The purification and characterization of biological 6-sulphatoxymelatonin and comparison with synthetic 6-sulphatoxymelatonin

: 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.