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.