Simultaneous determination of phospholipid hydroperoxides and cholesteryl ester hydroperoxides in human plasma by high-performance liquid chromatography with chemiluminescence detection

A method was developed for the simultaneous determination of phosphatidylcholine hydroperoxides (PCOOH) and cholesteryl ester hydroperoxides (CEOOH). Lipid hydroperoxides (LOOH) were quantitatively extracted from human plasma with a mixture of n-hexane and ethyl acetate, and separated by column-switching high-performance liquid chromatography using one aminopropyl column and two octyl columns followed by chemiluminescence detection. LOOHs could be completely separated from each other and detected at picomole levels. The results of method validation tests were satisfactory. This method was then applied to determine LOOH in normal human plasma; the levels of PCOOH and CEOOH found were 36.0+/-4.0 nM (mean+/-S.D., n=6) and 12.3+/-3.1 nM (mean+/-S.D., n=6), respectively.