Development of Simultaneous Analytical Method of Three Polypeptide Toxins α-Amanitin, β-Amanitin and Phalloidin in Poisonous Mushrooms and Human Serum Using UHPLC-MS/MS

Abstract

Accidental ingestion of toxic mushrooms remains a global public health concern because of the presence of highly potent peptide toxins such as α-amanitin, β-amanitin, and phalloidin. These compounds exhibit strong hepatotoxicity and can lead to acute liver failure and death. However, their rapid detection in biological and food matrices remains analytically challenging. Existing methods often require extensive sample preparation and are not suitable for urgent diagnostic applications. This study presents the development and validation of a rapid and sensitive analytical method for the simultaneous quantitation of α-amanitin, β-amanitin, and phalloidin in poisonous mushrooms and human serum. Among several preparation strategies evaluated, a method following direct extraction with 1% formic acid in methanol was selected for its speed, simplicity, and effectiveness in minimizing matrix interference. The method demonstrated excellent linearity (r² ≥ 0.99), low quantitation limits (10-50 ng/mL), and satisfactory recovery (72%-117%) and precision (RSD ≤ 19%) in both food and biological matrices. When applied to field-collected Amanita virosa, α-amanitin and β-amanitin were detected at 39 and 145 mg/kg, respectively, whereas no toxins were found in Amanita volvata. These findings demonstrate that the established method enables rapid and reliable detection of lethal peptide toxins with minimal sample preparation. The protocol is suitable for forensic investigations, clinical toxicology, and food safety monitoring. Its applicability in emergency settings underscores its potential as a practical tool for improving public health responses to mushroom poisoning incidents.

Keywords: UHPLC–MS/MS; mushroom; quantitative analysis; serum; toxins.

FIGURE 1

Structures of amanitins and phalloidin.

FIGURE 2

Schematic comparison of the five sample preparation methods evaluated in this study: (1) dSPE with C18 sorbent, (2) dSPE with C18/PSA mixture, (3) PRiME HLB cartridge with and (3) without liquid–liquid extraction (LLE), and (4) direct injection.

FIGURE 3

Comparison of recovery rates by five sample preparation methods (concentration: 50 ng/g, n = 3). *RSD: relative standard deviation.

FIGURE 4

Chromatograms of three mushroom toxins by recovery test.

FIGURE 5

Optimal preparation for mushroom and human serum samples.

FIGURE 6

Chromatograms for detected toxins in actual Amanita virosa.