Comparative analysis of salivary cortisol measurements using different assay methods in relation to serum-free cortisol measurement

Salivary cortisol reflects the biologically active form of serum cortisol, offering a noninvasive evaluation method for the diurnal rhythm of the hypothalamic–pituitary–adrenal (HPA) axis. While liquid chromatography–tandem mass spectrometry (LC–MS/MS) is known for its specificity, immunoassays (IA) are commonly used because of their simplicity. This study aimed to assess the performance of salivary cortisol measurement using both IA and LC–MS/MS in comparison to serum free cortisol measurement. and Methods: Assay results for 188 saliva and 94 serum samples from 47 participants were analyzed. Salivary samples collected at different time points were analyzed using IA and LC–MS/MS. Serum samples were analyzed for cortisol, cortisol-binding globulin, and serum free cortisol. The statistical analyses included correlations and method comparisons. The diurnal salivary cortisol profiles exhibited a comparable circadian rhythm pattern; however, the concentrations measured using IA were consistently higher than those measured using LC–MS/MS. The correlation analysis revealed robust associations among salivary cortisol (IA), salivary cortisol (LC–MS/MS), and serum free cortisol levels (LC-MS/MS). However, the method comparison revealed a systematic bias between IA and LC–MS/MS in salivary cortisol measurement. This study contributes to the ongoing debate on assay techniques by affirming the suitability of IA and LC–MS/MS for salivary cortisol measurement to assess dynamic changes in HPA axis activity. The identified systematic bias emphasizes the importance of selecting methods based on specific research or clinical requirements.