Abstract
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 characterized by a charge state of -4. HA oligosaccharides from 32- to 40-mers exist as [M-5H](-5) anions. Furthermore, for smaller HA species, from 4/6- to 18/20-mers, ESI-MS revealed, generally in low relative abundance, anions related to the loss of one/two monosaccharide unit(s) from the oligomers, and no odd-numbered anions were produced for HA species greater than 20-mers.
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