Detection of Serotonin, Melatonin, and Their Metabolites in Honey

Abstract

Melatonin and serotonin, products of tryptophan metabolism, are endogenous neurotransmitters and hormones. We have identified and quantified these metabolites in natural honey from Australia, USA, and Poland using a Xevo G2 XS qTof LC-MS. To help ensure correct product identification, some samples were prepurified by RP-HPLC based on the retention times of standards, prior to LC-MS. The concentrations of the metabolites of interest depended on the source of the honey. For Australian honey, levels for melatonin and 2-hydroxymelatonin were 0.91 and 0.68 ng/g, respectively. Melatonin was detected in one brand of US commercial honey at 0.48 ng/g, while a second brand contained serotonin at 88.2 ng/g. In Polish natural honey, 20.6 ng/g of serotonin and 40.8 ng/g of N-acetylserotonin (NAS) were detected, while in Polish commercial honey 25.9 ng/g of serotonin and 7.30 ng/g of NAS were present. We suggest that addictive and health-related properties of honey may be in part dependent on the presence of serotonin, melatonin, and their metabolites, and that these compounds may play a role in the colony activities of bees.

Keywords: 2-hydroxymelatonin; N-acetylserotonin; bees; honey; melatonin; serotonin.

Figure 1.

Detection of melatonin, 2-hydroxymelatonin, serotonin, and NAS in Australian honey. The honey was extracted with methylene chloride and analyzed after being prepurified on a C18 column (A) or directly (B) using a Zorbax Eclipse Plus C18 column connected to a Xevo G2 XS equipped with an ACQUITY UPLC I-Class System (Waters, Milford, MA). The extracted ion chromatograms (EICs) were obtained using m/z = 255.1 [M + Na]⁺, 249.1 [M + H]⁺, 160.1 [M + H – NH₃]⁺ and 160.1 [M + H – NH₂CH₃CO]⁺ for melatonin, 2-hydroxymelatonin, serotonin, and NAS, respectively.

Figure 2.

Detection of melatonin, serotonin, and NAS in commercial honey sample 1. The honey was extracted with methylene chloride and analyzed after prepurification of fractions of interest in a C18 column (A) or directly (B) using a Zorbax Eclipse Plus C18 column connected to a Xevo G2 XS equipped with an ACQUITY UPLC I-Class System (Waters, Milford, MA). The EICs were obtained using m/z = 255.1 [M + Na]⁺ or 233.1 [M + H]⁺, 160.1 [M + H – NH₃]⁺ and 160.1 [M + H – NH₂CH₃CO]⁺ for melatonin, serotonin, and NAS, respectively.

Figure 3.

Detection of melatonin and serotonin in commercial honey sample 2. The honey was extracted with ethyl acetate and analyzed after prepurification on a C18 column (A) or directly (B) using a Zorbax Eclipse Plus C18 column connected to a Xevo G2 XS equipped with an ACQUITY UPLC I-Class System (Waters, Milford, MA). The EICs were obtained using m/z = 174.1 [M + H – NH₂CH₃CO]⁺ and 160.1 [M + H – NH₃]⁺ for melatonin and serotonin, respectively.

Figure 4.

Measurement of serotonin and NAS in Polish honey. The honey was extracted with ethyl acetate and analyzed directly using a Zorbax Eclipse Plus C18 column connected to a Xevo G2 XS equipped with an ACQUITY UPLC I-Class System (Waters, Milford, MA). The extracted ion chromatograms (EICs) were obtained using m/z = 160.1 [M + H – NH₃]⁺ for serotonin and 160.1 [M + H – NH₂CH₃CO]⁺ for NAS. (A) Polish natural honey; (B) Polish commercial honey.