G1311a quatpump

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.

CHYS was dissolved in methanol and filtered through a 0.45 μm filter (Microgen, Laguna Hills, CA, USA) before high performance liquid chromatography (HPLC) analyses. The HPLC system consisted of Agilent G1311A QuatPump, G1313A Auto-Sampler, and Agilent G1315B diode array detector. HPLC analysis was performed using a Phenomenex Luna C18 column (4.6×250 mm, particle size 5 μm) with acetonitrile (as Solvent A): 0.5% phosphoric acid (as Solvent B) as mobile phase at a flow rate of 1.0 mL/min at the column temperature of 30°C. A linear gradient elution was applied from 5% of Solvent A starting from 0 to 10 min, 5–30% of Solvent A starting from 10 to 80 min, 30–100% of Solvent A starting from 80 to 120 min. Pure standards including protocatechuic acid (PA), chlorogenic acid (CA), calycosin 7-O-β-D-glucoside (CG), formononetin and dioscin were purchased from the National Institutes for Food and Drug Control (Beijing, China) and were used as external standards in the HPLC analysis. Identification of HPLC peak fractions was carried out by comparing retention times and UV spectra with the standards. Five major bioactive compounds in the three batches of CHYS included PA (0.424–0.434 μg/mg), CA (0.158–0.162 μg/mg), CG (1.702–1.738 μg/mg), formononetin (0.004–0.005 μg/mg), and dioscin (2.070–2.114 μg/mg) (Figure 1).