Rapid Annotation Strategy for in Vivo Phase II Metabolites of Anabolic-Androgenic Steroids Using Liquid...

Doping control laboratories are responsible for the precise measurement of anabolic-androgenic steroids (AASs) and determination of athlete usage. Intact phase II AASs are difficult to analyze due to their low abundance in complex biological matrices and their structural similarities that convolute tandem mass spectrometry interpretation. Discovery efforts of unknown phase II metabolites of new-to-the-field steroids have been challenging due to these deficiencies in current analytical techniques. Several methods for determining unknown conjugated AAS compounds have been developed that include deuterium tagging, fractionation, derivatization, and utilization of synthesized standards. Ion mobility (IM), a rapid gas-phase separation, allows for improved molecular differentiation and provides additional information for analyzing intact phase II AASs without sacrificing throughput. Here, candidate metabolites were putatively identified for oxymetholone (OXM) and methyl-1-testosterone (M1T) utilizing liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) and two independent data analysis strategies: a fully untargeted approach using mass defect analysis and collision cross section (CCS) filtering and a pseudotargeted approach using the biologically anticipated isotopic envelope in conjunction with CCS filtering, temporal profiling, and tandem mass spectrometry confirmation. A proof-of-concept time-course study was conducted using the urine from healthy male individuals after steroid administration. The fully untargeted approach reduced the number of original features by >85% while the pseudotargeted approach reduced original features by >99%, yielding 11 possible novel phase II AAS candidates for OXM and 23 for M1T.