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GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N(5)-Carboxy-ornithine and N(6)-Carboxy-lysine

Derivatization of amino acids by 2 M HCl/CH(3)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...

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Autores principales: Baskal, Svetlana, Bollenbach, Alexander, Tsikas, Dimitrios
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071523/
https://www.ncbi.nlm.nih.gov/pubmed/33921162
http://dx.doi.org/10.3390/molecules26082301
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author Baskal, Svetlana
Bollenbach, Alexander
Tsikas, Dimitrios
author_facet Baskal, Svetlana
Bollenbach, Alexander
Tsikas, Dimitrios
author_sort Baskal, Svetlana
collection PubMed
description Derivatization of amino acids by 2 M HCl/CH(3)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)(m)-(PFP)(n). This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (d(3)Me)(m)-(PFP)(n), from synthetic amino acids and 2 M HCl/CD(3)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 N(5)-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 N(6)-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 (N,O)-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP)(4) and hCit-(PFP)(4) are esterified on their C(1)-Carboxylic groups and on their activated N(ureido) groups. Procedure B also allows in situ preparation of (d(3)Me)(m)-(PFP)(n) 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 pubmed-80715232021-04-26 GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N(5)-Carboxy-ornithine and N(6)-Carboxy-lysine Baskal, Svetlana Bollenbach, Alexander Tsikas, Dimitrios Molecules Article Derivatization of amino acids by 2 M HCl/CH(3)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)(m)-(PFP)(n). This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (d(3)Me)(m)-(PFP)(n), from synthetic amino acids and 2 M HCl/CD(3)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 N(5)-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 N(6)-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 (N,O)-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP)(4) and hCit-(PFP)(4) are esterified on their C(1)-Carboxylic groups and on their activated N(ureido) groups. Procedure B also allows in situ preparation of (d(3)Me)(m)-(PFP)(n) 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. MDPI 2021-04-15 /pmc/articles/PMC8071523/ /pubmed/33921162 http://dx.doi.org/10.3390/molecules26082301 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Baskal, Svetlana
Bollenbach, Alexander
Tsikas, Dimitrios
GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N(5)-Carboxy-ornithine and N(6)-Carboxy-lysine
title GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N(5)-Carboxy-ornithine and N(6)-Carboxy-lysine
title_full GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N(5)-Carboxy-ornithine and N(6)-Carboxy-lysine
title_fullStr GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N(5)-Carboxy-ornithine and N(6)-Carboxy-lysine
title_full_unstemmed GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N(5)-Carboxy-ornithine and N(6)-Carboxy-lysine
title_short GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N(5)-Carboxy-ornithine and N(6)-Carboxy-lysine
title_sort gc-ms discrimination of citrulline from ornithine and homocitrulline from lysine by chemical derivatization: evidence of formation of n(5)-carboxy-ornithine and n(6)-carboxy-lysine
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071523/
https://www.ncbi.nlm.nih.gov/pubmed/33921162
http://dx.doi.org/10.3390/molecules26082301
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