Column-switching high-performance liquid chromatographic system with a laser-induced fluorimetric detector for direct, automated assay of salivary cortisol.

In order to measure human stress, an easy and rapid, fully automated method for the determination of cortisol in saliva has been developed, using column-switching high-performance liquid chromatography with laser-induced fluorescence detection, which involves post-column labeling with sulfuric acid. The developed system requires only 0.1 ml of saliva, and a simple pretreatment consisting of dilution and filtration is sufficient. The column-switching system consisted of a Polymer-Coated Mixed-Functional silica (PCMF) column for deproteinization, and a CN column for frontal concentration and separation. An ODS column in place of the CN provided a better separation, but required a post-column make-up of water for safe reaction. Detection limit of cortisol was 8 fmol (signal-to-noise ratio = 3), which is adequate for routine determination of normal levels of cortisol (1-20 pmol/ml). The analysis time was about 40 min and reproducibility was excellent with an R.S.D. of less than 5%.