LC-MS/MS Compares Hair Hormone Sampling Methods

Scientists often test hair samples to measure hormone levels built up over time, a method often used in research on stress, mood, and hormones. But there's been an open question: What's the best way to actually collect the hair to get accurate results? Some researchers snip one thicker strand from a single spot, while others gather several thinner strands from different spots nearby and combine them. A team at the University of Siegen in Germany decided to directly compare these two collection methods to see which one gives more reliable results. Hair samples used in the study were analyzed for cortisol, cortisone, progesterone, dehydroepiandrosterone, and endocannabinoids via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Their findings were published in the journal Psychoneuroendocrinology.¹

Why Might Combining Hair from Several Nearby Scalp Spots Give More Accurate Hormone Results?

Researchers have generally used one of two ways to collect hair samples: either they snip one thicker chunk of hair, roughly the width of half a pencil, or they take several thinner snips from different spots and combine them to get enough hair. That second approach is mainly done for appearance's sake, as it's less noticeable than cutting one visible chunk, which likely makes people more willing to participate.²That kind of loose, inconsistent approach to sampling would only be fine if both methods gave basically the same results. But forensic research suggests otherwise; levels of certain substances in hair (like ethyl glucuronide and caffeine) can differ quite a bit depending on exactly where on the scalp the sample was taken, even when the spots are right next to each other.^3,4

Similar patterns have shown up in stress-hormone research, too. In one study, researchers looked at cortisol levels across five different spots on the scalp in 14 people. They found noticeable differences depending on which region the hair came from (levels varied by about 30.5% on average), and even samples taken close together within the same small area showed some variation (about 15.6% on average).⁵ This matches what the researchers conducting the study found in their own recent research; they report similar levels of variation in cortisol within a single small area of the scalp, whether the hair came from the back-of-head "posterior vertex" spot (16.90% variation) or the "occipital" area lower on the back of the head (19.08% variation).⁶ Since this amount of variation is much higher than what would be expected just from small inconsistencies in the laboratory testing process itself, it suggests the differences are real — meaning cortisol levels genuinely do vary between hair samples taken from spots close together, not just due to measurement error.⁷

“This makes it conceivable,” write the authors of the paper,¹ “that accumulating hair material across nearby scalp positions may not only offer cosmetic advantages but may also improve results by averaging out regional differences in concentrations and thereby yielding more homogeneous samples that better reflect underlying long-term endocrine analyte concentrations.”

Which Hair-Sampling Method Produced More Reliable Hormone Measurements?

For this study, the researchers collected hair from 53 people, gathering samples twice, three months apart. Each time, they took six samples total (three using the "single strand" method and three using the "combined subsamples" method) all snipped from a small, carefully mapped-out area at the back of the head to keep the collection spots consistent. The team then compared the two methods on several fronts: how much the results varied from sample to sample within the same person, the average hormone levels each method picked up, how consistent the results were over the three-month gap, and how well each method's results lined up with other health markers like body mass index (BMI), waist-to-hip ratio, and self-reported stress levels.¹

The researchers found that the combined-subsample method turned out to be more consistent; for most of the substances measured, results didn't vary as much from sample to sample within the same person compared to the single-strand method. This method also showed a slightly stronger link between stress hormones and BMI. Otherwise, the two methods were similar: average hormone levels and how reliable the results were over time (the three-month retest) came out about the same either way. One extra finding was reported: when researchers dug into the single-strand samples more closely, they noticed that where exactly on the head the hair was taken from seemed to affect the levels of certain substances.¹

“Our findings,” write the authors of the paper,¹ “suggest that the accumulation of smaller subsamples may help to detect more consistent hair endocrine and endocannabinoid measures. The observed position-related differences within a small scalp area also further highlight the importance of careful sampling protocols and methodological standardization in PNE hair research.”

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References

1. Huthsteiner, K.; Finke, J. B.; Klucken, T. et al. A Systematic Comparison of Hair Sampling Methods in Endocrine and Endocannabinoid Hair Analysis: Is One Sample Enough or Should Hair Be Accumulated? Psychoneuroendocrinoloy 2026, 193, 107985. DOI: 10.1016/j.psyneuen.2026.107985
2. Cooper, G. A.; Kronstrand, R.; Kintz, P. et al. Society of Hair Testing Guidelines for Drug Testing in Hair. Forensic Sci Int. 2012, 218 (1-3), 20-4. DOI: 10.1016/j.forsciint.2011.10.024
3. Dussy, F.; Carson, N.; Hangartner, S. et al. Is One Hair Lock Really Representative? Drug Test Anal. 2014, 6 (Suppl 1), 5-8. DOI: 10.1002/dta.1627
4. Meier, U.; Briellmann, T.; Scheurer, E. et al. Distribution Pattern of Ethyl Glucuronide and Caffeine Concentrations over the Scalp of a Single Person in a Forensic Context. Drug Test Anal. 2017, 9 (10), 1594-1603. DOI: 10.1002/dta.2186
5. Sauvé, B.; Koren, G.; Walsh, G. et al. Measurement of Cortisol in Human Hair as a Biomarker of Systemic Exposure. Clin Invest Med. 2007, 30 (5), E183-191. DOI: 10.25011/cim.v30i5.2894
6. Huthsteiner, K.; Finke, J. B.; Peters, E. M. J. et al. What is the Best Sampling Region for Endocrine Hair Analysis? A Comparison Between the Posterior Vertex and Occipital Region and Recommendation for Standardization. Psychoneuroendocrinology 2025, 177, 107457. DOI: 10.1016/j.psyneuen.2025.107457
7. Gao, W.; Stalder, T.; Foley, P. et al. Quantitative Analysis of Steroid Hormones in Human Hair Using a Column-Switching LC-APCI-MS/MS Assay. J Chromatogr B Analyt Technol Biomed Life Sci. 2013, 928, 1-8. DOI: 10.1016/j.jchromb.2013.03.008