Cortecs uplc t3

The chromatography analysis was performed using Waters^® CORTECS^® UPLC^® T3 (1.6 µm, 2.1 mm × 100 mm) at 30°C. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid in water (B). The gradient elution with the following program was carried out as follows: 0–3 min, 0% A; 3–7 min, 0%–5% A; 7–30 min, 5%–13% A; 30–55 min, 13%–25% A; 55–67 min, 25%–40% A; 67–72 min, 40%–95% A; 72–75 min, 95% A; 75–75.1 min, 95%–0% A; 75.1–78 min, 0% A. The flow rate was set at 0.3 mL min⁻¹, the detection wavelength was 190–400 nm, and the injection volume was 2–5 μL. The MS detection was applied in an ESI-negative/positive ion mode.

To identify the constituents of the EEP extracts UPLC-Q-TOF-MS was conducted using standard compounds [55 (link)]. The UPLC apparatus used was an Agilent 1290 (Agilent Technologies, Inc., Santa Clara, CA, USA) with Agilent Q-TOF 6545 LC/MS (Agilent Technologies, Inc., Santa Clara, CA, USA). A Waters CORTECS UPLC T3 (2.1 × 100 mm, 1.6 µm). The mobile phase ratio and flow rates were acetonitrile (A) and a 0.1% aqueous solution of formic acid (B). The gradient program was set as follows: 0–3 min, 5% A and 95% B; 3–38 min, 5–25% A and 95–75% B; 38–45 min, 25–40% A and 75–60% B; 45–70 min, 40–55% A and 60–45% B; 70–75 min, 55–70% A and 45–30% B; 75–90 min, 70–85% A and 30–15% B; 90–95 min, 85–95% A and 15–5% B; 95–98 min, 95% A and5% B; 98–98.1 min, 95–5% A and 5–95% B; 98.1–100 min, 5% A and 95% B. Mass spectrometry detection mode by ESI-negative/positive ion mode, mass range 50–1500, gas temp (°C) was 320, drying gas (L/min) was 8, nebulizer (psi) was 35, sheath gas temp (°C) was 350, sheath gas flow (L/min) was 11, Vcap (V) was 4000, nozzle voltage (V) was 1000, Fragmentor (V) was 175, and Skimmer (V) was 65.

For the LC/MS analysis, an Q Exactive Orbitrap high resolution mass spectrometer equipped with a heated electrospray ionization (HESI) source, was coupled to a Thermo Dionex Ultimate 3000 UPLC system (consisting of an autosampler, a diode array detector, a column oven and a dual pump connected to an online degasser). The data were recorded and processed using Xcalibur, Metworks and Mass Frontier 6.0 software packages (Thermo Fisher Scientific).
UPLC chromatographic separations were executed on a Waters CORTECS UPLC T3 (2.1 × 100 mm, 1.6 μm) column thermostated at 40°C. The mobile phases consisted of water with 0.1% formic acid (A) and acetonitrile (B), and the gradient program was conducted as follows: 0–1 min (5% B), 1–20 min (5–95% B), 20–21 min (95–95% B), 21–21.1 min (95–5% B), and 21.1–22 min (5–5% B). The sample flow rate was 0.3 mL/min and the injection volume was 2 μL.
The MS conditions were as follows: alternate switching (−)/(+) ESI full scan mode, the capillary temperature was 300°C, auxiliary temperature was 250°C, positive spray voltage was set at +3.5 kV, negative spray voltage was set at −3.0 kV, shealth gas (N₂) flow was 35 Arb, aux gas flow rate was 10 Arb. Full MS scans were acquired in the range of m/z 100–1,500, the collision energy was set at 20, 30, 40 eV. The MS/MS experiments were set as data-dependent scans.

Waters H-Class UPLC system (Waters Technology Co., Ltd.) coupled with an AB Sciex Triple TOF^® 4,600 high-resolution mass spectrometer (SCIEX company) were used for UPLC-Q-TOF-MS analyses. The UPLC separation was performed on a Waters CORTECS^®UPLC^® T3 (2.1 × 100 mm, 1.6 μm). The mobile phases were acetonitrile (A) and distilled water containing 0.1% formic acid (B) at a flow rate of 0.3 ml/min. The column oven temperatures were set at 30°C, while the injection volumes for all samples were 2 μl. The mass spectrometer was operated in ESI-Negative/Positive ion mode. The Supplementary material shows gradient conditions and Mass parameters (Supplementary Tables 1, 2).