HPLC analysis of ADP extract

ADP extract was characterized using HPLC on a Waters 515 HPLC Pump system (Milford, USA) equipped with a Waters 2998 PDA detector, a Waters column temperature controller, a pump control module coupled with an empower chromatography workstation. For chromatographic analysis, an XBridge C₁₈ Reverse Phase column (4.6 × 250 mm, 5 μm), with gradient elution as the mobile phase, was adopted. The mobile phase consisted of a mixture of water (Solvent A) and methanol-acetonitrile (Solvent B) which was applied as a gradient for 40 min. The standard β-D-glucan and ADP extract were dissolved in HPLC grade water and filtered through a 0.45 μm membrane filter and the injection volume was 10 μL. The flow rate was 1.0 mL/min and the column temperature was set at 30 °C. The gradient of mobile phase was as follows: 80% A, 20% B for 0–6 min; 70% A, 30% B for 6–12 min, 30% A, 70% B for 12–18 min; 20% A, 80% B for 18–25 min; 20% A, 80% B for 25–30 min; 80% A, 20% B for 30–35 min and 90% A, 10% B for 35–40 min. HPLC was monitored at 254 nm to provide real-time chromatograms of both standard and ADP extract.