100 mm acquity uplc beh c18 1.7 μm column

To extract the secondary metabolites, streptomycetes strains were grown in 15 mL of TSB in a 100 mL flask for 2 days, and 1 mL of preculture was inoculated into 100 mL of production medium in a 500 mL flask. The strains were grown for 7 days at 28 °C and 180 rpm in an Infors multitron shaker (Infors AG, Basel, Switzerland). After cultivation, the secondary metabolites were extracted with an ethyl acetate and acetone:methanol (1:1) mixture. The obtained extracts were evaporated using an IKA RV-8 rotary evaporator (IKA, Staufen, Germany) at 40 °C and dissolved in 1 mL of methanol. The extracts were analysed on a Dionex Ultimate 3000 UPLC system (ThermoFisher Scientific, Waltham, MA, USA) coupled to a PDA detector using a 100 mm ACQUITY UPLC BEH C₁₈ 1.7 μm column (Waters Corporation, Milford, MA, USA). The extracts were separated by a linear gradient (from 5% to 95%) of water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B) as the mobile phase at a flow rate of 0.6 mL/min for 18 min. Mass analysis was performed on Bruker Amazon Speed (Bruker, Billerica, MA, USA) and Thermo LTQ Orbitrap XL (ThermoFisher Scientific, Waltham, MA, USA) mass spectrometers using the positive mode of ionization and a range detection of 200–2000 m/z. Data were analysed using Compass Data Analysis v. 4.2 (Bruker) and Xcalibur v. 3.0 (ThermoFisher Scientific).