A multicommuted flow-based procedure with detection by chemiluminescence for the determination of total and free cholesterol without changes in the flow manifold is proposed. Cholesterol esterase and cholesterol oxidase were both immobilized on glass beads via glutaraldehyde/(3-aminopropyl)-triethoxysilane and mini-columns containing the enzymes were used for online sample treatment. Cholesterol esters were cleaved to cholesterol and fatty acids at the packed reactor containing cholesterol esterase. The reactor containing cholesterol oxidase converted cholesterol to cholest-4-en-3-one also yielding hydrogen peroxide. Detection was based on the chemiluminescence produced by H2O2 in the hexacyanoferrate(III)-luminol system. Influence of both chemical and hydrodynamic variables on the chemiluminescence signals were investigated. The analytical curves were linear from 250 to 2500 mg L-1 and from 500 to 4000 mg L-1, for free and total cholesterol, respectively. Detection limits for both analytes were estimated as 60 mg L-1 at 99.7% confidence level. The sampling rate was 55 h-1 and reagent consumption was 350 µg of luminol and 2.6 mg of potassium hexacyanoferrate(III) per determination. The procedure developed was successfully applied for determination of cholesterol in eggs and in human blood serum with results in agreement with the reference spectrophotometric method at the 95% confidence level.
flow analysis; multicommutation; chemiluminescence; cholesterol; cholesterol esterase; cholesterol oxidase
