高级检索
    邓文红, 赵欣蕊, 张俊琦, 郭惠红. UPLC-MS/MS测定植物组织中植物激素的含量[J]. 北京林业大学学报, 2019, 41(8): 154-160. DOI: 10.13332/j.1000-1522.20190052
    引用本文: 邓文红, 赵欣蕊, 张俊琦, 郭惠红. UPLC-MS/MS测定植物组织中植物激素的含量[J]. 北京林业大学学报, 2019, 41(8): 154-160. DOI: 10.13332/j.1000-1522.20190052
    Deng Wenhong, Zhao Xinrui, Zhang Junqi, Guo Huihong. Determination of plant hormones in plant tissues by UPLC-MS/MS[J]. Journal of Beijing Forestry University, 2019, 41(8): 154-160. DOI: 10.13332/j.1000-1522.20190052
    Citation: Deng Wenhong, Zhao Xinrui, Zhang Junqi, Guo Huihong. Determination of plant hormones in plant tissues by UPLC-MS/MS[J]. Journal of Beijing Forestry University, 2019, 41(8): 154-160. DOI: 10.13332/j.1000-1522.20190052

    UPLC-MS/MS测定植物组织中植物激素的含量

    Determination of plant hormones in plant tissues by UPLC-MS/MS

    • 摘要:
      目的植物激素在植物生长发育和响应生物和非生物胁迫过程中起着非常重要的作用,研究植物激素的合成、运输、代谢和分子作用机理,需要对植物激素进行定量分析。针对植物激素在大多数植物组织中的含量很低,性质不稳定,与其共存的次生代谢产物背景干扰严重,此外有些植物材料非常珍稀,材料量少的问题,以毛白杨叶片为材料,建立了超高效液相色谱−串联质谱(UPLC-MS/MS)同时测定植物组织中吲哚乙酸(IAA)、脱落酸(ABA)、赤霉素(GA3)、茉莉酸(JA)和水杨酸(SA)的新方法,为植物激素的研究提供技术支撑。
      方法在用异丙醇、水和盐酸(2∶1∶0.002,vvv)混合液4 ℃提取激素时,定量加入IAA、ABA、GA3、JA和SA内标(2H5-IAA、2H6-ABA、2H-JA和2H5-SA各10 ng,2H2-GA3 20 ng),激素提取液经二氯甲烷萃取后,UPLC-MS/MS定量测定。乙腈−0.1%乙酸水溶液为流动相,梯度洗脱,流速 0.8 mL/min,柱温30 ℃,C18色谱柱分离,多反应监测(MRM)、电喷雾电离源(ESI)负离子模式下进行分析,内标曲线法定量。
      结果5种植物激素在5 min内实现完全分离,检出限为0.01 ~ 0.05 pg/g,定量限为0.05 ~ 0.15 pg/g。在实验所采用的浓度范围内线性相关系数r在0.999 4 ~ 0.999 9之间,精密度和重现性均较好,峰面积的相对标准偏差为1.1% ~ 3.9%。经检测毛白杨叶片中IAA、ABA、GA3、JA、SA的含量分别为(74.38 ± 4.62)ng/g、(193.80 ± 6.04)ng/g、(674.67 ± 18.08)ng/g、(286.62 ± 7.48)ng/g、(746.02 ± 13.93)ng/g。
      结论本法操作简单,灵敏度高,分析时间短,适用于多种植物激素的测定。

       

      Abstract:
      ObjectivePlant hormones have pivotal roles in plant growth, development and response to biotic and abiotic stress. Quantitative analysis is required for studying the chemical synthesis, transportation, metabolism and molecular regulation of plant hormones. However, hormones are trace level and unstable in most tissues of plants. Moreover, the analysis of plant hormones is seriously interfered by co-existed secondary metabolites. In addition, some plant materials are rare and their amounts are low. To address above-mentioned questions, a method was developed for simultaneously determining multiple plant hormones in Populus tomentosa leaves, including indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellins (GA3), jasmonic acid (JA), and salicylic acid (SA) using ultra-highperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), which could provide technical support for better studying plant hormones.
      MethodLeaching solution of 2-propanol, water, and hydrochloric acid (2∶1∶0.002, v/v/v) was used to extract IAA, ABA, GA3, JA, and SA, and five internal standards (2H5-IAA/2H6-ABA/2H-JA/2H5-SA, 10 ng per each, 2H2-GA3 20 ng) were simultaneously introduced into the samples at 4 ℃. Crude extract was re-extracted by dichloromethane and then analyzed by UPLC-MS/MS. The target analytes were separated by Agilent SB-C18 column, with gradient elution using acetonitrile and 0.1% acetic acid in water as mobile phases. The flow rate was 0.8 mL/min and the column temperature was 30 ℃. Negative electrospray ionization with multiple reaction monitoring (MRM) mode was used for detection. Internal standard curve method was used for quantitative analysis.
      ResultFive plant hormones were separated thoroughly and detected within 5 mins. The limits of detection were 0.01−0.05 pg/g, and the limits of quantitation were 0.05−0.15 pg/g. The correlation coefficients (r) of internal calibration curves ranged from 0.999 4 to 0.999 9. The relative standard deviation (RSD) of peak areas was 1.1%−3.9%. The results showed that the contents of IAA, ABA, GA3, JA, and SA in Populus tomentosa leaves were (74.38 ± 4.62) ng/g, (193.80 ± 6.04) ng/g, (674.67 ± 18.08) ng/g, (286.62 ± 7.48) ng/g, and (746.02 ± 13.93) ng/g, respectively.
      ConclusionThe established method is easy-to-operate, high sensitivity and time-saving, which is suitable for the simultaneous identification and quantification of multiple hormones in plant tissues.

       

    /

    返回文章
    返回