Determination of protein-bound α-amanitin in mouse plasma: A potential new indicator of poisoning with the mushroom toxin α-amanitin

Approximately 70%∼90% of mushroom poisoning deaths are caused by the class of mushroom toxins known as amatoxins. However, the rapid elimination of amatoxins from plasma within 48 h after mushroom ingestion limits the practical value of plasma amatoxin analysis as a diagnostic indicator of Amanita mushroom poisoning. To increase the positive detection rate and extend the detection window of amatoxin poisoning, we developed a new method to detect protein-bound α-amanitin based on the hypothesis that RNAP II-bound α-amanitin released from the tissue into the plasma could be degraded by trypsin hydrolysis and then detected by conventional liquid chromatography-mass spectrometry (LC‒MS). Toxicokinetic studies on mice intraperitoneally injected with 0.33 mg/kg α-amanitin were conducted to obtain and compare the concentration trends, detection rates, and detection windows of both free α-amanitin and protein-bound α-amanitin. By comparing detection results with and without trypsin hydrolysis in the liver and plasma of α-amanitin-poisoned mice, we verified the credibility of this method and the existence of protein-bound α-amanitin in plasma. Under the optimized trypsin hydrolysis conditions, we obtained a time-dependent trend of protein-bound α-amanitin in mouse plasma at 1-12 days postexposure. In contrast to the short detection window (0-4 h) of free α-amanitin in mouse plasma, the detection window of protein-bound α-amanitin was extended to 10 days postexposure, with a total detection rate of 53.33%, ranging from the limit of detection to 23.94 μg/L. In conclusion, protein-bound α-amanitin had a higher positive detection rate and a longer detection window than free α-amanitin in mice.