HILIC-based high-resolution mass spectrometry method for comprehensive characterization of hyaluronic acid hydrogel degradation products.

Hyaluronic acid (HA) hydrogels combine HA's inherent biocompatibility with tunable properties conferred by chemical crosslinking. However, precise characterization of hydrogel degradation kinetics is critical for safe and predictable clinical performance. The study presents an HILIC-HRMS profiling method for chemically crosslinked HA degradation products, including glucuronic acid, N-acetylglucosamine, the hyaluronic acid tetrasaccharide (HA4), hexasaccharide (HA6), and octasaccharide (HA8). Chromatographic separation was performed on a BEH Z-HILIC column using a 10 mM ammonium formate buffer and water-acetonitrile mobile phases, and two degradation protocols, acid hydrolysis and enzymatic digestion, were compared over time. Method demonstrated high accuracy, precision, linearity, and freedom from interference in methanol, enabling reliable profiling of HA fragments. Qualitative and quantitative comparative analyses revealed two distinct degradation mechanisms: acid hydrolysis rapidly produced monosaccharides and small oligomers, whereas hyaluronidase selectively generated even-numbered oligomers, consistent with enzyme-specific cleavage. Validated HILIC-HRMS approach enables robust analysis of HA degradation and supports quality control of HA-based biomaterials in biomedical applications.