Sialic Acid Speciation Using Capillary Electrophoresis: Optimization of Analyte Derivatization and Separation

Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) was employed for rapid sialic acid speciation, facilitating the quantitative determination of <i>N</i>-glycolylneuraminic acid (Neu5Gc) and <i>N</i>-acetylneuraminic acid (Neu5Ac) on glycoproteins. Derivatization of the sialic acids with 2-aminoacridone (2-AMAC), using classical reductive amination in a nonaqueous solvent, led to the spontaneous decarboxylation of the sialic acid residues as determined by CE-LIF and offline mass spectrometric analysis. Modification of both the labeling conditions, to drive the decarboxylation reaction to completion and the CE-LIF parameters to separate the neutral species by complexation with a neutral coated capillary and borate reversed polarity, led to a robust platform for the rapid, sensitive, and quantitative speciation of sialic acids. The method can readily be used for quality control of recombinant biopharmaceuticals