Alliance chromatographic system

Cultures of selected strains or mutants were extracted with ethyl acetate containing 1% formic acid (to enhance the extraction of compounds containing ionizing groups) and analysed by reverse phase chromatography with an Acquity UPLC instrument fitted with a BEH C18 column (1.7 μm, 2.1 × 100 mm; Waters, Barcelona, Spain), using acetonitrile (AcN) and aqueous 0.1% trifluoroacetic acid (TFA) as eluents. The program uses an isocratic hold of 10% AcN for 1 min, followed by a linear gradient up to 100% AcN over 7 min, at a flow rate of 0.5 ml min⁻¹ and a column temperature of 35°C.
For HPLC‐MS analysis, an Alliance chromatographic system coupled to a ZQ4000 mass spectrometer and a SunFire C18 column (3.5 μm, 2.1 × 150 mm; Waters) was used. Solvents were the same as above, and elution was performed with an initial isocratic hold with 10% AcN during 4 min followed by a linear gradient of AcN (10% to 88%) over 30 min, all at 0.25 ml min⁻¹. MS analysis was carried out by positive mode electrospray ionization (ESI), with a capillary voltage of 3 kV and a cone voltage of 20 V. Spectral identification and characterization of peaks were performed in both cases by photodiode array detection at 330 nm, using Empower software (Waters) to extract bidimensional chromatograms at different wavelengths, depending on the spectral characteristics of the desired compound.

Whole cultures were extracted with ethyl acetate containing 1% formic acid (to enhance the extraction of compounds containing ionizing groups) and analysed by reversed phase chromatography in an Acquity UPLC instrument fitted with a BEH C18 column (1.7 μm, 2.1 × 100 mm; Waters), with acetonitrile and 0.1% trifluoroacetic acid (TFA) as solvents. Samples were eluted with 10% acetonitrile for 1 min, followed by a linear gradient from 10% to 100% acetonitrile over 7 min, at a flow rate of 0.5 ml min⁻¹ and a column temperature of 35°C. For HPLC-MS analysis, an Alliance chromatographic system coupled to a ZQ4000 mass spectrometer and a SunFire C18 column (3.5 μm, 2.1 × 150 mm; Waters) was used. Solvents were the same as above and elution was performed with an initial isocratic hold with 10% acetonitrile during 4 min followed by a linear gradient from 10% to 88% acetonitrile over 26 min, at 0.25 ml min⁻¹. MS analysis were done by electrospray ionization in the positive mode, with a capillary voltage of 3 kV and a cone voltage of 20 V. Detection and spectral characterization of peaks was performed in both cases by photodiode array detection in the range from 200 nm to 500 nm, using empower software (Waters) to extract bidimensional chromatograms at different wavelengths, depending on the spectral characteristics of the desired compound.

Cultures of selected strains or mutants were extracted with ethyl acetate containing 1% formic acid (to enhance the extraction of compounds containing ionizing groups) and analysed by reverse phase chromatography with an Acquity UPLC instrument fitted with a BEH C18 column (1.7 µm, 2.1 × 100 mm, Waters), using acetonitrile (AcN) and aqueous 0.1% trifluoroacetic acid (TFA) as eluents. The program uses an isocratic hold of 10% AcN for 1 min, followed by a linear gradient up to 100% AcN over 7 min, at a flow rate of 0.5 mL/min and a column temperature of 35 °C.
For HPLC–MS analysis, an Alliance chromatographic system coupled to a ZQ4000 mass spectrometer and a SunFire C18 column (3.5 µm, 2.1 × 150 mm, Waters) was used. Solvents were the same as above and elution was performed with an initial isocratic hold with 10% AcN during 4 min followed by a linear gradient of AcN (10–88%) over 30 min, all at 0.25 mL/min. MS analysis was done by positive mode electrospray ionization (ESI), with a capillary voltage of 3 kV and a cone voltage of 20 V. Spectral identification and characterization of peaks was performed in both cases by photodiode array detection at 330 nm, using Empower software (Waters) to extract bidimensional chromatograms at different wavelengths, depending on the spectral characteristics of the desired compound.