Ca. Street et al., THE PURIFICATION AND CHARACTERIZATION OF BIOLOGICAL 6-SULPHATOXYMELATONIN AND COMPARISON WITH SYNTHETIC 6-SULPHATOXYMELATONIN, Journal of pineal research, 20(2), 1996, pp. 98-114
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.