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Anal Biochem. 1999 Sep 10;273(2):149-55.

Hydroperoxide assay with the ferric-xylenol orange complex.

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School of Biological Sciences, Macquarie University, Sydney, 2109, Australia.


A range of hydroperoxides were reduced by ferrous ions in acid solutions and the amount of ferric product was measured as a xylenol orange complex at 560 nm. Dilute sulfuric acid, 50% acetic acid, and acidified 90% methanol proved to be suitable solvents. The color developed within 15 min and was stable for several hours in most solvents. The apparent molar absorption coefficients (epsilon(app)) of H(2)O(2) and of the t-butyl, cumene, bovine serum albumin, and linoleate hydroperoxides were measured, using known hydroperoxide concentrations determined independently by an iodometric assay. The epsilon(app) values differed significantly and depended on the hydroperoxide, the solvent, and the source of the xylenol orange. The numbers of Fe(3+) ions formed by a range of hydroperoxides in different solvents showed that H(2)O(2) gave 2.5, t-butyl and cumene hydroperoxides 5, and the other hydroperoxides 2 Fe(3+) ions per -OOH group. This general finding allows the determination of approximate hydroperoxide concentrations even in chemically complex systems. Accurate measurements require knowledge of the nature of the hydroperoxide and its epsilon(app) and careful control of the assay conditions. However, the convenience of the assay makes it potentially useful in a variety of applications.

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