Quantification of Sialic Acid by Enzymatic Reaction and Fluorescence Detection

To evaluate total SA content, an acid hydrolysis was performed: 200 μL of sample was mixed with 800 μL of 0.05 M H₂SO₄, heated (60 min at 80°C), cooled to room temperature and centrifuged (13,000 × g for 30 min at 4°C). For free SA determination, 1 mL of sample was centrifuged (13,000 × g for 30 min at 4°C), the aqueous phase was collected, and 400 μL of the supernatant was purified by solid phase extraction using strong anionic exchange cartridges (OnGuard II-A; Dionex, Sunnyvale, CA, USA) that had been previously activated with 10 mL deionized water. After sample loading, each cartridge was washed with 10 mL of distilled water and SA was eluted with 10 mL of 100 mM NaCl. Dried samples were reconstituted in 500 μL of deionized water, vortexed, and sonicated to assure complete solubilization. SA (expressed as Neu5AC) concentrations were assessed to determine free and total SA in each sample using an enzymatic commercial kit with fluorescence detection (ab83375; Abcam, Cambridge, UK) as described by the manufacturer. Subsequently, the quantity of bound SA was calculated as the difference between total and free SA concentrations.