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 phytosterols are important indicators of lipoprotein oxidation and lipoprotein metabolism respectively. We describe a simple, sensitive and reproducible method for the simultaneous measurement of these sterols in human lipoprotein samples by capillary column gas liquid chromatography. The method is suitable for clinical studies as small quantities of lipoprotein are required. Sterols are analysed after extraction from lipoprotein samples obtained by sequential flotation ultracentrifugation. The method involves briefly: extraction from lipoprotein samples using chloroform-methanol, saponification of sterol esters using cold potassium hydroxide, purification and derivatisation to trimethylsilyl ethers using BSTFA and 1% TMCS. Oxidation is prevented by drying under nitrogen 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 confirmed by GC-MS. Phytosterols and 7-ketocholesterol are present at low concentration in all the major lipoproteins. Using [3,4-13C]cholesterol and GC-MS we present evidence that cholesterol oxidation does not occur during the processing of lipoproteins using this technique.