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

Authors: VOLLMER, ALINE - Saarland University; FECHER-TROST, CLAUDIA - Saarland University; BEVER, CANDACE - Former ARS Employee; Tam, Christina; WAGMANN, LEA - Saarland University; MEYER, MARKUS - Saarland University

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/7/2024
Publication Date: 9/13/2024
Citation: Vollmer, A.C., Fecher-Trost, C., Bever, C.S., Tam, C.C., Wagmann, L., Meyer, M.R. 2024. Analysis of amatoxins in human urine after magnetic bead-based immunoprecipitation and liquid chromatography high-resolution tandem mass-spectrometry. Scientific Reports. 14(1). Article 21397. https://doi.org/10.1038/s41598-024-72463-3.
DOI: https://doi.org/10.1038/s41598-024-72463-3

Interpretive Summary: Plants and mushrooms can contain a wide variety of toxic peptides and proteins, which may lead to severe or lethal intoxications after exposure with some of them being potential bioterror threats. Proteins and peptides such as ricin, abrin, or amanitin are well known for their potent toxicity. Amatoxin-containing mushrooms contribute to many fatalities each year. Fast, simple, and sensitive methods are needed to detect these toxins in human biosamples especially in urine for early diagnosis and initiation of supportive care. We developed and optimized an approach to sensitively detect alpha-, beta-, and gamma-amanitin using an antibody-coupled to a magnetic bead to pull down these toxins from clinical samples followed by high-resolution mass spectrometry. This approach was able to sensitively detect amatoxin in urine samples as low as ng/mL levels. This study expands on the use of this technology to the clinical toxicology field thus potentially enabling early diagnosis and support care treatments after ingestion of amatoxin-containing mushrooms.

Technical Abstract: Mushrooms contain a wide variety of different toxic substances leading to severe or lethal intoxications. Especially amatoxins are of great importance as these cyclic peptides contribute to high fatalities each year. Early diagnosis and treatment decision of patients is urgently required. Therefore, fast, simple, and sensitive analytical methods need to be developed. Here, we enlarge the portfolio of extracting amatoxins from urine applicable in clinical toxicology. The amatoxins a,-ß-, and y-amanitin were included in the development of the analytical approach. A magnetic bead-based immunoprecipitation using a monoclonal amanitin antibody (AMA9G3) was established for extraction of the target compounds followed by Orbitrap high-resolution mass spectrometry. Two different crosslinkers, disuccinimidyl suberate and tosylactivated beads were investigated for re-usage of AMA9G3. The method was validated qualitatively according to international recommendations. A proof of concept was performed by analyzing 51 suspected amatoxin poisonings submitted to the author´s laboratory for toxicological analysis. Antibody coupling followed by immunoprecipitation as sample preparation allowed for the detection of a,-ß-, and y-amanitin in human urine down to 1 ng/mL. An amount of 20 µg of AMA9G3 was needed for enrichment of a- and y-amanitin, at least 50 µg for ß-amanitin. Coupled antibodies could not be reused as crosslinking experiments indicated no covalent binding.