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

Amatoxin-containing mushrooms, which contribute to many intoxications each year are of particular interest for clinicians and toxicologists as patients require special treatment in hospital. To confirm the presence of amatoxins, approaches for their fast, sensitive, and reliable identification must be available. 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. Nevertheless, time-consuming preparatory steps as well as expensive equipment is required. Therefore, a lateral flow immunoassay (LFIA) for trace detection of α-, β-, and γ-amanitin was established and evaluated using dog urine. In this study, we answered the questions whether this LFIA can be transferred to human urine samples, and whether this LFIA can be used as a supporting tool prior to LC-HRMS/MS confirmation. Result interpretation by eye and using digitally-acquired pixel intensity ratios was investigated with respect to analytical sensitivity. The LFIA detects amatoxins in human urine after visual evaluation to as little as 5 ng/mL (α-amanitin - 10 ng/mL, β-amanitin - 50 ng/mL, γ-amanitin - 5 ng/mL). After digital analysis, pixel intensity ratios were determined to evaluate the LFIA as positive, negative, or trace result. Detection limits were redefined ranging from 1 ng/mL (α- and γ-amanitin) to 3 ng/mL (β-amanitin). For the proof-of-concept, 73 human urine samples submitted to the authors´ laboratory for toxicological analysis were analyzed using the LFIA and LC-HRMS/MS. Only three out of 73 urine samples were tested false positive with the LFIA as LC-HRMS/MS confirmation revealed no detection of amatoxins. Sixteen urine samples were evaluated as trace results and confirmed negative using LC-HRMS/MS except for one case which was positive for α-amanitin but negative for β-amanitin. Although particularly positive and trace results of the LFIA still need to be confirmed, the negative LFIA results correlated well with LC-HRMS/MS.