Reversed-phase (RP) chromatography LC-MS/MS was utilized to characterize ceramide (CER) profiles. The same UPLC-ESI-QTOF-MS system (Agilent 1290; Agilent 6540; Agilent Technologies, Santa Clara, CA, USA) was used for the analysis. The separation of ceramides was carried out on an RP C18 column (Acquity BEH Shield 2.1 × 100 mm; 1.7 μm; Waters, Milford, MA, USA). The mobile phase consisted of water with 20 mM ammonium formate at pH 5 (A) or methanol (B). The solvent gradient started at 70% eluent B held for 1 min, linearly increasing to 100% within 75 min, and returning to initial composition over a final 5 min period. Flow rate was 0.5 mL/min. The MS analysis was performed in positive-ion mode. Electrospray voltage set to 3.5 kV; the drying and sheath gas temperatures set to 300 °C, and the drying and sheath gas flow rates set to 6 and 8 L/min, respectively, were the typical ESI conditions. Data was acquired in DDA mode. Ceramides were identified according to the presence of the [M + H]+ molecular ion, retention time, and characteristic fragmentation patterns, as described previously [33 (link)].
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