Eclipse plus c8 column

C17 S1P and C17 Sph were quantified using a ThermoFisher Scientific Quantum Access triple quadrupole mass spectrometer, operated in positive ion mode, coupled to a 2.1 × 100 mm Agilent Eclipse Plus C8 column (1.8 μm pore size). Total HPLC time was 7 min per sample at a flow rate of 0.2 mL/min, using the following gradient: 0 min, 20:80 A/B; 3.75 min, 13.5:86.5 A/B; 6.5 min, 13.5:86.5 A/B; and 7 min, 20:80 A/B. Solvent A: 0.2% formic acid, 2 mM ammonium formate in MilliQ water; Solvent B: 0.2% formic acid, 1 mM ammonium formate in methanol. C17 S1P, C17 Sph, dhS1P (d18:0) and dhSph (d18:0) were analysed in multiple reaction monitoring mode, scanning for the following transitions: m/z 286.1 to 268.0 (C17 Sph), 302.5 to 284.1 (dhSph), 366.1 to 250.1 (C17 S1P), and 382.2 to 284.2 (dhS1P). The scan time for each event was 0.35 seconds. C17 S1P and C17 sphingosine were quantified as ratios to their respective internal standard (dhS1P or dhSph), using external calibration curves.

CAMAG TLC scanner, High-performed TLC Alum sheets plated with 60 F254 (0.25 mm) (Merck, DE), and R201 Shang. Shen. Biot. Lim. Co. Camag-Linomat IV applicator were instrumentations in HP-TLC methodology.
1200 infinity series LC (Agilent Technologies), 1260 infinity UV–VIS detector (Agilent Technologies), and Eclipse plus C8 column (15, 4.6 and 5 µm) were instrumentations in RP-HPLC methodology.

LC-UV was performed using a Shimadzu LC-20A with 20AT binary pump and M20A PDA detector (Kyoto, Japan). Three different columns from Agilent were compared: the ZORBAX Eclipse plus C₁₈ column, Eclipse plus C₈ column and Eclipse XDB-CN column (250 × 4.6 mm, 5 μm). LC-HRMS was performed using a Thermo Fisher Vanquish Q-Exactive Plus system, which had a quaternary pump and an orbitrap mass analyzer (San Jose, CA, USA). An XP205 balance from Mettler Toledo (Greifensee, Switzerland) was used to weigh compounds.

Lipids were extracted using a 1:1 mixture of methanol and butanol containing a panel of internal lipid standards (Avanti^® Polar Lipids). Sphingolipids were quantified using targeted lipidomics on a TSQ Altis triple quadrupole mass spectrometer, following lipid separation using an Agilent Eclipse Plus C8 column on a Vanquish^™ ultra high-performance liquid chromatography system [35 (link)]. Peaks were integrated using Xcalibur [35 (link)]. Absolute concentrations were determined by normalization against corresponding internal standards and tissue weight. Hierarchical clustering was employed to group similar patterns of lipid changes using Euclidean distance and Ward’s algorithm, where Z-scores were calculated to measure the relative deviation of individual lipid concentrations from the mean values (Metaboanalyst 5.0).

We explored the phospholipid profile in the plasma of patients with SSc using a lipidomics approach utilizing a high-performance liquid chromatography coupled to an ion mobility quadrupole time-of-flight (HPLC-IM-QTOF). The HPLC utilized an Eclipse Plus C8 column (3.5 μm, 3.0 mm × 150 mm, Agilent, Waldbronn, Germany) equipped with a C18 guard column (4 × 3.0 mm, Phenomenex). For measurement, a 1290 Infinity HPLC (Agilent Technologies) coupled to a 6560 IMS-QTOF-MS (Agilent Technologies) was used. For chromatographic separation, solvent A consisted of 60% 18 MΩ-water and 40% acetonitrile with 10 mM ammonium acetate and 1 mM acetic acid, and solvent B was 90% isopropanol, and 10% acetonitrile with 10 mM ammonium acetate and 1 mM acetic acid. The used HPLC gradient started with 60% solvent A and 40% solvent B for 8 min followed by 11 min of 30% A and 70% B and a final step to 10% A and 90% B for 9 min. The flow was 0.7 mL/min, and the injection volume was set to 3 µL. The mass spectrometer was equipped with a Dual Agilent Jet Stream Electrospray Ionization (Dual AJS-ESI, Agilent, Waldbronn, Germany) source, which was operated in positive mode. The scan range was 100 to 1200 m/z. Ion mobility measurement was completed by using N₂ as drift gas and 4-bit multiplexing with a trap fill time of 3900 µs and a trap release time of 250 µs.

Lipids were extracted from mouse liver tissues or Huh7 cells in 1 mL methanol containing a cohort of internal lipid standards (Avanti^® Polar Lipids, Alabaster, USA). The liver tissues isolated from the first 8 animals of each genotype were analysed. Hepatic levels of TG, CE, and phospholipids were quantified using untargeted lipidomic profiling on a Q Exactive HF-X mass spectrometer, following lipid separation on a Waters Acquity C18 UPLC column [53 ]. LipidSearch software was used for lipid annotation, chromatogram alignment and peak integration [75 (link)]. In contrast, FFAs, DG, FC, and sphingolipids were determined by targeted lipidomics on a TSQ Altis triple quadrupole mass spectrometer, following lipid separation on an Agilent Eclipse Plus C8 column [28 (link), 53 ]. Peaks were integrated using Xcalibur (Thermo Fisher, Waltham, USA) [28 (link)].