2.5. GC–MS analysis of fatty acid compositions
GC-MS was used for fatty acid composition of RBO analysis. The sample pretreatment was carried out according to the method reported by Zhang et al. (2010). GC-MS analysis of FAMEs according to the method of China National Standard GB 5009.168-2016. The analysis were performed by a QP2010 gas chromatography/mass spectrometer (Shimadzu, Kyoto, Japan), equipped with a TR-WAXMS capillary column (30 m × 0.25 mm × 0.25 μm). The specific operating conditions were as follows: injector temperature 250 ℃; carrier gas was high purity nitrogen; flow rate 1 mL/min; split ratio 1:10; the column temperature from 80 ℃ to 210 ℃ at the rate of 15 ℃/min; finally increased to 240 ℃ at the rate of 2 ℃/min; ion source temperature 200 ℃; detector temperature 240 ℃; mass range 40-500 m/z. The components of oil were determined by comparison of their retention indices with those of authentic standards of FAMEs.
2.6.Bioactive compounds analysis
2.6.1. Chromatographic analysis of vitamin E
The content of vitamin E in oil samples was determined by HPLC. Oil was diluted with n-heptane and analyzed using Agilent Technologies 1260-HPLC apparatus (Santa Clara, CA, USA) equipped with a fluorescent detector from the same manufacturer. Separation was carried on a Venusil XBP Silica column (4.6 × 250 mm × 5 μm). The mobile phase was a 3.85% tetrahydrofuran in n-heptane solution with a flow rate of 1 mL/min. The fluorescence detector was set at λexcitation= 294 nm and λemission = 330 nm.
2.6.2. Gas chromatography analysis of sterols
According to the modified method of Nyam, Tan, Che Man, Lai & Long (2009), the content of sterols was determined by the gas chromatography. Oil samples and 5α-cholestane solution were refluxed with ethanolic potassium hydroxide solution for 80 min. The unsaponifiables were first extracted with diethylether for three times, the ether layers were washed twice with 0.5 M KOH and deionized water, and then dried with anhydrous sodium sulphate. The extract was evaporated in a vacuum evaporator at 25 °C. N, O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) was added to the dry extracts, and the mixtures were heated at 60 °C for 60 min. The analysis was carried out using the GC-2010 PLUS (Shimadzu, Kyoto, Japan). Sterols were separated on a HP-1 capillary column (25 m × 0.32 mm × 0.17 μm; Agilent, Santa Clara, CA, USA). The temperatures were as follows: injector 250 °C, column 190 °C increased to 230 °C at 20 °C/min, then to 255 °C at 40 °C/min, and maintained for 17 min.