A Comparison of Three Analytical Procedures for Oxandrolone Long-Term Metabolite Determination in Urine Samples

In contemporary antidoping analysis, doping control laboratories are confronted with a wide range of evolving analytical challenges. Continuous methodological refinement is required in this dynamic field, particularly with respect to analytical strategies for the detection and confirmation of prohibited substances. The present study focuses on the comparative evaluation of three analytical strategies for the determination of long-term metabolites (LTMs) of the anabolic androgenic steroid (AAS) oxandrolone (Oxa) in human urine. The identification and implementation of LTMs in routine doping control have substantially enhanced the detection capabilities of antidoping laboratories, leading to an increased number of adverse analytical findings, particularly for AAS compounds. The primary objective of this work was to develop and validate novel confirmatory analytical procedures for Oxa LTMs and to compare their performance with that of an already established method. The investigated approaches comprise the following: (1) gas chromatography-mass spectrometry (GC-MS) following enzymatic hydrolysis of phase II metabolites and subsequent silylation, (2) liquid chromatography-high-resolution mass spectrometry (LC-HRMS) of methylated phase II metabolites, and (3) LC-HRMS analysis of hydrolyzed phase II metabolites. The first two approaches are reported here for the first time in the context of LTM analysis in antidoping applications. These methodologies represent innovative analytical strategies that may be transferable to other target analytes and thereby contribute to further improvements in sports drug testing.