Citation: XU Xiaomin, ZHANG Jingshun, CAI Zengxuan, MENG Zhen, HUANG Baifen, CHEN Qing. Determination of trace α-amanitin in urine of mushroom poisoning patient by online solid phase extraction-liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, ;2020, 38(11): 1281-1287. doi: 10.3724/SP.J.1123.2020.03010 shu

Determination of trace α-amanitin in urine of mushroom poisoning patient by online solid phase extraction-liquid chromatography-tandem mass spectrometry

Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China

Corresponding author: XU Xiaomin, CHEN Qing,
Received Date: 12 March 2020

Fund Project: Zhejiang Provincial Natural Science Foundation of China (No. LGF18H260007).

An analytical method was established for the determination of trace α-amanitin in the urine of patients suffering from mushroom poisoning by online solid phase extraction-liquid chromatography-tandem mass spectrometry (online SPE-LC-MS/MS). The sample was protein precipitated with formic acid acidified acetonitrile-methanol (5:1, v/v). Reversed-phase liquid-liquid microextraction was used to remove the organic solvent from the sample extract. The toxin was purified by online SPE using an ODS micro column (5 mm×2.1 mm, 5 μm), and separated on an XBridge^TM BEH C₁₈ column (150 mm×3.0 mm, 2.5 μm). Finally, the toxin was measured by MS/MS in the negative electrospray ionization (ESI^-) mode. Multiple reaction monitoring (MRM) was used, and the conditions were m/z 917.4>205.1 (quantitative ion transition) and m/z 917.4>257.1. Collision energy for both transitions was 55 eV. A fast valve-switching technique with a quantitative loop was used as an interface between the online SPE and LC-MS/MS modules. The two modules were independent, neither the mobile phase nor the pressure would interfere with each other, thus ensuring the stability of the system. Precise purification by the online system could effectively eliminate the matrix effects in the subsequent MS detection. Weak matrix suppression effects were found, with results of 88.7%-96.5%. The linear range of α-amanitin in urine was 0.1-50 μg/L with a correlation coefficient (r²) of 0.9983. The limit of detection (LOD) and limit of quantification (LOQ) in the sample matrix were 0.03 μg/L and 0.1 μg/L, respectively. The average recoveries at three spiked levels (0.1, 2.0 and 20 μg/L) were 84.3%-91.7% with relative standard deviations (RSDs) of 3.8%-7.2%. The accuracy and precision were evaluated using quality control samples with toxin contents of 0.1 μg/L (LOQ), 0.2 μg/L (2-fold LOQ), 2.0 μg/L (medium level), and 20 μg/L (high level). The calculated average intra-day accuracy was 85.1%-96.0% with the precision of 4.1%-7.8%. The inter-day accuracy was 82.9%-94.8% with the precision of 5.0%-9.5%. The specificity of the method was verified by negative samples derived from patients who suffered only gastroenteritis poisoning, without hepatotoxic symptoms. α-Amanitin was found in urine samples from nine mushroom poisoning patients with hepatotoxic symptoms. The sampling time ranged from 19 h to 92 h. The toxin contents were 0.11-53.1 μg/L. For patients with a high intake of poisonous mushrooms, the toxin content was 53.1 μg/L in a patient's urine sampled 19 h after accidental consumption and 0.19 μg/L in another patient's urine sampled 92 h after poisoning. The content of α-amanitin was only 0.53 μg/L in the urine sample obtained 23 h after consumption for a patient with low intake and 0.11 μg/L in the urine sampled from another patient 40 h after poisoning. Amatoxins can metabolize rapidly in vivo. The laboratory identification of amatoxin poisoning requires a method for trace-level analysis in the biological matrix. It is proved that this method is simple, accurate and sensitive by the application to the analysis of actual samples. The protein precipitation and reversed-phase liquid-liquid microextraction steps are fast and simple. Hence, they can be used as a rapid and effective pre-treatment method for online SPE-LC-MS/MS analysis of water-soluble toxins in biomaterial matrix. Highly sensitive analysis of α-amanitin in urine can be obtained using a precise purification technology via online SPE in this study. The problem of qualitative confirmation of the toxin at trace levels (0.03 μg/L) after poisoning can be solved. The laboratory identification time for amatoxin poisoning in some patients exceeds 90 h. The developed analytical method at trace level (0.1 μg/L of LOQ) can provide reliable technical support for establishing the dose-response relationship of α-amanitin in vivo. It can satisfy for the determination of trace α-amanitin in urine samples from patients with hepatotoxic mushroom poisoning.
- liquid chromatography-tandem mass spectrometry (LC-MS/MS),
- online solid phase extraction (online SPE),
- α-amanitin,
- urine,
- trace level,
- mushroom poisoning,
- foodborne disease
1. [1]
2. [2]
3. [3]
4. [4]
5. [5]
6. [6]
7. [7]
8. [8]
9. [9]
10. [10]
11. [11]
12. [12]
13. [13]
14. [14]
15. [15]
16. [16]
17. [17]
18. [18]
19. [19]
20. [20]
21. [21]
22. [22]
23. [23]
1. [1]
  Siwei Hou , Yaxin Niu , Guanglu Zhang , Yanmei Yang , Xu Wang , Zhenzhen Chen . Application of Solid-Phase Microextraction and Mass Spectrometry in Environmental Detection. University Chemistry, 2026, 41(3): 297-306. doi: 10.12461/PKU.DXHX202504078
2. [2]
  Yanhui Zhong , Ran Wang , Zian Lin . Analysis of Halogenated Quinone Compounds in Environmental Water by Dispersive Solid-Phase Extraction with Liquid Chromatography-Triple Quadrupole Mass Spectrometry. University Chemistry, 2024, 39(11): 296-303. doi: 10.12461/PKU.DXHX202402017
3. [3]
  Zunxiang Zeng , Yuling Hu , Yufei Hu , Hua Xiao . Analysis of Plant Essential Oils by Supercritical CO₂Extraction with Gas Chromatography-Mass Spectrometry: An Instrumental Analysis Comprehensive Experiment Teaching Reform. University Chemistry, 2024, 39(3): 274-282. doi: 10.3866/PKU.DXHX202309069
4. [4]
  Haiyuan Wang , Xiaoning Jin , Yajing Sun , Zhen Zhang , Wentao Zhao , Yi Li . Practical Exploration of High-Performance Liquid Chromatography Experiment Teaching Reform Empowered by Artificial Intelligence. University Chemistry, 2026, 41(4): 45-51. doi: 10.12461/PKU.DXHX202505076
5. [5]
  Yifan Xie , Liyun Yao , Ruolin Yang , Yuxing Cai , Yujie Jin , Ning Li . Exploration and Practice of Online and Offline Hybrid Teaching Mode in High-Performance Liquid Chromatography Experiment. University Chemistry, 2025, 40(11): 100-107. doi: 10.12461/PKU.DXHX202412133
6. [6]
  Jiayao Wang , Guixu Pan , Ning Wang , Shihan Wang , Yaolin Zhu , Yunfeng Li . Preparation of donor-π-acceptor type graphitic carbon nitride photocatalytic systems via molecular level regulation for high-efficient H₂O₂ production. Acta Physico-Chimica Sinica, 2025, 41(12): 100168-0. doi: 10.1016/j.actphy.2025.100168
7. [7]
  Mingyang Men , Jinghua Wu , Gaozhan Liu , Jing Zhang , Nini Zhang , Xiayin Yao . Sulfide Solid Electrolyte Synthesized by Liquid Phase Approach and Application in All-Solid-State Lithium Batteries. Acta Physico-Chimica Sinica, 2025, 41(1): 100004-0. doi: 10.3866/PKU.WHXB202309019
8. [8]
  Chongjing Liu , Yujian Xia , Pengjun Zhang , Shiqiang Wei , Dengfeng Cao , Beibei Sheng , Yongheng Chu , Shuangming Chen , Li Song , Xiaosong Liu . Understanding Solid-Gas and Solid-Liquid Interfaces through Near Ambient Pressure X-Ray Photoelectron Spectroscopy. Acta Physico-Chimica Sinica, 2025, 41(2): 100013-0. doi: 10.3866/PKU.WHXB202309036
9. [9]
  Lingyu Chang , Yanfang Lang , Yuyan Zhu , Jie Wang , Ying Guo , Die Wang , Peng Ding , Yueming Zhou , Zhixiang Gong , Shujuan Liu . Machine Learning-Optimized Microcolumn Ion Exchange Chromatography for Trace Arsenic Determination. University Chemistry, 2026, 41(1): 76-84. doi: 10.12461/PKU.DXHX202506023
10. [10]
  Runjie Li , Hang Liu , Xisheng Wang , Wanqun Zhang , Wanqun Hu , Kaiping Yang , Qiang Zhou , Si Liu , Pingping Zhu , Wei Shao . 氨基酸的衍生及手性气相色谱分离创新实验. University Chemistry, 2025, 40(6): 286-295. doi: 10.12461/PKU.DXHX202407059
11. [11]
  Zian Lin , Yingxue Jin . Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) for Disease Marker Screening and Identification: A Comprehensive Experiment Teaching Reform in Instrumental Analysis. University Chemistry, 2024, 39(11): 327-334. doi: 10.12461/PKU.DXHX202403066
12. [12]
  Ke Qiu , Fengmei Wang , Mochou Liao , Kerun Zhu , Jiawei Chen , Wei Zhang , Yongyao Xia , Xiaoli Dong , Fei Wang . A Fumed SiO₂-based Composite Hydrogel Polymer Electrolyte for Near-Neutral Zinc-Air Batteries. Acta Physico-Chimica Sinica, 2024, 40(3): 2304036-0. doi: 10.3866/PKU.WHXB202304036
13. [13]
  Shunü Peng , Huamin Li , Zhaobin Chen , Yiru Wang . Simultaneous Application of Multiple Quantitative Analysis Methods in Gas Chromatography for the Determination of Active Ingredients in Traditional Chinese Medicine Preparations. University Chemistry, 2025, 40(10): 243-249. doi: 10.12461/PKU.DXHX202412043
14. [14]
  Yajie Li , Bin Chen , Yiping Wang , Hui Xing , Wei Zhao , Geng Zhang , Siqi Shi . Inhibiting Dendrite Growth by Customizing Electrolyte or Separator to Achieve Anisotropic Lithium-Ion Transport: A Phase-Field Study. Acta Physico-Chimica Sinica, 2024, 40(3): 2305053-0. doi: 10.3866/PKU.WHXB202305053
15. [15]
  Gaoyan Chen , Chaoyue Wang , Juanjuan Gao , Junke Wang , Yingxiao Zong , Kin Shing Chan . Heart to Heart: Exploring Cardiac CT. University Chemistry, 2024, 39(9): 146-150. doi: 10.12461/PKU.DXHX202402011
16. [16]
  Xiaowu Zhang , Pai Liu , Qishen Huang , Shufeng Pang , Zhiming Gao , Yunhong Zhang . Acid-Base Dissociation Equilibrium in Multiphase System: Effect of Gas. University Chemistry, 2024, 39(4): 387-394. doi: 10.3866/PKU.DXHX202310021
17. [17]
  Chunai Dai , Yongsheng Han , Luting Yan , Zhen Li , Yingze Cao . Ideological and Political Design of Solid-liquid Contact Angle Measurement Experiment. University Chemistry, 2024, 39(2): 28-33. doi: 10.3866/PKU.DXHX202306065
18. [18]
  Ke Zhao , Zhen Liu , Luyao Liu , Changyuan Yu , Jingshun Pan , Xuguang Huang . Functionalized Reflective Structure Fiber-Optic Interferometric Sensor for Trace Detection of Lead Ions. Acta Physico-Chimica Sinica, 2024, 40(4): 2304029-0. doi: 10.3866/PKU.WHXB202304029
19. [19]
  Yiru Wang , Chanzi Ruan , Juanjuan Song , Yongxian Fan , Dongcheng Liu , Yanping Ren , Xiuqiong Zeng , Faqiong Zhao , Wenwei Zhang , Mei Shi , Min Hu , Wan Li , Xiuyun Wang , Weihong Li , Xiaohang Qiu , Yong Fan , Jianrong Zhang , Shuyong Zhang . Solid-Liquid Separation and Suggestions on the Operation Standards (I): Decantation and Centrifugation with Centrifuge Operation. University Chemistry, 2026, 41(3): 122-129. doi: 10.12461/PKU.DXHX202507037
20. [20]
  Yiru Wang , Zhiqiang Dong , Juanjuan Song , Yongxian Fan , Dongcheng Liu , Yanping Ren , Xiuqiong Zeng , Faqiong Zhao , Wenwei Zhang , Mei Shi , Min Hu , Wan Li , Xiuyun Wang , Weihong Li , Xiaohang Qiu , Yong Fan , Jianrong Zhang , Shuyong Zhang . Solid-Liquid Separation and Suggestions on the Operation Standards (II): Gravity Filtration and Vacuum Filtration with Vacuum Pump Operation. University Chemistry, 2026, 41(3): 130-142. doi: 10.12461/PKU.DXHX202507042

Get Citation

PDF

XML

Metrics

PDF Downloads(14)
Abstract views(1121)
HTML views(151)

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142