衍生化UPLC-MS法测定酸性植物激素  

Determination of Acidic Plant Hormones by Derivative UPLC-MS

作　　者：戴琛 汪瑾[1] 卢亚萍[1] Chen Dai;Jin Wang;Yaping Lu(Experimental Center of Life Sciences,College of Life Sciences,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学生命科学学院,生命科学实验中心,南京210095

出　　处：《植物学报》2022年第4期500-507,共8页Chinese Bulletin of Botany

摘　　要：为有效利用超高效液相-高分辨率飞行时间质谱(UPLC-TOF-MS)对酸性植物激素进行定性定量分析,选取几种有机酸衍生化试剂:2-溴苯乙酮(BP)、2-二甲氨基乙胺(DMED)、1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC)和溴代丙酮基三甲基溴化铵(BTA),分别与脱落酸(ABA)、赤霉素(GA_(3))、吲哚乙酸(IAA)、茉莉酸(JA)和水杨酸(SA)进行衍生化反应。通过比较上述各植物激素的质谱响应值,判断衍生化试剂的效果。将植物激素标准品进行浓度梯度稀释,并与衍生化试剂反应,测定它们的定量限。用衍生效果最佳的试剂与植物粗提液反应,检验其应用效果。结果显示,几种衍生化试剂均可大幅度提高酸性植物激素的质谱响应值,其中DMED衍生后植物激素的质谱灵敏度提高幅度最大,且反应稳定、重复性好。DMED衍生后, ABA、GA3、IAA、JA和SA的定量限分别为0.05、0.2、0.1、0.1和0.5 ng·mL^(-1),与未衍生化相比,分别降低100、25、50、50和10倍,即DMED衍生化使几种植物激素的质谱灵敏度提高10–100倍,显著高于目前报道的灵敏度。将该方法应用于水稻(Oryza sativa)、小麦(Triticum aestivum)和蚕豆(Vicia faba)等植物材料中,有效测定了其酸性植物激素,且灵敏度较衍生前显著提高。该研究建立了一种简单、快速且重现性好的基于衍生化的UPLC-TOF-MS测定方法,大幅提高了多种酸性植物激素测定的灵敏度。This study aims to establish a rapid and efficient method for qualitative and quantitative determination of plant acid hormones by ultra-high performance liquid chromatography/high resolution time-of-flight mass spectrometry(UPLC-TOF-MS). Several derivatization reagents, 2-bromoacetophenone(BP), 2-dimethylaminoethylamine(DMED), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide(EDC) and 3-bromoactonyltrimethylammonium bromide(BTA) were selected for derivatization with abscisic acid, gibberellic acid, indoleacetic acid, jasmonic acid and salicylic acid, respectively. The effect of derivatization reagent was evaluated by comparing the response value of the derivative hormones. The plant hormone standards were diluted in gradient and reacted with the derivatization reagent, and the detection limit and quantitation limit of the derivative hormones were determined. Reagent with the best derivatization effect was reacted with the plant crude extract to test its application effect. All the four derivatized reagents could improve the MS response value of acidic hormone, among which the MS sensitivity of hormone derived by DMED is the best. Reaction with DMED was stable and reproducible. The quantitation limits of ABA-DMED, GA3-DMED, IAA-DMED, JA-DMED and SA-DMED were 0.05, 0.2, 0.1, 0.1 and 0.5 ng·mL^(-1), respectively. Compared with non-derivatized hormones, the quantitative limits of ABA,GA3, IAA, JA and SA were reduced by 100, 25, 50, 50 and 10 times, respectively. So, DMED derivatization increased the mass spectrometry sensitivity of several hormones by 10–100 times. The method was applied to the determination of endogenous acidic hormones in rice, wheat and broad bean, and the sensitivity was significantly higher than that before derivation. A simple, rapid and reproducible UPLC-TOF-MS method based on derivatization was established, which greatly improved the sensitivity of the determination of acidic hormones in plants.

关 键 词：衍生化 高分辨质谱 酸性植物激素 定量测定 

分 类 号：O657.63[理学—分析化学] Q946[理学—化学]