A peroxyoxalate chemiluminescence-based assay for the evaluation of hydrogen peroxide scavenging activity employing 9,10-diphenylanthracene as the fluorophore

Introduction: A simple, rapid, sensitive, and enzyme-free analytical method for estimating scavenging of hydrogen peroxide (H2O2) was developed. Methods: Peroxyoxalate chemiluminescence (POCL) was measured, using 9,10-diphenylanthracene as fluorophore. Results: The chemiluminescence signal was found to be linear in response to increasing amounts of H2O2 in ethyl acetate/acetonitrile (9:1) (r(2) = .9990), within a range of concentrations varying from 9.0 to 72.0 muM. In contrast, acetonitrile was highly unsuitable because of poor linearity (r(2) = .3736) and poor signal stability. The linearity of POCL inhibition, as a measure of H2O2 scavenging, was tested employing well-known, lipid-soluble antioxidants, including beta-carotene, butylated hydroxytoluene (BHT) and alpha-tocopherol, and also the more polar flavonol quercetin, and the water-soluble L-ascorbic acid (AA). Under the experimental conditions, the corresponding values of H2O2 scavenging activity (SA(HP)) for quercetin, beta-carotene, alpha-tocopherol, and L-AA were 19.1 +/- 0.4, 70.9 +/- 20.1, 8.4 +/- 0.4, and 44.8 +/- 5.6 x 10(-3) muM(-1). Discussion: The data establish the assay as a method for assessing the H2O2 quenching activity of lipid-soluble antioxidants. (C) 2003 Elsevier Inc. All rights reserved.