Lct premier orthogonal accelerated time of flight mass spectrometer

For LC-MS analysis, sera were randomly sub-pooled into 4 groups of men and 4 groups of women, homogeneous for biometric parameters and HPTLC quantitative profiles. SL extracts were analyzed in the presence of internal standard^{52 (link)}, prepared as described by Merrill et al.^{59 (link)}, including an alkaline hydrolysis step to remove phospholipids, and analyzed by Waters Aquity Ultra Performance Liquid Chromatography (UPLC) system connected to a Waters LCT Premier orthogonal accelerated time of flight mass spectrometer (Waters, Millford, MA), operating in positive electrospray ionization mode. Spectra were acquired according to^{52 (link)}. Positive identification of compounds was based on the accurate mass measurement with an error <5 ppm and its LC retention time, compared to that of a standard (±2%). Mass spectra were analyzed by MassLynx™ 4.1 Software.
LC-MS/MS analyses of sphingosine, S1P and dhS1P were carried out on an Acquity UPLC system coupled with a Xevo TQ-MS triple quadrupole mass spectrometer. The mass spectrometer was operated in the positive ESI mode, and analytes were quantified by multiple reaction monitoring (MRM). Transitions considered were Q1 300.4- > Q3 264.4 for Sph, Q1 380.4 - > Q3 264.4 for S1P and Q1 382.4- > Q3 284.4 for dhS1P.

A total of five samples (biological replicates) of each extraction were randomly analyzed; one sample per set of extraction was also analyzed in triplicate (instrumental replicates). In total, 16 chromatograms were obtained (eight for each extraction batch). QCs and blanks were also interspersed in the chromatographic sequence. LC-MS analysis was carried out with a Waters Acquity UPLC system coupled to a Waters LCT Premier orthogonal accelerated time-of-flight mass spectrometer (Waters), operated in both positive (ESI+) and negative (ESI-) electrospray ionization modes. Full-scan spectra from 50 to 1500 Da were acquired at a scan cycle time of 0.3 s. The chromatographic method employed was described previously [25 (link)]. Briefly, an RP C8 Acquity UPLC bridged ethylene hybrid (Waters) column of 100 mm × 2.1 mm i.d. (1.7 μm) was employed. Mobile phases were: (A) MeOH 1 mM NH₄Form and 0.2% HForm; (B) H₂O 2 mM NH₄Form and 0.2% HForm. The solvent elution gradient started at 80%A, increased until 90%A at 3 min, held at 90%A until minute 6 min, increased to 99%A at 15 min, and held at 99% until 18 min. Then, the column was re-equilibrated during 2 min. Flow rate, injection volume and column temperature were set at 0.3 mL min⁻¹, 10 μL and 30 °C, respectively.

Lipids from homogenate samples were extracted with methanol:chloroform isolation (1:2, v/v). Mass spectrometry analysis of lipid species was performed in the Research Unit on Bioactive Molecules at the Institute of Advanced Chemistry of Catalonia (IQAC). Cells or tissue homogenates were pelleted, washed in PBS, and transferred to glass vials. Sphingolipid extracts were spiked with internal standards (N-dodecanoylsphingosine, N-dodecanoylglucosylsphingosine, and N-dodecanoylsphingosylphosphorylcholine, 0.2nmol each) and analysed in a Waters Aquity UPLC system connected to a Waters LCT Premier orthogonal accelerated time of flight mass spectrometer (Waters, Millford, MA) operated in positive electrospray ionisation mode. The analytical column was a 100mm x 2.1mm i.d., 1.7μm C8 Acquity UPLC BEH (Waters). The two mobile phases were phase A: water/formic acid (500/1 v/v); phase B: methanol/formic acid (500/1 v/v), both also contained 5mM ammonium formate. A linear gradient was programmed— 0.0min: 80% B; 3min: 90% B; 6min: 90% B; 15min: 99% B; 18min: 99% B; 20min: 80% B. The flow rate was 0.3mL/min. The column was maintained at 30°C. Quantification was carried out using the extracted ion chromatogram of each compound, using 50mDa windows. The linear dynamic range was determined by injecting standard mixtures.