Gc fid system

Metabolite analysis was performed on a Waters Acquity ultraperformance liquid chromatography machine with diode array detection (UPLC-DAD) using methods and software described in the Waters Corporation user manual. The manual was adapted for oilseed rape tissue by [34 (link), 35 (link)]. The AccQtag method was used to quantify amino acids and the integration software Empower (Waters Corporation, Milford, USA) was used for analysis. Samples were resuspended in 100 mL distilled water. Subsequently, 5 mL were derivatized using AccQTag Ultra Derivatization Kit, according to the manufacturer’s recommendations. An external standard of 100 mmol/L of each amino acid was run every 10 samples. Quantification of sugars was performed using a gas chromatography-flame ionization detector (GC-FID) System from Agilent Technologies (Santa Clara, CA, USA) according to [36 (link)]. The integrated Agilent software ChemStation Rev.B.04.02 was used for data analysis. Samples were resuspended in 50 mL pyridine (100%) with methoxamine hydrochloride (240 mmol/L), then derivatized with 50 mL MSTFA (N-methyl-N-(trimethylsilyl)trifluoro acetamide) (100%). An external standard containing 400 mmol/L of each sugar, sugar alcohol and organic acid was run every 10 samples.

The analysis of fatty acids was performed using GC-FID system (Agilent Technologies) with H₂ as a carrier gas. Methylation was performed on the samples (∼5 mg) in advance by adding 3 mL of methanol & 5% H₂SO₄ at 100 °C for 1 hour. Then, the methylated products were extracted using hexane that contained C15:0 methyl ester as an internal standard. The samples were then run through Nukol™ column (30 m × 0.53 mm × 1.0 μm, Supelco) with a split ratio of 0.1 : 1 and split flow of 3.55 mL min⁻¹. The oven temperature profile was 90 °C to 200 °C at 44.08 °C min⁻¹ and held for 7.5 minutes. In this study, the total amount of fatty acids was assumed as the amount of lipid.