Synthesis and characterization of stable polymer-coated C8 stationary phase with high durability under extreme pH conditions for high-performance liquid chromatography.

A highly chemically stable polymer-coated silica-based C8 stationary phase was developed by combining modification with octyl groups and a polymer coating technology. The stationary phase was prepared by the following procedure: (1) introduction of octyl groups to the silica surface; (2) coating the C8 silica with a silicone polymer. 29Si solid-state NMR spectra indicated that a silicone polymer reacted not only with residual silanol groups on the silica surface, but with those generated from silanes used for the introduction of octyl groups. Column durability was evaluated with an acidic mobile phase (60 degrees C, pH 1) and a basic mobile phase (50 degrees C, pH 10) in accelerated damaging conditions. The C8 phase showed a high durability under both conditions.

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.