Solvent extraction of 2AP from brown rice
The rice grain samples were husked by hand to yield brown rice seeds on the day of experiment. The brown rice seeds were ground and then screened through a 35-mesh sieve where crude particles were filtered out. Five grams of the ground rice powder was weighed and three replications of each sample were carried out. The samples were then placed to 125-ml flasks containing 50 ml of 0.25 mg/l 2,4,6-trimethylpyridine internal standard solutions. Each mixture was stirred for 30 min at room temperature. After filtration of the rice seed particles, 40 ml of the filtrate was transferred to a 125-ml pear-shaped separatory funnel, followed by the addition of approximately 1.2 ml of 5.0 M NaOH to make the solution slightly basic. Then, 50 ml of dichloromethane was immediately added as an organic solvent. The extraction was carried out twice and 90.0 ml of the combined dichloromethane portions collected. After being dried with anhydrous sodium sulfate, this extract was concentrated to 1 ml by removal of dichloromethane using a rotary evaporator under reduced pressure at a temperature of 28 °C. The resulting concentrated extract was transferred to a V-shaped vial and left open to the air at room temperature until its volume decreased to 0.1 ml before it was subjected to quantitative analysis by capillary GC with a flame ionization detector (FID). The whole experimental process was repeated for the extraction and analysis of standard 2-acetyl-1-pyrroline of known amounts in a dilution series in order to obtain a standard calibration curve.