One gram of rice flour was placed in a screw-top tube containing
5 mL distilled water. The mixture was vigorously mixed at
125 rpm for 90 min at 70 C and centrifuged at 8000g at 4 C for
5 min. The supernatant (200 lL) was freeze-dried, and the residue
was dissolved in 40 lL of an ethanol–water–triethylamine (2:2:1)
solution, and 60 lL of an ethanol–water–triethylamine–phenyl
isothiocyanate (PITC) solution (7:1:1:1) was added. This mixture
remained at room temperature for 20 min to allow the formation
of PITC-GABA and was then filtered through a 0.45-lm filter and
analyzed by HPLC according to the method described by Cho,
Chang, and Chang (2007) with slight modifications as follows: A
Shimadzu HPLC instrument (Shimadzu Co., Kyoto, Japan) was
equipped with a Mightysil RP-18 column (GP250-4.6, 5 lm, Kanro
Chemical, Tokyo, Japan), LC-10AD pumps, and an SPD-10AV UV–
visible detector at 254 nm. The elution solvent system was (A)
1.4 mM sodium acetate, 0.1% triethylamine, and 6% acetonitrile
(pH 6.1), and (B) 60% acetonitrile. The column was eluted with a
linear gradient of 0–100% at a flow rate of 1.0 mL/min with (B)
for 50 min. Authentic GABA was used as a control and to make a
standard curve for calculating the amount of GABA in the samples.
All measurements were conducted in triplicate.