Coulometric detection in high-performance liquid chromatographic analysis of cholesteryl ester hydroperoxides

A highly sensitive and simple method for the determination of cholesteryl ester hydroperoxides (CHE-OOH) was developed using high-performance liquid chromatography (HPLC) with coulometric electrochemical detection. The lowest detectable level by this technique was 2 pmol for cholesteryl linoleate hydroperoxides at the signal-to-noise ratio of 3. This method was applied to the determination of ChE-OOH presumably present in human plasma. Although ChE-OOH could not be detected, the ChE-OOH level in the fluid was estimated to be less than 27 nM. It was found that the extraction efficiency of an internal standard, cholesteryl nervonate, was decreased by lowering its amount spiked to the plasma. The concentration of ChE-OOH in human plasma and plasma lipoprotein, which were peroxidized with a radical initiator in vitro, could be determined by use of this standard. HPLC-coulometric technique is, therefore, useful to measure the peroxidation of plasma lipids in vitro.