Targeted Lipidomics from Serum Samples

For targeted lipidomics, a semi-quantitative approach was used. Lipids were extracted from 100 µL serum samples using simple protein precipitation in pre-chilled isopropanol (IPA) at 4 °C. The samples were mixed with lipid internal standard, after which 500 µL of pre-chilled IPA was added, vortexed for 1 min, placed at −20 °C for 10 min, and vortexed again for 10 min. After 2 h at 4 °C, the mixture was centrifuged at 10,300× g for 10 min at 4 °C, and the supernatant was removed. All analyses were performed in electron spray ionization (ESI) mode using a Waters iclass-Xevo TQ-S ultra-high-performance liquid chromatography–tandem mass spectrometry system (Waters, Milford, MA, USA). The dwell time for each lipid species was 3 ms. The source nitrogen temperature was 120 °C, and the flow rate was 150 L/h. The desolvation gas temperature was 500 °C, and the flow rate was 1000 L/h. The capillary voltage was 2.8 kV in the positive mode and 1.9 kV in the negative mode. The autosampler operated at 4 °C and the column chamber at 45 °C during the analysis.
Lipid species were separated using a Waters Acquity UPLC BEH Amide column (1.7 μm, 2.1 × 100 mm). Solvent A was 95% acetonitrile containing 10 mM ammonium acetate, and solvent B was 50% acetonitrile containing 10 mM ammonium acetate. The mobile-phase gradient was 0.1–20% B for 2 min, followed by 20–80% B for 3 min and 3 min re-equilibration, with a flow rate of 0.6 mL/min. Mass spectrometry multiple-reaction monitoring (MRM) was established for the identification and quantitative analysis of various lipids. Individual lipids were quantitated relative to their respective internal standards, including d7-phosphatidylcholine (15:0/18:1), d7-phosphatidylethanolamine (15:0/18:1), d7-phosphatidylglycerol (15:0/18:1), d7-phosphatidylinositol (15:0/18:1), d7-lysophosphatidylcholines (15:0/18:1), d7-lysophosphatidylethanolamine (15:0/18:1), d7-diacylglycerols (15:0/18:1), d7- triacylglycerols (15:0/18:1), d9-sphingomyelin (18:1), d7-cholesteryl esters (18:1), and d7-monoacylglycerols (18:1) obtained from Avanti Polar Lipids, and d7-phosphatidic acids (15:0/18:1) from Sigma-Aldrich.