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.
Hempen
Christel
C
Chemical Analysis Group and MESA Institute for Nanotechnology, University of Twente, PO Box 217, 7500, AE Enschede, The Netherlands.
van Leeuwen
Suze M
SM
Luftmann
Heinrich
H
Karst
Uwe
U
D002384:Catalysis; D002853:Chromatography, Liquid / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006861:Hydrogen Peroxide / Q000737:chemistry*; D010084:Oxidation-Reduction; D010655:Phenylenediamines / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D010655:Phenylenediamines; C034193:1,2-diaminobenzene; D006861:Hydrogen Peroxide