Trace Analysis of Alpha-Amanitin in Human Urine: Implications for Poisoning Detection

Trace Analysis of Alpha-Amanitin in Human Urine: Implications for Poisoning Detection

Roma Perlowska1;  Rafal Szewczyk1,2; Katarzyna Krupczyńska-Stopa1,2; Maciej Stopa1,2; Adrian Sobon2; Anastasiia Shyian2 1 – LabExperts sp. z o.o., Gdańsk, Poland; 2 – Bioanalytic sp z o.o., Gdańsk, Poland

Introduction

Alpha-amanitin is a cyclic peptide toxin produced by fungi of the genus Amanita, including the death cap mushroom (Amanita phalloides). Ingestion of this toxin has severe consequences, causing liver cell damage that can lead to liver failure, potentially resulting in multiple organ failure, coagulopathy, and death. Early detection of poisoning significantly increases the chances of survival and reduces the risk of severe organ damage. Due to the complexity and variability of urine sample composition across different patients, the detection of alpha-amanitin at very low concentrations using LC-MS analysis is challenging. This study focuses on the development of a reproducible and sensitive method for detecting alpha-amanitin in human urine samples.

Methods

Sample preparation was performed using solid-phase extraction (SPE) with DAU columns (200 mg/10 mL). The urine sample was diluted with phosphate buffer (pH 6.0) before application to the SPE column, which was conditioned with methanol, water, and phosphate buffer (pH 6.0). After applying the sample, the matrix was washed with water, a 0.1% acetic acid solution, and a methylene chloride-methanol solution. Alpha-amanitin was eluted with methanol. The eluate was evaporated under nitrogen and reconstituted in an injection-ready solvent. Data acquisition was performed using QTRAP 6500+ (SCIEX) mass spectrometer coupled with LC-40XR (Shimadzu) and processed with SciexOS software. The LC-MS/MS analysis was conducted in positive ionization MRM mode during an 8-minute reversed-phase separation on a Zorbax SB-C18 column (Agilent).

Preliminary data

Human urine is a complex biological matrix with significant interpatient variability in its composition. Factors like diet, health status, medications, physical activity, hormonal imbalances, and sample collection timing influence its composition. These variations pose challenges in the accurate analysis of compounds in urine. Given this complexity, efficient sample extraction and purification are necessary to achieve reliable results. Compared to the widely used ELISA test for determining alpha-amanitin, mass spectrometry offers superior accuracy in both the identification and quantification of alpha-amanitin, even at concentrations well below the detection limits of the ELISA method. Furthermore, the LC-MS method eliminates false positives or negatives, with data confirming or excluding the target substance in urine providing a more precise assessment of alpha-amanitin concentrations. In contrast, the ELISA test may exhibit reduced reliability, particularly at very low concentrations. The development of a sensitive and reproducible method for alpha-amanitin detection in urine was complicated because of the lack of commercially available stable isotope-labeled analogues and other compounds suitable as internal standards. Consequently, method development was significantly focused on the optimization of SPE extraction procedure to achieve sensitivity, specificity, linearity and reproducibility without the use of internal standards. Carefully selection of appropriate SPE columns and solvents, all criteria were met: LLOQ below 0.25 ng/ml (S/N ≥ 10, peak-to-peak), linearity in the range of 0.25 – 100 ng/ml (R = 0.998), reproducibility below 5% RSD and no interferences at analyte’s RT. These preliminary data demonstrate the effectiveness of the developed method in detecting alpha-amanitin at very low concentrations in human urine.

Novel aspect

A highly sensitive method enables early detection of alpha-amanitin in urine, allowing for faster poisoning diagnosis.

ASMS 2025 – Baltimore, USA, 1-5.06.2025

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