Tocols in the grain, digesta, and micellar fraction were identified and quantified using a SpectraSystem HPLC instrument (Thermo Separation Products, Inc., Waltham, MA, USA) equipped with a quaternary gradient pump (P4000), an autosampler (AS3000), and an FL detector (FL3000). Tocol separation was achieved with the isocratic elution of the mobile phase on two sequentially connected C18 columns: a Vydac 201TP54 column (5 µm, 4.6 × 150 mm; Hichrom, Reading, UK) and a Zorbax RX-C18 column (5 µm, 4.6 × 150 mm; Agilent Technologies, Santa Clara, CA, USA). The aforementioned columns were protected by a Supelguard Discovery C18 guard column (5 µm, 4 × 20 mm; Supelco, Bellefonte, PA, USA). The mobile phase used was an acetonitrile:dichloromethane:methanol (75:20:5, v/v/v) solution containing 0.1% BHT and 0.05% triethylamine. The flow rate was 1.8 mL/min and 30 µL of the sample was injected. The tocols were monitored at an extinction of 290 nm and an emission of 330 nm.
Tocols were identified by comparing their retention times and were quantified via external standardization with calibration curves, using commercially available standards (Sigma-Aldrich, St. Louis, MO, USA; purity ≥ 96%; r2 ≥ 0.98 for all tocols). The total tocol content was calculated by summing the contents of the individual tocols.
Tocols were identified by comparing their retention times and were quantified via external standardization with calibration curves, using commercially available standards (Sigma-Aldrich, St. Louis, MO, USA; purity ≥ 96%; r2 ≥ 0.98 for all tocols). The total tocol content was calculated by summing the contents of the individual tocols.