Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany; Invite GmbH, Formulation Technology, 42096 Wuppertal, Germany; Bayer AG, Research & Development, Pharmaceuticals, Analytical Development API, 42096 Wuppertal, Germany.
J Chromatogr A. 2023 Oct 11;1708:464349. doi: 10.1016/j.chroma.2023.464349. Epub 2023 Sep 3.
Enantioselective amino acid analysis is gaining increasing importance in pharmaceutical, biomedical and food sciences. While there are many methods available for enantiomer separation of amino acids, the simultaneous analysis of all chiral proteinogenic amino acids by a single method with one column and a single condition is still challenging. Herein, we report an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS) assay using Chiralpak QN-AX as chiral column. With 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC) as derivatization reagent, efficient enantioselective separation of D- and L-amino acids using HPLC has become possible. Thiol-containing amino acids like Cys are alkylated prior to AQC-labelling. A protocol for automated sample preparation including both derivatization step and calibrator preparation is presented. For compensating matrix effects, u-CN-labelled internal standards (IS) were employed. The method was validated and applied to the enantioselective analysis of amino acids in a bacterial fermentation broth.
对映体氨基酸分析在药物、生物医学和食品科学领域的重要性日益增加。虽然有许多方法可用于氨基酸的对映体分离，但用单一方法、单一柱和单一条件同时分析所有手性蛋白氨基酸仍然具有挑战性。本文报道了一种使用 Chiralpak QN-AX 作为手性柱的对映选择性高效液相色谱-串联质谱（LC-MS）分析方法。采用 6-氨基喹啉基-N-羟基琥珀酰亚胺基碳酸酯（AQC）作为衍生化试剂，使 HPLC 能够高效地对 D-和 L-氨基酸进行对映选择性分离。半胱氨酸等含巯基的氨基酸在 AQC 标记前先进行烷基化。本文提出了一种自动化样品制备的方案，包括衍生化步骤和标准品制备。为了补偿基质效应，使用 u-CN 标记的内标（IS）。该方法经过验证后，应用于细菌发酵液中氨基酸的对映体分析。
