Gc 2010 plus instrument

Seeds from each developmental stage were weighted and dried overnight at 60 °C, then were grounded, extracted with 1 mL of hexane for 1 h, centrifuged at 13,000 rpm for 10 min, and the upper suspensions were transferred to new tubes. This process was repeated for three times. To determine the profiles of fatty acids, extracted lipids were trans-methylated in 0.8 mL petroleum ether and 0.5 mL 5% H₂SO₄ (v/v, H₂SO_4: methanol) and the resulting fatty acids were analyzed using GC-MS method (GC-2010, plus instrument, Shimadzu, Japan) with a flame ionization detector on a DB-23 column (60 mm × 0.32 mm ID× 0.25 μm df, Agilent Technologies, Waldbronn, Germany) with the following parameters: column oven temperature 170 °C and flame ionization detector set as 280 °C. Fatty acid content was expressed as percentage of total fatty acids.

The oxysterol concentration was measured according to previous reports^{14 (link),37 (link)}. Briefly, lipids were extracted using chloroform/methanol (2:1, v/v) containing butylated hydroxytoluene. After overnight saponification, unsaponified lipids were extracted with hexane. The extracted lipids were applied to a Sep-Pak Silica Vac cartridge (Nihon Waters) to separate oxysterols from chol. After evaporating the oxysterol-containing solvent fraction under N₂, dried residues were converted into trimethylsilyl ethers. Oxysterol was quantified by gas chromatography–mass spectrometry using a Shimadzu GC-2010 Plus instrument (Shimadzu Corporation, Kyoto, Japan) coupled with an Inert Cap 5MS/NP capillary column (30 m × 0.25 mm i.d., 0.25 μm thick, GL Sciences Inc, Tokyo, Japan.) connected to a QP2020 series mass-selective detector (Shimadzu). The concentrations of individual oxysterols were measured using 19-hydroxycholesterol (Steraloids, Inc. Newport, RI, USA) as the internal standard. The oxysterols analyzed in this study are listed in Table S4.

A Shimadzu GC-2010 Plus instrument coupled to a Shimadzu QP2010 Ultra mass spectrometer (Shim-pol, Warsaw, Poland) was used for GC-MS analyses. The chromatograph was equipped with a fused-silica capillary column ZB-5 MS (30 m, 0.25 mm i.d.) with a film thickness of 0.25 mm (Phenomenex, Torrance, CA, USA). The oven temperature program was started at 50 °C, held for 3 min, then increased at the rate of 8–250 °C/min, and held for a further 2 min. The MS was operated in EI mode; the scan range was 40–500 amu, the ionization energy was 70 eV, and the scan rate was 0.20 s per scan. The injector (250 °C), interface (250 °C), and ion source (220 °C) temperatures were set. Split injection was performed with a split ratio of 1:20. Helium was the carrier gas at a 1.0 mL/min flow rate. Each of the 9 EOs samples were prepared by diluting 2 µL of EO in 1 mL of hexane. An internal standard was added to each sample. Three parallel measurements were done. The relative percentages of each component present in the EOs were calculated. The retention indices were determined in relation to a homologous series of n-alkanes (C₈–C₂₄) under the same operating conditions. The compounds were identified with computer-assisted spectral libraries (MassFinder 2.1 Hamburg, Germany; NIST 2011, Gaithersburg, MD, USA).