用改进的SPE-LC-ESI-MS/MS法测定小麦叶片ABA含量  

Determination of ABA Content in Wheat Leaves with the Method of Improved SPE-LC-ESI-MS/MS

作　　者：苏适[1] 李瑞航[1] 郎丹莹 王军卫[2,3] 郭蔼光[1,3] 徐虹[1,3] 

机构地区：[1]西北农林科技大学生命科学学院,杨凌712100 [2]西北农林科技大学农学院,西安712100 [3]旱区作物逆境生物学国家重点实验室西北农林科技大学,西安712100

出　　处：《分子植物育种》2015年第8期1850-1856,共7页Molecular Plant Breeding

基　　金：国家自然基金面上项目(31371541)资助

摘　　要：为了优化液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)定量分析脱落酸(ABA)的方法,实现快速、准确测定小麦叶片内源ABA含量,本研究建立以超纯水作为溶剂、4℃避光抽提12 h、C18小柱固相萃取(SPE)富集ABA、LC-ESI-MS/MS系统检测、以甲醇/0.2%甲酸水溶液(v/v=4:1)作为LC流动相等度洗脱的定量方法。电喷雾离子源(ESI)采用负离子模式,ABA的质谱(MS)检测应用多反应监测模式(MRM),选择的监测离子对为262.8-152.7。利用ABA标准品检测结果显示:在7.5~60 ng/m L浓度范围内,ABA峰面积与ABA含量线性关系良好,检出限(S/N=3)为4.7 ng/m L。20 ng加标水平的平均回收率达到了98.61%。用该体系测定了小麦返白系及其对照材料矮变1号在常温与低温培养条件下,苗期叶片中内源ABA的含量,结果揭示了两个材料间的差异。本研究结果表明,优化的SPE-LC-ESI-MS/MS体系可以用于小麦叶片中脱落酸的分析,具有样品前处理简便、回收率高、检测灵敏的优点。Here we present a optimize liquid phase chromatography with electro spray tandem mass spectrometry(LC-ESI-MS/MS) method for fast and highly sensitive determination of endogenous ABA content in wheat leaves.The ultrapure water was used as solvent,and the leaf tissues were extracted in dark at 4℃ for 12 h. Then,ABA fraction was enriched by C18 solid phase extraction column and was quantified by LC-ESI-MS/MS system. The methanol/0.2% formic acid(4:1,v/v) were used as mobile phase for isocratic elution of C18 reversed-phase column.The electro spray ionization(ESI) operated in negative ion mode,and the monitor mode was multiple reaction monitoring(MRM),themonitoredion pairswas 262.8 152.7. ABA standard test indicated good linearityin7.5~60 ng/m L and 4.7 ng/m L detection limits on a signal-to-noise ratio of three. We got the average recovery rate of this system98.61% by the method of 20 ng ABA standards addition. Using this system,we determined the endogenous ABA content in the leaves of wheat albino line and parent variety Aibian1 seedlings at room and low temperature,and with exogenous ABA treatment. The results showed the differences between two cultivars. So,we can conclude that the optimized SPE-LC-ESI-MS/MS system can be used for the analysis of trace amounts of ABA in wheat leaves,and has the advantages of simple sample pretreatment,higher recovery and sensitive detection.

关 键 词：脱落酸定量检测 液相色谱-电喷雾串联质谱法 小麦返白系 苗期叶片 

分 类 号：S512.1[农业科学—作物学]