Characterization of derivatization of sialic acid with 2-aminoacridone anddetermination of sialic acid content in glycoproteins by capillary electrophoresis and high performance liquid chromatography - ion trap mass spectrometry

A simple and highly sensitive capillary electrophoresis (CE) method for det ermining the content of N-acetylneuraminic acid (Neu5Ac) in glycoproteins w as developed. Neu5Ac was derivatized with 2-aminoacridone (AMAC) by reducti ve amination, and the AMAC-Neu5Ac adduct could be readily separated from th e other 11 AMAC-derivatized neutral and acidic monosaccharides usually pres ent in glycoproteins by CE in a 0.3 mol/L berate buffer, pH 10.5, and detec ted at 260 nm. The derivatization of Neu5Ac was achieved at 55 degrees C fo r 4 h. AMAC-Neu5Ac was stable at 20 degrees C in the dark for at least 12 h while at room temperature it spontaneously converted into another substanc e with a lower electrophoretic mobility, which was identified as decarboxyl ated AMAC-Neu5Ac by high performance liquid chromatography - ion trap mass spectrometry (HPLC-ITMS). Concentration and mass of Neu5Ac as low as 1 mu m ol/L and 35 fmol could be detected. The linear correlation coefficient betw een the ratio of peak area to migration time of AMAC-Neu5Ac and the concent ration of Neu5Ac ranging from 10 to 120 mu mol/L was 0.9978 (n=8). This met hod was successfully applied to the analysis of sialic acid in human urinar y trypsin inhibitor (hu-UTI), bovine alpha(1)-acid glycoprotein (alpha(1)-A GP) and recombinant human erythropoietin (rhu-EPO). By combination of CE an d HPLC-ITMS we found that N-glycolylneuraminic acid (Neu5Gc) was present in bovine alpha(1)-AGP in addition to Neu5Ac, with a quantity comparable to t hat of the latter.