Determination of cholesterol in egg yolk by high performance liquid chromatography using an automated precolumn-switching procedure

A direct injection high-performance liquid chromatographic method is develo ped for the determination of cholesterol in egg-yolk. The described method involved an on-line sample dean-up procedure using column-switching techniq ue and protein-coated RP-18 precolumn. The egg-yolk was diluted with tris b uffer, pH 8.5 to produce homogenous sample and render it suitable for injec tion. On the precolumn, cholesterol was preseparated from endogenous compon ents of the egg-yolk with tris buffer, pH 8.5 for 4 minutes. To complete th e precolumn washing cycle, the solvent selector is automatically changed fr om tris buffer to 0.05 M phosphate buffer, pH 4.8 for 2 min, followed by 15 % methanol in 0.05 M phosphate buffer, pH 4.8 for 1 min further. After column switching, the elute fraction containing cholesterol was furth er separated by reversed-phase chromatography using Spherisorb (ODS) column with a mobile phase consisting of acetonitrile, isopropanol, and phosphate buffer, pH 4 (40:50:10, v/v). The average cholesterol recoveries ranged fr om 97.70 to 100.50% and the relative standard deviations ranged from 2.70 t o 3.84%. The method allows accurate and fast daily routine monitoring of ch olesterol in large numbers of chicken eggs and is proven to be highly appli cable in studying nutritional effects on chicken egg cholesterol.