Uplc esi qtof ms system

The RP-LC-MS/MS was applied to obtain the CER profiles. The same Agilent UPLC-ESI-QTOF-MS system (Agilent 1290; Agilent 6540; Agilent Technologies, Santa Clara, CA, USA) was applied for analysis. Chromatographic separation of CERs was performed on RP-C18 column (Acquity BEH Shield 2.1 × 100 mm; 1.7 μm; Waters, Milford, MA, USA) in gradient elution with the mixture of water (20 mM ammonium formate, pH 5) and methanol. The QTOF mass spectrometer was operated in positive (electrospray voltage 3.5 kV) ion mode with a capillary temperature of 300 °C and a sheath gas flow of 8 L/min, as previously described in details [27 (link)].

Ceremide profiles were obtained by using the same Agilent UPLC-ESI-QTOF-MS system as in the case of phospholipid profiling (Agilent 1290; Agilent 6540; Agilent Technologies, Santa Clara, CA, USA). Ceremides were separated by RPLC on the RP C18 column (Acquity BEH Shield 2.1 × 100 mm; 1.7 μm; Waters, Milford, MA, USA) using methanol and water with 20 mM ammonium formate pH 5. The QTOF operating parameters and identification of ceramide species was previously described in detail^{29 (link)}.

The urine, feces, and microsome samples were analyzed by the UPLC-ESI-QTOF/MS system (Agilent, Santa Clara, CA, USA). Metabolites showed good separation in the Agilent 1290 infinity UPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with an XDB-C18 column (2.1 mm × 100 mm, 1.8 µM). The column temperature was maintained at 45 °C, and the flow was set at 0.3 mL/min. Elution was performed using gradient elution ranging from 2% to 98% acetonitrile, containing 0.1% formic acid for 16 min. The injection volume was 5 µL, and the mass signals of ions were collected in both positive (ESI +) and negative (ESI −) modes with electrospray ionization. Nitrogen was used as the collision gas and drying gas, which was set at 350 °C and 9 L/min. Nebulizer pressure was set at 35 psi, and the capillary voltage was set at 3.5 kV. The structures of metabolites were identified by the accurate mass measurements compared to the fragmentary mode of the parent compound, and the MS/MS chromatogram of metabolites was obtained using four collision energy, 10, 15, 20, and 30 eV. The MS was calibrated using the ESI-L Low-Concentration Tuning Mix (Agilent, Santa Clara, CA, USA).