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Drug testing and analysis2021; 14(4); 676-689; doi: 10.1002/dta.3210

Defining the specificity of recombinant human erythropoietin confirmation in equine samples by liquid chromatography-tandem mass spectrometry.

Abstract: The proteotypic human EPO peptides YLLEAK (T4), SLTTLLR (T11), TITADTFR (T14), and VYSNFLR (T17) are often used to confirm the presence of recombinant human EPO (rhEPO) in equine samples. Each of these peptides contains one or more isomeric leucine or isoleucine amino acids, raising the possibility that a simple leucine/isoleucine substitution could lead to a false identification when compared with a rhEPO reference standard. To examine this possibility variants of these four peptides were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These studies indicate that confirmation of rhEPO in equine samples by immuno-affinity capture and LC-MS/MS analysis is true and accurate. It was also found that chromatography played a greater role in determining LC-MS/MS specificity than tandem mass spectrometry and that, in the case of more hydrophilic peptides, the accuracy of peptide identification could be enhanced by the inclusion of C and N labelled peptide internal standards.
© 2021 John Wiley & Sons, Ltd.
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Publication Date: 2021-12-21 PubMed ID: 34898016DOI: 10.1002/dta.3210Google 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.

This study researched the accuracy of using certain peptides to detect the presence of recombinant human EPO (rhEPO) in horse samples, through a process known as liquid chromatography-tandem mass spectrometry. It found that the method is accurate, and that chromatography has more influence on accuracy than the mass spectrometry aspect of the process.

Peptides used for Identification

  • The research is based on the recognition of recombinant human erythropoietin (rhEPO), a hormone that controls red blood cell production, in equine samples using four proteotypic human EPO peptides – YLLEAK (T4), SLTTLLR (T11), TITADTFR (T14), and VYSNFLR (T17).
  • These particular peptides are often used to confirm the presence of rhEPO in samples, but there is concern that they may give false positives due to isomeric leucine or isoleucine amino acids they contain. This means there is a possibility of mistakenly identifying a substance as rhEPO if there is a simple substitution of the leucine/isoleucine amino acids.

Analysis by Liquid Chromatography-Tandem Mass Spectrometry

  • To test the accuracy of the identification process, the researchers analysed variations of these four peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a technique commonly used to separate, identify and quantify each component in a mixture.
  • The researchers established that the identification of rhEPO in equine samples through the combination of immuno-affinity capture and LC-MS/MS analysis is both true and accurate, thereby validating the use of these peptides for rhEPO detection.

Role of Chromatography and Tandem Mass Spectrometry

  • Furthermore, they found that chromatography, the part of the process where separation of the mixture occurs, has a greater influence on the specificity of LC-MS/MS detection than the mass spectrometry phase, which is used for the identification and quantification of the components.
  • In particular, the accuracy of peptide identification can be improved with the inclusion of C and N labelled peptide internal standards for more hydrophilic peptides. This means that the process can be fine-tuned for more accurate results by utilising peptides that are attracted to water.

Cite This Article

APA
Timms M, Steel R. (2021). Defining the specificity of recombinant human erythropoietin confirmation in equine samples by liquid chromatography-tandem mass spectrometry. Drug Test Anal, 14(4), 676-689. https://doi.org/10.1002/dta.3210

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 14
Issue: 4
Pages: 676-689

Researcher Affiliations

Timms, Mark
  • Biological Research Unit, Racing Analytical Services Ltd, Flemington, Victoria, Australia.
Steel, Rohan
  • Biological Research Unit, Racing Analytical Services Ltd, Flemington, Victoria, Australia.

MeSH Terms

  • Animals
  • Chromatography, Liquid / methods
  • Erythropoietin / analysis
  • Horses
  • Humans
  • Isoleucine
  • Leucine
  • Recombinant Proteins / analysis
  • Tandem Mass Spectrometry / methods

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Citations

This article has been cited 1 times.
  1. Jamieson CA, Baillie SL, Johnson JP. Blood Transfusion in Equids-A Practical Approach and Review. Animals (Basel) 2022 Aug 23;12(17).
    doi: 10.3390/ani12172162pubmed: 36077883google scholar: lookup
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