Validated electrospray liquid chromatographic-mass spectrometric assay for the determination of the mushroom toxins alpha- and beta-amanitin in urine after immunoaffinity extraction

Specific detection of amanitins in body fluids is necessary for an early diagnosis of an intoxication with amanita mushrooms. In this paper, a liquid chromatographic-mass spectrometric assay after immunoaffinity extraction (IAE-LC-MS) is described for the determination of alpha- and beta-amanitin in urine. The method has been validated according to the criteria established by the Journal of Chromatography B. The assay was found to be selective. The calibration curves for alpha- and beta-amanitin were linear from 5 to 75 ng/ml. Intra- and inter-day accuracy and precision were inside the required limits. Amatoxins in frozen urine samples or immunoaffinity extracts were stable for more than 6 months, and the IAE columns could be used more than fifty times without remarkable loss in performance. LOD for alpha- and beta-amanitin was 2.5 ng/ml and LOQ for both was 5.0 ng/ml. The absolute recoveries of alpha- and beta-amanitin were 63% and 58% for the low quality control and 61% and 57% for the high quality control. The absolute recovery for the internal standard gamma-amanitin methyl ether at 25 ng/ml was 60%. The analysis of 5 authentic urine samples from patients intoxicated by amanita mushrooms showed a good correlation between the results measured by radioimmunoassay and the IAE-LC-MS assay. A partial validation showed that the assay was also suitable for plasma analysis.