A method for the simultaneous determination of free cholesterol and cholesteryl esters using high-performance liquid chromatography has been developed. Total lipids extracted from tissue were separated into three fractions, which were cholesteryl ester, triacylglycerol and free cholesterol fractions, with a Sep-Pak Alumina-N cartridge. Free cholesterol and cholesteryl ester fractions were combined and introduced to a reversed-phase high-performance liquid chromatographic system provided with a Puresil C18 column and a UV detector monitoring at 210nm. With this system, free cholesterol and 12 cholesteryl esters could be separated and the maximum detection limit was 10pmol for cholesteryl arachidonate. This method was applied to the determination of cholesterol and cholesteryl esters in arterial walls of cholesterol-fed rabbits and human serum.
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