Detection and identification of hyaluronan oligosaccharides derived from free-radical depolymerization via reverse-phase ion-pair HPLC/mass spectrometry

Hyaluronan (HA) degradation through free-radical depolymerization is associated with numerous pathological processes such as osteoarthritis and rheumatoid arthritis. However, the precise chemical structures of HA oligosaccharides (o-HAs) generated via this process remain largely unclear. In this study, we employed reverse-phase ion-pair high-performance liquid chromatography (HPLC) coupled with high-resolution mass spectrometry to investigate partially depolymerized HA after the Fenton reaction. Among the reaction products, four o-HA series with different degrees of polymerization that maintained their original structures were identified as the main products. Further purification using strong anion exchange HPLC revealed minor o-HAs products exhibiting at least nine mass differences following regular patterns relative to the original o-HAs. These differences corresponded to structural modifications derived from GlcA and GlcNAc that occurred at either or both the reducing and non-reducing ends of o-HAs. By focusing on 16 types of disaccharides, we elucidated the proposed structures of terminal residues, which were primarily characterized by carboxyl-rich structures derived from ring-opened sugar moieties. These modifications arise from the oxidative cleavage of glycosidic bonds or C–C bonds in sugar rings, driven by hydroxyl radical-initiated cascade reactions. This study provides novel insights into the free-radical depolymerization mechanism of HA and highlights the structural diversity of o-HAs.