Zorbax sb aq c18 column

The phytochemical constituents of CEE were analyzed using an Agilent 1600 series HPLC (Agilent Technologies, CA, United States), and compared to the reference compounds brazilin and protosappanin B (Chengdu Desite, Sichuan, China). The analytical column that was used was an Agilent Zorbax SB-Aq C₁₈ column (4.6 mm inner diameter × 250 mm length, 5 μm particle size), which was maintained at 25°C. The mobile phase consisted of water (A) and acetonitrile (B). The following gradient elution program was used: 0–30 min, 5–10% B; 30–45 min, 10–16% B; 45-60 min, 16–18%; 60–90 min 18–23%; 90–105 min, 23–30%; 105–120 min, 30–40%. The analysis was performed at a flow rate of 0.8 mL/min with the UV detection wavelength at 285 nm (Chu et al., 2013 (link)). CEE and the reference compounds (brazilin and protosappanin B) were dissolved in acetonitrile. After filtration through a 0.25-μm membrane, a 10-μL aliquot was injected into the HPLC for analysis. The identities of the resulting main chromatographic peaks were confirmed by comparing the retention times and UV spectra with those of the reference standards.

The analysis was performed using the Shimadzu HPLC system (Chiyoda-Ku, Kyoto, Japan). Analytes were separated on an Agilent ZORBAX SB-Aq C₁₈ column (250 mm × 4.6 mm, 5 μm, Santa Clara, CA, USA) equipped with an Agilent analytical guard column (12.5 mm × 4.6 mm, 5 μm, Santa Clara, CA, USA).
The gradient mobile phase system, consisting of ACN (A)-H₂O (B) and 0.05% phosphoric acid as modifier, was used to analyze the samples. The gradient elution method was as follows: 0–4.5 min: 3% A; 4.5–8 min: 3% A→15% A, 8–15 min: 15% A→30% A; 15–20 min: 30% A→30% A; 20–25 min: 3% A.

To determine the ginsenoside content, the ginseng extracts were diluted to 1 mg/mL using 100% MeOH [64 (link),65 (link),66 (link)]. Using a SIL-9A auto-injector, a volume of 10 L was injected into the HPLC system (Hitachi L-6200 pump, Tokyo, Japan) connected to a Sedex 75 ELSD (Sedere, Vitry-sur-Seine, France) (Shimadzu, Japan). All separations were performed using an Agilent Technologies (Palo Alto, CA, USA) Zorbax SB-Aq C18 column (4.6 mm 150 mm, 5 m particle size). The high-pressure liquid chromatographic (HPLC) conditions were as follows: solvent A, water; solvent B, acetonitrile. B 20–22% (0–5 min); 22–25% (5–7 min); 26–30% (27–30 min); 30–35% (30–40 min); 50–70% (45–60 min); 70–85% (60–61 min); 85–100% (61–90 min). Nebulizer for nitrogen gas was adjusted to 2.5 bar, and ELSD was set to a probe temperature of 75 °C. A total of 5 μg of each ginsenoside standard was injected for HPLC analysis [66 (link)].

Analysis of PD concentration was carried out using an Agilent 1200 series high-performance liquid chromatography (HPLC) system coupled with an Applied Biosystem API 4000 triple quadrupole mass spectrometer. Chromatographic separation was performed using an Agilent Zorbax SB-Aq C18 column (150 mm × 2.1 mm i.d., 3.5 μm). The mobile phase consisted of acetonitrile (A) and 0.1% formic acid in Milli Q water (B) with the following linear gradient elution program: 0.0–1.0 min 5% A; 1.0–5.0 min 5%–90% A; 5.0–7.0 min 90% A; 7.0–7.1 min 90%–5% A; 7.1–15 min 90% A.
The mass analysis was carried out under the negative electrospray ionization mode. The optimum conditions of multiple reaction monitoring (MRM) were carried out at the following parameters: ion spray voltage (IS), −4500 V; ion source gas (GS1 and GS2), 65 and 65 psi, respectively. The transitions of m/z 1223.9→469.3 was used for quantification, and of m/z 1223.9→681.5 was used for identification (Figure S2).

Qualitative analysis of recovered saponins was performed on an HPLC chromatographic system (Agilent, Palo Alto, CA, USA) coupled with an LTQ-Qrbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The chromatographic with a ZORBAX SB-Aq C₁₈ column (250 × 4.6 mm, 5 µm, Agilent, Palo Alto, CA, USA). The mobile phase were methyl alcohol (C) and 0.01% formic acid water (D); the flow rate was 0.4 mL/min. Gradient elution was conducted as follows: 0–30 min, 50–70% C; 30–60 min, 70–80% C; 60–70 min, 80–50% C. Injection volume was 10 µL and injection temperature was 30 °C. MS parameters were as follows: capillary temperature, 250 °C; auxiliary gas heater temperature, 200 °C; spray voltage, 1.5 kV; scanning range, 300–2000 m/z.