The isolation, purification, and characterisation of the principal urinary metabolites of melatonin.

Melatonin is metabolised by hydroxylation to form 6-hydroxy-melatonin and by demethylation to form N-acetyl-serotonin, which are excreted as sulphate and glucuronide conjugates. We required these metabolites as pure powders and therefore undertook their isolation and characterisation. Three volunteers ingested 1 g each of melatonin, and their urine was collected and pooled. For the sulphate conjugates, a Lichoprep column was used to concentrate the metabolites and to remove most of the urea. The sulphate conjugates were separated from the glucuronides on a Florisil column and further purified on a fractogel column. They were separated by high-performance liquid chromatography (HPLC) resulting in white powders of 6-hydroxy-melatonin sulphate (SaMT) and N-acetyl-serotonin sulphate (SNAS). For the glucuronide conjugates, an aliquot of the pooled urine was taken to dryness, the residue was dissolved in methanol, and the solution was filtered. The methanol filtrate was taken to dryness, and the residue was applied to a Florisil column. The isolated glucuronide conjugates were recrystallized prior to separation by HPLC, which gave pure white powders of N-acetyl-serotonin glucuronide (GNAS) and 6-hydroxy-melatonin glucuronide (GaMT). Characterisation was achieved by using infrared and ultraviolet spectroscopy, thin-layer chromatography (TLC), and gas chromatography-mass spectrometry (GCMS). These techniques unambiguously confirmed the assigned structures for SaMT and SNAS and fully supported the assigned structures for GNAS and GaMT. Three TLC solvent systems were used, and in each case the individual conjugated metabolite appeared as a discreet spot. Purity, as assessed by GCMS, was shown to be greater than 95% for SNAS, SaMT, and GaMT and to be 88% for GNAS.