One of the major challenges in doping control testing in recent years for both human and animal sports has been the analysis of bioactive peptides in biological samples. Bioactive peptides are considered to have a high potential for abuse owing to their high potency and low toxicity.  This paper describes a sensitive method for the detection of 32 bioactive peptides (at low ppb 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 32 target peptides and/or their in vitro metabolites from horse urine. The final extract was analysed using UHPLC-HRMS with positive electrospray ionization (ESI) under both full-scan and data independent acquisition (DIA) modes. The estimated limits of detection (LoD) for most of the target peptides could reach down to 10 pg/mL in horse urine. This method was fully validated for qualitative identification purposes. The validation data, including method specificity, sensitivity, extraction recovery, precision and matrix effect will be presented. The applicability of this method was demonstrated by the positive identification of N-acetylated Leu-Lys-Lys-Thr-Glu-Thr-Gln and its metabolite N-acetylated Leu-Lys in urine obtained after subcutaneous administration of TB-500 to two thoroughbred geldings. A comprehensive 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 also been incorporated into our current method for controlling the misuse of GnRHs. Preliminary investigation showed that the method was also capable of detecting additional bioactive peptides, including alexmorelin, carpomorelin, deslorelin, GHRP-3, GHRP-4, GHRP-5, gonadorelin (luteinizing hormone releasing hormone), histrelin, MK677, ornipressin and peforelin, suggesting that the method has good potential to accommodate additional target peptides.