Oxidation of Hydroxy- and Dihydroxybenzoic Acids Under the Udenfriend's Conditions. An HPLC Study

Mónika Kuzma1, Nikoletta Kovács1, Lilla Sziva1, Gábor Maász2, Péter Avar2, Pál Perjési1, *
1 Institute of Pharmaceutical Chemistry, University of Pécs, H-7624 Pécs, Rókus str. 2, Hungary
2 Department of Biochemistry and Medical Chemistry, University of Pécs, H-7624 Pécs, Szigeti str. 12, Hungary

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© 2018 Kuzma et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4. 0 International Public License (CC-BY 4. 0), a copy of which is available at: https://creativecommons. org/licenses/by/4. 0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Institute of Pharmaceutical Chemistry, University of Pécs, H-7624 Pécs, Rókus str. 2, Hungary; Tel: +3672503626; Fax: +3672503627; E-mail:



Non-enzymatic hydroxylation of aromatic compounds to the respective phenolic derivatives is a possible metabolic pathway of xenobiotics. The formed metabolites can undergo consecutive oxidative reactions with free radicals to form potential toxic molecules.


Development of HPLC methods to separate, identify and quantitate the main products formed from salicylic acid, 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid under in vitro hydroxylation conditions (Udenfriend's system).


An RP-HPLC-UV-Vis method was developed to separate salicylic acid and isomeric dihydroxybenzoic acids formed in the Udenfriend's system. Confirmation of structures of the oxidized products of salicylic acid, 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid was performed by HPLC-ESI-MS/MS method.


The HPLC-UV-Vis method was evaluated for a number of validation characteristics (selectivity, repeatability and intermediate precision, LOD, LOQ and calibration range). It was found that oxidation of salicylic acid resulted in the formation of 2,3- and 2,5-dihydroxybenzoic acids. Furthermore, the hydroxylated metabolites can be further metabolized under the Udenfriend’s conditions.


The results give evidence for possible involvement of the oxidized metabolites of salicylic acid in the development of biological action of salicylates at the site of inflammation, where high hydroxyl radical level can be detected.

Keywords: Udenfriend's system, Hydroxyl radical, Salicylic acid, Dihydroxybenzoic acid, HPLC-UV-Vis, HPLC-ESI-MS.