Lateral flow immunoassay for amatoxins detection in human urine compared to liquid chromatography-high-resolution tandem mass spectrometry

Authors: VOLLMER, ALINE - Saarland University; BAMBAUER, THOMAS - Johannes Gutenberg University; BEVER, CANCACE - Former ARS Employee; Tam, Christina; WAGMANN, LEA - Saarland University; MEYER, MARKUS - Saarland University

Submitted to: Journal of Analytical Toxicology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/29/2025
Publication Date: 5/2/2025
Citation: Vollmer, A.C., Bambauer, T.P., Bever, C.S., Tam, C.C., Wagmann, L., Meyer, M.R. 2025. Lateral flow immunoassay for amatoxins detection in human urine compared to liquid chromatography-high-resolution tandem mass spectrometry. Journal of Analytical Toxicology. Article bkaf018. https://doi.org/10.1093/jat/bkaf018.
DOI: https://doi.org/10.1093/jat/bkaf018

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. 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 compared the sensitivity of a lateral flow immunoassay developed previously to detect alpha-, beta-, and gamma-amanitin in suspected human clinical urine samples to the gold-standard mass spectrometry method. The study results indicated that the LFIA can be used as a supporting tool prior to mass spectrometry but confirmation is still needed. 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: Poisonings caused by amatoxin-containing mushrooms are of particular interest for clinicians and toxicologists since there are no antidotes for amatoxin poisoning. Currently, solid-phase extraction followed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) is widely applied for analysis of amatoxins as this combination provides suitable sensitivity, specificity, and mass accuracy. However, this assay is time-consuming, requiring specially trained personnel and expensive equipment. Rapid, easy-to-use, and inexpensive detection methods are critically needed for rapid confirmation of amatoxin poisoning and initiation of supportive care. Recently, a lateral flow immunoassay (LFIA) for the trace detection of a-, ß-, and '-amanitin in dog urine was described. In this study, we tested the applicability of this LFIA’s use in the detection of amatoxins from human urine samples, and whether this LFIA can be used as a supporting tool prior to LC-HRMS/MS confirmation or as a complete replacement. The LFIA detects amatoxins in human urine after visual evaluation to as little as 5 ng/mL (a-amanitin – 10 ng/mL, ß-amanitin – 50 ng/mL, '-amanitin – 5 ng/mL). However, visual evaluations are subjective, and we developed a digital analysis methodology based on the relative pixel intensity levels of the control and test lines to objectively determine a positive, negative, or trace result. Using these pixel intensity ratios, detection limits were ranged from 1 ng/mL (a- and '-amanitin) to 3 ng/mL (ß-amanitin), which were lower LODs than LC-HRMS/MS, with pixel intensity ratios between 0.0 – 0.21. Analysis of 68 suspected amatoxin intoxications further revealed that this LFIA can be used as a supporting tool prior to LC-HRMS/MS but confirmation is still required.