Luna 5u nh2 100a 250 4.6 column

In the course of an LC-MS assay V. longisporum ELV43 and P. polymyxa Sb3-1 were co-incubated in order to exchange their VOCs without direct contact with one another. The assay was performed in three replicates. A 4-day old growth plate with V. longisporum ELV43 was placed on top of the P. polymyxa Sb3-1 plate and sealed to facilitate accumulation of VOCs. For the negative control, a fungal and a bacterial plate were incubated with a non-inoculated PDA plate. Cell lysis was performed using Ribolyser FastPrep-24 (MP Biomedicals, Santa Ana, California, USA) for two times 30 s at 6 m s⁻¹ in 90% methanol. The cell free extract was stored at −70°C. The bacterial and fungal metabolite extracts were analyzed with a combined HPLC-hybrid quadrupole-orbitrap mass spectrometer (Q Exactive; Thermo Scientific, Bremen, Germany). A Luna 5u NH2 100A 250 × 4.6 column (Phenomenex, Aschaffenburg, Germany) was used to separate different metabolites from the cell extracts as described by Cernava et al. (2015b (link)). Identification of the soluble compounds was performed with the XCalibur 2.2 and SIEVE 2.2 (Thermo Scientific, Bremen, Germany) and manual comparison of the spectra with corresponding spectra from literature as well as such from mzCloud (HighChem LLC, Bratislava, Slovakia).

Samples were analyzed in nine biological/technical replicates with a combined HPLC-hybrid quadrupole-orbitrap mass spectrometer (Q Exactive; Thermo Scientific, Bremen, Germany). A Luna 5u NH2 100A 250 × 4.6 column (Phenomenex, Aschaffenburg, Germany) was used to separate different metabolites from the cell extracts. Formic acid (0.1%, v/v) in acetonitrile was used as solvent A and aqueous formic acid (0.1%, v/v) as solvent B. Starting conditions for the gradient elution were 10% A and 90% B. The conditions were gradually changed to 80% A and 20% B within 15 min. This step was followed by 5 min at 10% A and 90% B for readjustment to initial conditions. The eluent flow was maintained at 0.8 mL/min together with a column temperature of 25°C. Sample analysis was carried out with negative ion ESI detection. ESI conditions were set to 3.2 kV spray voltage and 350°C capillary temperature. Scans were recorded in the range 100.0–300.0 m/z with the AGC target set to 500,000 and maximal accumulation time of 200 ms. The resolution was adjusted to 200,000. Altering full MS-SIM and targeted MS² cycles were employed and a specific inclusion mass of 146.16517 amu was selected. Standard calibration was obtained with 0, 0.02, 0.03, 0.04, 0.05, 0.1, and 0.2 μM spermidine standard (Duchefa Biochemie, Haarlem, The Netherlands) diluted in 0.2 mM HCL.