Microwave-assisted saponification of animal greases for cholesterol determination Original Research Article

An alternative method for the hydrolysis of cholesterol esters in animal grease samples has been developed. The method consists of the microwave-assisted treatment of 4 g of animal fat or oil with 40 ml of an ethanolic KOH solution inside a 115 ml closed reactor in which samples were irradiated for 2.5 min at 50% power level of an exit power of 700 W. After extraction with petroleum ether and dissolution of the unsaponifiables in toluene, total cholesterol was determined spectrophotometrically by using p-anisidine as a test reagent of the enzymatic reaction of cholesterol through a bienzymic reactor in which cholesterol oxidase and horseradish peroxidase were noncovalently immobilised in controlled pore glass beads. The method provided a quantitative recovery of spiked free cholesterol (100% recovery for an added concentration level of 36 mg g−1) and cholesteryl palmitate (98.7% recovery for an added concentration of 37 mg g−1) and results comparable to those obtained after saponification with ethanolic KOH at 90°C under reflux conditions for 1 h, saving a considerable time with a comparable figures of merit which involve a limit of detection of 1×10−6 M total cholesterol an average interday relative standard deviation of 3.5% for three independent analysis of a series of different samples.