| MS Description
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Water, isopropanol, and acetonitrile were … Water, isopropanol, and acetonitrile were purchased from Fisher Optima. Methanol was purchased from J.T. Baker. Ammonium formate, ammonium acetate, formic acid, and methyl tert-butyl ether (MTBE) were purchased from Sigma-Aldrich. Odd chain and deuterated lipids used as internal standards were purchased from Avanti Polar Lipids, CDN Isotopes, Cayman Chemical, and Sigma-Aldrich.
<LC Method>
The LC system consisted of an Agilent 1290 system (Agilent Technologies Inc.) with a pump (G4220A), a column oven (G1316C), and an autosampler (G4226A). Lipids were separated on an Acquity UPLC CSH C18 column (100 x 2.1 mm; 1.7 µm) coupled to an Acquity UPLC CSH C18 VanGuard precolumn (5 x 2.1 mm; 1.7 µm) (Waters, Milford, MA, USA). The column was maintained at 65°C at a flow-rate of 0.6 mL/min. For LC-ESI(+)-MS analysis the mobile phases consisted of (A) 60:40 (v/v) acetonitrile:water with ammonium formate (10 mM) and formic acid (0.1%) and (B) 90:10:0.1 (v/v/v) isopropanol:acetonitrile:water with ammonium formate (10 mM) and formic acid (0.1%). For LC-ESI(-)-MS analysis the organic solvents for mobile phases were the same with the exception of using ammonium acetate (10 mM) as mobile-phase modifier. A sample volume of 3 µL was used for the injection in both ESI(+) and ESI(-). The separation was conducted under the following gradient in ESI(+): 0 min 15% (B); 0-2 min 30% (B); 2-2.5 min 48% (B); 2.5-11 min 82% (B); 11-11.5 min 99% (B); 11.5-12 min 99% (B); 12-12.1 min 15% (B); and 12.1-15 min 15% (B). The separation was conducted under the following gradient in ESI(-): 0 min 15% (B); 0-2 min 30% (B); 2-2.5 min 48% (B); 2.5-9.5 min 76%; 9.5-9.6 min 99% (B); 9.6-10.5 min 99% (B); 10.5-10.6 min 15% (B); 10.6-13.5 min 15% (B). Sample temperature was maintained at 4°C.
<MS Method>
Mass spectrometric detection of lipids was performed on a quadrupole/time-of-flight mass spectrometer TripleTOF 6600 (SCIEX, Framingham, MA, USA). All analyses were performed at the high resolution mode in MS1 (~35,000 full width at half maximum (FWHM)) and at the high sensitivity mode (~15,000 FWHM) in MS2. For ESI(+), the SWATH parameters were MS1 accumulation time, 100 ms; MS1 mass range, m/z 100-1700; MS2 accumulation time, 10 ms; collision energy, 45 eV; collision energy spread, 15 eV; cycle time, 550 ms; Q1 window, 20 Da; SWATH mass range, m/z 300-1100; number of SWATH experiments, 40; MS2 mass range: m/z 80-1100. Other parameters were curtain gas, 35; ion source gas 1, 60; ion source gas 2, 60; temperature, 350°C; ion spray voltage floating, 4.5 kV; declustering potential, 80 V. For ESI(-), the SWATH parameters were MS1 accumulation time, 100 ms; MS1 mass range, m/z 100-1700; MS2 accumulation time, 10 ms; collision energy, -50 eV; collision energy spread, 10 eV; cycle time, 550 ms; Q1 window, 15 Da; SWATH mass range, m/z 400-1000; number of SWATH experiments, 40; MS2 mass range: m/z 80-1000. Other parameters were curtain gas, 35; ion source gas 1, 60; ion source gas 2, 60; temperature, 350°C; ion spray voltage floating, -4.5 kV; declustering potential, -80 V. The mass calibration was automatically performed using an APCI positive/negative calibration solution via a calibration delivery system (CDS). n via a calibration delivery system (CDS).
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