Smrm pro builder

Lipid analysis was performed by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) using a targeted approach using predefined MRM transitions (Sciex sMRM Pro Builder, Framingham, MA, USA) and in-house chromatographic retention time windows. Plasma and serum samples (10 μL) were placed in a 96-well plate, to which 90 μL extraction solvent (propanol-2-ol) containing stable-isotope-labelled internal standards (Lipidyzer^TM Internal Standards Kit from Sciex (Framingham, MA, USA), SPLASH LipidoMIX^TM, Lyso PI 17:1, Lyso PG 17:1, and Lyso PS 17:1 (Avanti Polar Lipids, Alabaster, AL, USA)) was added. Samples were shaken for 10 min and stored at −20 °C for 20 min before being centrifuged at 3900× g for 15 min. Supernatant was then transferred to a 96-well plate for analysis. For quality control (QC), an independent pooled sample underwent repeat extractions and was injected onto the system at frequent intervals throughout the run. The analytical platform is described in detail in the Supplementary Materials.

Lipidomic analysis of the plasma sample extracts was performed using a targeted tandem mass spectrometry (MS/MS) approach using predefined multiple reaction monitoring (MRM) transitions (Sciex sMRM Pro Builder, Framingham, MA, USA) and in-house defined chromatographic retention time windows.
The analytical system consisted of a Sciex ExionLC ^TM coupled to a Triple Quadrupole Linear Ion Trap (QTRAP 6500+) (SCIEX, Concord, ON, Canada). Lipid metabolites were chromatographically separated using a Waters Acquity BEH C18 reverse phase column (1.7 µm, 100 × 2.1 mm particle size; Waters Corp., Milford, MA, USA), maintained at 60 °C.
The mobile phase consisted of water, acetonitrile and isopropanol (all Optima grade), which were all purchased from Thermo Fisher Scientific (Malaga, Western Australia). Mobile phase composition and gradients are described in detail in the Supplementary Materials, along with additional instrument settings. Specific MS settings, MRM transitions and chromatographic retention times for the lipids of interest are reported in the results section and Tables S2–S4 are included in Table S4.