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.

An enzyme immunoassay for 6-sulphatoxy-melatonin in human urine.

We describe a newly developed enzyme immunoassay (EIA) for the determination of 6-sulphatoxy-melatonin (aMT6s) in human urine, using a aMT6s-bovine serum albumin-horseradish peroxidase (aMt6s-BSA-HRP) conjugate as the enzyme label. The assay incorporates a highly specific antibody raised in rabbits. The EIA has a sensitivity of 2 pg/well (40 pg/ml) with intraassay coefficients of variation of 2.

HPLC assay of melatonin in plasma with fluorescence detection.

We report an HPLC assay for melatonin that incorporates automated injection, methanol/water mobile phase, and fluorescence detection. Plasma samples were extracted by solid and liquid phases. Recovery was > 70% for 1-10 mL of plasma extracted, approximately 40 pg-250 ng of melatonin.