A capillary zone electrophoresis method for determining N ‐acetylneuraminic acid in glycoproteins and blood sera

A simple capillary zone electrophoresis (CZE) method for the determination of the content of the major sialic acid form N ‐acetylneuraminic acid (Neu5Ac) in glycoproteins was established. The present method utilizes a simplified hydrolysis‐purification procedure consisting of mild acid hydrolysis (25 m M trifluoroacetic acid for 2 h at 80°C) to release Neu5Ac and ultrafiltration on Centricon‐3 membrane to remove the obtained asialoglycoproteins and other macromolecules present in biologic samples. Derivatization with benzoic anhydride at 80°C for 20 min resulted in complete conversion of Neu5Ac to per‐ O ‐benzoylated Neu5Ac. CZE analysis was performed using the operating buffer 25 m M phosphate, pH 3.5, containing 50% (v/v) acetonitrile as organic modifier at 30 kV, and detection of the per‐ O ‐benzoylated Neu5Ac at 231 nm. The method showed excellent repeatability (RDS < 1.98%) and a linearity range from 5 µg/mL to 5 mg/mL with a detection limit of 2 µ M . Application of the method to microanalysis of human α 1 ‐acid glycoprotein and blood serum samples showed excellent agreement with previously published values, suggesting a high precision for the developed CZE method. Copyright © 2002 John Wiley & Sons, Ltd.