Generation of an Active Intermediate(s) during the Storage of a Peroxyoxalate Chemiluminescence Reagent Solution under the Presence of Strong Carboxylic Acids

Abstract

Catecholamines have been measured using a fully-automated HPLC-ethylenediamine condensation reaction-peroxy-oxalate chemiluminescence (CL) detection system with both high sensitivity and selectivity. When a CL solution containing trifluoroacetic acid was kept at room temperature, 4°C or − 20°C, and then used for the CL detection of catecholamines, the CL intensity was significantly increased. With the solutions stored at − 20°C, the maximum CL intensity was observed, even after being stored for one year. This increase in the CL intensity was observed with CL solutions prepared using either bis(2,4,6-trichlorophenyi)oxalate (TCPO) or bis[4-nitro-2-(3,6,9-trioxadecyloxycarbon-yl)phenyl]oxalate (TDPO) in either dioxane/ethyl acetate or acetonitrile. A certain strong carboxylic acid in the CL solution, such as trifluoroacetic acid, trichloroacetic acid or heptafluorobutyric acid, was necessary in order to increase the CL intensity. This increase in the CL intensity was also observed even after decomposition of all the oxalate in the CL solution, suggesting that an active intermediate(s) for generating CL is produced in a strongly acidic solution during storage.