Analytical Strategies for Natural Mono- and Disaccharide Isomer Differentiation: Techniques and Applications

The precise structural differentiation of natural mono- and disaccharide isomers is fundamental for understanding carbohydrate bioactivity. However, this task remains challenging due to their minimal structural variances, high polarity, and lack of chromophores. This review traces the evolution of analytical technologies designed to overcome these hurdles. Early derivatization - GC-MS methods provided the foundation for isomer separation. Modern liquid chromatography-mass spectrometry (LC-MS), particularly with advanced chiral columns, now delivers high-resolution isomer profiling. Ion mobility spectrometry (IM-MS) further distinguishes conformers by their collision cross-section (CCS) values. Recent breakthroughs in non-derivatization strategies like UHPLC, coupled with the definitive structural validation offered by 2D-NMR, have revolutionized the field. We critically evaluate these methods' detection limits and throughput for practical applications in food chemistry, clinical glycomics, and pharmaceutical analysis. Looking forward, emerging directions such as AI-assisted spectral interpretation and integrated microfluidic systems promise to propel glycan analysis toward rapid, precise, and intelligent diagnostics. This review provides a practical roadmap for selecting and advancing analytical techniques for real-world isomer characterization.