Pegasus 4d

The amount of 10.0 mg of the extracted sample was mixed with 100 µL of methyl nonadecanoate solution, which was used as the internal standard in anhydrous tert-butyl methyl ether (0.2 mg/mL) and 100 µL of trimethylsulfonium hydroxide solution ~0.25 M in methanol. The solution was incubated at 80 °C for 30 min, and 1 µL of the output mixture was subjected to GC-MS analysis. The GC-MS determination was performed with a gas chromatograph coupled with a time-of-flight mass spectrometry detector (Pegasus 4D, LECO, St. Joseph, MI, USA).
The operating parameters were set up as follows: Stabilwax-DA capillary column (length 20 m, internal diameter 0.18 mm and 0.18-µm film thickness; Restek Corp., Bellefonte, PA, USA). The oven temperature program was 50–245 °C at 4° min⁻¹, carrier gas helium and flow rate 1 mL min⁻¹. Identification of the lipids was based on the comparison of their mass spectra with data available from the commercial database, and the retention times of the methyl esters were compared with the standards (37 Component FAME Mix, Supelco, St. Louis, MO, USA; Cat. No. CRM47885). The contents of the individual components were expressed as a percentage of the total extracted lipids.

Volatile fingerprinting of the fecal samples was performed using an Agilent 7890B (Agilent Technologies, United States) gas chromatograph coupled to a Pegasus 4D (LECO, United States) time of flight mass spectrometer. Volatiles were collected using a solid-phase microextraction (SPME) fiber with divinylbenzene/carboxen/polydimethylsiloxane coating from Supelco (United States). Data acquisition and initial data processing were carried out using instrumental SW ChtomaTOF by LECO (United States).

The aroma in fruits was comprehensively analysed using static headspace extraction coupled with separation by two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS, LECO Pegasus 4D, Castle Hill, Australia). Briefly, sample (500 mg) was weighed into 20 mL silicon capped GC headspace vials (Restek, Germany) and kept at −80 °C until further analysis. 2.5 mL headspace syringe was used to collect 1.5 mL of sample headspace after sample agitation of 10 min at 80 °C. An empty vial was used as blank and quality assurance (QA) standard was prepared by mixing all the samples. The program settings, conditions and parameters are provided in Table S2 in Supplementary Materials [41 (link)]. LECO ChromaTOF 4.50 software was used to process the GC×GC-TOFMS data for pre-processing baseline correction and identification was conducted by library matching (NIST 11 v2.0) and from authentic reference standards created in an in-house library. The similarity of ≥80% with the NIST library was defined as putative identification (when standards were not available) [42 (link),43 (link),44 (link)].

Six replicate samples, mutants and their Ds donor lines were obtained from unique dishes. Metabolites were extracted from 50 mg of frozen plant material with CHCl₃:MeOH:H₂O (2:6:2) that included 10 stable isotope reference compounds. Metabolites were derivatized by methoxyamination for 22 h, using a 20 mg/ml solution of MeONH₃ in pyridine. The derivatives were subsequently trimethylsilated with N-methyl-N-trimethylsilyl-trifluoacetamide for 1 h. An n-heptane mixture (30 μl) was used for the determination of retention time indices. Samples were injected in splitless mode (2 μl/sample) and analyzed using GC-TOF/MS (Pegasus 4D; Leco, St. Joseph, MI, USA). Artifact peaks were manually identified and removed from the obtained peaks. Remaining peaks in each mass spectrum were transformed using × 1,000 and + 1 in order to form a normal distribution. Peak intensities were transformed using a logarithmic scale with a factor of 10.

The comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOF-MS) analyses were performed on an Agilent 7890 gas chromatograph equipped with a TOF-MS (Pegasus 4D, LECO Corporation, Joseph, MI, USA) detector and a LECO ChromaTOF program. The chromatographic separation was achieved using two different polar capillary columns, a conventional nonpolar column DB-5MS (29.950 m × 0.25 mm × 0.25 mm) from J&W Scientific (Folsom, CA, USA) and a medium column DB-17HT (1.640 m × 0.1 mm × 0.1 mm) from the same company. The connection between the two GC columns is a typical combination in GC × GC, which could produce ordered and orthometric chromatograms. According to the previous studies, the initial oven temperature was set to 60 °C for 1 min, then the temperature increased by 5 °C/min up to 165 °C and then increased again by 25 °C/min until it reached the final temperature of 280 °C and then held for 14 min. The mass spectrometer was operated at an acquisition rate of 100 spectra per second, ranging from 20 to 400 u. The electron impact ionization energy was 70 eV and the acquisition voltage was 1700 V. The temperature for the ion source was set to 220 °C.

The GC/MS analysis was performed using a 7890 GC (Agilent Technologies) coupled to a time of flight (TOF) MS (LECO Pegasus 4D, St. Joseph, MI, USA). The isolate was analyzed on two alternate column chemistries, namely DB-5 and DB-Wax. For the DB-5 analysis analogous column and oven conditions were as previously described. For the DB-Wax (60 m × 0.25 mm i.d. × 0.25 µm film thickness, Agilent Technologies) the GC conditions were as follows: 0.5 µL was injected into an inlet heated to 250 °C. The GC oven temperature program was as follows: initial conditions 40 °C followed by a 5 °C/min ramp to 250 °C and then held for 10 min, flow at 1 mL/min.
Quantification was carried out using five-point calibration curves for each of the 18 compounds in the following concentration ranges (µg/kg) listed, hexanal (50–800), 2-methylpyrazine (55–80), 2,3-dimethylpyrazine (51–815), 2,5-dimethylpyrazine (54–860), 2-methyl-2thiazoline (61–975), 2-pentylfuran (43.5–775), 2-ethyl-3,5-dimethylpyrazine (52.5–840), 3-hydroxy-2-methyl-4H-pyran-4-one (44–700), 2-methylphenol (60–965), 2-acetyl-2-thiazoline (61–975), (E,E)-2,4-decadienal (92.5–1480), 2-methoxy-4-vinylphenol (438–7000), 4-hydroxy-3-methoxybenzaldehyde (612–10000), all curves had high linearity (R² > 0.98 for all compounds) as similarly descripted by Trikusuma et al. [30 (link)].