Spectrophotometric determination of phenolic compounds by enzymatic and chemical methods--a comparison of...

A spectrophotometric determination of phenolic compounds by a peroxidase-catalyzed enzymatic (PE) method and the Folin-Ciocalteu (FC) chemical method was compared for their structure-activity relationship. In the PE method, the reaction time of 19 phenolic compounds with different chemical structures was found to be within 15 min, with those having bulky substituents showing slower reactivity. The responses of the phenolic compounds toward the PE method in terms of molar absorbance were positively correlated with the nucleophilicity of their corresponding phenoxyl radicals. An increase in the nucleophilicity by substitution of methoxyl and hydroxyl (electron-donating) groups enhanced the responses; while a decrease in nucleophilicity by substitution of the allyl carboxylic (electron-withdrawing) groups lowered the responses of phenolic compounds toward the enzymatic assay. The responses of phenolic compounds to the enzymatic method were found to be independent of the degree of hydroxylation while those to the FC assay were affected by both the position and degree of hydroxylation. Interfering chemicals found in the FC assay, including vitamin C, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (a vitamin E analogue), and tert-butylhydroquinone (a commercial antioxidant) were insensitive to the enzymatic assay. The PE method appears to have a higher specificity toward phenolic compounds and subject to less interferences from other antioxidants than the FC method.