HPLC and LC-MS studies of hydroxylation of phenylalanine as an assay for hydroxyl radicals generated from Udenfriend's reagent.

An HPLC assay method and an LC-MS method were used to study the Udenfriend reaction and its variations by using phenylalanine as the hydroxylation substrate. The results indicate that (1). citric acid can replace EDTA as the promoter for the production of hydroxyl radicals in the Undenfriend reaction, albeit in a somewhat less efficient way, (2). dihydroxylation of the hydroxylation substrate, phenylalanine, readily occurs with the Udenfriend systems (with either EDTA or citric acid), and (3). a novel oxidative degradation pathway may exist for o-tyrosine. It is cautioned that dihydroxylation needs to be accounted for when interpreting hydroxylation results in HPLC-based HO(z.rad;) assay systems with phenylalanine as the substrate.