A steroidomic approach for biomarkers discovery in doping control

Anti-doping authorities have high expectations of the athlete steroidal passport (ASP) for anabolic-androgenic steroids misuse detection. However, it is still limited to the monitoring of known well-established compounds and might greatly benefit from the discovery of new relevant biomarkers candidates. In this context, steroidomics opens the way to the untargeted simultaneous evaluation of a high number of compounds. Analytical platforms associating the performance of ultra-high pressure liquid chromatography (UHPLC) and the high mass-resolving power of quadrupole time-of-flight (QTOF) mass spectrometers are particularly adapted for such purpose. An untargeted steroidomic approach was proposed to analyse urine samples from a clinical trial for the discovery of relevant biomarkers of testosterone undecanoate oral intake. Automatic peak detection was performed and a filter of reference steroid metabolites mass-to-charge ratio (m/z) values was applied to the raw data to ensure the selection of a subset of steroid-related features. Chemometric tools were applied for the filtering and the analysis of UHPLC-QTOF-MS(E) data. Time kinetics could be assessed with N-way projections to latent structures discriminant analysis (N-PLS-DA) and a detection window was confirmed. Orthogonal projections to latent structures discriminant analysis (O-PLS-DA) classification models were evaluated in a second step to assess the predictive power of both known metabolites and unknown compounds. A shared and unique structure plot (SUS-plot) analysis was performed to select the most promising unknown candidates and receiver operating characteristic (ROC) curves were computed to assess specificity criteria applied in routine doping control. This approach underlined the pertinence to monitor both glucuronide and sulphate steroid conjugates and include them in the athletes passport, while promising biomarkers were also highlighted.     

From population- to subject-based limits of T/E ratio to detect testosterone abuse in elite sports

In elite sports, indirect testing of testosterone abuse is mainly based on the testosterone over epitestosterone (T/E) ratio. Since this marker is characterized by a small ratio of intra- to inter-individual variation, it is surprising that current anti-doping strategy uses a screening test based on a population-based limit. From a database of more than 15,000 steroid profiles obtained from routine controls, the collection of steroids profiles of 11 elite athletes followed during 2 years, and a longitudinal study involving 17 amateur athletes, 8 of which were orally administrated testosterone undecanoate pills, we selected 12 case studies to represent the possible scenarios to which the anti-doping laboratories are confronted. Various detection strategies at the disposal of the laboratories are employed and discussed, including isotope ratio mass spectrometry (IRMS) analysis and a Bayesian interpretation of the T/E-time profile. The weak sensitivity versus specificity relation of a population-based limit for the T/E ratio is outlined. As a result, we propose a Bayesian screening test whose T/E threshold progressively evolves from a population basis to a subject basis as the number of individual test results increases. We found that this screening test heightens drastically the capacity to detect testosterone abuse, at no additional financial and administrative expenses for anti-doping authorities.