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Laurentbernard Fay - One of the best experts on this subject based on the ideXlab platform.

  • Simultaneous measurement of C-13- and N-15-isotopic Enrichments of threonine by mass spectrometry
    Rapid Communications in Mass Spectrometry, 2009
    Co-Authors: Jean-philippe Godin, Laurentbernard Fay, Anne-france Mermoud, Didier Remond, Magali Faure, Denis Breuillé, Gary Williamson, Emma Peré-trepat, Ziad Ramadan, Sunil Kochhar
    Abstract:

    Under conditions of high isotopic dilution, e.g. in a tracer study, the ability to determine accurately and quantitatively small variations in isotopic Enrichments of differently labelled chemical compounds (e.g. C-13 and N-15 in threonine) in a single run by gas chromatography/mass spectrometry (GC/MS) is desirable but remains a technological challenge. Here, we report a new, rapid and simple GC/MS method for simultaneously measuring the isotopic Enrichments of doubly labelled threonine ([(UC)-C-13] and N-15) with isotopic Enrichment lower than 1.5 Molar Percent Excess (MPE). The long-term reproducibility measured was around 0.09 MPE for both tracers (throughout a 6 week period). The intra-day repeatability was lower than 0.05 and 0.06 MPE for [(UC)-C-13]-Thr and N-15-Thr, respectively. To calculate both isotopic Enrichments, two modes of calculations were used: one based on work by Rosenblatt et al. in 1992 and the other one using a matrix approach. Both methods gave similar results (ANOVA, P >0.05) with close precision for each mode of calculation. The GC/MS method was then used to investigate the differential utilization of threonine in different organs according to its route of administration in minipigs after administration of both tracers. In plasma samples, the lowest isotopic Enrichment measured between two successive time points was at 0.01 and 0.02 MPE for [(UC)-C-13]-Thr and N-15-Thr, respectively. Moreover, the accuracy of GC/MS C-13-isotopic Enrichment measured was validated by analyzing the same plasma samples by gas chromatography/combustion/isotope ratio mass spectrometry-(GC/C/IRMS). Statistical analysis showed that both techniques gave the same results (ANOVA, P >0.05). This new GC/MS method offers the possibility to measure C-13- and N-15-isotopic Enrichments with higher throughput, and using a lower amount of sample, than using GC/C/IRMS.

  • Liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of C-13-valine isotopic ratios in complex biological samples
    Journal of Mass Spectrometry, 2008
    Co-Authors: Jean-philippe Godin, Denis Breuillé, Christiane Obled, Isabelle Papet, Henk Schierbeek, Gerard Hopfgartner, Laurentbernard Fay
    Abstract:

    On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision (13)C isotopic Enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure (13)C isotopic Enrichment of underivatised amino acids (Asp, Thr-Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(delta(13)C) reported with this method was found to be below 1 per thousand . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic Enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (delta(13)C= -12.3 to 150.8 per thousand), the calculated root-mean-square (rms) of SD was 0.38 and 0.46 per thousand and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic Enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (delta(13)C) and absolute (atom%, APE and MPE) isotopic Enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic Enrichment of protein-bound (13)C-Val in tibial epiphysis in a tracer study performed in rats. Isotopic Enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p>0.05). The results of this work indicate that the LC-IRMS was successful for high-precision (13)C isotopic measurements in tracer studies giving (13)C isotopic Enrichment similar to the GC-C-IRMS but without the step of GC derivatisation. Therefore, for clinical studies requiring high-precision isotopic measurement, the LC-IRMS is the method of choice to measure the isotopic ratio.

  • high throughput simultaneous determination of plasma water deuterium and 18 oxygen Enrichment using a high temperature conversion elemental analyzer with isotope ratio mass spectrometry
    Rapid Communications in Mass Spectrometry, 2004
    Co-Authors: Myriam Richelle, C Darimont, Christelle Piguetwelsch, Laurentbernard Fay
    Abstract:

    This paper presents a high-throughput method for the simultaneous determination of deuterium and oxygen-18 (18O) Enrichment of water samples isolated from blood. This analytical method enables rapid and simple determination of these Enrichments of microgram quantities of water. Water is converted into hydrogen and carbon monoxide gases by the use of a high-temperature conversion elemental analyzer (TC-EA), that are then transferred on-line into the isotope ratio mass spectrometer. Accuracy determined with the standard light Antartic precipitation (SLAP) and Greenland ice sheet precipitation (GISP) is reliable for deuterium and 18O Enrichments. The range of linearity is from 0 up to 0.09 atom percent excess (APE, i.e. -78 up to 5725 delta per mil (dpm)) for deuterium Enrichment and from 0 up to 0.17 APE (-11 up to 890 dpm) for 18O Enrichment. Memory effects do exist but can be avoided by analyzing the biological samples in quintuplet. This method allows the determination of 1440 samples per week, i.e. 288 biological samples per week.

Christian Jeanthon - One of the best experts on this subject based on the ideXlab platform.

  • Cultivated anaerobic acidophilic/acidotolerant thermophiles from terrestrial and deep-sea hydrothermal habitats
    Extremophiles, 2005
    Co-Authors: Maria I. Prokofeva, Tatyana V. Kolganova, Tatyana P. Tourova, Ilya V. Kublanov, Olivier Nercessian, Alexander V. Lebedinsky, Elizaveta A. Bonch-osmolovskaya, Stefan Spring, Christian Jeanthon
    Abstract:

    Metabolic and phylogenetic diversity of cultivated anaerobic microorganisms from acidic continental hot springs and deep-sea hydrothermal vents was studied by molecular and microbiological methods. Anaerobic organotrophic Enrichment cultures growing at pH 3.5–4.0 and 60 or 85°C with organic energy sources were obtained from samples of acidic hot springs of Kamchatka Peninsula (Pauzhetka, Moutnovski Volcano, Uzon Caldera) and Kunashir Island (South Kurils) as well as from the samples of chimneys of East Pacific Rise (13°N). The analyses of clone libraries obtained from terrestrial Enrichment cultures growing at 60°C revealed the presence of archaea of genus Thermoplasma and bacteria of genus Thermoanaerobacter . Bacterial isolates from these Enrichments were shown to belong to genera Thermoanaerobacter and Thermoanaerobacterium , being acidotolerant with the pH optimum for growth at 5.5–6.0 and the pH minimum at 3.0. At 85°C, domination of thermoacidophilic archaea of genus Acidilobus in terrestrial Enrichments was found by both molecular and microbiological methods. Five isolates belonging to this genus possessed some phenotypic features that were new for this genus, such as flagellation or the ability to grow on monosaccharides or disaccharides. Analyses of clone libraries from the deep-sea thermoacidophilic Enrichment cultures showed that the representatives of the genus Thermococcus were present at both 60 and 85°C. From the 60°C deep-sea Enrichment, a strain belonging to Thermoanaerobacter siderophilus was isolated. It grew optimally at pH 6.0 with the minimum pH for growth at 3.0 and with salinity optimum at 0–2.5% NaCl and the maximum at 7%, thus differing significantly from the type strain. These data show that fermentative degradation of organic matter may occur at low pH and wide temperature range in both terrestrial and deep-sea habitats and can be performed by acidophilic or acidotolerant thermophilic prokaryotes.

  • cultivated anaerobic acidophilic acidotolerant thermophiles from terrestrial and deep sea hydrothermal habitats
    Extremophiles, 2005
    Co-Authors: Maria I. Prokofeva, Tatyana V. Kolganova, Tatyana P. Tourova, Ilya V. Kublanov, Olivier Nercessian, Alexander V. Lebedinsky, Stefan Spring, Elizaveta A Bonchosmolovskaya, Christian Jeanthon
    Abstract:

    Metabolic and phylogenetic diversity of cultivated anaerobic microorganisms from acidic continental hot springs and deep-sea hydrothermal vents was studied by molecular and microbiological methods. Anaerobic organotrophic Enrichment cultures growing at pH 3.5–4.0 and 60 or 85°C with organic energy sources were obtained from samples of acidic hot springs of Kamchatka Peninsula (Pauzhetka, Moutnovski Volcano, Uzon Caldera) and Kunashir Island (South Kurils) as well as from the samples of chimneys of East Pacific Rise (13°N). The analyses of clone libraries obtained from terrestrial Enrichment cultures growing at 60°C revealed the presence of archaea of genus Thermoplasma and bacteria of genus Thermoanaerobacter. Bacterial isolates from these Enrichments were shown to belong to genera Thermoanaerobacter and Thermoanaerobacterium, being acidotolerant with the pH optimum for growth at 5.5–6.0 and the pH minimum at 3.0. At 85°C, domination of thermoacidophilic archaea of genus Acidilobus in terrestrial Enrichments was found by both molecular and microbiological methods. Five isolates belonging to this genus possessed some phenotypic features that were new for this genus, such as flagellation or the ability to grow on monosaccharides or disaccharides. Analyses of clone libraries from the deep-sea thermoacidophilic Enrichment cultures showed that the representatives of the genus Thermococcus were present at both 60 and 85°C. From the 60°C deep-sea Enrichment, a strain belonging to Thermoanaerobacter siderophilus was isolated. It grew optimally at pH 6.0 with the minimum pH for growth at 3.0 and with salinity optimum at 0–2.5% NaCl and the maximum at 7%, thus differing significantly from the type strain. These data show that fermentative degradation of organic matter may occur at low pH and wide temperature range in both terrestrial and deep-sea habitats and can be performed by acidophilic or acidotolerant thermophilic prokaryotes.

Jean-philippe Godin - One of the best experts on this subject based on the ideXlab platform.

  • Simultaneous measurement of C-13- and N-15-isotopic Enrichments of threonine by mass spectrometry
    Rapid Communications in Mass Spectrometry, 2009
    Co-Authors: Jean-philippe Godin, Laurentbernard Fay, Anne-france Mermoud, Didier Remond, Magali Faure, Denis Breuillé, Gary Williamson, Emma Peré-trepat, Ziad Ramadan, Sunil Kochhar
    Abstract:

    Under conditions of high isotopic dilution, e.g. in a tracer study, the ability to determine accurately and quantitatively small variations in isotopic Enrichments of differently labelled chemical compounds (e.g. C-13 and N-15 in threonine) in a single run by gas chromatography/mass spectrometry (GC/MS) is desirable but remains a technological challenge. Here, we report a new, rapid and simple GC/MS method for simultaneously measuring the isotopic Enrichments of doubly labelled threonine ([(UC)-C-13] and N-15) with isotopic Enrichment lower than 1.5 Molar Percent Excess (MPE). The long-term reproducibility measured was around 0.09 MPE for both tracers (throughout a 6 week period). The intra-day repeatability was lower than 0.05 and 0.06 MPE for [(UC)-C-13]-Thr and N-15-Thr, respectively. To calculate both isotopic Enrichments, two modes of calculations were used: one based on work by Rosenblatt et al. in 1992 and the other one using a matrix approach. Both methods gave similar results (ANOVA, P >0.05) with close precision for each mode of calculation. The GC/MS method was then used to investigate the differential utilization of threonine in different organs according to its route of administration in minipigs after administration of both tracers. In plasma samples, the lowest isotopic Enrichment measured between two successive time points was at 0.01 and 0.02 MPE for [(UC)-C-13]-Thr and N-15-Thr, respectively. Moreover, the accuracy of GC/MS C-13-isotopic Enrichment measured was validated by analyzing the same plasma samples by gas chromatography/combustion/isotope ratio mass spectrometry-(GC/C/IRMS). Statistical analysis showed that both techniques gave the same results (ANOVA, P >0.05). This new GC/MS method offers the possibility to measure C-13- and N-15-isotopic Enrichments with higher throughput, and using a lower amount of sample, than using GC/C/IRMS.

  • liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of 13c valine isotopic ratios in complex biological samples
    Journal of Mass Spectrometry, 2008
    Co-Authors: Jean-philippe Godin, Denis Breuillé, Christiane Obled, Isabelle Papet, Henk Schierbeek, Gerard Hopfgartner
    Abstract:

    On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision (13)C isotopic Enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure (13)C isotopic Enrichment of underivatised amino acids (Asp, Thr-Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(delta(13)C) reported with this method was found to be below 1 per thousand . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic Enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (delta(13)C= -12.3 to 150.8 per thousand), the calculated root-mean-square (rms) of SD was 0.38 and 0.46 per thousand and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic Enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (delta(13)C) and absolute (atom%, APE and MPE) isotopic Enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic Enrichment of protein-bound (13)C-Val in tibial epiphysis in a tracer study performed in rats. Isotopic Enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p>0.05). The results of this work indicate that the LC-IRMS was successful for high-precision (13)C isotopic measurements in tracer studies giving (13)C isotopic Enrichment similar to the GC-C-IRMS but without the step of GC derivatisation. Therefore, for clinical studies requiring high-precision isotopic measurement, the LC-IRMS is the method of choice to measure the isotopic ratio

  • Liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of C-13-valine isotopic ratios in complex biological samples
    Journal of Mass Spectrometry, 2008
    Co-Authors: Jean-philippe Godin, Denis Breuillé, Christiane Obled, Isabelle Papet, Henk Schierbeek, Gerard Hopfgartner, Laurentbernard Fay
    Abstract:

    On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision (13)C isotopic Enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure (13)C isotopic Enrichment of underivatised amino acids (Asp, Thr-Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(delta(13)C) reported with this method was found to be below 1 per thousand . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic Enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (delta(13)C= -12.3 to 150.8 per thousand), the calculated root-mean-square (rms) of SD was 0.38 and 0.46 per thousand and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic Enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (delta(13)C) and absolute (atom%, APE and MPE) isotopic Enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic Enrichment of protein-bound (13)C-Val in tibial epiphysis in a tracer study performed in rats. Isotopic Enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p>0.05). The results of this work indicate that the LC-IRMS was successful for high-precision (13)C isotopic measurements in tracer studies giving (13)C isotopic Enrichment similar to the GC-C-IRMS but without the step of GC derivatisation. Therefore, for clinical studies requiring high-precision isotopic measurement, the LC-IRMS is the method of choice to measure the isotopic ratio.

Maria I. Prokofeva - One of the best experts on this subject based on the ideXlab platform.

  • Cultivated anaerobic acidophilic/acidotolerant thermophiles from terrestrial and deep-sea hydrothermal habitats
    Extremophiles, 2005
    Co-Authors: Maria I. Prokofeva, Tatyana V. Kolganova, Tatyana P. Tourova, Ilya V. Kublanov, Olivier Nercessian, Alexander V. Lebedinsky, Elizaveta A. Bonch-osmolovskaya, Stefan Spring, Christian Jeanthon
    Abstract:

    Metabolic and phylogenetic diversity of cultivated anaerobic microorganisms from acidic continental hot springs and deep-sea hydrothermal vents was studied by molecular and microbiological methods. Anaerobic organotrophic Enrichment cultures growing at pH 3.5–4.0 and 60 or 85°C with organic energy sources were obtained from samples of acidic hot springs of Kamchatka Peninsula (Pauzhetka, Moutnovski Volcano, Uzon Caldera) and Kunashir Island (South Kurils) as well as from the samples of chimneys of East Pacific Rise (13°N). The analyses of clone libraries obtained from terrestrial Enrichment cultures growing at 60°C revealed the presence of archaea of genus Thermoplasma and bacteria of genus Thermoanaerobacter . Bacterial isolates from these Enrichments were shown to belong to genera Thermoanaerobacter and Thermoanaerobacterium , being acidotolerant with the pH optimum for growth at 5.5–6.0 and the pH minimum at 3.0. At 85°C, domination of thermoacidophilic archaea of genus Acidilobus in terrestrial Enrichments was found by both molecular and microbiological methods. Five isolates belonging to this genus possessed some phenotypic features that were new for this genus, such as flagellation or the ability to grow on monosaccharides or disaccharides. Analyses of clone libraries from the deep-sea thermoacidophilic Enrichment cultures showed that the representatives of the genus Thermococcus were present at both 60 and 85°C. From the 60°C deep-sea Enrichment, a strain belonging to Thermoanaerobacter siderophilus was isolated. It grew optimally at pH 6.0 with the minimum pH for growth at 3.0 and with salinity optimum at 0–2.5% NaCl and the maximum at 7%, thus differing significantly from the type strain. These data show that fermentative degradation of organic matter may occur at low pH and wide temperature range in both terrestrial and deep-sea habitats and can be performed by acidophilic or acidotolerant thermophilic prokaryotes.

  • cultivated anaerobic acidophilic acidotolerant thermophiles from terrestrial and deep sea hydrothermal habitats
    Extremophiles, 2005
    Co-Authors: Maria I. Prokofeva, Tatyana V. Kolganova, Tatyana P. Tourova, Ilya V. Kublanov, Olivier Nercessian, Alexander V. Lebedinsky, Stefan Spring, Elizaveta A Bonchosmolovskaya, Christian Jeanthon
    Abstract:

    Metabolic and phylogenetic diversity of cultivated anaerobic microorganisms from acidic continental hot springs and deep-sea hydrothermal vents was studied by molecular and microbiological methods. Anaerobic organotrophic Enrichment cultures growing at pH 3.5–4.0 and 60 or 85°C with organic energy sources were obtained from samples of acidic hot springs of Kamchatka Peninsula (Pauzhetka, Moutnovski Volcano, Uzon Caldera) and Kunashir Island (South Kurils) as well as from the samples of chimneys of East Pacific Rise (13°N). The analyses of clone libraries obtained from terrestrial Enrichment cultures growing at 60°C revealed the presence of archaea of genus Thermoplasma and bacteria of genus Thermoanaerobacter. Bacterial isolates from these Enrichments were shown to belong to genera Thermoanaerobacter and Thermoanaerobacterium, being acidotolerant with the pH optimum for growth at 5.5–6.0 and the pH minimum at 3.0. At 85°C, domination of thermoacidophilic archaea of genus Acidilobus in terrestrial Enrichments was found by both molecular and microbiological methods. Five isolates belonging to this genus possessed some phenotypic features that were new for this genus, such as flagellation or the ability to grow on monosaccharides or disaccharides. Analyses of clone libraries from the deep-sea thermoacidophilic Enrichment cultures showed that the representatives of the genus Thermococcus were present at both 60 and 85°C. From the 60°C deep-sea Enrichment, a strain belonging to Thermoanaerobacter siderophilus was isolated. It grew optimally at pH 6.0 with the minimum pH for growth at 3.0 and with salinity optimum at 0–2.5% NaCl and the maximum at 7%, thus differing significantly from the type strain. These data show that fermentative degradation of organic matter may occur at low pH and wide temperature range in both terrestrial and deep-sea habitats and can be performed by acidophilic or acidotolerant thermophilic prokaryotes.

Gerard Hopfgartner - One of the best experts on this subject based on the ideXlab platform.

  • liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of 13c valine isotopic ratios in complex biological samples
    Journal of Mass Spectrometry, 2008
    Co-Authors: Jean-philippe Godin, Denis Breuillé, Christiane Obled, Isabelle Papet, Henk Schierbeek, Gerard Hopfgartner
    Abstract:

    On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision (13)C isotopic Enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure (13)C isotopic Enrichment of underivatised amino acids (Asp, Thr-Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(delta(13)C) reported with this method was found to be below 1 per thousand . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic Enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (delta(13)C= -12.3 to 150.8 per thousand), the calculated root-mean-square (rms) of SD was 0.38 and 0.46 per thousand and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic Enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (delta(13)C) and absolute (atom%, APE and MPE) isotopic Enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic Enrichment of protein-bound (13)C-Val in tibial epiphysis in a tracer study performed in rats. Isotopic Enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p>0.05). The results of this work indicate that the LC-IRMS was successful for high-precision (13)C isotopic measurements in tracer studies giving (13)C isotopic Enrichment similar to the GC-C-IRMS but without the step of GC derivatisation. Therefore, for clinical studies requiring high-precision isotopic measurement, the LC-IRMS is the method of choice to measure the isotopic ratio

  • Liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of C-13-valine isotopic ratios in complex biological samples
    Journal of Mass Spectrometry, 2008
    Co-Authors: Jean-philippe Godin, Denis Breuillé, Christiane Obled, Isabelle Papet, Henk Schierbeek, Gerard Hopfgartner, Laurentbernard Fay
    Abstract:

    On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision (13)C isotopic Enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure (13)C isotopic Enrichment of underivatised amino acids (Asp, Thr-Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(delta(13)C) reported with this method was found to be below 1 per thousand . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic Enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (delta(13)C= -12.3 to 150.8 per thousand), the calculated root-mean-square (rms) of SD was 0.38 and 0.46 per thousand and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic Enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (delta(13)C) and absolute (atom%, APE and MPE) isotopic Enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic Enrichment of protein-bound (13)C-Val in tibial epiphysis in a tracer study performed in rats. Isotopic Enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p>0.05). The results of this work indicate that the LC-IRMS was successful for high-precision (13)C isotopic measurements in tracer studies giving (13)C isotopic Enrichment similar to the GC-C-IRMS but without the step of GC derivatisation. Therefore, for clinical studies requiring high-precision isotopic measurement, the LC-IRMS is the method of choice to measure the isotopic ratio.