Ultimate 3 000 uplc system

Single colonies were inoculated into 2 ml Eppendorf tubes containing 1.8 ml LB broth supplemented with appropriate antibiotics and incubated at 37°C overnight with shaking at 900 rpm. The seed cultures (1%, v/v) were transferred into 50 ml M9 broth in 250 ml Erlenmeyer flask and incubated at 30°C with shaking at 200 rpm for 24 h before 2% XAD-16 resin was added, then the incubation was continued for another 48 h. The resin was collected by centrifugation and resuspended with 50 ml of methanol. The mixtures were shaken at 30°C, 200 rpm for 2 h. The methanol was removed by evaporation in vacuo, and residues were dissolved with 1 ml of methanol. After filtering with 0.22 μm membrane, the crude extracts were analyzed by UPLC-MS (UltiMate 3,000 UPLC system combined with Bruker amazon SL Ion Trap mass spectrometer). The C18 column (2.1 × 100 mm. 2.2 μm, Thermo) was utilized to analyze the crude extracts at a 0.3 ml/min flow rate using the following program: 0–3 min 5% solvent B; 3–22 min, 5 95% with linear gradient; then, 22–25 min, 5% solvent B (Solvent A, Milli Q water supplemented with 0.1% formic acid; Solvent B, acetonitrile supplemented with 0.1% formic acid). Mass spectra were acquired in positive ion mode.

The purified samples were loaded on a PGC column (100 mm × 0.32 mm, 5 µm particle size, Thermo Scientific, Waltham, MA, USA) with 10 mM ammonium bicarbonate as the aqueous solvent A and 80% acetonitrile in solvent A as solvent B, as described in [28 (link)]. In brief, 5.5 min after sample application at 1% B, a gradient from 8 to 22% solvent B was developed over 52.5 min followed by an increase up to 68% B at a flow rate of 6 µL/min. Detection was performed with an ion trap mass spectrometer (amaZon speed ETD; Bruker, Bremen, Germany) equipped with the standard ESI source directly linked to the Thermo Ultimate 3000 UPLC system. MS scans were recorded in positive and/or negative mode from 400–1600 m/z. Standard source settings (capillary voltage 4.5 kV, nebulizer gas pressure 0.5 bar, drying gas 5 L/min, 200 °C) were used. Instrument tuning was optimized for a low mass range (around 1500–2000 Da). MS/MS was carried out in data-dependent acquisition mode (switching to MS/MS mode for eluted peaks). Data interpretation was done with DataAnalysis 4.0 (Bruker, Bremen, Germany).

The reaction mixture was concentrated in vacuo, then the residue was reconstituted by sonication in 1.5 ml CH₂Cl₂–methanol (2:1). An aliquot of 150 µl was transferred from each sample and concentrated in vacuo. The dried extract was resuspended in 1 ml of HPLC-grade MeOH–H₂O (1:1) and diluted for analysis. LC–MS/MS analysis was performed using a Thermo UltiMate 3000 UPLC system coupled to a high-resolution Q-ToF mass spectrometer (Bruker Daltonics MaXis HD). A polar C18 column (Kinetex polar C18, 100 × 2.1 mm, 2.6 µm particle size, 100 Å pore size; Phenomenex) was used as the stationary phase, and a high-pressure binary gradient pump was used to deliver the mobile phase, which consisted of solvent A (100% H₂O + 0.1 % formic acid (FA)) and solvent B (100% acetonitrile + 0.1% FA). The flow rate was set to 0.5 ml min^–1 and the injection volume for each sample was 10 µl. Following injection, samples were eluted using the following gradient: 0–1.0 min, 5% B; 9.0–11.0 min, 100%; 11.5–14.0 min, 5%.