2.4. Tocopherols analysis
Approximately 50 mg of hempseed oil was dissolved in 0.5 ml ofn- hexane. After homogenization, the solution was filtered through a 0.2 μm nylon filter and 2.5 μl was injected in a HPLC 1200 series (Agilent Technologies, Palo Alto, California, USA) equipped with a fluorimeter detector (Agilent, Palo Alto, CA, USA). The excitation wavelength was 290 nm and the emission wavelength was 325 nm. The separation of tocopherols was performed by a HILIC Poroshell 120 column (100 mm × 3 mm and 2.7μm particle size; Agilent Technologies, USA), in isocratic conditions, using an n -hexane/ethyl acetate/acetic acid (97.3:1.8:0.9 v/v/v) mobile phase. The flow rate was 0.8 ml/min. Tocopherols were identified by co-elution with the respective standards. The calibration curve used for quantification was constructed with α-tocopherol standard solutions. Tocopherols composition was measured in 2 replicates for each lipid extract (n = 4) and expressed in mg/100g of oil; each analysis lasted 8 minutes.