Confirmation of ethanol administration in racing greyhounds by LC-MS-MS

Ethanol is a prohibited substance in professional animal racing as its administration causes physiological effects such as depression of the central nervous system. Regulation of potential doping agents, including those that inhibit performance, is critical to ensure integrity and animal welfare in greyhound racing, but the detection of ethanol is complicated by dietary and/or environmental exposure. In response, a reliable analytical method capable of detecting recent ethanol administration in greyhound urine samples was validated and implemented. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) was used to investigate the variation in urinary ethanol metabolites; ethyl-β-D glucuronide (EG; ${\overline{\gamma }}_{\mathrm{EG}}$ = 1.0 μg/ml, $s_{\mathrm{EG}}$ = 3.3 μg/ml) and ethyl sulfate (ES; ${\overline{\gamma }}_{\mathrm{ES}}$ = 0.9 μg/ml, $s_{\mathrm{ES}}$ = 1.9 μg/ml) levels from a reference population of 202 racing greyhounds. These were compared to urine samples collected following administration of ethanol to one male and one female greyhound. Results were used to establish a threshold within the national rules of greyhound racing: ${\overline{\gamma }}_{\mathrm{EG}}$ and ${\overline{\gamma }}_{\mathrm{ES}}$ > 20 μg/ml in urine are defensible criteria to confirm ethanol administration to greyhounds. Case studies of competition samples are provided to demonstrate the forensic translation of this work.