Anion-exchange high-performance liquid chromatographic assay of plasma lipoproteins

A fast, accurate, and precise high-performance liquid chromatographic (HPLC) assay method for human plasma lipoproteins has been developed. The method includes complete separation of high-, low-, and very-low-density lipoproteins within 20 min using a DEAE-glucomannan gel with stepwise elution, enzymatic oxidation of cholesterol (following ester hydrolysis) followed by a two-step reaction to form a fluorogenic product, and fluorometric detection as the total cholesterol (TC) level of each lipoprotein. The calibration range is 5-1000 micrograms/ml for the TC level of 20-microliters plasma samples and the relative standard deviation (n = 5) of the precision at 5 micrograms/ml is less than 10%. Each lipoprotein level determined by this optimized HPLC method agrees well with levels determined by ultracentrifugation or precipitation methods. This new method was successfully applied to assays of plasma lipoproteins in various forms of hyperlipidemia.