4me 16 0 diether dg

Neutral lipids (TAGs, DAGs and CEs) were analyzed using a modified version of reverse phase HPLC/ESI/MS/MS described previously [51 (link)]. Briefly, separation of lipids aforementioned was carried out on a Phenomenex Kinetex 2.6μ-C18 column (i.d. 4.6×100mm) using an isocratic mobile phase chloroform:methanol:0.1M ammonium acetate (100:100:4) at a flow rate of 150 μl/min for 22 min. Using neutral loss-based MS/MS techniques, the levels of TAG were calculated as relative contents to the spiked d5-TAG 48:0 internal standard (CDN isotopes), DAG species were quantified using 4ME 16:0 Diether DG as an internal standard (Avanti Polar Lipids, Alabaster, AL, USA) and CE species were quantified using d6-CE (CDN isotopes) as internal standard.

Glycerol lipids (phosphatidylcholines, triacylglycerides and diacylglycerides) were analyzed using a modified version of reverse phase (RP)-HPLC/ESI/MS/MS described previously^{23 (link)} on a Shimadzu Exion UPLC coupled with SCIEX 6500 QTRAP Plus system. Briefly, samples for neutral lipid analysis were prepared by dissolving oil with chloroform : methanol (1 : 1) solvent. The phosphatidylcholines (PCs) were concentrated for analysis by extraction with analytical grade ethanol. Separation of the aforementioned lipids was carried out on a Phenomenex Kinetex 2.6μ-C18 column (internal diameter 4.6 × 100 mm) using an isocratic mobile phase chloroform : methanol : 0.1 mol L⁻¹ ammonium acetate (100 : 100 : 4) at a flow rate of 160 μL min⁻¹ for 20 min. Using neutral loss-based MS/MS techniques, the levels of TAGs were calculated as relative contents to the spiked d5-TAG 48:0 internal standard (CDN Isotopes), while DAG species were quantified using 4ME 16:0 diether DG as an internal standard (Avanti Polar Lipids, Alabaster, AL, USA).

Lipids were extracted from wandering 3^rd instar larval fat body as previously described [60 (link)]. The lipidomic analyses were carried out on an analytical system comprising an Agilent HPLC 1260 coupled with a SCIEX 5500 QTRAP. Separation of individual classes of polar lipids by normal phase HPLC was carried out using a Phenomenex Luna 3u silica column (i.d. 150x2.0 mm). Multiple reaction monitoring (MRM) transitions were set up for quantitative analysis of various polar lipids. Individual lipid species were quantified by referencing to spiked internal standards. PC-14:0/14:0, LPC-C20, PE-14:0/14:0, PS-14:0/14:0, PA-17:0/17:0, PG-14:0/14:0 were obtained from Avanti Polar Lipids and dioctanoyl phosphatidylinositol (PI, 16:0-PI) was obtained from Echelon Biosciences, Inc. Separation of glycerol lipids (DAG and TAG) by reverse phase HPLC/ESI/MS/MS was carried out on a Phenomenex Kinetex 2.6μ-C18 column (i.d. 4.6x100mm). Using neutral loss-based MS/MS techniques, the levels of TAG were calculated as relative contents to the spiked d5-TAG 48:0 internal standard (CDN Isotopes), while DAG species were quantified using 4ME 16:0 Diether DG as an internal standard (Avanti Polar Lipids). Free cholesterols and ergosterols were analyzed using HPLC/APCI/MS/MS with the corresponding d6-Cho (CDN Isotopes) as the internal standard.