Lcms 9030

LC-QTOF/MS analysis was performed by Nexera X2 (Shimadzu) coupled with LCMS-9030 (Shimadzu). LC conditions were identical to that of LC-MS analysis. LCMS-9030 was operated under the following conditions; ionization, electrospray ionization; nebulizer gas flow, 2.0 ​mL ​min⁻¹; heating gas flow, 10 ​L/min; interface temperature, 300 ​°C; drying gas flow, 10 ​L/min; desolvent line temperature, 300 ​°C; heat block temperature, 400 ​°C; MS1 scan range, 70–1000; MS2 scan range, 70–350; precursor m/z, 339.1; Q1 resolution, low; collision energy, 35; collision energy spread, 17; collision gas, argon.

MS and MS/MS spectra were recorded on an LC/Q-TOF system consisting of a Nexera UHPLC and aquadrupole time-of-flight mass spectrometer (LCMS-9030,Shimadzu Corporation, Kyoto, Japan). LC conditions were as follows; column, Kinetex C8 (2.1 mmI.D. × 150 mm, 2.6 µm) Phenomenex, USA; solvent, isocratic, 30% of 20 mM ammonium formate-containing and 70% acetonitrile/isopropanol (1:1, v/v); flow rate, 0.4 mL/min; and column temperature, 40 °C. MS conditions were as follows: ionization mode, ESI positive/negative; capillary voltage, 4.5 kV (positive)/− 3.5 kV (negative); drying gas, 10 L/min; nebulizer gas, 2.0 L/min, heating gas, 10 L/min; interface temperature, 300 °C.

The NBT fraction was analyzed using an LCMS‐9030 (Shimadzu) equipped with a Shim‐pack GIST‐C18 column (2 μm, 2.1 mm × 100 mm). Water containing 0.1% formic acid (A) and acetonitrile (B) were used as the elution solvents. The elution program was the following: 0–2 min, 5% B; and 2–10 min, 5%–25% B. The flow rate was 0.2 μl/min, and the sample loading was 2 μl. A full MS scan was used with m/z 50–1000. The MS parameters of MS were set as follows: spray voltage, 3.3 kV; capillary temperature, 320°C; heater temperature, 350°C; auxiliary gas flow, 8.0 L/min; sheath gas flow rate, 32.0 L/min; sweep gas, 4.0 L/min; and S‐lens RF level, 50%.

¹H and ¹³C NMR data were obtained using the Avance Bruker 300 spectrophotometer (Bruker Biospin, Mannheim, Germany) operating at a frequency of 300 MHz for hydrogen and 75 MHz for carbon, respectively, and the spectra were measured in acetone-d6 (Merck Group, DE, Darmstadt, Germany). Mass data were obtained in an ultra-high-pressure liquid chromatography (UPLC) coupled with a quadrupole time-of-flight mass spectrometer detector (QTOF), Shimadzu (LCMS-9030) (Kioto, Japan) by direct injection. O-CH₃ and OH positions were obtained using UV-VIS 230 to 500 nm scanning (Jenway 6405 UV-VIS, Staffordshire, UK) with displacement reagents (AlCl₃, HCl, AcONa, MeONa, and H₃BO₃) (Merck Group, DE, Darmstadt, Germany).

Metabolites were analysed using a LC-MS-9030 mass spectrometer coupled with a Nexera-x2 ultra-performance liquid chromatography system (Shimadzu, Kyoto, Kyoto, Japan) as described by Abshirini et al. (22 (link)). Briefly, chromatographic separations of polar and non-polar metabolites were conducted by injecting 5 μL of samples onto Accucore^™ HILIC column, 2.1 mm × 100 mm, 2.6 μm particle size (Thermo Fisher Scientific, Waltham, MA, United States), and 2 μL of samples onto the CSH-C18 column, 2.1 × 100 mm, 1.7 μm particle size (Waters, Milford, MA, United States), respectively. The mobile phases used for chromatographic separations were 10 mM ammonium formate in water (solvent A) and 0.1% of formic acid in acetonitrile (solvent B) for polar metabolites and 10 mM of ammonium formate in water/acetonitrile/isopropanol (5:3:2 v/v) (solvent A) and 10 mM of ammonium formate in water/acetonitrile/isopropanol (1:9:90 v/v) (solvent B) for non-polar metabolites. The mass spectral detection for polar metabolites was performed in positive and negative ionisation modes. In contrast, for non-polar metabolites, mass spectral detection was only performed in positive ionisation mode, as it captures most lipids.

The dried residue of the lower phase was dissolved in 100 µL of chloroform containing 1 µL of Equisplash (Avanti polar lipids, Birmingham, AL, USA). After centrifugation at 6000 × g for 5 min, 50 µL of this supernatant was transferred to a vial. Lipids were analyzed using a Nexela X2 equipped with ACQUITY UPLC Peptide BEH C18 (50 mm, 2.1 mm, 1.7 µm, Waters, Milford, MA, USA) coupled to a quadrupole time-of-flight (QTOF)/MS (LCMS-9030, Shimadzu) operated in data-dependent acquisition mode as described previously^{35 (link),36 (link)}. MS-DIAL ver. 3.98 was used to identify lipid molecule species^{37 (link)}.