Direct determination of endogenous melatonin in human saliva by column-switching semi-microcolumn liquid chromatography/mass spectrometry with on-line analyte enrichment

An analytical method that enables direct and sensitive determination of endogenous melatonin (MLT) in human saliva was developed by means of column-switching semi-microcolumn liquid chromatography (i.d.: 1-2 mm)/mass spectrometry (LC/MS). The system allows direct injection analysis of a 400-microL aliquot of saliva with minimal sample pretreatment (internal standard (IS) addition and vortex mixing) and a relatively short run-time (10 min). The system consists of three columns to attain large volume injection and on-line analyte enrichment. A pre-column packed with a silica-based mixed-functional C8 (4.0 mm i.d. x 20 mm) was used for on-line sample cleanup. MLT and an IS, the d7 isomer of MLT (d7-MLT), were heart-cut by valve switching and enriched at the top of the intermediate trapping column packed with a silica-based C18 (4.0 mm i.d. x 10 mm). Subsequently, the analytes were backflushed into a semi-micro C18 silica column (2.0 mm i.d. x 150 mm) for the final separation. MLT and IS were ascertained by positive electrospray ionization and selected ion monitoring (SIM). MLT was monitored based on its fragment ion at m/z 174.1 by in-source collision-induced dissociation (CID). The validation of this method revealed a detection limit of 2.5 pg mL(-1) at a signal-to-noise (S/N) ratio of 5. The linearity of the method was established in the ranges 5-250 and 100-2500 pg mL(-1) with a coefficient of determination of greater than 0.998. Accuracies, evaluated at five levels in the range 5-1000 pg mL(-1), were between 81 and 108% with a relative standard deviation (RSD) ranging from 1.3-20%. The method was successfully applied for the endogenous saliva MLT monitoring of two healthy subjects.