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RESEARCH PRODUCT

Microwave-assisted saponification of animal greases for cholesterol determination

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Abstract 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.