State-of-the-art chromatographic and mass spectrometric techniques in heparin structural analysis

Heparin is the most extensively used anticoagulant in clinical practice. It is a highly sulfated, linear polysaccharide composed of repeating disaccharide units. As a member of the glycosaminoglycan (GAG) family, heparin's complex structure features significant molecular weight variability, diverse sugar residues, and variable sulfation patterns. Low molecular weight heparins (LMWHs), produced through chemical or enzymatic depolymerization, are distinguished by their reduced molecular weight and offer therapeutic advantages, including lower bleeding risks, reduced immunogenicity, and higher bioavailability following subcutaneous administration. The structural intricacy of heparin-based drugs presents major challenges for quality control, clinical safety, process optimization, and therapeutic expansion. Advanced analytical methods, particularly LC and MS, remain at the forefront of efforts to elucidate the detailed structures of these drugs. This review highlights recent progress in chromatographic and MS-based analysis techniques for heparin and its derivatives, including the application of computational algorithms for structural elucidation. The focus is on the analytical methodologies, their innovations, and limitations, while also exploring how machine learning and bioinformatics tools are shaping the future of heparin quality control and therapeutic application. This comprehensive review provides a reference point for researchers engaged in the structural analysis of heparin-based drugs and offers insights into the future development of novel analytical strategies for improving the safety and efficacy of these critical anticoagulants.