Acquity uplc cshtm c18 column

Cell samples were produced in the manner previously described (Zhu et al., 2022b (link)). Lipidomic analysis was conducted using LC-MS (QExactive Plus Orbitrap mass spectrometer, Thermo Scientific). Acetonitrile:MilliQ water (6:4 v/v) and isopropanol:acetonitrile (9:1 v/v) were used as solvents A and B, respectively; both solvents contained 10 mm ammonium acetate. Column chromatography was performed using the Waters ACQUITY UPLC CSHTM C18 column. The gradient profile was as follows: 32%–100% solvent B over 24 min, back to 32% solvent B and 6 min before the next injection, and equilibrate the column. LipidSearch program v4.1.16 (Thermo Scientific) was used to identify the type of lipids. A pool of all lipid extracts was prepared and used for quality control. SIMCA-P (Sweden) was used to import the raw MS data, and an orthogonal projection was performed for latent structures-discriminant analysis (OPLS-DA). The resulting heatmap plots were drawn using R software.

The UHPLC-Q-TOF-MS/MS system is comprised of an Agilent 1290 UHPLC instrument (Agilent Technologies Inc., Palo Alto, CA, USA) and Agilent 6520 Q-TOF mass spectrometer (Agilent Corporation, Santa Clara, CA, USA). Chromatographic separation was performed on a Waters ACQUITY UPLC^®®CSHTM C18 column (2.1 × 100 mm, 1.7 µm) at a temperature of 30 °C. The mobile phase consisted of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) at a flow rate of 0.2 mL/min, with a gradient elution program of 5–14% B at 0–5 min, 14–18% B at 5–12 min, 18–34% B at 12–14 min, 34–72% B at 14–22 min, 72–86% B at 22–23 min, 86–95% B at 23–31 min, and 95–95% B at 31–35 min. The injection volume for each sample was 5 μL.
The mass spectrometer was operated in both positive and negative modes with a scanning range of m/z 50–1500 and a scanning rate of 1 spectra/s. High resolution (4 GHz, High Res Mode) was used. The optimized instrumental parameters were as follows: capillary temperature, 350 °C; drying gas (N₂) flow rate, 8 L/min; nebulizer pressure, 25 psi; collision energy, 30 V; fragment voltage, 135 V.

A. rugosa was purchased from Kwangmyongdang Pharmaceutical Co. (Ulsan, Korea). A voucher specimen (no. 2-19-0365) was deposited in the Korean Herbarium of Standard Resources in the Korea Institute of Oriental Medicine (KIOM). A. rugosa (1.0 kg) was ultrasonicated in 70% EtOH (v/v) for 2 h. The extract was filtered through chromatography paper (46 × 57 cm) and evaporated in vacuo. The yield of the 70% EtOH extract of A. rugosa was 18.94% (w/w); the extract was stored at 4 °C. A. rugosa extract (2.5 mg/mL) was dissolved in 50% methanol and filtered through a 0.2-μm syringe filter. ACQUITY UPLC^® CSH^TM C18 column (2.1 × 100 mm, 1.7 μm, Waters Corporation, Milford, MA, USA) comprising a photodiode array detector (PDA eλ detector, sample manager with flow-through needle (FTN-H), and quaternary solvent manager (Waters Co.) was used. The mobile phase was 0.05% formic acid in distilled water (A) and acetonitrile (B). The UPLC was run in a gradient mode from 85% A→40% A for 9 min. The flow rate was 0.3 mL/min and injected volume was 2 μL at 35 °C (column temperature). The UV wavelength was monitored from 210 to 400 nm and detected at 330 nm.