Accela uplc system

Chromatographic analysis was performed using a Thermo Fisher Accela UPLC system (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a quaternary pump solvent management system, an online degasser, a diode-array detector (DAD), a column compartment, and an autosampler using a Phenomenex UPLC Kinetex C₁₈ column (2.1 × 100 mm, 1.7 μm). The flow rate was maintained at 0.2 mL/min with a volume of injection of 3 μL. The mobile phase consisted of an aqueous solution of 0.1% formic acid (A) and acetonitrile (B) with an elution gradient of 5%-25% B from 0 to 5 min, 25%-60% B from 5 to 28 min, 60%-90% B from 28 to 38 min, and 90% B between 38 and 42 min. Detection wavelengths were set at 214, 254, and 280 nm, with a column oven temperature set at 25°C.

S. albus strains were cultivated in 25 mL TSB medium for 48 h at 28 °C. The main cultures containing 50 mL of SG were inoculated with 2 mL of preculture. After 5 days of cultivation at 28 °C, the secreted metabolites were extracted with ethyl acetate and butanol, followed by solvent evaporation. The dry extracts were dissolved in 0.5 mL methanol, and 1 μL of the dissolved sample was separated in a Dionex Ultimate 3000 RSLC system using a BEH C18, 100 × 2.1 mm, 1.7 μm dp column (Waters Corporation, Milford, MA, USA). The separation of a 1 μl sample was achieved via a linear gradient with a mobile phase of water/acetonitrile, each containing 0.1% formic acid, with a gradient from 5–95% acetonitrile applied over 9 min at a flow rate of 0.6 mL/min and 45 °C. High-resolution mass spectrometry was performed on an Accela UPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a LTQ Orbitrap XL mass spectrometer ( Thermo Fisher Scientific, Waltham, MA, USA) operating in positive or negative ionization modes. Data were collected and analyzed with Thermo Xcalibur software, version 3.0 (Thermo Scientific, Waltham, MA, USA). The monoisotopic mass was searched in a natural product database.

Chromatographic separation was performed on an Accela UPLC system (Thermo Scientific) equipped with a Kinetex PFP column (100 × 4.6 mm, particle size 2.6 μm; Phenomenex). The elution was performed with the same HPLC-DAD gradient mode as described previously using water (solvent A), MeOH (solvent B), and ACN (solvent C), all acidified with 0.1% formic acid. The LC flow rate was 1 mL/min; the injection volume was 5 μL. The column oven temperature was 45°C and the sample tray temperature 4°C.
Eluted compounds were detected in negative mode in full mass scan (m/z 120 to 900) using a Thermo Scientific Orbitrap Mass Spectrometer Q Exactive equipped with a heated electrospray ionization source (HESI-II). Instrument parameters were as follows: sheath gas 60, auxiliary gas 20 (both arbitrary units), spray voltage 4 kV, capillary temperature 275°C, capillary voltage −60 V, tube lens voltage −135 V, skimmer voltage −20 V, and source temperature 300°C.
Mass spectra were recorded at a resolution of 50,000 with an automatic gain control (AGC) target of 500,000 and a maximum injection time of 500 ms.