title: Concerted derivatization and concentration method with dispersive liquid-liquid microextraction for liquid chromatographic analysis of 5-hydroxyindoles in human serum.
abstract: We developed a concerted derivatization and concentration method based on dispersive liquid-liquid microextraction (DLLME) for the liquid chromatography (LC) determination of 5-hydroxyindoles (5-HIs; serotonin, 5-hydroxyindole-3-acetic acid, N-acetylserotonin, and 5-hydroxytryptohol). Concerted derivatization and concentration could be affected by adding a mixture of an ionic liquid (1-hexyl-3-methylimidazolium hexafluorophosphate, extraction solvent), methanol (disperser), and water containing fluorescence derivatization reagents [benzylamine and potassium hexacyanoferrate(III)] into the sample. The resulting sedimented phase was injected into a reversed-phase LC column using a mixture of acetonitrile and 250 mM acetate buffer (pH 4.3) as the mobile phase for gradient elution, and the derivatives obtained were fluorometrically detected at excitation and emission wavelengths of 345 nm and 452 nm, respectively. The derivatization (reagent concentrations and pH) and extraction (extraction and disperser solvent type) conditions were optimized simultaneously. The limits of detection of the 5-HIs were in the range of 0.08-0.33 nM. The method was validated for 10 and 50 pmol/mL human serum levels, and the recovery of 5-HIs was between 66% and 98%, within a relative standard deviation of 9.5%. The proposed method is well suited for the highly sensitive analysis of trace amounts of 5-HIs in human serum samples.
authors.affiliation: Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Hayama
authors.forename: Yurika
authors.initials: Y
authors.lastname: Yabuuchi
authors.forename: Tomomi
authors.initials: T
authors.lastname: Iwamatsu
authors.forename: Erina
authors.initials: E
authors.lastname: Tamashima
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kawami
authors.forename: Miki
authors.initials: M
authors.lastname: Itoyama
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Yoshida
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Yamaguchi
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nohta
mesh_terms: D000097:Acetonitriles; D001596:Benzylamines / Q000737:chemistry; D002021:Buffers; D056148:Chromatography, Reverse-Phase; D005292:Ferricyanides / Q000737:chemistry; D006801:Humans; D006897:Hydroxyindoleacetic Acid / Q000097:blood*; D006917:Hydroxytryptophol / Q000097:blood*; D007093:Imidazoles / Q000737:chemistry; D052578:Ionic Liquids / Q000737:chemistry; D057230:Limit of Detection; D059627:Liquid Phase Microextraction / Q000379:methods; D008297:Male; D000432:Methanol; D015203:Reproducibility of Results; D012701:Serotonin / Q000031:analogs & derivatives* / Q000097:blood*
chemical_list: D000097:Acetonitriles; D001596:Benzylamines; D002021:Buffers; D005292:Ferricyanides; D007093:Imidazoles; D052578:Ionic Liquids; D006917:Hydroxytryptophol; D012701:Serotonin; D006897:Hydroxyindoleacetic Acid; C030796:benzylamine; C006389:N-acetylserotonin; C028033:potassium ferricyanide; D000432:Methanol; C032159:acetonitrile
keywords: 5-Hydroxyindoles; Benzylamine; Derivatization; Dispersive liquid–liquid microextraction; Human serum; Liquid chromatography