Comprehensive solid-phase extraction of multitudinous bioactive peptides from equine plasma and urine for doping detection.
Abstract: The ability to analyze biological samples for multitudinous exogenous peptides with a single analytical method is desired for doping control in horse racing. The key to achieving this goal is the capability of extracting all target peptides from the sample matrix. In the present study, theory of mixed-mode solid-phase extraction (SPE) of peptides from plasma is described, and a generic mixed-mode SPE procedure has been developed for recovering multitudinous exogenous peptides with remarkable sequence diversity, from equine plasma and urine in a single procedure. Both the theory and the developed SPE procedure have led to the development of a novel analytical method for comprehensive detection of multitudinous bioactive peptides in equine plasma and urine using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Thirty nine bioactive peptides were extracted with strong anion-exchange mixed-mode SPE sorbent, separated on a reversed-phase C column and detected by HRMS and data-dependent tandem mass spectrometry. The limit of detection (LOD) was 10-50 pg mL in plasma for most of the peptides and 100 pg mL for the remaining. For urine, LOD was 20-400 pg mL for most of the peptides and 1-4 ng mL for the others. In vitro degradation of the peptides in equine plasma and urine was examined at ambient temperature; the peptides except those with a D-amino acid at position 2 were unstable not only in plasma but also in urine. The developed method was successful in analysis of plasma and urine samples from horses administered dermorphin. Additionally, dermorphin metabolites were identified in the absence of reference standards. The developed SPE procedure and LC-HRMS method can theoretically detect virtually all peptides present at a sufficient concentration in a sample. New peptides can be readily included in the method to be detected without method re-development. The developed method also generates such data that can be retrospectively analyzed for peptides unknown at the time of sample analysis. It is the first generic analytical method for comprehensive detection of multitudinous exogenous peptides in biological samples, to the authors' knowledge.
Copyright © 2017 Elsevier B.V. All rights reserved.
Publication Date: 2017-07-17 PubMed ID: 28864198DOI: 10.1016/j.aca.2017.07.005Google Scholar: Lookup
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- Journal Article
Summary
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The study demonstrates the development of a new solid-phase extraction (SPE) method for detecting various bioactive peptides in equine plasma and urine, useful in doping controls in horse racing. The method uses mixed-mode SPE and liquid chromatography, coupled with high-resolution mass spectrometry. This provides a comprehensive doping detection tool capable of identifying a multitude of different peptides.
Method Development
- The researchers describe a theory for mixed-mode solid-phase extraction (SPE) of peptides from plasma. The methodology revolves around the extraction of all target peptides from the sample for analysis.
- The team developed a generic mixed mode SPE procedure that can recover a multitude of exogenous peptides with remarkable sequence diversity from horse plasma and urine in a single procedure.
- This theory and developed procedure formed the basis for the development of a new analytical method that allows comprehensive detection of a multitude of peptides in horse plasma and urine.
Analysis by LC-HRMS
- The method uses Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC-HRMS) to analyze the extracted peptides.
- The study demonstrated that 39 bioactive peptides could be extracted effectively employing solid phase extraction and could be detected by LC-HRMS and data-dependent tandem mass spectrometry.
Peptide Stability
- The stability of the peptides under in vitro conditions in both plasma and urine were also tested. This was found to be varied, with peptides having a D-amino acid at position 2 being the only stable peptides in both plasma and urine.
Practical Application
- The developed method was successful in detecting the presence of the biomarkers in plasma and urine samples from horses that were administered a drug.
- The method can theoretically detect almost all peptides present at sufficient concentration in a sample.
- New peptides can be easily incorporated into the detection method without the need for method redevelopment.
Conclusion
- According to the authors, this is the first universal analytical method for comprehensive detection of multiple exogenous peptides in biological samples.
- The method also has the advantage of being able to retrospectively analyze data for peptides unknown at the time of sample analysis.
Cite This Article
APA
Guan F, Robinson MA.
(2017).
Comprehensive solid-phase extraction of multitudinous bioactive peptides from equine plasma and urine for doping detection.
Anal Chim Acta, 985, 79-90.
https://doi.org/10.1016/j.aca.2017.07.005 Publication
Researcher Affiliations
- 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. Electronic address: guanf@vet.upenn.edu.
- 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
- Doping in Sports
- Horses
- Mass Spectrometry
- Peptides / blood
- Peptides / urine
- Solid Phase Extraction
Citations
This article has been cited 4 times.- Gómez-Guerrero NA, González-López NM, Zapata-Velásquez JD, Martínez-Ramírez JA, Rivera-Monroy ZJ, García-Castañeda JE. Synthetic Peptides in Doping Control: A Powerful Tool for an Analytical Challenge. ACS Omega 2022 Nov 1;7(43):38193-38206.
- 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.
- Ueda T, Tozaki T, Nozawa S, Kinoshita K, Gawahara H. Identification of metabolomic changes in horse plasma after racing by liquid chromatography-high resolution mass spectrometry as a strategy for doping testing. J Equine Sci 2019 Sep;30(3):55-61.
- Thomas A, Walpurgis K, Naumann N, Piper T, Thevis M. Bioanalytical methods in doping controls: a review. Bioanalysis 2025 Mar;17(5):359-370.
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