Analytical Sciences

Simultaneous Liquid Chromatographic Measurement of Melatonin and Related Indoles through Post-column Electrochemical Demethylation and Fluorescence Derivatization

Kenichiro TODOROKI,* Kousaku ISHIMARU,* Hideyuki YOSHIDA,* Takashi YOSHITAKE,** Hitoshi NOHTA,* and Masatoshi YAMAGUCHI*

*Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan
**School of Pharmaceutical Sciences, International University of Health and Welfare, 2600-1 Kitakanemaru, Ootawara, Tochigi 324-8501, Japan

In this paper we describe a highly sensitive and selective liquid chromatographic method for the determination of 5-methoxyindoles (5-methoxyindole-3-acetic acid, 5-methoxytryptamine, 5-methoxytryptophol, and melatonin) using a post-column technique involving electrolytic demethylation followed by fluorescence derivatization with benzylamine. We separated these compounds within 30 min by reversed-phase liquid chromatography using acetate buffer (pH 6.5)-acetonitrile-methanol [8:1:1 (v/v); isocratic elution] and then demethylated them, using a commercial coulometric system, to give the corresponding 5-hydroxyindoles. Next, we converted the 5-hydroxyindole products into fluorescent derivatives by their reactions with benzylamine in the presence of potassium hexacyanoferrate(III). We detected the derivatives spectrofluorometrically at 480 nm upon excitation at 345 nm. The detection limits (signal-to-noise ratio = 3) of the 5-methoxyindoles were in the range from 12 to 93 fmol per 20-µL injection.

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