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 MS ). 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).
© 2020 John Wiley & Sons, Ltd.
Publication Date: 2020-07-17 PubMed ID: 32558326DOI: 10.1002/dta.2880Google Scholar: Lookup
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- Journal Article
- Validation Study
- Biochemistry
- Diagnosis
- Diagnostic Technique
- Disease Diagnosis
- Doping
- Equestrian Sports
- Equine Diseases
- Equine Health
- Gonadotropin-Releasing Hormone
- High-performance Liquid Chromatography (HPLC)
- Horses
- Infection
- Laboratory Methods
- Metabolites
- Metabolomics
- Peptides
- Performance Horses
- Urine Analysis
- Veterinary Medicine
- Veterinary Research
Summary
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The research presents a highly precise method for detecting certain bioactive peptides in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry, which can help prevent the misuse of these substances in equine sports.
Overview
- The study focuses on the detection of bioactive peptides in horse urine as a measure to prevent doping in equine sports. Bioactive peptides, including gonadotrophin-releasing factors (GnRHs), are believed to be used as doping agents to enhance performance in animal sports. The scientists used an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS) approach to detect these substances accurately.
Methodology
- The method involved using a mixed-mode cation exchange solid-phase extraction (SPE) cartridge to extract 42 targets and/or their in vitro metabolites from horse urine.
- These extracts were then analysed by UHPLC/HRMS in positive electrospray ionization (ESI) mode under both full scan and data independent acquisition (DIA) for mass spectrometry (MS).
- The limits of detection (LoD) for most targets could reach down to 10 pg/mL in horse urine, indicating high sensitivity of the method.
- The method was validated for qualitative detection purposes, and parameters such as method specificity, sensitivity, extraction recovery, precision, and the matrix effect were reported.
In Vitro Study
- Four specific bioactive peptides related to Gonadotrophin-Releasing Hormone (GnRH), namely leuprorelin, buserelin, goserelin, and nafarelin, were studied in vitro using the S9 fraction which was isolated from horse liver.
- The identified in vitro metabolites were incorporated into the method to control the misuse of GnRHs effectively.
Application
- The applicability of this method was tested by identifying leuprorelin and one of its metabolites, Leu M4, in urine obtained from a castrated horse after the intramuscular administration of leuprorelin.
- The successful detection demonstrated that the method can be effectively used to monitor and prevent the misuse of bioactive peptides in equine sports.
Cite This Article
APA
Kwok KY, Choi TLS, Kwok WH, Lau MY, Leung EMK, Leung GNW, Wong JKY, Wan TSM, Adrian FF, Prabhu A, Ho ENM.
(2020).
Detection of bioactive peptides including gonadotrophin-releasing factors (GnRHs) in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS).
Drug Test Anal, 12(9), 1274-1286.
https://doi.org/10.1002/dta.2880 Publication
Researcher Affiliations
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
- Department of Veterinary Clinical Services, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
- Department of Veterinary Regulation, Welfare and Biosecurity Policy, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
MeSH Terms
- Animals
- Chromatography, High Pressure Liquid / methods
- Doping in Sports
- Gonadotropin-Releasing Hormone / analysis
- Gonadotropin-Releasing Hormone / urine
- Horses
- Humans
- Leuprolide / analysis
- Leuprolide / urine
- Limit of Detection
- Male
- Mass Spectrometry / methods
- Peptides / analysis
- Peptides / urine
- Reproducibility of Results
- Solid Phase Extraction
- Substance Abuse Detection / methods
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Citations
This article has been cited 2 times.- Salehpour N, Bayatloo MR, Nojavan S. Magnetic solid-phase extraction of high molecular weight peptides using stearic acid-functionalized magnetic hydroxyapatite nanocomposite: determination of some hypothalamic agents in biological samples.. Anal Bioanal Chem 2021 Dec;413(30):7609-7623.
- Phetsanthad A, Vu NQ, Yu Q, Buchberger AR, Chen Z, Keller C, Li L. Recent advances in mass spectrometry analysis of neuropeptides.. Mass Spectrom Rev 2023 Mar;42(2):706-750.
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