Absciex 4000 qtrap triple quadrupole mass spectrometer

Frozen plasma samples were thawed for nearly 20 min. Lipids were extracted using chloroform and methanol as previously described [18 (link),19 (link)]. Briefly, 10 μL of plasma was added to 200 μL of chloroform/methanol (2:1, v/v) along with 30 μL of internal lipid standards (ISTD) in Eppendorf tubes, vortexed for 10 min, and sonicated in a water bath at room temperature (RT) for 30 min. The mixture was allowed to settle for 20 min after which centrifuged at 20,000× g for 20 min at RT. The lipid-containing top layer was later transferred to a new Eppendorf tube and dried under a gentle stream of nitrogen gas (∼1 h). The dried samples were then resuspended in 50 μL of water-saturated 1-butanol and sonicated for 10 min, followed by adding 50 μL of 10 mM ammonium formate in methanol. Finally, the extract was spun (10,000× g for 10 min at RT), and the supernatant (80 µL) was transferred into glass vials with inserts. The samples were randomized prior to LC/MS analysis. The lipidomic profiling was performed using a Prominence chromatographic system (Shimadzu Corporation, Canby, OR, USA) coupled with an AbSciex 4000 QTRAP triple quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA). The entire LC/MS methodology for this work, including details of ISTD, quality control, and data processing, are described in detail in our previously published work [18 (link)].