COLUMN-SWITCHING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC SYSTEM WITH A LASER-INDUCED FLUOROMETRIC 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 mi 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%.