GENERATION OF AN ACTIVE INTERMEDIATE(S) DURING THE STORAGE OF A PEROXYOXALATE CHEMILUMINESCENCE REAGENT SOLUTION UNDER THE PRESENCE OF STRONG CARBOXYLIC-ACIDS

Catecholamines have measured using a fully-automated HPLC-ethylenediam ine condensation reaction-peroxy-oxalate chemiluminescence (CL) detect ion system with both high sensitivity and selectivity. When a CL solut ion containing trifluoroacetic acid was kept at room temperature, 4 de grees C or -20 degrees C, and then used for the CL detection of catech olamines, the CL intensity was significantly increased. With the solut ions stored at-20 degrees C, the maximum CL intensity was observed, ev en after being stored for one year. This increase in the CL intensity was observed with CL solutions prepared using either bis(2,4,6-trichlo rophenyl)oxalate (TCPO)or tro-2-(3,6,9-trioxadecyloxycarbonyl)phenly]o xalate (TDPO) in either dioxane/ethyl acetate or acetonitrile. A certa in strong carboxylic acid in the CL solution, such as trifluoroacetic acid, trichloroacetic acid or heptafluorobutyric acid, was necessary i n order to increase the CL intensity. This increase in the CL intensit y was also observed even after decomposition of all the oxalate in the CL solutions, suggesting that an active intermediate(s) for generatin g CL is produced in a strongly acidic solution during storage.