Methanol toluene

Lipids from whole-worm lysates were extracted using a biphasic separation with methyl tert-butyl ether, methanol and water^{149 (link)}. LC–MS/MS grade reagents were used for lipidomics. Briefly, 298 μl of ice-cold methanol and 2 μl of internal standard (equiSPLASH, Avanti Polar Lipids, 330731) were added to 50 μl of worm lysate. The mixture was vortexed for 20 s and 1,000 μl of ice-cold methyl tert-butyl ether was added. The mixture was incubated under agitation for 30 min at 4 °C. After the addition of 250 μl water, the samples were vortexed for 1 min and centrifuged at 14,000g for 10 min at room temperature. The upper phase containing the lipids was collected and dried under nitrogen. The dry extracts were reconstituted with 300 μl of 9:1 methanol:toluene (Fisher Scientific) with 10 mM ammonium acetate (Sigma Aldrich) and centrifuged at 14,000g for 5 min. Water extracted using the same protocol was used as a blank control. Samples were randomized during lipid extraction.