Identification of the colored guaiacol oxidation product produced by peroxidases.

Oxidation of guaiacol by peroxidases in the presence of H2O2 is the basis for a widely used colorimetric assay. However, the nature of the assay product, which has an absorption maximum around 470 nm, had not been determined. In the present study, we combined HPLC with a rapid scanning uv-visible detector and observed a single product with a spectrum identical to the assay product from the reaction catalyzed by lactoperoxidase. Analysis of the reaction product using on-line HPLC with atmospheric pressure chemical ionization detection (LC-APCI/MS) yielded a mass spectrum consistent with 3,3 '-dimethoxy-4,4'-biphenylquinone. A minor reaction product was observed with mass spectrum consistent with 3,3'-dimethoxy-4,4'-dihydroxybiphenyl. The presence of a catechol impurity in guaiacol was previously shown to yield an additional product from peroxidase-mediated oxidation based on its visible absorption (Taurog et al., 1992 Anal. Biochem. 205, 271-277). When such an incubation mixture was analyzed using LC-APCI/MS, a product with mass spectrum consistent with 3-methoxy-2',3',4-trihydroxybiphenyl was observed. Identification of such a heterodimeric product supports the previously proposed mechanism for catechol interference in the guaiacol assay as well as the radical nature of peroxidase-catalyzed oxidation of phenols.