Determination of biuret and allantoin in rice seedlings
At harvest, the rice roots were rinsed for 3 mins, three times with 100
mL of distilled water. Several seedlings were combined into a single
sample, blotted and dried with paper towels, separated into shoots and
roots, weighed, and freeze-dried. After determining the dry weights, the
samples were ground into a powder using a ball mill.
About 10 mg of the powdered sample was extracted with 250 µL of
distilled water. After centrifugation, a 35 µL aliquot of the
supernatant was mixed with 465 µL of acetonitrile and centrifuged again.
A 20 µL aliquot of the supernatant was injected into the HPLC system
(LC-10AS; UV detector: SPD-10A, Shimadzu, Kyoto, Japan) equipped with a
hydrophilic interaction chromatography (HILIC) column (YMC-Triart
Diol-HILIC, 5µL, 4.6 x 250 mm, YMC Co. Ltd., Kyoto, Japan). The
isocratic eluent was a mixture of 930 mL of HPLC-grade acetonitrile and
70 mL of distilled water. In some experiments, we modified the eluent to
a mixture of 940 mL of acetonitrile and 60 mL of distilled water to
improve the separation. In this case, a 94:6 mixing ratio was used for
sample preparation. Elution was performed at a flow rate of 0.5 mL
min-1, and the effluent was monitored at 190 nm. The
colorimetric determination of allantoin was performed as described by
Young and Conway (1942).