Development and validation of a UHPLC-HRMS method for the simultaneous determination of the endogenous anabolic...

Being performance enhancing hormones, endogenous anabolic androgenic steroids (EAAS) are banned from most competitive sports by the World Anti-doping Agency (WADA). In anti-doping control laboratories, routine assays are mainly performed on urine samples of athletes in and out of competitions. Serum constitutes a promising alternative to urine as it is less subjected to manipulation or contamination that may influence the method sensitivity. The simultaneous determination of EAAS including conjugated metabolites using LC-MS is very challenging due to their contradicting chemical behaviors at the ionization interface of the mass spectrometer. This may prejudice their detection or limit the method sensitivity. Herein, we have addressed these challenges and developed a new method for the simultaneous determination of unconjugated, sulphate- and glucuronide-conjugated EAAS (Androsterone, Etiocholanolone, testosterone, epitestosterone, dihydrotestosterone, dehydroepiandrosterone, androstenedione and 17a-hydroxyprogesterone) in human serum using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The use of mass spectrometric detection in full scan mode facilitated the study of the most versatile adducts for detection and quantitation. A solid phase extraction method was developed for the sample preparation prior to analysis. The method limits of quantitation ranged from 0.006 to 7.904 ng/mL and the recoveries ranged from 70.2% to 96.5%. The method calibration was performed in untreated serum representing realistic matrix composition with correlation coeffecients ranged from 0.9859 to 0.9988. Finally, the serum-levels of the investigated steroids were determined in 4 male and 1 female human subjects to provide estimates of baseline levels based on individual values.