Lipidyzertm internal standards kit

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 was completed using an established method previously described [49 (link)], resulting in the quantification of 856 lipid species from 10 μL of rat plasma. Sample extraction was completed using a Biomek i5 sample automation system (Beckman Coulter, Mount Waverley, VIC Australia). To each 10 μL plasma sample, 90 μL of isopropanol (Fisher Scientific, Malaga, Western Australia) containing stable isotope-labelled internal standards (LipidyzerTM Internal Standards Kit from Sciex, Framingham, MA, USA, and SPLASH LipidoMIXTM, Lyso PI 17:1, Lyso PG 17:1, and Lyso PS 17:1, Avanti Polar Lipids, Alabaster, AL, USA) was added, and then mixed for 20 min. Samples were then centrifuged (3500× g for 10 min at 4 °C). Supernatant aliquots of 70 μL were then transferred into an Eppendorf 350 μL 96-well plate for LC-MS analysis. Samples were analysed within 24 h of preparation. QC samples were prepared using a commercial pooled human plasma sample (BioIVT, New York, NY, USA) and underwent replicate extraction (n = 5) using the method described. These replicate samples were periodically analysed throughout the analytical sequence.