Mass Spectrometry-Based Analytical Methods of Amatoxins in Biological Fluids to Monitor Amatoxin-Induced Mushroom Poisoning

Abstract

Amatoxin-induced mushroom poisoning starts with nonspecific symptoms of toxicity but hepatic damage may follow, resulting in the rapid development of liver insufficiency and, ultimately, coma and death. Accurate detection of amatoxins, such as α-, β-, and γ-amanitin, within the first few hours after presentation is necessary to improve the therapeutic outcomes of patients. Therefore, analytical methods for the identification and quantification of α-, β-, and γ-amanitin in biological samples are necessary for clinical and forensic toxicology. This study presents a literature review of the analytical techniques available for amatoxin detection in biological matrices, and established an inventory of liquid chromatography (LC) techniques with mass spectrometry (MS), ultraviolet (UV) detection, and electrochemical detection (ECD). LC-MS methods using quadrupole tandem mass spectrometry, time-of-flight mass spectrometry, and orbitrap MS are powerful analytical techniques for the identification and determination of amatoxins in plasma, urine, serum, and tissue samples, with high sensitivity, specificity, and reproducibility compared to LC with UV and ECD, enzyme-linked immunoassay, and capillary electrophoresis methods.

Introduction

Mushrooms are consumed worldwide as an ingredient of many meals, but severe syndromes and even death can be caused by the misidentification of wild poisonous mushrooms as edible.^1-6 Mushrooms such as Psilocybe species and Amantia muscaria are intentionally abused because of their psychoactive activities.⁷ There is some evidence that mushroom poisoning may be increasing, and that exotic species are entering new areas and countries, thereby expanding the range of mushroom poisoning symptoms observed in presenting patients.⁸ White et al.⁹ classified mushroom toxins based on the clinical types of mushroom poisoning, as follows: primary hepatotoxicity (amatoxins), primary nephrotoxicity (AHDA, orellanine), neurotoxicity (psilocybins, muscarines, ibotenic acid, muscimol), mytotoxicity (saponaceolide B), metabolic/endocrine toxicity (gyromitrins, coprines, trichothecenes, polyporic acid), gastrointestinal irritants, and miscellaneous (entinan, acromelic acid).

Although global data are not available, the absolute number and incidence of mushroom poisoning cases may be increasing based on local studies.^9-12 An emerging mushroom poisoning risk in Europe may be the result of the large migrant influx, a subset of whom forage for food because of poor economic circumstances. This results in the consumption of mushrooms not known to the migrants and an increased incidence of amatoxin-type mushroom poisoning.^3,10 Because most toxic syndromes caused by mushroom toxins start with unspecific symptoms, diagnostic difficulties are most common during the critical first hours after presentation.³ Therefore, suspected poisonings should be confirmed or excluded to ensure that therapy starts as soon as possible, and to prevent an inappropriate therapy being implemented. The analytical strategies used to identify poisonous mushroom toxins include spore analysis (if mushroom leftovers or gastric content are available) and the identification of various toxins and their metabolites in human biological samples.^3,14,15 It is therefore necessary to develop sensitive, selective, and rapid analytical methods for the identification and quantification of mushroom toxins and their metabolites in the human biological matrix.

The purpose of this study was to review the bioanalytical methods used for the diagnosis of amatoxin-induced mushroom poisoning, which is the main cause of fatal mushroom poisoning.^3,10,13

Amatoxin

Amatoxins are highly toxic bicyclic octapeptides (Figure 1). They are the most toxic compounds among mushroom toxins, and are found in the Amanita, Galerina, and Lepiota mushroom species.^4-6 Among these species, Amanita phalloides has the most toxin components/weight and is responsible for most cases of fatal poisoning.^15-18 These toxins are classified as neutral substances (α-amanitin, γ-amanitin, amaninamide, amanullin, and proamanullin) or acidic substances (β-amanitin, ε-amanitin, amanine, and amanullic acid), which differ in terms of the number of hydroxyl groups and amide carboxyl exchange (Figure 1).^19,20 Amatoxins are water-soluble, heat-stable, and resistant to enzyme and acid degradation. Therefore, these substances remain unchanged during freezing, drying, and cooking (including frying, grilling, boiling, steaming, and other processing operations, such as digestive processing), and are resistant to gastrointestinal inactivation and metabolic processes.^18,21-23 However, amatoxins can be degraded slowly when stored in an open and aqueous solution, or exposed to sun or neon light for periods of about 7−8 months, which could potentiate the toxicity of amatoxins upon exposure in vivo.^17,20 The content of amatoxins varies among Amanita species, but α- and β-amanitin are the most abundant substances. For example, an amatoxin content of 9.3 mg/g was observed in dried mushrooms, while α- and β-amanitin accounted for 56% of the toxins found in dried powder from A. phalloides.²⁴ It has been reported that α- and β-amanitin account for 82% of toxins (2.87 mg of amanitin/3.49 mg peptide toxins/g dried powder) in A. exitialis.^25,26 According to Yilmaz et al.,²⁷ an oral intake of approximately 50 g of fresh A. phalloides, equivalent to a dose of 0.32 mg/kg of amatoxins, can be lethal.

Figure 1. Chemical structures of amatoxins.

The LD₅₀ value of α-amanitin was reported to be 0.3–0.6 mg/kg in mice and 4.0 mg/kg in rats (intraperitoneal injection), 0.1 mg/kg in humans (oral administration), and 0.1 mg/kg in dogs (intravenous injection).^17,28,29 The LD₅₀ values of β-amanitin, γ-amanitin, ε-amanitin, amanitin, and amaninamide were reported to be 0.5, 0.2−0.5, 0.3−0.6, 0.5, and 0.5 mg/kg, respectively, in mice following intraperitoneal injection.¹⁷ The LD₅₀ values for orally administered amanullin, amanullinic acid, and proamanullin in mice were > 20 mg/kg, but these levels are not toxic to humans.^17,28

Amanita phalloides poisoning can cause acute hepatitis, leading to the rapid development of liver insufficiency and, ultimately, coma and death.^5,15,17 However, nephrotoxicity has been less frequently reported.³⁰ The main toxicity mechanism of amatoxins is the inhibition of RNA polymerase II, which leads to the inhibition of messenger RNA synthesis and protein synthesis.^31-33 Other toxic mechanisms have been suggested, including oxidative stress-related damage via the increased formation of reactive oxygen species induced by an increase in superoxide dismutase activity and inhibition of catalase activity;^18,34,35 and amatoxin-induced apoptosis caused by the translocation of p53 to the mitochondria, leading to alteration of mitochondrial membrane permeability through the formation of a complex with Bcl-xL and Bcl-2.^17,18,36-39 The cytotoxicity of α-amanitin is 10-fold greater than that of β-amanitin in MCF-7 cells.⁴⁰ The main toxicological studies of amatoxins have focused on α- and β-amanitin; therefore, no conclusions have been drawn regarding potential toxicity differences between neutral and acid amatoxins.^15,17

Toxicokinetic studies of α- and β-amanitin after the intravenous, intraperitoneal, and oral administration of amatoxin in rats and mice have been reported.^{14,18,20,21,41,42} Low absolute bioavailability of α-amanitin (3.5–4.8%) and β-amanitin (7.3–9.4%), and substantial transport thereof to the intestines, kidneys, and liver, were observed after they were orally administered to mice at doses of 2, 5, or 10 mg/kg.^41,42 α- and β-amanitin show similarities in terms of the elimination process; they are both eliminated in urine without significant metabolism.^21,41,42 α- and β-amanitin show OATP1B1- and OATP1B3-mediated hepatic uptake, but only β-amanitin shows OAT3-mediated kidney uptake.⁴² α- and β-amanitin were detected in serum, plasma, urine, liver, and fecal samples of amatoxin poisoning patients.^15,43–46 Among 43 amatoxin-intoxicated patients, 11 showed plasma concentrations of 8−190 ng/mL for α-amanitin and 23.5−162 ng/mL for β-amanitin.⁴⁶ In total, 35 urine, 12 feces, and 4 liver and kidney samples were obtained from 43 amatoxin-intoxicated patients, with ranges of 0.03–3.29 mg for α-amanitin and 0.05–5.21 mg for β-amanitin in 24 of the urine samples; 8.4–152 μg for α-amanitin and 4.2-6270 μg for β-amanitin in 10 fecal samples; and 10–19 ng/g and 122–1719 ng/g for α-amanitin, and 170.8–3298 ng/g and 1017–1391 ng/g for β-amanitin, in liver and kidney samples, respectively, for three of patients.⁴⁶ Although cellular uptake (mediated by hepatic or renal transporters) is approximately two-fold higher for β-amanitin than α-amanitin, as is liver and kidney accumulation, the contribution of β-amanitin to in vivo amatoxin toxicity may be lower than that of α-amanitin because of differences in their cytotoxicity.⁴⁰

Benzylpenicillin, silibinin, and N-acetylcysteine have been used for the treatment of amatoxin-induced mushroom poisoning.^3,17 The therapeutic effects may be attributed to the hepatoprotective and anti-oxidative activities of silibinin and N-acetylcysteine, and the reduced hepatic distribution of α- and β-amanitin resulting from the inhibition of OATP1B3 by benzylpenicillin, silibinin, and cyclosporine.^17,40,47-49

Analytical methods of amatoxins in biological fluids

Confirmation of the intake of mushrooms containing amatoxins is needed by detecting amatoxins such as α-, β-, and γ-amanitin in biological fluids to avoid expensive and time-consuming treatment for every suspected intoxication case.¹⁵ Sufficient analytical sensitivity is also necessary because hospitalization often occurs late after intake such that only trace amounts of toxins can be found.^15,44,46 Several methods have been reported for the qualification and quantification of amatoxins in biological fluids using liquid chromatography (LC) combined with mass spectrometry (MS),^41-45,50-70 ultraviolet (UV) detection,^{71,72,75,77,78} or electrochemical detection (ECD),^71,73,74,76 as well as capillary zone electrophoresis (CZE),^79,80 radioimmunoassay (RIA),^81,82 enzyme-linked immunosorbent assay (ELISA),^83,84 and lateral flow immunoassay (LFA).⁸⁵ However, each method has drawbacks. The ELISA and LFA methods have been used for the screening of α-, β-, or γ-amanitin in clinical toxicology, but compared to LC-MS methods they have the disadvantages of low sensitivity (3−10 ng/mL), high workload, false-negative and -positive results, and the requirement for additional confirmation in forensic cases.

The LC–MS methods have the advantages of high specificity, sensitivity, resolution, and rapidity relative to other analytical methods, making them suitable for routine clinical and forensic toxicological analysis of amatoxins. The LC-MS methods that have been developed for the analysis of α-, β-, and γ-amanitin in various biological fluids are summarized in Table 1. High-performance liquid chromatography (HPLC) with UV detection and ECD methods for the quantification of α- and β-amanitin in plasma, urine, liver, and kidney are summarized in Table 2; these methods have drawbacks such as low sensitivity and laborious sample preparation.

Table 1. The LC-MS and LC-MS/MS methods used for the determination of amatoxins in various biological matrices.

LLE: liquid-liquid extraction; SPE: solid-phase extraction; SCX: strong cation exchanger; LOD: limit of detection; ESI: electrospray ionization; PRM: parallel reaction monitoring; SIM: selected ion monitoring; SRM: selected reaction monitoring; MRM: multiple reaction monitoring; TOF: time of flight; IS: internal standard.

Table 2. The HPLC and capillary zone electrophoresis methods for the determination of amatoxins in various biological matrices.

LLE: liquid-liquid extraction; SPE: solid-phase extraction; SCX: strong cation exchanger; LOD: limit of detection

Sample preparation

Blood, plasma, serum, urine, bile, and tissue samples have been used for clinical purposes, forensic toxicology, and toxicokinetics of amatoxins.^41-45,51-80 Because the amatoxin concentrations in urine are usually higher than those in serum and plasma,^26,45,46 urine is considered as the biological sample of choice. However, major drawbacks of urine sampling include reduced output in the case of decreased renal function and acute renal failure, which can occur in some amatoxin and other mushroom poisoning cases, and the greater intra- and interindividual variability in the urine as a biomatrix. If therapeutic measures like fluid replacement or forced diuresis are applied, the low amounts of amatoxins in urine could be further diluted. Therefore, blood, plasma, and serum samples are more commonly used in clinics than urine samples for the determination of amatoxins.

For the determination of α-, β-, and γ-amanitin in human and animal plasma, serum, urine, and tissue samples using HPLC, LC-MS, liquid chromatography-tandem mass spectrometry (LC-MS/MS), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and CZE, several sample preparation techniques have been developed, including protein precipitation with acetonitrile, methanol, or perchloric acid, 41,42,51,52,59,64,71 liquid-liquid extraction (LLE),⁷⁸ solid-phase extraction (SPE) with reverse-phase, cation exchange or immunoaffinity cartridges,^{43,50,54,56,58,60-62,66-68,70,71,73,74,76} SPE of the aqueous phase obtained after LLE with dichloromethane or chloroform,⁵⁵ SPE of the aqueous phase obtained after protein precipitation of the biomatrix with acetonitrile and LLE of the supernatant with chloroform,^{44,45,65,69,72,77} and online column switching technique,^63,75 and simple dilution in CZE^79,80 (Tables 1 and 2). These methods use different volumes of biological matrix samples, as follows: serum, 100−5000 μL;^{54,60,67,69,72,76-78} plasma, 5−3000 μL;^{41,42,44,50-53,57-60,67,68,74,75} and urine, 50−10000 μL^{41,42,43,45,50,54-58,60-67,70,73,76-80} (Tables 1 and 2). Two or three sample preparation procedures have been combined in an attempt to avoid the matrix effect, but this has disadvantages such as high labor requirements and a long turnaround time (~24 h).

LC-MS methods

Reverse-phase chromatography using C₁₈, C₈, C₄, or phenyhexyl columns is the most common technique for chromatographic analysis of α-, β-, and γ-amanitin in biological fluids. Gradient elution of mobile phase A (ammonium acetate or formic acid) and mobile phase B (acetonitrile or methanol) has been used as the mobile phase for LC-MS methods (Table 1), whereas isocratic elution is used in HPLC methods (Table 2). Hydrophilic interaction chromatography has been used for the simultaneous determination of α-, β-, and γ-amanitin, and six mushroom toxins in human urine, to increase the retention and ionization efficiency.^55,56

Positive and negative electrospray ionization (ESI) modes have been used for the ionization of amatoxins when applying LC-MS methods (Table 1). Negative ESI mode has higher sensitivity and smaller matrix effects compared to positive ESI mode.^{41,42,45,50-53,64} MALDI-TOF MS has been used for qualitative analysis of α-amanitin, β-amanitin, and phalloidin in human urine.⁶⁶

For the quantification of α-, β-, and γ-amanitin, selective ion monitoring mode with quadrupole MS,^43,68,70 multiple reaction monitoring (MRM) or selected reaction monitoring (SRM) mode with triple quadrupole tandem MS (MS/MS),^{45,50,51,53,54,56,58-60,65,67} and parallel reaction monitoring (PRM) mode using orbitrap MS^{41,42,44,55,62,63} have been used (Table 1). LC-MS/MS methods using quadrupole MS/MS and orbitrap MS are powerful techniques with high sensitivity (lower limit of quantification [LLOQ] = 0.02–50 ng/mL plasma for α-, β-, and γ-amanitin), reproducibility, and specificity. Recently, LC-MS/MS methods performed in PRM mode and protein precipitation of plasma samples (5 mL) for sample clean-up showed good sensitivity (LLOQ 0.5 ng/mL for α- and β-amanitin), selectivity, and speed.^41,42,52

Conclusions

Because the incidence of amatoxin-induced mushroom poisoning has increased globally, early detection of amatoxins in cases of suspected mushroom poisoning is necessary to improve patient outcomes through aggressive and immediate supportive care among other potential therapies. Early diagnosis of amatoxins has been achieved using LC-MS/MS methods. These methods may be suitable for routine clinical and forensic toxicological analysis of amatoxins in plasma, serum, urine, and tissue samples due to the high specificity, sensitivity, and reproducibility relative to other analytical methods. However, protein precipitation, LLE, and SPE have been combined for sample preparation, to minimize matrix effects and achieve high sensitivity, but with high labor requirements and a long turnaround time. There is a need to improve sample preparation procedures for rapid clinical and forensic toxicological analyses.