SIMULTANEOUS MEASUREMENT OF PHYTOSTEROLS (CAMPESTEROL AND BETA-SITOSTEROL) AND 7-KETOCHOLESTEROL IN HUMAN LIPOPROTEINS BY CAPILLARY COLUMN GAS-CHROMATOGRAPHY

7-Ketocholesterol (a major cholesterol oxidation product) and phytoste rols are important indicators of lipoprotein oxidation and lipoprotein metabolism respectively. We describe a simple, sensitive and reproduc ible method for the simultaneous measurement of these sterols in human lipoprotein samples by capillary column gas liquid chromatography. Th e method is suitable for clinical studies as small quantities of lipop rotein are required. Sterols are analysed after extraction from lipopr otein samples obtained by sequential flotation ultracentrifugation. Th e method involves briefly: extraction from lipoprotein samples using c hloroform-methanol, saponification of sterol esters using cold potassi um hydroxide, purification and derivatisation to trimethylsilyl ethers using BSTFA and 1% TMCS. Oxidation is prevented by drying under nitro gen and the use of powerful antioxidants. Separation is achieved using a DB-1 capillary column and a two-stage temperature ramp from 180-250 degrees C and detection using FID. The identity of sterols can be con firmed by GC-MS. Phytosterols and 7-ketocholesterol are present at low concentration in all the major lipoproteins. Using [3,4-C-13]choleste rol and GC-MS we present evidence that cholesterol oxidation does not occur during the processing of lipoproteins using this technique.