title: The purification and characterization of biological 6-sulphatoxymelatonin and comparison with synthetic 6-sulphatoxymelatonin.
abstract: We have purified the major metabolite of melatonin, 6-sulphatoxymelatonin, from urine and compared it to its synthetic counterpart. For preparation of the biological material, oral melatonin was administered to human volunteers and their urine extracted onto Amberlite XAD-2 resin to remove urea; the glucuronide metabolites of melatonin were removed by silica chromatography; and 6-sulphatoxymelatonin was separated from N-acetyl serotonin sulphate, the other sulphate metabolite of melatonin, by preparative thin-layer chromatography. Synthetic 6-sulphatoxymelatonin was produced by reacting 6-hydroxymelatonin with chlorosulphonic acid in dimethylformamide; the reaction mixture was purified on Florisil and preparative thin-layer chromatography was used to remove indolic by-products of the reaction. Elemental and X-ray microanalysis of the biological and synthetic products showed that classical methods used for their purification introduced inorganic impurities, such as silicon- and chlorine-containing compounds, which were not detectable by thin-layer chromatography, infrared spectroscopy, nuclear magnetic resonance spectroscopy, or gas chromatography-mass spectrometry. We introduced further purification steps to remove these inorganic impurities, monitoring the process using elemental and X-ray microanalysis. Extensive characterization of the resulting purified products showed that the biological and synthetic compounds were identical.
authors.affiliation: Academic Unit of Obstetrics, Gynaecology and Reproductive Physiology, London Hospital Medical College, Whitechapel, England.
authors.forename: C A
authors.initials: CA
authors.lastname: Street
authors.forename: W L
authors.initials: WL
authors.lastname: Di
authors.forename: J F
authors.initials: JF
authors.lastname: Peniston-Bird
authors.forename: S
authors.initials: S
authors.lastname: Patel
authors.forename: P
authors.initials: P
authors.lastname: Sadler
authors.forename: R E
authors.initials: RE
authors.lastname: Silman
mesh_terms: D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D006801:Humans; D017633:Magnesium Silicates; D009682:Magnetic Resonance Spectroscopy; D008550:Melatonin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification / Q000652:urine; D008854:Microscopy, Electron; D012117:Resins, Synthetic; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet
chemical_list: D017633:Magnesium Silicates; D012117:Resins, Synthetic; C005013:Florisil; C054513:6-sulfatoxymelatonin; C046797:amberlite; D008550:Melatonin