Acepromazine, a rapid-acting tranquiliser, is frequently administered as an equine veterinary medicine for its anxiolytic effects or in combination with anaesthesia for sedation during medical procedures. Its increased use in Australia for managing the behaviour of nervous or difficult horses has drawn recent attention. Within the context of the horseracing industry, its presence in race-day samples is controlled by International Screening Limits (ISL) due to its potential to alter performance, the associated dangers of racing a horse under its influence as well as other ethical and welfare concerns.
Following administration, acepromazine is rapidly and extensively metabolised and excreted primarily as the urinary metabolite 2-(1-hydroxyethyl)promazine sulphoxide (HEPS), this metabolite has been used to establish the ISL for acepromazine in urine. HEPS consists of four stereoisomers and is a structural isomer of another major metabolite, hydroxyethylhydroxypromazine (HEHP)1. Due to their structural similarity, HEPS and HEHP exhibit similar chromatographic behaviour and share similar fragment ions. During liquid chromatography-mass spectrometry analysis, these compounds may co-elute and appear as a single peak or as multiple partially resolved peaks depending on the chromatographic method employed, complicating their identification and reliable application of the ISL.
Accurate application of the ISL for acepromazine in urine relies on sufficient separation and correct identification of these multiple isomers. Insufficient separation or misidentification may lead to potential over- or underestimation of the concentration of HEPS in a sample. This work explores the analytical challenges involved in accurate identification of HEPS in race-day urine samples, with the aim of improving consistent application of the ISL during routine screening and confirmatory analysis.