GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of <i>N</i><sup>5</sup>-Carboxy-ornithine and <i>N</i><sup>6</sup>-Carboxy-lysine
Derivatization of amino acids by 2 M HCl/CH<sub>3</sub>OH (60 min, 80 °C) followed by derivatization of the intermediate methyl esters with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) is a useful two-step derivatization procedure (procedure A) for their quantit...
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MDPI AG
2021-04-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/26/8/2301 |
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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 | Derivatization of amino acids by 2 M HCl/CH<sub>3</sub>OH (60 min, 80 °C) followed by derivatization of the intermediate methyl esters with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) is a useful two-step derivatization procedure (procedure A) for their quantitative measurement in biological samples by gas chromatography-mass spectrometry (GC-MS) as methyl ester pentafluoropropionic (PFP) derivatives, (Me)<sub>m</sub>-(PFP)<sub>n</sub>. This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (d<sub>3</sub>Me)<sub>m</sub>-(PFP)<sub>n</sub>, from synthetic amino acids and 2 M HCl/CD<sub>3</sub>OD for use as internal standards. However, procedure A converts citrulline (Cit) to ornithine (Orn) and homocitrulline (hCit) to lysine (Lys) due to the instability of their carbamide groups under the acidic conditions of the esterification step. In the present study, we investigated whether reversing the order of the two-step derivatization may allow discrimination and simultaneous analysis of these amino acids. Pentafluoropropionylation (30 min, 65 °C) and subsequent methyl esterification (30 min, 80 °C), i.e., procedure B, of Cit resulted in the formation of six open and cyclic reaction products. The most abundant product is likely to be <i>N</i><sup>5</sup>-Carboxy-Orn. The second most abundant product was confirmed to be Orn. The most abundant reaction product of hCit was confirmed to be Lys, with the minor reaction product likely being <i>N</i><sup>6</sup>-Carboxy-Lys. Mechanisms are proposed for the formation of the reaction products of Cit and hCit via procedure B. It is assumed that at the first derivatization step, amino acids form (<i>N</i>,<i>O</i>)-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP)<sub>4</sub> and hCit-(PFP)<sub>4</sub> are esterified on their <i>C</i><sup>1</sup>-Carboxylic groups and on their activated <i>N</i><sup>ureido</sup> groups. Procedure B also allows in situ preparation of (d<sub>3</sub>Me)<sub>m</sub>-(PFP)<sub>n</sub> from synthetic amino acids for use as internal standards. It is demonstrated that the derivatization procedure B enables discrimination between Cit and Orn, and between hCit and Lys. The utility of procedure B to measure simultaneously these amino acids in biological samples such as plasma and urine remains to be demonstrated. Further work is required to optimize the derivatization conditions of procedure B for biological amino acids. |
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| spelling | doaj.art-b920d1ddac694ef6b8caec4cadaf0a9e2023-11-21T15:46:30ZengMDPI AGMolecules1420-30492021-04-01268230110.3390/molecules26082301GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of <i>N</i><sup>5</sup>-Carboxy-ornithine and <i>N</i><sup>6</sup>-Carboxy-lysineSvetlana Baskal0Alexander Bollenbach1Dimitrios Tsikas2Core Unit Proteomics, Institute of Toxicology, Hannover Medical School, 30625 Hannover, GermanyCore Unit Proteomics, Institute of Toxicology, Hannover Medical School, 30625 Hannover, GermanyCore Unit Proteomics, Institute of Toxicology, Hannover Medical School, 30625 Hannover, GermanyDerivatization of amino acids by 2 M HCl/CH<sub>3</sub>OH (60 min, 80 °C) followed by derivatization of the intermediate methyl esters with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) is a useful two-step derivatization procedure (procedure A) for their quantitative measurement in biological samples by gas chromatography-mass spectrometry (GC-MS) as methyl ester pentafluoropropionic (PFP) derivatives, (Me)<sub>m</sub>-(PFP)<sub>n</sub>. This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (d<sub>3</sub>Me)<sub>m</sub>-(PFP)<sub>n</sub>, from synthetic amino acids and 2 M HCl/CD<sub>3</sub>OD for use as internal standards. However, procedure A converts citrulline (Cit) to ornithine (Orn) and homocitrulline (hCit) to lysine (Lys) due to the instability of their carbamide groups under the acidic conditions of the esterification step. In the present study, we investigated whether reversing the order of the two-step derivatization may allow discrimination and simultaneous analysis of these amino acids. Pentafluoropropionylation (30 min, 65 °C) and subsequent methyl esterification (30 min, 80 °C), i.e., procedure B, of Cit resulted in the formation of six open and cyclic reaction products. The most abundant product is likely to be <i>N</i><sup>5</sup>-Carboxy-Orn. The second most abundant product was confirmed to be Orn. The most abundant reaction product of hCit was confirmed to be Lys, with the minor reaction product likely being <i>N</i><sup>6</sup>-Carboxy-Lys. Mechanisms are proposed for the formation of the reaction products of Cit and hCit via procedure B. It is assumed that at the first derivatization step, amino acids form (<i>N</i>,<i>O</i>)-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP)<sub>4</sub> and hCit-(PFP)<sub>4</sub> are esterified on their <i>C</i><sup>1</sup>-Carboxylic groups and on their activated <i>N</i><sup>ureido</sup> groups. Procedure B also allows in situ preparation of (d<sub>3</sub>Me)<sub>m</sub>-(PFP)<sub>n</sub> from synthetic amino acids for use as internal standards. It is demonstrated that the derivatization procedure B enables discrimination between Cit and Orn, and between hCit and Lys. The utility of procedure B to measure simultaneously these amino acids in biological samples such as plasma and urine remains to be demonstrated. Further work is required to optimize the derivatization conditions of procedure B for biological amino acids.https://www.mdpi.com/1420-3049/26/8/2301amino acidsderivatizationesterificationGC-MSpentafluoropropionic anhydrideureide |
| spellingShingle | Svetlana Baskal Alexander Bollenbach Dimitrios Tsikas GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of <i>N</i><sup>5</sup>-Carboxy-ornithine and <i>N</i><sup>6</sup>-Carboxy-lysine Molecules amino acids derivatization esterification GC-MS pentafluoropropionic anhydride ureide |
| title | GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of <i>N</i><sup>5</sup>-Carboxy-ornithine and <i>N</i><sup>6</sup>-Carboxy-lysine |
| title_full | GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of <i>N</i><sup>5</sup>-Carboxy-ornithine and <i>N</i><sup>6</sup>-Carboxy-lysine |
| title_fullStr | GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of <i>N</i><sup>5</sup>-Carboxy-ornithine and <i>N</i><sup>6</sup>-Carboxy-lysine |
| title_full_unstemmed | GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of <i>N</i><sup>5</sup>-Carboxy-ornithine and <i>N</i><sup>6</sup>-Carboxy-lysine |
| title_short | GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of <i>N</i><sup>5</sup>-Carboxy-ornithine and <i>N</i><sup>6</sup>-Carboxy-lysine |
| title_sort | gc ms discrimination of citrulline from ornithine and homocitrulline from lysine by chemical derivatization evidence of formation of i n i sup 5 sup carboxy ornithine and i n i sup 6 sup carboxy lysine |
| topic | amino acids derivatization esterification GC-MS pentafluoropropionic anhydride ureide |
| url | https://www.mdpi.com/1420-3049/26/8/2301 |
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