The new method is detailed in Application Note 266: Determination of Sialic Acids Using UHPLC with Fluorescence Detection and demonstrates that N-acetylated sialic acids can be determined and O-acetylated sialic acids can be identified in infant formula samples using high-performance liquid chromatography (HPLC) with fluorescence detection following acid hydrolysis and 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB) derivatization. By using a water:acetonitrile gradient, high resolution of the sialic acids can be obtained in a 20 minute analysis time, compared to the common 40 minute isocratic method.
The described assay uses a short-format Acclaim® RSLC 120 C18 column that allows fast run times. The lower flow rate and shorter run time requires less acetonitrile than other published methods. The sensitivity of the Dionex FLD-3400RS Fluorescence Detector easily allows determination of sialic acids in the infant formula that are present in the pmol range. The sensitivity provides for simple determination of Neu5Ac, Neu5Gc, and O-acetylated sialic acids in the derivatized samples.