GNPS - HILIC LC-MS/MS of sputum from CF patients treated with tobramycin

LC-MS/MS HILIC Analysis. The methanol metabolite extracts from three patients for the oxygen experiments with Aspergillus reads (CF157, CF146 and CF353) and three without (CF178, CF243, CF69) were additionally used for the HILIC chromatography analysis to more efficiently detect tobramycin and its metabolites. These samples were extracted further using a water/methanol 4:1 extract solution for 5 min, followed by centrifugation to pellet debris prior to mass spectrometry analysis. The analysis was conducted on a Vanquish UHPLC (Thermo Fisher Scientific, Waltham, MA) connected to Orbitrap (Q Exactive, Thermo Fisher Scientific, Waltham, MA) mass spectrometer equipped with HESI-II probe source. The separation was conducted using a 100 x 2.1 mm Kinetex 1.7 ?M, HILIC, 100Å column (Phenomenex, Torrance, CA). The column was held at 40°C during the analysis. The mobile phases used were: A 99.9 % HPLC grade water 0.1% formic acid (Thermo Fisher Scientific, Optima LC/MS), and B 99.9% HPLC grade acetonitrile 0.1% formic acid (Fisher Scientific, Optima LC/MS). Following gradient steps were used: 0-1 min 100% B, 1-4 min 100-90% B, 4-14 min 90-0% B, 14.0-14.9 min 100% A, 14.9-17.0 min 100% B. Flow rate was set at 0.350 mL/min. For the MS acquisition, following settings were used: positive ion mode, Spray voltage of 3500 V, ion source temperature of 270°C, Capillary temperature 250°C, S-lens RF level of 50 Hz, Sheath gas (N2) pressure of 45 psi, Auxiliary gas pressure (N2) of 10 psi, and Aux gas heater temp. at 410°C. 5 ?l of the sample was injected. The data were acquired in a data dependent acquisition (DDA) mode with both MS1 full scan (150-1500 m/z) and up to 5 MS2 scans of the most abundant ions per duty cycle. The resolution of Orbitrap mass analyzer was set at 30,000. The MS2 precursor selection window was set to 1.5 m/z with 0.5 m/z offset. The normalized collision energy was set to stepwise increase from 20 to 30 to 40 units with z = 1 as default charge state. The MS2 acquisition was set to be automatically triggered at the apex of a peak within 2 to 15 s from their first occurrence with the dynamic exclusion time of 5 s.