High Performance Liquid Chromatography of Free Catecholamines in Urine Using 1,2-Diphenylethylenediamine As New Precolumn Fluorescent Derivatization Reagent

Abstract

A simple, rapid and highly sensitive method for the determination of free catecholamines(norepinephrine, epinephrine and dopamine) in urine is described which employs high performance liquid chromatography with fluorescence detection. Catecholamines and isoproterenol added as an internal standard in 100 yl of urine, after chromatography on a small column of CM-Sephadex C25, are converted into the corresponding fluorescent compounds by the reaction with 1,2-diphenylethylenediamine in aqueous ethanol. These compounds are separated within 7 min on a reversed phase column, TSK-gel ODS-120T, with isocratic elution using aqueous dioxane containing a Tris hydrochloric acid buffer (pH 7.0). The detection limits of norepinephrine, epinephrine and dopamine are 20, 30 and 40 fmol in a 50 µ injection volume, respectively. © 1984, The Japan Society for Analytical Chemistry. All rights reserved.