Chemiluminescence determination of catecholamines in human blood plasma using 1,2-bis(3-chlorophenyl)ethylenediamine as pre-column derivatizing reagent for liquid chromatography Original Research Article

A highly sensitive method for the determination of the human blood plasma catecholamines (norepinephrine, epinephrine and dopamine) is described which employs liquid chromatography (LC) with chemiluminescence detection. Solid-phase extraction using cation exchange cartridges was used for selective and quantitative isolation of the catecholamines and isoproterenol (an internal standard) from 20 μl of human plasma containing 0.1 pmol ml−1 isoproterenol. The amines and isoproterenol, derivatized with 1,2-bis(3-chlorophenyl)ethylene diamine, were separated on a reversed-phase LC column with isocratic elution using a mixture of imidazole buffer (120 mM, pH 5.8), methanol, acetonitrile (13 : 4 : 18, v/v/v). The eluate was detected by a post-column chemiluminescence reaction system using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate and hydrogen peroxide. The detection limits (a signal-to-noise ratio = 3) for each catecholamine are 40–120 amol in a 100 μl injection volume.