Use of graphitised carbon negative ion LC-MS to analyse enzymatically digested glycosaminoglycans

Capillary liquid chromatography–mass spectrometry using graphitised carbon stationary phase and ion trap mass spectrometry was shown to be a powerful technique for analysing glycosaminoglycans digested with endoglycosidases. Commonly found disaccharides from heparin/heparan sulphate digests at sub nanomole levels were found to be separated by mass and/or retention time and detected by negative ion electrospray mass spectrometry predominantly as [M − H]− ions using a standard electrospray interface and flow rate between 6–10 μL/min. Graphitised carbon liquid chromatography–fragmentation mass spectrometry provided sequence data of disaccharides and oligosaccharides. Sequence information was obtained from either collision of the [M − H]− ions (low sulphated disaccharides) or of the [M + Na − 2H]− ions (highly sulphated disaccharides). This separation and identification method of endoglycosidase digestion and sample preparation using a combination of cation exchange and graphitised carbon, was used to successfully analyse digests of keratan sulphate (keratanase) and heparin (heparinase) standards, and hyaluronic acid (hyaluronidase) from synovial fluid samples.9 page(s