Determination of Melatonin and Its Metabolites in Biological Fluids and Eggs Using High-Performance Liquid Chromatography with Fluorescence and Quadrupole-Orbitrap High-Resolution Mass Spectrometry

Abstract

Melatonin is a hormone found in animals, plants, fungi, and bacteria, which can be used as a sleep aid and in the treatment of sleep disorders. Here, a rapid and accurate method was developed for the determination of melatonin in plasma, milk, and egg using high-performance liquid chromatography with fluorimetric detection (HPLC-FLD) and ultra high performance liquid chromatography–quadrupole-orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). Sample was extracted with methanol and cleaned by passing through hydrophilic–lipophilic balanced (HLB) solid phase extraction cartridge. The method validation results showed that recovery for melatonin was between 80.3 and 105.1 % with relative standard deviations less than 9.8 %. UHPLC-Q-Orbitrap HRMS was employed as a further means of confirmation to assure accuracy of the results. Limits of detection for melatonin were 0.13 ng g−1 and 0.14 ng mL−1 for solid and liquid samples, respectively. Thus, the proposed method can be applied to detect melatonin in milk, plasma, and egg samples. Meanwhile, the metabolites of melatonin were identified using Q-Orbitrap HRMS. The approach identified five metabolites; among them, a novel metabolite, named mono-oxygenation-demethylation-melatonin, was found in plasma.