Acclaim rslc 120 c18 2.1 mm 100 mm 2 2 μm column

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.

The extracted total lipid was dried under nitrogen gas and immediately re-dissolved with acetonitrile/2-propanol/H₂O (65:30:5). The samples were capped and stored in −20 °C to prevent evaporation until LC/MS Ion-Trap analysis (Bruker Corporation). Reverse phase HPLC contained solution A: ACN:H₂O (60:40), 10 mM ammonium formate, 0.1% formic acid and solution B: IPA:ACN (90:10), 10 mM ammonium formate, 0.1% formic acid for the elution gradient from 60% solution A/40% solution B to 100% solution B in 25 min and maintained 100% solution B until 45 min. The elution was in an Acclaim RSLC 120 C18 2.1mm × 100 mm 2.2 μm column (Thermo) at a flow rate of 0.2 mL/min at 55 °C. Data were further analyzed by Bruker DataAnalysis (ver.4.1). The extract ion current (XIC) of each cardiolipin species was quantitated by their area ratio of XIC to internal standard. The total CL is the sum of all quantitated cardiolipin species. The percentage of CL was the ratio of XIC of each cardiolipin species to total XIC. Standard deviations were calculated by the STDEV function in Microsoft Excel for the error bars of the histograms and t-tests are applied to all triplicated data