1290 hplc

These details have been described fully in Sung et al. [1 (link)]. However, briefly, plasma samples were extracted in CHCl3:MeOH (2:1) together with an internal standard mix containing non-physiological or stable isotope-labelled lipid standards, as previously described [17 (link)]. Lipidomic analysis was performed by UHPLC ESI-MS/MS, using an Agilent 1290 HPLC coupled to an Agilent 6490 triple quadrupole mass spectrometer. Results from the chromatographic data were analyzed using Mass Hunter Quant where relative lipid abundances were calculated by relating the area under the chromatogram for each lipid species to the corresponding internal standard. Correction factors were applied to adjust for different response factors, where these were known [18 (link)]. Species that were chromatographically separated were labelled as such (e.g., PC(16:0–22:6) and PC(18:2–20:4)), whereas species that were mixed isomers were given the standard phospholipid notation (e.g., PC(40:8) was a mixture of 20:4/20:4 and 18:2–22:6) [18 (link)]. Where structural details were sufficient, lipids were manually annotated as containing long-chain omega-3 components (i.e., 20:5 EPA, 22:5 DPA, and 22:6 DHA).

Wild-type male flies were loaded on to standard cornmeal-yeast-agar food containing either 0 mM (control) or 50 mM threonine, and then entrained in LD cycles at 25°C for 4 days before harvest. Extracts were prepared from 100 fly heads per condition and the relative levels of free amino acids were measured using ion exchange chromatography as described previously (Sonn et al., 2018 (link)). For quantification of energy metabolites, fly heads were homogenized in 400 µl of chloroform/methanol (2/1, v/v) and clarified by centrifugation. The supernatant was dried by vacuum centrifugation, and then reconstituted with 50 μL of 50% acetonitrile prior to liquid chromatography-tandem mass spectrometry analysis using 1290 HPLC (Agilent), Qtrap 5500 (ABSciex), and a reverse phase column (Synergi fusion RP 50 × 2 mm).