Abstract：
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-1, and 8.36-9.03 µg.kg-1 for α-amanitin and, 0.32-1.69 µg.kg-1 and 0.55-5.62 µg.kg-1 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.
摘要：
食用含有二环amanitin八肽的错误识别的觅食蘑菇是全球公共卫生和兽医问题，被认为是由于急性肝功能衰竭（ALF）导致的最致命的意外人类和犬类食物摄入之一。从胆囊中明确清除积聚的富含amatoxin的胆汁后，可以实现晚期ALF的逆转和临床完全恢复。定量吸入胆汁中amanitin含量的准确方法是，因此,迫切需要。在我们先前的工作基础上，验证了一种检测和量化肝尸检组织中amanitin的方法，我们开发了一种准确的方法来测量抽吸胆囊胆汁中的α-和β-amanitin，以评估这种急诊手术作为中毒患者临床治疗的有效性。优化了固相萃取（SPE）程序，然后基于超高效液相色谱与质谱联用（UHPLC-MS）进行检测。通过UHPLC-MS/MS（三重四极杆MS）和UHPLC-ToF-MS（飞行时间MS）的验证，将低分辨率质谱（LRMS）与高分辨率（HRMS）进行了比较。两种方法均能够检测胆汁中的阿莫毒素，检出限和定量范围为2.71至3.46µg。kg-1和8.36-9.03µg。kg-1用于α-amanitin和，0.32-1.69µg。kg-1和0.55-5.62µg。kg-1为β-amanitin，分别。通过线性评估完成验证，特异性，鲁棒性，recovery,和精确度遵循ICH指南和CIR808/2021。最终将经过验证的方法应用于从越南接受胆囊引流程序的4名阿马毒素中毒患者摄入后48-96小时获得的胆汁样品，加拿大,和加州。amatoxin蘑菇中毒患者的胆囊胆汁含有显著的amanitin含量，即使在摄入后几天吸气，从而证实肠肝循环在amatoxin肝毒性中的重要作用。这项工作代表了抗菌肽中毒的高而独特的分析能力，可以有效地应对这种致命的健康问题。
