Tocochromanols were determined using an HPLC (Agilent 1200 series, Palo Alto, CA, USA) equipped with a fluorimetric detector (FLD) as previously described by Ben Lajnef et al. [11 (link)]. A total of 50 mg of oil was dissolved in 0.5 mL of n-hexane, and then the extracts were filtered using a 0.45 µm nylon filter. The chromatographic separation was carried out using a HILIC Poroshell 120 column (100 mm × 3 mm and 2.7 μm particle size; Agilent Technologies, Palo Alto, CA, USA) under isocratic conditions with n-hexane/ethyl acetate/acetic acid (97.3:1.8:0.9 v/v/v) as the mobile phase and with a flow rate of 0.8 mL/min.
The identification of tocochromanols was conducted by setting the excitation wavelength at 290 nm and the emission wavelength at 325 nm. Tocopherols were identified via co-elution with the standards, whereas tocotrienols were identified by comparing their retention times with that of tocotrienols in a barley extract obtained via hot saponification. α-Tocopherol standard solutions (from 1 to 100 μg/mL) were used for calibration curve construction for quantification. Two replicates for each lipid extract (n = 4) were performed.
The identification of tocochromanols was conducted by setting the excitation wavelength at 290 nm and the emission wavelength at 325 nm. Tocopherols were identified via co-elution with the standards, whereas tocotrienols were identified by comparing their retention times with that of tocotrienols in a barley extract obtained via hot saponification. α-Tocopherol standard solutions (from 1 to 100 μg/mL) were used for calibration curve construction for quantification. Two replicates for each lipid extract (n = 4) were performed.