Department of Pharmacology, University of Oxford, Oxford, United Kingdom.
Department of Pharmacology, University of Oxford, Oxford, United Kingdom; Institute of Basic Medical Sciences, Department of Nutrition, University of Oslo, Norway.
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Sep 1;1229:123893. doi: 10.1016/j.jchromb.2023.123893. Epub 2023 Oct 1.
Accurate quantification of amino acids (AA) is essential for several applications, including clinical research, food analysis, and pharmaceutical studies. In this study, we developed an analytical method based on liquid chromatography with electrospray ionization coupled to tandem mass spectrometry detection (LC-ESI-MS/MS). This method was devised to accurately quantify a spectrum of amino acids, notably taurine, creatinine, glutathione (GSH), and sulfur-containing amino acids (SAAs) such as methionine, cysteine, and homocysteine, using only 10 μL of human plasma. A stable isotope derivative of each AA is used as an internal standard (IS) for accurate quantification. For retention and separation on a C18 column, heptafluorobutyric acid (HFBA) was employed as an ion pair agent. Multiple reaction monitoring (MRM) in positive mode with the precursor-to-product ion transitions at m/z is used for quantification. The method showed excellent linearity for all AA with a high correlation coefficient (r > 0.9927). The linear fit indicates that the detector response is linear over the tested range of standard concentrations. The accuracy and precision of the method were within the acceptable range of 92-110% and < 15%, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were in the range of 0.001-1.80 µM and 0.004-6.0 µM, respectively. No significant ion suppression or carry over was observed. In conclusion, the assay was validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The assay has been successfully applied to the analysis of human plasma.
准确测定氨基酸（AA）对于多种应用至关重要，包括临床研究、食品分析和药物研究。在这项研究中，我们开发了一种基于液相色谱-电喷雾电离串联质谱检测（LC-ESI-MS/MS）的分析方法。该方法旨在准确测定多种氨基酸，包括牛磺酸、肌酸、谷胱甘肽（GSH）和含硫氨基酸（SAAs），如蛋氨酸、半胱氨酸和同型半胱氨酸，仅需 10μL 人血浆。每种 AA 的稳定同位素衍生物用作准确定量的内标（IS）。采用庚氟丁酸（HFBA）作为离子对试剂，在 C18 柱上进行保留和分离。采用正模式下的多重反应监测（MRM），以 m/z 的前体到产物离子转换进行定量。该方法对所有 AA 均表现出优异的线性关系，相关系数（r>0.9927）高。线性拟合表明，检测器的响应在测试的标准浓度范围内呈线性。该方法的准确度和精密度均在可接受范围内，分别为 92-110%和<15%。检测限（LOD）和定量限（LOQ）分别为 0.001-1.80μM 和 0.004-6.0μM。未观察到明显的离子抑制或交叉污染。总之，该测定方法经过验证，具有足够的准确性、精密度、线性、灵敏度和选择性。该方法已成功应用于人血浆分析。
