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Home / Equine Research Bank / Drug Test Anal / A comprehensive approach to detecting multitudinous bioactiv...
Drug testing and analysis2019; 11(9); 1308-1325; doi: 10.1002/dta.2671

A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry.

Abstract: Bioactive peptides possess pharmacological effects and can be illicitly used in sports. To deter such misuse, an untargeted method using high resolution mass spectrometry (HRMS) has been developed for comprehensive detection of multitudinous exogenous peptides in equine plasma and urine. Forty-four peptides were extracted using mixed-mode solid-phase extraction (SPE) from plasma and urine, separated with a hydrophilic interaction liquid chromatography (HILIC) column, and detected on an HRMS instrument. Ammonium formate as a mobile phase additive had effects on HILIC retention and charge state distribution of the peptides. The acetonitrile percentage in the reconstitution solution affected the solubility of peptide neat standards and peptides in plasma and urine extracts differently. The stability of the peptides in plasma at ambient temperature was assessed. The limit of detection (LOD) was 10-50 pg/mL for most of the peptides in plasma, and ≤ 500 pg/mL for the remaining. LOD was 100-400 pg/mL for the majority of the analytes in urine, and ≤ 4000 pg/mL for the others. The method was used successfully to analyze incurred plasma and urine samples from research horses administered dermorphin. Even in the absence of reference standards, dermorphin metabolites (aFGYPS-NH , YaFG, and YaF) were identified. These results demonstrate that data generated with this method can be retrospectively reviewed for peptides that are unknown at the time of sample analysis without requiring re-analysis of the sample. This method provides a powerful novel tool for detection of numerous bioactive peptides and their metabolites in equine plasma and urine for doping control.
© 2019 John Wiley & Sons, Ltd.
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Publication Date: 2019-07-25 PubMed ID: 31250565DOI: 10.1002/dta.2671Google Scholar: Lookup
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Summary

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This research article presents a method for detecting a wide range of bioactive peptides in horse blood and urine using a specific type of liquid chromatography and high resolution mass spectrometry. The method was successful in identifying peptides, even those that were previously unknown at the time of the sample analysis, which indicates its potential in deterring misuse of such substances in sports.

Understanding the Detection Method

The central focus of this research is the development and application of a innovative, comprehensive approach to detect exogenous peptides in horses that can be potentially abused in sports. This method uses mixed-mode solid-phase extraction (SPE) to extract 44 different peptides from blood and urine.

  • SPE exploits the differences in the physico-chemical properties of the peptides. In simpler terms, the method isolates the peptides from the rest of the elements in the plasma or urine samples.
  • The extracted peptides are then separated by a hydrophilic interaction liquid chromatography (HILIC) column, a popular technique for the separation of polar compounds, and detected by a high resolution mass spectrometry (HRMS) instrument.

Role of Ammonium Formate and Acetonitrile Percentage

The researchers found that ammonium formate, used as an additive, affected the HILIC retention and charge state distribution of the peptides, indicating that it plays a vital role in the separation process. Similarly, the acetonitrile percentage in the reconstitution solution had different effects on the solubility of neat standards and peptides in plasma and urine extracts.

Stability of Peptides and Limit of Detection

The study also assessed the stability of peptides in plasma at room temperature. This is a key consideration for real-world applications as it determines how the samples need to be stored. The limit of detection (LOD) ranged from 10-50 pg/mL for most peptides in plasma, and up to 500 pg/mL for the rest. For urine, the LOD was between 100-400 pg/mL for most peptides, and up to 4000 pg/mL for the others.

Practical Application

The method was successfully used to analyze samples from research horses administered with dermorphin. Despite the absence of reference standards, the researchers were able to identify dermorphin metabolites. This ability to retrospectively review data for peptides previously unknown at the time of sample analysis prevents the need for re-analysis, providing an efficient tool for doping control in the equine sports industry.

Cite This Article

APA
Guan F, You Y, Li X, Robinson MA. (2019). A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry. Drug Test Anal, 11(9), 1308-1325. https://doi.org/10.1002/dta.2671

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 11
Issue: 9
Pages: 1308-1325

Researcher Affiliations

Guan, Fuyu
  • Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, 382 West Street Road, Kennett Square, PA, 19348, USA.
  • Pennsylvania Equine Toxicology and Research Laboratory, 220 East Rosedale Avenue, West Chester, PA, 19382, USA.
You, Youwen
  • Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, 382 West Street Road, Kennett Square, PA, 19348, USA.
  • Pennsylvania Equine Toxicology and Research Laboratory, 220 East Rosedale Avenue, West Chester, PA, 19382, USA.
Li, Xiaoqing
  • Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, 382 West Street Road, Kennett Square, PA, 19348, USA.
  • Pennsylvania Equine Toxicology and Research Laboratory, 220 East Rosedale Avenue, West Chester, PA, 19382, USA.
Robinson, Mary A
  • Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, 382 West Street Road, Kennett Square, PA, 19348, USA.
  • Pennsylvania Equine Toxicology and Research Laboratory, 220 East Rosedale Avenue, West Chester, PA, 19382, USA.

MeSH Terms

  • Animals
  • Chromatography, Liquid / methods
  • Doping in Sports
  • Horses / blood
  • Horses / urine
  • Hydrophobic and Hydrophilic Interactions
  • Limit of Detection
  • Peptides / blood
  • Peptides / urine
  • Solid Phase Extraction / methods
  • Substance Abuse Detection / methods
  • Tandem Mass Spectrometry / methods

Grant Funding

  • N/A / Pennsylvania Department of Agriculture State Horse Racing Commission

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Citations

This article has been cited 2 times.
  1. Zhang W, Feng Y, Pan L, Zhang G, Guo Y, Zhao W, Xie Z, Zhang S. Silica microparticles modified with ionic liquid bonded chitosan as hydrophilic moieties for preparation of high-performance liquid chromatographic stationary phases. Mikrochim Acta 2023 Apr 6;190(5):176.
    doi: 10.1007/s00604-023-05755-6pubmed: 37022499google scholar: lookup
  2. Thomas A, Walpurgis K, Naumann N, Piper T, Thevis M. Bioanalytical methods in doping controls: a review. Bioanalysis 2025 Mar;17(5):359-370.
    doi: 10.1080/17576180.2025.2460951pubmed: 39916648google scholar: lookup
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