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R. Lobinski - One of the best experts on this subject based on the ideXlab platform.
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Focus on Determination of Selenocysteine and Selenomethionine in Foodstuffs of Animal Origin by 2D Size-Exclusion Reversed-Phase HPLC-ICP-MS
2015Co-Authors: Katarzyna Bierla, R. Lobinski, Simon Godin, Joanna SzpunarAbstract:The chapter, after summarizing the state-of-the art for selenium analysis and selenium status of different aliments, describes an analytical procedure that was developed for the quantitative recovery of selenomethionine (SeMet) and selenocysteine (SeCys) from foodstuffs of animal origin. It points out all the crucial steps of raw sample treatment and gives the details of all the steps that had to be optimized to achieve efficient protein extraction (protein unfolding/denaturation by urea) and ensure lack of losses (carbamidomethylation of the aminoacid residues using iodoacetamide) due to Se-compound degradation. The seleno-aminoacids were specifically determined by ion-paring Reversed-Phase HPLC-ICP MS and quantified by the method of standard addition after proteolysis followed by their isolation from the postreaction mixture by size-exclusion LC. In addition, formal identification by ESI MS/MS was carried out for compounds for which standards are not commercially available. Determination and quantification of separate selenoamino acids gives us information about specific or nonspecific incorporation of selenium into proteins.
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determination of selenocysteine and selenomethionine in edible animal tissues by 2d size exclusion Reversed Phase HPLC icp ms following carbamidomethylation and proteolytic extraction
Analytical and Bioanalytical Chemistry, 2008Co-Authors: Katarzyna Bierla, R. Lobinski, Joanna Szpunar, Mihaly Dernovics, Veronique Vacchina, Gerard BertinAbstract:A method was developed for the simultaneous determination of selenomethionine (SeMet) and selenocysteine (SeCys) in meat (chicken and lamb muscles) and different offal tissues (heart, liver, kidney). The analytical procedure was based on the protein extraction with urea under reducing conditions (dithiothreitol), derivatization of SeCys and SeMet by carbamidomethylation with iodoacetamide (IAM) followed by quantitative proteolysis. The mixture of the derivatized Se-amino acids was purified by size-exclusion liquid chromatography (LC) and analysed by ion-paring Reversed-Phase HPLC–inductively coupled plasma mass spectroscopy (ICP MS). The quantification of SeCys and SeMet was carried out by the method of standard additions. 77SeMet was used to control the SeMet derivatization efficiency and recovery. The method was validated by the determination of the Se mass balance. The Se-amino acids accounted for 91 ± 8% of the total selenium (mean of 95 samples of seven tissues analysed over a period of 18 months). The method was applied to the discrimination of the contribution of selenoproteins (containing SeCys) and other Se-containing proteins (containing SeMet) in tissues of animals during supplementation studies (dose–effect and tolerance).
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Determination of cobalamins and cobinamides by microbore Reversed-Phase HPLC with spectrophotometric, ion-spray ionization MS and inductively coupled plasma MS detection
Analytica Chimica Acta, 1998Co-Authors: H. Chassaigne, R. LobinskiAbstract:Microbore Reversed-Phase HPLC was optimized for the separation of cyanocobalamin, 5'-desoxyadenosylcobalamin (coenzyme B12), methylcobalamin, hydroxocobalamin, aquocobalamin and cobinamide dicyanide isoforms. Various detection modes: UV at 278 nm, ion-spray (IS)-MS and direct-injection nebulization ICP-MS were examined and compared. IS-MS allows one to identify, unambiguously, the eluted compounds, whereas ICP MS was found to be more sensitive than any other on-line detection techniques described for the cobalamin analogues, so far allowing to achieve detection limits down to ca. 10 ng ml-1. The usefulness of the methods developed was demonstrated for the determination of cobalamins present in pharmaceutical preparations.
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Speciation of metal complexes with biomolecules by Reversed-Phase HPLC with ion-spray and inductively coupled plasma mass spectrometric detection
Fresenius' Journal of Analytical Chemistry, 1998Co-Authors: H. Chassaigne, R. LobinskiAbstract:Complementarity of ion-spray MS and ICP-MS as detection techniques in Reversed-Phase HPLC for the characterization of metals complexed by biomacromolecules is discussed by the example of the speciation of metallothionein-bound cadmium. The commercially purified rabbit liver MT-2 isoform is eluted from a microbore C8 column with a gradient of up to 50% methanol in acetate buffer (pH 6.0) to give one major and three minor peaks detected at 254 nm. The preparation is further characterized by using an ICP mass spectrometer interfaced with HPLC via a direct injection nebulizer which allows for the specific detection of cadmium down to the 10 ng mL-1 level. On-line detection by mass spectrometry with an ion-spray (pneumatically-assisted electrospray) ion source further allows the determination of the molecular masses of the eluted compounds. © Springer-Verlag 1998.
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Polymorphism and Identification of Metallothionein Isoforms by Reversed-Phase HPLC with On-Line Ion-Spray Mass Spectrometric Detection
Analytical Chemistry, 1998Co-Authors: H. Chassaigne, R. LobinskiAbstract:Microbore Reversed-Phase HPLC with on-line ion-spray mass spectrometric detection is proposed for a study of polymorphism of rabbit liver metallothionein (MTRL) and its major isoforms MT-1 and MT-2. Separation conditions had been optimized until each Chromatographic peak could be attributed to a single metalloprotein species, of which the molecular mass could be determined by ion-spray MS. At the optimized conditions (elution with the gradient 5-8% of acetonitrile within 50 min using a 5 mM acetate buffer at pH 6), the chromatogram of MTRL showed five peaks, whereas those of MT-1 and MT-2 showed nine and seven different peaks, respectively. The on-line determination of the molecular masses (±1 Da) of the compounds eluted permits the unambiguous cataloguing and further referencing of putative and true MT subisoforms. The results obtained are compared with those obtained by direct ion-spray MS of the MT preparations at different pHs, with a goal to identify possible Chromatographic artifacts. Metals (Cd, Cu) in the eluted complexes were studied by HPLC with on-line ICP MS detection.
H. Chassaigne - One of the best experts on this subject based on the ideXlab platform.
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The coupling of Reversed-Phase HPLC with ICP-MS in bioinorganic analysis : Metals and biomolecules
Analusis, 1998Co-Authors: H. Chassaigne, J. SzpunarAbstract:The coupling of Reversed-Phase HPLC with ICP-MS offers an attractive orthogonal approach to size-exclusion HPLC-ICP-MS for the characterisation of complex bioinorganic systems. An interface between microbore RP-HPLC and ICP-MS using a direct injection nebulizer which allows the introduction of mobile Phases up to 50% methanol is described, its applications for the analysis of cobalamine and metallothioneins are illustrated.
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Determination of cobalamins and cobinamides by microbore Reversed-Phase HPLC with spectrophotometric, ion-spray ionization MS and inductively coupled plasma MS detection
Analytica Chimica Acta, 1998Co-Authors: H. Chassaigne, R. LobinskiAbstract:Microbore Reversed-Phase HPLC was optimized for the separation of cyanocobalamin, 5'-desoxyadenosylcobalamin (coenzyme B12), methylcobalamin, hydroxocobalamin, aquocobalamin and cobinamide dicyanide isoforms. Various detection modes: UV at 278 nm, ion-spray (IS)-MS and direct-injection nebulization ICP-MS were examined and compared. IS-MS allows one to identify, unambiguously, the eluted compounds, whereas ICP MS was found to be more sensitive than any other on-line detection techniques described for the cobalamin analogues, so far allowing to achieve detection limits down to ca. 10 ng ml-1. The usefulness of the methods developed was demonstrated for the determination of cobalamins present in pharmaceutical preparations.
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Speciation of metal complexes with biomolecules by Reversed-Phase HPLC with ion-spray and inductively coupled plasma mass spectrometric detection
Fresenius' Journal of Analytical Chemistry, 1998Co-Authors: H. Chassaigne, R. LobinskiAbstract:Complementarity of ion-spray MS and ICP-MS as detection techniques in Reversed-Phase HPLC for the characterization of metals complexed by biomacromolecules is discussed by the example of the speciation of metallothionein-bound cadmium. The commercially purified rabbit liver MT-2 isoform is eluted from a microbore C8 column with a gradient of up to 50% methanol in acetate buffer (pH 6.0) to give one major and three minor peaks detected at 254 nm. The preparation is further characterized by using an ICP mass spectrometer interfaced with HPLC via a direct injection nebulizer which allows for the specific detection of cadmium down to the 10 ng mL-1 level. On-line detection by mass spectrometry with an ion-spray (pneumatically-assisted electrospray) ion source further allows the determination of the molecular masses of the eluted compounds. © Springer-Verlag 1998.
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Polymorphism and Identification of Metallothionein Isoforms by Reversed-Phase HPLC with On-Line Ion-Spray Mass Spectrometric Detection
Analytical Chemistry, 1998Co-Authors: H. Chassaigne, R. LobinskiAbstract:Microbore Reversed-Phase HPLC with on-line ion-spray mass spectrometric detection is proposed for a study of polymorphism of rabbit liver metallothionein (MTRL) and its major isoforms MT-1 and MT-2. Separation conditions had been optimized until each Chromatographic peak could be attributed to a single metalloprotein species, of which the molecular mass could be determined by ion-spray MS. At the optimized conditions (elution with the gradient 5-8% of acetonitrile within 50 min using a 5 mM acetate buffer at pH 6), the chromatogram of MTRL showed five peaks, whereas those of MT-1 and MT-2 showed nine and seven different peaks, respectively. The on-line determination of the molecular masses (±1 Da) of the compounds eluted permits the unambiguous cataloguing and further referencing of putative and true MT subisoforms. The results obtained are compared with those obtained by direct ion-spray MS of the MT preparations at different pHs, with a goal to identify possible Chromatographic artifacts. Metals (Cd, Cu) in the eluted complexes were studied by HPLC with on-line ICP MS detection.
Katarzyna Bierla - One of the best experts on this subject based on the ideXlab platform.
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Focus on Determination of Selenocysteine and Selenomethionine in Foodstuffs of Animal Origin by 2D Size-Exclusion Reversed-Phase HPLC-ICP-MS
2015Co-Authors: Katarzyna Bierla, R. Lobinski, Simon Godin, Joanna SzpunarAbstract:The chapter, after summarizing the state-of-the art for selenium analysis and selenium status of different aliments, describes an analytical procedure that was developed for the quantitative recovery of selenomethionine (SeMet) and selenocysteine (SeCys) from foodstuffs of animal origin. It points out all the crucial steps of raw sample treatment and gives the details of all the steps that had to be optimized to achieve efficient protein extraction (protein unfolding/denaturation by urea) and ensure lack of losses (carbamidomethylation of the aminoacid residues using iodoacetamide) due to Se-compound degradation. The seleno-aminoacids were specifically determined by ion-paring Reversed-Phase HPLC-ICP MS and quantified by the method of standard addition after proteolysis followed by their isolation from the postreaction mixture by size-exclusion LC. In addition, formal identification by ESI MS/MS was carried out for compounds for which standards are not commercially available. Determination and quantification of separate selenoamino acids gives us information about specific or nonspecific incorporation of selenium into proteins.
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determination of selenocysteine and selenomethionine in edible animal tissues by 2d size exclusion Reversed Phase HPLC icp ms following carbamidomethylation and proteolytic extraction
Analytical and Bioanalytical Chemistry, 2008Co-Authors: Katarzyna Bierla, R. Lobinski, Joanna Szpunar, Mihaly Dernovics, Veronique Vacchina, Gerard BertinAbstract:A method was developed for the simultaneous determination of selenomethionine (SeMet) and selenocysteine (SeCys) in meat (chicken and lamb muscles) and different offal tissues (heart, liver, kidney). The analytical procedure was based on the protein extraction with urea under reducing conditions (dithiothreitol), derivatization of SeCys and SeMet by carbamidomethylation with iodoacetamide (IAM) followed by quantitative proteolysis. The mixture of the derivatized Se-amino acids was purified by size-exclusion liquid chromatography (LC) and analysed by ion-paring Reversed-Phase HPLC–inductively coupled plasma mass spectroscopy (ICP MS). The quantification of SeCys and SeMet was carried out by the method of standard additions. 77SeMet was used to control the SeMet derivatization efficiency and recovery. The method was validated by the determination of the Se mass balance. The Se-amino acids accounted for 91 ± 8% of the total selenium (mean of 95 samples of seven tissues analysed over a period of 18 months). The method was applied to the discrimination of the contribution of selenoproteins (containing SeCys) and other Se-containing proteins (containing SeMet) in tissues of animals during supplementation studies (dose–effect and tolerance).
Joanna Szpunar - One of the best experts on this subject based on the ideXlab platform.
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Focus on Determination of Selenocysteine and Selenomethionine in Foodstuffs of Animal Origin by 2D Size-Exclusion Reversed-Phase HPLC-ICP-MS
2015Co-Authors: Katarzyna Bierla, R. Lobinski, Simon Godin, Joanna SzpunarAbstract:The chapter, after summarizing the state-of-the art for selenium analysis and selenium status of different aliments, describes an analytical procedure that was developed for the quantitative recovery of selenomethionine (SeMet) and selenocysteine (SeCys) from foodstuffs of animal origin. It points out all the crucial steps of raw sample treatment and gives the details of all the steps that had to be optimized to achieve efficient protein extraction (protein unfolding/denaturation by urea) and ensure lack of losses (carbamidomethylation of the aminoacid residues using iodoacetamide) due to Se-compound degradation. The seleno-aminoacids were specifically determined by ion-paring Reversed-Phase HPLC-ICP MS and quantified by the method of standard addition after proteolysis followed by their isolation from the postreaction mixture by size-exclusion LC. In addition, formal identification by ESI MS/MS was carried out for compounds for which standards are not commercially available. Determination and quantification of separate selenoamino acids gives us information about specific or nonspecific incorporation of selenium into proteins.
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determination of selenocysteine and selenomethionine in edible animal tissues by 2d size exclusion Reversed Phase HPLC icp ms following carbamidomethylation and proteolytic extraction
Analytical and Bioanalytical Chemistry, 2008Co-Authors: Katarzyna Bierla, R. Lobinski, Joanna Szpunar, Mihaly Dernovics, Veronique Vacchina, Gerard BertinAbstract:A method was developed for the simultaneous determination of selenomethionine (SeMet) and selenocysteine (SeCys) in meat (chicken and lamb muscles) and different offal tissues (heart, liver, kidney). The analytical procedure was based on the protein extraction with urea under reducing conditions (dithiothreitol), derivatization of SeCys and SeMet by carbamidomethylation with iodoacetamide (IAM) followed by quantitative proteolysis. The mixture of the derivatized Se-amino acids was purified by size-exclusion liquid chromatography (LC) and analysed by ion-paring Reversed-Phase HPLC–inductively coupled plasma mass spectroscopy (ICP MS). The quantification of SeCys and SeMet was carried out by the method of standard additions. 77SeMet was used to control the SeMet derivatization efficiency and recovery. The method was validated by the determination of the Se mass balance. The Se-amino acids accounted for 91 ± 8% of the total selenium (mean of 95 samples of seven tissues analysed over a period of 18 months). The method was applied to the discrimination of the contribution of selenoproteins (containing SeCys) and other Se-containing proteins (containing SeMet) in tissues of animals during supplementation studies (dose–effect and tolerance).
Gerard Bertin - One of the best experts on this subject based on the ideXlab platform.
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determination of selenocysteine and selenomethionine in edible animal tissues by 2d size exclusion Reversed Phase HPLC icp ms following carbamidomethylation and proteolytic extraction
Analytical and Bioanalytical Chemistry, 2008Co-Authors: Katarzyna Bierla, R. Lobinski, Joanna Szpunar, Mihaly Dernovics, Veronique Vacchina, Gerard BertinAbstract:A method was developed for the simultaneous determination of selenomethionine (SeMet) and selenocysteine (SeCys) in meat (chicken and lamb muscles) and different offal tissues (heart, liver, kidney). The analytical procedure was based on the protein extraction with urea under reducing conditions (dithiothreitol), derivatization of SeCys and SeMet by carbamidomethylation with iodoacetamide (IAM) followed by quantitative proteolysis. The mixture of the derivatized Se-amino acids was purified by size-exclusion liquid chromatography (LC) and analysed by ion-paring Reversed-Phase HPLC–inductively coupled plasma mass spectroscopy (ICP MS). The quantification of SeCys and SeMet was carried out by the method of standard additions. 77SeMet was used to control the SeMet derivatization efficiency and recovery. The method was validated by the determination of the Se mass balance. The Se-amino acids accounted for 91 ± 8% of the total selenium (mean of 95 samples of seven tissues analysed over a period of 18 months). The method was applied to the discrimination of the contribution of selenoproteins (containing SeCys) and other Se-containing proteins (containing SeMet) in tissues of animals during supplementation studies (dose–effect and tolerance).
