1,2-Diarylethylenediamines as sensitive pre-column derivatizing reagents for chemiluminescence detection of...

Chemiluminescence detection in high-performance liquid chromatography for derivatives of catecholamines (norepinephrine, epinephrine and dopamine) and isoproterenol was studied on the basis of the peroxyoxalate chemiluminescence reaction. The amines and isoproterenol, derivatized with 1,2-diarylethylenediamines, were separated on a reversed-phase HPLC column (TSK gel ODS-120T) with isocratic elution using a mixture of imidazole buffer (pH 5.8, 120 mM)-methanol-acetonitrile (6:2:9, 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. Of the 141,2-diarylethylenediamines investigated, it was found that 1,2-bis(3-chlorophenyl)ethylenediamine, 1,2-bis(3,4-dichlorophenyl)-ethylenediamine and 1,2-bis(4-chlorophyenyl)ethylenediamine were the most sensitive derivatives for all catecholamines. The derivatization and peroxyoxalate chemiluminescence reaction conditions were optimized for 1,2-bis(3-chlorophenyl)-ethylenediamine. The chromatographic detection limits for catecholamines were approximately 40-120 amol for an injection volume of 100 microliters (signal-to-noise ratio of 3).