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 °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 °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 (150×1.5 mm I.D., packed with C18 silica, 5 μm) 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-μl injection.