Izumi Yoshihiro, Okazawa Atsushi, Bamba Takeshi, Kobayashi Akio, Fukusaki Eiichiro
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.
Anal Chim Acta. 2009 Aug 26;648(2):215-25. doi: 10.1016/j.aca.2009.07.001. Epub 2009 Jul 4.
In recent plant hormone research, there is an increased demand for a highly sensitive and comprehensive analytical approach to elucidate the hormonal signaling networks, functions, and dynamics. We have demonstrated the high sensitivity of a comprehensive and quantitative analytical method developed with nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-IT-MS/MS) under multiple-reaction monitoring (MRM) in plant hormone profiling. Unlabeled and deuterium-labeled isotopomers of four classes of plant hormones and their derivatives, auxins, cytokinins (CK), abscisic acid (ABA), and gibberellins (GA), were analyzed by this method. The optimized nanoflow-LC-ESI-IT-MS/MS method showed ca. 5-10-fold greater sensitivity than capillary-LC-ESI-IT-MS/MS, and the detection limits (S/N=3) of several plant hormones were in the sub-fmol range. The results showed excellent linearity (R(2) values of 0.9937-1.0000) and reproducibility of elution times (relative standard deviations, RSDs, <1.1%) and peak areas (RSDs, <10.7%) for all target compounds. Further, sample purification using Oasis HLB and Oasis MCX cartridges significantly decreased the ion-suppressing effects of biological matrix as compared to the purification using only Oasis HLB cartridge. The optimized nanoflow-LC-ESI-IT-MS/MS method was successfully used to analyze endogenous plant hormones in Arabidopsis and tobacco samples. The samples used in this analysis were extracted from only 17 tobacco dry seeds (1mg DW), indicating that the efficiency of analysis of endogenous plant hormones strongly depends on the detection sensitivity of the method. Our analytical approach will be useful for in-depth studies on complex plant hormonal metabolism.
在最近的植物激素研究中，对于一种高度灵敏且全面的分析方法来阐明激素信号网络、功能及动态变化的需求日益增加。我们已经证明了一种采用纳流液相色谱 - 电喷雾电离 - 离子阱质谱（LC - ESI - IT - MS/MS）在多反应监测（MRM）模式下开发的全面定量分析方法在植物激素分析中的高灵敏度。通过该方法分析了四类植物激素及其衍生物、生长素、细胞分裂素（CK）、脱落酸（ABA）和赤霉素（GA）的未标记和氘标记的同位素异构体。优化后的纳流 - LC - ESI - IT - MS/MS方法的灵敏度比毛细管 - LC - ESI - IT - MS/MS高约5 - 10倍，几种植物激素的检测限（S/N = 3）在亚飞摩尔范围内。结果显示所有目标化合物具有出色的线性（R²值为0.9937 - 1.0000）以及洗脱时间的重现性（相对标准偏差，RSDs，<1.1%）和峰面积的重现性（RSDs，<10.7%）。此外，与仅使用Oasis HLB柱净化相比，使用Oasis HLB和Oasis MCX柱进行样品净化显著降低了生物基质的离子抑制效应。优化后的纳流 - LC - ESI - IT - MS/MS方法成功用于分析拟南芥和烟草样品中的内源植物激素。本分析中使用的样品仅从17粒烟草干种子（1mg干重）中提取，这表明内源植物激素的分析效率在很大程度上取决于该方法的检测灵敏度。我们的分析方法将有助于深入研究复杂的植物激素代谢。
