A Validated Method for the Simultaneous Determination of Oxytocin and Cortisol in Human Saliva

Oxytocin and cortisol (OXY and CORT) are hormones related to stress, cognitive, and social behaviors. Their detection is relevant to epidemiological studies aimed at investigating the effects of stressor factors on human life. The aim of this study was to develop and validate an assay for the measurement of OXY and CORT in saliva samples using liquid chromatogra- phy/tandem mass spectrometry (LC-MS/MS) in the presence of deuterated analogs. A 500 mL aliquot of oral fluid, obtained by the centrifugation of a chewed swab, was purified by solid-phase extraction. Analytes were then separated using C18 reversed-phase chromatography, subjected to positive electrospray ionization, and then quantified using a triple-quadrupole mass detector in multiple-reaction monitoring mode. The limits of quantification and the linear dynamic ranges were 2.0 × 10−3 and 0.5 nmol/L, and up to 1.0 × 10−1 and 20 nmol/L for OXY and CORT, respectively. Inter- and intra-run precision, expressed as relative standard deviation, was <7%, and accuracy was within 93–104% of the theoretical concentrations. The evaluation of matrix effects showed that the use of internal standards controlled sources of bias. The high sensitivity of the method allowed the quantification of OXY and CORT in the salivary samples of both adults and children: levels of CORT ranged from 0.6 to 18.5 nmol/L, while OXY levels were two orders of magnitude lower (from 1.7 × 10−3 to 1.1 × 10−2 nmol/L). To our knowledge, this is the first method that can analyze, in the same chromatographic run, both hormones in saliva samples.