HPLC method for quantitation of cholesterol and four of its major oxidation products in muscle and liver tissues

A fast, sensitive, high performance liquid chromatographic method was developed for the quantitation of cholesterol and four of its major oxidation products: 3β-hydroxycholest-5-en-7-one (7-ketocholesterol), cholest-5-ene-3β, 7α-diol (7α-hydroxycholesterol), cholest-5-ene-3β,7β-diol (7β-hydroxycholesterol), and cholest-5-ene-3β,25-diol (25-hydroxycholesterol). In this procedure 2:1 chloroform:methanol (v/v) extracts of tissue homogenate were combined, dried over anhydrous Na₂SO₄, filtered, evaporated to dryness under N₂ and dissolved with a mobile phase of either 97:3 or 93:7 hexane:isopropanol (v/v). After membrane filtration and without further purification, aliquots were directly injected onto a 10-μm pore size, 30×0.39 cm μ-Porasil normal phase column. The separation of cholesterol and its oxidation products was monitored by a UV detector at 206 and 233 nm. This method was successfully applied to pork muscle as well as mouse liver tissues and was able to detect cholesterol oxidation products (COP) in the ppm range. The identity of the COP was confirmed by mass spectroscopy.