High performance anion exchange chromatography coupled to an ion trap mass spectrometer

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High performance anion exchange chromatography (HPAEC) allows for a very efficient separation of diverse anionic substances. The most important applications include the analysis of inorganic anions, organic acids, as well as oligo- and polymeric carbohydrates. The latter are among the most important biomolecules and their versatile functionality is highly dependent on their structure. For carbohydrate analysis, HPAEC provides a very good separation which cannot be achieved by other techniques. For example, some oligo- and polysaccharides can be resolved as individual peaks up to a degree of polymerization of 60. Detection is usually achieved by applying pulsed amperometric detection (PAD) which allows for a specific and very sensitive detection. However, this method does not give any information about structural composition of the carbohydrates. This limitation can be overcome by coupling HPAEC to a mass spectrometer (MS) which requires an appropriate suppressor to desalt the strongly alkaline eluents. For the mass spectrometric analysis of carbohydrates, ion trap MS is a very suitable method because it allows for a multi-stage fragmentation (MSn). This approach includes the collection of a selected molecular ion in the ion trap and its targeted decomposition by using a collision gas. Subsequently, a selected ion is collected from the obtained fragments and further fragmented. The detection of diagnostic fragments can yield detailed information on the structure of the analyzed oligosaccharides: The type of the monosaccharide units, the sequence of the monosaccharide units within the oligosaccharide, and the glycosidic linkages. Therefore, both HPAEC-PAD and ion trap MS have a high potential in different areas of carbohydrates analysis. Consequently, we apply for an HPAEC-PAD/MSn system which combines the individual benefits of the two methods. Because alkali-labile bonds such as esters are hydrolyzed during HPAEC-PAD analysis, the chromatography system needs to include a separate independent high performance liquid chromatography (HPLC) channel which allows for measurements with other stationary phases. By connecting the MS to one of the two channels, a quick and easy change of the separation method can be achieved. Furthermore, the second channel can also be used for the analysis of glycopeptides and other glycoconjugates. For a universal and ionization independent detection of these compounds, a diode array detector needs to be included into the second channel. A detailed characterization of carbohydrates and carbohydrate conjugates is part of several different research projects which will strongly benefit from the unique possibilities offered by the described HPAEC-PAD/MSn system.