Simultaneous determination of 5-hydroxyindoles and catechols by high-performance liquid chromatography with fluorescence detection following derivatization with benzylamine and 1,2-diphenylethylenediamine.
A highly selective and sensitive method for the simultaneous determination of 5-hydroxyindoles and catechols (serotonin, norepinephrine, dopamine and related compounds) by high-performance liquid chromatography with fluorescence detection is described. The method is based on the two-step precolumn derivatization of 5-hydroxyindoles and catechols with benzylamine (BA) and 1,2-diphenylethylenediamine (DPE), respectively, resulting in highly fluorescent and stable benzoxazole derivatives. The first derivatization with BA proceeds at room temperature (ca. 23 degrees C) for 2 min in a mixture of 0.3 M 3-cyclohexylamino-1-propanesulfonic acid buffer (pH 10.0) and methanol in the presence of potassium hexacyanoferrate(III). The subsequent second derivatization with DPE is carried out at 50 degrees C for 20 min in the presence of glycine. The resulting fluorescent derivatives of five 5-hydroxyindoles and seven catechols are separated on a reversed-phase column (150 x 1.5 mm I.D., packed with C18 silica, 5 microm) with isocratic elution using a mixture of acetonitrile-15 mM acetate buffer (pH 4.5) (34:66, v/v) containing 1 mM octanesulfonic acid sodium salt, and are detected spectrofluorimetrically at 480 nm with excitation at 345 nm. The detection limits (signal-to-noise ratio of 3) of the related compounds are 80 amol to 86 fmol for a 20-microl injection.
Fujino
Kaoru
K
Chemicals Evaluation and Research Institute, 3-822 Ishii Machi, Hita, Oita 877-0061, Japan.
Yoshitake
Takashi
T
Kehr
Jan
J
Nohta
Hitoshi
H
Yamaguchi
Masatoshi
M
D001596:Benzylamines / Q000737:chemistry*; D002396:Catechols / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005029:Ethylenediamines / Q000737:chemistry*; D007211:Indoles / Q000032:analysis*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D001596:Benzylamines; D002396:Catechols; D005029:Ethylenediamines; D007211:Indoles; C026785:5-hydroxyindole; C030796:benzylamine; C007305:1,2-dianilinoethane