G1329a als

The HPLC analysis was performed on an Agilent 1200 system equipped with a G1311A QuatPump (Agilent Technologies, Inc., Santa Clara, CA, USA), a G1322A degasser, a G1315D diode array detector (DAD), and a G1329A ALS with a 20 μL loop. An Hypersil ODS-C18 (250 mm × 4.6 mm i.d., 5 μm, Agilent Technologies, Inc., Santa Clara, CA, USA) column was used. The mobile phase (phase A) was 0.1% formic acid solution (v/v)—phase B was methanol. A gradient program was used as follows: 0–10 min, 20–30% B; 10–15 min, 30% B; 15–35 min, 30–95% B; 35–45 min, 95% B. The flow rate was 0.8 mL/min, and the injection volume was 10 μL. The chromatograms were recorded at 254 nm.
In addition, stock standard solutions of PUE, 3-MPR and PRX were prepared by dissolving in the analytical grade methanol for quantification.

The monosaccharide compositions of GLP-1 and GLP-2 were analyzed by high-performance liquid chromatography (HPLC), as described in a previous study [23 (link)], but with some modifications. A polysaccharide sample (2 mg) was hydrolyzed with 2 M trifluoroacetic acid (1 mL) at 110 °C for 6 h, followed by derivatization with 0.5 M PMP. The PMP derivatives were analyzed on an Agilent 1200 Series HPLC system (G1322A Degasser, G1311A Quat Pump, G1329A ALS, G1315D DAD, Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with an Eclipse XDB-C18 column (250 mm × 4.6 mm × 5 µm, Agilent, Santa Clara, CA, USA) at 30 °C. The detection wavelength was set at 250 nm, and the flow rate was 0.8 mL/min. The mobile phase was a mixture of phosphate-buffered saline (0.1 M, pH 6.5) and acetonitrile (84:16, v/v). Rhamnose, ribose, fucose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid, and galacturonic acid were used as standards.

HPLC analysis was performed on an Agilent 1200 system including a G1311A QuatPump, a G1322A degasser, a G1315D diode array detector (DAD), a G1329A ALS with a 20 μL loop. In addition, the HPLC column used was Hypersil ODS-C18 (250mm × 4.6mm i.d., 5μm, Agilent). The mobile phase consisted of water containing 0.1% formic acid (phase A) and acetonitrile (phase B). The gradient program was as follows: 0–12 min, 10–22% B, 12–38 min, 22–30% B, 38–50 min, 30–45% B, 50–55 min, 45–90% B, 55–65min, 90% B. The flow rate was 0.8 mL/min, and the wavelengths were 254 nm (for TIIA and CYT) and 280 nm (for SAB).
Calibration curves were established for SAB, TIIA and CYT by plotting the nominal concentrations of standard solutions versus peak areas. The linear ranges, linear regression equations and related details are listed in Table S1 in Supporting Information.