Tc c18

The LC-ESI-MSⁿ analysis was performed with an Agilent 1100 LC system with an LC/MSD Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). A TC-C18 (4.6 mm × 250 mm, 5 μm, Agilent) column was used for analysis. The column temperature was kept at 30 °C. The mobile phases consisted of methanol (A) and water containing 1% formic acid (B). The following gradient condition was used: 0–10 min, 10% A; 10–12 min, 10%–30% A; 12–30 min, 30%–35% A; 30–35 min, 35%–40% A; 35–40 min, 40%–55% A; 40–50 min, 55%–66% A; and 50–60 min, 66%–100% A. The mobile phase flow rate was 0.8 mL/min and the sample injection volume was 10 μL. The detected wavelength was 254 nm. Mass spectra were acquired in both positive and negative ion modes with an ESI source in the range of m/z 100 to 1000. The ESI-MS conditions were: the nebulizer pressure at 45 psi and nitrogen as the drying gas at a flow rate of 10 L/min with a temperature of 350 °C. The capillary voltage was set at 3500 V. Data were acquired by use of Agilent Chemstation software (Agilent Technologies).

For the DBF samples, 0.2 g was extracted in 20 × volumes of 70% methanol by supersonic bath for 30 min, with HQ and DG served as the positive controls. These extractions were then filtered through 0.45 μm thick organic membranes and directly submitted to the HPLC analysis. To do this, we used Agilent equipment (Agilent, 1200 series, United States), with a C₁₈ column (Agilent, TC-C18, 250 mm × 4.6 mm, 5 μm) as the solid phase. The mobile phase contained 0.2% formic acid aqueous solution (A) and chromatographic pure acetonitrile (B), and following this procedure: 0–15 min, B: 5–45%; 15–60 min, B: 45–95%. An ultraviolet detector measured the ultraviolet absorption of the elution at the wavelengths of 254 and 316 nm, against HQ and DG, respectively. Authentic standard compounds, FA and C-7-G, were dissolved in 70% methanol and pure methanol, respectively, and diluted to a concentration of 10 μg/mL. For each sample solution and standard solution, 10 μL was injected for the HPLC assay under the conditions already described above.

The yield of (S)-CHBE was analyzed by HPLC using an Agilent LC 1260 Infinity II: the column was an Agilent TC-C18 (packing size of 5 μm, column length of 250 mm × column inner diameter of 4.6 mm) with an injection volume of 10 μL, and the finalized mobile phases were selected as 80% water (0.1% formic acid) and 20% acetonitrile. The column temperature was set at 25 °C, the flow rate was 0.8 mL/min, and the wavelength of the VWD detector was 220 nm. The retention time of (S)-CHBE was 15.9 min.
The extent of the reaction was expressed by the yield (chemical yield, %), defined as follows: $$Yield=\frac{\mathrm{F}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{u}\mathrm{b}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}\times 100\%$$

The concentration of mycotoxins (DON and two acetylated DONs) and their metabolites in the supernatant from incubation samples was analyzed and quantified. The mycotoxin stock solutions were diluted in acetonitrile and freshly prepared. Calibration curves were built from stock solutions by creating nine standards as follows: 1, 2.5, 5, 10, 25, 50, 100, 250, and 500 µM. The analysis was undertaken using an Agilent 1220 Infinity II system connected to a photodiode array detector (Agilent Technologies, Palo Alto, California, CA, USA). The column was an Agilent TC-C18 (250 mm × 4.6 mm). The flow rate was 1.0 mL/min. 20 μL of each sample was injected with a mobile phase composed of nanopure water (A) and acetonitrile (B). The gradient elution program was as follows: starting condition 100% A, 1.0 min 20% A, 20.0 min 20% A, 25.0 min 100% A, 30.0 min 100% A. The concentration of mycotoxins and their metabolites was obtained by comparing the peak areas to the corresponding built calibration curves at a wavelength of 237 nm.

Using the Agilent 1290 ultra-high performance liquid chromatography system (Agilent Instruments, USA), we identified the major components in PDL. The Agilent TC-C₁₈ (4.6 mm 100 mm, 2.7 µm) chromatographic column was used, and the mobile phase was 0.2 percent acetic acid (A)-acetonitrile (B), with a flow rate of 0.5 mL/min and a column temperature of 30 °C. The detective wavelength of 289 nm was employed.

Equal volumes of samples were mixed well with methanol stored at −20°C. Before HPLC analysis, samples were filtered using 0.22-μm filters.
The HPLC system (Shimadzu, Kyoto, Japan) consisted of an LC-20AT pump and an SPD-20A UV/VIS detector. A reverse phase column (Agilent TC-C18, 4.6 mm × 250 mm, 80 Å, 5 μm) was used. To detect DON and its derivatives, the mobile phase comprised methanol and water (15:85, v/v) at a flow rate of 1.0 ml/min. To detect 3-keto-DON, the mobile phase comprised methanol and water (1:2, v/v). UV/VIS detection was performed at a wavelength of 220 nm. The column was heated to 40°C.

The monosaccharide composition analysis of non-irradiated and 25 kGy-irradiated PSs was carried out according to the method of Honda et al. as modified by Yang et al. [53 (link),54 (link)]. In brief, the PSs were hydrolyzed (4M TFA, 8 h at 110 °C) and then the released monosaccharides were derivatized employing 1-phenyl-3-methyl-5-pyrazolone (PMP) to UV-absorbing products. The resulting PMP derivatives were separated on an Agilent 1220 HPLC system with a UV detector (250 nm). The separation was conducted on an Agilent TC-C18 (4.6 × 250 mm, 5 μm) column with a mobile phase consisting of 50 mM phosphate buffer (Na₂HPO₄-NaH₂PO₄, pH 6.9) and acetonitrile using a gradient elution [54 (link)].