Abstract：
Two new analytical methods, applying absolute 1H qNMR, were developed to monitor product yield and quantify unreacted carbohydrate and fatty acid reactants, in the synthesis of carbohydrate fatty acid esters (CFAE). These methods provide a mass balance of the crude reaction mixtures and diversify the analytical screening and quantitation approaches available within the synthesis of these molecules. Both methods were validated for the model reaction of methyl α-d-glucopyranoside (MAG) and lauric acid (LA) to form the mono ester product, methyl 6-O-dodecanoyl-α-d-glucopyranoside. Analysis in CD3OD by 1H qNMR, with fumaric acid (FA) as an internal standard (IS), allowed monitoring of all reaction components. Alternatively, using CDCl3 and (E)-stilbene as IS enabled the analysis of CFAE and fatty acid. Parameters calculated for method validation included specificity and selectivity, linearity, accuracy, intermediate precision, limit of detection (LOD), limit of quantification (LOQ) and robustness. Both methods provided excellent linearity with R2 > 0.997. The accuracy, precision, and robustness of the method in CD3OD was <2 % uncertainty making it suitable for complete reaction analysis. The method completed in CDCl3 resulted in accuracy, intermediate precision, and robustness of <5 %, except for accuracy in the lowest levels of concentration (>5 %). For all related analytes in the CD3OD and CDCl3 methods, the LOD and LOQ were determined to ensure applicability for the intended use in the assessment of reaction crude composition. Finally, the system suitability was assessed in a scaled lipase catalysed CFAE synthetic reaction. The determined qNMR product yields were verified against isolated purified product yields with <5 % uncertainty.
摘要：
两种新的分析方法，应用绝对1HqNMR，被开发用于监测产品收率和量化未反应的碳水化合物和脂肪酸反应物，在碳水化合物脂肪酸酯（CFAE）的合成中。这些方法提供了粗反应混合物的质量平衡,并且使这些分子的合成中可用的分析筛选和定量方法多样化。两种方法都验证了甲基α-d-吡喃葡萄糖苷（MAG）和月桂酸（LA）形成单酯产物的模型反应，甲基6-O-十二烷酰基-α-d-吡喃葡萄糖苷。通过1HqNMR在CD3OD中分析，以富马酸（FA）为内标（IS），允许监测所有反应组分。或者，使用CDCl3和(E)-二苯乙烯能够分析CFAE和脂肪酸。方法验证计算的参数包括特异性和选择性，线性度准确度,中间精度,检测限（LOD），量化限(LOQ)和稳健性。两种方法均提供优异的线性,R2>0.997。准确性,精度,并且该方法在CD3OD中的鲁棒性<2％不确定度，使其适用于完整的反应分析。在CDCl3中完成的方法导致了准确性，中间精度,和<5%的鲁棒性，除了在最低水平的浓度（>5％）的准确性。对于CD3OD和CDCl3方法中的所有相关分析物，确定LOD和LOQ以确保反应粗组合物评估中预期用途的适用性。最后,在缩放脂肪酶催化的CFAE合成反应中评估了系统的适用性.确定的qNMR产物产率针对分离的纯化产物产率进行验证,不确定度<5%。
