title: Selective determination of native fluorescent bioamines through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection.
abstract: In this paper, we introduce a novel approach for the highly selective and sensitive analysis of native fluorescent bioamines (indoleamines and catecholamines). This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the bioamines' amino groups. In this detection process, we monitored the FRET from the native fluorescent moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 15 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated the FRET most effectively. The OPA derivatives of the native fluorescent bioamines emitted OPA fluorescence (445 nm) through an intermolecular FRET process when they were excited at the excitation maximum wavelengths of the native fluorescent bioamines (280 nm). The generation of FRET was confirmed through comparison with the analysis of a nonfluorescent amine (isoleucine) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the indoleamines and catecholamines when performing LC on an ODS column. The detection limits (signal-to-noise ratio, 3) for the indoleamines and catecholamines, at a 20-muL injection volume, were 17-120 and 28-200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than those of systems that take advantage of both native fluorescence detection (i.e., without derivatization) and the conventional detection of OPA derivatives. Furthermore, this method provides enough selectivity and sensitivity for the determination of the indoleamines present in the urine of healthy humans.
authors.affiliation: Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Johnan, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Yoshitake
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nohta
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Yoshida
authors.forename: Takashi
authors.initials: T
authors.lastname: Yoshitake
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Todoroki
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Yamaguchi
mesh_terms: D001679:Biogenic Amines / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D015394:Molecular Structure; D012680:Sensitivity and Specificity
chemical_list: D001679:Biogenic Amines