Sb aq column

The glucose concentration was measured using an SBA-40D biosensor analyzer (Institute of Biology of Shandong Province Academy of Sciences, Shandong, China). The cell concentration was determined by measuring the absorbance at 600 nm (OD₆₀₀) using a spectrophotometer (V-1100D; Mapada Instruments, Shanghai, China). The dry cell weight (DCW) per liter was calculated using an experimentally determined formula: DCW (g/L) = 0.27 × OD₆₀₀ [77 (link)]. The amino acids in the culture supernatant were determined using high-performance liquid chromatography with a Zorbax Eclipse XDB-C₁₈ column (4.6 mm × 150 mm, 5 μm; Agilent) at 40 °C and 360 nm after derivatization with 2,4-dinitrofluorobenzene. Mobile phase A was 55% (v/v) acetonitrile, and mobile phase B consisted of 40 mmol/L KH₂PO₄ at pH 7.0–7.2. The flow rate of the mobile phase was 1 mL/min. The organic acids in the culture supernatant were determined using high-performance liquid chromatography with a SB-Aq column (4.6 × 250 mm; 5 μm; Agilent) at 40 °C and 210 nm. Mobile phase A was acetonitrile, and mobile phase B consisted of 20 mmol/L KH₂PO₄ at pH 2.3. The flow rate of the mobile phase was 1 mL/min.

50 adult aphids were feeding on plant for 0 and 48 h, five biological replicates were used, and a previously established method was employed (Qi et al., 2016 (link)). In brief, samples were extracted with 80% methanol and then analyzed on an Agilent 1200 Rapid Resolution Liquid Chromatography (RRLC) system equipped with a ZORBAX SB-Aq column (2.1 × 100 mm, 1.8 μm) followed by an Agilent 6510 Q-TOF running in positive ionization mode. The column temperature was set at 50°C, and the flow rate was 0.3 mL min⁻¹. All metabolites were identified based on pure standards, except some Bxs: DIMBOA was confirmed by comparing the Bx profiles between maize inbred line H88 and bx1 mutant (GRMZM2G085381); DIMBOA-Glc, DIM₂BOA-Glc, HDMBOA-Glc, and MBOA were confirmed with purified standards, and the other putative Bxs were identified by their molecular masses. The abundance of each metabolite was estimated from the peak area. Peak detection and matching were performed using bioconductor XCMS and CAMERA packages (http://www.bioconductor.org/.) Samples (metabolites after integration) with P < 0.05 were considered to be differentially regulated. The acquired data were introduced to the SIMCA-P software package (v11.5, Umetric, Umea, Sweden) for Principal Component Analysis (PCA). The data was filtered by orthogonal signal correction (OSC) to remove variations from non-correlated factors.

Cell density was measured by determining the absorbance at 600 nm (OD₆₀₀) using a spectrophotometer (V-1100D; Mapada Instruments, Shanghai, China). The concentration of glucose was assayed using an enzyme electrode analyzer (SBA-40D; Institute of Biology, Shandong, China). After dilution and filtration of the culture supernatant and cell extract with ddH₂O, nucleoside metabolites were determined using a high-performance liquid chromatography (HPLC) system (Agilent, USA) with an SB-AQ column (4.6 × 250 mm, 5 μm, Agilent) at 33 °C. Mobile phase A was 100% (v/v) methanol, whereas mobile phase B consisted of 0.5% (W/V) KH₂PO₄ at a pH of 4.5. HPLC was performed using a ratio of 90% A and 10% B at 1 mL/min with a monitor at 360 nm. All measurements were performed at least in triplicate and standard deviations (SD) were calculated from three independent experiments.

Samples were analyzed with the QTRAP 5500 LC/MS/MS system (AB Sciex, Foster City, CA, USA) with the 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA) including a degasser, an autosampler, and a binary pump. The LC separation was performed on an Agilent SB-Aq column (4.6 mm × 50 mm, 5 µ) (Santa Clara, CA, USA) with mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile). The flow-rate was 0.35 mL/min. The linear gradient was as follows: 0–1 min, 100% A; 8–11 min, 25% A; 12–18 min, 100% A.
The autosampler was set at 5 °C and the injection volume is 5 µL. Positive mass spectrometry was achieved with electrospray ionization (ESI). The ion spray voltage was 5500 V. The source temperature was 400 °C. The curtain gas, ion source gas 1, and ion source gas 2 were 32, 65, and 50, respectively. Multiple reaction monitoring (MRM) was used to quantify O⁶ hydroxyethyldeoxyguanosine (m/z 312.2 àm/z 110.1), N⁷ hydroxyethylguanine (m/z 196.1 àm/z 110.1), and the internal standard deuterated O⁶ methylguanine (m/z 169.1 àm/z 70.1) (Figure 1).

The FP was dissolved in pure methanol and filtered through a 0.22 μm membrane before HPLC-Q-TOF-MS analysis. The liquid chromatography separation was performed on an Agilent SB-Aq column (250 mm × 4.6 mm, 4.5 μm, 400 bar) at 30°C. For HPLC analysis, 0.1% formic acid/water (v/v) and 0.1% formic acid/methanol were used as the mobile phases A and B, respectively. The gradient elution was programmed as follows: 0–110 min (35–90% B) and 110–120 min (90% B). The flow rate was 1.0 mL/min and the injected sample volume was 10 μL. The Q-TOF-MS scan range was set at m/z 100–1200 in positive modes. The dry gas (N₂) flow rate was 9.0 L/min, the dry gas temperature was 350°C, the nebulizer gas was set at 30 psi, the capillary voltage was 3500 V, the fragmentor was 175 V, and the skimmer was 65 V.
Standard pinocembrin, kaempferol, isosakuranetin, pinobanksin-3-O-acetate, 12-acetoxyviscidone, galangin, and chrysin (purity > 98% by HPLC) were purchased from the National Institutes for Food and Drug Control of China. They were mixed and dissolved in pure methanol at the concentration of 2 μM per compound prior to use.
Data analysis was performed on an Agilent MassHunter Workstation Software-Qualitative Analysis (version B.04.00, Build 4.0.479.5, Service Pack 3, Agilent Technologies, Inc., 2011).