Improved HPLC assay for lipid peroxides in human plasma using the internal standard of hydroperoxide.

We have developed a sensitive reversed-phase chemiluminescence HPLC approach for simultaneous quantitative and qualitative analyses of hydroperoxides of cholesteryl ester and TG in human plasma. Standard hydroperoxides of cholesteryl ester and TG and a novel internal standard (1-tetradecanyl 3-octadecenoyloxy-5beta-cholan-24-oate monohydroperoxide) (I.S.) were chemically synthesized and the standard curves confirmed to be linear throughout the calibration range (1-1000 pmol). Within-day and between-day CV were less than 7%, and the recoveries were within the range of 84-93%. With sample size minimized to 0.1 mL of plasma for each run, plasma cholesteryl ester hydroperoxide levels were 189 +/- 87 nM (mean +/- SD) in healthy young (22-25 yr old; n = 15, male/female = 6:9) and 210 +/- 69 nM in healthy elderly (39-60 yr old; n = 6, male/female = 3:3). TG hydroperoxide was not detected in healthy subjects. In patients with advanced liver failure (36-67 yr old; n = 4, male/female = 2:2), hydroperoxide levels of plasma cholesteryl ester and TG were 11,903 +/- 9,553 nM and 3,318 +/- 1,590 nM, respectively, indicating an involvement of lipid oxidation. Sensitive and specific monitoring of plasma lipid peroxides using the present chemiluminescence HPLC approach with the synthesized I.S. may help our understanding of chemical and pathophysiological aspects of lipid peroxidation.