Chemiluminescence determination of catecholamines in human blood plasma and urine using 1,2-diphenylethylenediamine as pre-column derivatization reagent in liquid chromatography

A chemiluminescence detection of the fluorescent derivatives of catecholamines (norepinephrine, epinephrine and dopamine) and isoproterenol as an internal standard is described for the highly sensitive liquid Chromatographic determination of these compounds. The amines were converted by reaction with 1,2-diphenylethylenediamine into the corresponding fluorescent derivatives, which were separated on a reversed-phase column (TSK gel ODS-120T) with isocratic elution. The derivatives in the column eluate were detected by the post-column chemiluminescence reaction system using bis[4-nitro-2-(3,6,9-trioxade-cyloxycarbonyl)phenyl] oxalate and hydrogen peroxide. The method allowed the determination of catecholamines in 50 μl of human blood plasma and 10 μl of 20 times diluted urine. The detection limits for the amines were 150–450 amol per 100-μl injection volume at a signal-to-noise ratio of 3.