Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstr. 20, 38106, Braunschweig, Germany.
Department of Biochemistry, Physikalisch-Technische-Bundesanstalt, Bundesallee 100, 38116, Braunschweig, Germany.
Anal Bioanal Chem. 2023 Apr;415(10):1817-1828. doi: 10.1007/s00216-023-04622-w. Epub 2023 Mar 3.
The methyl substitution along and among the polymer chains of methyl cellulose (MC) is commonly analyzed by ESI-MS after perdeuteromethylation of the free-OH groups and partial hydrolysis to cello-oligosaccharides (COS). This method requires a correct quantification of the molar ratios of the constituents belonging to a particular degree of polymerization (DP). However, isotopic effects are most pronounced for H/D since their mass difference is 100%. Therefore, we investigated whether more precise and accurate results could be obtained for the methyl distribution of MC by MS of CH instead of CD-etherified O-Me-COS. Internal isotope labeling with CH makes the COS of each DP chemically and physically much more similar, reducing mass fractionation effects, but at the same time requires more complex isotopic correction for evaluation. Results from syringe pump infusion ESI-TOF-MS with CH and CD as isotope label were equal. However, in the case of LC-MS with a gradient system, CH was superior to CD. In the case of CD, the occurrence of a partial separation of the isotopologs of a particular DP resulted in slight distortion of the methyl distribution since the signal response is significantly dependent on the solvent composition. Isocratic LC levels this problem, but one particular eluent-composition is not sufficient for a series of oligosaccharides with increasing DP due to peak broadening. In summary, CH is more robust to determine the methyl distribution of MCs. Both syringe pump and gradient-LC-MS measurements are possible, and the more complex isotope correction is not a disadvantage.
甲基纤维素（MC）的聚合物链上和链间的甲基取代通常通过 ESI-MS 进行分析，方法是对游离-OH 基团进行全氘代甲基化，并对其进行部分水解得到纤维二糖低聚糖（COS）。该方法需要对属于特定聚合度（DP）的组分的摩尔比进行正确的定量。然而，对于 H/D，同位素效应最为明显，因为它们的质量差为 100%。因此，我们研究了通过 MS 对 CH 而不是 CD-醚化 O-Me-COS 进行分析，是否可以获得更精确和准确的 MC 甲基分布结果。用 CH 进行内部同位素标记使每个 DP 的 COS 在化学和物理上更加相似，从而减少质量分馏效应，但同时需要更复杂的同位素校正进行评估。用 CH 和 CD 作为同位素标记的注射器泵注入 ESI-TOF-MS 的结果是相等的。然而，在具有梯度系统的 LC-MS 的情况下，CH 优于 CD。在 CD 的情况下，由于溶剂组成对信号响应有显著影响，特定 DP 的同位素的部分分离会导致甲基分布的轻微扭曲。等度 LC 解决了这个问题，但由于峰展宽，对于一系列 DP 增加的低聚糖，一个特定的洗脱液组成是不够的。总之，CH 更适合用于确定 MC 的甲基分布。注射器泵和梯度 LC-MS 测量都是可行的，并且更复杂的同位素校正不是缺点。
