The Experts below are selected from a list of 225 Experts worldwide ranked by ideXlab platform
Osamu Suzuki - One of the best experts on this subject based on the ideXlab platform.
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determination of Ibotenic Acid and muscimol the amanita mushroom toxins in human serum by liquid chromatography tandem mass spectrometry
Forensic Toxicology, 2013Co-Authors: Koutaro Hasegawa, Kunio Gonmori, Hiroki Fujita, Yoshito Kamijo, Hideki Nozawa, Itaru Yamagishi, Kayoko Minakata, Kanako Watanabe, Osamu SuzukiAbstract:In our previous article, we reported the analysis of Ibotenic Acid and muscimol in Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The levels of Ibotenic Acid and muscimol in the mushroom were as high as 210 and 107 μg/g, respectively. We have since tried to measure the same toxins in human serum obtained from a poisoned subject, who ingested the Amanita mushrooms, by the same method. However, the levels of the toxins in the human serum were about three orders of magnitude lower than those in Amanita mushrooms. In addition, the recovery rates for Ibotenic Acid and muscimol in human serum were found to be much lower than those in the previous study for the mushrooms. Therefore, we optimized the solid-phase extraction procedure again, and reevaluated the data for validation at much lower levels of Ibotenic Acid and muscimol in human serum. A 100-μl aliquot of human serum containing the target toxins was mixed with 100 ng of acivicin as internal standard (IS), 200 μl of distilled water, and 100 μl of 0.5 % ammonium hydroxide in distilled water, and vortexed well for 10 s. The mixture was loaded on an Oasis MAX 3cc (60 mg) extraction cartridge. The cartridge was washed with 0.5 ml of distilled water and 1.0 ml of methanol. The target compounds and IS were eluted with 4 ml of 0.05 % trifluoroacetic Acid in methanol. The eluate was evaporated to dryness and reconstituted in methanol, and its small volume was subjected to LC–MS–MS analysis with the same TSK-GEL Amide-80 separation column. The LC elution was made in the gradient and isocratic modes. The selected reaction monitoring chromatograms showed clear peaks at 5.3, 3.5, and 3.6 min for Ibotenic Acid, muscimol, and IS, respectively; the blank serum sample without the target compounds or IS gave no peaks at the respective retention times except for an impurity peak at 6.5 min. There was good linearity from 10 to 1,000 ng/ml for both Ibotenic Acid and muscimol with correlation coefficients not <0.999. The detection limits (signal-to-noise ratio = 3) were 1.0 and 2.5 ng/ml for Ibotenic Acid and muscimol, respectively. The recovery rates of the target compounds in sera at five different concentrations were 87.9–103 %. The intraday and interday accuracy and precision data were also generally satisfactory. Using the modified method, the actual concentrations of Ibotenic Acid and muscimol were measured for a serum sample obtained from an ill patient thought to have ingested Amanita ibotengutake; they were 95.9 and 105 ng/ml, respectively. To our knowledge, this is the first report of analysis of Ibotenic Acid and muscimol in human serum by an MS technique, which we believe will be very useful in forensic and clinical toxicology.
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Determination of Ibotenic Acid and muscimol, the Amanita mushroom toxins, in human serum by liquid chromatography–tandem mass spectrometry
Forensic Toxicology, 2013Co-Authors: Koutaro Hasegawa, Kunio Gonmori, Hiroki Fujita, Yoshito Kamijo, Hideki Nozawa, Itaru Yamagishi, Kayoko Minakata, Kanako Watanabe, Osamu SuzukiAbstract:In our previous article, we reported the analysis of Ibotenic Acid and muscimol in Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The levels of Ibotenic Acid and muscimol in the mushroom were as high as 210 and 107 μg/g, respectively. We have since tried to measure the same toxins in human serum obtained from a poisoned subject, who ingested the Amanita mushrooms, by the same method. However, the levels of the toxins in the human serum were about three orders of magnitude lower than those in Amanita mushrooms. In addition, the recovery rates for Ibotenic Acid and muscimol in human serum were found to be much lower than those in the previous study for the mushrooms. Therefore, we optimized the solid-phase extraction procedure again, and reevaluated the data for validation at much lower levels of Ibotenic Acid and muscimol in human serum. A 100-μl aliquot of human serum containing the target toxins was mixed with 100 ng of acivicin as internal standard (IS), 200 μl of distilled water, and 100 μl of 0.5 % ammonium hydroxide in distilled water, and vortexed well for 10 s. The mixture was loaded on an Oasis MAX 3cc (60 mg) extraction cartridge. The cartridge was washed with 0.5 ml of distilled water and 1.0 ml of methanol. The target compounds and IS were eluted with 4 ml of 0.05 % trifluoroacetic Acid in methanol. The eluate was evaporated to dryness and reconstituted in methanol, and its small volume was subjected to LC–MS–MS analysis with the same TSK-GEL Amide-80 separation column. The LC elution was made in the gradient and isocratic modes. The selected reaction monitoring chromatograms showed clear peaks at 5.3, 3.5, and 3.6 min for Ibotenic Acid, muscimol, and IS, respectively; the blank serum sample without the target compounds or IS gave no peaks at the respective retention times except for an impurity peak at 6.5 min. There was good linearity from 10 to 1,000 ng/ml for both Ibotenic Acid and muscimol with correlation coefficients not
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Analysis of Ibotenic Acid and muscimol in Amanita mushrooms by hydrophilic interaction liquid chromatography–tandem mass spectrometry
Forensic Toxicology, 2012Co-Authors: Kunio Gonmori, Koutaro Hasegawa, Hiroki Fujita, Yoshito Kamijo, Hideki Nozawa, Itaru Yamagishi, Kayoko Minakata, Kanako Watanabe, Osamu SuzukiAbstract:We have established the most modern method for analysis of Ibotenic Acid and muscimol in toxic Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Acivicin, an Ibotenic Acid analog and antitumor agent, was used as internal standard (IS). A target mushroom sample was homogenized in water/methanol (1:1) and mixed with a fixed concentration of IS. The target compounds and IS were purified with an Oasis MAX 3cc (60 mg) extraction cartridge. The eluate was subjected to hydrophilic interaction (TSK-GEL Amide-80 3 μm, 150 × 2.0 mm i.d. column) LC–MS–MS. The quantitation was made by multiple reaction monitoring (MRM). The ion transitions were: m/z 179→133.1 for IS, m/z 159→113.1 for Ibotenic Acid, and m/z 115→98.1 for muscimol. The elution was made in the gradient mode with 0.5 % formic Acid aqueous solution (A) and 0.5 % formic Acid in acetonitrile (B) from 90 % B to 80 % B in 1.85 min, and then in the isocratic elution mode with 20 % A/80 % B up to 10 min. The MRM chromatograms gave clear and symmetrical peaks for Ibotenic Acid, muscimol, and IS. Their recovery rates were 84.6–107 %. There was good linearity from 10 to 500 μg/g for both Ibotenic Acid and muscimol with correlation coefficients not
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analysis of Ibotenic Acid and muscimol in amanita mushrooms by hydrophilic interaction liquid chromatography tandem mass spectrometry
Forensic Toxicology, 2012Co-Authors: Kunio Gonmori, Koutaro Hasegawa, Hiroki Fujita, Yoshito Kamijo, Hideki Nozawa, Itaru Yamagishi, Kayoko Minakata, Kanako Watanabe, Osamu SuzukiAbstract:We have established the most modern method for analysis of Ibotenic Acid and muscimol in toxic Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Acivicin, an Ibotenic Acid analog and antitumor agent, was used as internal standard (IS). A target mushroom sample was homogenized in water/methanol (1:1) and mixed with a fixed concentration of IS. The target compounds and IS were purified with an Oasis MAX 3cc (60 mg) extraction cartridge. The eluate was subjected to hydrophilic interaction (TSK-GEL Amide-80 3 μm, 150 × 2.0 mm i.d. column) LC–MS–MS. The quantitation was made by multiple reaction monitoring (MRM). The ion transitions were: m/z 179→133.1 for IS, m/z 159→113.1 for Ibotenic Acid, and m/z 115→98.1 for muscimol. The elution was made in the gradient mode with 0.5 % formic Acid aqueous solution (A) and 0.5 % formic Acid in acetonitrile (B) from 90 % B to 80 % B in 1.85 min, and then in the isocratic elution mode with 20 % A/80 % B up to 10 min. The MRM chromatograms gave clear and symmetrical peaks for Ibotenic Acid, muscimol, and IS. Their recovery rates were 84.6–107 %. There was good linearity from 10 to 500 μg/g for both Ibotenic Acid and muscimol with correlation coefficients not <0.99. Intraday and interday accuracy and precision were also generally satisfactory. Using the above new method, the concentrations of Ibotenic Acid and muscimol were actually measured for a mushroom (most probably Amanita ibotengutake) obtained from a poisoning case; they were 210 and 107 μg/g, respectively. The novel points of our method are no requirement for derivatization before LC–MS–MS analysis, the use of anion exchange solid-phase extraction, the use of hydrophilic interaction column for LC separation, and the use of acivicin as IS.
C Nitsch - One of the best experts on this subject based on the ideXlab platform.
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Composition of Ibotenic Acid-induced calcifications in rat substantia nigra.
Brain research, 1998Co-Authors: G Herrmann, H Stünitz, C NitschAbstract:Agonists of the excitatory neurotransmitter glutamate have neurotoxic properties and are, therefore, frequently used to place locally circumscript brain lesions. In certain vulnerable brain areas, especially the substantia nigra and globus pallidus, the ensuing neurodegeneration is accompanied by the formation of calcium deposits. In the present study, we investigated the structure and chemical composition of calcium deposits formed in rat substantia nigra upon local application of Ibotenic Acid. Using scanning and transmission electron microscopy in combination with X-ray analysis and analysis of the electron diffraction patterns, we demonstrate that the inorganic components of the calcifications consist of calcium and phosphate. The calcium phosphate is deposited in a polycrystalline manner in degenerating neurons and in a matrix surrounding the degenerated complexes. New matrix is continuously added around the enlarging calcium deposits. Content of inorganic material is always higher in the center of the deposits than in the margin, but in every case the diffraction pattern reveals that the calcium phosphates are present in the form of hydroxyapatite. Thus, organic and inorganic components of the calcifications are subject to a continuous process of growth and maturation. The Ibotenic Acid-induced calcium deposits in rat substantia nigra provide a reliable model system to study the pathogenesis of non-arteriosclerotic calcifications.
Koutaro Hasegawa - One of the best experts on this subject based on the ideXlab platform.
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determination of Ibotenic Acid and muscimol the amanita mushroom toxins in human serum by liquid chromatography tandem mass spectrometry
Forensic Toxicology, 2013Co-Authors: Koutaro Hasegawa, Kunio Gonmori, Hiroki Fujita, Yoshito Kamijo, Hideki Nozawa, Itaru Yamagishi, Kayoko Minakata, Kanako Watanabe, Osamu SuzukiAbstract:In our previous article, we reported the analysis of Ibotenic Acid and muscimol in Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The levels of Ibotenic Acid and muscimol in the mushroom were as high as 210 and 107 μg/g, respectively. We have since tried to measure the same toxins in human serum obtained from a poisoned subject, who ingested the Amanita mushrooms, by the same method. However, the levels of the toxins in the human serum were about three orders of magnitude lower than those in Amanita mushrooms. In addition, the recovery rates for Ibotenic Acid and muscimol in human serum were found to be much lower than those in the previous study for the mushrooms. Therefore, we optimized the solid-phase extraction procedure again, and reevaluated the data for validation at much lower levels of Ibotenic Acid and muscimol in human serum. A 100-μl aliquot of human serum containing the target toxins was mixed with 100 ng of acivicin as internal standard (IS), 200 μl of distilled water, and 100 μl of 0.5 % ammonium hydroxide in distilled water, and vortexed well for 10 s. The mixture was loaded on an Oasis MAX 3cc (60 mg) extraction cartridge. The cartridge was washed with 0.5 ml of distilled water and 1.0 ml of methanol. The target compounds and IS were eluted with 4 ml of 0.05 % trifluoroacetic Acid in methanol. The eluate was evaporated to dryness and reconstituted in methanol, and its small volume was subjected to LC–MS–MS analysis with the same TSK-GEL Amide-80 separation column. The LC elution was made in the gradient and isocratic modes. The selected reaction monitoring chromatograms showed clear peaks at 5.3, 3.5, and 3.6 min for Ibotenic Acid, muscimol, and IS, respectively; the blank serum sample without the target compounds or IS gave no peaks at the respective retention times except for an impurity peak at 6.5 min. There was good linearity from 10 to 1,000 ng/ml for both Ibotenic Acid and muscimol with correlation coefficients not <0.999. The detection limits (signal-to-noise ratio = 3) were 1.0 and 2.5 ng/ml for Ibotenic Acid and muscimol, respectively. The recovery rates of the target compounds in sera at five different concentrations were 87.9–103 %. The intraday and interday accuracy and precision data were also generally satisfactory. Using the modified method, the actual concentrations of Ibotenic Acid and muscimol were measured for a serum sample obtained from an ill patient thought to have ingested Amanita ibotengutake; they were 95.9 and 105 ng/ml, respectively. To our knowledge, this is the first report of analysis of Ibotenic Acid and muscimol in human serum by an MS technique, which we believe will be very useful in forensic and clinical toxicology.
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Determination of Ibotenic Acid and muscimol, the Amanita mushroom toxins, in human serum by liquid chromatography–tandem mass spectrometry
Forensic Toxicology, 2013Co-Authors: Koutaro Hasegawa, Kunio Gonmori, Hiroki Fujita, Yoshito Kamijo, Hideki Nozawa, Itaru Yamagishi, Kayoko Minakata, Kanako Watanabe, Osamu SuzukiAbstract:In our previous article, we reported the analysis of Ibotenic Acid and muscimol in Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The levels of Ibotenic Acid and muscimol in the mushroom were as high as 210 and 107 μg/g, respectively. We have since tried to measure the same toxins in human serum obtained from a poisoned subject, who ingested the Amanita mushrooms, by the same method. However, the levels of the toxins in the human serum were about three orders of magnitude lower than those in Amanita mushrooms. In addition, the recovery rates for Ibotenic Acid and muscimol in human serum were found to be much lower than those in the previous study for the mushrooms. Therefore, we optimized the solid-phase extraction procedure again, and reevaluated the data for validation at much lower levels of Ibotenic Acid and muscimol in human serum. A 100-μl aliquot of human serum containing the target toxins was mixed with 100 ng of acivicin as internal standard (IS), 200 μl of distilled water, and 100 μl of 0.5 % ammonium hydroxide in distilled water, and vortexed well for 10 s. The mixture was loaded on an Oasis MAX 3cc (60 mg) extraction cartridge. The cartridge was washed with 0.5 ml of distilled water and 1.0 ml of methanol. The target compounds and IS were eluted with 4 ml of 0.05 % trifluoroacetic Acid in methanol. The eluate was evaporated to dryness and reconstituted in methanol, and its small volume was subjected to LC–MS–MS analysis with the same TSK-GEL Amide-80 separation column. The LC elution was made in the gradient and isocratic modes. The selected reaction monitoring chromatograms showed clear peaks at 5.3, 3.5, and 3.6 min for Ibotenic Acid, muscimol, and IS, respectively; the blank serum sample without the target compounds or IS gave no peaks at the respective retention times except for an impurity peak at 6.5 min. There was good linearity from 10 to 1,000 ng/ml for both Ibotenic Acid and muscimol with correlation coefficients not
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Analysis of Ibotenic Acid and muscimol in Amanita mushrooms by hydrophilic interaction liquid chromatography–tandem mass spectrometry
Forensic Toxicology, 2012Co-Authors: Kunio Gonmori, Koutaro Hasegawa, Hiroki Fujita, Yoshito Kamijo, Hideki Nozawa, Itaru Yamagishi, Kayoko Minakata, Kanako Watanabe, Osamu SuzukiAbstract:We have established the most modern method for analysis of Ibotenic Acid and muscimol in toxic Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Acivicin, an Ibotenic Acid analog and antitumor agent, was used as internal standard (IS). A target mushroom sample was homogenized in water/methanol (1:1) and mixed with a fixed concentration of IS. The target compounds and IS were purified with an Oasis MAX 3cc (60 mg) extraction cartridge. The eluate was subjected to hydrophilic interaction (TSK-GEL Amide-80 3 μm, 150 × 2.0 mm i.d. column) LC–MS–MS. The quantitation was made by multiple reaction monitoring (MRM). The ion transitions were: m/z 179→133.1 for IS, m/z 159→113.1 for Ibotenic Acid, and m/z 115→98.1 for muscimol. The elution was made in the gradient mode with 0.5 % formic Acid aqueous solution (A) and 0.5 % formic Acid in acetonitrile (B) from 90 % B to 80 % B in 1.85 min, and then in the isocratic elution mode with 20 % A/80 % B up to 10 min. The MRM chromatograms gave clear and symmetrical peaks for Ibotenic Acid, muscimol, and IS. Their recovery rates were 84.6–107 %. There was good linearity from 10 to 500 μg/g for both Ibotenic Acid and muscimol with correlation coefficients not
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analysis of Ibotenic Acid and muscimol in amanita mushrooms by hydrophilic interaction liquid chromatography tandem mass spectrometry
Forensic Toxicology, 2012Co-Authors: Kunio Gonmori, Koutaro Hasegawa, Hiroki Fujita, Yoshito Kamijo, Hideki Nozawa, Itaru Yamagishi, Kayoko Minakata, Kanako Watanabe, Osamu SuzukiAbstract:We have established the most modern method for analysis of Ibotenic Acid and muscimol in toxic Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Acivicin, an Ibotenic Acid analog and antitumor agent, was used as internal standard (IS). A target mushroom sample was homogenized in water/methanol (1:1) and mixed with a fixed concentration of IS. The target compounds and IS were purified with an Oasis MAX 3cc (60 mg) extraction cartridge. The eluate was subjected to hydrophilic interaction (TSK-GEL Amide-80 3 μm, 150 × 2.0 mm i.d. column) LC–MS–MS. The quantitation was made by multiple reaction monitoring (MRM). The ion transitions were: m/z 179→133.1 for IS, m/z 159→113.1 for Ibotenic Acid, and m/z 115→98.1 for muscimol. The elution was made in the gradient mode with 0.5 % formic Acid aqueous solution (A) and 0.5 % formic Acid in acetonitrile (B) from 90 % B to 80 % B in 1.85 min, and then in the isocratic elution mode with 20 % A/80 % B up to 10 min. The MRM chromatograms gave clear and symmetrical peaks for Ibotenic Acid, muscimol, and IS. Their recovery rates were 84.6–107 %. There was good linearity from 10 to 500 μg/g for both Ibotenic Acid and muscimol with correlation coefficients not <0.99. Intraday and interday accuracy and precision were also generally satisfactory. Using the above new method, the concentrations of Ibotenic Acid and muscimol were actually measured for a mushroom (most probably Amanita ibotengutake) obtained from a poisoning case; they were 210 and 107 μg/g, respectively. The novel points of our method are no requirement for derivatization before LC–MS–MS analysis, the use of anion exchange solid-phase extraction, the use of hydrophilic interaction column for LC separation, and the use of acivicin as IS.
G Herrmann - One of the best experts on this subject based on the ideXlab platform.
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Composition of Ibotenic Acid-induced calcifications in rat substantia nigra.
Brain research, 1998Co-Authors: G Herrmann, H Stünitz, C NitschAbstract:Agonists of the excitatory neurotransmitter glutamate have neurotoxic properties and are, therefore, frequently used to place locally circumscript brain lesions. In certain vulnerable brain areas, especially the substantia nigra and globus pallidus, the ensuing neurodegeneration is accompanied by the formation of calcium deposits. In the present study, we investigated the structure and chemical composition of calcium deposits formed in rat substantia nigra upon local application of Ibotenic Acid. Using scanning and transmission electron microscopy in combination with X-ray analysis and analysis of the electron diffraction patterns, we demonstrate that the inorganic components of the calcifications consist of calcium and phosphate. The calcium phosphate is deposited in a polycrystalline manner in degenerating neurons and in a matrix surrounding the degenerated complexes. New matrix is continuously added around the enlarging calcium deposits. Content of inorganic material is always higher in the center of the deposits than in the margin, but in every case the diffraction pattern reveals that the calcium phosphates are present in the form of hydroxyapatite. Thus, organic and inorganic components of the calcifications are subject to a continuous process of growth and maturation. The Ibotenic Acid-induced calcium deposits in rat substantia nigra provide a reliable model system to study the pathogenesis of non-arteriosclerotic calcifications.
Kunio Gonmori - One of the best experts on this subject based on the ideXlab platform.
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determination of Ibotenic Acid and muscimol the amanita mushroom toxins in human serum by liquid chromatography tandem mass spectrometry
Forensic Toxicology, 2013Co-Authors: Koutaro Hasegawa, Kunio Gonmori, Hiroki Fujita, Yoshito Kamijo, Hideki Nozawa, Itaru Yamagishi, Kayoko Minakata, Kanako Watanabe, Osamu SuzukiAbstract:In our previous article, we reported the analysis of Ibotenic Acid and muscimol in Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The levels of Ibotenic Acid and muscimol in the mushroom were as high as 210 and 107 μg/g, respectively. We have since tried to measure the same toxins in human serum obtained from a poisoned subject, who ingested the Amanita mushrooms, by the same method. However, the levels of the toxins in the human serum were about three orders of magnitude lower than those in Amanita mushrooms. In addition, the recovery rates for Ibotenic Acid and muscimol in human serum were found to be much lower than those in the previous study for the mushrooms. Therefore, we optimized the solid-phase extraction procedure again, and reevaluated the data for validation at much lower levels of Ibotenic Acid and muscimol in human serum. A 100-μl aliquot of human serum containing the target toxins was mixed with 100 ng of acivicin as internal standard (IS), 200 μl of distilled water, and 100 μl of 0.5 % ammonium hydroxide in distilled water, and vortexed well for 10 s. The mixture was loaded on an Oasis MAX 3cc (60 mg) extraction cartridge. The cartridge was washed with 0.5 ml of distilled water and 1.0 ml of methanol. The target compounds and IS were eluted with 4 ml of 0.05 % trifluoroacetic Acid in methanol. The eluate was evaporated to dryness and reconstituted in methanol, and its small volume was subjected to LC–MS–MS analysis with the same TSK-GEL Amide-80 separation column. The LC elution was made in the gradient and isocratic modes. The selected reaction monitoring chromatograms showed clear peaks at 5.3, 3.5, and 3.6 min for Ibotenic Acid, muscimol, and IS, respectively; the blank serum sample without the target compounds or IS gave no peaks at the respective retention times except for an impurity peak at 6.5 min. There was good linearity from 10 to 1,000 ng/ml for both Ibotenic Acid and muscimol with correlation coefficients not <0.999. The detection limits (signal-to-noise ratio = 3) were 1.0 and 2.5 ng/ml for Ibotenic Acid and muscimol, respectively. The recovery rates of the target compounds in sera at five different concentrations were 87.9–103 %. The intraday and interday accuracy and precision data were also generally satisfactory. Using the modified method, the actual concentrations of Ibotenic Acid and muscimol were measured for a serum sample obtained from an ill patient thought to have ingested Amanita ibotengutake; they were 95.9 and 105 ng/ml, respectively. To our knowledge, this is the first report of analysis of Ibotenic Acid and muscimol in human serum by an MS technique, which we believe will be very useful in forensic and clinical toxicology.
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Determination of Ibotenic Acid and muscimol, the Amanita mushroom toxins, in human serum by liquid chromatography–tandem mass spectrometry
Forensic Toxicology, 2013Co-Authors: Koutaro Hasegawa, Kunio Gonmori, Hiroki Fujita, Yoshito Kamijo, Hideki Nozawa, Itaru Yamagishi, Kayoko Minakata, Kanako Watanabe, Osamu SuzukiAbstract:In our previous article, we reported the analysis of Ibotenic Acid and muscimol in Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The levels of Ibotenic Acid and muscimol in the mushroom were as high as 210 and 107 μg/g, respectively. We have since tried to measure the same toxins in human serum obtained from a poisoned subject, who ingested the Amanita mushrooms, by the same method. However, the levels of the toxins in the human serum were about three orders of magnitude lower than those in Amanita mushrooms. In addition, the recovery rates for Ibotenic Acid and muscimol in human serum were found to be much lower than those in the previous study for the mushrooms. Therefore, we optimized the solid-phase extraction procedure again, and reevaluated the data for validation at much lower levels of Ibotenic Acid and muscimol in human serum. A 100-μl aliquot of human serum containing the target toxins was mixed with 100 ng of acivicin as internal standard (IS), 200 μl of distilled water, and 100 μl of 0.5 % ammonium hydroxide in distilled water, and vortexed well for 10 s. The mixture was loaded on an Oasis MAX 3cc (60 mg) extraction cartridge. The cartridge was washed with 0.5 ml of distilled water and 1.0 ml of methanol. The target compounds and IS were eluted with 4 ml of 0.05 % trifluoroacetic Acid in methanol. The eluate was evaporated to dryness and reconstituted in methanol, and its small volume was subjected to LC–MS–MS analysis with the same TSK-GEL Amide-80 separation column. The LC elution was made in the gradient and isocratic modes. The selected reaction monitoring chromatograms showed clear peaks at 5.3, 3.5, and 3.6 min for Ibotenic Acid, muscimol, and IS, respectively; the blank serum sample without the target compounds or IS gave no peaks at the respective retention times except for an impurity peak at 6.5 min. There was good linearity from 10 to 1,000 ng/ml for both Ibotenic Acid and muscimol with correlation coefficients not
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Analysis of Ibotenic Acid and muscimol in Amanita mushrooms by hydrophilic interaction liquid chromatography–tandem mass spectrometry
Forensic Toxicology, 2012Co-Authors: Kunio Gonmori, Koutaro Hasegawa, Hiroki Fujita, Yoshito Kamijo, Hideki Nozawa, Itaru Yamagishi, Kayoko Minakata, Kanako Watanabe, Osamu SuzukiAbstract:We have established the most modern method for analysis of Ibotenic Acid and muscimol in toxic Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Acivicin, an Ibotenic Acid analog and antitumor agent, was used as internal standard (IS). A target mushroom sample was homogenized in water/methanol (1:1) and mixed with a fixed concentration of IS. The target compounds and IS were purified with an Oasis MAX 3cc (60 mg) extraction cartridge. The eluate was subjected to hydrophilic interaction (TSK-GEL Amide-80 3 μm, 150 × 2.0 mm i.d. column) LC–MS–MS. The quantitation was made by multiple reaction monitoring (MRM). The ion transitions were: m/z 179→133.1 for IS, m/z 159→113.1 for Ibotenic Acid, and m/z 115→98.1 for muscimol. The elution was made in the gradient mode with 0.5 % formic Acid aqueous solution (A) and 0.5 % formic Acid in acetonitrile (B) from 90 % B to 80 % B in 1.85 min, and then in the isocratic elution mode with 20 % A/80 % B up to 10 min. The MRM chromatograms gave clear and symmetrical peaks for Ibotenic Acid, muscimol, and IS. Their recovery rates were 84.6–107 %. There was good linearity from 10 to 500 μg/g for both Ibotenic Acid and muscimol with correlation coefficients not
-
analysis of Ibotenic Acid and muscimol in amanita mushrooms by hydrophilic interaction liquid chromatography tandem mass spectrometry
Forensic Toxicology, 2012Co-Authors: Kunio Gonmori, Koutaro Hasegawa, Hiroki Fujita, Yoshito Kamijo, Hideki Nozawa, Itaru Yamagishi, Kayoko Minakata, Kanako Watanabe, Osamu SuzukiAbstract:We have established the most modern method for analysis of Ibotenic Acid and muscimol in toxic Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Acivicin, an Ibotenic Acid analog and antitumor agent, was used as internal standard (IS). A target mushroom sample was homogenized in water/methanol (1:1) and mixed with a fixed concentration of IS. The target compounds and IS were purified with an Oasis MAX 3cc (60 mg) extraction cartridge. The eluate was subjected to hydrophilic interaction (TSK-GEL Amide-80 3 μm, 150 × 2.0 mm i.d. column) LC–MS–MS. The quantitation was made by multiple reaction monitoring (MRM). The ion transitions were: m/z 179→133.1 for IS, m/z 159→113.1 for Ibotenic Acid, and m/z 115→98.1 for muscimol. The elution was made in the gradient mode with 0.5 % formic Acid aqueous solution (A) and 0.5 % formic Acid in acetonitrile (B) from 90 % B to 80 % B in 1.85 min, and then in the isocratic elution mode with 20 % A/80 % B up to 10 min. The MRM chromatograms gave clear and symmetrical peaks for Ibotenic Acid, muscimol, and IS. Their recovery rates were 84.6–107 %. There was good linearity from 10 to 500 μg/g for both Ibotenic Acid and muscimol with correlation coefficients not <0.99. Intraday and interday accuracy and precision were also generally satisfactory. Using the above new method, the concentrations of Ibotenic Acid and muscimol were actually measured for a mushroom (most probably Amanita ibotengutake) obtained from a poisoning case; they were 210 and 107 μg/g, respectively. The novel points of our method are no requirement for derivatization before LC–MS–MS analysis, the use of anion exchange solid-phase extraction, the use of hydrophilic interaction column for LC separation, and the use of acivicin as IS.
