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.
Targeted Lipidomics from Serum Samples
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.
Corresponding Organization : Beijing Sport University
Other organizations : Capital Institute of Pediatrics, Umeå University
Variable analysis
- Lipid extraction method using pre-chilled isopropanol (IPA) at 4 °C
- Addition of lipid internal standard
- Centrifugation at 10,300× g for 10 min at 4 °C
- Identification and quantitative analysis of various lipids
- Sample volume (100 µL serum)
- Use of pre-chilled IPA
- Vortexing (1 min, 10 min)
- Incubation at -20 °C for 10 min
- Incubation at 4 °C for 2 h
- Electron spray ionization (ESI) mode
- Waters iclass-Xevo TQ-S ultra-high-performance liquid chromatography–tandem mass spectrometry system
- Dwell time (3 ms)
- Source nitrogen temperature (120 °C), flow rate (150 L/h)
- Desolvation gas temperature (500 °C), flow rate (1000 L/h)
- Capillary voltage (2.8 kV in positive mode, 1.9 kV in negative mode)
- Autosampler temperature (4 °C)
- Column chamber temperature (45 °C)
- Lipid separation using Waters Acquity UPLC BEH Amide column (1.7 μm, 2.1 × 100 mm)
- Mobile-phase gradient (0.1–20% B for 2 min, 20–80% B for 3 min, 3 min re-equilibration, flow rate of 0.6 mL/min)
- Quantitation using respective internal standards
- 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)
- D7-monoacylglycerols (18:1)
- D7-phosphatidic acids (15:0/18:1)
- None specified
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