Determination of neuraminidase-suseeptible and total N-acetylneuraminic acid in cells by selected ion monitoring

N-acetylneuraminic acid (NANA) is released from glycoproteins by neuraminidase or acid hydrolysis and quantified by monitoring the protonated molecular ions of fully silylated NANA ( m e = 814) and neuraminic acid β-methylglycoside (internal standard, m e = 714) in a gas chromatograph-mass spectrometer system using isobutane ionization. Detection limit is 200 pg (0.6 pmol, underivatized weight) of NANA injected. In biological samples 5 ng (15 pmol) of NANA can be detected in 50 μl of hydrolysate. Only 1 to 50 μl of hydrolysate is needed, sample preparation is simple, NANA is positively identified in every analysis, 2-deoxy carbohydrates and other sialic acids do not interfere, only free NANA is determined, and the internal standard increases reliability. The NANA content of neuraminidase-treated human leukemic cells was on the order of 0.3μmol 10⁹ cells. NANA was quantified using as few as 5 × 10⁴ cells, in contrast to the conventional colorimetric (thiobarbituric) technique which requires 2.5 × 10⁷ cells.