Determination of selected endogenous anabolic androgenic steroids and ratios in urine by ultra high performance liquid chromatography tandem mass spectrometry and isotope pattern deconvolution

•Robust determination of selected endogenous steroids in urine by LC-IDMS and isotope pattern deconvolution.•Accuracy and precision were successfully validated for T,E, A and Etio using deuterated analogs and two CRMs.•Method meets the WADA uncertainty requirements for T, E and T/E for Athlete Biological Passport assessment.•T/E uncertainty was well below usual variability in longitudinal profiles, improving profiling studies.•The developed method is a potential alternative to traditional GC- and calibration-based quantifications.

An isotope dilution mass spectrometry (IDMS) method for the determination of selected endogenous anabolic androgenic steroids (EAAS) in urine by UHPLC-MS/MS has been developed using the isotope pattern deconvolution (IPD) mathematical tool. The method has been successfully validated for testosterone, epitestosterone, androsterone and etiocholanolone, employing their respective deuterated analogs using two certified reference materials (CRM). Accuracy was evaluated as recovery of the certified values and ranged from 75% to 108%. Precision was assessed in intraday (n = 5) and interday (n = 4) experiments, with RSDs below 5% and 10% respectively. The method was also found suitable for real urine samples, with limits of detection (LOD) and quantification (LOQ) below the normal urinary levels. The developed method meets the requirements established by the World Anti-Doping Agency for the selected steroids for Athlete Biological Passport (ABP) measurements, except in the case of androsterone, which is currently under study.