1290 series pump

Reversed phase samples from the lipid fraction were randomized in the worklist and run randomly in triplicate using an Agilent 1290 series pump with an Agilent Zorbax Rapid Resolution HD (RRHD) SB-C18, 1.8 micron (2.1 × 100 mm) analytical column and an Agilent Zorbax SB-C18, 1.8 micron (2.1 × 5 mm) guard column. The autosampler tray temperature was set at 4 °C, column temperature was set at 60 °C, and the sample injection volume was 8 µL for BAL and 4 µL for plasma. The flow rate was 0.7 mL/min with the following mobile phases: mobile phase A was water with 0.1% formic acid, and mobile phase B was 60:36:4 isopropyl alcohol:acetonitrile:water with 0.1% formic acid. Gradient elution was as follows: 0–0.5 min 30–70% B, 0.5–7.42 min 70–100% B, 7.42–10.4 min 100% B, 10.4–10.5 min 100–30% B, 10.5–15.1 min 30% B. The lipid fraction MS conditions were as follows: Agilent 6545 Quadrupole Time-of-Flight mass spectrometer (QTOF-MS) in positive ionization mode with dual AJS ESI source, mass range 50–1700 m/z, scan rate 2.00, gas temperature 300 °C, gas flow 12.0 L/min, nebulizer 35 psi, sheath gas temperature 275 °C, skimmer 65 V, capillary voltage 3500 V, fragmentor 120 V, reference masses 121.050873 and 922.009798 (Agilent reference mix).

All samples were randomized prior to instrument analysis for both the hydrophobic and hydrophilic fraction. The hydrophobic fraction was analyzed using an Agilent Zorbax Rapid Resolution HD SB-C18, 1.8 micron, 2.1 × 100 mm analytical column on an Agilent 1290 series pump. Injection volume was 4 µL. HPLC flow rate was 0.7 mL/min with the following mobile phases: mobile phase A was water with 0.1% formic acid and mobile phase B was 60:36:4 isopropyl alcohol:acetonitrile:water with 0.1% formic acid. The gradient was as follows for positive mode: 0.0–1.0 min 30–70% B, 1.0–7.92 min 70–100% B, 7.92–10.4 min 100% B, 10.4–10.5 min 100–30% B, 10.5–15.1 min 30% B. Autosampler tray temperature was set to 4 °C and column temperature was set to 60 °C.
The hydrophilic fraction was analyzed on an Agilent 1200 series pump using a Phenomenex Luna NH₂ HILIC, 5 µm, 2 × 250 mm analytical column. 2 µL was injected with a flow rate of 0.5 mL/min. Mobile phase A was 50% ACN with 20 mM ammonium acetate pH 9.45 and mobile phase B was 100% ACN. Gradient elution was as follows: 0–4 min 90% B, 4.0–15.0 min 90–28% B, 15.0–15.01 min 28–0% B, 15.01–20.0 min 0% B, 20.0–20.01 min 0–90% B, 20.01–30.0 min 90% B. Autosampler tray temperature was set to 4 °C and column temperature was set to 20 °C.