FEMTOMOLE ANALYSIS OF 9-OXONONANOYL CHOLESTEROL BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

9-Oxononanoyl cholesterol, a cholesterol core-aldehyde formed during l ipoprotein oxidation, was recently identified in advanced human athero sclerotic lesions. Here we present a rapid and sensitive HPLC method f or 9-oxononanoyl cholesterol analysis, 9-Oxononanoyl cholesterol was c onverted to the corresponding fluorescent decahydroacridine derivative by reaction with l,3-cyclohexanedione. The derivatives formed were pu rified by solid-phase extraction on C-18 columns, separated by reverse d phase HPLC with isocratic elution, and detected by their fluorescenc e. Decahydroacridine derivatives of 9-oxononanoyl cholesterol were sta ble for at least 160 h, The limit of quantitation of the method presen ted is at the low (approximate to 50) femtomole level, with an absolut e limit of detection (signal: noise = 6) of 15 fmol, Intra-assay varia tion was less than or equal to 5%, while interassay variations were be tween 5 and 15%, depending on the concentration of the analyte. Standa rd curves were linear over nearly three orders of magnitude (50 fmol-1 2.5 pmol). 9-Oxononanoyl l cholesterol proved to be the major choleste rol core-aldehyde formed during t-BuOOh/FeSO4 oxidation of cholesteryl linoleate and Cu2+-induced LDL oxidation, findings confirmed by atmos pheric pressure chemical ionization-mass spectrometry, Analysis of lip id extracts obtained from advanced human atherosclerotic lesions revea led the presence of 9-oxononanoyl cholesterol in all tissue samples an alyzed (28 +/- 14 mu mol/mol cholesterol, n = 9) despite the presence of a-tocopherol (4 +/- 1.2 mmol/mol cholesterol, n = 9).