Efficient approach for the comprehensive detection of unknown anabolic steroids and metabolites in human urine by liquid chromatography-electrospray-tandem mass spectrometry.

The detection of new anabolic steroid metabolites and new designer steroids in urine is a challenge in doping analysis. An approach based on precursor ion scanning for the detection of unknown anabolic steroids and metabolites is proposed. The study of the MS/MS spectra of selected anabolic steroids revealed different fragmentation pathways at low and medium collision energy depending on the steroid structure. However, after analysis at high collision energy three common ions at m/z 105, m/z 91, and m/z 77 were found for all studied anabolic steroids. These ions can be explained by the fragmentation of the steroid structure and corresponded to the methyl tropylium, tropylium, and phenyl ions, respectively. Because of the theoretical low specificity of these ions, the simultaneous presence of all of them was used as a starting point to consider a substance as a possible anabolic steroid. Hence, the developed approach is based on the simultaneous acquisition of the precursor ion scan of m/z 105, 91, and 77. The specificity of this approach has been checked by the injection of several doping agents including beta-agonists, corticosteroids, beta-blockers, and diuretics. In general, only compounds with a steroidal structure showed a signal at all three selected m/z values although some exceptions have been found. The applicability of the method was tested for three different scenarios: the detection of steroid metabolites, the detection of unknown steroids, and the analysis of prohormones. In metabolic studies, several recently reported fluoxymesterone metabolites were also found using this method. For detection of unknown steroids, some negative urine samples were spiked with the designer steroid THG and 33 other anabolic steroids and treated as blind samples. Finally, the applicability of the developed approach for the analysis of dietary supplements was checked by the analysis of a prohormone where several impurities and/or degradation products were found.