University of Coimbra, Faculty of Pharmacy, Health Science Campus, Azinhaga de Santa Comba, Coimbra 3000-548, Portugal; National Institute for Agricultural and Veterinary Research (INIAV), Rua dos Lágidos, Lugar da Madalena, Vila do Conde 4485-655, Portugal; REQUIMTE/LAQV, R. D. Manuel II, Apartado, Porto 55142, Portugal.
National Institute for Agricultural and Veterinary Research (INIAV), Rua dos Lágidos, Lugar da Madalena, Vila do Conde 4485-655, Portugal; REQUIMTE/LAQV, R. D. Manuel II, Apartado, Porto 55142, Portugal.
J Pharm Biomed Anal. 2024 Sep 1;247:116253. doi: 10.1016/j.jpba.2024.116253. Epub 2024 May 24.
Consumption of misidentified foraged mushrooms containing bicyclic amanitin octapeptides is a worldwide public health and veterinary problem, being considered one of the deadliest accidental human and canine food ingestion due to acute liver failure (ALF). Reversal of advanced ALF and complete clinical recovery can be achieved following definitive removal of accumulated amatoxin laden bile from the gallbladder. An accurate means of quantifying amanitin content in aspirated bile is, therefore, urgently needed. Building on our prior work validating a method to detect and quantify amanitin in hepatic autopsy tissue, the development of an accurate method of measuring α- and β-amanitin in aspirated gallbladder bile was performed to evaluate the efficiency of this emergency procedure applied as a clinical treatment for intoxicated patients. A solid-phase extraction (SPE) procedure was optimized followed by detection based on ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). Low resolution mass spectrometry (LRMS) was compared with high resolution (HRMS) by the validation of UHPLC-MS/MS (triple quadrupole MS) and UHPLC-ToF-MS (time-of-flight MS). Both methods were able to detect amatoxins in bile with limits of detection and quantification ranging from 2.71 to 3.46 µg.kg, and 8.36-9.03 µg.kg for α-amanitin and, 0.32-1.69 µg.kg and 0.55-5.62 µg.kg for β-amanitin, respectively. Validation was completed with the evaluation of linearity, specificity, robustness, recovery, and precision following the ICH guidelines and CIR 808/2021. The validated methods were finally applied to bile samples obtained 48-96 hours + post-ingestion from 4 amatoxin poisoning patients who underwent gallbladder drainage procedures in Vietnam, Canada, and California. Gallbladder bile from patients with amatoxin mushroom poisoning contained significant amanitin content, even when aspirated several days post-ingestion, thus confirming the important role of enterohepatic circulation in amatoxin hepatotoxicity. This work represents a high and unique analytical throughput in amanitin poisoning allowing to efficiently respond to this fatal health problem.
食用被误认的野生蘑菇中含有的双环鹅膏肽八肽是一个全球性的公共卫生和兽医问题，由于急性肝衰竭（ALF），它被认为是最致命的意外人类和犬类食物摄入中毒之一。通过从胆囊中彻底清除累积的鹅膏毒素胆汁，可以逆转晚期 ALF 并实现完全临床康复。因此，迫切需要一种准确测量抽吸胆汁中鹅膏毒素含量的方法。在验证了一种检测和量化肝组织中鹅膏毒素的方法的基础上，我们开发了一种准确测量抽吸胆囊胆汁中α-和β-鹅膏毒素的方法，以评估将这种紧急程序作为中毒患者的临床治疗的应用效率。优化了固相萃取（SPE）程序，然后基于超高效液相色谱-质谱联用（UHPLC-MS）进行检测。通过验证 UHPLC-MS/MS（三重四极杆 MS）和 UHPLC-ToF-MS（飞行时间 MS）对低分辨率质谱（LRMS）与高分辨率（HRMS）进行了比较。两种方法都能够检测胆汁中的鹅膏毒素，α-鹅膏毒素的检测限和定量限分别为 2.71-3.46μg.kg 和 8.36-9.03μg.kg，β-鹅膏毒素的检测限和定量限分别为 0.32-1.69μg.kg 和 0.55-5.62μg.kg。根据 ICH 指南和 CIR 808/2021，通过评估线性、特异性、稳健性、回收率和精密度完成了验证。最后，将验证后的方法应用于从 4 名在越南、加拿大和加利福尼亚接受胆囊引流术的鹅膏毒素中毒患者中获得的 48-96 小时+摄入后胆汁样本。来自鹅膏毒素蘑菇中毒患者的胆囊胆汁中含有大量的鹅膏毒素，即使在摄入后几天抽吸，也证实了肠肝循环在鹅膏毒素肝毒性中的重要作用。这项工作在鹅膏毒素中毒方面代表了一种高且独特的分析通量，能够有效地应对这一致命的健康问题。
