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Drug testing and analysis2020; 12(9); 1274-1286; doi: 10.1002/dta.2880

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.
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Publication Date: 2020-07-17 PubMed ID: 32558326DOI: 10.1002/dta.2880Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

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

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 12
Issue: 9
Pages: 1274-1286

Researcher Affiliations

Kwok, Karen Y
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
Choi, Timmy L S
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
Kwok, Wai Him
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
Lau, Ming Yip
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
Leung, Elvis M K
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
Leung, Gary N W
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
Wong, Jenny K Y
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
Wan, Terence S M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
Adrian, Farrington F
  • Department of Veterinary Clinical Services, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
Prabhu, Anil
  • Department of Veterinary Regulation, Welfare and Biosecurity Policy, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T, Hong Kong, China.
Ho, Emmie N M
  • 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

References

This article includes 27 references
  1. Fields K, Falla TJ, Rodan K, Bush L. Bioactive peptides: signaling in the future.. J Cosmet Dermatol 2009;8(1):8-13.
  2. Fosgerau K, Hoffman T. Peptide therapeutics: current status and future directions.. Drug Discov Today 2015;20(1):122-128.
  3. Craik DJ, Fairlie DP, Liras S, Price D. The future of peptide-based drugs.. Chem Biol Drug Des 2013;81(1):136-147.
  4. Sato AK, Viswanathan M, Kent RB, Wood CR. Therapeutic peptides: technological advances driving peptides into development.. Curr Opin Biotechnol 2006;17(6):638-642.
  5. Smith RG. Development of growth hormone secretagogues.. Endocr Rev 2005;26(3):346-360.
  6. Ghigo E, Arvat E, Muccioli G, Camanni F. Growth hormone-releasing peptides.. Eur J Endocrinol 1997;136(5):445-460.
  7. Sanchis-Gomar F, Martinez-Bello VE, Nascimento AL. Desmopressin and hemodilution: implications in doping.. Int J Sports Med 2010;31(1):5-9.
  8. Broccardo M, Erspamer V, Falconieri G. Pharmacological data on dermorphins, a new class of potent opioid peptides from amphibian skin.. Br J Pharmocol 1981;73:625-631.
  9. Negri L, Lattanzi R, Melchiorri P. Production of antinociception by peripheral administration of [Lys]dermorphin, a naturally occurring peptide with high affinity for mu-opioid receptors.. Br J Pharmocol 1995;114:57-66.
  10. World Anti-Doping Agency (WADA). The 2020 prohibited list - international standard. Available at: https://www.wada-ama.org/sites/default/files/wada_2020_english_prohibited_list_0.pdf [Accessed on 17 Jan 2020]
  11. International Federation of Horseracing Authorities (IFHA). Article 6E, International Agreement on Breeding, Racing and Wagering (IABRW). Available at: https://www.ifhaonline.org/resources/ifAgreement.pdf [Accessed on 17 Jan 2020]
  12. World Anti-Doping Agency (WADA). 2018 Anti-Doping Testing Figures Report. Available at: https://www.wada-ama.org/sites/default/files/resources/files/2018_testing_figures_report.pdf [Accessed on 17 Jan 2020]
  13. Ho ENM, Kwok WH, Wong ASY, Wan TSM. Doping control analysis of TB500, a synthetic version of an active region of thymosin β4, in equine urine and plasma by liquid chromatography/mass spectrometry.. J Chromatogr A 2012;1265:57-69.
  14. Kwok WH, Ho ENM, Lau MY, Leung GNW, Wong ASY, Wan TSM. Doping control analysis of seven bioactive peptides in horse plasma by liquid chromatography-mass spectrometry.. Anal Bioanal Chem 2013;405(8):2595-2606.
  15. Leung GNW, Choi TLS, Kwok WH, Lau MY, Wong JKY, Wan TSM. Doping control analysis of bioactive peptides in equine urine and plasma by liquid chromatography - high resolution mass spectrometry.. Proceedings of 20th International Conference of Racing Analysts and Veterinarians (ICRAV) 2015:114-124.
  16. Mazzarino M, Calvaresi V, de la Torre X, Parrotta G, Sebastianelli C, Botrè F. Development and method validation of a liquid chromatography-mass spectrometry procedure after solid phase extraction for detection of 19 doping peptides in human urine.. Forensic Toxicol 2015;33:321-337.
  17. Steel R, Timms M, Levina V, Vine J. A high throughput screen for 17 dermorphin peptides in equine and human urine and equine plasma.. Drug Test Anal 2014;6(9):909-921.
  18. Guan F, Robinson MA. Comprehensive solid-phase extraction of multitudinous bioactive peptides from equine plasma and urine for doping detection.. Anal Chim Acta 2017;8:79-90.
  19. Timms M, Hall N, Levina L, Vine J, Steel R. A high throughput LC-MS/MS screen for GHRP in equine and human urine, featuring peptide derivatization for improved chromatography.. Drug Test Anal 2014;6(10):985-995.
  20. Semenistaya E, Zvereva I, Krotov G, Rodchenkov G. Solid-phase extraction of small biologically active peptides on cartridges and microelution 96-well plates from human urine.. Drug Test Anal 2015;8:940-949.
  21. Thomas A, Görgens C, Guddat S. Simplifying and expanding the screening for peptides < 2 kDa by direct urine injection, liquid chromatography, and ion mobility mass spectrometry.. J Sep Sci 2016;39(2):333-341.
  22. Judák P, Grainger J, Goebel C, Van Eenoo P, Deventer K. DMSO assisted electrospray ionization for the detection of small peptide hormones in urine by dilute-and-shoot-liquid-chromatography-high resolution mass spectrometry.. J Am Soc Mass Spectrom 2017;28(8):1657-1665.
  23. Sardela VF, Martucci MEP, de Araújo ALD. Comprehensive analysis by liquid chromatography-Q-Orbitrap mass spectrometry: fast screening of peptides and organic molecules.. J Am Soc Mass Spectrom 2018;53:476-503.
  24. Handelsman DJ, Idan A, Grainger J, Goebel C, Turner L, Conway AJ. Detection and effects on serum and urine steroid and LH of repeated GnRH analog (leuprolide) stimulation.. J Steroid Biochem Mol Biol 2014;141:113-120.
  25. Wong JKY, Tang FPW, Wan TSM. In vitro metabolic studies using homogenised horse liver in place of horse liver microsomes.. Drug Test Anal 2011;3(6):393-399.
  26. Zvereva I, Dudko G, Dikunets M. Determination of GnRH and its synthetic analogues’ abuse in doping control: small bioactive peptide UPLC-MS/MS method extension by addition of in vitro and in vivo metabolism data; evaluation of LH and steroid profile parameter fluctuations as suitable maker.. Drug Test Anal 2018;10(4):711-722.
  27. Palmer D, Rademaker K, Martin I, Hessell J, Howitt R. Identification of gonadotropin-releasing hormone metabolites in greyhound urine.. Drug Test Anal 2017;9:1499-1505.

Citations

This article has been cited 4 times.
  1. 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.
    doi: 10.1007/s00216-021-03725-6pubmed: 34668043google scholar: lookup
  2. 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.
    doi: 10.1002/mas.21734pubmed: 34558119google scholar: lookup
  3. Okyem S, Tan Y, Romanova E, Sweedler JV. Neuropeptide Characterization Workflow from Sampling to Data-Independent Acquisition Mass Spectrometry. J Vis Exp 2025 Aug 8;(222).
    doi: 10.3791/68741pubmed: 40853859google scholar: lookup
  4. Chang W, Yan S, Yan X, Wang Z, Gu B, Liu Y, Zhang Y, Yang S. The sensitive detection of low molecular mass peptide drugs in dried blood spots by solid-phase extraction and LC-HRMS. Anal Bioanal Chem 2024 Nov;416(26):5655-5669.
    doi: 10.1007/s00216-024-05480-wpubmed: 39180594google scholar: lookup
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