Core Unit Proteomics, Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany.
Molecules. 2021 Apr 15;26(8):2301. doi: 10.3390/molecules26082301.
Derivatization of amino acids by 2 M HCl/CHOH (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)-(PFP). This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (dMe)-(PFP), from synthetic amino acids and 2 M HCl/CDOD 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 -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 -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 (,)-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP) and hCit-(PFP) are esterified on their -Carboxylic groups and on their activated groups. Procedure B also allows in situ preparation of (dMe)-(PFP) 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.
将氨基酸用 2 M HCl/CHOH（60 分钟，80°C）衍生化，然后用五氟丙酸酐（PFPA）在乙酸乙酯中衍生化中间甲酯（30 分钟，65°C）是一种有用的两步衍生化程序（程序 A），用于通过气相色谱-质谱法（GC-MS）对生物样品中这些氨基酸进行定量测量，作为甲酯五氟丙酸（PFP）衍生物，（Me）-（PFP）。该程序允许从合成氨基酸和 2 M HCl/CDOD 原位制备氘代甲酯 PFP 衍生物（dMe）-（PFP），用作内标。然而，由于酰胺基团在酯化步骤的酸性条件下不稳定，程序 A 将瓜氨酸（Cit）转化为鸟氨酸（Orn），将同型瓜氨酸（hCit）转化为赖氨酸（Lys）。在本研究中，我们研究了反转两步衍生化的顺序是否可以允许对这些氨基酸进行区分和同时分析。五氟丙酰化（30 分钟，65°C）和随后的甲酯化（30 分钟，80°C），即程序 B，Cit 产生了六个开放和环状反应产物。最丰富的产物可能是β-羧基-Orn。第二个最丰富的产物被确认为 Orn。hCit 的最丰富的反应产物被确认为 Lys，次要的反应产物可能是β-羧基-Lys。通过程序 B 提出了 Cit 和 hCit 反应产物形成的机制。假设在第一步衍生化步骤中，氨基酸形成包括混合酸酐的（，）-PFP 衍生物。在第二步衍生化步骤中，Cit-（PFP）和 hCit-（PFP）在其β-羧基和活化基团上酯化。程序 B 还允许从合成氨基酸原位制备（dMe）-（PFP），用作内标。结果表明，衍生化程序 B 能够区分 Cit 和 Orn，以及 hCit 和 Lys。程序 B 用于同时测量生物样品（如血浆和尿液）中这些氨基酸的实用性仍有待证明。需要进一步的工作来优化程序 B 对生物氨基酸的衍生化条件。
