Reversed-Phase High-Performance Liquid Chromatography: A Robust Method for Quantitation of Fluorescently Labeled and Derivatized Sialic Acids Isolated from Mouse Liver

Sialic acids, such as N-acetylneuraminic acid and N-glycolylneuraminic acid, are negatively charged carbohydrate moieties found at distal ends of oligosaccharide chains on mammalian cell surface.

To quantify relative amounts of these sialic acids in mouse liver tissue using reversed-phase high-performance liquid chromatography or RP-HPLC, first, treat the isolated sialic acid mixture with derivatization agent. The derivatization agent interacts with the functional groups of sialic acids, generating stable, fluorescent sialic acid derivatives.

Assemble an RP-HPLC column, connected to a fluorescence detector. The column comprises porous silica-based stationary phase bonded to non-polar, hydrophobic, alkyl chain ligands to facilitate selective interactions with sialic acid derivatives during chromatographic run.

Equilibrate the column using aqueous polar mobile phase containing low concentrations of acetonitrile, a less polar organic solvent, for optimal column conditioning.

Load the derivatized sialic acid mixture into the column.

N-acetylneuraminic acid, containing a hydrophobic N-acetyl group, adsorbs more strongly to the hydrophobic ligands of the stationary phase than N-glycolylneuraminic acid with a hydrophilic N-glycolyl group.

Analyze the samples, using a standard HPLC system connected to an online fluorescence detector. Use a reversed-phase C-18 column with a 250-millimeter length and 4.6-millimeter diameter for the analysis.

After preparing solvent A and solvent B, and setting up the HPLC run as detailed in the text protocol, inject 50 microliters of the sample into the HPLC system.

Monitor eluents, using the fluorescence detector excitation wavelength of 373 nanometers, and an emission wavelength of 448 nanometers.

Finally, calculate the relative amount of Neu5Gc as described in the text protocol.