Evaluation of sulfate metabolites as markers of intramuscular testosterone administration in Caucasian and Asian populations.

The introduction of alternative markers to the steroid profile can be an effective approach to improving the screening capabilities for the detection of testosterone (T) misuse. In this work, endogenous steroid sulfates were evaluated as potential markers to detect intramuscular (IM) T administration. Fourteen sulfate metabolites were quantified using mixed-mode solid-phase extraction and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples after a single IM injection (100 mg) of T cypionate to six Caucasian and six Asian healthy male volunteers were analyzed. Principal component analysis (PCA) was used to characterize the sample cohort and to obtain the most useful markers for discrimination between pre- and post-administration samples. For Caucasian volunteers, a separation between pre- and post-administration samples was observed in PCA, whereas for Asian volunteers no separation was obtained. Seventeen ratios between sulfate metabolites were selected and further considered. Detection times (DTs) of each marker were evaluated using individual thresholds for each volunteer. The best results were obtained using ratios involving T and epitestosterone (E) sulfates in the denominator. The best marker was the ratio androsterone sulfate/testosterone sulfate (A-S/T-S) which prolonged the DT 1.2-2.1 times in respect to those obtained using T/E ratio in all Caucasian volunteers and 1.3-1.5 times in two Asian volunteers. Other ratios between A-S or etiocholanolone sulfate and E-S, and sulfates of etiocholanolone, dehydroandrosterone or epiandrosterone, and T-S were also found adequate. These ratios improve the DT after IM T administration and their incorporation to complement the current steroid profile is recommended.

Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part I: Transdermal administration.

Although the introduction by the World Anti-Doping Agency (WADA) of the steroid module of the athlete biological passport (ABP) marked an important step forward in the screening of testosterone (T) misuse, it still remains one of the most difficult challenges in doping control analysis. The urinary determination of alternative markers has been recently reported as a promising tool for improving the screening of T oral administration. However, their evaluation for other, commonly used, administration routes is still required. The main goal of this study is the evaluation of the potential of 2 groups of metabolites (cysteinyl conjugated and glucuronoconjugated) after transdermal and intramuscular administration of T. Their suitability was evaluated in individuals with both low basal (L-T/E) and medium basal (M-T/E) values of T/E. In this Part I, we evaluated the urinary excretion profile of these 2 groups of T metabolites after the administration of 3 doses of T gel to 12 volunteers (6 L-T/E and 6 M-T/E) for 3 consecutive days. For this purpose, 9 different concentration ratios (5 cysteinyl conjugated and 4 glucuronoconjugated markers) were studied. Both, the intra-individual variability and the detection windows (DW) obtained by each ratio were evaluated. Cysteinyl conjugates showed a general low intra-individual variability and DWs that were shorter than any other tested marker. Despite the relatively large intra-individual variability, the DWs reached by glucuronoconjugates (2-3 days) were similar to those obtained by markers currently included in the ABP. Overall; this evaluation advises for the introduction of additional glucuronoconjugated markers in the screening of transdermal T administration.

Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part II: Intramuscular administration.

In the fight against doping, the introduction of alternative markers to the steroid profile can be considered as an effective approach to improve the screening capabilities for the detection of testosterone (T) misuse. The aim of this study was to evaluate the potential of several T metabolites (cysteinyl conjugated and glucuronoconjugated resistant to enzymatic hydrolysis) to detect both the transdermal and the intramuscular administration of T. In Part I of the study, we studied the potential of these metabolites for the detection of T transdermal administration. Results revealed that resistant glucuronides can be a suitable complement to the current steroid profile. In this, Part II, dedicated to the intramuscular administration, we studied the potential of cysteinyl conjugated, resistant glucuronoconjugated and 1-cyclopentenoylglycine (1-CPG) for the detection of a single intramuscular injection of T cypionate. Possible differences in the excretion profile of all markers were explored between individuals with low basal (n=6) and medium basal (n=6) values of the testosterone/epitestosterone ratio (T/E). The results showed that all tested markers presented low intra-individual stability in basal conditions. Despite this, all glucuronoconjugated markers and 1-CPG, but not the cysteinyl conjugated markers, provided detection windows that were similar or longer than those obtained by markers currently included in the steroid profile. Based on the results obtained from the 2 parts of this study and from previously reported data, the potential applicability and the limitations of including these markers in the steroid profile are discussed.

LC-MS/MS Determination of Testolactone in Human Urine / 中国运动医学杂志

Objecave To develop a sensitive and specific LC-MS/MS method for determination of testolactone in human urine.Methods A C_(18 )column(2.1×50mm,3.5μm) was used.The mobile phase Was a mixture of acetonitrile and the buffer solution(ammonium acetate-water solution adjusted with formic acid to pH 3.5)at a flow rate of 0.5ml/min.A mass spectrometer equipped with electrospray ionization source was used as a detector and operated in the positive mode.In multiple reaction monitoring(MRM)mode,the ion transitions of m/z 301→121 and m/z 301→25 was used to qualify and quantify the testolactone,respectively.Results Chromatograms showed no endogenous interfering peaks with the urine blank sample.Each analysis was completed within 7min The calibration wag linear in the concentration range within 0.1～50μg/ml.The intra-batch and inter-batch RSD were less than 10%.The recovery rate of the extraction was about 60%.Conclusions The method is proved to meet the requirements of WADA and be suitable for routine screening.

A Survey of Medication Taken by Chinese Athletes / 中国运动医学杂志

Objective To make a survey of medication taken by Chinese athletes and a comparison between Chinese athletes and athletes from other countries in order to get information about how to improve Chinese athletes' performance. Method The information came from the forms"Doping Control Sample Collection" in which athletes answered the question: "What medications have you taken in the past 3 days?" The medicines taken by athletes were classified and statistically analyzed.Results 2,330 athletes and 25 kinds of sports were involved in. Medicines were statistically analyzed with 4 classes: profiling of declaration, vitamins and minerals, medicines for treatments, alternative medicine. Conclusion The survey recorded the types of supplements and medications taken by athletes in China in 1999. Chinese athletes took less vitamins and more alternative medicines than athletes from other countries.

Statistical Analysis of the Urine Steroid Profile in Chinese Athletes / 中国运动医学杂志

Objective To investigate the steroid levels of urine in Chinese athletes. Methods Subjects were 1217 Chinese athlete, 8 kinds of steroids named androsterone (An), etiocholanolone (Etio), dihydrotestosterone (DHT), dehydroepiandrosterone (DHET), Testosterone (T), Epitestosterone (ET), 5?-androstane-3?, 17?-diol (5?-diol), 5?-androstane-3?, 17?-diol (5?-diol) were measured by gas chromatography combined with mass spectrometry (GC/MS) after the urine sample being treated with chemical pre-extraction, hydrolysis, extraction and derivatization. Results The urine steroid levels of the Chinese male athlete were higher than those of the female athlete, but both male and female athlete had a lower urine steroids especially testosterone and 5?-androstane-3?,17?-diol, 4～7 times lower than other country's athletes. Some ratios of urine steroids such as An/Etio, T/ET, 5?-diol/5?-diol were relatively steady, which were affected less by gender.