Nonaqueous reverse phase liquid chromatographic analysis for cholesterol in milk chocolate

A method using liquid chromatography was developed for the analysis of cholesterol in milk chocolate products. The method involves saponification of the sample with methanolic KOH followed by extraction with ether. Potentially interfering components are eliminated through the use of a silica Sep-Pak cleanup step before injection. The nonaqueous reverse phase LC system consists of a C18 column and an isopropanol-hexane mobile phase with direct detection at 205 nm. Recoveries of 1, 3, and 5 mg cholesterol added to 1 g sample of milk chocolate were 88.6, 102.8, and 110.1%, respectively. Studies conducted with [4-14C]-cholesterol were undertaken to further document the accuracy of the method.