Polyamine 2 column

Disaccharide compositions of various GAGs were analyzed by high HPLC as described previously (21 (link)). Briefly, to determine the disaccharide composition of commercial Hep, HS, or Hep/HS in the GAGs extracted from various cells or organs the GAG preparations obtained above were individually digested with by Hepases (I, II, and III) followed by labeling with 2-aminobenzamide. The labeled samples were analyzed by anion exchange HPLC on a YMC-Pack Polyamine II column (YMC-Pack) eluted with a linear gradient from 16 mM to 550 mM NaH₂PO₄ over 60 min at a flow rate of 1.0 ml/min at room temperature using a fluorescence detector with excitation and emission wavelengths of 330 and 420 nm, respectively. In the case of CS/DS disaccharide assay, CS/DS sample was digested with chondroitinase ABC (CSase ABC) followed by 2-aminobenzamide labeling and analyzed by anion exchange HPLC on a YMC Pack PA-G column as described above. The percentage of each component was calculated based on its peak area.

The depolymerized oligosaccharide mixture was quantitatively analyzed by HPLC on an Agilent 1260 instrument equipped with YMC-Pack Polyamine II column (4 mm × 250 mm, S-5 μm, 12 nm) and UV detector at 30°C. The flow rate of mobile phase (acetonitrile: 100 mM NH₄H₂PO₄ = 1:9, v/v) was 0.5 ml/min.
The pure chondroitin disaccharide (CH2), tetrasaccharide (CH4), hexasaccharide (CH6), octasaccharide (CH8) and decasaccharide (CH10) (0.25, 0.5, 1.0, 1.5, and 2.0 g/L) were used to create the standard curves by in-house standards made of the relationship between oligosaccharide peak area with mass concentration by HPLC. The conversion rate of oligosaccharides refers to the mass concentration percentage of oligosaccharides converted from large M_w chondroitin substrate.