A method for quantitative analysis of monosaccharides including N-acet
ylneuraminic acid derived from sialic acid-containing oligosaccharides
and glycoproteins is presented. The analysis is based on the combinat
ion of chemical and enzymatic methods coupled with capillary electroph
oretic (CE) separation and laser-induced fluorescence (LIE) detection.
The present method utilizes a simplified acid hydrolysis procedure co
nsisting of mild hydrolysis (0.1 M TFA) to release sialic acid and str
ong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. A
mino sugars released from strong acid hydrolysis of oligosaccharides a
nd glycoproteins were reacetylated and derivatized with 8-aminopyrene-
1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of
sodium cyanoborohydride to yield quantitatively the highly stable flu
orescent APTS adducts, N-acetylneuraminic acid (Neu5Ac), a major compo
nent of most mammalian glycoproteins, was converted in a fast specific
reaction by the action of neuraminic acid aldolase (N-acylneuraminate
pyruvatelyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvat
e. ManNAc was then derivatized with APTS in the same manner as the oth
er monosaccharides, This method was demonstrated for the quantitation
of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'
-sialyl-N-acetyllactosamine and bovine fetuin glycan. Quantitative rec
overy of the N-acetylmannosamine was obtained from a known amount of N
eu5Ac in a mixture of seven other monosaccharides or from the sialylat
ed oligosaccharides occurring in glycoproteins, The sequence of proced
ures consists of acid hydrolysis, enzymatic conversion and APTS deriva
tization which produced quantitative recovery of APTS-monosaccharide a
dducts, The detection Limits for sugars derivatized with APTS and dete
cted by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugar
s.