Modification of the Ferrous Oxidation‐Xylenol Orange Method for Determination of Peroxide Value in Highly Pigmented Sea Cucumber Viscera Lipid

Abstract A ferrous oxidation‐xylenol orange (FOX) method was modified to measure the levels of hydroperoxides in highly pigmented sea cucumber ( Cucumaria frondosa ) viscera lipid. With the FOX method, oil hydroperoxides oxidize Fe(II) to Fe(III) ions, which in turn bind to xylenol orange (XO) to form a complex that has a maximum absorbance at 560 nm. When applied to lipid extracts containing high levels of carotenoid pigments, the carotenoids interfere in the absorbance measurement because the XO‐complex and carotenoids absorb at the same wavelength. To avoid this, an approach was developed where carotenoids were removed from the solution containing the XO‐complex before absorbance measurements were made. This was accomplished by aqueous extraction of the reaction solution, leaving an organic layer containing the carotenoid pigment. The absorbance of the aqueous layer was subsequently measured at 560 nm allowing for determination of the lipid hydroperoxide content (mmol kg −1 ) using a cumene hydroperoxide calibration curve. The method was validated using oxidized, unpigmented fish oils. The modified FOX method described herein was linear, accurate, and precise, and was validated over a hydroperoxide concentration range of 5–35 meq kg −1 lipid. This modified FOX method for hydroperoxide determination of highly pigmented lipids is a valuable alternative to the methods presently available for determination of peroxide values.