Separation of Hydroxy and Hydroperoxy Polyunsaturated Fatty Acids By High-Pressure Liquid Chromatography

Lipid peroxidation is an autocatalytic free radical-mediated chemical mechanism in which polyunsaturated fatty acids (PUFA) undergo oxidation to form lipid hydroperoxide (LHP). Increased lipid peroxidation has been implicated in many disease conditions including acute myocardial infarction (
1
), stroke (
2
), diabetes mellitus (
3
), hepatic disorders (
4
), and toxicity (
5
) by certain drugs, pesticides, and metals. Study of lipid peroxidation in disease requires simple, discriminative, and sensitive methods for LHP measurement because of their low concentration and because of the multitude of isomeric and esterified forms. Methods have been developed that use spectrophotometry, fluorometry, chemiluminescence, and chromatography as well as enzymatic and non-enzymatic techniques (
6
–
8
). These methods determine total hydroperoxides as well as interfering substances particular to each assay. High pressure liquid chromatography (HPLC) techniques remove interfering substances from the analysis of LHP as well as separate hydroperoxides of different lipid classes. Because lipid peroxidation occurs predominantly in PUFA, esterified to cholesterol and glycerol-based lipids, assessment of the extent of lipid peroxidation can be made through direct measurement of total PUFA hydroperoxides and indirectly by measurement of their hydroxy derivative. HPLC-ultraviolet (UV) detection of PUFA hydroperoxides, based upon the absorbance of the conjugated diene at 234 nm, has been criticized owing to its inability to differentiate LHP from the corresponding alcohol-reduction products that co-elute in procedures reported to date (
9
,
10
).