Use of Size Exclusion Chromatography to study the protective effect of radical scavengers on oxygen free radical-induced degradation of hyaluronic acid

A size exclusion chromatography (SEC) method has been established for the study of hyaluronic acid depolymerization induced by oxygen free radicals and of the protective effect of two widely employed anti-inflammatory agents, piroxicam and tenoxicam. Molecular weight distribution of hyaluronic acid was determined by aqueous SEC equipped with absolute online detectors, light scattering detector and a viscometer. Ten min exposure of the macromolecules to a flux of hydroxyl radicals generated by xanthine/xanthine oxidase/Fe 2+ system caused massive depolymerization (Mw fell from 4.7 · 105 to 8.7 · 104). Both drugs exhibited a dose-dependent protective effect on hyaluronic acid degradation due to their radical scavenging properties. Tenoxicam was far more active than piroxicam in quenching highly reactive hydroxyl radicals (minimal effective concentrations 25 vs. 250 μM). This method gives a more complete analytical profile of the macromolecules with respect to conventional techniques.