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

高效阴离子交换层析(HPAEC)可以非常有效地分离各种阴离子物质。最重要的应用包括无机阴离子、有机酸以及低聚碳水化合物的分析。后者是最重要的生物分子之一，它们的多功能高度依赖于它们的结构。对于碳水化合物的分析，HPAEC提供了其他技术无法实现的非常好的分离。例如，一些寡糖和多糖可以被分解为聚合度高达60的单个峰。检测通常是通过应用脉冲安培检测(PAD)实现的，这允许进行特定和非常灵敏的检测。然而，这种方法没有给出任何关于碳水化合物结构组成的信息。这一限制可以通过将HPAEC耦合到质谱仪(MS)来克服，质谱仪需要适当的抑制器来脱去强碱性洗脱液的盐分。对于碳水化合物的质谱分析，离子陷阱MS是一种非常合适的方法，因为它允许多阶段裂解(MSN)。这种方法包括在离子陷阱中收集选定的分子离子，并通过使用碰撞气体进行定向分解。随后，从获得的碎片中收集选择的离子并进一步碎裂。对诊断片段的检测可以获得有关所分析的低聚糖结构的详细信息：单糖单元的类型、低聚糖中单糖单元的序列以及糖苷键。因此，HPAEC-PAD和离子陷阱MS在不同的碳水化合物分析领域都具有很高的潜力。因此，我们申请HPAEC-PAD/MSN系统，它结合了两种方法的各自优点。由于酯等碱不稳定键在HPAEC-PAD分析过程中会被水解，因此层析系统需要包括单独的独立高效液相色谱(HPLC)通道，以便与其他固定相一起进行测量。通过将MS连接到两个通道中的一个，可以快速且容易地改变分离方法。此外，第二通道还可用于糖肽和其他糖偶联物的分析。对于这些化合物的通用和独立于电离的检测，需要在第二通道中包括二极管阵列检测器。碳水化合物和碳水化合物结合物的详细表征是几个不同研究项目的一部分，这些项目将从所描述的HPAEC-PAD/MSN系统提供的独特可能性中受益匪浅。

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.