| MS Description
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Water was purchased from VWR International … Water was purchased from VWR International, isopropanol from Merck, and acetonitrile from Honeywell. MTBE, ammonium formate, ammonium acetate, formic acid, and acetic acid were purchased from Sigma-Aldrich. Odd chain and deuterated lipids used as internal standards were purchased from Avanti Polar Lipids, Chromsystems, Cayman Chemical, and Sigma-Aldrich.
<LC Method>
The LC system consisted of a Vanquish UHPLC system (Thermo Fisher Scientific, Bremen, Germany) with a pump, a column oven and an autosampler. Lipids were separated on an Acquity UPLC BEH C18 column (50 x 2.1 mm; 1.7 µm) coupled to an Acquity UPLC BEH 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) and acetic acid (0.1%) as mobile-phase modifiers. A sample volume of 2 µL and 3 µL was used for the injection in ESI(+) and ESI(-), respectively. Separation was conducted under the following gradient for LC-ESI(+): 0 min 15% (B); 0-1 min 30% (B); 1-1.3 min from 30% to 48% (B); 1.3-5.5 min from 48% to 82% (B); 5.5-5.8 min from 82% to 99% (B); 5.8-6 min 99% (B); 6-6.1 min from 99% to 15% (B); 6.1-7.5 min 15% (B). For LC-ESI(-), the following gradient was used: 0 min 15% (B); 0-1 min 30% (B); 1-1.3 min from 30% to 48% (B); 1.3-4.8 min from 48% to 76% (B); 4.8-4.9 min from 76% to 99% (B); 4.9-5.3 min 99% (B); 5.3-5.4 min from 99% to 15% (B); 5.4-6.8 min 15% (B). Sample temperature was maintained at 4°C.
<MS Method>
Mass spectrometric detection of lipids was performed on a quadrupole/orbital ion trap mass spectrometer Q Exactive Plus with a HESI-II ion source (Thermo Fisher Scientific, Bremen, Germany). Simultaneous MS1 and MS/MS (data-dependent MS/MS) acquisition was used. The parameters were as follows: sheath gas pressure, 60; aux gas flow, 25; sweep gas flow, 2; spray voltage, 3.6 kV and -3.0 kV for ESI(+) and ESI(-), respectively; capillary temperature, 300°C; aux gas heater temperature, 370°C; MS1 mass range, m/z 200-1700; MS1 resolving power, 35,000 FWHM (m/z 200); number of data-dependent scans per cycle: 3; MS/MS resolving power, 17,500 FWHM (m/z 200); MS1 ion time: 100 ms; MS2 ion time: 50 ms; normalized collision energy, 20% for ESI(+) and 10, 20, 30% for ESI(-). The instrument was tuned using a Thermo positive and negative ion mode calibration solutions. d negative ion mode calibration solutions.
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