Comparison of urine analysis and dried blood spot analysis for the detection of ephedrine and methylephedrine in doping control.

When the misuse of stimulants is determined in doping control tests conducted during the in-competition period, athletes are asked to account for the violation of the rules. This study was designed to evaluate whether the urinary threshold values (10 µg/mL) for ephedrine and methylephedrine set by the World Anti-Doping Agency (WADA) can be exceeded after the oral administration of each substance (25 mg). In addition, the study describes the validity of a liquid chromatography-tandem mass spectrometric method using dried blood spot testing to detect ephedrine and methylephedrine by comparing it to a quantitative laboratory urine assay. After administration of ephedrine, the urinary concentration of ephedrine did not exceed the threshold at 4-10 h in two subjects, whereas the threshold was exceeded in both the subjects at 12 h after administration. For methylephedrine, the urinary concentrations of all the subjects failed to reach the threshold for up to 10 h after administration. The concentrations reached the threshold at 12-24 h after administration in some volunteers. In contrast, the blood concentrations of ephedrine and methylephedrine reached their maximum levels at 2-8 h after administration. The blood concentrations showed a low inter-individual variability, and the results suggested that the urinary excretion of ephedrine and methylephedrine can be strongly affected by urine pH and/or urine volume. These facts suggest that urinary concentrations cannot reflect the psychoactive level of ephedrines in circulation. Thus, dried blood analysis might be suitable for the adequate detection of stimulants during in-competition testing.

Determination of mepitiostane metabolites in human urine by liquid chromatography/tandem mass spectrometry for sports drug testing.

Mepitiostane (2α,3α-epithio-17ß-(1-methoxycyclopentyloxy)-5α-androstane), which is a prodrug of epitiostanol (2α,3α-epitio-5α-androstane-17ß-ol), is an epitiosteroid having anti-estrogenic and weak androgenic anabolic activities. The World Anti-Doping Agency prohibits the misuse of mepitiostane by athletes. Detection of the urinary metabolites epitiostanol sulfoxide and epitiostanol was studied using liquid chromatography/mass spectrometry (LC-MS) for doping control purposes. The use of LC-MS provided advantages over gas chromatography/mass spectrometry for detecting heat labile steroids because epitiostanol and epitiostanol sulfoxide were primarily pyrolized to 5α-androst-2-en-17ß-ol. The method consists of enzymatic hydrolysis using ß-glucuronidase (Escherichia coli), liquid-liquid extraction, and subsequent ultra-performance liquid chromatography/electrospray-tandem mass spectrometry. Epitiostanol sulfoxide was determined at urinary concentrations of 0.5-50ng/mL, recovery was 76.2-96.9%, and assay precision was calculated as 0.9-1.7% (intra-day) and 2.0-6.6% (inter-day). Epitiostanol was determined at urinary concentrations of 0.5-50ng/mL, recovery was 26.1-35.6% and assay precision was calculated as 4.1-4.6% (intra-day) and 3.3-8.5% (inter-day). The limits of detection for epitiostanol sulfoxide and epitiostanol were 0.05ng/mL and 0.10ng/mL, respectively. Epitiostanol sulfoxide and epitiostanol, as their gluco-conjugates, were identified in human urine after oral administration of 10mg mepitiostane. Epitiostanol sulfoxide and epitiostanol could be detected up to 48h and 24h after administration, respectively. The results showed that the detection window of epitiostanol is much shorter than that of epitiostanol sulfoxide. The LC-MS detection of urinary epitiostanol sulfoxide, a specific metabolite with a sulphur atom in its molecular structure, is likely to be able to identify the abuse of mepitiostane.