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

高性能阴离子交换色谱法（HPAEC）可以非常有效地分离各种阴离子物质。最重要的应用包括分析无机阴离子，有机酸，以及低聚和聚合碳水化合物。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，一些低聚和多糖可以分解为单个峰，聚合度可达60。检测通常是通过应用脉冲安培检测（PAD），它允许一个特定的和非常敏感的检测。然而，这种方法不能提供任何有关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，这需要适当的抑制剂来淡化强碱性洗脱液。对于碳水化合物的质谱分析，离子阱质谱是一种非常合适的方法，因为它允许多级破碎（MSn）。这种方法包括在离子阱中收集选定的分子离子，并通过使用碰撞气体对其进行目标分解。随后，从获得的碎片中收集选定的离子并进一步破碎。诊断片段的检测可以获得所分析寡糖结构的详细信息：单糖单元的类型，寡糖内单糖单元的序列，以及糖苷键。因此，HPAEC-PAD和离子阱质谱在碳水化合物分析的不同领域都具有很高的潜力。因此，我们申请了一个HPAEC-PAD/MSn系统，它结合了两种方法的各自优点。由于在HPAEC-PAD分析过程中，碱不稳定键（如酯）会被水解，因此色谱系统需要包括一个独立的高效液相色谱（HPLC）通道，以便与其他固定相进行测量。通过将质谱连接到两个通道中的一个，可以实现快速简便地改变分离方法。此外，第二通道也可用于糖肽和其他糖缀合物的分析。对于这些化合物的普遍和电离独立的检测，一个二极管阵列探测器需要包括在第二通道。碳水化合物和碳水化合物缀合物的详细表征是几个不同研究项目的一部分，这些研究项目将从所描述的HPAEC-PAD/MSn系统提供的独特可能性中受益。

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.