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.

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.

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.

Application to several suspected poisoning cases of a validated analytical method for the determination of muscarine in human biological matrices using liquid chromatography with high-resolution mass spectrometry detection.

Despite prevention efforts, many cases of mushroom poisoning are reported around the world every year. Among the different toxins implicated in these poisonings, muscarine may induce parasympathetic neurological damage. Muscarine poisonings are poorly reported in the current literature, implying a lack of available data on muscarine concentrations in human matrices. A validated liquid chromatography with high-resolution mass spectrometry detection (Orbitrap technology) method was developed to determine muscarine concentrations in human urine, plasma, and whole blood samples. Muscarine was determined using 100 µL of biological fluids, and precipitation was used for sample preparation. Liquid chromatography-mass spectrometry was performed using an Accucore Phenyl-X analytical column with the electrospray source in positive ion mode. Muscarine was quantitated in parallel reaction monitoring (PRM) mode with D9-muscarine as the internal standard. The method was validated successfully over the concentration range 0.1-100 µg/L for plasma and whole blood and 1-100 µg/L for urine, with acceptable precision and accuracy (<13.5%), including the lower limit of quantification. Ten real cases of suspected muscarine poisoning were successfully confirmed with this validated method. Muscarine concentrations in these cases ranged from 0.12 to 14 µg/L in whole blood,

GC/MS determination of ibotenic acid and muscimol in the urine of patients intoxicated with Amanita pantherina.

Ibotenic acid and muscimol are substances which mostly participate in psychotropic properties of Amanita pantherina and Amanita muscaria. They are rapidly absorbed from the gastrointestinal tract and readily excreted in urine. The poisoning with A. pantherina is in the majority of cases accidental because it can be easily mistaken for the edible species (Amanita rubescens, Amanita spissa and Macrolepiota procera). Intoxication with A. muscaria is mostly intentional for recreational purposes. Prognosis of the poisoning is generally good; lethal cases are rare. Mushroom poisoning is often proved by microscopic examination of spores in the stomach or intestinal content. Authors of this article introduce an instrumental method of proving A. pantherina or A. muscaria poisoning. The article describes the isolation of ibotenic acid and muscimol from urine, the derivatization step and the determination of these compounds by gas chromatography/mass spectrometry. Isolation of these alkaloids from urine was performed on a strong cation exchanger (Dowex® 50W X8), and the elution and derivatization of the alkaloids were made in one step with ethyl chloroformate in aqueous solution of sodium hydroxide with the addition of ethanol and pyridine. Cycloserine was used as internal standard. By this method, concentrations of ibotenic acid and muscimol in the urine of four persons intoxicated with A. pantherina were determined. In this study, mass spectra of derivatized ibotenic acid and muscimol are shown, and validation of the method is described.

Further development of a liquid chromatography-high-resolution mass spectrometry/mass spectrometry-based strategy for analyzing eight biomarkers in human urine indicating toxic mushroom or Ricinus communis ingestions.

Recently, we presented a strategy for analysis of eight biomarkers in human urine to verify toxic mushroom or Ricinus communis ingestions. However, screening for the full panel is not always necessary. Thus, we aimed to develop a strategy to reduce analysis time and by focusing on two sets of analytes. One set (A) for biomarkers of late-onset syndromes, such as phalloides syndrome or the syndrome after castor bean intake. Another set (B) for biomarkers of early-onset syndromes, such as pantherine-muscaria syndrome and muscarine syndrome. Both analyses should be based on hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry (MS)/MS (HILIC-HRMS/MS). For A, urine samples were prepared by liquid-liquid extraction using dichloromethane and subsequent solid-phase extraction of the aqueous supernatant. For B urine was precipitated using acetonitrile. Method A was validated for ricinine and α- and ß-amanitin and method B for muscarine, muscimol, and ibotenic acid according to the specifications for qualitative analytical methods. In addition, robustness of recovery and normalized matrix factors to matrix variability measured by urinary creatinine was tested. Moreover, applicability was tested using 10 urine samples from patients after suspected mushroom intoxication. The analytes α- and ß-amanitin, muscarine, muscimol, and ibotenic acid could be successfully identified. Finally, psilocin-O-glucuronide could be identified in two samples and unambiguously distinguished from bufotenine-O-glucuronide via their MS2 patterns. In summary, the current workflow offers several advantages towards the previous method, particularly being more labor-, time-, and cost-efficient, more robust, and more sensitive.

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.

[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.

[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.

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.

Development and application of a strategy for analyzing eight biomarkers in human urine to verify toxic mushroom or ricinus communis ingestions by means of hydrophilic interaction LC coupled to HRMS/MS.

The analytical proof of a toxic mushroom and/or plant ingestion at an early stage of a suspected intoxication can be crucial for fast therapeutic decision making. Therefore, comprehensive analytical procedures need to be available. This study aimed to develop a strategy for the qualitative analysis of α- and ß-amanitin, psilocin, bufotenine, muscarine, muscimol, ibotenic acid, and ricinine in human urine by means of hydrophilic interaction liquid chromatography-high resolution MS/MS (HILIC-HRMS/MS). Urine samples were prepared by hydrophilic-phase liquid-liquid extraction using dichloromethane and subsequent solid-phase extraction and precipitation, performed in parallel. Separation and identification of the biomarkers were achieved by HILIC using acetonitrile and methanol as main eluents and Orbitrap-based mass spectrometry, respectively. The method was validated as recommended for qualitative procedures and tests for selectivity, carryover, and extraction recoveries were included to also estimate the robustness and reproducibility of the sample preparation. Limits of identification were 1 ng/mL for α- and ß-amanitin, 5 ng/mL for psilocin, bufotenine, muscarine, and ricinine, and 1500 ng/mL and 2000 ng/mL for ibotenic acid and muscimol, respectively. Using γ-amanitin, l-tryptophan-d5, and psilocin-d10 as internal standards, compensation for variations of matrix effects was shown to be acceptable for most of the toxins. In eight urine samples obtained from intoxicated individuals, α- and ß-amanitin, psilocin, psilocin-O-glucuronide, muscimol, ibotenic acid, and muscarine could be identified. Moreover, psilocin-O-glucuronide and bufotenine-O-glucuronide were found to be suitable additional targets. The analytical strategy developed was thus well suited for analyzing several biomarkers of toxic mushrooms and plants in human urine to support therapeutic decision making in a clinical toxicology setting. To our knowledge, the presented method is by far the most comprehensive approach for identification of the included biomarkers in a human matrix.

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.

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.

A Simple and High-Throughput Analysis of Amatoxins and Phallotoxins in Human Plasma, Serum and Urine Using UPLC-MS/MS Combined with PRiME HLB µElution Platform.

Amatoxins and phallotoxins are toxic cyclopeptides found in the genus Amanita and are among the predominant causes of fatal food poisoning in China. In the treatment of Amanita mushroom poisoning, an early and definite diagnosis is necessary for a successful outcome, which has prompted the development of protocols for the fast and confirmatory determination of amatoxins and phallotoxins in human biological fluids. For this purpose, a simple, rapid and sensitive multiresidue UPLC-MS/MS method for the simultaneous determination of α-amanitin, ß-amanitin, γ-amanitin, phalloidin (PHD) and phallacidin (PCD) in human plasma, serum and urine was developed and validated. The diluted plasma, serum and urine samples were directly purified with a novel PRiME technique on a 96-well µElution plate platform, which allowed high-throughput sample processing and low reagent consumption. After purification, a UPLC-MS/MS analysis was performed using positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. This method fulfilled the requirements of a validation test, with good results for the limit of detection (LOD), lower limit of quantification (LLOQ), accuracy, intra- and inter-assay precision, recovery and matrix effects. All of the analytes were confirmed and quantified in authentic plasma, serum and urine samples obtained from cases of poisoning using this method. Using the PRiME µElution technique for quantification reduces labor and time costs and represents a suitable method for routine toxicological and clinical emergency analysis.

[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.

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.

Analysis of amatoxins alpha-amanitin and beta-amanitin in toadstool extracts and body fluids by capillary zone electrophoresis with photodiode array detection.

Over 90% of the lethal cases of mushroom toxin poisoning in man are caused by a species of amanita. The amatoxins (especially alpha- and beta-amanitin) found in amanita deserve special attention, because of their high pharmacological potency, their high natural concentration and their high chemical and thermal stability. Measures can be taken to improve the survival rates (aggressive gastroenteric decontamination, liver protection therapy) if the poisoning is diagnosed correctly and as early as possible. The standard assay for alpha-amanitin is a radioimmunoassay (RIA). Among other reagents, this assay uses 125I-labelled alpha-amaintin, which has a low shelf life. The assay is therefore not available at all hospitals and all year round. In this paper, a first attempt to employ capillary zone electrophoresis (CZE) to quantify amatoxins alpha- and beta-amanitin in urine samples of afflicted patients and in toadstool extracts is described. Diode array detection is used for identification of the resolved substances in the electropherogram. An analysis requires 20 min. The detection limit is 1 microgram/ml, i.e., 5 pg absolute. Relative standard deviations are between 1 and 2% for the calibration standards (peak height and area) and ca. 7.5% for the real samples. Advantages of the CZE over the RIA include lower cost, the possibility of quantifying several toxins in one analysis, less consumption of potentially harmful reagents (no radio-labelled substances, no addition of alpha-amanitin as reagent) and, most importantly, all-year-round availability of the assay. The detection limit is still somewhat high and does not cover the entire clinically relevant range. Attempts to lower the detection limit by the necessary order of magnitude are currently under way in our laboratory. These include application of laser-induced fluorescence detection, liquid chromatography-CZE and CZE-mass spectrometry techniques.

Validation of a high performance liquid chromatographic method for alpha amanitin determination in urine.

The objective of the present study was to develop and validate a liquid chromatographic method with electrochemical detection to measure alpha amanitin concentrations in urine after sample pretreatment with double mechanism (reversed phase/cation exchange) solid-phase extraction cartridges. The urine samples (10 ml) were purified and concentrated to 1 ml with elimination of matrix contaminants. The extracts were then separated by isocratic reversed-phase chromatography using a C18 column (4.6 mm x 25 cm) with a mobile phase composed of 0.005 M phosphate buffer (pH 7.2) and acetonitrile (90:10). Coulometric detection was performed by applying an oxidation potential of +500 mV to a porous graphite electrode in a low-volume analytical cell. The limit of quantitation was 10 ng/ml with a signal-to-noise ratio = 25. The linearity studied on spiked urine was satisfactory (r = 0.9966) from 10 ng/ml to 200 ng/ml. The average extraction recovery of alpha amanitin was 78%, determined using spiked urine samples ranging from 10-300 ng/ml. The intra-assay precision was checked at 10, 50 and 100 ng/ml levels (n = 10) in spiked urine samples, with resulting coefficients of variation of 3.6%, 2% and 1.5%, respectively.