UHPLC-MS/MS Measures Stress Hormones in Wolf Hair

Hair is an easy-to-collect sample that does not require harming or disturbing an animal, and it can reveal how wild animals have been affected by long-term stress from their environment or from human activity. Researchers tested and confirmed a an ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method which can accurately measure two stress-related hormones, cortisol and its byproduct cortisone, at the same time in hair samples taken from wild grey wolves. A paper based on this research was published in the journal Molecules.¹

What Are Cortisol and Cortisone, and Why Are They Important?

Cortisol and cortisone are two closely related hormones that are commonly used to measure stress levels in both humans and animals. These hormones play key roles in how the body manages energy, responds to stress, and controls the immune system. Cortisol is the "active" form of the hormone, made in the adrenal glands (small glands that sit on top of the kidneys) and released into the body during stressful situations. This release is triggered by a chain reaction involving the brain and adrenal glands known as the stress-response system. Once released, cortisol travels through the bloodstream to the rest of the body, where it helps regulate metabolism, reduces inflammation, and can suppress immune system activity.^2-6

Did the New Hair-Based Hormone Testing Method Work, and Could It Be Useful for Monitoring Wolf Stress and Welfare in the Wild?

The research team soaked wolf hair samples in a chemical solvent to extract the hormones, then cleaned up the samples using a filtering technique to remove unwanted substances that could interfere with the results. They also used reference markers to make sure the hormone measurements were accurate and comparable across samples. The team reports that their testing method worked well: it reliably measured hormone levels across a wide range of amounts, and results were consistent when repeated (with less than 15% variation). The method was also sensitive enough to detect very small amounts (as low as about 4 picograms of cortisol and about 2.5 picograms of cortisone per milligram of hair; a picogram is a trillionth of a gram). When the researchers tested real wolf hair samples, cortisol levels ranged from undetectable up to about 12 picograms per milligram, and cortisone levels ranged from undetectable up to about 3.7 picograms per milligram. Finally, the cortisol results closely matched those from an antibody-based test, confirming that the new method is accurate and reliable.¹

The research team believes that this testing method can be used to check how stress affects the health and wellbeing of wolves. It could serve as a helpful tool for tracking Europe's recovering wolf populations, especially as they spread into new areas that may not be ideal habitats and come with fresh challenges.¹

“Overall,” write the authors of the paper,¹ “this method represents a useful analytical tool for future studies on stress physiology and welfare assessment in wild carnivores.”

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References

1. Jastrzębski, A.; Ożga-Wybranowska, K.; Łopucki, R. et al. A Validated LC-MS/MS Method for Simultaneous Determination of Cortisol and Cortisone in Grey Wolf Hair for Application in Ecological Studies. Molecules 2026, 31 (14), 2420. DOI: 10.3390/molecules31142420
2. Balasamy, S.; Atchudan, R.; Arya, S. et al. Cortisol: Biosensing and Detection Strategies. Clin Chim Acta 2024, 562, 119888. DOI: 10.1016/j.cca.2024.119888
3. Meyer, J. S.; Novak, M. A. Minireview: Hair Cortisol: A Novel Biomarker of Hypothalamic-Pituitary-Adrenocortical Activity. Endocrinology 2012, 153 (9), 4120-7. DOI: 10.1210/en.2012-1226
4. Botía, M.; Escribano, D.; Martínez-Subiela, S. et al. Different Types of Glucocorticoids to Evaluate Stress and Welfare in Animals and Humans: General Concepts and Examples of Combined Use. Metabolites 2023, 13 (1), 106. DOI: 10.3390/metabo13010106
5. Talha Iqbal, T.; Adnan Elahi, A.; William Wijns, W. et al. Cortisol Detection Methods for Stress Monitoring in Connected Health. Health Sci. Rev. 2023, 6, 100079, DOI: 10.1016/j.hsr.2023.100079
6. Lee, D. Y.; Kim, E.; Choi, M. H. Technical and Clinical Aspects of Cortisol as a Biochemical Marker of Chronic Stress. BMB Rep. 2015, 48 (4), 209-216. DOI: 10.5483/bmbrep.2015.48.4.275