Uhplc 1290 infinity series

For LC-ESI-MS analytics, the ethyl acetate extracts were dried in vacuo and resolved in 15 ml 40% aqueous acetonitrile + 0.1% formic acid and 4 µl were injected onto the UHPLC (ultrahigh performance liquid chromatography) column. LC-ESI-MS analytics were performed using an Agilent6410 Triple Quadrupole LC/MS system in negative ionization mode coupled to an UHPLC 1290 Infinity-Series (Agilent Technologies, Waldbronn, Germany). A VisionHT C18 Column 50 mm×2 mm, 1.5 µm (Grace, Grace GmbH & Co KG, Worms, Germany) was used for separation. Samples were analysed by linear gradient elution using H₂O + 0.1% formic acid as solvent A and acetonitrile + 0.1% formic acid as solvent B. The gradient was from 5% to 100% solvent B in 5 min with a 1.4 min isocratic elution at 100% for solvent B. Mass spectrometry parameters were optimized with commercial bacillibactin (EMC microcollections GmbH; Tübingen, Germany): fragmentor voltage: 80 V; cell accelerator voltage: 3 V; capillary voltage: 3000 V; nozzle voltage: 500 V. The optimized method was used for product ion scans (collision energy: 30 eV (collision-induced dissociation, CID)) with bacillibactin ([M-H]⁻  =  881 Da) as precursor ion.

All measurements for the analysis of crude extracts of pellet and supernatant were performed using an Agilent UHPLC 1290 Infinity-Series system containing an Eclipse Plus C18 column (2.1 × 50 mm) coupled to an ESI-Triple-Quadrupol mass spectrometer (6460 Series, Agilent Technologies, Waldbronn, Germany). For chromatographic separation, a mobile phase H₂O (solvent A)/ACN (solvent B) each with 0.1 % formic acid (v/v) was used. The gradient started at 5 % solvent B to reach 100 % in 4 min and was held constant for 3 min at 100 % solvent B. For MS scan, MS² and multiple reaction monitoring (MRM) analysis of enniatin with an exact mass [M] = 639.4095 Da, all measurements were performed in the positive mode. The three most abundant fragments of MS² experiments were used for quantification in MRM by fragmentation of precursor ion m/z 640 as well the characteristic mass transitions m/z 527.4 as quantifier and m/z 427.3 and m/z 196.2 as qualifier ion. For relative quantification, we used a calibration curve of enniatin B and integrated the measured peak areas accordingly. Calibration curves comprising five concentrations in the range of 0.586-9.375 μg/mL were measured before and after the measurements of enniatin B from B. subtilis extracts (Fig. S1).