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 mM trifluoroacetic acid for 2h at 80 degrees 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 degrees C for 20 min resulted in complete conversion of Neu5Ac to per-O-benzoylated Neu5Ac. CZE analysis was performed using the operating buffer 25mM 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 microg/mL to 5mg/mL with a detection limit of 2 microM. Application of the method to microanalysis of human alpha(1)-acid glycoprotein and blood serum samples showed excellent agreement with previously published values, suggesting a high precision for the developed CZE method.
Strousopoulou
K
K
Department of Chemistry, Section of Organic Chemistry, Biochemistry and Natural Products, Laboratory of Biochemistry, University of Patras, 26110 Patras, Greece.
Militsopoulou
M
M
Stagiannis
K
K
Lamari
F N
FN
Karamanos
N K
NK
D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D019158:N-Acetylneuraminic Acid / Q000032:analysis* / Q000097:blood; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D006023:Glycoproteins; D019158:N-Acetylneuraminic Acid