Extraction was performed according to Pascual et al. (2013)
with slight modification of an increase of the number of extractions.
In brief, 2 g of rice flour (exact to 0.0001 g) was mixed with
30 mL of HPLC grade methanol for c-oryzanol extraction and
MeOH:hexane (50:50) for tocols extraction in a capped test tube.
The mixture was vortexed for 1 min, sonicated for 15 min and then
centrifuged at 4600 g for 15 min. All steps were performed under a
controlled temperature at 25 C, and the supernatant was collected.
The extraction was performed three times with the same
conditions. The supernatant was combined, and the fraction was
evaporated at 30 C until complete dryness was achieved. The residue
was redissolved with 3 mL of HPLC grade MeOH and sonicated
in a sonicated water bath (Branson 1800, USA) at 25 C for 10 min
to increase the solubility of tocols and c-oryzanol in MeOH. Then,
the aliquot was filtered through a 0.22 lm membrane filter and
transferred into an HPLC vial for HPLC analysis.