Total tocopherols and tocotrienols content were measured by a Waters HPLC system equipped with a Waters 2487 ultraviolet detector set at an absorbance of 290 nm and a Luna Silica column (150 mm × 4.6 mm, 3 μm particle size) used at 35 °C. The test method was improved based on the method Boso et al. [35 (link)] used. The mobile phase consisted of 0.2% IPA (Isopropanol) and 0.8% acetic acid in n-hexane flowing at 1.0 mL/min. Extracted oil samples (1 g) were diluted with 10 mL n-hexane and injected into the HPLC system.
Preparation of unsaponifiable fraction was used to determine the phytosterols composition following the method used by Iafflice et al. [9 (link),36 (link)]. GC/MS analysis was performed using an Agilent DB-5 capillary column (30 m × 0.25 mm × 0.25 μm). Helium was used as the carrier gas with a flow rate of 1 mL/min. The initial oven temperature was held at 200 °C for 0.5 min then raised to 300 °C at 10 °C/min for 20 min. The transfer line temperature and ion source temperature were 280 °C and 250 °C, respectively. A sample injection of 1 μL was performed in a split mode of 100:1 at 250 °C. Detection was performed in the full scan mode from m/z 50 to 650. Compounds were identified by matching mass spectra and retention times with those pure compounds. NIST Mass Spectra Library was used as a reference.
Preparation of unsaponifiable fraction was used to determine the phytosterols composition following the method used by Iafflice et al. [9 (link),36 (link)]. GC/MS analysis was performed using an Agilent DB-5 capillary column (30 m × 0.25 mm × 0.25 μm). Helium was used as the carrier gas with a flow rate of 1 mL/min. The initial oven temperature was held at 200 °C for 0.5 min then raised to 300 °C at 10 °C/min for 20 min. The transfer line temperature and ion source temperature were 280 °C and 250 °C, respectively. A sample injection of 1 μL was performed in a split mode of 100:1 at 250 °C. Detection was performed in the full scan mode from m/z 50 to 650. Compounds were identified by matching mass spectra and retention times with those pure compounds. NIST Mass Spectra Library was used as a reference.