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Salivary cortisol is an important biomarker for the assessment of stress-related disorders and endocrine function. Due to the noninvasive and convenient nature of saliva sampling, it has considerable potential for clinical application. However, the low concentration of cortisol in saliva, typically at the nanograms-per-milliliter level, together with the complexity of the salivary matrix, poses substantial challenges to analytical sensitivity and selectivity. In this study, an analytical method for salivary cortisol determination was developed based on immunomagnetic bead enrichment and purification coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cortisol monoclonal antibodies were conjugated to carboxyl-modified nanomagnetic beads, and the target analyte was extracted and purified through immunoaffinity adsorption followed by ethanol elution. The method exhibited good linearity over the ranges of 0.10-1.00 ng/mL and 1.00-20.00 ng/mL, with a limit of detection (LOD) of 0.04 ng/mL and a lower limit of quantification (LLOQ) of 0.10 ng/mL. The method also showed satisfactory accuracy, with spiked recoveries ranging from 80% to 110%. In addition, salivary cortisol concentrations showed a strong positive correlation with serum cortisol concentrations (Pearson correlation coefficient, r = 0.91636). These results indicate that the proposed method provides a sensitive, selective, and reliable approach for salivary cortisol analysis and shows promise for clinical application.