Validated electrospray liquid chromatographic–mass spectrometric assay for the determination of the mushroom toxins α- and β-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 α- and β-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 α- and β-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 α- and β-amanitin was 2.5 ng/ml and LOQ for both was 5.0 ng/ml. The absolute recoveries of α- and β-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 γ-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.