Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Center for Molecular Signaling (PZMS), Saarland University, 66421, Homburg, Germany.
Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Center for Molecular Signaling (PZMS), Saarland University, 66421, Homburg, Germany.
Talanta. 2020 Jun 1;213:120847. doi: 10.1016/j.talanta.2020.120847. Epub 2020 Feb 14.
The analytical proof of a toxic mushroom and/or plant ingestion at an early stage of a suspected intoxication can be crucial for fast therapeutic decision making. Therefore, comprehensive analytical procedures need to be available. This study aimed to develop a strategy for the qualitative analysis of α- and β-amanitin, psilocin, bufotenine, muscarine, muscimol, ibotenic acid, and ricinine in human urine by means of hydrophilic interaction liquid chromatography-high resolution MS/MS (HILIC-HRMS/MS). Urine samples were prepared by hydrophilic-phase liquid-liquid extraction using dichloromethane and subsequent solid-phase extraction and precipitation, performed in parallel. Separation and identification of the biomarkers were achieved by HILIC using acetonitrile and methanol as main eluents and Orbitrap-based mass spectrometry, respectively. The method was validated as recommended for qualitative procedures and tests for selectivity, carryover, and extraction recoveries were included to also estimate the robustness and reproducibility of the sample preparation. Limits of identification were 1 ng/mL for α- and β-amanitin, 5 ng/mL for psilocin, bufotenine, muscarine, and ricinine, and 1500 ng/mL and 2000 ng/mL for ibotenic acid and muscimol, respectively. Using γ-amanitin, l-tryptophan-d5, and psilocin-d10 as internal standards, compensation for variations of matrix effects was shown to be acceptable for most of the toxins. In eight urine samples obtained from intoxicated individuals, α- and β-amanitin, psilocin, psilocin-O-glucuronide, muscimol, ibotenic acid, and muscarine could be identified. Moreover, psilocin-O-glucuronide and bufotenine-O-glucuronide were found to be suitable additional targets. The analytical strategy developed was thus well suited for analyzing several biomarkers of toxic mushrooms and plants in human urine to support therapeutic decision making in a clinical toxicology setting. To our knowledge, the presented method is by far the most comprehensive approach for identification of the included biomarkers in a human matrix.
在疑似中毒的早期阶段，对有毒蕈类和/或植物的分析证明对于快速治疗决策至关重要。因此，需要有全面的分析程序。本研究旨在开发一种通过亲水相互作用液相色谱-高分辨率 MS/MS（HILIC-HRMS/MS）定性分析人尿中α-和β-鹅膏蕈碱、裸盖菇素、蟾蜍色胺、毒蕈碱、麦斯卡林、异噁唑丙酸和黎芦碱的策略。尿样通过使用二氯甲烷的亲水相液-液萃取和随后的固相萃取和沉淀进行预处理，这两个步骤同时进行。通过使用乙腈作为主要洗脱剂的亲水作用色谱和基于轨道阱的质谱法分别实现了生物标志物的分离和鉴定。该方法按照定性程序的建议进行了验证，并进行了选择性、交叉污染和提取回收率的测试，以评估样品制备的稳健性和重现性。α-和β-鹅膏蕈碱的鉴定限为 1ng/mL，裸盖菇素、蟾蜍色胺、毒蕈碱和黎芦碱的鉴定限为 5ng/mL，异噁唑丙酸和麦斯卡林的鉴定限分别为 1500ng/mL 和 2000ng/mL。使用γ-鹅膏蕈碱、l-色氨酸-d5 和裸盖菇素-d10 作为内标，证明了对大多数毒素的基质效应变化进行补偿是可以接受的。在从 8 名中毒个体获得的尿样中，鉴定出了α-和β-鹅膏蕈碱、裸盖菇素、裸盖菇素-O-葡糖苷酸、麦斯卡林、异噁唑丙酸和毒蕈碱。此外，还发现了裸盖菇素-O-葡糖苷酸和蟾蜍色胺-O-葡糖苷酸是合适的附加靶标。因此，所开发的分析策略非常适合分析人尿中毒蕈类和植物的几种生物标志物，以支持临床毒理学环境中的治疗决策。据我们所知，该方法是目前为止在人基质中鉴定所包括生物标志物的最全面的方法。
