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 N-glycolylneuraminic acid (Neu5Gc) and N-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.
Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, Massachusetts 02115, United States.
Zoltan
Z
Szabo
Jonathan
J
Bones
Andras
A
Guttman
James
J
Glick
Barry L
BL
Karger
D000609:Aminoacridines / Q000737:chemistry; D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary; D006801:Humans; D007834:Lasers; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D009438:Neuraminic Acids / Q000032:analysis; D009961:Orosomucoid / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
D000609:Aminoacridines; D009438:Neuraminic Acids; D009961:Orosomucoid; C032592:N-glycolylneuraminic acid; C072515:2-aminoacridone; D019158:N-Acetylneuraminic Acid