GC/MS determination of ibotenic acid and muscimol in the urine of patients intoxicated with Amanita pantherina.

Ibotenic acid and muscimol are substances which mostly participate in psychotropic properties of Amanita pantherina and Amanita muscaria. They are rapidly absorbed from the gastrointestinal tract and readily excreted in urine. The poisoning with A. pantherina is in the majority of cases accidental because it can be easily mistaken for the edible species (Amanita rubescens, Amanita spissa and Macrolepiota procera). Intoxication with A. muscaria is mostly intentional for recreational purposes. Prognosis of the poisoning is generally good; lethal cases are rare. Mushroom poisoning is often proved by microscopic examination of spores in the stomach or intestinal content. Authors of this article introduce an instrumental method of proving A. pantherina or A. muscaria poisoning. The article describes the isolation of ibotenic acid and muscimol from urine, the derivatization step and the determination of these compounds by gas chromatography/mass spectrometry. Isolation of these alkaloids from urine was performed on a strong cation exchanger (Dowex® 50W X8), and the elution and derivatization of the alkaloids were made in one step with ethyl chloroformate in aqueous solution of sodium hydroxide with the addition of ethanol and pyridine. Cycloserine was used as internal standard. By this method, concentrations of ibotenic acid and muscimol in the urine of four persons intoxicated with A. pantherina were determined. In this study, mass spectra of derivatized ibotenic acid and muscimol are shown, and validation of the method is described.

Isolation and identification of the Amanita muscaria and Amanita pantherina toxins in human urine.

OBJECTIVES: Ibotenic acid, muscimol and muscarine were recognized as responsible for psychotropic effects of A. muscaria and A. pantherina. Demand for their specific and sensitive identification and quantitation in biological material lead to effort to develop reliable analytical method, but satisfactory solution is still lacking. Presented article describes liquid chromatography-mass spectrometry method suitable for isolation and identification of principal toxins of A. muscaria and A. pantherina in urine. METHODS AND RESULTS: Dedicated liquid chromatography-mass spectrometry method is reported. Technique consists of an extraction of analytes on Strata X-CW and Discovery SCX SPE cartridges and separation is achieved using a Gemini C18 column (150 mm x 2.0 mm, 5 micron) and 8 mM heptafluorobutyric acid as mobile phase. Detection at m/z 159 for ibotenic acid, m/z 115 for muscimol and m/z 174 for muscarine was used. Retention times and LODs are 2.6 min and 50 ng.ml-1 for ibotenic acid, 4.6 min and 40 ng.ml-1 for muscimol and 14.2 min and 3 ng.ml-1 for muscarine. CONCLUSION: A sensitive and specific liquid chromatography-mass spectrometry assay was developed for analysis of principal toxins of A. muscaria and A. pantherina in urine. Method was found to be sufficiently sensitive and specific for analysis of urine of intoxicated patients.

Determination of muscarine in human urine by electrospray liquid chromatographic-mass spectrometric.

A liquid chromatography-mass spectrometry based method for determination of muscarine in human urine was developed and validated. The method involved a solid phase extraction of muscarine from urine using Strata X-CW column. Separation of muscarine was achieved within 16.0 min on a reversed phase Gemini C18 analytical column (150 mm × 2.0mm i.d., 5 µm) with a mobile phase consisted of aqueous 8 mmol/L heptafluorobutyric acid and acetonitrile in a gradient mode. Mass spectrometric detection was performed at m/z 174 and m/z 216 for muscarine and acetylmuscarine (internal standard), respectively. The linearity was satisfactory with a coefficient of determination (R(2)) 0.9993 at concentration range from 0.3 ng/mL to 2.0 µg/mL, LOD and LOQ for muscarine was 0.09 ng/mL and 0.3 ng/mL, respectively. The found out recoveries of muscarine were 96% or 95% for concentration 0.3 ng/mL and 0.2 µg/mL or 2.0 µg/mL, respectively. The precision in the intra-assay-study varied from 0.48% to 1.39% and in the inter-assay-study from 2.39% to 5.49%. The accuracy ranged from -3.3% to -6%. The validation results demonstrated that the method fulfilled satisfactory requirements for precision and accuracy across the calibration curve. The applicability of the method has been demonstrated by analyzing clinical urine samples. The method offers the fast objective identification of intoxication by muscarine and can become a routine screening alternative to more difficult microscopic examination of spores in the gastric content in clinical practice.