NMR Spectroscopy in Heparin Research: Structural Characterization, Quality Control, and Contaminant Screening

Heparin, a highly sulfated glycosaminoglycan and widely used anticoagulant, exhibits remarkable structural heterogeneity due to variations in sulfation patterns, monosaccharide composition, and molecular weight. This complexity presents major challenges for structural characterization and quality control. Nuclear magnetic resonance (NMR) spectroscopy has become a key analytical tool because it provides comprehensive, non-destructive structural information from native samples. This review summarizes recent advances in NMR-based heparin analysis, including structural assignment, conformational characterization, quantitative compositional analysis, molecular weight determination, and detection of nitrogen-containing structural motifs. We further highlight the critical role of NMR in heparin quality control, including identification of adulterants, process-related impurities, species origin, and biosimilar comparability. The integration of NMR with chemometric methods, such as principal component analysis, partial least squares regression, and machine learning, has enabled high-throughput spectral fingerprinting, predictive modeling, and automated batch consistency evaluation. Moreover, combining NMR with chromatography, mass spectrometry, and electrophoresis provides a multidimensional framework linking structural characterization with regulatory requirements. Finally, current challenges and future perspectives, including sensitivity enhancement, analytical standardization, and real-time process monitoring, are discussed. These advances underscore the indispensable role of NMR in improving the structural characterization, quality assurance, and safety of heparin therapeutics.