• ISSN 1008-505X
  • CN 11-3996/S
卢玉秋, 宋阿琳, 唐治玉, 李艳玲, 董炜灵, 王恩召, 唐治喜, 范分良. UPLC-MS/MS同时测定土壤中19种植物激素方法的建立和验证[J]. 植物营养与肥料学报, 2019, 25(6): 953-962. DOI: 10.11674/zwyf.18129
引用本文: 卢玉秋, 宋阿琳, 唐治玉, 李艳玲, 董炜灵, 王恩召, 唐治喜, 范分良. UPLC-MS/MS同时测定土壤中19种植物激素方法的建立和验证[J]. 植物营养与肥料学报, 2019, 25(6): 953-962. DOI: 10.11674/zwyf.18129
LU Yu-qiu, SONG A-lin, TANG Zhi-yu, LI Yan-ling, DONG Wei-ling, WANG En-zhao, TANG Zhi-xi, FAN Fen-liang. Establishment and validation of UPLC-MS/MS method for simultaneous determination of 19 phytohormones in soil[J]. Journal of Plant Nutrition and Fertilizers, 2019, 25(6): 953-962. DOI: 10.11674/zwyf.18129
Citation: LU Yu-qiu, SONG A-lin, TANG Zhi-yu, LI Yan-ling, DONG Wei-ling, WANG En-zhao, TANG Zhi-xi, FAN Fen-liang. Establishment and validation of UPLC-MS/MS method for simultaneous determination of 19 phytohormones in soil[J]. Journal of Plant Nutrition and Fertilizers, 2019, 25(6): 953-962. DOI: 10.11674/zwyf.18129

UPLC-MS/MS同时测定土壤中19种植物激素方法的建立和验证

Establishment and validation of UPLC-MS/MS method for simultaneous determination of 19 phytohormones in soil

  • 摘要:
    目的 本文旨在建立土壤中多种植物激素的提取及超高效液相色谱串联质谱法,并用此方法同时测定土壤中茉莉酸、吲哚-3-乙酸、反式玉米素,玉米素核苷、异戊烯基腺嘌呤、吲哚-3-丁酸、N6-异戊烯基腺嘌呤、独脚金内酯、玉米素、二氢玉米素核苷、吲哚-3-丙酸、吲哚-3-乙酸甲酯、二氢玉米素、吲哚-3-羧酸、茉莉酸甲酯、油菜素内酯、脱落酸、赤霉素、水杨酸共19种植物激素含量。
    方法 以根标土壤为试验材料,采用异丙醇︰水︰甲酸 (80︰19︰1,V/V/V) 作为提取剂,经过超声、离心后得到植物激素提取液,提取液于常温条件下真空浓缩至干,采用甲醇进行复溶,得到植物激素待测液。采用Waters ACQUITYUPLC® HSST3色谱柱对19种植物激素进行分离,以0.30 mmol/L甲酸铵水溶液 (含0.01%甲酸) 作为流动相A和0.30 mmol/L甲酸铵乙腈 (含0.01%甲酸) 作为流动相B进行梯度洗脱,流速为0.3 mL/min,柱温为30℃;采用超高效液相色谱串联质谱,多反应监测离子模式进行定性定量分析,其中,茉莉酸、吲哚-3-乙酸、反式玉米素、玉米素核苷、异戊烯基腺嘌呤、吲哚-3-丁酸、N6-异戊烯基腺嘌呤、独脚金内酯、玉米素、二氢玉米素核苷、吲哚-3-丙酸、吲哚-3-乙酸甲酯、二氢玉米素、吲哚-3-羧酸、茉莉酸甲酯、油菜素内酯采用正离子模式扫描,脱落酸、赤霉素、水杨酸采用负离子模式扫描;采用外标法测定植物激素回收率。
    结果 在本试验浓度范围内,上述19种植物激素浓度与对应峰面积的相关系数 (r) 均大于0.99,检出限介于0.02~1.06 ng/g之间,19种植物激素的加标回收率为70.2%~117%,相对标准偏差介于0.20%~7.3%之间。采用优化后的实验方法测定土壤中的植物激素,结果检测出吲哚-3-乙酸、吲哚-3-羧酸、吲哚-3-乙酸甲酯、水杨酸和独角金内酯5种植物激素,含量为0.55~5.79 ng/g。
    结论 本方法前处理不需要过夜浸提,加入二氯甲烷后,超声离心浓缩复溶后可直接进样检测,大大缩短了样品的前处理时间,超高效液相色谱串联质谱法操作简单,选择性好,灵敏度高,可实现土壤样品中多种植物激素的同时检测,为土壤中植物激素的深入研究提供了一种有效的研究方法。

     

    Abstract:
    Objectives The purpose of this study is to establish an effective method for simultaneously extracting a total of 19 of phytohormones in rhizosphere soil by using ultra performance liquid chromatography tandem mass spectrometry (UPLS-MS/MS). These phytohormones include ABA, GA3, JA, IAA, tZ, tZR, IPR, IBA, IP, SL, cZ, DZR, IPA, MeIAA, SA, DZ, ICA, MeJA, and BL.
    Methods The rhizosphere soil were extracted by the solution of isopropyl alcohol∶ water∶ formic acid (80∶19∶1, V/V/V). After ultrasonic treatment and centrifugation, the phytohormones extracts were concentrated by drying at room temperature, and redissolved with methanol. The Waters ACQUITYUPLC ® HSST3 chromatographic column was used for separation of 19 phytohormones, 0.30 mmol/L of ammonium formate aqueous solution (containing 0.01% formic acid) was used as mobile phase A and 0.30 mmol/L of ammonium formate acetonitrile (containing 0.01% formic acid) was used as mobile phase B, accompanying a gradient elution, the flow rate was 0.3 mL/min and the column temperature was maintained at 30℃. Then the phytohormones were identified and quantified with ultra performance liquid chromatography tandem mass spectrometry through a multiple reaction-monitoring mode. The JA, IAA, tZ, tZR, IPR, IBA, IP, SL, cZ, DZR, IPA, MeIAA, DZ, ICA, MeJA, and BL were scanned with the positive ion mode, while the ABA, GA3, SA were scanned by the negative ion mode. External standard method was used to quantify the recovery.
    Results The concentration and corresponding peak area correlation coefficients (r) of 19 kinds of phytohormones such as ABA, GA3, JA, IAA, tZ, tZR, IPR, IBA, IP, SL, cZ, DZR, IPA, MeIAA, SA, DZ, ICA, MeJA, and BL were more than 0.99 in the concentration range of this experiment. The detection limits were ranged from 0.02 ng/g to 1.06 ng/g, the recoveries of 19 phytohormones were between 70.2%–117%, and the relative standard deviations were ranged from 0.2% to 7.3%. The phytohormones in rhizosphere soil were determined by the optimized experimental method, and the detected five phytohormones (IAA, ICA, MeIAA, SA, SL) were ranged from 0.55 to 5.79 ng/g in rhizosphere soil.
    Conclusions In the modified procedure, the phytohormones do not require overnight extraction, they can be effectively extracted using dichloromethane through a series of centrifugation, concentration and resolution. The modified procedure could produce better selectivity and higher sensitivity for the 19 main phytohormones in soil.

     

/

返回文章
返回