Simultaneous analysis of heparosan oligosaccharides by isocratic liquid chromatography with charged aerosol detection/mass spectrometry

• A novel isocratic PGC-HPLC-CAD method was developed for separation and quantification of heparosan oligosaccharides.
• The retention and selectivity of PGC material to heparosan oligosaccharide were significantly affected by addition of small fraction of methanol to mobile phase and column temperature.
• PGC-HPLC–MS was conducted for on-line identification of heparosan oligomers with high efficiency under the same chromatographic condition.
• Such a PGC-HPLC-CAD method combined methods allow further studies on the biological role of heparosan and its related enzymes.

Uncovering the biological roles of heparosan oligosaccharides requires a simple and robust method for their separation and identification. We reported on systematic investigations of the retention behaviors of synthetic heparosan oligosaccharides on porous graphitic carbon (PGC) column by HPLC with charged aerosol detection. Oligosaccharides were strongly retained by PGC material in water-acetonitrile mobile phase, and eluted by trifluoroacetic acid occurring as narrow peaks. Addition of small fraction of methanol led to better selectivity of PGC to oligosaccharides than acetonitrile modifier alone, presumably, resulting from displacement of methanol to give different chemical environment at the PGC surface. Van’t-Hoff plots demonstrated that retention behaviors highly depended on the column temperature and oligosaccharide moieties. By implementing the optimal MeOH content and temperature, a novel isocratic elution method was successfully developed for baseline resolution and identification of seven heparosan oligosaccharides using PGC-HPLC–CAD/MS. This approach allows for rapid analysis of heparosan oligosaccharides from various sources.