Measurement of glycated protein by a rapid and specific method for absolute quantification of lysine-bound glucose

We modified the liquid-chromatographic assay of Schleicher and Wieland (J Clin Chem Clin Biochem 1981; 19:81-7) for measuring lysine-bound glucose in serum proteins, increasing its performance and practicality. After precipitating serum proteins from 10- to 50-microL samples with ethanol (700 mL/L) and hydrolyzing these in 6 mol/L HCl, we inject 20 microL of the diluted hydrolysate directly into the chromatograph, which consists of an acid-resistant C18 precolumn combined with a high-resolution C18 main column. The eluent is 3.5 mmol/L H3PO4 solution containing 30 mL of acetonitrile per liter. These modifications increase sensitivity, provide excellent resolution and longevity of stationary phases, shorten assay times to 15 to 20 min, and are suited for automation. The assay is highly sensitive and highly specific, quantifying nanomoles of lysine-bound glucose per milligram of protein. A precision (CV) of 5.1% is achievable at physiological and supra-physiological glucose concentrations, and analytical recovery is 99%. This inexpensive method has been applied to serum albumin, bulk serum proteins, and preparations of low-density lipoproteins and immunoglobulins.