Simple and rapid detection of three amatoxins and three phallotoxins in human body fluids by UPLC-MS-MS and its application in 15 poisoning cases.

Amatoxins and phallotoxins are toxic cyclopeptides found in the genus Amanita and are among the predominant causes of foodborne sickness and poisoning-related fatalities in China. This study introduces and validates a simple, rapid and cost-effective ultra-performance liquid chromatography-mass spectrometry method for the simultaneous determination and quantification of α-amanitin, ß-amanitin, γ-amanitin, phallisacin, phallacidin and phalloidin in human blood and urine. Quick therapeutic decision-making is supported by a 9 min chromatographic separation performed on a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm) using a gradient of high-performance liquid chromatography (HPLC)-grade water and methanol:0.005% formic acid. The analyte limit of quantification was 1-3 ng/mL in blood and 0.5-2 ng/mL in urine. Calibrations curves, prepared by spiking drug-free blood and urine, demonstrated acceptable linearity with mean correlation coefficients (r) greater than 0.99 for all phallotoxins and amatoxins. Acceptable intraday and interday precision (relative standard deviation <15%) and accuracy (bias, -4.8% to 13.0% for blood and-9.0% to 14.7% for urine) were achieved. The validated method was successfully applied to analyze 9 blood samples and 2 urine samples testing positive for amatoxins and/or phallotoxins. Amatoxins and/or phallotoxins were identified in each whole blood sample at a range of 1.12-5.63 ng/mL and in two urine samples from 1.01-9.27 ng/mL. The method has the benefits of simple sample preparation (protein precipitation) and wide analyte coverage, making it suitable for emergency quantitative surveillance toxicological analysis in clinics and forensic poisoning practice.

A label-free electrochemical immunosensor based on decorated cellulose nanofibrous membrane for point-of-care diagnosis of amanitin poisoning via human urine.

α-Amanitin (AMN) is one of the deadliest toxins from mushrooms, present in the deadly mushroom species Amanita phalloides. It is a bicyclic octapeptide and represents up to 40% of the amatoxins in mushrooms, damaging the liver and kidneys. Current methods of detecting amatoxins are time-consuming and require the use of expensive equipment. A novel label-free electrochemical immunosensor was successfully developed for rapid detection of α-amanitin, which was fabricated by immobilization of anti-α-amanitin antibodies onto a functionalized cellulose nanofibrous membrane-modified carbon screen-printed electrode. An oxidation peak of the captured amanitin on the tethered antibodies was observed at 0.45 V. The performance of the nanofibrous membrane on the electrode and necessary fabrication steps were investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Due to its unique structural features and properties such as high specific surface area and microporous structure, the nanofibrous membrane as an immunosensor matrix for antibody tethering improved the electrochemical performance of the immunosensor by more than 3 times compared with cast membranes. Under the optimal conditions, the assembled immunosensor exhibited high sensitivity toward α-amanitin detection in the range of 0.009-2 ng mL-1 with a limit of detection of 8.3 pg mL-1. The results clearly indicate that the fabricated nanofiber-based-immunosensor is suitable for point-of-care detection of lethal α-amanitin in human urine without any pretreatment within 30 min.

Production of highly sensitive monoclonal antibody and development of lateral flow assays for phallotoxin detection in urine.

Phallotoxins, toxic cyclopeptides found in wild poisonous mushrooms, are predominant causes of fatal food poisoning. For the early and rapid diagnosis mushroom toxin poisoning, a highly sensitive and robust monoclonal antibody (mAb) against phallotoxins was produced for the first time. The half-maximum inhibition concentration (IC50) values of the mAb-based indirect competitive ELISAs for phallacidin (PCD) and phalloidin (PHD) detection were 0.31 ng mL-1 and 0.35 ng mL-1, respectively. In response to the demand for rapid screening of the type of poisoning and accurate determination of the severity of poisoning, colloidal gold nanoparticle (GNP) and time-resolved fluorescent nanosphere (TRFN) based lateral flow assays (LFA) were developed. The GNP-LFA has a visual cut-off value of 3.0 ng mL-1 for phallotoxins in human urine sample. The TRFN-LFA provides a quantitative readout signal with detection limit of 0.1 ng mL-1 in human urine sample. In this study, urine samples without pretreatment were used directly for the LFA strip tests, and both two LFAs were able to accomplish analysis within 10 min. The results demonstrated that LFAs based on the newly produced, highly sensitive, and robust mAb were able to be used for both rapid qualitative screening of the type of poisoning and accurate quantitative determination of the severity of poisoning after accidental ingestion by patients of toxic mushrooms.

Analysis of α- and ß-amanitin in Human Plasma at Subnanogram per Milliliter Levels by Reversed Phase Ultra-High Performance Liquid Chromatography Coupled to Orbitrap Mass Spectrometry.

Amatoxins are known to be one of the main causes of serious to fatal mushroom intoxication. Thorough treatment, analytical confirmation, or exclusion of amatoxin intake is crucial in the case of any suspected mushroom poisoning. Urine is often the preferred matrix due to its higher concentrations compared to other body fluids. If urine is not available, analysis of human blood plasma is a valuable alternative for assessing the severity of intoxications. The aim of this study was to develop and validate a liquid chromatography (LC)-high resolution tandem mass spectrometry (HRMS/MS) method for confirmation and quantitation of α- and ß-amanitin in human plasma at subnanogram per milliliter levels. Plasma samples of humans after suspected intake of amatoxin-containing mushrooms should be analyzed and amounts of toxins compared with already published data as well as with matched urine samples. Sample preparation consisted of protein precipitation, aqueous liquid-liquid extraction, and solid-phase extraction. Full chromatographical separation of analytes was achieved using reversed-phase chromatography. Orbitrap-based MS allowed for sufficiently sensitive identification and quantification. Validation was successfully carried out, including analytical selectivity, carry-over, matrix effects, accuracy, precision, and dilution integrity. Limits of identification were 20 pg/mL and calibration ranged from 20 pg/mL to 2000 pg/mL. The method was applied to analyze nine human plasma samples that were submitted along with urine samples tested positive for amatoxins. α-Amanitin could be identified in each plasma sample at a range from 37-2890 pg/mL, and ß-amanitin was found in seven plasma samples ranging from <20-7520 pg/mL. A LC-HRMS/MS method for the quantitation of amatoxins in human blood plasma at subnanogram per milliliter levels was developed, validated, and used for the analysis of plasma samples. The method provides a valuable alternative to urine analysis, allowing thorough patient treatment but also further study the toxicokinetics of amatoxins.

Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine.

Globally, mushroom poisonings cause about 100 human deaths each year, with thousands of people requiring medical assistance. Dogs are also susceptible to mushroom poisonings and require medical assistance. Cyclopeptides, and more specifically amanitins (or amatoxins, here), are the mushroom poison that causes the majority of these deaths. Current methods (predominantly chromatographic, as well as antibody-based) of detecting amatoxins are time-consuming and require expensive equipment. In this work, we demonstrate the utility of the lateral flow immunoassay (LFIA) for the rapid detection of amatoxins in urine samples. The LFIA detects as little as 10 ng/mL of α-amanitin (α-AMA) or γ-AMA, and 100 ng/mL of ß-AMA in urine matrices. To demonstrate application of this LFIA for urine analysis, this study examined fortified human urine samples and urine collected from exposed dogs. Urine is sampled directly without the need for any pretreatment, detection from urine is completed in 10 min, and the results are read by eye, without the need for specialized equipment. Analysis of both fortified human urine samples and urine samples collected from intoxicated dogs using the LFIA correlated well with liquid chromatography-mass spectrometry (LC-MS) methods.

A case study of Lepiota brunneoincarnata poisoning with endoscopic nasobiliary drainage in Shandong, China.

The most frequently reported fatal Lepiota ingestions are due to L. brunneoincarnata. We present a case of L. brunneoincarnata poisoning with endoscopic nasobiliary drainage known to be the first in China. The patient suffered gastrointestinal symptoms 9 h post ingestion of mushrooms. The patient was hospitalized 4 days after eating the mushrooms with jaundice. The peak ALT, AST, APTT, TBIL and DBIL values of the patient were as follow: ALT, 2980 U/L (day 4 post ingestion); AST, 1910 U/L (day 4 post ingestion); APTT, 92.8 seconds (day 8 post ingestion), TBIL, 136 µmol/L (day 10 post ingestion), DBIL 74 µmol/L (day 10 post ingestion). UPLC-ESI-MS/MS was used to detect the peptide toxins in the mushroom and biological samples from the patient. We calculated that the patient may have ingested a total of 29.05 mg amatoxin from 300 g mushrooms, consisting of 19.91 mg α-amanitin, 9.1 mg ß-amanitin, and 0.044 mg γ-amanitin. Amatoxins could be detected in bile even on day 6 after ingestion of L. brunneoincarnata. With rehydration, endoscopic nasobiliary drainage and intravenous infusion of Legalon SIL, the patient recovered after serious hepatotoxicity developed.

Potential value of urinary amatoxin quantification in patients with hepatotoxic mushroom poisoning.

BACKGORUND & AIMS: Mushroom poisoning with Amanita phalloides or similar species can lead to liver failure with 10-30% mortality rates. We aimed at defining the prognostic value of urinary amatoxin quantification in patients with hepatotoxic mushroom poisoning. METHODS: Data from 32 patients with hepatotoxic mushroom poisoning (Hospital Clínic Barcelona, 2002-16) in whom urinary amatoxins were determined (ELISA) were retrospectively reviewed. Correlations between urinary amatoxin and collected baseline variables with outcomes including hepatotoxicity (ALT>1000 U/L), severe acute liver injury (ALI, prothrombin <50%), acute liver failure (ALF, ALI and encephalopathy), transplantation/death and hospital length-of-stay, were evaluated. RESULTS: 12/32 patients developed increased aminotransferase activity. Among the 13/32 amatoxin negative patients, 1 developed ALI and 12/13 no hepatotoxicity. Among the 19/32 amatoxin positive patients, 8/19 (42%) developed hepatotoxicity, including 5 who progressed to severe ALI, of whom 3 developed ALF (2 deaths, 1 transplantation). Urinary amatoxin and prothrombin were independent predictors of hepatotoxicity, ALT peak values (along with age) and hospital length-of-stay. In positive amatoxins patients, urinary concentrations > 55 ng/ml (or a baseline prothrombin ≤ 83%), were associated to hepatotoxicity (presented by 8/9 patients with ALT>1000 U/L). Among 5 patients with urinary amatoxin ≥ 70 ng/ml, 4 developed severe ALI. CONCLUSIONS: In patients with hepatotoxic mushroom poisoning, a negative urinary amatoxin quantification within 72h of intake ruled out the risk of hepatotoxicity in 92% of patients, whereas positive urinary amatoxins were associated with hepatotoxicity and severe ALI. Concentrations >55 ng/ml and ≥ 70 ng/ml were predictive of hepatotoxicity and severe ALI, respectively.

Detection of α-, ß-, and γ-amanitin in urine by LC-MS/MS using 15N10-α-amanitin as the internal standard.

The majority of fatalities from poisonous mushroom ingestion are caused by amatoxins. To prevent liver failure or death, it is critical to accurately and rapidly diagnose amatoxin exposure. We have developed a liquid chromatography tandem mass spectrometry method to detect α-, ß-, and γ-amanitin in urine to meet this need. Two internal standard candidates were evaluated, including an isotopically labeled 15N10-α-amanitin and a modified amanitin methionine sulfoxide synthetic peptide. Using the 15N10-α-amanitin internal standard, precision and accuracy of α-amanitin in pooled urine was ≤5.49% and between 100 and 106%, respectively, with a reportable range from 1-200 ng/mL. ß- and γ-Amanitin were most accurately quantitated in pooled urine using external calibration, resulting in precision ≤17.2% and accuracy between 99 and 105% with calibration ranges from 2.5-200 ng/mL and 1.0-200 ng/mL, respectively. The presented urinary diagnostic test is the first method to use an isotopically labeled α-amanitin with the ability to detect and confirm human exposures to α-, ß-, and γ-amanitin.

Amanitinas / Amanitines

La intoxicación por consumo de hongos es un fenómeno estacional que se produce con relativa frecuencia en áreas geográficas donde es habitual su consumo, en especial de especies silvestres. Dependiendo del tipo de hongo ingerido pueden aparecer distintos cuadros clínicos (gastrointestinal, nefrotóxico, alucinatorio, etc.). El cuadro más grave es el hepatotóxico, asociado a una alta mortalidad, y causado por hongos que contienen amatoxinas (síndrome ciclopeptídico). Presentamos una revisión actualizada de las características de las amatoxinas, su cinética y mecanismo de acción, los métodos utilizados para su determinación analítica, así como las diferentes opciones para el tratamiento de la intoxicación (AU)

Mushroom poisoning is a seasonal phenomenon that occurs relatively frequently in geographical areas where its consumption is common. Depending on the type of fungus ingested different clinical symptoms (gastrointestinal, nephrotoxic, hallucinatory, etc.) can occur. Hepatotoxic syndrome caused by fungi containing amatoxins is the most serious condition, associated to high mortality. We present an updated review of amatoxins characteristics, kinetics, mechanism of action, methods used for analytical determination, as well as the different options for the treatment of poisoning (AU)

Simultaneous determination of mushroom toxins α-amanitin, ß-amanitin and muscarine in human urine by solid-phase extraction and ultra-high-performance liquid chromatography coupled with ultra-high-resolution TOF mass spectrometry.

This paper presents a method for the simultaneous determination of α-amanitin, ß-amanitin and muscarine in human urine by solid-phase extraction (SPE) and ultra-high-performance liquid chromatography coupled with ultra-high-resolution TOF mass spectrometry. The method can be used for a diagnostics of mushroom poisonings. Different SPE cartridges were tested for sample preparation, namely hydrophilic modified reversed-phase (Oasis HLB) and polymeric weak cation phase (Strata X-CW). The latter gave better results and therefore it was chosen for the subsequent method optimization and partial validation. In the course of validation, limits of detection, linearity, intraday and interday precisions and recoveries were evaluated. The obtained LOD values of α-amanitin and ß-amanitin were 1ng/mL and of muscarine 0.09ng/mL. The intraday and interday precisions of human urine spiked with α-amanitin (10ng/mL), ß-amanitin (10ng/mL) and muscarine (1ng/mL) ranged from 6% to 10% and from 7% to 13%, respectively. The developed method was proved to be a relevant tool for the simultaneous determination of the studied mushroom toxins in human urine after mushroom poisoning.

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.

Direct analysis of the mushroom poisons α- and ß-amanitin in human urine using a novel on-line turbulent flow chromatography mode coupled to liquid chromatography-high resolution-mass spectrometry/mass spectrometry.

Poisonings with Amanita phalloides toxins require fast diagnosis in order to avoid expensive and unnecessary therapies. Initial clinical assessment in combination with urinary amanitin analysis is necessary for a definite diagnosis. Therefore, a simple, fast, and robust method was developed for reliable detection of α- and ß-amanitin as well as for fully validated quantification of α-amanitin in human urine. After simple dilution and centrifugation of the urine sample, a fast on-line extraction using a Transcend TLX-II system based on turbulent flow chromatography (TurboFlow) was established. A new TurboFlow mode was introduced, the pseudo quick elute mode (PQEM), which had more options for method optimization than the generic quick elute mode (QEM). It allowed running several modes in one valve arrangement. The PQEM showed better practicability in routine and emergency analysis than the previously used methods. After extraction, the fast 15min LC-high resolution (HR)-MS/MS analysis allowed reliable identification of α- and ß-amanitin based on fragments identified using so-called HR pseudo MS(3) experiments. According to international recommendations, the requirements for full validation including the parameters selectivity, calibration, accuracy, precision, recovery, matrix effects, and stability were fulfilled for α-amanitin. The method was successfully applied to the analysis of authentic urine samples containing amatoxins. In conclusion, this method allowed the determination of amatoxins using the novel PQEM in a faster, robust, and more reliable way than existing methods, making it suitable for daily routine and especially emergency toxicological analysis.

Development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry for determination of amanitins in urine and liver samples.

Amanitins, highly toxic cyclopeptides isolated from various Amanita species, are the most potent poisons accounting for the hazardous effects on intestinal epithelium cells and hepatocytes, and probably the sole cause of fatal human poisoning. The present study was focused on the development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), following urine and liver sample preparation by protein precipitation with organic solvents, and solid phase extraction (SPE) procedure, for the determination of the amatoxins, α- and ß-amanitin. Linearity, detection and quantification limits, selectivity, sensitivity, intra and inter-assay precision and recovery were studied, in order to guarantee reliability in the analytical results. The developed method proved to be specific and selective, with LOD (Limit of Detection) values for α- and ß-amanitin of 0.22 and 0.20 ng mL(-1) in urine and 10.9 and 9.7 ng g(-1) in liver, respectively. LOQ (Limit of Quantification) values ranged from 0.46 to 0.57 ng mL(-1) in urine and 12.3-14.7 ng g(-1) in tissue, for both amanitins. Linearity, in the range of 10.0-200.0 ng mL(-1) or ng g(-1), shows that coefficients of correlation were greater than 0.997 for α-amanitin and 0.993 for ß-amanitin. Precision was checked at three levels during three consecutive days with intra-day and inter-day coefficients of variation not greater than 15.2%. The extraction recovery presents good results for the concentrations analyzed, with values ranging from 90.2 to 112.9% for both matrices. Thus, the proposed analytical method is innovative, presents a high potential in the identification, detection and determination of α- and ß-amanitins in urine and tissue samples, as well as in other biological samples, such as kidney and 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.

[Amanitin determination in mushroom poisoning diagnostics]. / Oznaczanie amanityny w diagnostyce zatruc grzybami.

There are some serious poisonings with toxic mushroom species in Poland every year. Good prognostics in the cases is correlated to short time from mushroom consumption to hospitalization, correct distinguish not specific gastrointestinal and Amanita phalloides syndrome and immediately specific treatment. The purpose of the paper was to make appraisal of usefulness of amanitin blood and urine determination and transaminases activity determination (ALT, AST) in diagnostics of mushroom poisoned patients up to three days after mushroom consumption. The material was twenty two retrospective histories of mushroom poisoned patients treated in the years 2007-2008. Amanitin blood and urine determinations were made by ELISA method. Urine amanitin results in samples collected within 40 h from mushroom consuming were positive in all Amanita phalloides syndrome cases. Serum amanitin determination was not useful for the diagnostics. Trans-aminases activity determinations let to distinguish Amanita phalloides syndrome on the second and the third day after mushroom consumption. In the first poisoning phase (within 24 h), the ALT and AST activities were in normal ranges and only amanitin urine determination let to confirm or exclude Amanita phalloides poisoning. Amanitin urine determinations were useful to take fast decision about specific treatment and avoid internal organs dysfunctions.

Isolation and identification of the Amanita muscaria and Amanita pantherina toxins in human urine.

OBJECTIVES: Ibotenic acid, muscimol and muscarine were recognized as responsible for psychotropic effects of A. muscaria and A. pantherina. Demand for their specific and sensitive identification and quantitation in biological material lead to effort to develop reliable analytical method, but satisfactory solution is still lacking. Presented article describes liquid chromatography-mass spectrometry method suitable for isolation and identification of principal toxins of A. muscaria and A. pantherina in urine. METHODS AND RESULTS: Dedicated liquid chromatography-mass spectrometry method is reported. Technique consists of an extraction of analytes on Strata X-CW and Discovery SCX SPE cartridges and separation is achieved using a Gemini C18 column (150 mm x 2.0 mm, 5 micron) and 8 mM heptafluorobutyric acid as mobile phase. Detection at m/z 159 for ibotenic acid, m/z 115 for muscimol and m/z 174 for muscarine was used. Retention times and LODs are 2.6 min and 50 ng.ml-1 for ibotenic acid, 4.6 min and 40 ng.ml-1 for muscimol and 14.2 min and 3 ng.ml-1 for muscarine. CONCLUSION: A sensitive and specific liquid chromatography-mass spectrometry assay was developed for analysis of principal toxins of A. muscaria and A. pantherina in urine. Method was found to be sufficiently sensitive and specific for analysis of urine of intoxicated patients.

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.

Amatoxin poisoning: a 15-year retrospective analysis and follow-up evaluation of 105 patients.

INTRODUCTION: Fatalities due to mushroom poisonings are increasing worldwide, with more than 90% of deaths resulting from ingestion of amatoxin-containing species. METHODS: A retrospective evaluation of the history and clinical outcome of each patient treated from 1988 to 2002 in the Toxicological Unit of Careggi General Hospital (University of Florence, Italy) for amatoxin poisoning. Data included the biological parameters monitored, the treatment protocols used (intensive fluid and supportive therapy, restitution of the altered coagulation factors, multiple-dose activated charcoal, mannitol, dexamethasone, glutathione, and penicillin G), and outpatient follow-up evaluations. RESULTS: The clinical data of 111 patients were evaluated; their biological parameters were monitored every 12-24 hours until discharge. Two patients died; both were admitted to the hospital more than 60 hours after mushroom ingestion. Of all the laboratory parameters evaluated, the evolution of hepatic transaminases and prothrombin activity over four days were the most predictive indicators of recovery or death. Our follow-up evaluation of 105 patients demonstrated that our survivors recovered completely. CONCLUSIONS: Our experience indicates that the protocol used in our Toxicologicy Unit is effective for amatoxin poisoning, and that all patients treated within 36 hours after mushroom ingestion were cured without sequelae.

[Phalloidin syndrome: role of Elisa-based assay for the detection of alpha- and gamma-amanitins in urine. Preliminary results]. / Syndrome phalloïdien: quelle est la place du dosage des alpha- et gamma-amanitines par ELISA (Bühlmann)? Résultats préliminaires.

UNLABELLED: After consumption of mushrooms containing amatoxins (Amanita, Lepiota, and Galerina species), symptoms usually develop after a long delay (>6 h). Initial symptoms start as severe gastroenteritis, progressing to liver failure and possibly death as a result of hepatic coma. Since the survival rate of poisoned patients is claimed to depend on the time of beginning of efficient treatment, fast and reliable assays for amatoxins in biological fluids are essential. Described analytical methods for amatoxins include high performance liquid chromatography and radioimmunoassay (RIA). Recently, a new enzyme-linked immunosorbent assay (Bühlmann Amanitin ELISA kit) has been introduced as an alternative method to RIA. This ELISA-based assay offers several advantages: no complex extraction procedure is required (vs. HPLC) and no safety precautions concerning radioactivity have to be taken (vs. RIA). From August 2004 to October 2005, a pilot study was performed to test the practicability and the clinical utility of this method in emergency situations. RESULTS: ten urines, 9 serums and 1 faeces from 10 patients suffering from acute gastroenteritis after mushroom ingestions (7 contaminated meals) were analyzed. Definitive diagnosis of amatoxin poisoning was made in 4 cases (3 contaminated meals) on the basis of the anamnesis, laboratory results, and clinical course. A patient developed a severe amatoxin poisoning with urinary amanitins level < 1.5 microg/L (urines were collected more than 72 h after mushroom ingestion). Two patients were paucisymptomatic with urinary amanitins levels >10 microg/L (urines were collected before the 36th hour). CONCLUSION: Urine is the sample of choice for the determination of amatoxins. The most critical factor to invalidate the usefulness of this analysis is time. After 36 h, the sensitivity is unreliable.