Samples were analyzed on an Ultra-Performance Liquid Chromatography (UPLC) system coupled to an iFunnel quadrupole time of flight (QTOF) Agilent 6550 spectrometer (Agilent Technologies, CA, USA). The chromatographic separation of lipids was carried out using an Acquity UPLC C18 CSH chromatographic column (100 × 2.1 mm, 1.8 µm) from Waters (Wexford, Milford, MA, USA). The UPLC-MS method employed was previously described by Alcoriza et al.20 (link). Briefly, for ESI(+) mode, the mobile phases consisted of (A) 10 mM ammonium formate in 60:40 (v/v) acetonitrile:water and (B) 10 mM ammonium formate in 90:10 (v/v) isopropanol:acetonitrile and a flow rate of 0.4 ml/min; for ESI(−) mode, ammonium acetate was used as modifier, and the flow rate employed was 0.6 ml/min. Autosampler and column temperatures were set to 4 °C and 65 °C, respectively, and the injection volume was 5 µl.
Samples and QC were acquired using Full scan MS data from 50 to 1700 m/z with a scan frequency of 6 Hz. QC were also acquired using dependent data acquisition (DDA), by Auto MS/MS mode, and data-independent acquisition (DIA), by using the all-ion fragmentation mode, both using 0 and 40 V as collision energies.
Samples and QC were acquired using Full scan MS data from 50 to 1700 m/z with a scan frequency of 6 Hz. QC were also acquired using dependent data acquisition (DDA), by Auto MS/MS mode, and data-independent acquisition (DIA), by using the all-ion fragmentation mode, both using 0 and 40 V as collision energies.