Direct Characterization of Polysaccharides with In-Source Derivatization and Tandem Mass Spectrometry

Abstract Polysaccharide structural characterization is challenging due to the complex heterogenous nature and the limitations of time-consuming sample pretreatment and analytical methods. This study introduces an ambient ionization mass spectrometry strategy enabling direct, high-throughput characterization of polysaccharides with online methylation with an excited-state molecular beam in a direct analysis in real time (DART) ion source. Rapid in situ methylation and concurrent in-source fragmentation of polysaccharides was achieved with the use of tetramethylammonium hydroxide (TMAH), generating characteristic methylated ion patterns resolved by high-resolution tandem mass spectrometry (MS/MS). Characteristic fragmentation patterns from MS/MS analysis of key precursor ions allowed for the differentiation of various polysaccharides, including isomers of identical monomeric units. The established workflow, validated for linearity (R2 = 0.9899− 0.9983), sensitivity (LODs: 0.05− 0.43 μg mL−1), and reproducibility (RSD <12%), was applied to complex food and plant matrices and enabled the identification of specific polymeric components. This work demonstrates that DART-MS/MS with online methylation provides a rapid, robust, and powerful platform for the direct structural characterization of polysaccharides in complex samples, circumventing the need for extensive hydrolysis, separation, or offline derivatization at the analysis step.