Agilent 6540

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 ceramides 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 ceramide 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 [71 (link)].

Cryotome (Thermo Shandon As620 Cryotome, Cheshire, UK), Cryogen (Thermo Shandon, Cheshire, UK), Non-fluorescent polyethylene terephthalate (PET) microscope steel frame slide (76 × 26 mm, 1.4 μm, Leica Microsystems, Bensheim, Germany), Leica Laser microdissection 7000 system, 500 μL micro-centrifuge tube (Leica), Centrifuge (Centrifuge 5417R, Eppendorf, Hamburg, Germany), Ultrasonic instrument (CREST 1875HTAG Ultrasonic Processor, CREST, Trenton, NJ), HPLC grade vial (1.5 mL, Grace, Hong Kong), Glass-lined pipe with plastic ring (400 μL, Grace, Hong Kong), Electronic balance (Mettler Toledo MT5 style), Agilent 6540 ultra-definition accurate mass quadrupole time-of-flight spectrometer equipped with a mass hunter workstation software (Agilent version B.06.00 series, Agilent Technologies, USA), Acquity UPLC BEH C₁₈ column (2.1 mm × 100 mm, 1.7 μm) coupled with a C₁₈ pre-column (2.1 mm × 5 mm, 1.7 μm, Waters, USA).

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)].

NMR spectra of the synthesized molecules were recorded in the Bruker Avance 400 NMR spectrometer, Germany, using deuterated solvents. All the mass spectra were recorded in Agilent 6540, Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA) connected with Agilent 1290 UPLC using DI water and acetonitrile along with 0.1% TFA as mobile phase with a gradient solvent system. The analytes 4% sodium hypochlorite solution was purchased from Qualigens (cat no. 7681-52-9) and 50% Hydrogen Peroxide solution from Fisher Scientific (cat no. 7722-84-1). Using an Agilent Cary 8454 UV-Vis diode array spectrophotometer, all the absorption spectra were measured, whereas the emission spectra were measured in HORIBA Fluorolog-3 spectrofluorometer (Model: FL3-2-IHR). The scan slits for excitation and emission were adjusted to 2 nm. A Waters Alliance System (Milford, MA, USA) equipped with an e2695 separation module, and a 2998 photodiode-array detector was used for the HPLC experiments. The cells were incubated in a Thermo Fisher CO₂ incubator, and imaging was performed with Nikon confocal microscope.

UHPLC–QTOF-MS analysis was performed on an Agilent 6540 ultra-high definition accurate mass quadrupole time-of-flight spec-trometer with UHPLC (UHPLC–QTOF-MS, Agilent Technologies, U.S.A.). A UPLC C₁₈ analytical column (2.1 mm × 100 mm, I.D. 1.7 μm, ACQUITY UPLC®BEH, Waters, U.S.A.) was used for separation, coupled with a C₁₈ pre-column (2.1 mm × 5 mm, I.D. 1.7 μm, Van-GuardTM BEH, Waters, U.S.A.) at room temperature of 20 °C. The mobile phase was a mixture of water (A) and acetonitrile (B), both containing 0.1% formic acid, with an optimized linear gradient elution as follows: 0–5 min, 10–35% B; 5–25 min, 35–55% B; 25–28 min, 55–85% B; 28–30 min, 85–100% B. The injection volume was 4 μL. The flow rate was set at 0.35 mL/min. The mass spectra were acquired in negative mode by scanning from 100 to 1700 in mass to charge ratio (m/z). The MS analysis was performed under the following operation parameters: dry gas temperature 300 °C, dry gas (N₂) flow rate 5 L/min, nebulizer pressure 30 psi, Vcap 3000, nozzle voltage 500 V, and fragmentor voltage 200 V.