Utilizing the DNA Aptamer to Determine Lethal α-Amanitin in Mushroom Samples and Urine by Magnetic Bead-ELISA (MELISA).

Amanita poisoning is one of the most deadly types of mushroom poisoning. α-Amanitin is the main lethal toxin in amanita, and the human-lethal dose is about 0.1 mg/kg. Most of the commonly used detection techniques for α-amanitin require expensive instruments. In this study, the α-amanitin aptamer was selected as the research object, and the stem-loop structure of the original aptamer was not damaged by truncating the redundant bases, in order to improve the affinity and specificity of the aptamer. The specificity and affinity of the truncated aptamers were determined using isothermal titration calorimetry (ITC) and gold nanoparticles (AuNPs), and the affinity and specificity of the aptamers decreased after truncation. Therefore, the original aptamer was selected to establish a simple and specific magnetic bead-based enzyme linked immunoassay (MELISA) method for α-amanitin. The detection limit was 0.369 µg/mL, while, in mushroom it was 0.372 µg/mL and in urine 0.337 µg/mL. Recovery studies were performed by spiking urine and mushroom samples with α-amanitin, and these confirmed the desirable accuracy and practical applicability of our method. The α-amanitin and aptamer recognition sites and binding pockets were investigated in an in vitro molecular docking environment, and the main binding bases of both were T3, G4, C5, T6, T7, C67, and A68. This study truncated the α-amanitin aptamer and proposes a method of detecting α-amanitin.

In vivo and in vitro α-amanitin metabolism studies using molecular networking.

Amanitin poisonings are among the most life-threatening mushroom poisonings, and are mainly caused by the genus Amanita. Hepatotoxicity is the hallmark of amanitins, powerful toxins contained in these mushrooms, and can require liver transplant. Among amatoxins, α-amanitin is the most studied. However, the hypothesis of a possible metabolism of amanitins is still controversial in this pathophysiology. Therefore, there is a need of clarification using cutting-edge tools allowing metabolism study. Molecular network has emerged as powerful tool allowing metabolism study through organization and representation of untargeted tandem mass spectrometry (MS/MS) data in a graphical form. The aim of this study is to investigate amanitin metabolism using molecular networking. In vivo (four positive amanitin urine samples) and in vitro (differentiated HepaRG cells supernatant incubated with α-amanitin 2 µM for 24 h) samples were extracted and analyzed by LC-HRMS/MS using a Q Exactive™ Orbitrap mass spectrometer. Using molecular networking on both in vitro and in vivo, we have demonstrated that α-amanitin does not undergo metabolism in human. Thus, we provide solid evidence that a possible production of amanitin metabolites cannot be involved in its toxicity pathways. These findings can help to settle the debate on amanitin metabolism and toxicity.

[Determination of trace α-amanitin in urine of mushroom poisoning patient by online solid phase extraction-liquid chromatography-tandem mass spectrometry].

An analytical method was established for the determination of trace α-amanitin in the urine of patients suffering from mushroom poisoning by online solid phase extraction-liquid chromatography-tandem mass spectrometry (online SPE-LC-MS/MS). The sample was protein precipitated with formic acid acidified acetonitrile-methanol (5:1, v/v). Reversed-phase liquid-liquid microextraction was used to remove the organic solvent from the sample extract. The toxin was purified by online SPE using an ODS micro column (5 mm×2.1 mm, 5 µm), and separated on an XBridgeTM BEH C18 column (150 mm×3.0 mm, 2.5 µm). Finally, the toxin was measured by MS/MS in the negative electrospray ionization (ESI-) mode. Multiple reaction monitoring (MRM) was used, and the conditions were m/z 917.4>205.1 (quantitative ion transition) and m/z 917.4>257.1. Collision energy for both transitions was 55 eV. A fast valve-switching technique with a quantitative loop was used as an interface between the online SPE and LC-MS/MS modules. The two modules were independent, neither the mobile phase nor the pressure would interfere with each other, thus ensuring the stability of the system. Precise purification by the online system could effectively eliminate the matrix effects in the subsequent MS detection. Weak matrix suppression effects were found, with results of 88.7%-96.5%. The linear range of α-amanitin in urine was 0.1-50 µg/L with a correlation coefficient (r2) of 0.9983. The limit of detection (LOD) and limit of quantification (LOQ) in the sample matrix were 0.03 µg/L and 0.1 µg/L, respectively. The average recoveries at three spiked levels (0.1, 2.0 and 20 µg/L) were 84.3%-91.7% with relative standard deviations (RSDs) of 3.8%-7.2%. The accuracy and precision were evaluated using quality control samples with toxin contents of 0.1 µg/L (LOQ), 0.2 µg/L (2-fold LOQ), 2.0 µg/L (medium level), and 20 µg/L (high level). The calculated average intra-day accuracy was 85.1%-96.0% with the precision of 4.1%-7.8%. The inter-day accuracy was 82.9%-94.8% with the precision of 5.0%-9.5%. The specificity of the method was verified by negative samples derived from patients who suffered only gastroenteritis poisoning, without hepatotoxic symptoms. α-Amanitin was found in urine samples from nine mushroom poisoning patients with hepatotoxic symptoms. The sampling time ranged from 19 h to 92 h. The toxin contents were 0.11-53.1 µg/L. For patients with a high intake of poisonous mushrooms, the toxin content was 53.1 µg/L in a patient's urine sampled 19 h after accidental consumption and 0.19 µg/L in another patient's urine sampled 92 h after poisoning. The content of α-amanitin was only 0.53 µg/L in the urine sample obtained 23 h after consumption for a patient with low intake and 0.11 µg/L in the urine sampled from another patient 40 h after poisoning. Amatoxins can metabolize rapidly in vivo. The laboratory identification of amatoxin poisoning requires a method for trace-level analysis in the biological matrix. It is proved that this method is simple, accurate and sensitive by the application to the analysis of actual samples. The protein precipitation and reversed-phase liquid-liquid microextraction steps are fast and simple. Hence, they can be used as a rapid and effective pre-treatment method for online SPE-LC-MS/MS analysis of water-soluble toxins in biomaterial matrix. Highly sensitive analysis of α-amanitin in urine can be obtained using a precise purification technology via online SPE in this study. The problem of qualitative confirmation of the toxin at trace levels (0.03 µg/L) after poisoning can be solved. The laboratory identification time for amatoxin poisoning in some patients exceeds 90 h. The developed analytical method at trace level (0.1 µg/L of LOQ) can provide reliable technical support for establishing the dose-response relationship of α-amanitin in vivo. It can satisfy for the determination of trace α-amanitin in urine samples from patients with hepatotoxic mushroom poisoning.

Highly sensitive α-amanitin sensor based on molecularly imprinted photonic crystals.

α-amanitin is the most toxic amanita in mushrooms with lethal dose to humans around 0.1 mg. Kg-1. Hence, early identification of the poison would improve survival rates and prevent lethal poisoning cases. In this study, molecularly imprinted photonic crystal (MIPC) sensor was prepared by combining molecular imprinting with photonic crystal templates and tested towards the detection of α-amanitin. In this process, synthesized moiety of α-amanitin was utilized as template, dispersed SiO2 colloidal photonic crystal as carrier, methacrylic acid (MAA) as functional monomer, and ethylene glycol dimethacrylate (EDGMA) as crosslinker. The adsorption behavior of MIPC towards α-amanitin in ethanol solution showed shifts in diffraction peaks of MIPC upon binding with α-amanitin molecules. The reflection peak wavelength varied linearly with α-amanitin concentration according to the correlation formula: λ = 15.417c+489.17 (R2 = 0.9985). The recognition process was accompanied by gradual color change in MIPC film. The prepared MIPC sensor possessed wide linear range (10-9-10-3 mg L-1), change in visual color, low detection limit (10-10 mg L-1), short response time (2 min), and good reusability. The MIPC film was then tested towards the detection of α-amanitin in real biological samples (mushroom, urine, and serum) and showed reasonable shift in diffraction peaks and color change upon soaking in solutions spiked with α-amanitin at 10-6 mg L-1 and 10-3 mg L-1, suggesting the suitability of the film for the rapid identification of α-amanitin in complex sample matrices. Overall, the proposed sensor looks promising for the rapid identification of α-amanitin in clinical analysis and food poisoning.

[Amanitine determination as an example of peptide analysis in the biological samples with HPLC-MS technique].

Introduction: Routine toxicological analysis is mostly focused on the identification of non-organic and organic, chemically different compounds, but generally with low mass, usually not greater than 500­600 Da. Peptide compounds with atomic mass higher than 900 Da are a specific analytical group. Several dozen of them are highly-toxic substances well known in toxicological practice, for example mushroom toxin and animal venoms. In the paper the authors present an example of alpha-amanitin to explain the analytical problems and different original solutions in identifying peptides in urine samples with the use of the universal LC MS/MS procedure. Materials and methods: The analyzed material was urine samples collected from patients with potential mushroom intoxication, routinely diagnosed for amanitin determination. Ultra filtration with centrifuge filter tubes (limited mass cutoff 3 kDa) was used. Filtrate fluid was directly injected on the chromatographic column and analyzed with a mass detector (MS/MS). Results: The separation of peptides as organic, amphoteric compounds from biological material with the use of the SPE technique is well known but requires dedicated, specific columns. The presented paper proved that with the fast and simple ultra filtration technique amanitin can be effectively isolated from urine, and the procedure offers satisfactory sensitivity of detection and eliminates the influence of the biological matrix on analytical results. Another problem which had to be solved was the non-characteristic fragmentation of peptides in the MS/MS procedure providing non-selective chromatograms. It is possible to use higher collision energies in the analytical procedure, which results in more characteristic mass spectres, although it offers lower sensitivity. Conclusions: The ultra filtration technique as a procedure of sample preparation is effective for the isolation of amanitin from the biological matrix. The monitoring of selected mass corresponding to transition with the loss of water molecule offers satisfactory sensitivity of determination.

Amatoxins (α- and ß-Amanitin) and phallotoxin (Phalloidin) analyses in urines using high-resolution accurate mass LC-MS technology.

Mycotoxin intoxications can result from the consumption of amatoxins like α- and ß-amanitin or of phallotoxin, present in several toxic mushrooms like Amanita phalloides. To identify and quantify amatoxins and phallotoidin in biological matrixes, we developed a method using liquid chromatography coupled with an ultra-high-resolution and accurate mass instrument (liquid chromatography-high-resolution-mass spectrometry, LC-HR-MS), Q Exactive™ (Thermo Fisher). The method includes a simple solid-phase extraction of urine samples spiked with flurazepam as internal standard (IS), using Bond Elut Agilent Certify cartridges (C18, 200 mg, 3 mL). LC separation was performed on a C18 Accucore column (100 × 2.1 mm, 2.6 µm) using a gradient of 10 mM ammonium acetate buffer containing 0.1% (v/v) formic acid and of acetonitrile with 0.1% (v/v) formic acid. Separation of analytes was obtained in 7 min, with respective retention times for α-amanitin, ß-amanitin, phalloidin and IS of 1.9, 1.7, 3.5 and 3.8 min, respectively. Quantitation on the LC-HR-MS system was performed by extracting the exact mass value of each protonated species using a 5-p.p.m. mass window, which was 919.3614, 920.3455, 789.3257 and 388.1586 for α-amanitin, ß-amanitin, phalloidin and IS, respectively. Calibration curves were obtained by spiking drug-free urine at 1-100 ng/mL. Mean correlation coefficients, r(2), were above 0.99 for each amatoxins and phalloidin. According to currently accepted validation procedures, the method was tested for selectivity, calibration, accuracy, matrix effect, precision and recovery. Authentic urine samples from 43 patients suffering from a suspected intoxication with mushrooms were analyzed by LC-HR-MS, and the results were compared with ELISA competitive immunoassay. The LC-HR-MS presented large benefits over immunoassay of being specific, faster and more sensitive, making it suitable for daily emergency toxicological analysis.

Amanitin intoxication in two beef calves in California.

Two 2- and 3-month-old beef calves from 2 separate herds, locations, and times were found dead and were submitted to the veterinary diagnostic laboratory for diagnostic work-up. In both cases, no premonitory signs were seen by the owners. Histopathology revealed acute panlobular hepatic necrosis in both calves. In addition, calf A had copper and selenium deficiency, and calf B had oxalate nephrosis, and selenium and zinc deficiencies. Alpha-amanitin was detected in the urine from calf A, and in the liver and rumen contents from calf B using liquid chromatography-mass spectrometry. The cause of panlobular hepatic necrosis and death of both calves was determined to be amanitin toxicosis from ingestion of amanitin-containing mushrooms based on microscopic changes and toxicological analysis of tissues. In cases of sudden death in cows with histopathological findings of panlobular hepatic necrosis, toxicological analysis for amanitin is needed for a definitive diagnosis of poisoning by amanitin-containing mushrooms.

The enterohepatic circulation of amanitin: kinetics and therapeutical implications.

BACKGROUND: Amatoxin poisoning induces a delayed onset of acute liver failure which might be explained by the prolonged persistence of the toxin in the enterohepatic circulation. Aim of the study was to demonstrate amanitin kinetics in the enterohepatic circulation. METHODS: Four pigs underwent α-amanitin intoxication receiving 0.35 mg/kg (n=2) or 0.15 mg/kg (n=2) intraportally. All pigs remained under general anesthesia throughout the observation period of 72 h. Laboratory values and amanitin concentration in systemic and portal plasma, bile and urine samples were measured. RESULTS: Amanitin concentrations measured 5h after intoxication of 219±5ng/mL (0.35 mg/kg) and 64±3 (0.15 mg/kg) in systemic plasma and 201±8ng/mL, 80±13ng/mL in portal plasma declined to baseline levels within 24h. Bile concentrations simultaneously recorded showed 153±28ng/mL and 99±58ng/mL and decreased slightly delayed to baseline within 32 h. No difference between portal and systemic amanitin concentration was detected after 24h. CONCLUSIONS: Amanitin disappeared almost completely from systemic and enterohepatic circulation within 24 h. Systemic detoxification and/or interrupting the enterohepatic circulation at a later date might be poorly effective.

Determination of alpha- and beta-amanitin in clinical urine samples by Capillary Zone Electrophoresis.

Amanitins are toxins found in species of the mushroom genera Amanita, Lepiota and Galerina. Intoxication after ingestion of these mushrooms can be fatal with an estimated 20% of mortality rate. An early diagnosis is necessary in order to avoid invasive and expensive therapy and to improve patient's prognosis. In this paper, a Capillary Zone Electrophoresis method was developed and validated to determine alpha- and beta-amanitin in urine in less than 7 min using 5 mM, pH 10 borate buffer as background electrolyte. The separation conditions were: capillary: 75 microm I.D., 41 cm effective length, 48 cm total length, 25 degrees C, 20 KV and PDA detection at 214 nm. Sample treatment for analysis only required urine dilution in background electrolyte. The method was validated following established criteria and was found to be selective, linear in the range 5-100 ng/ml. Intra- and inter-day precision and accuracy were within required limits. Limit of detection (LOD) and limit of quantification (LOQ) were 1.5 and 5 ng/ml, respectively. Eight urine samples from suspected cases of intoxication with amanitins were analyzed after 2 years of storage at -20 degrees C, and beta-amanitin was determined in two samples with concentrations of 53 and 65 ng/ml, respectively. The method here described includes the use of non-aggressive reagents to the capillary or the system and is the first Capillary Electrophoresis method used to determine amanitins in clinical samples.