Hydrophilic interaction 10 μm I.D. porous layer open tubular columns for ultratrace glycan analysis by liquid chromatography–mass spectrometry

The sensitivity of glycan analysis using nano-liquid chromatography interfaced with electrospray ionization mass spectrometry (ESI–MS) increases with the decrease of the mobile phase flow rate, accompanied by reduced ion suppression. In this study, we describe the preparation and performance of high efficiency 10 μm I.D. amine-bonded poly(vinylbenzyl chloride-divinylbenzene) hydrophilic interaction (HILIC) porous layer open tubular (PLOT) columns operated at 20 nL/min for the separation and analysis of glycan mixtures. HILIC-PLOT columns with a uniform porous polymer layer were reproducibly prepared (∼4% RSD in retention time from column-to-column) via in situ polymerization, followed by one step modification with ethylenediamine. When coupled on-line with negative ESI–MS, low detection limits (0.3 fmol) for a 3-sialyl-tetrasaccharide were achieved using a 2.5 m × 10 μm I.D. HILIC-PLOT column. A dextran ladder standard was used to evaluate the performance of the column, and high efficiency separation was achieved with detection of the dextrans up to G22 from ∼50 fmol amounts injected. As an example of the high sensitivity of the column, MS6 characterization of glycan structures was possible from the injection of 10 fmol of a neutral and sialylated glycan. As another example of high sensitivity LC–MS analysis of 3 ng of a PNGase F digest of ovalbumin allowed 28 N-linked glycans to be confidently identified from a single analysis. High quality MS/MS spectra for each ovalbumin glycan were acquired and manually interpreted for structure analysis. The HILIC-PLOT column is a very promising approach for LC–MS analysis of glycans at the ultratrace level.