Lipidomic Analysis of Serum Samples

Lipidomic analysis was performed according to Simcox et al^{43 (link),44 (link)}; briefly, 40μl aliquots of sera were combined with 250μls PBS and 225μls ice-cold MeOH containing internal standards (Avanti Splash cat#3307–07), and homogenized. Samples were then mixed with 750μls of ice-cold MTBE (methyl tert-butyl ether), rehomogenized, and separated by centrifugation (17,000g for 5 mins/4°C). The upper phase was transferred to a new tube, lyophilized and resuspended in 150 μls of isopropanol. Lipids were analyzed by UHPLC/MS/MS in positive and negative ion modes, at a dilution suitable to eliminate saturation issues. Extracts were separated on an Agilent 1260 Infinity II UHPLC system using an Acquity BEH C18 column (Waters 186009453; 1.7 μm 2.1 × 100 mm) maintained at 50 C with VanGuard BEH C18 precolumn (Waters 18003975), using the chromatography gradients described by Jain et al. The UHPLC system was connected to an Agilent 6546 Q-TOF MS dual AJS ESI mass spectrometer and run in both positive and negative modes as described. Samples were injected in a random order and scanned between 100 and 1,500 m/z. Tandem MS was performed at a fixed collision energy of 25 V. The injection volume was 2 μl for positive mode and 5 μl for negative mode.