Toward Routine Analysis of Anabolic Androgenic Steroids in Urine Using Ion Mobility-Mass Spectrometry

Anabolic androgenic steroids (AAS) make up one of the most prevalent classes of performance-enhancing drugs banned by the World Anti-Doping Agency (WADA) due to the competitive advantage they can afford athletes. Mass spectrometry-based methods coupled with chromatographic separations have become the gold standard for AAS analysis because of the superior sensitivity and selectivity provided. However, emerging analytical techniques including ion mobility spectrometry (IMS) have been demonstrated in recent applications as a means to further characterize and identify potential unknowns while simultaneously delivering improved sensitivity by filtering noise. Herein we outline the next crucial steps in bringing IMS to the routine drug testing workflow by combining it with established chromatographic and mass spectrometry methods (i.e., LC-IM-MS) for the detection of AAS in human urine. In addition to robust measurement of collision cross sections which can be used for identification purposes, functional group microtrends provide a structural basis on which to elucidate the structure of future novel anabolic agents. Lastly, the developed workflow is tested by analysis of testosterone in a realistic matrix (human urine) and demonstrates a limit of detection of 524 pg/mL, which surpasses the WADA Minimum Required Performance Levels for anabolic steroids. This work is expected to pave the way toward routine incorporation of IMS into analytical drug testing workflows to augment both qualitative and quantitative measure of performance enhancing drugs in the future.