Determination of Brassinolide by HPLC equipped with Fluoresence Detector in Rice(Oriza sativa L.)

HPLC 형광분석법을 통한 벼에서 Brassinolide의 검정

To determine brassinolide in rice(Oriza sativa L.) using HPLC equipped with fluoresence detector, a highly sensitive fluorescence reagent. 1-cyanoisoindole-2-m-phenylboronic acid, was synthesized from the reaction of o-phthaldehyde, m-phenylboronic acid and KCN, then was reacted with brassinolide. The formation ratio of brassinolide boronate exhibited 90% up at the ratio of $20\;:\;1({\mu}g/{\mu}g)$ of 1-cyanoisoindole-2-m-phenylboronic acid and brassinolide respectively. The detection limit of brassinolide boronate with fluoresence detector was 0.16 ng. Brassinolide was detected in heading stage(biomass : 10 g) and panicle formation stage(biomass : 100 g) of the rice(Oryza sativa L.) with quantity of $0.8\;{\mu}g\;and\;0.2\;{\mu}g$respectively. However, brassinolide was not detected in blooming and elongation stage.

벼의 생육 단계별 brassinolide의 검정을 위하여 HPLC 형광분석법을 개발하였다. 형광분석에 민감한 1-cyanoisoindole-2-m-phenylboronic acid를 o-phthalaldehyde와 m-phenylbornic acid 및 KCN과 반응시켜 합성하여 brassinolide의 1, 2-diol과 유도체화 시켰다. Brassinolide $1\;{\mu}g$에 대한 1-cyanoisoindole-2-m-phenylboronic acid의 brassinolide boronate의 형성정도는 $20\;{\mu}g$에서 90% 이상의 유도체화를 보였고 형광검출법에 의한 brassinolide의 검출한계는 0.16 ng이었다. 벼의 생육단계별 brassinolide는 분얼기 및 신장기에서는 검출이 되지 않았으며 유수형성기에는 $0.8\;{\mu}g$(생체 10g)이 그리고 출수개화기에는 $0.2\;{\mu}g$(생체 10g)이 검출되었다.