Development of a rapid quantitative method of analysis of carbohydrate fatty acid ester reaction mixtures using ¹H qNMR

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.