Acquity beh c18 column

The LC‐MS analysis of isoflavones was performed as described previously by Wyspiańska et al. (2017). Isoflavones identification was performed on an Acquity ultraperformance liquid chromatography (UPLC) system, coupled with a quadrupole time‐of‐flight (Q‐TOF) MS instrument (UPLC/Synapt Q‐TOF MS; Waters Corp., Milford, MA, USA), with an electrospray ionization (ESI) source. Separation was achieved on the Acquity TM BEH C18 column (100 × 2.1 mm i.d., 1.7 μm; Waters). The mobile phase was a mixture of 4.5% v/v aq. formic acid (A) and acetonitrile (B). The gradient program was as follows: initial conditions, 1% B; 12 min, 25% B; 12.5 min, 100% B; 13.5 min, 1% B (return to initial conditions). The flow rate was 0.45 ml/min, and the injection volume was 5 ml. The column was operated at 30°C. The major operating parameters for the Q‐TOF MS were consistent with the description of Wyspiańska et al. (2017). The run was monitored at 254 nm.

The content of tanshinones ⅡA was determined using an ultra-high-performance liquid chromatography (UPLC) analysis [55 (link)]. Ten independent seedlings samples with the same growth were collected every five days after 15 days of growth. The roots were rinsed with running water, divided into three portions (three repetitions), and then dried to a constant weight in an oven at 60 °C. Then, 0.1 g of powder was extracted with 10 mL of 80% methanol under ultrasound for 30 min at 30 °C. Hereafter, the sample was filtered through a 0.22 µm microporous membrane filter. The Waters UPLC system (Waters, MA, USA) was used to determine the content of tanshinone ⅡA. Chromatographic separations were performed using the Waters^® ACQUITYTM BEH C₁₈ column (3.0 × 150 mm, 1.7  µm). Gradient elution was performed using a mobile phase of acetonitrile (A) H₂O containing 0.05% phosphoric acid (B), included 20%A (0–5 min), 20–30% A (5–10 min), 30–60% A (10–15 min), 60–70% A (15–20 min), 70–80% A (20–25 min), and 80–100% A (25–30 min). The sample injection volume was set at 4 µL, and the flow rate was 0.5 mL/min. The tanshinone ⅡA was detected using 280 nm PDA wavelengths.

Briefly, MHCC-97H cells cultured to around 80% confluence were washed in pre-cold PBS 3 times, quenched in 1 mL cold (-80 °C) 80% methanol (methanol/water, v/v) and were detached from the culture dish using a cell scraper. Quenched cells were centrifuged at 12,000 g for 15 min at 4 °C, and 0.8 mL supernatant was dried under nitrogen gas and dissolved in 100 μL aqueous acetonitrile water. The mixture was centrifuged at 12,000 g for 15 min and analyzed by LC-MS within 24 h. Sample separation and analysis were performed on a Waters Acquity TM BEH C18 column (2.1 mm×50 mm, particle size of 1.7 μm) using a gradient of buffer A [10 mM tributylamine, 15 mM acetic acid, 3% (v/v) methanol in water] and buffer B (methanol) and using multiple reaction monitoring (MRM) transitions.

ACQUITY^TM Ultra Performance Liquid Chromatography (UPLC) system equipped with ACQUITY^TM UPLC Photodiode Array Detector (PDA; Waters Corp, Milford, MA), and ACQUITY^TM BEH C₁₈ column (1.7 μm, 2.1 × 100) were used for UPLC analysis. In addition, Empower Chromatography Data software (Waters Corp, Milford, MA) was used to analyze the results. The sample was extracted with ultrasonicator (Branson Ultrasonics, Danbury, CT). The reagents, methanol (Junsei Chemical Co. Ltd., Tokyo, Japan), acetonitrile (BAKER, Centre Valley, PA), and water (tertiary distilled water) were all HPLC grade. The standard preparations were purchased from Extrasynthese (Genay Cedex, France) or Sigma-Aldrich (St. Louis, MO).

The method was described previously [27 (link)]. Identification of compounds was performed on the Acquity ultra-performance liquid chromatography (UPLC) system, coupled with a quadrupole-time of flight (q-TOF) MS instrument (UPLC/Synapt q-TOF MS, Waters Corp., Milford, MA, USA), with an electrospray ionization (ESI) source. Separation was achieved on the Acquity TM BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm; Waters). The mobile phase was a mixture of 4.5% aq. formic acid v/v (A) and acetonitrile (B). The gradient program was as follows: initial conditions—1% B in A, 12 min—25% B in A, 12.5 min—100% B, 13.5 min—1% B in A. The flow rate was 0.45 mL/min and the injection volume was 5 µL. The column was operated at 30 °C. UV-vis absorption spectra were recorded on-line during UPLC analysis, and the spectral measurements were made in the wavelength range of 200–600 nm, in steps of 2 nm. The major operating parameters for the q-TOF MS were set as follows: capillary voltage 2.0 kV, cone voltage 40 V, cone gas flow of 11 L/h, collision energy 28–30 eV, source temperature 100 °C, desolvation temperature 250 °C, collision gas, argon; desolvation gas (nitrogen) flow rate, 600 L/h; data acquisition range, m/z 100–2000 Da; ionization mode, negative and positive. The data were collected with Mass-Lynx V 4.1 software. The runs were monitored at the wavelength of 254 nm.