Development of a simple and reliable method for α‐amanitin detection in rat plasma and its application to a toxicokinetic study

Abstract

Rationale α‐Amanitin is a highly toxic peptide widely found in species of poisonous mushrooms. The matrix effect has been a major obstacle for accurate determination of α‐amanitin in plasma samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS). In this study, the strategy to eliminate the matrix effect of α‐amanitin with a one‐step dilution approach after deproteinization was applied. Methods Rat plasma samples were processed by protein precipitation with methanol followed by a nine‐fold dilution with pure water. The matrix effect value of α‐amanitin was 19.7%–22.2% by protein precipitation and then changed to 87.5%–88.7% after dilution. α‐Amanitin and the internal standard (roxithromycin) were analyzed on an ACQUITY UPLC® BEH C18 (50 mm × 2.1 mm, 1.7 μm) column within 3.0 min by gradient elution. Results The linear ranges were 0.90–600 ng/mL with a correlation coefficient r >0.9958. A lower limit of quantification (LLOQ) of 0.90 ng/mL was achieved using only 50 μL of rat plasma. The intra‐ and inter‐day precisions for the analyte ranged from 3.2% to 7.5% and 3.1% to 7.1%, respectively, and the accuracy ranged from −5.3% to −8.0%. Conclusions The matrix effect of α‐amanitin was reduced by sample dilution after plasma deproteinization. A reliable LC/MS/MS method for the determination of α‐amanitin in rat plasma was developed. This method was successfully applied for a toxicokinetic study of rats after intravenous injection of α‐amanitin with a subacute toxicity dose at 0.10 mg/kg.