Fatal Amanita muscaria poisoning in a dog confirmed by PCR identification of mushrooms.

Diagnosing mushroom poisoning in dogs can be difficult and often includes identification of suspect mushrooms. Visual identification may be hindered by mastication, oral medications, or poor quality of environmental mushroom samples. Other analytical techniques may thus be necessary to aid in mushroom identification. A 5-y-old neutered male Labrador Retriever dog developed acute onset of vomiting, diarrhea, tremors, seizures, and somnolence. The dog was treated at a veterinary clinic and was briefly stabilized, but died during transport to an emergency clinic. On postmortem examination at the University of Kentucky Veterinary Diagnostic Laboratory, the dog's stomach was full of mushrooms covered with activated charcoal. Mushrooms were damaged, fragmented, and discolored, precluding accurate visual identification. Mushroom pieces were sent to the Department of Plant Pathology at the University of California-Davis for PCR identification; the neurotoxic mushroom Amanita muscaria was identified. A qualitative liquid chromatography-mass spectrometry (LC-MS) method was developed to detect ibotenic acid and muscimol, the toxic compounds present in A. muscaria. Mushrooms, stomach contents, and urine were analyzed by LC-MS; ibotenic acid and muscimol were detected in all samples. Because identification of ingested mushrooms is sometimes necessary to confirm mushroom poisoning, PCR can identify ingested mushrooms when visual identification is unreliable.

Determination of mushroom toxins ibotenic acid, muscimol and muscarine by capillary electrophoresis coupled with electrospray tandem mass spectrometry.

The CE-ESI-MS/MS method for the identification, separation and determination of mushroom toxins, namely ibotenic acid, muscimol and muscarine, was developed. It proved to be sensitive and thus useful for the real sample analysis with omitting the labor and time consuming pretreatment step. The CE-ESI-MS/MS method was applied on the spiked human urine. The analytical characteristics of the proposed method, such as limits of detection, linearity and repeatability of the peak area and the migration time, were evaluated. The RSD of the migration time and peak area were from 0.93% to 1.60% and from 2.96% to 3.42%, respectively. The obtained LOD values were at the nanomolar concentration level, therefore the developed method is sufficient for the determination and quantification of studied toxins in human urine after mushroom intoxication.

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.