HPLC Analysis of Tocopherol Profiles

Analysis of tocopherols was performed by HPLC according to Brazaityte et al.’s [25 (link)] methodology with some modifications. About 1 mg of oil was weighed in an Eppendorf tube, and then 1 mL of n-hexane with 1% of BHT was added to the tube. Afterward, the samples were filtrated through a 0.45 µm polytetrafluoroethylene (PTFE) membrane syringe filter (VWR International, Radnor, PA, USA) and were analyzed by HPLC/FLD (fluorescence detector) (Agilent Technologies, Santa Clara, CA, USA). The HPLC measurements were performed using a normal phase column (Phenomenex Luna Silica, 5 μm, 250 mm × 4.6 mm). The HPLC 10A system, equipped with an RF-10A fluorescence detector (Shimadzu, Japan), was used for analysis. Peaks were detected at an excitation wavelength of 295 nm and an emission wavelength of 330 nm. The mobile phase (0.5% isopropanol in hexane) was used at a flow rate of 1 mL min⁻¹. The α-tocopherol, γ-tocopherol, and δ-tocopherol were identified according to the analytical standard. The α-tocopherol, γ-tocopherol, and δ-tocopherol content were expressed per 100 g of oil.