Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC–MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene Extracts
Analysis of amino acids by gas chromatography-mass spectrometry (GC–MS) requires at least one derivatization step to enable solubility in GC–MS-compatible water-immiscible organic solvents such as toluene, to make them volatile to introduce into the gas chromatograph and thermally stable enough for...
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| Format: | Article |
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MDPI AG
2021-03-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/26/6/1726 |
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| author | Svetlana Baskal Alexander Bollenbach Dimitrios Tsikas |
| author_facet | Svetlana Baskal Alexander Bollenbach Dimitrios Tsikas |
| author_sort | Svetlana Baskal |
| collection | DOAJ |
| description | Analysis of amino acids by gas chromatography-mass spectrometry (GC–MS) requires at least one derivatization step to enable solubility in GC–MS-compatible water-immiscible organic solvents such as toluene, to make them volatile to introduce into the gas chromatograph and thermally stable enough for separation in the GC column and introduction into the ion-source, and finally to increase their ionization by increasing their electronegativity using F-rich reagents. In this work we investigated the long-term stability of the methyl esters pentafluoropropionic (Me-PFP) derivatives of 21 urinary amino acids prepared by a two-step derivatization procedure and extraction by toluene. In situ prepared trideuteromethyl ester pentafluoropropionic derivatives were used as internal standards. GC–MS analysis (injection of 1 µL aliquots and quantification by selected-ion monitoring of specific mass fragments) was performed on days 1, 2, 8, and 15. Measured peak areas and calculated peak area ratios were used to evaluate the stability of the derivatives of endogenous amino acids and their internal standards, as well as the precision and the accuracy of the method. All analyses were performed under routine conditions. Me-PFP derivatives of endogenous amino acids and their stable-isotope labelled analogs were stable in toluene for 14 days. The peak area values of the derivatives of most amino acids and their internal standards were slightly higher on days 8 and 15 compared to days 1 and 2, yet the peak area ratio values of endogenous amino acids to their internal standards did not change. Our study indicates that Me-PFP derivatives of amino acids from human urine samples can easily be prepared, are stable at least for 14 days in the extraction solvent toluene, and allow for precise and accurate quantitative measurements by GC–MS using in situ prepared deuterium-labelled methyl ester as internal standard. |
| first_indexed | 2024-03-10T13:04:40Z |
| format | Article |
| id | doaj.art-3e9a93356e2842e8ac2ff68485321388 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T13:04:40Z |
| publishDate | 2021-03-01 |
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| record_format | Article |
| series | Molecules |
| spelling | doaj.art-3e9a93356e2842e8ac2ff684853213882023-11-21T11:13:18ZengMDPI AGMolecules1420-30492021-03-01266172610.3390/molecules26061726Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC–MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene ExtractsSvetlana Baskal0Alexander Bollenbach1Dimitrios Tsikas2Institute of Toxicology, Core Unit Proteomics, Hannover Medical School, 30625 Hannover, GermanyInstitute of Toxicology, Core Unit Proteomics, Hannover Medical School, 30625 Hannover, GermanyInstitute of Toxicology, Core Unit Proteomics, Hannover Medical School, 30625 Hannover, GermanyAnalysis of amino acids by gas chromatography-mass spectrometry (GC–MS) requires at least one derivatization step to enable solubility in GC–MS-compatible water-immiscible organic solvents such as toluene, to make them volatile to introduce into the gas chromatograph and thermally stable enough for separation in the GC column and introduction into the ion-source, and finally to increase their ionization by increasing their electronegativity using F-rich reagents. In this work we investigated the long-term stability of the methyl esters pentafluoropropionic (Me-PFP) derivatives of 21 urinary amino acids prepared by a two-step derivatization procedure and extraction by toluene. In situ prepared trideuteromethyl ester pentafluoropropionic derivatives were used as internal standards. GC–MS analysis (injection of 1 µL aliquots and quantification by selected-ion monitoring of specific mass fragments) was performed on days 1, 2, 8, and 15. Measured peak areas and calculated peak area ratios were used to evaluate the stability of the derivatives of endogenous amino acids and their internal standards, as well as the precision and the accuracy of the method. All analyses were performed under routine conditions. Me-PFP derivatives of endogenous amino acids and their stable-isotope labelled analogs were stable in toluene for 14 days. The peak area values of the derivatives of most amino acids and their internal standards were slightly higher on days 8 and 15 compared to days 1 and 2, yet the peak area ratio values of endogenous amino acids to their internal standards did not change. Our study indicates that Me-PFP derivatives of amino acids from human urine samples can easily be prepared, are stable at least for 14 days in the extraction solvent toluene, and allow for precise and accurate quantitative measurements by GC–MS using in situ prepared deuterium-labelled methyl ester as internal standard.https://www.mdpi.com/1420-3049/26/6/1726amino acidsderivatizationesterificationGC–MSpentafluoropropionic anhydridestability |
| spellingShingle | Svetlana Baskal Alexander Bollenbach Dimitrios Tsikas Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC–MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene Extracts Molecules amino acids derivatization esterification GC–MS pentafluoropropionic anhydride stability |
| title | Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC–MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene Extracts |
| title_full | Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC–MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene Extracts |
| title_fullStr | Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC–MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene Extracts |
| title_full_unstemmed | Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC–MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene Extracts |
| title_short | Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC–MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene Extracts |
| title_sort | two step derivatization of amino acids for stable isotope dilution gc ms analysis long term stability of methyl ester pentafluoropropionic derivatives in toluene extracts |
| topic | amino acids derivatization esterification GC–MS pentafluoropropionic anhydride stability |
| url | https://www.mdpi.com/1420-3049/26/6/1726 |
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