Sequential Injection Analysis of Cholesterol Using an Oxidation–Reduction Electrode Detector

A new automated method for the determination of cholesterol in serum was developed by combining sequential injection analysis (SIA) with potentiometric detection using a gold oxidation-reduction potential (ORP) electrode because serum cholesterol is an important indicator of abnormal lipid metabolism, arteriosclerosis, and hypertension in clinical diagnosis. The method is based on enzymatic hydrolysis of cholesterol esters by cholesterol esterase (CE) to yield free cholesterol, followed by oxidation with cholesterol oxidase (COD) to produce hydrogen peroxide. In the presence of horseradish peroxidase (HRP) and potassium ferrocyanide (K₄[Fe(CN)₆]), hydrogen peroxide oxidizes ferrocyanide to ferricyanide (K₃[Fe(CN)₆]), and the concentration ratio of ferri-/ferrocyanide is determined potentiometrically. Experimental conditions were optimized as follows: 5.0 mM K₄[Fe(CN)₆], 2 min reaction time, 0.5 units/mL HRP, 0.75 units/mL COD for free cholesterol, 1.5 units/mL COD and 10.0 units/mL CE for total cholesterol, and 5.0% (<i>w</i>/<i>v</i>) Triton X-100 with 5.0% (<i>v</i>/<i>v</i>) isopropanol as solubilizing agents. Under these conditions, the calibration curve for total cholesterol exhibited a Nernstian slope of 47.6 mV/decade over the range of 1.0 × 10^-5-1.0 × 10^-3 M, with no significant interference from common serum constituents. This method offers low reagent consumption, high automation, and simple operation, making it promising for clinical cholesterol analysis.