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HPLC method for quantitation of cholesterol and four of its major oxidation products in muscle and liver tissues
Author(s) -
Csallany A. Saari,
Kindom S. E.,
Addis P. B.,
Lee JooHee
Publication year - 1989
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/bf02535082
Subject(s) - chromatography , chemistry , anhydrous , high performance liquid chromatography , chloroform , cholesterol , methanol , sterol , diol , mass spectrometry , biochemistry , organic chemistry
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 Na 2 SO 4 , filtered, evaporated to dryness under N 2 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.