Melatonin (N-acetyl-5-methoxytryptamine) (Fig. 1) is a hormone isolated and identified for the first time in the pineal gland of vertebrates and later found in many species, such as bacteria, fungi, plants  and different animal species, including humans. Melatonin biosynthesis in pineal gland cells was identified by Axelrod : tryptophan acts as the precursor and is converted into 5-hydroxytryptophan by the enzyme tryptophan hydroxylase (TPH); by means of the aromatic amino acid decarboxylase (AAAD) serotonin is next obtained and then converted by the arylalkyl-amine N-acetyltransferase (AANAT) in N-acetylserotonin, eventually converted into melatonin by N acetylserotonin methyltransferase (ASMT). Once synthesized, melatonin is not stored in the pineal gland but secreted into blood capillaries and cerebrospinal fluids . Also owing to its amphipathic nature, melatonin is able to permeate all tissues and subcellular compartments. The enzymes responsible for biosynthesis of melatonin are also present in human lymphocytes  in the retina, skin, bone marrow and, in large quantities, in the gastrointestinal tract. In human, pineal melatonin production has a circadian rhythm, with high production overnight and low levels during the day; as the years passed, the pineal gland is reduced in size and melatonin production decreases.
Melatonin plays a very important role as an antioxidant against free radicals production either directly or through the stimulation of antioxidant enzymes (promoting transcription) and by reducing the activity of those pro-oxidants . This substance is involved in the regulation of the immune system [3,4,5] and in the glucose metabolism, as low levels of melatonin increase blood glucose levels [4, 6]. It also acts as modulator of gastrointestinal motility [4, 7,8,9] and protector of fibroblasts and myelin [10, 11].
The first evidence that melatonin exists in plants came from two independent groups in 1995 [12, 13], and in subsequent years, melatonin was also detected in roots, leaves, fruits, flowers, and seeds of a variety of vegetables, cereals, fruits, as well in medicinal herbs [14, 15]. The potent free-radical scavenger properties of melatonin and its broad-spectrum antioxidant activity in animals suggest that it acts similarly in plants .
The biosynthetic pathway of melatonin in plants is different from that in the pineal gland of vertebrates: tryptophan is converted to tryptamine by tryptophan decarboxylase, subsequently a hydroxylase converts into serotonin; the following steps en route to melatonin are the same that occur in mammals . In plants, roots could be a potential site of melatonin biosynthesis but the generality of this view is unproven. Currently, no melatonin receptors or binding sites have been identified in plants; alternatively, since melatonin has similar structure to auxin, it could possibly interact with the auxin receptor . Auxin is involved in modulating the development of roots and leaves, in the interactions between plant and pathogenic, and seems to protect against senescence. Melatonin might therefore agonistically mimic the properties of auxin, acting as a growth hormone. Melatonin can penetrate all intracellular compartments and protect all parts of the plant from oxidative stress and cell death, particularly germ and reproductive tissues [19, 20]. Melatonin can directly scavenge reactive oxygen species and stimulates the activity of antioxidant enzymes; it is also able to protect cells from lipid peroxidation and proteins and DNA from damage [21, 22]. It also acts as cell protector, modulator of cytoskeleton, growth promoter and protector against low and high temperatures as well as drought, and could also be involved in photosynthesis [19, 22].
From the first detection of melatonin in plant in 1995, numerous studies have shown the presence of melatonin in different plant species and tissues, with concentrations ranging from pg g−1 to μg g−1 of plant material [20, 23,24,25,26,27,28]. Melatonin concentrations in the plants differ not only from species to species but also among varieties within the same species. Moreover, the melatonin levels also vary within different organs or tissues of a given plant, and the highest contents have generally been reported in reproductive organs such as flowers, fruits and the seeds [13, 14].
Although the presence of melatonin seems to be a common trait of many plants, there is still lack of information on its occurrence hemp (Cannabis sativa L.), herbaceous annual dioecious plant bearing male and female flowers on separate plants. The taxonomy of hemp has always been controversial, but there seems now a general consensus to view it as a single species, Cannabis sativa L., sorted out into several varieties or chemotypes. To date, more than 480 constituents have been identified from of Cannabis, out of which almost 150 are classified as cannabinoids, enclosed in glandular tricomes of unfertilized flower heads of the female plant . However, many other natural product classes besides the cannabinoids been identified from aerial parts (flowers heads and leaves) of Cannabis sativa L. including monoterpenes, sesquiterpenes, flavonoids, stilbenes, steroids, fatty acids, and nitrogenous compounds [30,31,32,33].
Different analytical techniques to detect melatonin in plants have been reported, including radioimmunoassay , immunoprecipitation , HPLC combined with fluorescence detection, elettrochemical detection or mass spectrometry [19, 25, 26]. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) seems up to now the most reliable technique to determine melatonin in plants [34, 35]. However the complicated matrix of each selected plant and tissue extract and its low concentrations always require the set up of a specific extraction and purification procedure of endogenous melatonin prior the analysis.
The aim of this study was to develop a simple, reliable and validate analytical method for the extraction, purification and quantitation of melatonin in hemp (Cannabis sativa L.). The method was applied to investigate the amount of melatonin in different hemp varieties, belonging to fiber-type and drug-type chemotype. Inside each hemp variety, melatonin was determined in two organs, the aerial parts (flower heads and leaves) and the reproductive organ (seeds).