THE PURIFICATION AND CHARACTERIZATION OF BIOLOGICAL 6-SULPHATOXYMELATONIN AND COMPARISON WITH SYNTHETIC 6-SULPHATOXYMELATONIN

We have purified the major metabolite of melatonin, 6-sulphatoxymelato nin, from urine and compared it to its synthetic counterpart. For prep aration 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 fro m N-acetyl serotonin sulphate, the other sulphate metabolite of melato nin, by preparative thin-layer chromatography. Synthetic 6-sulphatoxym elatonin was produced by reacting 6-hydroxymelatonin with chlorosulpho nic acid in dimethylformamide; the reaction mixture was purified on Fl orisil and preparative thin-layer chromatography was used to remove in dolic by-products of the reaction. Elemental and X-ray microanalysis o f the biological and synthetic products showed that classical methods used for their purification introduced inorganic impurities, such as s ilicon- and chlorine- containing compounds, which were not detectable by thin-layer chromatography, infrared spectroscopy, nuclear magnetic resonance spectroscopy, or gas chromatography-mass spectrometry. We in troduced further purification steps to remove these inorganic impuriti es, monitoring the process using elemental and X-ray microanalysis. Ex tensive characterization of the resulting purified products showed tha t the biological and synthetic compounds were identical.