Determination of cortisol in human saliva by automated in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry.

We developed a simple, rapid, and sensitive method for determination of cortisol levels in human saliva. Cortisol was analyzed by on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC/MS). Cortisol was separated within 5 min by HPLC using an Eclipse ZDB-C8 column and 1% acetic acid/methanol (50/50, v/v) at a flow rate of 0.2 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for MS detection of cortisol. The optimum in-tube SPME conditions were 20 draw/eject cycles with a sample size of 40 microL using a Supel Q PLOT capillary column as the extraction device. The extracted compounds could be desorbed easily from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC/MS method, good linearity of the calibration curve (r=0.9977) was obtained in the concentration range 50-2000 pg/mL of cortisol in saliva, and the limit of detection (S/N=3) was 5 pg/mL. The method described here showed 48-fold higher sensitivity than the direct injection method (5 microL injection). The within-run and between-day precisions (relative standard deviations) were below 4.6% and 8.9% (n=5), respectively. This method was applied successfully to the analysis of saliva samples without interference peaks. The recoveries of cortisol spiked into saliva samples were above 95%, and the relative standard deviations were below 6.0%. This method was used to analyze the changes in salivary cortisol level according to stress load.