Determination of hydroperoxides by the ferric-xylenol orange method

Abstract

Organic hydroperoxides are some of the first semi-stable products of the interaction between free radicals (and other reactive oxygen species) with biological systems, so that they are potential indicators of the formation and effects of these reactive molecules. Many assays have been developed for the detection and measurement of hydroperoxides, but all have significant drawbacks. For example, while the acid iodometric technique is the only one giving indisputably quantitative results,¹ it requires anaerobic conditions, making it difficult to use in routine assays. A promising alternative method, with similar sensitivity but unaffected by oxygen, was developed for a wide range of hydroperoxides. It is based on the reaction between the hydroperoxide and ferrous iron in acid medium and the measurement of the ferric iron produced by formation of a complex with xylenol orange. The assay was initially used to measure H₂O₂, but later also in the measurement of a wide range of organic hydroperoxides, including some present in biological fluids.² In attempting to apply the assay to solutions of oxidized proteins, we found that accurate hydroperoxide concentrations could not be obtained by the recommended protocols. Therefore, we examined the variables affecting the assay and validated a new protocol able to provide more reliable results.