Assay of cardiolipin peroxidation by high-performance liquid chromatography

Commercial preparations of bovine cardiolipin (diphosphatidylglycerol) in chloroform solution contain substantial amounts of oxidation products. These oxidized derivatives, characterized by the presence of varying amounts of hydroperoxides and conjugated dienes, can be separated from unoxidized cardiolipin by normal phase high-performance liquid chromatography (HPLC) using UV detection. When purified cardiolipin is subjected to autoxidation in aqueous media, oxidation products of similar HPLC properties are produced. Storage of cardiolipin in chloroform induces both autoxidation and hydrolysis whereas storage in ethanol and other solvents does not. It is recommended not to use chloroform for the long-term storage of cardiolipin.