FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Drug Test Anal / Enhanced analysis of equine plasma for the presence of recom...
Drug testing and analysis2024; 17(6); 786-795; doi: 10.1002/dta.3785

Enhanced analysis of equine plasma for the presence of recombinant human erythropoietin – Implementation of an improved workflow.

Abstract: An improved screening workflow and a robust capillary flow LC-MS confirmatory method for the detection of recombinant human erythropoietin (rHuEPO) has been implemented to increase the sensitivity of rHuEPO detection and to reduce the number of suspect samples committed to confirmatory testing. The influence of repeated dosing of epoetin-β on the detection window of rHuEPO in equine plasma was assessed using the optimised method. Samples were initially assessed using an economical R&D Human EPO Duo-Set ELISA Development System. Samples indicating a result greater than the batch baseline were analysed using the complementary R&D Human EPO Quantikine IVD ELISA kit. All samples recording an abnormal screening result were subjected to confirmatory analysis. Confirmation of rHuEPO in plasma (≥2.5 ml) in the range of 4-13 mIU/ml (n = 6) was achieved using immunoaffinity enrichment, tryptic digestion, and capillary flow LC-MS/MS. Four horses were administered a single dose of epoetin-β (10,000 IU) via the subcutaneous and intravenous routes, on two occasions, seven days apart. The excretion profile was rapid with epoetin-β detection times of 48 to 72 h following each administration, with no appreciable difference observed between the two routes of administration. This workflow has been shown as an effective anti-doping strategy related to rHuEPO misuse and supports the use of out-of-competition testing of horses in the 2 to 3-day period prior to race-day.
© 2024 John Wiley & Sons Ltd.
Get Full Text
Publication Date: 2024-08-04 PubMed ID: 39097987DOI: 10.1002/dta.3785Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

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.

Overview

  • This study developed and implemented an improved method to detect recombinant human erythropoietin (rHuEPO) in horse blood plasma, enhancing sensitivity and reducing false positives.
  • The research also evaluated how repeated dosing of epoetin-β affects the detection window in horses, confirming the method’s usefulness for anti-doping testing before races.

Background and Purpose

  • Recombinant human erythropoietin (rHuEPO) is a synthetic hormone that stimulates red blood cell production and can enhance athletic performance.
  • In horse racing, misuse of rHuEPO is a doping concern, so sensitive and reliable detection methods are important for fair competition and animal welfare.
  • The study aimed to improve existing workflows for screening and confirming the presence of rHuEPO in equine plasma, increasing sensitivity and reducing unnecessary confirmatory testing.
  • It also sought to understand how repeated epoetin-β dosing affects how long rHuEPO can be detected in horse plasma.

Methods

  • Screening Workflow:
    • Used a cost-effective ELISA kit (R&D Human EPO Duo-Set ELISA Development System) for initial screening of plasma samples.
    • Samples with results above the baseline were further analyzed using a second ELISA kit (R&D Human EPO Quantikine IVD ELISA) for confirmation.
  • Confirmatory Testing:
    • All samples with abnormal screening results underwent confirmatory testing using a robust capillary flow liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
    • The confirmatory protocol involved immunoaffinity enrichment of rHuEPO, tryptic digestion of proteins, and sensitive mass spectrometry analysis.
    • This approach substantially enhanced detection sensitivity, allowing confirmation of rHuEPO levels as low as 4-13 mIU/ml in plasma volumes of ≥2.5 ml.
  • In Vivo Study:
    • Four horses were each given two doses of 10,000 IU epoetin-β—administered subcutaneously and intravenously—seven days apart.
    • Plasma samples taken at intervals were tested using the optimized workflow to assess detection windows and compare the routes of administration.

Results

  • The improved screening workflow was effective at detecting rHuEPO in equine plasma with greater sensitivity.
  • The confirmatory LC-MS/MS method reliably quantified rHuEPO at low concentrations, improving confidence in positive findings.
  • The detection window for epoetin-β was relatively short, with drugs detectable for 48 to 72 hours after administration via both subcutaneous and intravenous injection.
  • No significant difference in detection time was observed between the two routes of administration.

Implications and Applications

  • The improved testing workflow reduces false positives and allows for more focused and cost-effective confirmatory testing of suspect samples.
  • The short detection window suggests that out-of-competition testing within 2-3 days before race day is effective for identifying rHuEPO doping in horses.
  • This method supports anti-doping agencies and regulatory bodies in maintaining integrity and fairness in equine sports.
  • The approach can be adapted or serve as a model for detecting similar performance-enhancing substances in other animals or potentially in human anti-doping contexts.

Summary

  • By integrating an improved ELISA-based screening followed by sensitive capillary flow LC-MS/MS confirmation, the study established a robust and sensitive workflow for detecting recombinant human erythropoietin in horse plasma.
  • The in vivo dosing study demonstrated that detection windows are limited to around 2-3 days after epoetin-β administration regardless of injection method, guiding effective doping control testing schedules.
  • This research contributes to better anti-doping strategies and protection of equine athletes against illicit performance enhancement with rHuEPO.

Cite This Article

APA
Richards S, Palmer D, Cawley A, Wainscott M, Keledjian J. (2024). Enhanced analysis of equine plasma for the presence of recombinant human erythropoietin – Implementation of an improved workflow. Drug Test Anal, 17(6), 786-795. https://doi.org/10.1002/dta.3785

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 17
Issue: 6
Pages: 786-795

Researcher Affiliations

Richards, Stacey
  • Australian Racing Forensic Laboratory, Racing NSW, Sydney, NSW, Australia.
Palmer, David
  • New Zealand Racing Laboratory Services Ltd, Avondale, Auckland, New Zealand.
Cawley, Adam
  • Australian Racing Forensic Laboratory, Racing NSW, Sydney, NSW, Australia.
Wainscott, Martin
  • Harness Racing NSW, Bankstown, NSW, Australia.
Keledjian, John
  • Australian Racing Forensic Laboratory, Racing NSW, Sydney, NSW, Australia.

MeSH Terms

  • Animals
  • Erythropoietin / blood
  • Erythropoietin / administration & dosage
  • Horses / blood
  • Recombinant Proteins / blood
  • Recombinant Proteins / administration & dosage
  • Tandem Mass Spectrometry / methods
  • Humans
  • Chromatography, Liquid / methods
  • Substance Abuse Detection / methods
  • Substance Abuse Detection / veterinary
  • Enzyme-Linked Immunosorbent Assay / methods
  • Doping in Sports
  • Workflow
  • Limit of Detection

References

This article includes 38 references
  1. Lombardero M, Kovacs K, Scheithauer BW. Erythropoietin: a hormone with multiple functions. Pathobiology 2011;78(1):41‐53.
    doi: 10.1159/000322975google scholar: lookup
  2. Shaskey DJ, Green GA. Sports Haematology. Sports Med 2000;29(1):27‐38.
    doi: 10.2165/00007256-200029010-0003google scholar: lookup
  3. Reichel C, Gmeiner G. Erythropoietin and Analogs. 2010:540.
    doi: 10.1007/s00330-024-10933-ygoogle scholar: lookup
  4. Lavoie C, Diguet A, Milot M, Gareau R. Erythropoietin (rHuEPO) doping: effects of exercise on anaerobic metabolism in rats. Int J Sports Med 1998;19(4):281‐286.
    doi: 10.1005/s-2007-971919google scholar: lookup
  5. Plumb JO, Otto JM, Grocott MP. 'Blood doping' from Armstrong to prehabilitation: manipulation of blood to improve performance in athletes and physiological reserve in patients. Extrem Physiol Med 2016;5(5):1‐11.
    doi: 10.1186/s13728-016-0046-0google scholar: lookup
  6. McKeever KH, Agans JM, Geiser S, Lorimer PJ, Maylin GA. Low dose exogenous erythropoietin elicits an ergogenic effect in standardbred horses. Equine Vet J Suppl 2006;36:233‐238.
    doi: 10.1111/j.2024-3306.2006.tb05545.xgoogle scholar: lookup
  7. Piercy RJ, Swardson CJ, Hinchcliff KW. Erythroid hypoplasia and anemia following administration of recombinant human erythropoietin to two horses. J am Vet Med Assoc 1998;212(2):244‐247.
    doi: 10.2460/javma.1998.212.02.244google scholar: lookup
  8. Schwarzwald C, Hinchcliff KW. Recombinant human erythropoietin as a cause of non‐regenerative anemia in a stable of racing thoroughbreds. Proceedings of the 50th Convention American Association of Equine Practitioners, Denver 2004;270–71.
    doi: 10.1371/journal.pone.0274722google scholar: lookup
  9. Woods PR, Campbell G, Cowell RL. Non‐regenerative anaemia associated with administration of recombinant human erythropoietin to a thoroughbred racehorse. Equine Vet J 1997;29(4):326‐328.
    doi: 10.1111/j.2024-3306.1997.tb03132.xgoogle scholar: lookup
  10. International Agreement on breeding, racing and wagering. https://www.ifhaonline.org/resources/2018Agreement.pdf. Accessed February 16, 2023.
  11. Guan F, Uboh CE, Soma LR. LC‐MS/MS method for confirmation of recombinant human erythropoietin and Darbepoetin alpha in equine plasma. Anal Chem 2007;79(12):4627‐4635.
    doi: 10.1021/ac070135ogoogle scholar: lookup
  12. Yu NH, Ho EN, Wan TS, Wong AS. Doping control analysis of recombinant human erythropoietin, darbepoetin alfa and methoxy polyethylene glycol‐epoetin beta in equine plasma by nano‐liquid chromatography‐tandem mass spectrometry. Anal Bioanal Chem 2010;396(7):2513‐2521.
    doi: 10.1007/s00216-010-3455-8google scholar: lookup
  13. Guan F, Uboh CE, Soma LR, Maylin G, Jiang Z, Chen J. Confirmatory analysis of continuous erythropoietin receptor activator and erythropoietin analogues in equine plasma by LC‐MS for doping control. Anal Chem 2010;82(21):9074‐9081.
    doi: 10.1021/ac102031wgoogle scholar: lookup
  14. Scarth JP, Seibert C, Brown PR. UPLCMS/MS method for the identification of recombinant human erythropoietin analogues in horse plasma and urine. Chromatographia 2011;74(7‐8):593‐608.
    doi: 10.1007/s10337-011-2119-ygoogle scholar: lookup
  15. Timms M, Steel R, Vine J. Identification of recombinant human EPO variants in greyhound plasma and urine by ELISA, LC‐MS/MS and western blotting: a comparative study. Drug Test Anal 2016;8(2):164‐176.
    doi: 10.1002/dta.1835google scholar: lookup
  16. Lasne F, Martin L, Crepin C, de Ceaurrz J. False‐positive detection of recombinant human erythropoietin in urine following strenuous physical exercise. Anal Biochem 2002;311(2):119‐126.
    doi: 10.1016/s0003‐2697(02)00407‐4google scholar: lookup
  17. Wang H, Dou P, Lu C, Liu Z. Immuno‐magnetic beads‐based extraction‐capillary zone electrophoresis‐deep UV laser‐induced fluorescence analysis of erythropoietin. J Chromatogr a 2012;1246:48‐54.
    doi: 10.1016/j.chroma.2012.02.017google scholar: lookup
  18. Guan F, Uboh CE, Soma LR, Luo Y, Jahr JS, Driessen B. Confirmation and quantification of hemoglobin‐based oxygen carriers in equine and human plasma by hyphenated liquid chromatography tandem mass spectrometry. Anal Chem 2004;76(17):5127‐5135.
    doi: 10.1021/ac035430xgoogle scholar: lookup
  19. Thevis M, Schänzer W. Mass spectrometric identification of peptide hormones in doping‐control analysis. Analyst 2007;132(4):287‐291.
    doi: 10.1039/b618748jgoogle scholar: lookup
  20. Chang Y, Maylin GM, Matsumoto G, Neades SM, Catlin DH. Screen and confirmation of PEG‐epoetin β in equine plasma. Drug Test Anal 2011;3(1):68‐73.
    doi: 10.1002/dta.212google scholar: lookup
  21. Bailly‐Chouriberry L, Cormant F, Garcia Patrice, Lönnberg Maria, Szwandt Simon, Bondesson Ulf, Popot Marie‐Agnès, Bonnaire Yves. A new analytical method based on anti‐EPO monolith column and LC‐FAIMS‐MS/MS for the detection of rHuEPOs in horse plasma and urine samples. Analyst 2012;137(10):2445‐2453.
    doi: 10.1039/c2an15662hgoogle scholar: lookup
  22. Delcourt V, Garcia P, Chabot B. Screening and confirmatory analysis of recombinant human EPO for racing camels doping control. Drug Test Anal 2020;12(6):763‐770.
    doi: 10.1002/dta.2772google scholar: lookup
  23. Guan F, Uboh CE, Soma LR. Differentiation and identification of recombinant human erythropoietin and Darbepoetin alfa in equine plasma by LC‐MS/MS for doping control. Anal Chem 2008;80(10):3811‐3817.
    doi: 10.1021/ac800054tgoogle scholar: lookup
  24. Okano M, Sato M, Kageyama S. Identification of the long‐acting erythropoiesisstimulating agent darbepoetin alfa in human urine by liquid chromatographytandem mass spectrometry. Anal Bioanal Chem 2014;406(5):1317‐1329.
    doi: 10.1007/s00216‐013‐6836‐ygoogle scholar: lookup
  25. Vogel M, Thomas A, Schänzer W, Thevis M. EPOR‐based purification and analysis of erythropoietin mimetic peptides from human urine by Cys‐specific cleavage and LC/MS/MS. J am Soc Mass Spectrom 2015;26(9):1617‐1625.
    doi: 10.1007/s13361‐015‐1189‐8google scholar: lookup
  26. Vogel M, Blobel M, Thomas A. Isolation, enrichment, and analysis of erythropoietins in anti‐doping analysis by receptor‐coated magnetic beads and liquid chromatography‐mass spectrometry. Anal Chem 2014;86(24):12014‐12021.
    doi: 10.1021/ac5024765google scholar: lookup
  27. Dynabeads M‐280 tosylactivated manual https://assets.thermofisher.com/TFS-Assets/LSG/manuals/dynabeads_m280tosylactivated_man.pdf. Accessed August 1, 2015.
  28. Yeung YG, Stanley ER. Rapid detergent removal from peptide samples with ethyl acetate for mass spectrometry analysis. Curr Protoc Protein Sci 2010;59(1):16.12.1‐16.12.5.
    doi: 10.1002/0471140864.ps1612s59google scholar: lookup
  29. AORC, AORC Guidelines for the Minimum Criteria for Identification by Chromatography and Mass Spectrometry http://www.aorc-online.org/documents/aorc-ms-criteriaaccepted-5-jan-2015-modified-23-aug-2016-final.pdf. Accessed September 3, 2016.
  30. Quantikine IVS ELISA Human Erythropoietin Immunoassay DEP00 package insert. https://www.rndsystems.com/products/human-erythropoietin-quantikine-ivd-elisa-kit_dep00#product-datasheets. Accessed November 1, 2020
  31. Biotechne R&D Systems Human Erythropoietin/EPO DuoSet ELISA DY286 package insert. https://www.rndsystems.com/products/human-erythropoietin-epo-duoset-elisa_dy286-05#product-datasheets. Accessed November 1, 2020
  32. Lipman NS, Jackson LR, Trudel LJ. Weis‐Garcia F monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR j 2005;46(3):258‐268.
    doi: 10.1093/ilar.46.3.258google scholar: lookup
  33. Narat M. Production of antibodies in chickens. Food Technol Biotechnol 2003;1(3):259‐267.
  34. Bailly‐Chouriberry L, Noguier F, Manchon L. Identification of a blood RNA fingerprint to screen for EPO abuse in horseracing. Proceedings of the 17th International Conference of Racing Analysts and Veterinarians 2009;58–64.
    doi: 10.1002/dta.2401google scholar: lookup
  35. Zang N, Li L. Effects of common surfactants on protein digestion and matrix‐assisted laser desorption/ionisation mass spectrometric analysis of the digested peptides using two‐layer sample preparation. Rapid Commun Mass Spectrom 2004;18(8):889‐896.
    doi: 10.1002/rcm.1423google scholar: lookup
  36. Zang G, Neubert TA. Use of detergents to increase selectivity of immunoprecipitation of tyrosine phosphorylated peptides prior to identification by MALDI quadrupole‐TOF MS. Proteomics 2006;6(2):571‐578.
    doi: 10.1002/pmic.200500267google scholar: lookup
  37. Suelter CH, DeLuca M. How to prevent losses of protein by adsorption to glass and plastic. Anal Biochem 1983;135(1):112‐119.
    doi: 10.1016/0003‐2697(83)90738‐8google scholar: lookup
  38. Kovalchuk SI, Anikanov NA, Ivanova OM, Ziganshin RH, Govorun VM. Bovine serum albumin as a universal suppressor of non‐specific peptide binding in vials prior to nano‐chromatography coupled mass‐spectrometry analysis. Anal Chim Acta 2015;893:57‐64.
    doi: 10.1016/j.aca.2015.08.027google scholar: lookup

Citations

This article has been cited 0 times.
Subscribe to our newsletter
Join 99,000 horse owners like you who receive our email updates on horse health and nutrition.