Detection of bioactive peptides including gonadotrophin‐releasing factors (GnRHs) in horse urine using ultra‐high performance liquid chromatography–high resolution mass spectrometry (UHPLC/HRMS)

Abstract

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 two non‐peptide growth hormone secretagogues (< 2 kDa) at low pg/mL levels 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 analyzed using UHPLC/HRMS in positive electrospray ionization (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 purposes. 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).

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

“…In parallel experiments from the same paper, GnRH (identified as luteinizing hormone‐releasing hormone [LHRH]) produced a different pattern of N‐terminal catabolites when incubated with cytosolic proteases from the human liver. The GnRH analogues leuprorelin, buserelin, goserelin and nafarelin also produced N‐terminal catabolites when incubated with equine liver cytosols 14 . Given the contrasting results when peptides are incubated in plasma or with cytosolic liver proteases, there is a question of which model of catabolism best represents the metabolic processing that occurs in vivo after i.v.…”

“…In cases where the intact peptide is no longer detectable in the urine after administration, the detection of peptide catabolites (products of peptide catabolism) may be critical for controlling the use of these substances 13 . However, in the absence of administration data, there is a need to develop in vitro models of peptide catabolism to aid in the identification of the relevant urinary catabolites to target by mass spectrometry (MS) 12,14–18 . To this end, we compare the results of our own in vitro studies of GnRH catabolism with the analysis of urine from an administration study and urine samples collected in the course of routine analytical screening.…”

Doping control analysis of small peptides: A decade of progress

“…This pioneering work only included GHRPs, however, as the extension of the method to other chemically and pharmacologically different peptides (e.g. desmopressin, LHRH) [43] was gradually realized, it became the standard extraction protocol in many laboratories, as well as in the field of horse doping [44]. This prevalence is well illustrated in Table 2.…”

Recent advances in mass spectrometry analysis of neuropeptides

“…While DIA is increasingly being incorporated into proteomics analysis workflows, it is slow to be applied to neuropeptidomics, a field that would benefit greatly from a decrease in high-abundance bias. This is made evident by the work by Kwok et al (2020), where they developed a sensitive method for the detection of 42 bioactive peptides and hormones using DIA. A side-by-side comparison performed by Delaney and Li (2019b) demonstrated the utility and benefits of incorporating DIA over DDA into the neuropeptidomics workflow.…”