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Drug testing and analysis2023; doi: 10.1002/dta.3544

Detection of doping peptides and basic drugs in equine urine using liquid chromatography-mass spectrometry.

Abstract: The abuse of prohibited agents including peptides and basic small-molecule drugs is an area of great concern in horseracing due to their high potential to act as doping agents. These compound classes include agents such as growth hormone-releasing peptides, peptide analgesics, beta-2-adrenergic receptor agonists, and quaternary ammonium drugs that can be challenging to detect and regulate because of their chemical properties and potential rapid elimination following administration. The use of highly sensitive and selective analytical techniques such as liquid chromatography-mass spectrometry (LC-MS) is necessary to provide coverage of these substances and their potential metabolites. This study describes the development and validation of methodology capable of the detection of over 50 different peptide-based doping agents, related secretagogues, quaternary ammonium drugs, and other challenging small molecules in equine urine following solid-phase extraction using a mixed mode weak cation exchange sorbent. Following sample extraction, the compounds were analyzed using LC-MS with chromatographic separation via a reverse phase gradient and detection via selective reaction monitoring following introduction to a triple-stage quadrupole mass spectrometer using positive mode electrospray ionization. Validation parameters including limits of detection and quantitation, accuracy, precision, linear range, recovery, stability, and matrix effects were determined. Briefly, the limits of detection for most compounds were in the sub-ng/mL ranges with adequate precision and accuracy sufficient for an initial testing procedure. Stability studies indicated that most compounds were sufficiently stable to allow for effective screening using conditions commonly utilized in drug testing laboratories.
Drug Testing and Analysis© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.
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Publication Date: 2023-08-07 PubMed ID: 37548131DOI: 10.1002/dta.3544Google 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.

Introduction:

  • The misuse of prohibited substances like peptides and basic small-molecule drugs is a major concern in horseracing. These substances have a high potential to be used as doping agents.
  • They include growth hormone-releasing peptides, peptide analgesics, beta-2-adrenergic receptor agonists, and quaternary ammonium drugs.
  • Detecting and regulating these compounds can be difficult due to their chemical properties and the possibility of them being quickly eliminated from the body after being administered.

Analytical Techniques:

To detect these substances and their potential metabolites, it’s crucial to use highly sensitive and specific analytical methods. Liquid chromatography-mass spectrometry (LC-MS) is one such technique.

Study Objective:

This research focused on developing and validating a method that can detect over 50 different peptide-based doping agents, related secretagogues, quaternary ammonium drugs, and other challenging small molecules in horse urine.

Methodology:

  1. Samples were first prepared using solid-phase extraction with a mixed mode weak cation exchange sorbent.
  2. After extraction, the compounds were analyzed using LC-MS.
  3. Chromatographic separation was achieved through a reverse phase gradient.
  4. Detection was done using selective reaction monitoring after introducing the samples to a triple-stage quadrupole mass spectrometer using positive mode electrospray ionization.

Validation:

The validation process examined various parameters such as:

  • Limits of detection and quantitation.
  • Accuracy and precision.
  • Linear range.
  • Recovery rate.
  • Stability and matrix effects.

Results:

Most compounds could be detected at sub-ng/mL ranges, with acceptable precision and accuracy suitable for initial testing. Stability studies indicated that most substances remained stable enough for effective screening under conditions typically used in drug testing labs.

Conclusion:

The developed method provides an effective way to screen for a wide range of potential doping agents in horse urine, ensuring the integrity of horseracing and the well-being of the animals.

Cite This Article

APA
Flores L, Hargrave S, Clifford A, Alarcio G, Moeller BC. (2023). Detection of doping peptides and basic drugs in equine urine using liquid chromatography-mass spectrometry. Drug Test Anal. https://doi.org/10.1002/dta.3544

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English

Researcher Affiliations

Flores, Luis
  • KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.
Hargrave, Sabine
  • KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.
Clifford, Amel
  • KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.
Alarcio, Gwendolyne
  • KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.
Moeller, Benjamin C
  • KL Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.
  • Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.

Grant Funding

  • California Horse Racing Board

References

This article includes 48 references
  1. Fragkaki AG, Kioukia-Fougia N, Kiousi P, Kioussi M, Tsivou M. Challenges in detecting substances for equine anti-doping. Drug Test Anal 2017;9(9):1291-1303.
    doi: 10.1002/dta.2162google scholar: lookup
  2. Muttenthaler M, King GF, Adams DJ, Alewood PF. Trends in peptide drug discovery. Nat Rev Drug Discov 2021;20(4):309-325.
    doi: 10.1038/s41573-020-00135-8google scholar: lookup
  3. Gross NJ. Anticholinergic agents in asthma and COPD. Eur J Pharmacol 2006;533(1-3):36-39.
    doi: 10.1016/j.ejphar.2005.12.072google scholar: lookup
  4. Gross NJ. Ipratropium bromide. N Engl J Med 1988;319(8):486-494.
    doi: 10.1056/nejm198808253190806google scholar: lookup
  5. International Agreement on Breeding, Racing and Wagering. International Federation of Horseracing Authorities; 2021:167.
  6. Association of Racing Commissioners International. Uniform Classification Guidelines for Foreign Substances and Recommended Penalties and Model Rule; 2020:72.
  7. Guan F, Fay S, Li X, You Y, Robinson MA. Identification of ex vivo catabolites of peptides with doping potential in equine plasma by HILIC-HRMS. Drug Test Anal 2020;12(6):771-784.
    doi: 10.1002/dta.2781google scholar: lookup
  8. A.C. Commision. (Ed). Organised Crime and Drugs In Sport. Australia Crime Commission; 2013.
  9. Martínez-Sanz J, Sospedra I, Ortiz C, Baladía E, Gil-Izquierdo A, Ortiz-Moncada R. Intended or unintended doping? A review of the presence of doping substances in dietary supplements used in sports. Nutrients 2017;9(10):1093.
    doi: 10.3390/n鄁093google scholar: lookup
  10. Pugliese J, Boyce MC, Lawler NG, Coumbaros J, Le Dispersive TT. SPE, an alternative to traditional SPE for extraction of 43 doping peptides from equine urine prior to LC-MS screening. Forensic Toxicol 2020;38(2):365-377.
    doi: 10.1007/s11419-020-00524-zgoogle scholar: lookup
  11. Guan F, Robinson MA. Comprehensive solid-phase extraction of multitudinous bioactive peptides from equine plasma and urine for doping detection. Anal Chim Acta 2017;985:79-90.
    doi: 10.1016/j.aca.2017.07.005google scholar: lookup
  12. Ho ENM, Kwok WH, Lau MY, et al. Doping control analysis of TB-500, 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.
    doi: 10.1016/j.chroma.2012.09.043google scholar: lookup
  13. Robinson MA, Guan F, McDonnell S, Uboh CE, Soma LR. Pharmacokinetics and pharmacodynamics of dermorphin in the horse. J Vet Pharmacol Ther 2015;38(4):321-329.
    doi: 10.1111/jvp.12179google scholar: lookup
  14. 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.
    doi: 10.1002/dta.1585google scholar: lookup
  15. Wang CC, Hartmann-Fischbach P, Krueger TR, Wells TL, Feineman AR, Compton JC. Fast and sensitive analysis of dermorphin and HYP6-dermorphin in equine plasma using liquid chromatography tandem mass spectrometry. Drug Test Anal 2014;6(4):342-349.
    doi: 10.1002/dta.1487google scholar: lookup
  16. Ho ENM, Wan TSM, Wong ASY, Lam KKH, Stewart BD. Doping control analysis of insulin and its analogues in equine plasma by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2008;1201(2):183-190.
    doi: 10.1016/j.chroma.2008.04.060google scholar: lookup
  17. Xia X, Tao Q, Ma Q, Chen H, Wang J’, Yu H. Growth hormone-releasing hormone and its analogues: significance for MSCs-mediated angiogenesis. Stem Cells Int 2016;2016:8737589.
    doi: 10.1155/2016/8737589google scholar: lookup
  18. Werle M, Bernkop-Schnürch A. Strategies to improve plasma half life time of peptide and protein drugs. Amino Acids 2006;30(4):351-367.
    doi: 10.1007/s00726-005-0289-3google scholar: lookup
  19. Judák P, Esposito S, Coppieters G, van Eenoo P, Deventer K. Doping control analysis of small peptides: a decade of progress. J Chromatogr B 2021;1173:122551.
    doi: 10.1016/j.jchromb.2021.122551google scholar: lookup
  20. Thomas A, Delahaut P, Krug O, Schänzer W, Thevis M. Metabolism of growth hormone releasing peptides. Anal Chem 2012;84(23):10252-10259.
    doi: 10.1021/ac302034wgoogle scholar: lookup
  21. Thomas A, Höppner S, Geyer H, et al. Determination of growth hormone releasing peptides (GHRP) and their major metabolites in human urine for doping controls by means of liquid chromatography mass spectrometry. Anal Bioanal Chem 2011;401(2):507-516.
    doi: 10.1007/s00216-011-4702-3google scholar: lookup
  22. Semenistaya E, Zvereva I, Thomas A, Thevis M, Krotov G, Rodchenkov G. Determination of growth hormone releasing peptides metabolites in human urine after nasal administration of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin. Drug Test Anal 2015;7(10):919-925.
    doi: 10.1002/dta.1787google scholar: lookup
  23. Hostrup M, Jacobson GA, Jessen S, Lemminger AK. Anabolic and lipolytic actions of beta2-agonists in humans and antidoping challenges. Drug Test Anal 2020;12(5):597-609.
    doi: 10.1002/dta.2728google scholar: lookup
  24. Kwok KY, Choi TLS, Kwok WH, et al. 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 2020;12(9):1274-1286.
    doi: 10.1002/dta.2880google scholar: lookup
  25. Cox HD, Hughes CM, Eichner D. Detection of GHRP-2 and GHRP-6 in urine samples from athletes. Drug Test Anal 2015;7(5):439-444.
    doi: 10.1002/dta.1791google scholar: lookup
  26. Kwok WH, Choi TLS, Kwok KY, Chan GHM, Wong JKY, Wan TSM. Doping control analysis of 46 polar drugs in horse plasma and urine using a ‘dilute-and-shoot’ ultra high performance liquid chromatography-high resolution mass spectrometry approach. J Chromatogr A 2016;1451:41-49.
    doi: 10.1016/j.chroma.2016.05.002google scholar: lookup
  27. Ho ENM, Kwok WH, Wong ASY, Wan TSM. Detection of singly- and doubly-charged quaternary ammonium drugs in equine urine by liquid chromatography/tandem mass spectrometry. Anal Chim Acta 2012;710:94-101.
    doi: 10.1016/j.aca.2011.10.046google scholar: lookup
  28. Cox HD, Knussmann GN, Moore C, Eichner D. Detection of insulin analogues and large peptides >2 kDa in urine. Drug Test Anal 2022;14(7):1264-1272.
  29. Timms M, Hall N, Levina V, 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.
    doi: 10.1002/dta.1624google scholar: lookup
  30. Palmer D, Rademaker K, Martin I, Hessell J, Howitt R. Identification of gonadotropin-releasing hormone metabolites in greyhound urine. Drug Test Anal 2017;9(10):1499-1505.
    doi: 10.1002/dta.2164google scholar: lookup
  31. Garcia P, Paris AC, Gil J, Popot MA, Bonnaire Y. Analysis of β-agonists by HPLC/ESI-MSn in horse doping control. Biomed Chromatogr 2011;25(1-2):147-154.
    doi: 10.1002/bmc.1562google scholar: lookup
  32. 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.
    doi: 10.1007/s00216-012-6697-9google scholar: lookup
  33. Wood T, Weckman TJ, Henry PA, Chang S-L, Blake JW, Tobin T. Equine urine pH: normal population distributions and methods of acidification. Equine Vet J 1990;22(2):118-121.
    doi: 10.1111/j.2042-3306.1990.tb04222.xgoogle scholar: lookup
  34. Stejskal K, Potěšil D, Zdráhal Z. Suppression of peptide sample losses in autosampler vials. J Proteome Res 2013;12(6):3057-3062.
    doi: 10.1021/pr400183vgoogle scholar: lookup
  35. Association of Official Racing Chemists Guidelines for the Minimum Criteria for Identification by Chromatography and Mass Spectrometry. 2016.
  36. Okano M, Sato M, Ikekita A, Kageyama S. Determination of growth hormone secretagogue pralmorelin (GHRP-2) and its metabolite in human urine by liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 2010;24(14):2046-2056.
    doi: 10.1002/rcm.4619google scholar: lookup
  37. Boersema PJ, Mohammed S, Heck AJR. Hydrophilic interaction liquid chromatography (HILIC) in proteomics. Anal Bioanal Chem 2008;391(1):151-159.
    doi: 10.1007/s00216-008-1865-7google scholar: lookup
  38. Guo Y. Analysis of quaternary amine compounds by hydrophilic interaction chromatography/mass spectrometry (HILIC/MS). Journal of Liquid Chromatography & Related Technologies 2005;28(4):497-512.
    doi: 10.1081/jlc-200047200google scholar: lookup
  39. Memdouh S, Gavrilović I, Ng K, Cowan D, Abbate V. Advances in the detection of growth hormone releasing hormone synthetic analogs. Drug Test Anal 2021;13(11-12):1871-1887.
    doi: 10.1002/dta.3183google scholar: lookup
  40. Rumpler MJ, Sams RA, Colahan P. Regulatory control of glycopyrrolate in performance horses using validated UHPLC/MS-MS methods. J Chromatogr B 2012;889-890:130-137.
    doi: 10.1016/j.jchromb.2012.02.008google scholar: lookup
  41. Sardela VF, Martucci MEP, de Araújo ALD, et al. Comprehensive analysis by liquid chromatography Q-Orbitrap mass spectrometry: fast screening of peptides and organic molecules. J Mass Spectrom 2018;53(6):476-503.
    doi: 10.1002/jms.4077google scholar: lookup
  42. Jing J, Tian T, Wang Y, Xu X, Shan Y. Multi-analyte screening of small peptides by alkaline pre-activated solid phase extraction coupled with liquid chromatography-high resolution mass spectrometry in doping controls. J Chromatogr A 1676;2022:463272.
  43. Houston T, Chay S, Woods WE, et al. Phenylbutazone and its metabolites in plasma and urine of thoroughbred horses: population distributions and effects of urinary pH. J Vet Pharmacol Ther 1985;8(2):136-149.
    doi: 10.1111/j.1365-2885.1985.tb00937.xgoogle scholar: lookup
  44. Schott HC 2nd, Hodgson DR, Bayly WM. Haematuria, pigmenturia and proteinuria in exercising horses. Equine Vet J 1995;27(1):67-72.
    doi: 10.1111/j.2042-3306.1995.tb03035.xgoogle scholar: lookup
  45. Ryan D, McKemie DS, Kass PH, Puschner B, Knych HK. Pharmacokinetics and effects on arachidonic acid metabolism of low doses of cannabidiol following oral administration to horses. Drug Test Anal 2021;13(7):1305-1317.
    doi: 10.1002/dta.3028google scholar: lookup
  46. Judák P, Van Eenoo P, Deventer K. Adsorption effects of the doping relevant peptides Insulin Lispro, Synachten, TB-500 and GHRP 5. Anal Biochem 2017;537:69-71.
    doi: 10.1016/j.ab.2017.09.003google scholar: lookup
  47. Esposito S, Deventer K, Geldof L, van Eenoo P. In vitro models for metabolic studies of small peptide hormones in sport drug testing. J Pept Sci 2015;21(1):1-9.
    doi: 10.1002/psc.2710google scholar: lookup
  48. Timms M, Botteon A, Manos C, Griffin J, Levina V, Steel R. Detection of gonadotropin releasing hormone (GnRH) and its analogues in equine and canine urine by high-resolution data independent acquisition (DIA). Drug Test Anal 2023;1-13.
    doi: 10.1002/dta.3486google scholar: lookup

Citations

This article has been cited 1 times.
  1. Králík F, Kvíčalová A, Salaďáková A, Kuchař M, Setnička V. Growth Hormone-Releasing Peptides: Investigation of Their Secondary Structure, Thermal Stability, and Model Membrane Interactions. Chirality 2026 Feb;38(2):e70083.
    doi: 10.1002/chir.70083pubmed: 41555539google scholar: lookup
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