High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) for the sensitive determination of hyaluronan oligosaccharides

• A HPAEC-PAD method was developed to determine hyaluronan (HA) oligosaccharides.
• Sensitive analysis of oligosaccharides comprising 2–25 disaccharides is enabled.
• HPAEC-PAD plus CZE–ESI-TOF-MS revealed formation of odd-numbered oligosaccharides.
• The method closes a gap in the analytical toolbox for hyaluronan oligosaccharides.
• Combined methods allow studies on the biological role of HA and hyaluronidases.

High performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) was optimized for the analysis of oligosaccharides derived from the extracellular matrix component hyaluronan. Using this sensitive approach, the separation of oligosaccharides consisting of two (molecular weight ca. 0.8 kDa) up to 25–30 (molecular weight: ca. 9.5–11.4 kDa) disaccharide moieties was possible. Standard oligosaccharides (comprising 2–4 repetitive disaccharides) were detectable at very low amounts of 0.2–0.3 pmol (20–30 nM). Including 10 min of column equilibration, a complex mixture of low molecular weight hyaluronan can be analyzed within 40 min. The HPAEC method was successfully applied to the study of the size-dependency of both the action of bovine testicular hyaluronidase (BTH) and the precipitation of hyaluronan by cetyltrimethylammonium bromide (CTAB), a physicochemical reaction often used for the determination of hyaluronan and hyaluronidase activity.

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