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Yvan Gaillard - One of the best experts on this subject based on the ideXlab platform.
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a validated method for quantifying atractyloside and carboxyatractyloside in blood by hplc hrms ms a non fatal case of intoxication with atractylis gummifera l
Journal of Analytical Toxicology, 2014Co-Authors: Jeremy Carlier, Ludovic Romeuf, Jerome Guitton, Cedric Priezbarallon, Fabien Bevalot, Laurent Fanton, Yvan GaillardAbstract:: Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene glycosides that are responsible for the toxicity of several Asteraceae plants around the world. Mediterranean gum thistle (Atractylis gummifera L.) and Zulu impila (Callilepis laureola DC.), in particular, are notoriously poisonous and the cause of many accidental deaths, some suicides and even some murders. There is no current method for measuring the two toxins in biological samples that meet the criteria of specificity required in forensic medicine. We have endeavored to fill this analytical gap. Analysis was carried out using a solid-phase extraction and a high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry detection. The method was validated in the whole blood with quantification limits of 0.17 and 0.15 µg/L for ATR and CATR, respectively. The method was applied to a non-fatal case of intoxication with A. gummifera. To the best of the authors' knowledge, this is the first time that a concentration of ATR and CATR in blood (883.1 and 119.0 µg/L, respectively) and urine (230.4 and 140.3 µg/L, respectively) is reported. ATR and CATR were quantified in A. gummifera roots by the standard method addition (3.7 and 5.4 mg/g, respectively).
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A Validated Method for Quantifying Atractyloside and Carboxyatractyloside in Blood by HPLC-HRMS/MS, a Non-Fatal Case of Intoxication with Atractylis gummifera L.
Journal of Analytical Toxicology, 2014Co-Authors: Jeremy Carlier, Ludovic Romeuf, Jerome Guitton, Fabien Bevalot, Laurent Fanton, Cédric Priez-barallon, Yvan GaillardAbstract:: Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene glycosides that are responsible for the toxicity of several Asteraceae plants around the world. Mediterranean gum thistle (Atractylis gummifera L.) and Zulu impila (Callilepis laureola DC.), in particular, are notoriously poisonous and the cause of many accidental deaths, some suicides and even some murders. There is no current method for measuring the two toxins in biological samples that meet the criteria of specificity required in forensic medicine. We have endeavored to fill this analytical gap. Analysis was carried out using a solid-phase extraction and a high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry detection. The method was validated in the whole blood with quantification limits of 0.17 and 0.15 µg/L for ATR and CATR, respectively. The method was applied to a non-fatal case of intoxication with A. gummifera. To the best of the authors' knowledge, this is the first time that a concentration of ATR and CATR in blood (883.1 and 119.0 µg/L, respectively) and urine (230.4 and 140.3 µg/L, respectively) is reported. ATR and CATR were quantified in A. gummifera roots by the standard method addition (3.7 and 5.4 mg/g, respectively).
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o27 a validated method for quantifying atractyloside and carboxyatractyloside in blood and urine by hplc hrms ms a non fatal case of intoxication with atractylis gummifera l
Toxicologie Analytique et Clinique, 2014Co-Authors: Jeremy Carlier, Ludovic Romeuf, Jerome Guitton, Fabien Bevalot, Laurent Fanton, Yvan GaillardAbstract:Introduction Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene glycosides that are responsible for the toxicity of several Asteraceae plants around the world. Mediterranean gum thistle ( Atractylis gummifera L.) and Zulu impila ( Callilepis laureola DC.), in particular, are notoriously poisonous and the cause of many accidental deaths, some suicides and even some murders. When a case history is not available, it is not easy to determine ATR or CATR poisoning anatomically or histologically. There is no current method for measuring the two toxins in biological samples that meet the criteria of specificity required in forensic medicine. We have endeavoured to fill this analytical gap. Methods Analysis was carried out using an original technique of high-performance liquid chromatography coupled with highresolution mass spectrometry detection (HPLC-HRMS). Given the singular structure of ATR and CATR, it is difficult to achieve and maintain the conditions required for their chromatographic separation and perfect ionization for the mass spectrometry. Separation was thus performed using an XTerra ® phenyl column (length: 150 mm; internal diameter: 2,1 mm; particle size: 3,5 μm) (Waters) [Steenkamp et al. , Forensic Sci Int, 2006, 163: 81–92] with a gradient mobile phase composed of acetonitrile containing 10% of isopropyl alcohol and a 5 mM ammonium acetate buffer at pH = 4.5. The chromatographic run time was 12.5 min. Spectrometric detection was performed using a quadrupole-Orbitrap high-resolution detector (Q Exactive™; Thermo Scientific) after ionization by heated electrospray in negative-ion mode. The mass spectrometer operated in full-scan mode and targeted-MS 2 mode alternately. MS scans (288 – 292, 723 – 727 and 767 – 771 amu) were acquired with a mass resolution of 140000. The [M. – H] − and [M – H+1] − ions of ATR (725.2154 and 726.2188 amu ± 5 ppm) and CATR (769.2053 and 770.2086 amu ± 5 ppm) were used for quantification. The fullscan product ion spectrum of the compounds (resolution of 17500) was used to confirm the identity of the toxins. The processing of the biological sample (1 mL) consisted of a protein precipitation followed by solid phase extraction on Oasis ® HLB cartridges (Waters) at pH = 4.5. Results The method was validated in the whole blood with between- and within-day RSD (relative standard deviation) less than 5.8% and 5.2% for ATR (accuracy between 95.9% and 98.6%) and 5.4% and 9.8% for CATR (accuracy between 92.0% and 107.4%). The calibration curves were linear for concentrations ranging from 0.17 to 200 μg/L for ATR and 0.15 to 200 μg/L for CATR. The detection limits were 0.066 and 0,055 μg/L respectively. ATR and CATR were quantified in blood and urine samples from a non-fatal case of intoxication by A. gummifera . The concentrations were 883.1 and 119,0 μg/L respectively in peripheral blood and 230.4 and 140,3 mg/L in urine. ATR and CATR were quantified in dried A. gummifera roots by the standard addition method. The concentrations were 3.7 and 5,4 mg/g respectively. Conclusion We present the first validated method, applicable in forensic toxicology, for quantifying ATR and CATR in whole blood. The analysis is sensitive and quick.
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O27: A validated method for quantifying atractyloside and carboxyatractyloside in blood and urine by HPLC-HRMS/MS, a non-fatal case of intoxication with Atractylis gummifera L.
Toxicologie Analytique et Clinique, 2014Co-Authors: Jeremy Carlier, Ludovic Romeuf, Jerome Guitton, Fabien Bevalot, Laurent Fanton, Yvan GaillardAbstract:Introduction Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene glycosides that are responsible for the toxicity of several Asteraceae plants around the world. Mediterranean gum thistle ( Atractylis gummifera L.) and Zulu impila ( Callilepis laureola DC.), in particular, are notoriously poisonous and the cause of many accidental deaths, some suicides and even some murders. When a case history is not available, it is not easy to determine ATR or CATR poisoning anatomically or histologically. There is no current method for measuring the two toxins in biological samples that meet the criteria of specificity required in forensic medicine. We have endeavoured to fill this analytical gap. Methods Analysis was carried out using an original technique of high-performance liquid chromatography coupled with highresolution mass spectrometry detection (HPLC-HRMS). Given the singular structure of ATR and CATR, it is difficult to achieve and maintain the conditions required for their chromatographic separation and perfect ionization for the mass spectrometry. Separation was thus performed using an XTerra ® phenyl column (length: 150 mm; internal diameter: 2,1 mm; particle size: 3,5 μm) (Waters) [Steenkamp et al. , Forensic Sci Int, 2006, 163: 81–92] with a gradient mobile phase composed of acetonitrile containing 10% of isopropyl alcohol and a 5 mM ammonium acetate buffer at pH = 4.5. The chromatographic run time was 12.5 min. Spectrometric detection was performed using a quadrupole-Orbitrap high-resolution detector (Q Exactive™; Thermo Scientific) after ionization by heated electrospray in negative-ion mode. The mass spectrometer operated in full-scan mode and targeted-MS 2 mode alternately. MS scans (288 – 292, 723 – 727 and 767 – 771 amu) were acquired with a mass resolution of 140000. The [M. – H] − and [M – H+1] − ions of ATR (725.2154 and 726.2188 amu ± 5 ppm) and CATR (769.2053 and 770.2086 amu ± 5 ppm) were used for quantification. The fullscan product ion spectrum of the compounds (resolution of 17500) was used to confirm the identity of the toxins. The processing of the biological sample (1 mL) consisted of a protein precipitation followed by solid phase extraction on Oasis ® HLB cartridges (Waters) at pH = 4.5. Results The method was validated in the whole blood with between- and within-day RSD (relative standard deviation) less than 5.8% and 5.2% for ATR (accuracy between 95.9% and 98.6%) and 5.4% and 9.8% for CATR (accuracy between 92.0% and 107.4%). The calibration curves were linear for concentrations ranging from 0.17 to 200 μg/L for ATR and 0.15 to 200 μg/L for CATR. The detection limits were 0.066 and 0,055 μg/L respectively. ATR and CATR were quantified in blood and urine samples from a non-fatal case of intoxication by A. gummifera . The concentrations were 883.1 and 119,0 μg/L respectively in peripheral blood and 230.4 and 140,3 mg/L in urine. ATR and CATR were quantified in dried A. gummifera roots by the standard addition method. The concentrations were 3.7 and 5,4 mg/g respectively. Conclusion We present the first validated method, applicable in forensic toxicology, for quantifying ATR and CATR in whole blood. The analysis is sensitive and quick.
Scot A. Kelchner - One of the best experts on this subject based on the ideXlab platform.
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Phylogeny of South African Gnaphalieae (Asteraceae) based on two noncoding chloroplast sequences
American journal of botany, 2000Co-Authors: Randall J. Bayer, Christopher F. Puttock, Scot A. KelchnerAbstract:The Gnaphalieae are a group of sunflowers that have their greatest diversity in South America, Southern Africa, and Australia. The objective of this study was to reconstruct a phylogeny of the South African Gnaphalieae using sequence data from two noncoding chloroplast DNA sequences, the trnL intron and trnL/trnF intergenic spacer. Included in this investigation are the genera of the Gnaphalieae from the African basal groups, members of the subtribes Cassiniinae, Gnaphaliinae, and Relhaniinae, and African representatives from the large Old World genus Helichrysum. Results indicate that two Gnaphaloid genera, Printzia and Callilepis, should be excluded from the Gnaphalieae. In most trees the Relhaniinae s.s. (sensu stricto) and some of the basal taxa comprise a clade that is sister to the remainder of the tribe Gnaphalieae. The Relhaniinae, which are restricted to Africa, are not a monophyletic group as presently circumscribed, nor are the South African members of Helichrysum, the Cassiniinae and Gnaphaliinae. There is general agreement between our molecular analysis and that of morphology, particularly in the terminal branches of the trees.
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ASTERACEAE) BASED ON TWO NONCODING CHLOROPLAST SEQUENCES1
2000Co-Authors: Randall J. Bayer, Christopher F. Puttock, Scot A. KelchnerAbstract:The Gnaphalieae are a group of sunflowers that have their greatest diversity in South America, Southern Africa, and Australia. The objective of this study was to reconstruct a phylogeny of the South African Gnaphalieae using sequence data from two noncoding chloroplast DNA sequences, the trnL intron and trntiL/trnzF intergenic spacer. Included in this investigation are the genera of the Gnaphalieae from the African basal groups, members of the subtribes Cassiniinae, Gnaphaliinae, and Relhaniinae, and African representatives from the large Old World genus Helichrysun?. Results indicate that two Gnaphaloid genera, Printzia and Callilepis, should be excluded from the Gnaphalieae. In most trees the Relhaniinae s.s. (sensu stricto) and some of the basal taxa comprise a clade that is sister to the remainder of the tribe Gnaphalieae. The Relhaniinae, which are restricted to Africa, are not a monophyletic group as presently circumscribed, nor are the South African members of Helichrysumn, the Cassiniinae and Gnaphaliinae. There is general agreement between our molecular analysis and that of morphology, particularly in the terminal branches of the trees.
Randall J. Bayer - One of the best experts on this subject based on the ideXlab platform.
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Evolutionary relationships in the Asteraceae tribe Inuleae (incl. Plucheeae) evidenced by DNA sequences of ndhF; with notes on the systematic positions of some aberrant genera
Organisms Diversity & Evolution, 2005Co-Authors: Arne A. Anderberg, Randall J. Bayer, Pia Eldenäs, Markus EnglundAbstract:The phylogenetic relationships between the tribes Inuleae sensu stricto and Plucheeae are investigated by analysis of sequence data from the cpDNA gene ndhF. The delimitation between the two tribes is elucidated, and the systematic positions of a number of genera associated with these groups, i.e. genera with either aberrant morphological characters or a debated systematic position, are clarified. Together, the Inuleae and Plucheeae form a monophyletic group in which the majority of genera of Inuleae s.str. form one clade, and all the taxa from the Plucheeae together with the genera Antiphiona, Calostephane, Geigeria, Ondetia, Pechuel-loeschea, Pegolettia, and Iphionopsis from Inuleae s.str. form another. Members of the Plucheeae are nested with genera of the Inuleae s.str., and support for the Plucheeae clade is weak. Consequently, the latter cannot be maintained and the two groups are treated as one tribe, Inuleae, with the two subtribes Inulinae and Plucheinae. The genera Asteriscus, Chrysophthalmum, Inula, Laggera, Pentanema, Pluchea, and Pulicaria are demonstrated to be non-monophyletic. Cratystylis and Iphionopsis are found to belong to the same clade as the taxa of the former Plucheeae. Caesulia is shown to be a close relative of Duhaldea and Blumea of the Inuleae-Inulinae. The genera Callilepis and Zoutpansbergia belong to the major clade of the family that includes the tribes Heliantheae sensu lato and Inuleae (incl. Plucheeae), but their exact position remains unresolved. The genus Gymnarrhena is not part of the Inuleae, but is either part of the unresolved basal complex of the paraphyletic Cichorioideae, or sister to the entire Asteroideae.
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Phylogeny of South African Gnaphalieae (Asteraceae) based on two noncoding chloroplast sequences
American journal of botany, 2000Co-Authors: Randall J. Bayer, Christopher F. Puttock, Scot A. KelchnerAbstract:The Gnaphalieae are a group of sunflowers that have their greatest diversity in South America, Southern Africa, and Australia. The objective of this study was to reconstruct a phylogeny of the South African Gnaphalieae using sequence data from two noncoding chloroplast DNA sequences, the trnL intron and trnL/trnF intergenic spacer. Included in this investigation are the genera of the Gnaphalieae from the African basal groups, members of the subtribes Cassiniinae, Gnaphaliinae, and Relhaniinae, and African representatives from the large Old World genus Helichrysum. Results indicate that two Gnaphaloid genera, Printzia and Callilepis, should be excluded from the Gnaphalieae. In most trees the Relhaniinae s.s. (sensu stricto) and some of the basal taxa comprise a clade that is sister to the remainder of the tribe Gnaphalieae. The Relhaniinae, which are restricted to Africa, are not a monophyletic group as presently circumscribed, nor are the South African members of Helichrysum, the Cassiniinae and Gnaphaliinae. There is general agreement between our molecular analysis and that of morphology, particularly in the terminal branches of the trees.
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ASTERACEAE) BASED ON TWO NONCODING CHLOROPLAST SEQUENCES1
2000Co-Authors: Randall J. Bayer, Christopher F. Puttock, Scot A. KelchnerAbstract:The Gnaphalieae are a group of sunflowers that have their greatest diversity in South America, Southern Africa, and Australia. The objective of this study was to reconstruct a phylogeny of the South African Gnaphalieae using sequence data from two noncoding chloroplast DNA sequences, the trnL intron and trntiL/trnzF intergenic spacer. Included in this investigation are the genera of the Gnaphalieae from the African basal groups, members of the subtribes Cassiniinae, Gnaphaliinae, and Relhaniinae, and African representatives from the large Old World genus Helichrysun?. Results indicate that two Gnaphaloid genera, Printzia and Callilepis, should be excluded from the Gnaphalieae. In most trees the Relhaniinae s.s. (sensu stricto) and some of the basal taxa comprise a clade that is sister to the remainder of the tribe Gnaphalieae. The Relhaniinae, which are restricted to Africa, are not a monophyletic group as presently circumscribed, nor are the South African members of Helichrysumn, the Cassiniinae and Gnaphaliinae. There is general agreement between our molecular analysis and that of morphology, particularly in the terminal branches of the trees.
Manuela G. Neuman - One of the best experts on this subject based on the ideXlab platform.
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Mechanism of Impila (Callilepis laureola)-induced cytotoxicity in Hep G2 cells
Clinical biochemistry, 2002Co-Authors: Alpa Popat, Neil H. Shear, Izabella M. Malkiewicz, Stuart Thomson, Manuela G. NeumanAbstract:Abstract Objectives: To determine the mechanism(s) of Impila ( Callilepis laureola )-induced toxicity in human hepatoblastoma Hep G2 cells in vitro and the possible prevention of this toxicity by N -acetylcysteine (NAC). Design and methods: Cells were treated with an aqueous extract of Impila (10 mg/mL) for up to 24 h. NAC (5 mM) was administered either concomitantly with Impila or one hour post Impila treatment. Cytotoxicity was quantitated spectrophotometrically by the metabolism of the tetrazolium dye MTT. Total glutathione (GSH) was measured using the Tietze assay. Results: Impila produced cytotoxicity and depleted GSH in a concentration- and time-dependent manner. A significant depletion in GSH was observed after 15 min ( p p Impila -induced GSH depletion and resulted in a significant decrease in Impila -induced cytotoxicity ( p Conclusion: Our results suggest the mechanism of Impila -induced cytotoxicity in Hep G2 cells in vitro involves depletion of cellular GSH. Preventing GSH depletion by supplementing cells with NAC reduces cytotoxicity.
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The toxicity of Callilepis laureola, a South African traditional herbal medicine.
Clinical biochemistry, 2001Co-Authors: Alpa Popat, Vanessa Steenkamp, Michael J. Stewart, Neil H. Shear, Izabella M. Malkiewicz, Stuart Thomson, Manuela G. NeumanAbstract:Objectives: To review the literature on the toxicity of Callilepis laureola, and to assess the cytotoxicity of C. laureola in human hepatoblastoma Hep G2 cells in vitro. Design and methods: Cells were incubated for up to 48 h in the presence of increasing concentrations of an aqueous extract of C. laureola (0.3‐13.3 mg/mL). Cytotoxicity was quantitated spectrophotometrically by the metabolism of the tetrazolium dye MTT. Cytoviability of the control cells was considered to be 100%. Results: C. laureola produced cytotoxicity in a concentration-dependent manner. Cytotoxicity was significant at all concentrations tested (0.3‐2.5 mg/mL, p , 0.05 vs. controls and 3.3‐13.3 mg/mL, p , 0.0001 vs. controls). After 6 h, 100% toxicity was observed at a concentration of 6.7 mg/mL. Conclusion: C. laureola causes significant cytotoxicity in Hep G2 cells in vitro. These findings are in accordance with the observed hepatotoxicity in clinical cases of C. laureola poisoning. © 2001 The Canadian Society of Clinical Chemists. All rights reserved.
Jeremy Carlier - One of the best experts on this subject based on the ideXlab platform.
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a validated method for quantifying atractyloside and carboxyatractyloside in blood by hplc hrms ms a non fatal case of intoxication with atractylis gummifera l
Journal of Analytical Toxicology, 2014Co-Authors: Jeremy Carlier, Ludovic Romeuf, Jerome Guitton, Cedric Priezbarallon, Fabien Bevalot, Laurent Fanton, Yvan GaillardAbstract:: Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene glycosides that are responsible for the toxicity of several Asteraceae plants around the world. Mediterranean gum thistle (Atractylis gummifera L.) and Zulu impila (Callilepis laureola DC.), in particular, are notoriously poisonous and the cause of many accidental deaths, some suicides and even some murders. There is no current method for measuring the two toxins in biological samples that meet the criteria of specificity required in forensic medicine. We have endeavored to fill this analytical gap. Analysis was carried out using a solid-phase extraction and a high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry detection. The method was validated in the whole blood with quantification limits of 0.17 and 0.15 µg/L for ATR and CATR, respectively. The method was applied to a non-fatal case of intoxication with A. gummifera. To the best of the authors' knowledge, this is the first time that a concentration of ATR and CATR in blood (883.1 and 119.0 µg/L, respectively) and urine (230.4 and 140.3 µg/L, respectively) is reported. ATR and CATR were quantified in A. gummifera roots by the standard method addition (3.7 and 5.4 mg/g, respectively).
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A Validated Method for Quantifying Atractyloside and Carboxyatractyloside in Blood by HPLC-HRMS/MS, a Non-Fatal Case of Intoxication with Atractylis gummifera L.
Journal of Analytical Toxicology, 2014Co-Authors: Jeremy Carlier, Ludovic Romeuf, Jerome Guitton, Fabien Bevalot, Laurent Fanton, Cédric Priez-barallon, Yvan GaillardAbstract:: Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene glycosides that are responsible for the toxicity of several Asteraceae plants around the world. Mediterranean gum thistle (Atractylis gummifera L.) and Zulu impila (Callilepis laureola DC.), in particular, are notoriously poisonous and the cause of many accidental deaths, some suicides and even some murders. There is no current method for measuring the two toxins in biological samples that meet the criteria of specificity required in forensic medicine. We have endeavored to fill this analytical gap. Analysis was carried out using a solid-phase extraction and a high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry detection. The method was validated in the whole blood with quantification limits of 0.17 and 0.15 µg/L for ATR and CATR, respectively. The method was applied to a non-fatal case of intoxication with A. gummifera. To the best of the authors' knowledge, this is the first time that a concentration of ATR and CATR in blood (883.1 and 119.0 µg/L, respectively) and urine (230.4 and 140.3 µg/L, respectively) is reported. ATR and CATR were quantified in A. gummifera roots by the standard method addition (3.7 and 5.4 mg/g, respectively).
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o27 a validated method for quantifying atractyloside and carboxyatractyloside in blood and urine by hplc hrms ms a non fatal case of intoxication with atractylis gummifera l
Toxicologie Analytique et Clinique, 2014Co-Authors: Jeremy Carlier, Ludovic Romeuf, Jerome Guitton, Fabien Bevalot, Laurent Fanton, Yvan GaillardAbstract:Introduction Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene glycosides that are responsible for the toxicity of several Asteraceae plants around the world. Mediterranean gum thistle ( Atractylis gummifera L.) and Zulu impila ( Callilepis laureola DC.), in particular, are notoriously poisonous and the cause of many accidental deaths, some suicides and even some murders. When a case history is not available, it is not easy to determine ATR or CATR poisoning anatomically or histologically. There is no current method for measuring the two toxins in biological samples that meet the criteria of specificity required in forensic medicine. We have endeavoured to fill this analytical gap. Methods Analysis was carried out using an original technique of high-performance liquid chromatography coupled with highresolution mass spectrometry detection (HPLC-HRMS). Given the singular structure of ATR and CATR, it is difficult to achieve and maintain the conditions required for their chromatographic separation and perfect ionization for the mass spectrometry. Separation was thus performed using an XTerra ® phenyl column (length: 150 mm; internal diameter: 2,1 mm; particle size: 3,5 μm) (Waters) [Steenkamp et al. , Forensic Sci Int, 2006, 163: 81–92] with a gradient mobile phase composed of acetonitrile containing 10% of isopropyl alcohol and a 5 mM ammonium acetate buffer at pH = 4.5. The chromatographic run time was 12.5 min. Spectrometric detection was performed using a quadrupole-Orbitrap high-resolution detector (Q Exactive™; Thermo Scientific) after ionization by heated electrospray in negative-ion mode. The mass spectrometer operated in full-scan mode and targeted-MS 2 mode alternately. MS scans (288 – 292, 723 – 727 and 767 – 771 amu) were acquired with a mass resolution of 140000. The [M. – H] − and [M – H+1] − ions of ATR (725.2154 and 726.2188 amu ± 5 ppm) and CATR (769.2053 and 770.2086 amu ± 5 ppm) were used for quantification. The fullscan product ion spectrum of the compounds (resolution of 17500) was used to confirm the identity of the toxins. The processing of the biological sample (1 mL) consisted of a protein precipitation followed by solid phase extraction on Oasis ® HLB cartridges (Waters) at pH = 4.5. Results The method was validated in the whole blood with between- and within-day RSD (relative standard deviation) less than 5.8% and 5.2% for ATR (accuracy between 95.9% and 98.6%) and 5.4% and 9.8% for CATR (accuracy between 92.0% and 107.4%). The calibration curves were linear for concentrations ranging from 0.17 to 200 μg/L for ATR and 0.15 to 200 μg/L for CATR. The detection limits were 0.066 and 0,055 μg/L respectively. ATR and CATR were quantified in blood and urine samples from a non-fatal case of intoxication by A. gummifera . The concentrations were 883.1 and 119,0 μg/L respectively in peripheral blood and 230.4 and 140,3 mg/L in urine. ATR and CATR were quantified in dried A. gummifera roots by the standard addition method. The concentrations were 3.7 and 5,4 mg/g respectively. Conclusion We present the first validated method, applicable in forensic toxicology, for quantifying ATR and CATR in whole blood. The analysis is sensitive and quick.
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O27: A validated method for quantifying atractyloside and carboxyatractyloside in blood and urine by HPLC-HRMS/MS, a non-fatal case of intoxication with Atractylis gummifera L.
Toxicologie Analytique et Clinique, 2014Co-Authors: Jeremy Carlier, Ludovic Romeuf, Jerome Guitton, Fabien Bevalot, Laurent Fanton, Yvan GaillardAbstract:Introduction Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene glycosides that are responsible for the toxicity of several Asteraceae plants around the world. Mediterranean gum thistle ( Atractylis gummifera L.) and Zulu impila ( Callilepis laureola DC.), in particular, are notoriously poisonous and the cause of many accidental deaths, some suicides and even some murders. When a case history is not available, it is not easy to determine ATR or CATR poisoning anatomically or histologically. There is no current method for measuring the two toxins in biological samples that meet the criteria of specificity required in forensic medicine. We have endeavoured to fill this analytical gap. Methods Analysis was carried out using an original technique of high-performance liquid chromatography coupled with highresolution mass spectrometry detection (HPLC-HRMS). Given the singular structure of ATR and CATR, it is difficult to achieve and maintain the conditions required for their chromatographic separation and perfect ionization for the mass spectrometry. Separation was thus performed using an XTerra ® phenyl column (length: 150 mm; internal diameter: 2,1 mm; particle size: 3,5 μm) (Waters) [Steenkamp et al. , Forensic Sci Int, 2006, 163: 81–92] with a gradient mobile phase composed of acetonitrile containing 10% of isopropyl alcohol and a 5 mM ammonium acetate buffer at pH = 4.5. The chromatographic run time was 12.5 min. Spectrometric detection was performed using a quadrupole-Orbitrap high-resolution detector (Q Exactive™; Thermo Scientific) after ionization by heated electrospray in negative-ion mode. The mass spectrometer operated in full-scan mode and targeted-MS 2 mode alternately. MS scans (288 – 292, 723 – 727 and 767 – 771 amu) were acquired with a mass resolution of 140000. The [M. – H] − and [M – H+1] − ions of ATR (725.2154 and 726.2188 amu ± 5 ppm) and CATR (769.2053 and 770.2086 amu ± 5 ppm) were used for quantification. The fullscan product ion spectrum of the compounds (resolution of 17500) was used to confirm the identity of the toxins. The processing of the biological sample (1 mL) consisted of a protein precipitation followed by solid phase extraction on Oasis ® HLB cartridges (Waters) at pH = 4.5. Results The method was validated in the whole blood with between- and within-day RSD (relative standard deviation) less than 5.8% and 5.2% for ATR (accuracy between 95.9% and 98.6%) and 5.4% and 9.8% for CATR (accuracy between 92.0% and 107.4%). The calibration curves were linear for concentrations ranging from 0.17 to 200 μg/L for ATR and 0.15 to 200 μg/L for CATR. The detection limits were 0.066 and 0,055 μg/L respectively. ATR and CATR were quantified in blood and urine samples from a non-fatal case of intoxication by A. gummifera . The concentrations were 883.1 and 119,0 μg/L respectively in peripheral blood and 230.4 and 140,3 mg/L in urine. ATR and CATR were quantified in dried A. gummifera roots by the standard addition method. The concentrations were 3.7 and 5,4 mg/g respectively. Conclusion We present the first validated method, applicable in forensic toxicology, for quantifying ATR and CATR in whole blood. The analysis is sensitive and quick.
