Quantification of Hydroperoxides in Oils and Fats, Oil-in-Water Emulsions, and Food Products by Ferrous Oxidation–Xylenol Orange Method

Lipid hydroperoxides (HPX) are primary products of lipid oxidation and key markers of chemical alteration of dietary oils and fats. The ferrous ion–xylenol orange (FOX2) assay allows for HPX quantification in oils and lipid-containing food products. It is based on the oxidation of ferrous ions in acid and alcoholic medium by HPX, followed by the formation of a colored xylenol orange–ferric ion complex. The complex is finally detected and quantified by spectrophotometry at 560 nm. This method is rapid, is very sensitive (<0.5 mmol HPX/kg lipids), and applies to very small amounts of sample (about 10 mg lipids). Here, we detail the procedure to quantify HPX in dietary oils or lipid extracts, in oil-in-water (O/W) emulsions, or in complex food matrices. The steps for the optimization of HPX extraction from a new matrix and the factors to be carefully monitored throughout the execution of the method are also specified.