Spherisorb s5

Purified tetrasaccharides were quantified using SAX-HPLC coupling with an analytical Spherisorb S5 (4 × 250 mm; Waters, Milford, MA) at 232 nm. Mobile phase A: 1.8 mM monobasic sodium phosphate, pH 3.0. Mobile phase B: 1.8 mM monobasic sodium phosphate, 1 M sodium perchlorate, pH 3.0. Flow rate: 0.45 mL/min. Gradient: T_0–35 min, 30%–65% B; T_35–50 min, 65%–85% B. ΔUA2S-GlcNS6S with different concentrations (0.02–0.2 mg/mL) were used as the standard. The linearity was based on amount of peak areas in HPLC.

Preparation of 3-O-sulfated tetrasaccharide standards was described previously [23 (link)]. Briefly, 400 mg heparin sample was dissolved in 50 mM sodium phosphate buffer (pH 7.4) and was exhaustively digested by heparin lyase 2 (35 IU, activity against heparin). Buffer salts and disaccharides within the product mixture were removed by Bio-Rad P10 column (100 × 50 cm) eluted at 1.2 mL/min with 0.2 M NaCl. The remaining tetrasaccharides mixture was desalted on a Bio-Rad P2 column (100 × 20 cm) and lyophilized. The resulting mixture was fractionated on a semi-preparative strong anion exchange (SAX)-high performance liquid chromatography (HPLC) column Spherisorb S5 (20 × 250 mm Waters, Milford, MA, USA). A gradient of solution A (water, pH 3.5, adjusted with HCl) for 2 min and followed by a linear gradient 0–60% solution B (2.0 M NaCl, pH 3.5, adjusted with HCl) from 2 to 60 min was used at flow rate of 4.0 mL/min with absorbance detected at 232 nm. Individual peaks were desalted on a Bio- Rad P2 column and were further purified by repeated separation on the same SAX-HPLC column. The purified tetrasaccharide standards were quantified by carbazole analysis described previously [31 (link)].