Labeling conditions using a 2-aminobenzamide reagent for quantitative analysis of sialo-oligosaccharides

During 2-aminobenzamide (2-AB)-labeling of sialo-oligosaccharides the authors encountered a significant loss of sialic acids under the reported conditions, at 65 degrees C for 2 h. By examination of the relationship between labeling temperature and recovery of tetra-sialylated oligosaccharides, 2-AB-labeling at 37 degrees C for 16 h resulted in a maximum yield with practically no loss of sialic acid. Under the newly optimized conditions, the reproducibility of 2-AB-labeling was examined in triplicate with three lots of recombinant human erythropoietin (r-HuEPO). The present conditions gave analytical data comparable to those obtained by the established NaB[3H]4 labeling procedure. The modified method has been applied to oligosaccharide structural analysis of sialoglycoprotein therapeutics.