Determination of melatonin content of different varieties of hemp (Cannabis sativa L.) by liquid chromatography tandem mass spectrometry

Abstract: Melatonin (N-acetyl-5-methoxytryptamine), was first isolated in the bovine pineal gland, and then was discovered in bacteria, algae and higher plants. Melatonin concentrations in plants differ from species to species, among varieties within the same species and also within different organs or tissues of a given plant as roots, leaves, fruits, flowers, and seeds. Although the presence of melatonin in plants seems to be a universal trait, there is still lack of information on its occurrence in several plants, in particular in hemp (Cannabis sativa L.). The aim of this study was to develop a method to quantitate melatonin in the aerial parts of the plant, constituted of leaves and flower heads, and in the seeds, of four different hemp varieties using liquid chromatography–tandem mass spectrometry (LC–ESI/MS/MS). A sample preparation procedure with methanol extraction followed by solid-phase clean-up protocol was set up for melatonin extraction and a LC–ESI/MS/MS method in single reaction monitoring (SRM) was used for its determination. Chromatographic separation was achieved on a C18 column using a gradient elution with acetonitrile–buffer ammonium formate/formic acid system. The developed method, validated as per International Conference on Harmonization guidelines, was successfully applied to analyze hemp samples varieties belonging to fiber-type or drug-type chemotypes. The melatonin was found to accumulate highly in seeds in the range 13.43–30.40 ng g⁻¹ while the content in the aerial parts was assessed in the range 1.16–4.85 ng g⁻¹. No correlation was found between the melatonin levels detected in aerial parts and seeds in each hemp variety and the concentration of specific cannabinoids in the same variety. Graphic abstract: [Figure not available: see fulltext.].