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Home / Equine Research Bank / Drug Test Anal / The detection of ACTH and insulin in equine plasma by solid-...
Drug testing and analysis2024; 17(5); 589-600; doi: 10.1002/dta.3762

The detection of ACTH and insulin in equine plasma by solid-phase extraction and micro-flow LC/MSMS.

Abstract: Previous liquid chromatography/mass spectrometry (LC/MS) methods for the detection of insulin and other similar peptide hormones in equine plasma relied on the use of antibody affinity extraction. As a result, these methods were not suitable for routine high-throughput analysis. A solid-phase extraction (SPE) method incorporating size exclusion as well as reversed-phase interactions allows the selective extraction of peptide hormones such as adrenocorticotropic hormone (ACTH), insulin and their synthetic analogues from equine plasma with approximately 80% extraction efficiencies. This extraction was combined with on-column derivatisation with acetic anhydride, followed by tryptic digestion and analysis by micro-LC/MSMS for high-sensitivity peptide hormone detection. The analysis of tryptic peptides provides greater sensitivity and more robust chromatography compared with the analysis of intact insulin and ACTH. For quantitative analysis, isotopically labelled internal standards of target peptides can be prepared in the laboratory through the use of deuterated acetic anhydride. The utility of the method was assessed for the analysis of ACTH and insulin in samples from horses suffering from pituitary pars intermedia dysfunction (PPID).
© 2024 John Wiley & Sons Ltd.
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Publication Date: 2024-07-08 PubMed ID: 38978168DOI: 10.1002/dta.3762Google Scholar: Lookup
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  • 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 presents a new method to detect peptide hormones, specifically ACTH and insulin, in horse plasma using solid-phase extraction and micro-flow LC/MSMS to enable efficient and sensitive high-throughput analysis.

Background

  • Previous methods for detecting insulin and similar peptides in equine plasma depended on antibody affinity extraction, which limited their suitability for routine high-throughput applications.
  • Such antibody-based methods often are labor-intensive, costly, and less adaptable to processing large sample volumes efficiently.

Method Development

  • The researchers developed a solid-phase extraction (SPE) protocol that exploits both size exclusion and reversed-phase interactions to selectively isolate peptide hormones from plasma.
  • This SPE method achieved around 80% extraction efficiency for key hormones such as ACTH, insulin, and their synthetic analogues.
  • The isolation procedure enhances selectivity by removing interfering plasma components while concentrating the target peptides.

Analytical Workflow

  • After extraction, on-column derivatization with acetic anhydride was performed, chemically modifying peptides to improve analysis.
  • Subsequently, the peptides underwent tryptic digestion, breaking them into smaller fragments suitable for mass spectrometry.
  • The digested peptides were analyzed using micro-liquid chromatography coupled with tandem mass spectrometry (micro-LC/MSMS), offering high sensitivity and robust chromatographic performance.
  • Analyzing tryptic peptide fragments rather than intact hormones yields better sensitivity and chromatographic reproducibility.

Quantitative Analysis and Internal Standards

  • For accurate quantification, isotopically labelled internal standards were prepared using deuterated acetic anhydride, allowing synthesis of labelled target peptides.
  • These internal standards compensate for variability during sample preparation and instrumental analysis to enable more precise measurement of hormone levels.

Application and Utility

  • The method was tested on plasma samples from horses with pituitary pars intermedia dysfunction (PPID), a condition that often involves altered levels of ACTH and insulin.
  • The approach demonstrated effective detection and quantification of these hormones in clinically relevant samples.
  • This method supports improved diagnostic and research capabilities in equine endocrinology by allowing routine, sensitive, and high-throughput hormone measurement.

Cite This Article

APA
Steel R, Timms M, Bamford N, Spence R, Sillence M. (2024). The detection of ACTH and insulin in equine plasma by solid-phase extraction and micro-flow LC/MSMS. Drug Test Anal, 17(5), 589-600. https://doi.org/10.1002/dta.3762

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 17
Issue: 5
Pages: 589-600

Researcher Affiliations

Steel, Rohan
  • Biological Research Unit, Racing Analytical Services Ltd, Flemington, Australia.
Timms, Mark
  • Biological Research Unit, Racing Analytical Services Ltd, Flemington, Australia.
Bamford, Nicholas
  • Veterinary Biosciences, Melbourne University, Parkville, Australia.
Spence, Robert
  • Department of Biological and Environmental Sciences, Queensland University of Technology, Brisbane, Australia.
Sillence, Martin
  • Department of Biological and Environmental Sciences, Queensland University of Technology, Brisbane, Australia.

MeSH Terms

  • Horses / blood
  • Animals
  • Insulin / blood
  • Solid Phase Extraction / methods
  • Solid Phase Extraction / veterinary
  • Adrenocorticotropic Hormone / blood
  • Tandem Mass Spectrometry / methods
  • Chromatography, Liquid / methods
  • Pituitary Diseases / veterinary
  • Pituitary Diseases / blood
  • Pituitary Gland, Intermediate
  • Horse Diseases / blood

Grant Funding

  • LP180101000 / Australian Research Council

References

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