Overcoming Reductant Interference in Peroxidase-Based Assays for Hydrogen Peroxide Quantification

A problem commonly encountered when using peroxidase-based methods for hydrogen peroxide quantification in biobased matrixes is interference due to the presence of endogenous reductants. Such assays are typically based on the generation of an oxidized reporter molecule in direct proportion to the amount of hydrogen peroxide reduced in the peroxidase-catalyzed reaction. Endogenous reductants confound such assays by reducing the oxidized reporter molecule, thus resulting in underestimates of hydrogen peroxide content. In the present work, we demonstrate how this problem can be circumvented by selectively oxidizing offending compounds by treatment with the oxidized reporter molecule prior to initiating the peroxidase reaction for hydrogen peroxide quantification. The approach is demonstrated using horseradish peroxidase, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), as the reporter molecule and a representative garlic paste as the hydrogen peroxide-containing biobased matrix. The approach is expected to be generally applicable to a wide range of peroxidase-based assays when applied to complex biobased systems.