Determination of melatonin in saliva using automated solid‐phase extraction, high‐performance liquid chromatography and fluorescence detection

Abstract

A sensitive bioanalytical method for the determination of melatonin in saliva by solid‐phase extraction (SPE), high‐performance liquid chromatography (HPLC) and fluorescence detection has been developed and validated. Saliva was collected with a Salivette^® sampling device (Sarstedt) and a mixed‐mode SPE column was used for the extraction of melatonin and internal standard (N‐acetyl‐6‐methoxytryptamine) from the saliva. Chromatographic separation was performed using a HyPurity C18 LC column (150×2.1 mm) with mobile phase acetonitrile – ammonium hydrogen carbonate buffer, 0.015 M, pH 6.8 (23:77, v/v). Excitation and emission wavelengths were set to 285 nm and 345 nm, respectively. The within‐day precision for the method at 50 pmol/L was 7.9 % and the between‐day precision was 10.5 %. The limit of quantification was 50 pmol/L.

• Circadian rhythm
• HPLC
• indoleamine
• N‐acetyl‐5‐methoxytryptamine
• Salivette
• SPE

Acknowledgements

This study was funded by the Centrum för Klinisk Forskning (Center for Clinical Research) Landstinget Dalarna, Sparbanksstiftelsen Dalarna and the Marcus and Amelia Wallenberg Foundation.