超高效液相色谱串联质谱法同时测定作物中三种内源性植物激素

Application of ultra-performance liquid chromatography-tandem mass spectrometry for the simultaneous determination of three endogenous plant hormones in crops

采用超高效液相色谱串联质谱法(UPLC-MS/MS)对主粮及杂粮三种内源激素(脱落酸ABA、水杨酸SA、茉莉酸JA)进行了测定分析。以甲醇水为提取液,加入内标,在4℃条件下震荡提取。质谱条件采用了电喷雾离子源ESI、多反应监测模式测定、负离子检测模式及内标曲线法定量,相关系数R2为0.996 2~1.000 0,定量限范围为0.05~0.13 pg/g。该方法线性关系良好,灵敏度高。该文还利用该方法测定了玉米、大麦、小麦、谷子、荞麦等多种作物的激素,以及128份水稻种质资源激素,筛选出多个优质种质。该实验方法前处理过程操作简单,分析时间短,灵敏度高,适用于多种作物的激素检测。

[Objective]Selected appropriate hormone pretreatment and detection methods were utilized to determine the stress-resistant hormone content in various germplasm resources.High-quality germplasm resources were screened,which laid the groundwork for subsequent studies on crop hormone metabolism pathways and provided technical support.[Methods]The plant sample was ground into powder using liquid nitrogen,and 50 mg of the plant powder was accurately weighed and loaded into a 2.0 mL centrifuge tube.An extraction solution of 80%methanol,along with an internal standard,was added,and the extraction was conducted at 4°C and 300 rpm for 40 min.Following this,centrifugation was performed at 4°C and 14,000 rpm for 10 min.Upon the formation of two phases,the liquid supernatant was carefully removed and transferred to a new 2.0 mL centrifuge tube.The process was repeated a second time.Subsequently,the liquid was filtered using a 0.22μm organic nylon filter membrane and transferred to a brown vial for sampling.Finally,the sample was analyzed using a machine.The distribution of three endogenous hormones(abscisic acid,salicylic acid,and jasmonic acid)in staple grains and coarse grains was assessed through ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry(UPLC-MS/MS).Electrospray ion source ESI,multiple reaction monitoring mode,negative ion detection mode,and internal standard curve quantification were employed.The correlation coefficient(R2)ranged from 0.9962 to 1.0000.The limit of quantification ranged from 0.05 to 0.13 pg/g,indicating a good linear relationship and high sensitivity of the method.[Results]The extraction efficiency of methanol aqueous solutions with varying concentrations(100%,90%,80%,70%,60%)was examined,and the results revealed that 80%methanol–water yielded the optimal extraction of plant hormones.Additionally,the impact of formic acid content in the mobile phase was evaluated.The chromatographic peak response was highest when the formic acid content was 0.02%,and the tailing phenomenon was also mitigated.The Athena UHPLC C18 Column was chosen for the experiment.The negative ion mode[ESI(−)]was selected for this experiment.Additionally,characteristic ion pairs with higher abundance were chosen,and parameters such as desolvation temperature,collision energy,scan residence time,atomization gas flow,capillary voltage,and cone voltage were optimized to enhance sensitivity.Standard solutions with concentrations of 1,50,and 100 ng/mL were prepared and added to the treated samples,and three parallel samples were set to improve experimental accuracy.Calculation of the recovery rate and relative standard deviation across different treatment groups indicated the reliability of the pretreatment method and the absence of interference in the retention time area,confirming the robust reliability and selectivity of the method.According to the analytical method established in this paper,the levels of abscisic acid,salicylic acid,and jasmonic acid in leaves of different crops were determined,providing scientific and effective support for quality breeding.[Conclusions]This method was employed to determine the hormone levels in various crops such as corn,barley,wheat,millet,and buckwheat.Additionally,it was utilized to analyze 128 hormones in rice germplasm resources,leading to the screening of several high-quality germplasms.The method is characterized by a simple pretreatment process,short analysis time,and high sensitivity,making it suitable for the detection of multiple types of hormones in a variety of crops.