CHROMATOGRAPHIC-SEPARATION AND ELECTROCHEMICAL DETERMINATION OF CHOLESTEROL HYDROPEROXIDES GENERATED BY PHOTODYNAMIC-ACTION

Reverse-phase HPLC with electrochemical detection (HPLC-EC) was used to separate and quantitate photochemically generated cholesterol hydroperoxides. The EC measurements were performed in the reduction mode under anaerobic conditions. When cholesterol-containing liposomes were irradiated in the presence of a phthalocyanine dye, at least four major oxidation products of cholesterol were detected by HPLC-EC: 5α-hydroperoxide (5α-OOH), 6β-hydroperoxide (6β-OOH), 7α-hydroperoxide (7α-OOH), and 7β-hydroperoxide (7β-OOH). The detection limit for each compound was found to be ∼25 pmol. Product identification was based on matching HPLC and TLC behavior of standards and on physical indicators (melting points and NMR chemical shifts). The cholesterol hydroperoxides were barely separated from EC-silent diol derivatives, which could be detected by 210 nm absorbance after reduction of the hydroperoxides with triphenylphosphine. Irradiation of a dye-sensitized natural membrane, the human erythrocyte ghost, also resulted in formation of 5α-OOH, 6-OOH, and 7-OOH, as evidenced by HPLC-EC. Under the chromatographic conditions used, these species were well separated not only from one another but also from a family of at least six phospholipid hydroperoxides. These results illustrate the strenghts of HPLC-EC as a relatively convenient, sensitive, and selective means of analyzing cholesterol hydroperoxides in biological samples. © 1991.