ELIMINATING AMINO-ACID AND PEPTIDE INTERFERENCE IN HIGH-PERFORMANCE ANION-EXCHANGE PULSED AMPEROMETRIC DETECTION GLYCOPROTEIN MONOSACCHARIDE ANALYSIS

The monosaccharide content of a glycoprotein is often determined by ac id hydrolysis at elevated temperature and subsequent high pH chromatog raphy of the released, underivatized monosaccharides on pellicular ani on-exchange resin (HPAE) using pulsed amperometric detection (PAD). We have found that for glycoproteins with low levels of glycosylation, m onosaccharide quantitation can be compromised by amino acids fouling t he working electrode surface. Specifically, lysine elutes on the Carbo Pac PA1 column just prior to galactosamine, whereas remaining amino ac ids and most peptides elute after the monosaccharides and do not inter fere with monosaccharide quantification. A direct comparison of PAD vs Abs(215) detection of lysine using the CarboPac PA1 column as the sep arator reveals that lysine does not cleanly come off the working elect rode. The monosaccharide response inhibition caused by lysine could be corrected by the posthydrolysis addition of a rhamnose internal stand ard and the determination of ''correction factors.'' We have developed a guard column with an altered selectivity for amino acids which, whe n used with a new separator, causes lysine to elute after the monosacc harides and also causes hydrophobic amino acids to elute further after the monosaccharides. Together the new separator and guard columns sol ve the lysine fouling problem, reduce sample-related baseline noise, a nd reduce the magnitude of correction factors. (C) 1996 Academic Press , Inc.