Analysis of Oligosaccharides by HILIC-MS/MS.

The chromatographic separations were performed on an ACQUITY Glycoprotein BEH Amide column, 300 Å (1.7 μm, 2.1 mm × 150 mm, Waters Corporation, Milford, MA, USA) using the Ultimate 3000 UHPLC system (Thermo Scientific) coupled to an Orbitrap mass spectrometer (Thermo Scientific Orbitrap Fusion Lumos) as previously described.⁴ The composition of the two mobile phases was 10 mmol/L ammonium formate with 0.1% (volume fraction) formic acid (A) and 99.9% (volume fraction) ACN with 0.1% (volume fraction) formic acid (B). The acquisition time was 65 min, and the mobile phase had a flow rate of 400 μL/min, pH 4.5 with a column oven temperature of 35 °C. The injection volume was 10 μL. Mass spectra were acquired over a range of collisional energies in an HCD cell at NCE (normalized collision energies) values of 10, 15, 20, 40, and 50, and in an ion trap (FT-IT) at 35% NCE. Each sample was analyzed in triplicate. Commercial milk oligosaccharides standards, including nonfucosylated, fucosylated, and sialylated isomers were analyzed and used as standards.