Studies on the minor metabolite 6a-hydroxy-androstenedione for doping control purposes and its contribution to the...

Recent publications have shown that the concentrations of minor metabolites such as formestane and 6a-hydroxy-androstenedione (6aOHADION) are import parameters, capable of increasing the specificity and efficiency of steroid abuse screening. The importance of such minor metabolites has been recognized for some time, but setting up concentration thresholds is not that straightforward with a single quadrupole gas chromatograph mass spectrometer (GC-MS) because of the low concentrations; this is especially the case for 6aOH-ADION. The main aim of this study was to propose a concentration threshold above which the detected 6aOH-ADION is considered suspicious and isotope ratio mass spectrometry (IRMS) is recommended. Routine doping control samples (2128) from athletes that entered our lab and were not found suspicious for the intake of any doping substance were used to determine the baseline concentrations of 6a-OH-ADION. For this purpose, the more sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) was used, capable of quantifying these low concentrations with high reliability. A urinary concentration threshold of 5 ng/mL was set. Concentrations above this threshold are considered suspicious and are forwarded to IRMS for confirmation in routine practice. In addition, an IRMS method was developed, capable of determining the 13C value of 6aOH-ADION. If a urine sample has an elevated 6aOH-ADION concentration and normal 13C values for the traditional IRMS target compounds, we are still able to check the 13C value of 6aOH-ADION. Six excretion studies were executed to stress the applicability of the threshold by visualizing the concentration and δ13C value time profiles of 6aOH-ADION.