SIALIC ACIDS COMPOSITION ANALYSIS BY HPAEC

University Of Georgia, Athens GA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The samples, CN195-01-08-1 (1.4 mg), CN195-01-09-1 (1.3 mg) CN195-01-09-2 (1.4 mg) were weighed into pre-rinsed (with methanol) screw-cap tubes, whereas all of 06NT11-060317 was transferred from the original container into a pre-rinsed (with methanol) screw cap tube with nanopure water and dried under a stream of N2. The dried samples were hydrolyzed with 400 [unreadable]L of 2.0 M acetic acid at 80[unreadable]C for 3 h. The hydrolysates were dried under a stream of N2, resuspended with water H2O (CN195-01-08-1, 100 [unreadable]L;CN195-01-09-1, 100 [unreadable]L;CN195-01-09-2, 100 [unreadable]L;and 06NT11-060317, 70 [unreadable]L) sonicated for 7 min in ice and transferred to injection vials. However, sialic acid peak from the originally dissolved hydrolysates were too, hence, were diluted 100 times in 100-[unreadable]L solutions. A mix of standards for N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) with a known number of moles was hydrolyzed in the same manner and at the same time as the samples. Four concentration of standard mix (0.5, 1.0, 2.0, and 4.0 nmoles per injection) were prepared to establish a calibration equation. Quantification of the number of moles of each sialic acid per injection volume (10 [unreadable]L) was accomplished through Chromeleon software in a quadratic calibration type. Total number of moles per sample hydrolyzed was calculated by incorporating the dilution factor. Coefficient of determination was 99.8887% for NANA and 95.7791% for NGNA. The sialic acids were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The individual sialic acids were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The mobile phase solutions in the gradient program were 100 mM NaOH, and 1M sodium acetate in 100 mM NaOH and injections were made every 35 minutes. All methods were based on protocols described by Hardy and Townsend (Hardy, M. R., and Townsend, R. R., "High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates", 1994, Methods Enzymol. 230: 208-225).