Agilent 5973 ms

Monosaccharide composition analysis (as acetylated methyl glycoside) and determination of their absolute configuration (as octyl-glycosides derivatives) was performed on 1×10¹⁰ viral particles, as previously reported (De Castro et al. 2010 (link)). Gas chromatography-mass spectrometry (GC-MS) analyses were performed on an Agilent instrument (GC instrument Agilent 6850 coupled to MS Agilent 5973) equipped with a SPB-5 capillary column (Supelco, 30 m × 0.25 i.d., flow rate, 0.8 mL min^–1) and He as the carrier gas. Electron impact mass spectra were recorded with an ionization energy of 70 eV and an ionizing current of 0.2 mA. The temperature program used for analyses was as follows: 150°C for 5 min, 150 to 280°C at 3°C/min, 300°C for 5 min. Interpretation of these data is based on the following concept: each sugar that is derivatized as acetylated methyl-glycoside or octyl-glycoside is eluted in a specific range of the chromatogram and each peak corresponds to a specific fragmentation pattern, which enables identification of the monosaccharide (Lönngren and Svensson 1974 ). Elution time is compared with those of standard monosaccharides and allows discrimination between sugars of the same class (i.e. glucose and mannose) and between sugars with different absolute configuration (D or L).

The system used for sample analysis comprised an Agilent 6890 N GC connected to an Agilent 5973 MS and a deconvolution reporting system (version A.02.00; Agilent Technologies) for compound identification. The method for compound analysis and identification is identical to that described in detail by Thöming et al. (2014 (link)). The identity of six selected compounds [dimethyl disulfide, β-myrcene, o-xylene, 3-hexanol, (Z)-3-hexen-1-yl-acetate and 1,3-di-tert-butylbenzene] was verified by comparing mass spectra, Kovats index, and retention time with those obtained for synthetic standards (Sigma-Aldrich, Zwijndrecht, the Netherlands) on the same column. Relative amounts of identified compounds were calculated by dividing the peak area (using the area from the total ion chromatogram) by the area of the internal standard heptyl acetate. Only compounds found exclusively in treatments with D. radicum-infested plants were analyzed further, i.e., we excluded all compounds found in control samples (empty bags and soil) and UP.

GC-MS analysis was carried out using an Agilent 6890N coupled to an Agilent 5973 MS (Agilent, Massy, France). Samples were analyzed on a fused-silica capillary column HP-1 (polydimethylsiloxane, 50 m × 0.20 mm i.d. × film thickness 0.33 µm; Interchim) and INNOWAX (polyethyleneglycol, 50 m × 0.20 mm i.d. × film thickness 0.4 µm; Interchim). Operation conditions: carrier gas, helium constant flow 1 mL/min, injector temperature, 250 °C, split ratio, 1:100, temperature program, 45 °C to 250 °C or 230 °C, at 2 °C/min then held isothermal (20 min) at 250 °C (apolar column) or 230 °C (polar column), ion source temperature, 230 °C; transfer line temperature, 250 °C (apolar column) or 230 °C (polar column); ionization energy, 70 eV; electron ionization mass spectra were acquired over the mass range 35–400 amu.

Aliquots of freeze-dried maize material, corresponding to 2.0 g fresh weight, were extracted according to the protocol of Gobel et al. (2003) (link) and methylated with trimethylsilyl diazomethane (2 M in hexane, Sigma-Aldrich, Taufkirchen, Germany). As an internal standard, (6Z,9Z,11E,13S)-13-hydroxy-6,9,11-octadecatrienoic acid was added. Hydroxyl fatty acids were purified on reverse phase-HPLC equipped with ET250/2 Nucleosil 120-5 C18 column (Macherey-Nagel, Dueren, Germany) as described in Gobel et al. (2003) (link). Eluate fraction was collected between 8 and 13.5 min, evaporated to dryness and re-dissolved in 2 μL acetonitrile. After addition of 2 μL N,O-bis(trimethylsilyl)trifluoroacetamide (Sigma-Aldrich, Taufkirchen, Germany), analysis was carried out with an Agilent 6890 gas chromatograph equipped with a capillary DB-23 column (Agilent, Waldbronn, Germany, nominal diameter: 0.25 mm, length: 30 m, nominal film thickness: 0.25 μm) and coupled with an Agilent 5973 MS. Standard curves were constructed by plotting ion intensities vs. molar amounts of known hydroxyl fatty acids.

One Tenax sorbent cartridge from each animal was used for GC/MS analysis. A 50 mg sample of Tenax was extracted from each cartridge, and mixed with 0.5 mL hexane solvent. Each sample was sonicated for 10 minutes and the solvent then decanted into a GC vial. Analysis was performed using an Agilent 6890 GC coupled with an Agilent 5973 MS. Five microliters of sample solvent were injected into the GC in pulsed spitless mode. The inlet port temperature was 235°C, and the pulse pressure was 206.8 kPa (30 psi) for 0.5 minutes. The carrier gas was helium delivered with an average velocity of 59.0 cm/s. The column used was a DB-5 ms 30 m×250 µm column with a film thickness of 0.25 µm (J&W Scientific, Agilent Technologies, Santa Clara, CA, USA). Analytes were eluted from this column using a thermal gradient starting at 30°C and ramping at a rate of 5°C/1.0 min to a final temperature of 150°C. The total GC run time was 26.5 min. The temperature of the transfer line was 280°C. The MS was operated in positive ion mode, performing a total ion scan ranging from 10 to 550 m/z with a threshold of 150 m/z at a scan rate of 20 Hz. The MS source was operated at 230°C with the quad set to 150°C. Data were generated as raw Agilent.dat files.