Determination of hair cortisol by liquid chromatography coupled to mass spectrometry (LC-MS/MS) as biomarker of chronic stress and application to academic students

Hair cortisol concentration (HCC), a non-invasive biomarker reflecting long-term cortisol exposure, offers valuable insight into chronic stress, complementing established acute stress measures such as salivary or blood cortisol. At the moment, the most valuable use of hair cortisol bio-analysis is to study trends and associations within specific populations rather than establishing a single cut-off value for stress level assessment. In this frame, a rapid LC-MS/MS method for HCC determination has been developed and applied to measure chronic stress in university students with the aim to correlate the analytical results with the perceived stress during the preparation of the exams. A total of 100 students from different academic programs were recruited providing hair samples and data on academic and lifestyle stressors. The method has been validated in accordance with forensic toxicological guidelines ensuring high sensitivity (LOD 2 pg/mg; LOQ 5 pg/mg) and robust performance across selectivity, linearity, accuracy, and stability parameters. Method linearity was assessed in the range 5-50 pg/mg; accuracy and precision calculated on QC were always below 7 %; prepared samples were stable for 4 days at refrigerated temperature. HCC was detectable in 94 % of samples in the range 5--47.7 pg/mg. Students attending Law and Biology courses exhibited the highest mean HCC values. Dietary changes and smoking were associated with higher stress perception. Among academic stressors, balancing work and study, as well as difficulties in study organization, were linked to greater perceived stress. No statistically significant correlation was found between perceived stress and HCC, underscoring the complexity of chronic stress assessment and the value of combining subjective and physiological indicators.