UPLC-ESI-QTOF-MS Characterization of Ceramide Profiles

An Agilent UPLC-ESI-QTOF-MS system (Agilent 1290; Agilent 6540; Agilent Technologies, Santa Clara, CA, USA) was used to characterize the CER profiles. The mobile phase was composed of solvent A (water with 20 mM ammonium formate pH 5) and solvent B (methanol). Initially, 70% of B was held isocratically for 1 min, followed by a linear increase to 100% of B within 75 min, and return to initial conditions in 5 min. The CERs were separated on an RP C18 column (Acquity BEH Shield 2.1 × 100 mm; 1.7 μm; Waters, Milford, MA, USA) with a flow rate of 0.5 mL/min. The QTOF mass spectrometer was operated in positive ion mode (electrospray voltage 3.5 kV) with a capillary temperature of 300 °C and a sheath gas flow rate of 8 L/min. The data was collected in DDA mode. Identification of CER species was based on the presence of the [M + H]⁺ molecular ion, retention time and characteristic fragmentation patterns observed in MS/MS spectra, which was previously described in detail^{19 (link)}. Cermide internal standards, N-lignoceroyl-d-erythro-sphingosine and N-lignoceroyl-d-erythro-sphinganine (Avanti Polar Lipids, Alabaster, AL, USA) were used for quantification of CERs.