Briefly, alkaline hydrolysis of cracker
powder (5.00 g) was carried out in potassium hydroxide/methanol
solution with added antioxidant (BHT). The mixture was heated to
boiling in round flask with reflux condenser in dark and simmering
is continued for 20 min. Free tocopherols were extracted from the
reaction mixture using n-hexane, and the extract was washed with
water until free of alkalis. After evaporation to dryness, the
obtained extract was dissolved in HPLC grade methanol (10 mL),
and filtered through 0.45 mm pore size PTFE filters (RotilaboSpritzenfilter
13 mm, Roth, Karlsruhe, Germany) before injection
into the HPLC system.
HPLC analysis was performed using the same equipment and
column, as described for determination of phenolic compounds.
Methanol was used as a mobile phase at a flow-rate of 1.000 mL/
min. Separated compounds were detected at 295 nm with reference
wavelength set at 550/100 nm and the spectra were acquired
in the range 210e400 nm. Run time was 10 min, and the post time
5 min. The column temperature was 30 C, and 5 ml of the sample
was injected using autosampler. Identification of a-, g- and dtocopherol
was achieved by comparing their retention time and
spectra with those of standards. The external standard method was
used for quantification.