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

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 mu m, 150 x 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 mu g/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 mu g/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.