2.5.2. HPLC analysisThe tocols were

2.5.2. HPLC analysis
The tocols were quantified according to the method reported by Lampi et al. (2004) with some modification. Tocols were separated by a normal phase HPLC using a GRACE Altima HP Silica
150 _ 3 mm, 3 micron column and quantified with a fluorescence detector (NP-HPLC–FLD) with an excitation wavelength of 290 nm and an emission wavelength of 325 nm. The mobile phase
was 1,4-dioxane/n-hexane (2:98, v/v) at a flow rate of 1 mL/min. Separation of tocols was based on isocratic elution (Lampi et al., 2004). The quantity of the individual vitamin E isomers in samples was
calculated by the use of calibration curves for all standard isomers. Tocopherol (T) standards (a-, b-, c-, d-T) and tocotrienol (T3) standards (a-, b-, c-, d-T3) were purchased from Cayman Chemical,
USA, while n-hexane, ethyl acetate, ascorbic acid and DPPH (2,2- diphenyl-1-picrylhydrazyl) were from Chem-Supply Pty. Ltd., Australia. Standard stock solutions were prepared to a concentration
of 500 lg/mL in hexane. Calibration curves for all isomers were prepared over the concentration range of 1.0–25.0 lg/mL using GenStat 14 (Lawes Agricultural Trust; VSN International, Ltd., Hemel Hempstead, UK). Barley extractions and standard isomers were injected into the HPLC machine separately. Identification of peaks in barley samples was made based on the retention time when compared with standard peaks. Curves between standard peaks and standard contents
were used to calculate contents of isomers in barley samples. The vitamin E content, expressed in mg of a-tocopherol-equivalents (TE), was calculated according to Mclaughlin and Weihrauch
(1979) using biological activities of 1.0 for a-T, 0.3 for a-T3, 0.4 for b-T, 0.05 for b-T3, 0.1 for c-T, 0.01 for c-T3 and 0.01 for d-T (a- TE = a-T * 1.0 + a-T3 * 0.3 + b-T * 0.4 + b-T3 * 0.05 + c-T * 0.1 + c-T3
* 0.01 + d-T * 0.01).