不同种质柑橘果实各组织中类黄酮LC-MS检测   

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Original research article

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实验目的:定性及定量分析柑橘各种质果实中的差异类黄酮类。

关键词: 柑橘, 次生代谢, 类黄酮

材料与试剂

  1. 色谱柱 (shim-packVP-ODS C18 (pore size 5.0 μm, length 2 x 150 mm))
  2. 内标 (Lidocaine)
  3. 类黄酮标准品 (apigenin 6-C-glucoside, apigenin 7-O-glucoside, luteolin 7-O-glucoside等)
  4. 乙酸
  5. 甲醇 (色谱纯)
  6. 乙腈 (色谱纯)
  7. 超纯水

仪器设备

  1. Milli-Q s ULTRA purification system (Millipore, Milford, MA, USA)
  2. HPLC-ESI-QqTOF-MS/MS (用于定性分析,靶向MS2模式)
  3. LC-ESI-Q TRAP-MS/MS (用于定量分析,MRM模式)

实验步骤

  1. 组织提取物通过HPLC-ESI-QqTOF-MS/MS进行检测,且利用靶向MS2模式进行代谢物的定性分析。HPLC体系如下所述:色谱柱,shim-pack VP-ODS C18 (粒径5.0 μm,长度2 × 150 mm);流动相,水 (含有0.04%乙酸),乙腈 (含有0.04%乙酸);梯度洗脱条件,0 min时,95:5 V/V,20 min时,5:95 V/V,22 min,5:95 V/V,22.1 min,95:5 V/V,25 min时,95:5 V/V;流速,0.25 ml/min;柱温,40 °C;进样体积,2 μl。利用安捷伦6520精确飞行时间质谱 (Q-TOF) 法进行HPLC流出物的全时扫描,其中m/z范围为50-1,000,检测模式为正离子模式 (http://www.chem.agilent.com/en-US/products-services/Instruments-Systems/Mass-Spectrometry/Pages/default.aspx)。参数设置如下所示:雾化气体,氮气,40 psi;干燥气体,氮气,10 L/min,350 °C;毛细管电压,3500 V;碎裂电压,135 V;微光电压,65 V;八极杆射频电压峰值,750 V。在MS模式下,扫描质量范围为m/z 50-1,000,且采集速率和收集时间分别为2 spectra/s和500 ms/spectrum。对于MS2模式,碰撞电压设置为10,20和30 V,且隔离宽度为± 4 m/z。数据的记录和处理通过MassHunter软件执行。
  2. 代谢物的定量分析通过 LC-ESI-Q TRAP-MS/MS法多反应监测 (MRM) 模式进行分析。配备了ESI-Turbo离子喷雾接口的三重四极线性离子阱质谱仪 (Q TRAP),API 4000 Q TRAP LC/MS/MS体系用于进行正离子模式下的线性三重四极 (QQQ) 扫描分析,Analyst 1.5 software (AB Sciex)。ESI源参数设置如下:离子源,涡轮喷雾;离子源温度,500 °C;离子喷雾电压 (IS),5,500 V;离子源雾化气 (GSI),辅助气 (GSII) 以及帘气气压分别为55,60和25.0 psi;碰撞气压 (CAD) 设置为高。在线性三重四极扫描模式下,分别用10和100 mol/L聚丙烯乙二醇进行仪器调谐和质量校准。

溶液配方

  1. 乙酸水溶液
    1,000 ml纯水中加入0.4 ml乙酸
  2. 0.04%乙腈溶液
    1,000 ml乙腈中加入0.4 ml乙酸

参考文献

  1. Wang, S., Tu, H., Wan, J., Chen, W., Liu, X., Luo, J., Xu, J. and Zhang, H. (2016). Spatio-temporal distribution and natural variation of metabolites in citrus fruits. Food Chem 199: 8-17.
  2. Wang, S., Yang, C., Tu, H., Zhou, J., Liu, X., Cheng, Y., Luo, J., Deng, X., Zhang, H. and Xu, J. (2017). Characterization and metabolic diversity of flavonoids in citrus species. Sci Rep 7(1): 10549.
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:王守创, 涂虹, 徐娟. (2018). 不同种质柑橘果实各组织中类黄酮LC-MS检测. Bio-101: e1010208. DOI: 10.21769/BioProtoc.1010208.
How to cite: Wang, S. C., Tu, H. and Xu, J.  (2018). Detection of Flavonoids in Different Tissues from Different Population of Citrus Fruits by LC-MS. Bio-101: e1010208. DOI: 10.21769/BioProtoc.1010208.
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