Ultra high performance liquid chromatography/tandem mass spectrometry based identification of steroid esters in serum and plasma: An efficient strategy to detect natural steroids abuse in breeding and racing animals

Abstract

During last decades, the use of natural steroids in racing and food producing animals for doping purposes has been flourishing. The endogenous or exogenous origin of these naturally occurring steroids has since remained a challenge for the different anti-doping laboratories. The administration of these substances to animals is usually made through an intra-muscular pathway with the steroid under its ester form for a higher bioavailability and a longer lasting effect. Detecting these steroid esters would provide an unequivocal proof of an exogenous administration of the considered naturally occurring steroids. A quick analytical method able to detect at trace level (below 50pg/mL) a large panel of more than 20 steroid esters in serum and plasma potentially used for doping purposes in bovine and equine has been developed. Following a pre-treatment step, the sample is submitted to a solid phase extraction (SPE) before analysis with UPLC–MS/MS. The analytical method's efficiency has been probed through three different in vivo experiments involving testosterone propionate intra-muscular administration to three heifers, 17-estradiol benzoate intra-muscular administration to a bull and a heifer and nandrolone laurate intra-muscular administration to a stallion. The results enabled detecting the injected testosterone propionate and 17-estradiol benzoate 2 and 17 days, respectively, post-administration in bovine and nandrolone laurate up to 14 days post-administration in equine. The corresponding elimination profiles in bovine serum and equine plasma have been established. The first bovine experiment exhibited a maximal testosterone propionate concentration of 400pg/mL in one of the three heifer serum within 5h post-administration. The second bovine experiment reported a maximal 17-estradiol benzoate concentration of 480pg/mL in the same matrix recorded 9 days after its administration. The last equine experiment resulted in a maximal nandrolone laurate concentration of 440pg/mL in horse plasma 24h after administration.