IDENTIFICATION OF THE COLORED GUAIACOL OXIDATION-PRODUCT PRODUCED BY PEROXIDASES

Oxidation of guaiacol by peroxidases in the presence of H2O2 is the ba sis for a widely used colorimetric assay, However, the nature of the a ssay product, which has an absorption maximum around 470 nm. had not b een determined, In the present study, we combined HPLC with a rapid sc anning uv-visible detector and observed a single product with a spectr um identical to the assay product from the reaction catalyzed by lacto peroxidase, Analysis of the reaction product using on-line HPLC with a tmospheric 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 wi th 3,3'-dimethoxy-4,4'-dihydroxybiphenyl. The presence of a catechol i mpurity in guaiacol was previously shown to yield an additional produc t from peroxidase-mediated oxidation based on its visible absorption ( Taurog et al., 1992 Anal. Biochem. 205, 271-277). When such an incubat ion mixture was analyzed using LC-APCI/MS, a product with mass spectru m consistent with 3-methoxy-2',3',4-trihydroxybiphenyl was observed. I dentification 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 phenol s. (C) 1997 Academic Press.