DETECTION OF BIOACTIVE PEPTIDES INCLUDING GONADOTROPHIN-RELEASING FACTORS (GnRHs) IN HORSE URINE USING ULTRA-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-HIGH RESOLUTION MASS SPECTROMETRY (UHPLC/HRMS).

The use of bioactive peptides as a doping agent in both human and animal sports has become increasingly popular in recent years. As such, methods to control the misuse of bioactive peptides in equine sports have received attention. This paper describes a sensitive accurate mass method for the detection of 40 bioactive peptides and 2 non-peptide growth hormone secretagogues (<2 kDa) at low pg/mL level in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS). A simple mixed-mode cation exchange solid-phase extraction (SPE) cartridge was employed for the extraction of 42 targets and/or their in vitro metabolites from horse urine. The final extract was analysed using UHPLC/HRMS in positive electrospray ionisation (ESI) mode under both full scan and data independent acquisition (DIA, for MS2 ). The estimated limits of detection (LoD) for most of the targets could reach down to 10 pg/mL in horse urine. This method was validated for qualitative detection purpose. The validation data, including method specificity, method sensitivity, extraction recovery, method precision and matrix effect were reported. A thorough in vitro study was also performed on four gonadotrophin-releasing factors (GnRHs), namely leuprorelin, buserelin, goserelin and nafarelin, using the S9 fraction isolated from horse liver. The identified in vitro metabolites have been incorporated into the method for controlling the misuse of GnRHs. The applicability of this method was demonstrated by the identification of leuprorelin and one of its metabolites, Leu M4, in urine obtained after intramuscular administration of leuprorelin to a thoroughbred gelding (castrated horse).