Synapt g2 si q tof

The UPLC-PDA-HR-MS analyses were performed on an Acquity UPLC I Class (Waters, Milford, MA, USA). The column was an Acquity UPLC HSS T3 (100 × 2.1 mm, 1.8 μm) fitted with a VanGuard cartridge (Waters). The column temperature was 30 °C and the eluents were: A, water + 0.1% formic acid and B, acetonitrile + 0.1% formic acid. Isocratic separation: 10% of eluent B at 0.350 mL/min. Injection volume: 2 µL of 250 μM solution. The detector was an Acquity UPLC PDA Detector and the wavelength selected for the analysis was 254 nm. A Synapt G2-Si QTof (Waters) High Resolution mass spectrometer equipped with a Zspray ESI-probe was used in ESI negative ionization mode. Optimized source parameters: capillary −2.0 kV, cone 40, source temperature 120 °C, desolvatation temperature 150 °C, desolvatation gas flow rate 600 L/h, and full scan range 50–800 m/z. The lock mass compound was leucine enkephalin. The software was MassLynx^TM v4.2 software (Waters).
NMR spectra were performed using an Avance NEO 400 spectrometer (Bruker, Billerica, MA, USA) equipped with a “BBI 400 MHz S1” probe with Z gradient.

Lipid mediators were examined by LCMS essentially as described previously (Quehenberger et al., 2011 (link); Tam et al., 2013 (link); Tam et al., 2020 (link)). Before lipid metabolite isolation by solid phase extraction (SPE), deuterated standards (Cayman Chemical) were added to 0.9 mL of BAL Methanol was evaporates and the samples reconstituted in a minimal volume of water/acetonitrile (60/40) containing 0.02% v/v acetic acid. Eicosanoids were separated using a Waters Acquity UPLC BEH 1.7 μm 2.1 × 50 mm column using a 4 minute gradient of 99.9% A/B to 75/25 A/B followed by washing and reconditioning. Solvent A is 50/50 water/acetonitrile containing 0.02% acetic acid and solvent B is 50/50 acetonitrile/isopropanol. Eicosanoids were analyzed by a Waters Synapt G2Si QTOF operated in negative-ionization mode via MS^e. Data analysis was performed using UNIFI 1.6 (Waters), MS-DIAL4 (Tsugawa et al., 2020 (link)), and Mzmine 2.53 (Pluskal et al., 2010 (link)).

LESA CID MS/MS was carried out using a SYNAPT G2-Si QToF (Waters) and the LESA Advion TriVersa NanoMate was used as the ionization source^{60 (link)}. A surface extraction solvent of 95:5 methanol:water was used for all experiments. LESA parameters used for analysis were: solvent volume 4 µl, solvent depth 1 mm up from the bottom of the reservoir, dispensed 2 µl, delayed 2 s after dispensing, aspirated 3.5 µl, repeated mix two times, delayed 2 s after aspirate. Negative ion nano-ESI was used for the MS/MS analysis with spraying parameters of 0.3 psi gas pressure and 1.4 kV for applied voltage. The SYNAPT G2-Si was operated in negative ion MS/MS mode for precursor ion at m/z 464. A range of collision energies were used with the data averaged.

Metabolite analysis was achieved using a Waters ACQUITY UPLCTM I-Class chromatography system (Waters Corp, Milford, MA, USA) equipped with ion mobility mass spectrometry detector. The separation was performed on a Thermo Scientific™ Syncronis™ C18 (100 mm×2.1 mm, 1.7 mum particle size, Thermo Fisher Scientific) and the injection volume was 5 μL at 25° C. A gradient elution profile was employed using mobile phase A (0.1% formic acid in acetonitrile) and mobile phase B (0.1% formic acid in water). The following gradient was utilized: 0 min, 5% A; 0.5 min, 5% A; 3 min, 40% A; 10.5 min, 95% A. The flow rate was set to 0.40 mL/min.
The mass spectrometry was carried out using a SYNAPT G2-Si Q TOF (Waters Corp, Wilmslow, UK) connected to the ACQUITY UPLC I-Class System via an ESI interface. The mass spectrometry analysis was performed in the positive mode and negative mode, with the following parameters: Capillary 1.5 kV, sample voltage 30 V, cone voltages 5 V, source temperature 120° C, desolvation temperature 500° C. desolvation gas flow rate 800 L/h, cone gas flow rate 50 L/h. The MS was set with 0.1 second of scan time to acquire in sensitivity mode. The accurate mass precursor and fragment ion data was obtained using the selected mass range of m/z 100-1200.