High-performance liquid chromatography analysis of cholesterol linoleate hydroperoxide in oxidized low density lipoproteins: calibration by conjugated diene internal standard.

The cholesterol linoleate hydroperoxides formed in LDL after oxidant stress are measured by HPLC, with UV detection at 234 nm. Calibration is performed with a conjugated diene internal standard. This internal standard is synthesized by the transesterification of the methyl ester of conjugated diene linoleic acid with a long-chain alcohol, such as arachidyl alcohol (C20). Different long-chain alcohols can be used during the transesterification, to achieve internal standards with variable HPLC retention times. The method allows measurement of cholesterol linoleate hydroperoxide in LDL very early during attack with Cu2+ or other initiator, so that the kinetics of antioxidant loss and hydroperoxide formation can be concurrently monitored.