Analysis of ibotenic acid and muscimol in Amanita mushrooms by hydrophilic interaction liquid chromatography–tandem mass spectrometry

Abstract

We have established the most modern method for analysis of ibotenic acid and muscimol in toxic Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Acivicin, an ibotenic acid analog and antitumor agent, was used as internal standard (IS). A target mushroom sample was homogenized in water/methanol (1:1) and mixed with a fixed concentration of IS. The target compounds and IS were purified with an Oasis MAX 3cc (60 mg) extraction cartridge. The eluate was subjected to hydrophilic interaction (TSK-GEL Amide-80 3 μm, 150 × 2.0 mm i.d. column) LC–MS–MS. The quantitation was made by multiple reaction monitoring (MRM). The ion transitions were: m/z 179→133.1 for IS, m/z 159→113.1 for ibotenic acid, and m/z 115→98.1 for muscimol. The elution was made in the gradient mode with 0.5 % formic acid aqueous solution (A) and 0.5 % formic acid in acetonitrile (B) from 90 % B to 80 % B in 1.85 min, and then in the isocratic elution mode with 20 % A/80 % B up to 10 min. The MRM chromatograms gave clear and symmetrical peaks for ibotenic acid, muscimol, and IS. Their recovery rates were 84.6–107 %. There was good linearity from 10 to 500 μg/g for both ibotenic acid and muscimol with correlation coefficients not <0.99. Intraday and interday accuracy and precision were also generally satisfactory. Using the above new method, the concentrations of ibotenic acid and muscimol were actually measured for a mushroom (most probably Amanita ibotengutake) obtained from a poisoning case; they were 210 and 107 μg/g, respectively. The novel points of our method are no requirement for derivatization before LC–MS–MS analysis, the use of anion exchange solid-phase extraction, the use of hydrophilic interaction column for LC separation, and the use of acivicin as IS.