在线固相萃取-高效液相色谱-串联质谱法同时检测大豆不同部位的4种植物激素

An Automated Dual-gradient Liquid ChromatographyMS / MS Method for Simultaneous Determination of Four Kinds of Plants Hormones in Different Parts of Soya Beans and Its Application to a Real Analysis

采用双三元液相色谱（ Dual gradient liquid chromatography,DGLC）建立了在线固相萃取技术与电喷雾串联质谱联用方法（ Online SPE DGLC-ESI MS/MS）,并成功应用于实际样品检测。本方法同时检测大豆不同部位中的4种酸碱性植物激素（赤霉素（GA3）、吲哚乙酸（IAA）、玉米素（ZT）和脱落酸（ABA））。通过考察固相萃取富集柱、分析色谱柱、流动相对植物激素的保留和选择性的影响,获得较高的灵敏度、回收率、稳定性及精密度。大豆样品经液氮低温研磨,以80％甲醇溶液提取,再经离心稀释过滤后,进样分析。进样后样品经在线固相萃取Hypersep Retain AX柱洗脱保留,目标分析物依次转移至分析柱Acclaim PA2色谱柱,并以0.1％甲酸和甲醇溶液作为流动相进行梯度洗脱,采用选择反应监测离子模式（ SRM）同时采集正负离子通道进行定性分析,基质标准曲线外标法进行定量分析, GA3, IAA, ZT 在0.1～50μg/L 范围内线性良好,检出限为0.0002μg/g；ABA在0.5～50μg/L的范围内线性良好,其检出限为0.0010μg/g。以0.8,4.0和40μg/L分别为低、中、高浓度考察4种植物激素的回收率为76.1％～93.5％,RSD为0.8％～6.0％。结果表明,籽粒中含有的ABA浓度明显高于其它部位。本研究为快速准确地分离和测定大豆不同部位内源激素提供了有效方法。

There are some common analysis challenges in the hormone detection in agriculture science, including matrix interference, complicated sample preparation, poor reproducibility, trace analyte content. An automated on-line SPE and innovative fast polarity switch analysis method employing dual-gradient liquid chromatography （ DGLC ） coupled with tandem mass spectrometry （ DGLC-MS/MS ） was established and validated for the simultaneous determination of gibberellic acid （ GA3 ） , indole acetic acid （ IAA ） , zeatin （ ZT） and abscisic acid （ ABA） in the soybean plant （ leaf, grain and pod） . The method was applied in the actual sample detection successfully. In order to acquire higher sensitivity, recovery, stability and precision, some conditions including SPE column, analytical column, mobile phase, additive etc were optimized according to the selection and retain of hormone. Beans were cryogenically grinded by liquid nitrogen, extracted by 80% methanol, certrifugatel and dilluted with water, and then injected directly. Samples were transported and gradient eluted on the analytical column Acclaim PA2 by 0 . 1% formic acid in water and methanol, after retaining and separation on the SPE column Hypersep Retain AX. All analytes were detected in selection reaction monitoring （ SRM） mode in both positive and negative channels. The quantification was based on linear regression. The linear ranges of GA3, IAA and ZT were 0. 1-50 μg/L with the LOQ of 0. 0002 μg/g, and the linear of ABA was 0. 5-50μg/L with the LOQ of 0.0010μg/g. The recoveries of four kinds of plants hormones were 76 . 1%-93 . 5%, and RSDs were 0 . 82%-6 . 02% at low （ 0 . 8 μg/L ） , medium （4. 0μg/L） and high （40μg/L）. The results noted that the content of ABA in seeds was apparently higher than others. This method could be used for the rapid and accurate detection of hormone in different parts of soya beans.