Uhplc ms

THSWD was diluted 10 times with double-distilled water. The reference substances of gallic acid, ferulic acid, paeoniflorin, hydroxysafflor yellow A, amygdalin, and ligustilide were accurately weighed and dissolved in methanol to make the mass concentrations respectively 0.2037, 0.02625, 0.3159, 0.0565, 0.1287, 0.5000 mg/mL single reference solution. And the single reference solution was stored at − 20 ℃.
Samples were filtered with a 0.22 μm membrane before UHPLC-MS (Thermo Fisher, United States) analysis. Analytes were separated using a SunFire-C18 column (3.0 mm×150 mm, 3.5 μm) with a column temperature of 25℃ and a flow rate of 0.2 mL/min. Mobile phase B was acetonitrile, and the mobile phase A was water containing 0.5% formic acid. A gradient elution program was applied (0–10 min, 5–7% B; 10–35 min, 17–22% B; 35–40 min, 22–65% B; 40–45 min, 65–80% B; 45–50 min, 80% B). The detection wavelengths are, respectively, 210, 230, 328, and 403 nm.

We obtained an OFF1 of 4.6 g. The fraction OFF1 was selected and identified by ultra-high-performance liquid chromatography (thermoultimate 3000) coupled to mass spectrometry (UHPLC–MS, Thermo Scientific, Pleasanton, CA, USA) according to our previous report [23 (link)]. The chromatographic conditions were as follows: Hypersil GOLD aQ C18 (2.1 mm × 100 mm, 1.9 μm); column temperature: 40 °C; flow rate: 0.4 mL/min; mobile phase: acetonitrile (A) and water (B); gradient elution program: 2% A (0–2 min), 2–98% A (2–15 min). The mass spectrometric conditions were as follows: ion source: HESI-source (positive and negative ion mode); capillary temperature: 320 °C; auxiliary gas temperature: 350 °C; sheath gas: 40 arb; aux gas: 10 arb; spray voltage: 4.00 kV (+)/2.80 kV (−); resolution: 70,000 (full scan), 17,500 (dd-MS2); normalized collision energy: 20, 30, 40 Ev; scan range: 100–1500 m/z. We identified the main compounds in bioactive fraction by Predicted Compositions, mzCloud Search, MassList Search, mzVault Search and ChemSpider Search Library.

Cells used for the metabolomic analysis were cultured in a 10 cm dish until Huh7 reached 70% confluence, and the DFO was added to the medium to reach the desired concentration. Three days after DFO addition, adherent cells were detached and collected, washed 3 times with PBS, stored at −80 °C, and sent to Metabolon Inc. for further analysis (n = 3). Metabolomic and statistical analyses were carried out at Metabolon (Morrisville, NC, USA), following previously described methods [17 (link),18 (link)]. In summary, cellular pellets (5 × 10⁵ cells) were subjected to methanol extraction. The resulting extract was then subdivided into portions for analysis using ultrahigh performance liquid chromatography/mass spectrometry (UHPLC/MS,) (Thermo Scientific, Waltham, MA, USA) in positive, negative, or polar ion modes, as well as gas chromatography/mass spectrometry (GC/MS) (Thermo Scientific, Waltham, MA, USA). Identification of metabolites was accomplished by matching ion features against a reference library of chemical standards using an automated process, which was subsequently followed by visual inspection to ensure quality control. For statistical computations and data presentation, any absent values were treated as being below the detection thresholds, and these values were estimated using the lowest compound value available.