Eclipse plus

The ABA and proline extraction was performed on 10mg of freeze-dried leaf tissue as described by Forcat et al. (2008 (link)). The samples were analyzed for ABA and proline using LCMS/MS, and were filtered through a 0.45μm cellulose acetate syringe. The phytohormones separation was done using a C18 column (ZORBAX Eclipse Plus). An injection of 2 μl was loaded onto the C18 column (1.8μm particle size, 2.1mm inner diameter, and 50mm long) at a flow rate of 0.2 ml/min and the column temperature was kept at 35°C. The liquid chromatography was connected to an Agilent Technologies Mass Spectrometry (6420 Triple Quad detector). For elution, solvent A consists of formic acid (0.1%) with distilled water and solvent B consists of an LCMS grade acetonitrile were used. The analytical procedure was as follows.
Solvent A was used (5min), then the gradient from 0 to 100% solvent B was used (5–20min), after the solvent B was kept constant (5min) and at 25.1min solvent A was 100% was used for 30min. During the analysis with LC–MSMS only negative polarity mode was used for ABA and Proline analysis. For fragmentation, nitrogen gas was used. The capillary voltage was 4,000V, the gas flow was 8 L/min, the gas temperature was 300°C and the nebulizer pressure was 45 psi.

After removal of solvent using the GeneVac Series II and addition of 25% methanol to enhance solubility the polyene samples (candicidin, filipin) were analysed by electrospray mass spectrometry (ESI-MS) using an LTQ-FT (Thermo) mass spectrometer with a 7 T magnet at the Pinnacle Laboratory (University of Newcastle). Experiments were run with a parent/precursor scan at 100,000 resolution. MS/MS fragmentations were carried out in the ion-trap (LTQ) stage of the instrument. Streptothricin and candicidin were analyzed on an LC-MS (Bruker micrOTOF, Agilent 1260 HPLC system, Zorbax Eclipse Plus column (3.5 μm 100 × 4.6 mm).

The method from Thumkasem et al.^{4 (link)} was used for carotenoid extraction. The fermented broth (1.5 mL) was centrifuged at 7378×g for 5 min, and the resulting cell pellet was washed with distilled water and dried in a freeze-dryer (Gold-sim, Miami, USA). Carotenoids were then extracted using 850 µL of Dimethyl sulfoxide (DMSO) and 600 µL of acetone. The clear crude extract solution was recovered after centrifuging at 7378×g for 5 min, and β-carotene was quantified using High-Performance Liquid Chromatography (HPLC) (1260 Infinity II, Agilent Technology, Santa Clara, USA) using a modified method of Khumrangsee et al.²⁸ . The HPLC analysis was performed using a C18 reverse-phase column (ZORBAX Eclipse Plus, 150 mm × 4.6 mm × 5 µm) and a diode array detector at a wavelength of 450 nm. The mobile phase consisted of acetonitrile, dichloromethane, and methanol (7:2:1, v/v/v) at a flow rate of 1 mL/minute. The carotenoid concentration was evaluated using a calibration curve of a β-carotene standard curve (Sigma-Aldrich, USA).

Quantitation of extracted phenols was made by a reverse-phase HPLC (Agilent Technologies, Wilmington, DE, United States) equipped with a UV detector. Briefly, 20 μl injection volume was separated by a C18 column (ZORBAX Eclipse Plus, 95 Å, 4.6 × 250 mm, 5 μm, 959990-902) using isocratic elution program. CUR was detected at 425 nm and eluted with a mobile phase containing acetonitrile–water–acetic acid (50:49:1 v/v/v) at a flow rate of 1 ml/min and 20°C. CAP was UV detected at a wavelength of 281 nm and eluted using with a mobile phase containing methanol and water (80:20 v/v) at a flow rate of 1 ml/min and 20°C. Calibration curves was compiled based on seven samples of standard solutions at varying concentrations (R² = 0.9987), so as to enable quantification of CUR or CAP.

Type A Trichothecenes were detected using the method previously described by Pascale et al. [27 (link)]. T-2 and HT-2 toxins analysis was performed using a UHPLC (Agilent UHPLC system, 1290 Series). Both data acquisition and instrument control were performed by LC Openlab software (Agilent). For chromatographic separation, a reversed-phase column of C₁₈ (50 × 2.1 mm i.d., 1.8 μm, ZORBAX Eclipse Plus) was used. Analyses were performed in the gradient mode. Solvent A was water and solvent B acetonitrile. Gradient conditions were initiated by holding for the first 1.5 min with 30% B, and then solvent B was linearly increased to 35% in 0.5 min and kept constant for 2 min. The flow rate was 0.5 mL/min, and the injection volume was 10 μL. The column temperature was maintained at 50 °C, and the detector was set at 202 nm wavelength. Retention times were 1.97 min and 4.9 min for HT-2 and T-2, respectively. The mycotoxins were quantified by comparing peak areas with calibration curves obtained with standard solutions. The detection limit (LOD) based on a signal-to-noise ratio of 3:1 for both toxins was 0.24 μg/g.