The aims of this study were to extract the stress biomarkers from human hair samples and explore the optimal
procedure for this extraction. The investigation included a methodological variation related to the extraction time and
pulverization method in the procedure for the determination of stress biomarkers. Consequently, the three stress
biomarkers, cortisol, testosterone, and dehydroepiandrosterone were successfully extracted from the hair sample.
Moreover, the pulverization method had no impact on the determination procedure. Additionally, the extraction time
significantly affected the amount of the target substances. An optimal procedure based on the results of this study
was proposed.
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