A UHPLC-MS/MS method for the quantification of oxytocin in human saliva

Given the reported correlation between cerebrospinal fluid (CSF) and salivary oxytocin concentrations, salivary oxytocin has been proposed as a promising peripheral biomarker of central oxytocin activity. To date, oxytocin levels are commonly measured using enzyme immunoassays or radio-immunoassays; however, several challenges related to its accurate quantification have been reported, underscoring the need for a standardized and reliable analytical method. The present study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of oxytocin in human saliva, in accordance with EMA and FDA guidelines for bioanalytical method validation. Oxytocin levels were determined using the standard addition method. The chromatographic separation was performed through a gradient elution of two mobile phases (H₂O + 0.1% F.A. and MeOH: DMSO 97:3 (v/v) + 0.1% F.A.) using a Phenomenex Luna Omega 1.6 μm PS C18 100 Å LC Column 50 × 2.1 mm at 40°C with a column thermostat. It was connected to a tandem mass spectrometer, and the chromatographic run lasted 5 min. Additionally, in order to evaluate precision and accuracy of the method, real-world samples from caregivers working with people with dementia were analyzed. Method validation achieved satisfactory results in terms of robustness and reproducibility, especially at higher concentration values, while expected higher variability at lower concentrations was observed. However, real-world samples analysis confirmed the applicability of the method to real biological samples. Overall, the method was able to accurately quantify oxytocin levels in real-world samples, thus demonstrating its applicability in epidemiological studies.