6-Aminomethylphthalhydrazide as a highly sensitive chemiluminescence derivatization reagent for 5-hydroxyindoles in liquid chromatography

6-Aminomethylphthalhydrazide was synthesized as a highly sensitive and selective chemiluminescence derivatization reagent for 5-hydroxyindoles in liquid chromatography. 5-Hydroxytryptophan, serotonin and 5-hydroxyindole-3-acetic acid were used as model compounds to optimize the derivatization conditions. The reagent reacts selectively with the indoles in the presence of potassium hexacyanoferrate(III) to give highly chemiluminescent derivatives which produce chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline solution. The chemiluminescent derivatives of the three 5-hydroxyindoles can be separated within 35 min by reversed-phase liquid chromatography with isocratic elution, followed by chemiluminescence detection. The detection limits (signal-to-noise ratio = 3) for 5-hydroxyindoles are in the range 0.7–4 fmol for a 20 μl injection.