US EPA

1447283 

Journal Article 

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

Gonmori, K; Hasegawa, K; Fujita, H; Kamijo, Y; Nozawa, H; Yamagishi, I; Minakata, K; Watanabe, K; Suzuki, O 

2012 

Yes 

Forensic Toxicology
ISSN: 1860-8965
EISSN: 1860-8973 

30 

2 

168-172 

10.1007/s11419-012-0144-7 

WOS:000306427300011 

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. 

Ibotenic acid; Muscimol; Acivicin; Amanita mushrooms; Hydrophilic interaction; LC-MS-MS