4.3. Metabolomics

Each seed (weighted using a microbalance) was extracted with inox beads in an Eppendorf tube, with methanol:water (80:20, v:v) with ribitol as an internal standard. After centrifugation, 20 µL of supernatant were poured into a vial (with insert) and spin-dried at 39 °C. Gas chromatography/mass spectrometry (GC–MS) analyses were carried out using a GC–MS-Orbitrap Q Exactive (Thermo Scientific, Waltham, MA, USA). Samples were derivatized (automatically with a preparative robot) with 20 µL of methoxyamine (20 mg mL⁻¹ in pyridine; 90 min at 37 °C) and then 30 µL N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA) for 30 min at 37 °C. Prior to injection, 5 µL of alkane mix (14 alkanes from C₉ to C₃₆, 3 µg µL⁻¹, Connecticut n-Hydrocarbon Mix, Supelco) were added to each sample to compute the retention index. Analyses were performed by injecting 1 µL in splitless mode at 230 °C (injector temperature) in a TG-5 SILMS column (30 m × 0.25 mm × 0.25 µm; Thermo Scientific) set in a Trace 1310 Series GC (Thermo Scientific). Helium was used as gas carrier with a constant flow of 1 mL min⁻¹. After one minute at initial GC oven temperature (70 °C), temperature was raised to 325 °C at 15 °C min⁻¹ and finally kept at 325 °C for 4 min. MS analyses were operated in positive polarity in full MS scan mode with the following source settings: mass scan range 50–750 m/z, resolution 60,000, AGC target 1E6, MS transfer line 300 °C and filament delay 4.12 min. Ionisation by electron impact (70 eV) was performed at 250 °C ion source temperature. Metabolites were identified automatically using TraceFinder (Thermo Scientific) using retention time, major characteristic fragment (m/z ion) and a confirmation fragment, with a maximum tolerance of 0.00007 Da. Two (2) quality control (QC) samples (mix of all samples) were injected each 10 samples to check that GC-MS response was constant across sample batches. Additionally, within batches, samples were randomised to avoid any batch-related bias. Metabolomics data were normalised to the internal standard and seed weight.