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).