Flow injection determination of free and total cholesterol in animal greases using enzymes in non-aqueous media.
A non-covalently coimmobilized bienzymic reactor of horseradish peroxidase (HRP) and cholesterol oxidase (COD), operating in a continuous organic flowing stream of 1X10-3 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 X 10-3 M cholesterol and average recoveries of 99.5%, a high sensitivity, with a detection limit of 1 X 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 10d with 1mg of HRP, and 1 mg of COD, equivalent to 220 purpurogallin units and 23 U, respectively.
Departament de Química Analítica, Unversitat de València, Spain.
G
G
Piñeiro-Avila
A
A
Salvador
M
M
de la Guardia
D000818:Animals; D002417:Cattle; D002645:Chickens; D002784:Cholesterol / Q000032:analysis*; D005223:Fats / Q000737:chemistry*; D017022:Flow Injection Analysis; D013053:Spectrophotometry; D013552:Swine
D005223:Fats; D002784:Cholesterol