Shim pack gist c18 hp reversed phase column

Active fractions were analyzed for their purity using an analytical Shimadzu LC-2030 system with PDA detection (190–800 nm) with a Shimadzu Shim-pack GIST C18-HP reversed-phase column (3 μm, 4.6x100 mm) (Shimadzu's Hertogenbosch, the Netherlands). Besides determining the purity, the UV-VIS spectrum of the fractions was also obtained. Pure fractions were further analyzed by measuring the mass using a Shimadzu LC-2030C 3D plus system, sometimes followed by more accurate high-resolution mass spectrometry (HRMS) measured on an LCT instrument (MicroMass ltd, Manchester UK). For HRMS, the sample was mixed with sodium formate for the detection of sodium adduct ions. In addition, this procedure gave an internal calibrant to each sample to facilitate a more accurate measurement of the mass of the sample. Obtained masses and UV spectra were compared with previously identified samples and literature. If needed, further chemical analysis using NMR measurements was performed. For the NMR measurements, the fractions were dried in a speed-vac overnight and dissolved in DMSO-d₆ before measurements on a Bruker 600 MHz.

Secondary metabolites from 6-day-old A. parvulus or 5-day-old A. oryzae transformant liquid culture filtrates were isolated with a 1:1 volume of ethyl acetate (VWR Chemicals, Radnor, PA). After shaking on an orbital shaker for at least 1 h, the organic phase was transferred to a 50-mL tube and evaporated under nitrogen flow. The resulting solid was dissolved in acetonitrile. Organic extracts were analyzed with a Shimadzu LC-2030 3D-Prominence-i PDA system coupled to a Shimadzu LCMS-2020 mass spectrometer and equipped with a Shimadzu Shim-pack GIST C₁₈-HP reversed-phase column (3 μm, 4.6 by 100 mm). The following method was used: a linear gradient of buffer B (0 to 95%) for 20 min, 5 min of 95% buffer B, and then 100% buffer A for 5 min. Water with 0.1% trifluoroacetic acid (TFA) for high-performance liquid chromatography (HPLC) or 0.05% formic acid for mass spectrometry (MS)-coupled analyses was used as buffer A, and acetonitrile (LCMS grade) with 0.1% TFA for HPLC or 0.05% formic acid for MS-coupled analyses was used as buffer B. The flow rate was 1 mL/min or 0.5 mL/min for HPLC or MS-coupled analyses, respectively. The equipment was controlled and results were analyzed using Shimadzu LabSolutions LCMS software.

Liquid cultures or agar plates were extracted with ethyl acetate. The solvent was evaporated and the residue was dissolved in DMSO. Extracts were fractionated using a modular preparative high-performance liquid chromatography (HPLC) system (Shimadzu) using a C18 reversed phase Reprosil column (10 μm, 120 Å, 250 × 22 mm). The mobile phase was 0.1% trifluoroacetic acid in water (buffer A) and 0.1% trifluoroacetic acid in acetonitrile (buffer B) in a linear gradient. Fractions were collected, dried in an Eppendorf speedvac concentrator, dissolved in DMSO and tested for antimicrobial activities.
The identification procedure was performed as previously described^{20 (link)} using preparative HPLC (Shimadzu) using a Shimadzu Shim-pack GISTC18-HP reversed phase column (3 μm, 4.6 × 100 mm) coupled to a LCMS-2020 mass spectrometer (Shimadzu). High resolution mass spectrometry (HRMS) was measured on either a µQTOF instrument (Micromass Ltd) or an LCT instrument (Micromass Ltd). Samples were dissolved in DMSO-d₆ or CDCl₃ for NMR spectroscopy. ¹H-NMR, HSQC, HMBC and COSY spectra were measured at either 300 MHz, 400 MHz, 500 MHz or 600 MHz using either a Mercury-300, an Agilent-400, an INOVA-500 or a Bruker-600 spectometer. ¹³C-NMR was measured using the same instruments at either 100 MHz or in case the Bruker-600 instrument was used at 150 MHz.