Establishment of a reference range for hair cortisol using a HPLC-MS/MS method in a Northern Italian population

PURPOSE: Hair cortisol concentration (HCC) represents a promising biomarker for long-term hypothalamic-pituitary-adrenal (HPA) axis activity. However, its routine clinical application is currently limited by the lack of well-established reference ranges. We aimed to establish population-based reference intervals for HCC in healthy adults from Northern Italy, using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method following the Clinical and Laboratory Standards Institute (CLSI) EP28-A3c guidelines. METHODS: Hair samples were collected from 256 healthy volunteers selected using strict inclusion and exclusion criteria. Cortisol quantification was performed by LC-MS/MS using a deuterated internal standard, guaranteeing high sensitivity and specificity. The method was validated for linearity, limit of detection, extraction efficiency, matrix effect, robustness across different sample weights, and inter-batch reproducibility using an internal quality control sample. Reference intervals were defined nonparametrically (2.5th-97.5th percentiles). Associations with demographic, clinical, and lifestyle variables were assessed. RESULTS: The established reference interval was 0.84 to 8.07 pg/mg, with a median of 2.41 pg/mg. HCC was significantly associated with body mass index (ρ = 0.160, p = 0.010) and weakly correlated with perceived stress (ρ = 0.137, p = 0.028). However, no significant associations were observed with sex, age, smoking status, alcohol consumption, physical activity, hair dyeing, or hair washing frequency. CONCLUSION: This study provides, for the first time, a population-based reference interval for hair cortisol concentrations using LC-MS/MS in healthy adults. These findings offer a benchmark for future research, supporting the use of HCC as a biomarker in epidemiological and translational research contexts.