Detection of gonadotropin‐releasing hormone and its analogues in equine and canine urine by high‐resolution data‐independent acquisition

Abstract Gonadotropin‐releasing hormone (GnRH) and its synthetic analogues are considered banned substances by the racing industry. GnRH is used as a pharmaceutical to regulate the female oestrous cycle, but the hormone is also thought to increase the production of testosterone in male animals. Using liquid chromatography in conjunction with high‐resolution mass spectrometry (LC–HRMS) and data‐independent acquisition (DIA), a method is presented for the detection of intact and truncated peptides of GnRH and its analogues down to the low picogram level in equine urine. The study of the catabolism of GnRH and analogues in plasma, combined with the analysis of urine from administration studies, reveals a common pattern of peptide catabolites that can be used to guide the design of MS‐based screens for this class of drugs. This culminated in the successful detection of the peptide in two out‐of‐competition canine urine samples.