Rapid detection of α-amanitin and β-amanitin in rat plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its application to the toxicokinetic study of Lepiota brunneoincarnata

Purpose: Lepiota brunneoincarnata is a well-known poisonous mushroom and is responsible for fatal mushroom poisoning cases worldwide. α-Amanitin and β-amanitin are the main amatoxin compounds of Lepiota brunneoincarnata. However, there are no published toxicokinetic studies of Lepiota brunneoincarnata. To study the toxicokinetics of Lepiota brunneoincarnata, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determination of α-amanitin and β-amanitin in rat plasma.

Methods: UPLC-MS/MS analyses were performed with a triple quadrupole mass spectrometer in positive-ion mode. The sensitivity of α-amanitin and β-amanitin detection was increased by inhibiting the production of [M + Na]⁺ adducts. α-Amanitin and β-amanitin were separated and quantified on an UPLC octadecyl silyl column in only 2.5 min.

Results: The linear ranges were 3.0-3000 ng/mL for α-amanitin and 1.8-1800 ng/mL for β-amanitin with a correlation coefficient r > 0.99 for both analytes. The lower limit of quantification of 3.0 ng/mL for α-amanitin and 1.8 ng/mL for β-amanitin was achieved using only 50 μL of rat plasma. The accuracy of α-amanitin and β-amanitin was between - 9.5 and 7.0% with the precision ranged from 2.2 to 12.5%. The developed method was then applied for Lepiota brunneoincarnata toxicokinetic study after intravenous administration of Lepiota brunneoincarnata extracts.

Conclusions: Establishing UPLC-MS/MS method for quantifying amanitines in rat plasma successfully enabled toxicokinetic study of Lepiota brunneoincarnata extracts.