Unraveling the anabolic-androgenic steroid profile of untreated horses by UHPLC-MS/MS

Clean sport is a priority for the regulatory organs of both the horse racing industry (IFHA, International Federation of Horseracing Authorities) and the Equestrian sports (FEI, Fédération Equestre Internationale). Urinary and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AAS). Because of their importance as ergogenic drugs of abuse and their involvement in equine reproduction, AAS remain an important field of investigation. Consequently, based on the suspected endogenous origin of some forbidden AAS (ß-boldenone as the most elicit one) this study focused on non-treated, out of competition horses, to improve the knowledge on the natural endogenous AAS profile of horses in general. In total, 105 guaranteed non-treated horses (47 geldings, 53 mares and 5 stallions serving as a control) were screened for ß-boldenone and five related steroids: ADD (androstadienedione), AED (androstenedione), αT (alpha-testosterone), ßT (ß-testosterone) and P (progesterone) via UHPLC-MS/MS (Triple Quadripole). The extraction and detection method was validated according to EC guidelines for ß-boldenone, ADD, AED, αT and ßT. Correlations between the occurrences of these AAS were calculated and hypotheses for the alleged endogenous traces were formulated. By using in vitro digestive simulations we were able to test different hypotheses