Agilent 6545 quadrupole time of flight mass spectrometer

Extracts were reconstituted in ACN/ISP (150 μL, 7:3, v/v), followed by 20 s of probe sonication (Misonix, Farmingdale, NY, USA) and 5 min centrifugation (4 °C, 14,000 rpm) before analysis with an Agilent 1290 UHPLC coupled to an Agilent 6545 quadrupole time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was performed using a previously published reverse phase method employing a C8 column (Acquity Plus BEH, Waters, Milford, MA, 100 mm × 2.1 mm, 1.7 μm particle size) and an H₂O/ACN/ISP mobile phase with NH₄OAc and acetic acid modifiers [29 (link)]. The injection volume was 5 μL. The following ionization source parameters were utilized: spray voltage negative ion = −2000 V, ion transfer tube temperature = 300 °C, vaporizer temperature = 325 °C, sheath gas flow = 45 (arbitrary units), aux gas flow = 13 (arbitrary units), sweep gas flow = 1 (arbitrary units). The mass spectrometer was tuned and calibrated before the analysis, and the manufacturer’s reference mixture analyzed throughout every sample injection every second scan for real-time-of-flight calibration (as performed by the software). Pooled quality control samples (QC) were analyzed every 6^th injection, as well as process blanks ~25^th injection, to correct for instrument performance and remove persistent contaminant features from the data, respectively.

200 mL of samples supplemented with 100 nM levofloxacin as external standard were transferred into glass vials and 10 μL of samples and standards were injected into the UPLC in triplicates. A UPLC/HRMS system Agilent 1290 Infinity UPLC coupled to an Agilent 6545 quadrupole time-of-flight mass spectrometer was used to quantify the concentrations of compounds. Samples were processed in positive ion mode. An Eclipse plus C18 Rapid Resolution HD column (1.8 μm, 2.1 × 50 mm, Agilent, USA) was used for separation. The flow rate was 0.6 mL/min using the following gradient: 0 to 1 min (0- 20% ACN), 1 to 4 min (20- 80% ACN), 4 to 5.1 min (80–100% ACN), 5.1 to 6.30 (100% ACN). Solvents contained 0.1% formic acid to promote positive ion formation in the electrospray. The MS parameters were as follows: ion-source gas temperature, 325 °C; capillary voltage, 4000 V; fragmentor voltage, 180 V; m/z range, 50–1100; data acquisition rate, 4 GHz; and 2 spectra were recorded per second. Data were analyzed using Agilent Masshunter workstation quantitative analysis v. B.10 software.

Dried lipid extracts were resuspended in the appropriate volume of 9:1 methanol/toluene solution to obtain an equivalent protein concentration of 2 μg/μl per sample. A quality control (QC) sample was subsequently generated which was composed of equi‐volumes of each sample. This QC sample was analyzed in Auto‐MSMS mode, whereas each biological sample was analyzed in MS1‐only. The QC sample was injected after every eighth injection and at regular intervals. Samples were injected on a 1260 Infinity HPLC (Agilent Technologies Inc., Santa Clara, California, USA) onto an Accucore C30 column (2.6 μm, 100 × 2.1 mm) (Thermo Scientific, Waltham, Massachusetts, USA) for reverse‐phase separation which was heated at 50°C with a constant flow rate at 0.400 ml/min, using a gradient of mobile phase A (50:50, acetonitrile/water, 10 mM ammonium formate) and mobile phase B (90:10 isopropanol/acetonitrile, 10 mM ammonium formate). The gradient program was as follows: 0–5 min, 15%–40%; 5–30 min, 40%–95% B; 30–32 min, hold at 95% B; 10 min, equilibrate at 15% B. Positive and negative ion mode acquisition were utilized on an Agilent 6545 quadrupole time‐of‐flight mass spectrometer equipped with a JetStream ionization source (Agilent Technologies Inc., Santa Clara, California, USA) to profile the lipid extracts, using 4 and 10 μg equivalent protein concentration, respectively.