Cosmosil 5c18 paq column

Monosaccharide composition analysis was performed as previously described (Wang et al., 2020 (link)). Briefly, WGP (200 g) was hydrolysed in anhydrous methanol solution (1 ml) containing hydrochloric acid in nitrogen. Then, the sample was dried and hydrolysed in 2 M trifluoroacetate acid. After dried, the sample was dissolved using 0.3 M sodium hydroxide and added an equal volume of 0.5 M PMP (1-phenyl-3-methyl-5-pyrazolone) with thoroughly blending using pipettor. Placed the mixture (0.2 ml) for 30 min at 70°C, added 0.1 ml hydrochloric acid and 0.7 ml dichloromethane for extraction. The aqueous phase was filtered via 0.22 μm organic membrane. Conversion of monosaccharides with PMP were detected via high-performance liquid chromatography (HPLC). The sample (10 μL) was injected into a 4.6 mm × 250 mm COSMOSIL 5C18-PAQ column (Nacalai Tesque, Shanghai branch, China) controlled by an LC-20AT system (Shimadzu, Shanghai branch, China). HPLC was performed using a mobile phase composed of 19.5% Acetonitrile and 80.5% 0.1 M PBS (pH 7.0) at flow rate of 1 ml/min. The absorbance values at wavelength 245 nm were compared with those of monosaccharide standards including arabinose, fucose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, rhamnose, and xylose to determine the monosaccharide composition of WGP.

Test substances were desalted using ultrafiltration via an ultrafiltration filter unit, and 20 μg aliquots were hydrolyzed with 2.5 mol/L trifluoroacetic acid at 100°C for 3 h. The samples were then cooled, dried, and reconstituted in L-rhamnose solution. Neutral monosaccharide was analyzed using a Shin-pack ISA-07/S2504 column (0.7 μm ID and 250 mm in length, Shimadzu) with a 50-min linear gradient of 0.1–0.4 mol/L potassium buffer (pH 8.0–9.0) by the Reducing Sugar Analysis System (Shimadzu). For amino sugar determination, test substances were hydrolyzed with 4 mol/L HCl at 100°C for 3 h, cooled, dried, and reconstituted in 0.02 mol/L HCl. An amino acid analyzer JLC-500/V2 (JEOL, Tokyo, Japan) was used for the analysis. For sialic acid analysis, test substances were treated with 0.1 mol/L NaOH at 37°C for 30 min, neutralized with HCl, and then reacted with 0.035 mol/L HCl at 70°C for 140 min to release sialic acids. The released sialic acids were fluorescently labeled with 1,2-diamino-4,5-methylenedioxybenzene at 50°C for 150 min. The solutions were analyzed by RP-HPLC using a COSMOSIL 5C18-PAQ column (4.6 mm ID and 150 mm in length, Nacalai Tesque, Kyoto, Japan) with a methanol/acetonitrile/water solvent system and fluorescence detection (Ex: 373 nm, Em: 448 nm). Quantification was performed relative to the appropriate monosaccharide standard curve.