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THE ISOLATION OF AMINO ACIDS FROM MIXTURES AS THEIR N-2,4-DINITROPHENYL DERIVATIVES, METHIONINE, LEUCINE, PHENYLALANINE, ORNITHINE, LYSINE AND TYROSINE.

ABSTRACT: 1. The separation of the dinitrophenyl derivatives of methionine, leucine (together with isoleucine), phenylalanine, ornithine, lysine and tyrosine from each other, and from the dinitrophenyl derivatives of the other protein amino acids, by partition chromatography on columns is described. 2. The harmful effect on resolving power of using too close-fitting a packing tool in preparing these partition columns is indicated and the dimensions needed to obtain good resolution are given.

SUBMITTER: MATHESON NA

PROVIDER: S-EPMC1206582 | biostudies-other | 1965 Mar

REPOSITORIES: biostudies-other

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Project description:Derivatization of amino acids by 2 M HCl/CH3OH (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, (d3Me)m-(PFP)n, from synthetic amino acids and 2 M HCl/CD3OD 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 N5-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 N6-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 C1-Carboxylic groups and on their activated Nureido groups. Procedure B also allows in situ preparation of (d3Me)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.

Dataset Information

THE ISOLATION OF AMINO ACIDS FROM MIXTURES AS THEIR N-2,4-DINITROPHENYL DERIVATIVES, METHIONINE, LEUCINE, PHENYLALANINE, ORNITHINE, LYSINE AND TYROSINE.

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