Liquid chromatographic/mass spectrometric investigation on the reaction products in the peroxidase-catalyzed oxidation of o-phenylenediamine by hydrogen peroxide

Mass spectrometric evidence was obtained to confirm that the main reaction product of the horseradish peroxidase (POD)-catalyzed oxidation of o-phenylenediamine (OPD) by hydrogen peroxide is 2,3-diaminophenazine. Although this reaction is one of the most widespread detection schemes in enzyme-linked immunosorbent assays (ELISAs), the literature data on the identity of the reaction product(s) have been strongly contradictory throughout the last few decades. Liquid chromatography with UV/Vis and mass spectrometric detection as well as exact mass measurements after LC fraction collection have led to the unambiguous identification of 2,3-diaminophenazine as main reaction product. 2,2′-Diaminoazobenzene, which is frequently described in other publications to be the major reaction product, was not detected at all.