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

• Published in: Rapid communications in mass spectrometry Vol. 18; no. 12; pp. 1250 - 1258
• Main Authors: Motoyama, Akira, Kanda, Taketoshi, Namba, Ryujiro
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.01.2004
• Subjects: Adult; Antioxidants - analysis; Antioxidants - physiology; Child, Preschool; Chromatography, High Pressure Liquid - methods; Circadian Rhythm - physiology; Female; Humans; Male; Melatonin - analysis; Melatonin - physiology; Middle Aged; Online Systems; Reproducibility of Results; Saliva - chemistry; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization - methods
• ISSN: 0951-4198; 1097-0231
• DOI: 10.1002/rcm.1473

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‐μL 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. × 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. × 10 mm). Subsequently, the analytes were backflushed into a semi‐micro C18 silica column (2.0 mm i.d. × 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. Copyright © 2004 John Wiley & Sons, Ltd.