A simple assay for lipid hydroperoxides based on triphenylphosphine oxidation and high-performance liquid chromatography.

A simple and sensitive method was developed to measure total hydroperoxides. After the reduction of hydroperoxides with triphenylphosphine (TP) in cyclohexane at 30°C, the amount of triphenylphosphine oxide (TPO) produced is determined by reverse-phase or normal-phase high-performance liquid chromatography (HPLC) in combination with an ultraviolet (UV)-detector by measuring absorption at 220 or 260 nm. TPO was shown to be gener-ated stoichiometrically by reduction of known amounts of either cumene hydroperoxide or methyl 13-hydroper-oxyoctadecadienoate. For various lipids at low levels of oxidation, the peroxide values determined by this method were in good agreement with those obtained by conventional iodometry. The detection limit of TPO in HPLC using absorption at 220 nm was less than 10 pmol. Consequently, total hydroperoxides in lipids at levels corresponding to less than a peroxide value of 1 can be estimated by the TP method on a 10-mg sample.