Acquity beh hilic column

Quantitative analysis of sphingolipids was performed by using liquid chromatography and tandem mass spectrometry. Briefly, tissues were homogenized in water to give a final concentration of approximately 100 mg/mL (w/v). The homogenate was extracted with 1 mL of a solution of acetonitrile:methanol:water (97:2:1, v/v/v) at room temperature. Extracts were injected onto an Atlantis HILIC silica column (Waters Corp, Milford, MA) for separation of GlcCer and GalCer, and these molecules were detected by using multiple reaction monitoring (MRM) mode tandem mass spectrometry with an AB Sciex API‐5000 mass spectrometer (AB Sciex, Framingham, MA). For other lipid analysis, extracts were injected onto an Acquity BEH C8 column (Waters Corp., Milford, MA), and MRM mode detection was performed using an AB Sciex API‐5000 mass spectrometer. For GlcSph analysis, homogenate was extracted with 1 mL of acetonitrile:methanol:water (48.5:50.5:1, v/v/v), and extracts were injected onto an Acquity BEH HILIC column to resolve GlcSph from psychosine, (Waters Corp., Milford, MA) and detected using MRM mode with an Agilent 6490 mass spectrometer. Except for phosphatidylcholine, all analytes were quantitated against standards obtained from Matreya, LLC (Pleasant Gap, PA).

Samples were subjected to chromatographic separation through a Waters ACQUITY BEH HILIC column (3.0 mm × 150 mm, 1.7 μm) connected to a Waters ACQUITY BEH HILIC VanGuard Pre-Column (2.1 mm × 5 mm, 1.7 μm) using a Thermo Scientific UltiMate 3000 ultra-high performance liquid chromatrography (UHPLC) system. The column temperature was maintained at 40 °C, and the autosampler temperature was 10 °C. Injection volume was 10 μl. Eluent A was 20 mM ammonium acetate and 20 mM ammonium hydroxide in water. Eluent B was acetonitrile. The flow rate was 300 μl min⁻¹ using the following gradient: 0–1 min, 50% B; 1–2 min, linear gradient to 0% B; 2–10 min, 0% B; 10–10.5 min, linear gradient to 50% B; 10.5–15 min, 50% B. The eluate was directed to a Thermo Scientific Q Exactive Mass Spectrometer with a heated electrospray, HESI II source. Source settings were as follows: Spray Voltage, 3.5 kV; Capillary Temperature, 320 °C; Sheath Gas, 20; Aux Gas, 5; Spare Gas, 2; S-Lens RF level, 55. Full scan spectra was acquired over an m/z range of 70–1,000 Da at a resolution of 140,000. Both positive and negative polarities were acquired. Data-dependent MS2 spectra were acquired at a resolution of 17,500 using 30 V collision energy.