Methodology for a rapid and simultaneous determination of total cysteine, homocysteine, cysteinylglycine and glutathione in plasma by isocratic RP-HPLC
► Rapid and simultaneous determination of four total aminothiols levels in plasma. ► RP-HPLC isocratic elution within 6min of all analytes, including the IS. ► Validated method exhibits an excellent precision and recovery for all aminothiols. ► Method appropriated for high-throughput routine clinica...
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| Published in | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 911; pp. 15 - 20 |
|---|---|
| Main Authors | , , |
| Format | Journal Article |
| Language | English |
| Published |
Netherlands
Elsevier B.V
12.12.2012
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1570-0232 1873-376X 1873-376X |
| DOI | 10.1016/j.jchromb.2012.10.022 |
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| Abstract | ► Rapid and simultaneous determination of four total aminothiols levels in plasma. ► RP-HPLC isocratic elution within 6min of all analytes, including the IS. ► Validated method exhibits an excellent precision and recovery for all aminothiols. ► Method appropriated for high-throughput routine clinical analysis.
Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53mm×7mm I.D., 3μm) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r2) ≥0.99. The LOD for the four plasma thiols was lower than 0.10μmol/L, while LOQ varied from 0.5 to 15μmol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6min. By reducing the total run time, a larger number of analysis can be performed daily. |
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| AbstractList | Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53mm×7mm I.D., 3μm) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r²) ≥0.99. The LOD for the four plasma thiols was lower than 0.10μmol/L, while LOQ varied from 0.5 to 15μmol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6min. By reducing the total run time, a larger number of analysis can be performed daily. Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53mm×7mm I.D., 3μm) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r(2)) ≥0.99. The LOD for the four plasma thiols was lower than 0.10μmol/L, while LOQ varied from 0.5 to 15μmol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6min. By reducing the total run time, a larger number of analysis can be performed daily. Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53mm×7mm I.D., 3μm) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r(2)) ≥0.99. The LOD for the four plasma thiols was lower than 0.10μmol/L, while LOQ varied from 0.5 to 15μmol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6min. By reducing the total run time, a larger number of analysis can be performed daily.Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53mm×7mm I.D., 3μm) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r(2)) ≥0.99. The LOD for the four plasma thiols was lower than 0.10μmol/L, while LOQ varied from 0.5 to 15μmol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6min. By reducing the total run time, a larger number of analysis can be performed daily. Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53 mm X 7 mm I.D., 3 mu m) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r2) aY0.99. The LOD for the four plasma thiols was lower than 0.10 mu mol/L, while LOQ varied from 0.5 to 15 mu mol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6 min. By reducing the total run time, a larger number of analysis can be performed daily. ► Rapid and simultaneous determination of four total aminothiols levels in plasma. ► RP-HPLC isocratic elution within 6min of all analytes, including the IS. ► Validated method exhibits an excellent precision and recovery for all aminothiols. ► Method appropriated for high-throughput routine clinical analysis. Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53mm×7mm I.D., 3μm) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r2) ≥0.99. The LOD for the four plasma thiols was lower than 0.10μmol/L, while LOQ varied from 0.5 to 15μmol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6min. By reducing the total run time, a larger number of analysis can be performed daily. |
| Author | Ferin, Rita Baptista, José Pavão, Maria Leonor |
| Author_xml | – sequence: 1 givenname: Rita surname: Ferin fullname: Ferin, Rita email: rborges@uac.pt organization: Department of Technological Sciences and Development, University of the Azores, Rua da Mãe de Deus, 9501-801 Ponta Delgada, Azores, Portugal – sequence: 2 givenname: Maria Leonor surname: Pavão fullname: Pavão, Maria Leonor organization: Department of Technological Sciences and Development, University of the Azores, Rua da Mãe de Deus, 9501-801 Ponta Delgada, Azores, Portugal – sequence: 3 givenname: José surname: Baptista fullname: Baptista, José organization: Department of Technological Sciences and Development, University of the Azores, Rua da Mãe de Deus, 9501-801 Ponta Delgada, Azores, Portugal |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/23217300$$D View this record in MEDLINE/PubMed |
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| Keywords | Cysteine Cysteinylglycine Homocysteine Plasma aminothiols HPLC Glutathione |
| Language | English |
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| Snippet | ► Rapid and simultaneous determination of four total aminothiols levels in plasma. ► RP-HPLC isocratic elution within 6min of all analytes, including the IS. ►... Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely... |
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| SubjectTerms | acetonitrile Adult biomarkers blood plasma Chromatography, High Pressure Liquid - methods Chromatography, Reverse-Phase Cysteine Cysteine - blood Cysteinylglycine Dipeptides - blood Diseases Female fluorescence Glutathione Glutathione - blood Homocysteine Homocysteine - blood HPLC Humans Linear Models Linearity Male Methodology Middle Aged Phosphates Plasma aminothiols Reproducibility of Results reversed-phase high performance liquid chromatography Run time (computers) screening Sensitivity and Specificity Thiols |
| Title | Methodology for a rapid and simultaneous determination of total cysteine, homocysteine, cysteinylglycine and glutathione in plasma by isocratic RP-HPLC |
| URI | https://dx.doi.org/10.1016/j.jchromb.2012.10.022 https://www.ncbi.nlm.nih.gov/pubmed/23217300 https://www.proquest.com/docview/1237508603 https://www.proquest.com/docview/1283669924 https://www.proquest.com/docview/2000049817 |
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