Beh c8 column

The sample was analyzed using an Ultimate 3000UPLC system (Dionex, United States) combined with a BEH C8 column (2.1 × 100 mm, 1.7 µm) (Waters, United States) and a HSS T3 column (2.1 × 100 mm, 1.8 µm) (Waters, United States). The temperature of the column was kept at 30°C, the flow rate was 0.35 mL/min, and the sample injection volume was 5 μL. In the positive ion mode, the mobile phase was consisted of water with 0.1% formic acid (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The gradient profile was performed as follows: 5% B (0–1 min); 5%–100% B (1–24 min); 100% B (24.1–27.5 min); 5% B (7.6–30 min). In the negative ion mode, the mobile phase was a mixture of water with 6.5 mM ammonium bicarbonate (solvent A) and 95% methanol with 6.5 mM ammonium bicarbonate (solvent B). Gradient elution condition: 5% B (0–1 min); 5% B (1–18 min); 100% B (18.1–22 min); 5% B (22.1–25 min). The electrospray ionization (ESI) source was operated and optimized and the MS parameters were as follows: aux gas flow rate: 8 Arb; sheath gas flow rate: 35 Arb; capillary temperature: 320°C; aux gas heater temperature: 350°C; mass range: 70–1,050 m/z, the full MS resolution: 70000; MS/MS resolution: 17500. The collision energy was 20, 40% in NCE mode; the spray voltage was +3.8 kV (positive) or −3.0 kV (negative).

Tissues for analysis were removed quickly, frozen in liquid nitrogen, and stored at − 80 °C prior to quantification. Acylcarnitines were analyzed using an Acquity UPLC-TOF system (Waters) with a BEH C8 column (1.7 μm particle size, 100 mm × 2.1 mm, Waters). The two mobile phases were 1 mM ammonium formate in methanol (phase A) and 2 mM ammonium formate in H2O (phase B), both phases with 0.05 mM formic acid. The following gradient was programmed: 0 min, 65% A; 10 min, 90% A; 15 min, 99% A; 17 min, 99% A; 20 min, 65% A, and a flow rate of 0.3 mL min⁻¹. Quantification was carried out using the extracted ion chromatogram of each compound, with 50-mDa windows. The linear dynamic range was determined by injecting standard mixtures. Positive identification of compounds was based on the accurate mass measurement with an error < 5 ppm and their LC retention time was compared to that of a standard (± 2%).

Peptide composition and homogeneity of Transferon batches were determined by MS (mass spectrometry) using an ESI-Q-TOF (electrospray ionization-quadrupole-time of flying) mass detector, coupled to an Acquity chromatographic system (Waters). Transferon samples were filtered through a 0.10 µm PVDF membrane (Merck Millipore Co.), and 5 μL was injected into the chromatographic system. Sample desalination and separation were performed on a BEH C8 column (130 Å, 1.7 μm, 2.1 × 100 mm) (Waters) using aqueous formic acid (0.1%) (Sigma-Aldrich) (Solution A) and acetonitrile (Mallinckrodt Baker) (Solution B). Column temperature was maintained at 30°C, and the flow rate was set at 0.2 mL/min using a gradient configuration (1-2 min, 60% A; 3–40 min, 90% A; 41–45 min, 60% A). Mass spectrometric analysis was performed on a Xevo G2-SQ-TOF mass detector (Waters). Electrospray mass spectra data were recorded in a positive ion mode for a mass range from 100 m/z to 2000 m/z with a scan time of 0.1 s. The source temperature was set at 150°C with a cone gas flow of 20 L/h. Desolvation gas flow was set at 600 L/h and 250°C. The capillary was maintained at 3.5 kV, and the cone voltage was set to 20 V. Mass spectra data were analyzed using Mass Lynx™ (Waters).

The lipids extracted from the previous step were solubilised in 300 μl of a mixture of acetonitrile:isopropanol (7:3 v/v), vortexed and incubated 10 min in an ultrasonication bath. The samples were centrifuged for 5 min at 10,000 × g, and 100 μl aliquots were transferred into glass vials for further analysis (Salem et al., 2016 (link)). Lipids in 2 μl injection samples were separated on a reversed-phase Bridged Ethyl Hybrid (BEH) C8 column (100 mm × 2.1 mm containing 1.7 μm diameter particles, Waters, Manchester, United Kingdom) using a Waters Acquity UPLC system (Waters, Manchester, United Kingdom) (Hummel et al., 2011 (link)). The UPLC–FT–MS raw chromatograms were analysed and processed either by using Xcalibur (Version 2.10, Thermo-Fisher, Bremen, Germany) or ToxID (Version 2.1.1, Thermo-Fisher) as described in Hummel et al. (2011) (link).