Chemiluminescent determination of hydrogen peroxide with 9-acridinecarbonylimidazole and use in measurement of glucose oxidase and alkaline phosphatase activity.

Several activated derivatives of 9-acridinecarboxylic acid were prepared in order to investigate their utility for detection of hydrogen peroxide. One of these derivatives, 9-acridinecarbonylimidazole (I), is especially stable and is a useful reagent for measuring, by chemiluminescence, the activity of a number of enzymes that directly produce peroxide, including glucose oxidase. Other enzymes can also be assayed if an appropriate intermediate substrate exists that ultimately produces hydrogen peroxide after being acted upon by the enzyme. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and 3-indolyl phosphate (3-IP) are such substrates for alkaline phosphatase. The detection limits for both of these enzymes are in the 1-10 amol range. Other enzymes that can potentially be assayed using I include oxidases, hydrolases and dehydrogenases. Negative assays for compounds that consume or bind peroxide such as reducing agents, antioxidants, catalases and peroxidases are also feasible.