Rapid analysis of amatoxins in human urine by means of affinity column chromatography and liquid chromatography-high-resolution tandem mass spectrometry

Analysis of amatoxins is of great importance as these cyclic peptides contribute to a high number of fatalities each year. Development of analytical approaches needs to focus on rapid, sensitive, and reliable methods. By establishing an affinity column chromatography-based assay using the monoclonal amanitin antibody AMA9G3 and liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) for the trace detection of alpha-, beta-, and gamma-amanitin in human urine samples to confirm ingestion, we report the first approach that extents the current status of amatoxin analysis. The presented procedure allows detection of amatoxins in human urine down to 1 ng/mL. The method was successfully validated qualitatively for alpha- and gamma-amanitin according to international recommendations. A proof of concept was performed by analyzing 37 urine samples after suspected amatoxin consumption submitted for regular clinical toxicological analysis. Using this antibody-based enrichment strategy, acute amatoxin intoxications can be determined within 90 min and due to the high sensitivity and selectivity, a comparable approach using target specific antibodies may also be used for other toxicological relevant peptides.