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.