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Scientific reports2025; 15(1); 14987; doi: 10.1038/s41598-025-99470-2

Surrogate analyte approach for the determination of endogenous cortisol, cortisone, DHEA, DHEAS in horse hair and sheep wool by LC-HRMS/MS.

Abstract: Two new quantitative methods for the determination of four stress-related hormones (cortisol, cortisone, dehydroepiandrosterone and dehydroepiandrosterone sulfate) in horse hair and sheep wool by liquid chromatography coupled with hybrid high resolution mass spectrometry were developed and validated. Hormones were extracted overnight from ground samples with methanol after a washing step with the same solvent to remove external contamination. The extract was evaporated and dry residue was solubilized in an acid mixture of methanol and water. The methods were validated according to ICH guidelines for bioanalytical method validation within 5-100 pg/mg with LC-Q Exactive platform (except for DHEA: 100-2000 pg/mg for both matrices and 1-100 pg/mg for DHEAS in mane hair). The validated concentration range was 5-100 pg/mg for wool and 1-100 pg/mg (except for DHEA: 5-100 pg/mg) for mane hair samples with LC-Exploris 120 system. Satisfactory quantitative performances were obtained using isotopic dilution and surrogate analyte approach. After successful validation, the applicability of the methods were tested. The detected concentrations of investigated analytes in real samples collected from animals living in marginal areas were encompassing the interval 1.1-13 and 5.0-30.5 pg/mg in mane hair and wool samples, respectively.
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Publication Date: 2025-04-29 PubMed ID: 40301517PubMed Central: PMC12041554DOI: 10.1038/s41598-025-99470-2Google Scholar: Lookup
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Summary

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The research involves the development of two new methods for determining levels of four stress-related hormones in horse hair and sheep wool using liquid chromatography and high resolution mass spectrometry.

Research Objective

  • The main aim of the study was to develop and validate two quantitative methods for measuring levels of cortisol, cortisone, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) in horse hair and sheep wool.

Methodology

  • The research utilized liquid chromatography coupled with hybrid high-resolution mass spectrometry to analyze samples.
  • The process involved the extraction of hormones from ground samples of hair or wool using methanol following a washing process to remove external contaminants.
  • The extracted solution was then evaporated, and the resulting dry residue solubilized in a methanol-water acid mixture.

Validation Process

  • The newly developed techniques were evaluated according to International Council for Harmonisation (ICH) guidelines for bioanalytical method validation.
  • Validation ranges were established for each hormone in each material. For example, for DHEA, the range was 100-2000 pg/mg in both horse and sheep samples and 1-100 pg/mg for DHEAS in mane hair.
  • For the horse mane hair samples, the validated concentration range for DHEA was 5-100 pg/mg, while it was 1-100 pg/mg for DHEAS. The ranges for the wool samples were 5-100 pg/mg.

Test Results

  • The methods proved successful, with satisfactory quantitative performances achieved via isotopic dilution and the surrogate analyte approach.
  • These procedures were then used on real samples collected from animals in marginal areas with results within the expected intervals.

Conclusion

  • These new methods for measuring levels of stress-related hormones in hair and wool offer a reliable means of gauging stress levels in animals, with their applicability confirmed through testing on real samples.

Cite This Article

APA
(2025). Surrogate analyte approach for the determination of endogenous cortisol, cortisone, DHEA, DHEAS in horse hair and sheep wool by LC-HRMS/MS. Sci Rep, 15(1), 14987. https://doi.org/10.1038/s41598-025-99470-2

Publication

Scientific reports
ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 15
Issue: 1
Pages: 14987
PII: 14987

Researcher Affiliations

MeSH Terms

  • Animals
  • Horses
  • Hair / chemistry
  • Cortisone / analysis
  • Hydrocortisone / analysis
  • Sheep
  • Dehydroepiandrosterone / analysis
  • Tandem Mass Spectrometry / methods
  • Wool / chemistry
  • Chromatography, Liquid / methods
  • Dehydroepiandrosterone Sulfate / analysis
  • Reproducibility of Results

Grant Funding

  • GR-2021-12374382 / Ministero della Salute

Conflict of Interest Statement

Competing interests: The authors declare no competing interests.

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