Van guard beh

UHPLC–QTOF-MS analysis was performed on an Agilent 6540 ultra-high definition accurate mass quadrupole time-of-flight spec-trometer with UHPLC (UHPLC–QTOF-MS, Agilent Technologies, U.S.A.). A UPLC C₁₈ analytical column (2.1 mm × 100 mm, I.D. 1.7 μm, ACQUITY UPLC®BEH, Waters, U.S.A.) was used for separation, coupled with a C₁₈ pre-column (2.1 mm × 5 mm, I.D. 1.7 μm, Van-GuardTM BEH, Waters, U.S.A.) at room temperature of 20 °C. The mobile phase was a mixture of water (A) and acetonitrile (B), both containing 0.1% formic acid, with an optimized linear gradient elution as follows: 0–5 min, 10–35% B; 5–25 min, 35–55% B; 25–28 min, 55–85% B; 28–30 min, 85–100% B. The injection volume was 4 μL. The flow rate was set at 0.35 mL/min. The mass spectra were acquired in negative mode by scanning from 100 to 1700 in mass to charge ratio (m/z). The MS analysis was performed under the following operation parameters: dry gas temperature 300 °C, dry gas (N₂) flow rate 5 L/min, nebulizer pressure 30 psi, Vcap 3000, nozzle voltage 500 V, and fragmentor voltage 200 V.

UHPLC-QTOF-MS was conducted on an Agilent 1290 ultra-high definition accurate mass QTOF spectrometer with UHPLC (Agilent Technologies, AB Sciex, CA, USA). A UPLC C18-column (2.1 mm × 100 mm, ID 1.7 μm, ACQUITY UPLC^® BEH; Waters, Milford, MA, USA) was used for separation, together with a C18-pre-column (2.1 mm × 5 mm, ID 1.7 μm, VanGuardTM BEH; Waters) at room temperature (20 °C). The mobile phase consisted of 0.1% formic-acid–water (A) and 0.1% formic-acid–acetonitrile (B), with the following optimized linear gradient elution: 0–3.5 min, 5% B; 3.5–6 min, 1% B; 6–12 min, 30% B; 12–12.5 min, 70% B; 12.5–22 min, 100% B. The injection volume was 5 μL and the flow rate was of 400 μL/min. Mass spectra were acquired in positive mode and negative mode. Data analysis was performed using Progenesis QI. Retention time correction, peak identification, peak extraction, peak integration, and peak alignment were optimized by the software. The corresponding Chinese medicine metabolism library was also queried to identify compounds. Then, matching between the self-built secondary mass spectrometry database and the corresponding fragmentation regularity as used to identify the peaks based on MS/MS data.