Stability of Phase II long term sulfated steroid metabolites for their use in Quality Assessment exercises and routine doping control.

Code: 23A03JP

Sulfated phase II metabolites have gained great interest as long term metabolites of many anabolic steroids. The direct analysis of those sulfates using LC/MSMS has been extensively used as the research tool to finding new long-term metabolites. However, sulfates are known to be potentially unstable, and very particularly at acidic pH where catalysis occurs. Urine samples normally have acidic pH and the stability of those precious long-term metabolites in the biological matrix needs to be investigated. The preparation of reference urine samples to be used within the EQAS quality Assessment scheme has led us to obtaining shocking evidence on the instability of some of the analytes of interest in routine doping control. In particular, some long-term sulfated phase II steroid metabolites. As a good example, results obtained when assaying the stability of a excretion study of danazol, 7 days at different temperatures showed a huge increase in the concentration of the two metabolites (ethisterone and 2-OH-methyl-ethisterone). This increase is attributed to the hydrolysis of their sulfated phase II counterparts. The aim of the present project is the study of the stability of sulfated phase II metabolites using available excretion studies of danazol, mesterolone, clostebol and drostanolone, all having known sulfated long-term metabolites, as proof of concept. The idea is developing conditions to keep those sulfates stable for sample preparation and/or establish the conditions for a complete hydrolysis in order to target the analysis of the resulting compounds.