Multiplex Nontargeted Framework Enables Tracking Metabolic Profile of Oxymetholone and Methasterone In Vivo at Nanogram Level by GC-Orbitrap-HRMS for Antidoping Purpose

Abstract

Oxymetholone and methasterone are anabolic androgenic steroids prohibited by the World Anti-Doping Agency (WADA) for both in-competition and out-of-competition use. Detecting metabolites of exogenous steroids is crucial for establishing doping violations, making the study of these metabolites essential in antidoping efforts. This study investigated the urinary metabolic profiles of oxymetholone and methasterone using gas chromatography-orbitrap high-resolution mass spectrometry (GC-Orbitrap-HRMS) in nanogram level by utilizing a novel multiplex nontargeted framework protocol. Healthy volunteers each ingested one tablet of the drug, and urine samples were collected over 50 days in postadministration phase. The complete detection of the three fractions (free fraction, glucuronide fraction, and sulfate fraction) of the metabolites was carried out. The GC-Orbitrap-HRMS full-scan mode was employed to detect postadministration urine samples, comparing these with preadministration baseline urine samples to identify newly formed substances. Electron ionization (EI) mass spectra were used to infer possible metabolite structures, leading to the discovery of three novel metabolites of oxymetholone and two novel metabolites of methasterone. Notably, the newly identified oxymetholone metabolite, 2-methylene-17α-methyl-androstane-16ξ,17β-diol-3-one (O-M6), was detectable as a glucuronide conjugate up to 4 days after administration. The methasterone metabolite, 18-nor-17β-hydroxymethyl-2α,17α-dimethyl-5α-androst-13-en-3-one (M-M4), exhibited prolonged detectability as a glucuronide conjugate, being present in urine samples from both volunteers up to 50 days after administration. These findings have significant implications for antidoping purpose, providing extended detection windows for these anabolic steroids.