title: A validated UHPLC-MS/MS method to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses.
abstract: Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with β-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to the Association of Official Racing Chemists (AORC) and European Commission (EC) guidelines, proving the power of this new method. Low limits of detection (0.2 ng/mL), good reproducibility (percentage of standard deviation (%RSD) < 10%), high recovery (94.6 to 117.1%), selectivity and specificity, and a linear response (confirmed with R(2) > 0.99 and lack-of-fit analysis) were obtained for all included AASs. With this method, urine samples of 105 guaranteed untreated horses (47 geldings, 53 mares, and 5 stallions serving as a control) were screened for β-boldenone and five related natural steroids: androstadienedione (ADD), androstenedione (AED), alpha-testosterone (αT), beta-testosterone (βT), and progesterone (P). Progesterone, β-testosterone, and α-testosterone were detected in more than half of the horses at low concentrations (<2 ng/mL). Occasionally, not only testosterone and progesterone but also low concentrations of AED, ADD, and boldenone (Bol) were found (0.5-5 ng/mL). Graphical Abstract A sensitive, new and fully validated UHPLC-MS/MS method has been developed that is able to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses (mares and geldings).
authors.affiliation: Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis, Ghent University, 133 Salisburylaan, 9820, Merelbeke, Belgium.
authors.forename: Anneleen
authors.initials: A
authors.lastname: Decloedt
authors.forename: Ludovic
authors.initials: L
authors.lastname: Bailly-Chouriberry
authors.forename: Julie
authors.initials: J
authors.lastname: Vanden Bussche
authors.forename: Patrice
authors.initials: P
authors.lastname: Garcia
authors.forename: Marie-Agnes
authors.initials: MA
authors.lastname: Popot
authors.forename: Yves
authors.initials: Y
authors.lastname: Bonnaire
authors.forename: Lynn
authors.initials: L
authors.lastname: Vanhaecke
mesh_terms: D045930:Anabolic Agents / Q000652:urine*; D000728:Androgens / Q000652:urine*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004300:Doping in Sports; D005260:Female; D006736:Horses / Q000652:urine*; D057230:Limit of Detection; D008297:Male; D015203:Reproducibility of Results; D013256:Steroids / Q000652:urine*; D015813:Substance Abuse Detection / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013739:Testosterone / Q000031:analogs & derivatives* / Q000652:urine
chemical_list: D045930:Anabolic Agents; D000728:Androgens; D013256:Steroids; D013739:Testosterone; C013915:boldenone