Lc 2000 plus series

Each purified trichothecene was applied to analytical HPLC (LC-2000 plus series; JASCO, Corp., Tokyo, Japan; UV detection at 254 nm or 195 nm) equipped with a C₁₈ column (Pegasil ODS SP100 4.6φ × 250 mm). Trichothecenes were eluted in a mobile phase of H₂O-acetonitrile using an appropriate gradient mode at 40 °C at a flow rate of 1 mL/min. The concentration of each trichothecene was calculated from the corresponding peak area based on the standard curve, if available. If the standard curve was not available, each purified toxin was dried completely using a freeze dryer (TITEC Corp., Saitama, Japan) and weighted. In some cases, quantitative NMR was performed, as previously described [66 (link)].
For LC–MS/MS analysis, samples were separated using an Eksigent ekspert™ ultraLC 100-XL system (Dublin, CA, USA) with a C₁₈ reverse phase column (PEGASIL ODS SP100-3; 2φ × 100 mm) according to the following steps: a linear gradient of 10–95% of acetonitrile in 5 min in 0.1% ammonium formate at a flow rate of 0.3 mL/min. The ultraLC was connected to an AB SCIEX Triple TOF 4600 system (Framingham, MA, USA), with a DuoSpray source operated in electrospray ionization mode. The IDA and/or time-of-flight (TOF)–MS methods were used to obtain MS/MS spectra in the positive ion mode. Data were analyzed using the PeakView software version 1.2.0.3 (AB SCIEX).

Supernatant from the BSG fermentation (100 μl) was obtained by centrifugation (8,000 ×g, 5 min, 4°C), followed by dansylation at 80°C for 40 min after addition of 1 M sodium carbonate–sodium bicarbonate buffer (pH 10.0; 200 μl), dansyl chloride dissolved in acetone at 20 mg/ml (100 μl), and distilled water (600 μl). Then, 10%(v/v) acetic acid (100 μl) was added to terminate the reaction. After centrifugation, the reaction solution was filtered with a 0.2-μm syringe filter (Merck Millipore, USA) and injected into a high-performance liquid chromatography (HPLC) system for analysis at 254 nm and 30°C [29 (link)]. A JASCO HPLC system (JASCO LC-2000 Plus Series, Japan) with a photodiode array detector, an autosampler (JASCO AS-2055 Plus), and an AccQ-Tag C18 column (3.9 × 150 mm, 4 μm; Waters Corporation, UK) was used. Eluent A was prepared by mixing tetrahydrofuran, methanol, and 50 mM sodium acetate–acetic acid buffer (pH 6.2) at a ratio of 1:15:84 (v:v:v), and eluent B was methanol. Eluents A and B were applied at a flow-rate of 1 ml/min for 5 min, 16 min, and 4 min at ratios of 80:20, 45:55, and 0:100, respectively [29 (link)]. The GABA content was calculated using calibration curves determined at five concentrations with GABA standard material (Sigma-Aldrich, USA). All experiments were conducted three times.