Lc ms ion trap

The lipid extracts were dried by nitrogen gas, and re-dissolved with 400 µL of acetonitrile/2-propanol (1:9) with 0.1% formic acid and 10 mM ammonium formate. The sample vials were stored in −20 °C until the analysis by Bruker LC/MS Ion-Trap. Reverse phase HPLC gradient was operated in an Acclaim RSLC 120 C18 column at a flow rate of 0.2 mL/min at 55 °C. The gradient contained solution A: acetonitrile:water (60:40), 10 mM ammonium formate, 0.1% formic acid, and solution B: isopropanol: acetonitrile (90:10), 10 mM ammonium formate, 0.1% formic acid. The acquired mass spectrometry (MS) data were further analyzed by Bruker DataAnalysis (ver.4.1). The extract ion current (XIC) of each phospholipid species was quantitated by their relativity of XIC to internal standard. Each type of total phospholipid is the sum of all quantitated species of its type. The percentage of each phospholipid was the ratio of XIC of each phospholipid species to total XIC of its type.

The extracted total lipids were re-dissolved in 200 μL of Acetonitrile/Isopropanol/H₂O (63:30:5). 20 μL of the samples were analyzed by LC/MS Ion-trap (Bruker). There are two different HPLC mobile phases, including solution A: Acetonitrile:H₂O (60:40), 10 mM ammonium formate, 0.1% formic acid and solution B: Isopropanol:Acetonitrile (90:10), 10 mM ammonium formate, 0.1% formic acid. Gradient was from 60% solution A to 100% solution B in 25 min and preserved in 100% solution B until 40 min and then backed to 60% solution A in an Acclaim RSLC 120 C18 2.1 mm × 100 mm × 2.2 μm column (Thermo, USA) at a flow rate of 0.2 mL/min at 55 °C. Then, we calculated the integrated area of the extract ion current (XIC) in Bruker DataAnalysis (ver.4.1). All experiments were in triplicate and the standard deviations of the triplicated experiments were plotted as the error bars of the figures.