Detection of Testosterone Esters in Urine: Evidence of Trace‐Level Detectability in Doping Control Samples

The detection of exogenous testosterone (T) remains a critical challenge in antidoping analysis due to its identical chemical structure to endogenous testosterone. Testosterone is often administered as synthetic esters (T-esters), which are not naturally produced by the human body and serve as unequivocal evidence of doping. Although T-esters are traditionally detected in blood samples, their presence in urine has been largely unexplored due to their apolar nature and rapid hydrolysis. In this study, we developed and validated an ultrasensitive LC-MS/MS method capable of detecting T-esters in urine at sub-pg/mL concentrations. The method employs derivatization with Girard P reagent to enhance sensitivity and selectivity. Application of the method to doping control urine samples revealed the presence of T-esters in cases with elevated testosterone/epitestosterone (T/E) ratios, providing what is, to our knowledge, the first evidence of trace-level detectability of T-esters in human urine. This novel approach offers a complementary tool for identifying exogenous testosterone use.