Abstract: | A method for reactivation of inactivated horseradish peroxidase (HRP) was studied and exploited in an assay for hydrogen peroxide (H2O2). Addition of imidazole into a mobile phase made continuous determination of hydrogen peroxide (H2O2) possible by micro?ow injection based on horseradish‐catalysed luminol chemiluminescence. For reproducible determination of H2O2 with HRP, the inactivation of HRP via protonation of the active sites of HRP caused by reaction with H2O2 must be avoided. We successfully reactivated protonated HRP (inactive HRP) with exogenous imidazole in the mobile phase of the micro?ow injection system. The imidazole successfully removed the attached proton from the inactive sites of the HRP. This assay was reproducible (within‐run reproducibility, CV = 4.0%) and the detection limit for H2O2 was 5 pmol. Copyright © 2003 John Wiley & Sons, Ltd. |