Xcalibur quan software

Mice were euthanized with CO₂ followed by cervical dislocation. Spinal cord was rapidly dissected and snap-frozen in liquid nitrogen. Samples were sent to the Metabolomics core (VIB, Leuven, Belgium) to perform targeted metabolomic profiling by Liquid Chromatography - Mass Spectrometry (LC-MS). Separation of metabolites prior to MS measurement was performed using a Dionex UltiMate 3000 LC System (Thermo Scientific) in-line coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Scientific). Practically, 10 μl of the extract was injected on a C18 column (Acquity UPLC®HSS T3 1.8 μm 2.1x100mm, Waters) using solvent A (H₂O, 10 mM Tributyl-Amine, 15 mM acetic acid) and solvent B (100% Methanol). Chromatographic separation was achieved with a flowrate of 0.250 ml/min and the following gradient elution profile: 0 min, 0%B; 2 min, 0%B; 7 min, 37%B; 14 min, 41%B; 26 min, 100%B; 30 min, 100%B; 31 min, 0%B; 40 min, 0%B. The column was thermostatted at 40 °C throughout the analysis. The MS operated in full scan negative ion mode (m/z range: 70–1050 Th) using a spray voltage of 4.2 kV, capillary temperature of 320 °C, sheath gas at 50.0, auxiliary gas at 15.0. The AGC target was set at 3e6 and resolution at 140,000, with a maximum IT fill time of 512 ms. Data processing was performed using the Xcalibur Quan software (Thermo Scientific).