Ziegler Jörg, Hussain Hina, Neubert Reinard H H, Abel Steffen
Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle, Germany.
Department of Biopharmacy and Pharmaceutical Technology, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Methods Mol Biol. 2019;2030:365-379. doi: 10.1007/978-1-4939-9639-1_27.
A method for selective and sensitive quantification of amino acids is described. The combination of established derivatization procedures of secondary and primary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) and subsequent detection of derivatized amino acids by LC-ESI-MS/MS using multiple reaction monitoring provides high selectivity. The attachment of an apolar moiety enables purification of derivatized amino acids from matrix by a single solid-phase extraction step, which increases sensitivity by reduced ion suppression during LC-ESI-MS/MS detection. Additionally, chromatography of all amino acids can be performed on reversed-phase HPLC columns using eluents without additives, which are known to cause significant decreases in signal to noise ratios. The method has been routinely applied for amino acid profiling of low amounts of liquids and tissues of various origins with a sample throughput of about 50-100 samples a day. In addition to a detailed description of the method, some representative examples are presented.
本文描述了一种用于氨基酸选择性和灵敏定量分析的方法。仲氨基和伯氨基与9-芴甲氧羰基氯（Fmoc-Cl）的既定衍生化程序相结合，随后使用多反应监测通过LC-ESI-MS/MS检测衍生化氨基酸，可提供高选择性。非极性部分的连接使得能够通过单一固相萃取步骤从基质中纯化衍生化氨基酸，这通过减少LC-ESI-MS/MS检测期间的离子抑制来提高灵敏度。此外，所有氨基酸的色谱分析均可在反相HPLC柱上进行，使用不含添加剂的洗脱液，已知添加剂会导致信噪比显著降低。该方法已常规应用于各种来源的少量液体和组织的氨基酸谱分析，每天的样品通量约为50-100个样品。除了对该方法的详细描述外，还给出了一些代表性示例。
