On-line HPLC/ESI-MS separation and characterization of hyaluronan oligosaccharides from 2-mers to 40-mers.

A new method for the separation and identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with hyaluronidase (EC 3.2.1.35) using on-line high-performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI-MS) is presented. Reversed-phase ion pairing-HPLC, based on tributylamine salts and a volatile mobile phase, provided excellent chromatographic resolution and separation was achieved for HA oligosaccharides containing 2−40 monomers (from 2- to 40-mers). Using the on-line ion trap mass analyzer, complete identification and structural information for each HA oligomer species was obtained. In particular, a series of negatively charged species of different m/z ratios are seen for each oligosaccharide. Smaller HA species, from 2- to 4-mers, exhibit mainly [M − H]-1 anions, whereas the 6−10-mers exist predominantly as the charge state of −2. The HA oligomers from 12- to 18-mers are mainly represented by [M − 3H]-3 anions while species from 20- to 28/30-mers are ...