G6460 triple quadripole spectrometer

The lipid extract was dried and dissolved in 50 µL of MeOH then stored at −20 °C prior to analysis. Analysis were performed on an Agilent 1290 UPLC system coupled to a G6460 triple quadripole spectrometer (Agilent Technologies, Les Ulis, France) an Acquity UPLC BEH-C8 (Waters, Issy les Moulineaux, France), 100 × 2.1 mm, 1.7 μm) maintained at 35 °C. The mobile phases A and B were H₂O, FA (99.9:0.1; v/v), and ACN, FA (99.9:0.1, v/v), respectively. The gradient was as follows: 50% B at 0 min, 60% B at 2 min, 60% B at 3 min, 100% B at 4 min, 100% B at 8.5 min, and 50% B at 9 min. The flow rate of mobile phase was 0.3 mL/min and the injection volume was 5 µL. ESI was performed in positive ion mode at 300 °C. The collision gas was nitrogen. Needle voltage was set at + 4000 V. SRM transitions in neutral loss scan were used. For quantitative analysis, calibration samples (500 to 0.976 ng) were prepared with commercial sphingolipid standards. All of the quantitative calculations were based on the peak area ratios relative to the internal standards [adapted from Sikora [52 (link)].

Lipids were extracted from tumors 10 days after melanoma cell implantation, and sphingolipids were quantified by ultra-performance liquid chromatography, using an Agilent 1290 system coupled to a G6460 triple quadripole spectrometer (Agilent Technologies) [45 (link)]. Alternatively, the amount of extracellular S1P was evaluated as reported [46 (link)] after incubation of the cells with 0.45 μCi/ml, 1.5 μM D-erythro-[3-³H] sphingosine (Perkin-Elmer).

The lipid extract was dried, dissolved in 50 µL of MeOH then stored at −20 °C prior to analysis. Analysis were performed on an Agilent 1290 UPLC system coupled to a G6460 triple quadripole spectrometer (Agilent Technologies, Les Ulis, France)) using a Kinetex HILIC column (Phenomenex, Le Pecq, France), 50 × 4.6 mm, 2.6 µm). The column temperature was controlled at 40 °C. The mobile phases A and B were ACN and 10 mM AF in H₂O at pH 3.2, respectively. The gradient was as follows: from 10% to 30% B in 10 min; 10–12 min, 100% B; and, then back to 10% B at 13 min for 1 min. The flow rate of mobile phase was 0.3 mL/min and the injection volume was 5 µL. Electrospray ionization (ESI) was employed at 325 °C in positive (for Cer, PE, PC, and SM analysis) and negative ion mode (for PI and PS analysis). The collision gas was nitrogen. Needle voltage was set at +4000 V. SRM transitions were used for relative quantification with a precursor ion scan of 184 m/z, 241 m/z, and 264 m/z to PC/SM, PI, and Cer, respectively; and, a neutral loss scan of 141 m/z and 87 m/z to PE and PS, respectively. In each family, individual molecular species were scanned with suitable SRM scan mode, the area of each peak were measured via Mass Hunter software. The relative quantitative calculations were based on the peak area ratios relative to the internal standards [37 (link)].