Analytical method development for α-amanitin and β-amanitin in plasma at ultra-trace level by online solid phase extraction-high C performance liquid chromatography-triple quadrupole mass spectrometry and its application in poisoning events

alpha-Amanitin and beta-amanitin are the main fatal mushroom toxins. The toxins metabolize rapidly in blood and are reported hard to be detected 24 h after poisoning. The main challenge is that of developing a highly sensitive method at sub-pg mL(-1) level in blood to diagnose intoxication cause and to study the poisoning mechanism and blood toxicity kinetics. An analytical method for alpha-amanitin and beta-amanitin at ultra-trace level was developed in this study by online solid phase extraction-high performance liquid chromatography-triple quadrupole mass spectrometry (online SPE-LC-MS/MS). Simple protein precipitation and liquid-liquid extraction were introduced to resolve the sample preparation problem of the online SPE-LC-MS/MS system with large-volume injection. A quick valve-switching technique with a quantitative loop as interface was used in the online system. This design can ensure the independence of flow path and pressure between the SPE and LC-MS/MS modules and can obtain the precise cleanup of the toxins. The limits of detection for alpha-amanitin and beta-amanitin in plasma were both 0.02 ng mL(-1). The linear ranges were 0.05-20 ng mL(-1) with a correlation coefficient r >0.99. The average recoveries at three spiking levels were 82.9 %-92.2 % with the relative standard deviations (RSD) of 5.4 %-8.0 % for alpha-amanitin and 84.5 %-93.9 % with RSDs of 4.5 %-7.8 % for beta-amanitin. The composition and concentration of the toxins in plasma from 18 patients in 5 mushroom poisoning events caused by aminitins were studied. The developed method has high positive confirmation ability and can identify toxins in plasma 40 h after poisoning. (C) 2020 Elsevier B.V. All rights reserved.