Determination of Animal Fat in Margarine by High Pressure Liquid Chromatography of the Sterols

Abstract

The utility of high pressure liquid chromatography as a routine method to detect animal fats in vegetable margarines on the basis of sterol content was investigated. Cholesterol, which makes up almost 100% of the total sterol in animal fats, constitutes only 0-4% of the total sterols in vegetable oils. The major phytosterols in vegetable oils currently used for margarine production are B-sitosterol, stigmasterol and campesterol. Brassicasterol is less conunon but in rapeseed oil it is present in high proportion. The minor phytosterols are: ~ 5 -avenasterol, ~ 7 -stiqmasterol, 7 ~ -avenasterol, 24-methyl cholest-7-enol and cholesterol. A uBondapak c18 column and a yariable wavelength ultraviolet detector along with a variety of mobile phases, have been used throughout this investigation. A method was developed for isolating, extracting and derivatizing sterols from biological material. Optimum chromatographic conditions are described. Lard, soy bean, corn, cottonseed, safflower, sunflower, olive, walnut and rapeseed oils were analyzed. A good separation was obtained for free sterols from vegetable o·ils. Free cholesterol could be separated from the major phytosterols but not from the whole group and in a blend of 25% lard and 75% corn oil, cholesterol did not show up clearly. With benzoate derivatives a good separation was obtained for cholesterol from major phytosterols, but brassicasterol from rapeseed oil and some minor phytosterols from other oils interfered with the cholesterol peak. Because of this interference, the detectable ratio of animal fat in vegetable margarine is dependent on the raw material.· Fifty percent of animal fat can be easily detected in all kinds of margarines. In soy bean oil marqarine, 25% animal fat can be easily detected. The method is reproducible, rapid, and worth further investigation to find a column and solvent system capable of separating cholesterol completely from all phytosterols, so that very low levels of animal fat could be detected in vegetable oil.