Flow Injection Analysis (FIA) system was developed for the determination of cholesterol using immobilized cholesterol oxidase and cholesterol ester hydrolase. The enzymes were immobilized on controlled pore glass (CPG) by glutaraldehyde method. The glass columns packed with immobilized enzymes contained 3~5 U for each enzyme. A hydrogen peroxide sensitive electrode was constructed and applied to FIA system as a detector. The electrode had a wide dynamic range about 5 order of magnitude of hydrogen peroxide concentration at potential difference of + 850 mV vs. saturated calomel electrode. To achieve good analytical readouts under FIA system, the conditions for FIA response were investigated and optimized; such as, sampling volumes, flow rates and pH of carrier buffer. The linear range of response was up to 60 ~ 80 and 400 mg/dL for free cholesterol and cholesterol ester, respectively. All sample was analyzed with a good precision (＜2.5 % CV) and accuracy. About 23 samples were measured within an hour. Intermittent assays of more than 500 times caused 50 % decrease of response sensitivity. However, oxidizable substances in sample interfered with the assays of cholesterol.
