α- and β-amanitin with purity of ≥90% and ~90%, respectively, were purchased from Sigma-Aldrich (Taufkirchen, Germany) and γ-amanitin methyl ether was donated by Prof. Dr. Heinz Faulstich (Max-Planck-Institute for Cell Biology, Ladenburg, Germany). Methanol (MeOH), acetonitrile (ACN), dichloromethane (DCM), formic acid, and other chemicals were of analytical grade or better and were purchased from VWR (Darmstadt, Germany). All chemicals used for the preparation of eluents for LC-MS were of LC-MS grade except for acetic acid and ascorbic acid. Water was purified using a Milli-Q water purification system (Merck KGaA, Darmstadt, Germany) to reach a resistivity of 18.2 MΩ∙cm.
β amanitin
Manufactured by Merck Group
Sourced in United States, Germany
β-amanitin is a laboratory standard product used for research and analytical purposes. It is a cyclic peptide compound isolated from the death cap mushroom (Amanita phalloides). β-amanitin is commonly used as a reference standard in various analytical techniques, such as high-performance liquid chromatography (HPLC) and mass spectrometry, for the identification and quantification of similar compounds.
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Lab products found in correlation
α amanitin, by Merck Group (5 mentions)
Phallacidin, by Merck Group (3 mentions)
γ amanitin, by Merck Group (2 mentions)
Acetonitrile, by Merck Group (2 mentions)
Milli q water purification system, by Merck Group (1 mentions)
Formalin, by Sangon (1 mentions)
Hematoxylin and eosin, by Sangon (1 mentions)
Hplc grade water, by Merck Group (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
1100 series lc msd system, by Agilent Technologies (1 mentions)
6 protocols using β amanitin
1
Amanitin Toxins Quantification by LC-MS
2
Biochemical Analysis of Amanita Species
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Fourteen dried specimens were selected for biochemical analyses. They represented all four sections of A. subgen. Lepidella, with an emphasis on the species which were morphologically assigned in the section Phalloideae but were phylogenetically clustered outside the section in our analyses. The presence of α-amanitin, β-amanitin and phalloidin (standard samples provided by Sigma Chemical Co, USA) was evaluated through high-performance liquid chromatography (HPLC) with the method of Chen et al. [53 ].
3
Amanitin Quantification by HPLC
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All reagents and solvents were of analytical grade unless specified. The standards of α-amanitin (≥90% purity), β-amanitin (≈90% purity), and sodium acetate were acquired from Sigma–Aldrich (Steinheim, Germany). HPLC-grade methanol and acetonitrile were obtained from Merck (Darmstadt, Germany), and ultrapure water was supplied by a Milli-Q water purification apparatus (Millipore Lda, Bedford, MA, USA). All solutions prepared for HPLC were filtered using a 0.45 mm nylon filter. The Oasis® PRIME HLB (1 mL/30 mg) from Waters Corp, (Mildford, MA, USA) and a vacuum manifold system (VaElut 6 Manifold Processing Station, Agilent Technologies, Santa Clara, CA, USA) were used.
4
Extraction and Analysis of Amanita exitialis
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lyophilized Amanita exitialis (100 g) was provided by Professor Chen (College of Life Sciences, Hunan Normal University, Changsha, China). The Amanita exitialis powder was stored in a refrigerator at 2–8 °C. α-amanitin, β-amanitin, γ-amanitin, phallacidin, HPLC-grade water, acetonitrile, and ammonium acetate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Normal saline (0.9% NaCl), ethyl alcohol, formalin, and hematoxylin and eosin were obtained from Sangon Biotech (Shanghai, China).
5
Toxin Analysis of Dried Basidiomata
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Dried basidiomata of the target taxon have been used for toxin analyses using the method of Lüli et al. (2019) (link). Toxins were extracted from basidiomata, using methanol–water–0.01 M hydrochloric acid (5:4:1, vol/vol) as the extraction buffer. Dried material (0.05 g) was crushed into fine powder in a mortar and pestle with liquid nitrogen. Then, 1.5 mL aforementioned buffer was added, and the suspension transferred into 1.5-mL centrifuge tubes. The tubes were kept at room temperature for 30 min, followed by centrifugation (12,000 rpm) for 3 min. Finally, the supernatant was transferred into new centrifuge tubes for mass spectrometry analysis.
The presence of cyclic peptides, especially α-amanitin, β-amanitin, phalloidin, and phallacidin (standards provided by Sigma Chemical Co, United States), was evaluated through the liquid chromatography–high-resolution mass spectrometry (LC-HRMS) using 1290 Infinity II HPLC systems coupled with 6540 UHD precision mass Q-TOF instruments under the conditions listed inTable 2 .
The presence of cyclic peptides, especially α-amanitin, β-amanitin, phalloidin, and phallacidin (standards provided by Sigma Chemical Co, United States), was evaluated through the liquid chromatography–high-resolution mass spectrometry (LC-HRMS) using 1290 Infinity II HPLC systems coupled with 6540 UHD precision mass Q-TOF instruments under the conditions listed in
6
Extraction and LC-MS Analysis of Fungal Toxins
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Peptide toxins including α-amanitin, β-amanitin, phallacidin and phalloidin obtained from Sigma-Aldrich (St. Louis, MO, USA) were used as standard reference material. Two grams of mushroom samples were blended and extracted with 20 mL of methanol. The extract was incubated at 60°C for 10 min, followed by centrifugation at 8000 g for 5 min. The clear supernatant was decanted to dryness under a stream of nitrogen. LC methods for separation of amatoxins and phallotoxins were applied according to Chung et al. (2007) and Jansson et al. (2012) The separation of the peptide toxins as well as determination of molecular weight were performed using a reversed phase LC-MS method on Agilent Technologies 1100 series LC/MSD system (Foster City, CA, USA) equipped with a diode array detector and single quad MS detector (VL) with an electrospray source (ESI-MS). Mobile phases used included different mixtures of a buffer containing 10% of 2 mM ammonium formate with 5 mM formic acid and 90% of acetonitrile.
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