A sensitive method to determine melatonin in saliva by automated online in-tube solid-phase microextraction coupled with stable isotope-dilution liquid chromatography-tandem mass spectrometry

A simple, rapid, and sensitive method was developed for the determination of melatonin (MLT) in human saliva by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry. MLT was separated within 3 min by high performance liquid chromatography using an Inertsil ODS-3 column and water/acetonitrile (50/50, v/v) at a flow rate of 0.2 mL min(-1). The optimum in-tube SPME conditions were 25 draw/eject cycles with a sample size of 40 mL 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, with no carryover observed. The calibration curve for MLT using its stable isotope-labeled internal standard was linear (r = 0.9993) in the range of 0.2-50 pg mL(-1), and the limit of detection (S/N = 3) was 0.032 pg mL(-1). The method described here showed 40-fold higher sensitivity than the direct injection of 10 mL. The intra-day and inter-day precisions (relative standard deviations) were below 4.1% and 5.8% (n = 5), respectively. This method was applied successfully to the analysis of saliva samples without other pretreatment and interference peaks. The recoveries of MLT spiked into saliva samples were above 96%, and the relative standard deviations were below 7.1%. This method was used to analyze the changes in salivary MLT levels associated with light stimulation and lifestyle habits.