Comprehensive Lipidomic Analysis by UHPLC-qTOF/MS

Samples were analysed using an Agilent UHPLC 1290 Infinity Series coupled to an Agilent qTOF/MS 6550 Series (Agilent Technologies, Santa Clara, CA, USA). The chromatographic separation consists of an elution with a ternary mobile phase containing water with 10 mM ammonium formate and 0.1% formic acid (solvent A), methanol (solvent B), and 2-propanol (solvent C). The stationary phase was a C18 column (Kinetex EVO C18 Column, 2.6 µm, 2.1 mm × 100 mm) that allows the sequential elution of the more hydrophobic lipids such as lysophospholipids, sphingomyelins, phospholipids, diglycerides, triglycerides, and cholesteryl esters, among others. The flow rate was 0.6 mL/min, the injection volume was 2 µL, and the column temperature was set to 60 °C. The gradient employed was 0–0.5 min, 55–45% A + 10% B; 0.5–1.5 min, 45–42.8% A + 10–9.5% B; 1.5–1.6 min, 42.8–34% A + 9.5–7.5% B; 1.6–5 min, 34–31.8% A + 7.5–7% B; 5–5.1 min, 31.8–18.6% A + 7–4% B; 5.1–7.5 min, 18.6–16.4% A + 4–3.5% B; 7.5–9 min, 16.4% A + 3.5% B; 9–9.5 min, 16.4–0% A + 3.5–0% B; 9.5–11.5 min, 0% A + 0% B; 11.5–11.6 min, 0–45% A + 0–10% B; and 24.75–29.25, 55–45% A + 10% B. The qTOF operated in positive electrospray ionisation mode (ESI+), and mass spectra were recorded between m/z 300–1700 at 3 spectra/s. The source conditions were 35 psi for nebuliser gas, 225 °C for gas temperature, 11 L/min for gas flow, 300 °C for sheath gas temperature, 12 L/min for sheath gas flow, 3500 V for capillary voltage, and 500 V for nozzle voltage.