6520 quadrupole time of flight

Untargeted metabolomics measurements were performed on an Agilent 6520 Quadrupole Time-of-Flight (Q-TOF) LC/MS. Mass spectrometry analysis was performed using electrospray ionization (ESI) in negative mode. The dual ESI source parameters were set as follows, the gas temperature was set at 250 °C with a drying gas flow of 12 l/min and the nebulizer pressure at 20 psi. The capillary voltage was set to 3500 V and the fragmentor voltage was set to 100 V. Separation of metabolites was conducted on a Luna 5 μm C5 100 Å, LC Column 100 × 4.6 mm (Phenomenex 00D-4043-E0) with normal phase chromatography. Mobile phases were as follows: Buffer A, 95:5 water/methanol; Buffer B, 60:35:5 isopropanol/methanol/water with 0.1% ammonium hydroxide in both Buffer A and B for negative ionization mode. For 10 min runs, the LC gradient started at 95% A with a flow rate of 0.6 ml/min from 0 to 1 min. The gradient was then increased linearly to 95% B at a flow rate of 0.6 ml/min from 1 to 8 min. From 8 to 10 min, the gradient was maintained at 95% A at a flow rate of 0.6 ml/min. For 30 min runs, the LC gradient started at 95% A with a flow rate of 0.6 ml/min from 0 to 3 min. The gradient was then increased linearly to 95% B at a flow rate of 0.6 ml/min from 3 to 25 min. From 25 to 30 min, the gradient was maintained at 95% A at a flow rate of 0.6 ml/min.

The lipid fraction positive mode MS conditions for the BALF and plasma samples were as follows: Agilent 6210 Time-of-Flight (TOF-MS) with dual ESI source, scan rate 2.03 spectra/second, mass range 60–1600 m/z, gas temperature 300 °C, gas flow 12.0 L/min, nebulizer 30 psi, skimmer 60 V, capillary voltage 4000 V, fragmentor 120 V, reference masses 121.050873 and 922.009798 (Agilent reference mix). The negative mode conditions were as follows: Agilent 6210 Time-of-Flight (TOF-MS) with dual ESI source, scan rate 2.02 spectra/second, mass range 60–1600 m/z, gas temperature 300 °C, gas flow 12.0 L/min, nebulizer 30 psi, skimmer 60 V, capillary voltage 4000 V, fragmentor 140 V, reference masses 112.985628 and 966.000725 (Agilent reference mix).
The aqueous fraction MS conditions for the BALF and plasma samples were as follows: Agilent 6520 Quadrupole Time-of-Flight (Q-TOF-MS) in positive ionization mode with ESI source, mass range 50–1700 m/z, scan rate 2.22 spectra/second, gas temperature 300 °C, gas flow 10.0 L/min, nebulizer 30 psi, skimmer 60 V, capillary voltage 4000 V, fragmentor 120 V, reference masses 121.050873 and 922.009798 (Agilent reference mix).

LPs were detected on an Agilent 1260 HPLC instrument using an Agilent ZORBAX SB-C18 column (5 µm, 4.6 mm × 150 mm), eluted with methanol and water as the mobile phase, with DAD detection at 220 nm and a flow rate of 1.0 mL/min. The sample injection volume was 10 μL. The column was equilibrated with 20% (v/v) methanol. The gradient conditions were as follows: (ddH₂O (A) and methanol (B)) 0–2 min, 40% B; 2–12 min, 60% B; 12–22 min, 80% B; 22–25 min, 100% B; and 25–30 min, 100% B. The post-run time was set at 5 min.
LC/MS analysis was performed on a rapid resolution liquid chromatography–quadrupole-time-of-flight mass spectrometer (1200RRLC-6520 Accurate Mass Q-TOF LC/MS, Agilent) with electrospray ionization (ESI) in positive mode. High-resolution mass spectral (HRMS) data were displayed as m/z on an Agilent 6520 Quadrupole Time-of-Flight LC/MS delivered in positive and negative modes.