ANALYSIS OF THE N-ACETYLNEURAMINIC ACID AND N-GLYCOLYLNEURAMINIC ACIDCONTENTS OF GLYCOPROTEINS BY HIGH-PH ANION-EXCHANGE CHROMATOGRAPHY WITH PULSED AMPEROMETRIC DETECTION (HPAEC PAD)/

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a s ialylated glycoprotein's serum half-life and possibly its function, We evaluated the linearity, sensitivity, reproducibility, and accuracy o f a HPAEC/PAD method to determine its suitability for routine simultan eous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc), An eff ective internal standard for this analysis is 3-deoxy-d-glycero-d-gala cto-2-nonulosonic acid (KDN). We investigated the effect of the Au wor king electrode recession and determined that linear range and sensitiv ity were dependent on electrode recession, Using an electrode that was 350 mu m recessed from the electrode block, the minimum detection lim its of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, an d were reduced to 1, 2, and 0.5 pmol using a new electrode, The respon se of standards was linear from 10 to 500 pmol (r(2) > 0.99) regardles s of electrode recession, When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibil ity was unaffected when injections of glycoprotein neuraminidase and a cid digestions were interspersed with standard injections, Area RSDs o f Neu5Ac and Neu5Gc improved when the internal standard was used, We d etermined the precision and accuracy of this method for both a recesse d and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins, Results were consi stent with published values and independent of the working electrode, The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.