The abuse of performance-enhancing peptides is a threat to the integrity of both equine and human sports. Last year, a large number of post-race samples from different horse racing jurisdictions in the USA were reported positive for dermorphin (aka “frog venom”), a potent painkiller1. Besides, the Australian Crime Commission has recently reported the widespread use of peptides and hormones by professional athletes in Australia2. In order to tackle this challenge, the author’s laboratory has established a method to detect seven bioactive peptides and their in vitro metabolites in horse plasma3. This method has recently been expanded to cover a total of 16 bioactive peptides (namely, AOD9604, -casomorphin, bremelanotide, CJC-1295, dermorphin, desmopressin, GHRP-1, GHRP-2, GHRP-6, hexarelin, hyp6-dermorphin, ipamorelin, morphiceptin, N-acetylated LKKTETQ (active ingredient of TB-500), selank, and triptorelin). In addition, another method has been developed to detect 15 of the above peptides (excluding CJC-1295) and -casozepine in horse urine. This paper describes these two methods for the simultaneous screening of 16 bioactive peptides and some of their in vitro metabolites in horse urine or plasma. Urine and plasma extractions were performed using different mixed-mode ion exchange solid-phase extraction cartridges. The final extracts were subject to ultra-high performance liquid chromatographic separation on a C18 column and then analysed with LC/HRMS in positive ESI mode. The limits of detection for the target peptides were within 50 – 200 pg/mL in urine or plasma. The applicability of these multi-analyte methods was demonstrated by the identification of N-acetylated LKKTETQ and/or its metabolites in urine and plasma obtained after subcutaneous administration of TB-500 to two thoroughbred geldings. Method validation with respect to specificity, precision, recovery and ion suppression effect will be presented. A comprehensive in vitro study to identify potential metabolites useful for the control of these bioactive peptides will also be described.
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