Determination of cholesterol in milk and dairy products by high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method was developed to determine the content of cholesterol in milk and dairy products. To optimize separation of cholesterol, mobile phases including acetonitrile:2-propanol (8:1, v/v), acetonitrile: methanol (3:1. v/v). and acetonitrile:methanol:2-propanol (7:3:1, v/v/v) were compared. Acetonitrile/methanol/2-propanol was superior to the other mobile phase systems for separating cholesterol. Liquid-liquid extraction (LLE) of cholesterol was simplified using a non-polar solvent, hexane, to remove interfering compounds, and had an excellent recovers (100 +/-1.0%) of cholesterol. A solid phase extraction (SPE) method using Sep-pak Cis was developed and compared with LLE. The SPE method was rapid and highly reproducible. Both extraction methods were useful when used in combination with saponification of esterified cholesterol to facilitate total cholesterol determination. The detection limit of cholesterol was 0.01 mug. The newly developed HPLC method was rapid, simple, and accurate, and has advantages over the many methods commonly used.