title: Simultaneous liquid chromatographic measurement of melatonin and related indoles through post-column electrochemical demethylation and fluorescence derivatization.
abstract: 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-microL injection.
authors.affiliation: Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan.
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Todoroki
authors.forename: Kousaku
authors.initials: K
authors.lastname: Ishimaru
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Yoshida
authors.forename: Takashi
authors.initials: T
authors.lastname: Yoshitake
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nohta
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Yamaguchi
mesh_terms: D002021:Buffers; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D004563:Electrochemistry / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000032:analysis* / Q000737:chemistry; D013058:Mass Spectrometry; D008550:Melatonin / Q000032:analysis*; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry; D012680:Sensitivity and Specificity; D012972:Sodium Hydroxide / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
chemical_list: D002021:Buffers; D007211:Indoles; D012972:Sodium Hydroxide; C487413:5-methoxyindole; D008550:Melatonin; D010100:Oxygen