Quantifying Sialic Acids by HPLC

Derivatization and quantitation of sialic acids by HPLC was carried out as previously described [31 (link),83 (link)–86 (link)]. Briefly, total sialic acids were measured by first releasing any bound sialic acids using mild acetic acid hydrolysis (2 N acetic acid for 3 h at 80°C), followed by derivatization of total sialic acids with DMB as described below. Alternatively, free sialic acid levels were measured by DMB derivatization without prior acid hydrolysis. The derivatization reaction requires that sialic acids be in the monosaccharide form. Thus, the concentration of bound sialic acids is measured indirectly (bound = total − free). Reaction conditions for DMB derivatization were 7 mM DMB, 22 mM sodium thiosulfite, 0.75 M 2-mercaptoethanol, and 1.4 M acetic acid for 2 h at 50°C. Derivatized samples were injected into a Waters HPLC (Milford, MA, USA) equipped with a reverse-phase C18 column (Tosoh Bioscience, Tokyo, Japan) and eluted using isocratic conditions at 0.9 ml/min using 8% methanol, 7% acetonitrile in water. An online fluorescence detector (Waters) was set to excite at 373 nm and detect emission at 448 nm. Peak integrations were used to quantitate sialic acid content by referencing a standard curve of pure sialic acid (Neu5Ac; Sigma-Aldrich) derivatized in parallel.