping Agency for all sports competitions. In the years since then, about half of all positive doping cases in Switzerland have been related to cannabis consumption. In doping urine analysis, the target analyte is 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), the cutoff being 15ng/mL. However, the wide urinary detection window of the long-term metabolite of Δ9-tetrahydrocannabinol (THC) does not allow a conclusion to be drawn regarding the time of consumption or the impact on the physical performance. The purpose of the present study on light cannabis smokers was to evaluate target analytes with shorter urinary excretion times. Twelve male volunteers smoked a cannabis cigarette standardized to 70mg THC per cigarette. Plasma and urine were collected up to 8h and 11days, respectively. Total THC, 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), and THC-COOH were determined after hydrolysis followed by solid-phase extraction and gas chromatography/mass spectrometry. The limits of quantitation were 0.1-1.0ng/mL. Eight puffs delivered a mean THC dose of 45mg. Plasma levels of total THC, THC-OH, and THC-COOH were measured in the ranges 0.2-59.1, 0.1-3.9, and 0.4-16.4ng/mL, respectively. Peak concentrations were observed at 5, 5-20, and 20-180min. Urine levels were measured in the ranges 0.1-1.3, 0.1-14.4, and 0.5-38.2ng/mL, peaking at 2, 2, and 6-24h, respectively. The times of the last detectable levels were 2-8, 6-96, and 48-120h. Besides high to very high THC-COOH levels (245 ± 1,111ng/mL), THC (3 ± 8ng/mL) and THC-OH (51 ± 246ng/mL) were found in 65 and 98% of cannabis-positive athletes' urine samples, respectively. In conclusion, in addition to THC-COOH, the pharmacologically active THC and THC-OH should be used as target analytes for doping urine analysis. In the case of light cannabis use, this may allow the estimation of more recent consumption, probably influencing performance during competitions. However, it is not possible to discriminate the intention of cannabis use, i.e., for recreational or doping purposes. Additionally, pharmacokinetic data of female volunteers are needed to interpret cannabis-positive doping cases of female athletes. Figure Urine concentration ranges of delta-9-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-delta-9-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in athletes tested cannabispositive (15ng/mL THC-COOH or more; N=81
Hidden figures: Revisiting doping prevalence estimates previously reported for two major international sport events in the context of further empirical evidence and the extant literature
Background: High levels of admitted doping use (43.6% and 57.1%) were reported for two international sport events in 2011. Because these are frequently referenced in evaluating aspects of anti-doping, having high level of confidence in these estimates is paramount.
Objectives: In this study, we present new prevalence estimates from a concurrently administered method, the Single Sample Count (SSC), and critically review the two sets of estimates in the context of other doping prevalence estimates.
Methods: The survey featuring the SSC model was completed by 1,203 athletes at the 2011 World Championships in Athletics (WCA) (65.3% of all participating athletes) and 954 athletes at the 2011 Pan-Arab Games (PAG) (28.2% of all participating athletes). At WCA, athletes completed both UQM and SSC surveys in randomised order. At PAG, athletes were randomly allocated to one of the two surveys. Doping was defined as “having knowingly violated anti-doping regulations by using a prohibited substance or method.”
Results: Estimates with the SSC model for 12-month doping prevalence were 21.2% (95% CI: 9.69–32.7) at WCA and 10.6% (95% CI: 1.76–19.4) at PAG. Estimated herbal, mineral, and/or vitamin supplements use was 8.57% (95% CI: 1.3–16.11) at PAG. Reliability of the estimates were confirmed with re-sampling method (n = 1,000, 80% of the sample). Survey non-compliance (31.90%, 95%CI: 26.28–37.52; p < 0.0001) was detected in the WCA data but occurred to a lesser degree at PAG (9.85%, 95% CI: 4.01–15.69, p = 0.0144 and 11.43%, 95% CI: 5.31–11.55, p = 0.0196, for doping and nutritional supplement use, respectively). A large discrepancy between those previously reported from the UQM and the prevalence rate estimated by the SSC model for the same population is evident.
Conclusion: Caution in interpreting these estimates as bona fide prevalence rates is warranted. Critical appraisal of the obtained prevalence rates and triangulation with other sources are recommended over “the higher rate must be closer to the truth” heuristics. Non-compliance appears to be the Achilles heel of the indirect estimation models thus it should be routinely tested for and minimised. Further research into cognitive and behaviour aspects, including motivation for honesty, is needed to improve the ecological validity of the estimated prevalence rates.publishedVersio
Circulating miRNAs: a new generation of anti-doping biomarkers
MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. Cell-free miRNAs detected in blood plasma are used as specific and sensitive markers of physiological processes and some diseases. Circulating miRNAs are highly stable in body fluids, for example plasma. Therefore, profiles of circulating miRNAs have been investigated for potential use as novel, non-invasive anti-doping biomarkers. This review describes the biological mechanisms underlying the variation of circulating miRNAs, revealing that they have great potential as a new class of biomarker for detection of doping substances. The latest developments in extraction and profiling technology, and the technical design of experiments useful for anti-doping, are also discussed. Longitudinal measurements of circulating miRNAs in the context of the athlete biological passport are proposed as an efficient strategy for the use of these new markers. The review also emphasizes potential challenges for the translation of circulating miRNAs from research into practical anti-doping application
Statistical discrimination of steroid profiles with support vector machines in doping control
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