Development of mass spectrometry based methods for the detection of 11-ketotestosterone and 11-ketodihydrotestosterone

Code: 20A13MT

The detection of novel doping-related substances is challenging for doping control laboratories, especially if these compounds are also produced endogenously by the common human metabolism. In these cases not only the detection of these compounds or related metabolites is sufficient but (semi)quantification becomes necessary followed by confirmation of the exogenous source of the urinary metabolites by isotope ratio mass spectrometry. This procedure is well known for testosterone and its metabolites.

The novel doping agent under investigation in this study will be the 11-keto derivative of testosterone and its urinary metabolites. 11-Ketotestosterone (11KT) is described as a potent anabolic doping agent that is of special interest as it additionally showed the ability to lower cortisol levels. It is easily available via internet-based providers and at the moment no analytical strategy exists to detect its misuse. 11KT is closely related to a prohormone called adrenosterone (androste-4-ene-3,11,17-trione) but the proposed method for detection of adrenosterone will not be suitable for 11KT as it is only focusing on the major metabolites also produced in large amounts in the adrenal glands strongly limiting the sensitivity of this approach.

Within this research project, all currently known metabolites of 11KT will be taken into consideration to identify the most promising candidates for doping controls. First, the initial testing procedure will be developed to semi-quantitate the urinary amounts of all compounds of interest. Secondly, a confirmation procedure based on IRMS will be developed and validated. Both methods will be used to investigate an excretion study performed with 11KT in order to identify those metabolites most suitable for sports drug testing. This research will be completed by the investigation of routine doping control samples enabling to identify urinary baseline concentrations and carbon isotope ratios at natural abundance.

Main Findings

In this study, the human metabolism of KT was investigated in order to provide additional means for the detection of KT and its prohormone OHA4. Two volunteers (one female and one male) orally administered 20 mg of KT each, and urine samples were collected for 5 days. Urinary concentrations of KT and its metabolites were investigated, and a reference population encompassing 220 male and female athletes was investigated in order to elucidate preliminary thresholds. As confirmation procedure, an isotope ratio mass spectrometry-based method was developed in order to determine the CIR of KT and relevant metabolites. The developed methods enabled the detection of exogenous KT for more than 20 h after a single oral administration, which is comparable to a single oral testosterone administration.