Flow injection determination of free and total cholesterol in animal greases using enzymes in non-aqueous media

Abstract

A non-covalently coimmobilized bienzymic reactor of horseradish peroxidase (HRP) and cholesterol oxidase (COD), operating in a continuous organic flowing stream of 1 × 10^–3 M p-anisidine in buffer-saturated (pH 7.0) toluene, has been employed for cholesterol determination in animal greases, such as pig, beef, and chicken fat, and codfish liver oil. The method provides a good linear relationship up to 1.8 × 10^–3 M cholesterol and average recoveries of 99.5%, a high sensitivity, with a detection limit of 1 × 10^–6 M of cholesterol and a good precision (an interday RSD of 1.8% for the determination of total cholesterol in a codfish oil sample). The method permits the direct spectrophotometric determination of free cholesterol present in animal grease samples without any pre-treatment, and the total cholesterol determination after a microwave-assisted saponification. The bienzymic reactor exhibits a good stability in the water-restricted environment, being possible to perform more than 180 analyses during a period of 10 d with 1 mg of HRP, and 1 mg of COD, equivalent to 220 purpurogallin units and 23 U, respectively.