Simultaneous analysis of multiple steroidal biomarkers in saliva for objective stress assessment by on-line coupling of automated in-tube solid-phase microextraction and polarity-switching LC-MS/MS

To objectively and quantitatively assess the degree of stress, we developed a simple and sensitive method for the simultaneous determination of four steroidal stress biomarkers in saliva: cortisol (CRT), testosterone (TES), dehydroepiandrosterone (DHEA) and DHEA sulfate (DHAES), by on-line coupling of automated in-tube solidphase microextraction (IT-SPME) and polarity-switching liquid chromatography-tandem mass spectrometry (LCMS/MS). These four compounds were extracted from saliva and concentrated on Supel-Q PLOT capillary tubes by IT-SPME, followed by their separation and detection within 8 min by LC-MS/MS using a TSK-gel ODS-80Ts column. Calibration curves using their stable isotope-labeled internal standards showed good linearity ranging from 0.01 to 20 ng mL-1, with the limits of detection (S/N=3) of CRT, TES, DHEA and DHEAS being 0.58, 0.40, 8.5 and 0.47 pg mL-1, respectively. Moreover, intra- and inter-day variations were lower than 6.1 and 7.9% (n = 5), respectively. The recoveries of these compounds from saliva samples were in the range of 94.5-106.2%. The developed method is highly sensitive and specific and can easily measure these biomarkers in 50 & mu;L samples of saliva.