Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, 9700 RB, The Netherlands.
Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, 9700 RB, The Netherlands.
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 May 1;1238:124098. doi: 10.1016/j.jchromb.2024.124098. Epub 2024 Mar 18.
Trace amines are powerful neuromodulators influencing the release and reuptake of catecholamines. These low concentrated endogenous amines impact mood, cognition, and hormone regulation. Dysregulation of trace amines have been associated with a variety of diseases, such as schizophrenia, Parkinson's disease, migraine, depression and more. Succesfull simultaneous quantification of trace amines, their precursors and metabolites would benefit both research and patient care. Since these compounds have various functional groups and are present in biological matrices with large concentration difference, their simultaneous quantification is an analytical challenge. Our goal was to develop a highly sensitive LC-MS/MS assay to simultaneously quantify trace amines, their precursors and metabolites in plasma.
Our method is based on a simple two-step in-matrix derivatization protocol: propionic anhydride (PA) and 3-Ethyl-1-[3-(dimethylamino)propyl]carbodiimide (EDC) in combination with 2,2,2-trifluoroethylamine (TFEA) followed by online solid phase extraction combined with LC-MS/MS. Fifteen metabolites can be measured simultaneously, three precursors, eight trace amines and four metabolites. Validation of this method was performed according to international validation guidelines. The pre-analytical stability of trace amines was assessed.
This novel method was successful in quantifying trace amines, their precursors, and metabolites in plasma. Using just 50 µl human plasma, we were able to accomplish limit of quantification for 2-phenylethylamine and N-methyl-phenylethylamine of 0.2 nmol/L and 0.1 nmol/L for tyramine and n-methyltyramine. Inter-and intra-assay imprecision was < 15 % for all analytes. Stability assessment showed susceptibility of certain trace amines e.g. 2-phenylethylamine and N-methyl-phenylethylamine to enzymatic degradation in plasma. The addition of the monoamine oxidase inhibitor pargyline to plasma prevented this enzymatic degradation.
We developed a novel LC-MS/MS method that1) uses a new double derivatization technique, 2) is automated with online SPE, 3) uses far less sample volume then previous methods and 4) detects more components in the same sample (eight trace amines, three precursors, and four metabolites) with high specificity and selectivity. Furthermore, addition of MAO A/B inhibitor prevents degradation and guarantees more accurate quantification of trace amines.
痕量胺是影响儿茶酚胺释放和再摄取的强大神经调节剂。这些低浓度的内源性胺类物质会影响情绪、认知和激素调节。痕量胺的失调与多种疾病有关，如精神分裂症、帕金森病、偏头痛、抑郁症等。成功地同时定量痕量胺、其前体和代谢物将有益于研究和患者护理。由于这些化合物具有各种官能团，并且存在于具有大浓度差异的生物基质中，因此它们的同时定量是一个分析挑战。我们的目标是开发一种高灵敏度的 LC-MS/MS 测定法，以同时定量血浆中的痕量胺、其前体和代谢物。
我们的方法基于一种简单的两步基质衍生化方案：丙酸酐 (PA) 和 3-乙基-1-[3-(二甲氨基)丙基]碳二亚胺 (EDC) 与 2,2,2-三氟乙胺 (TFEA) 结合，然后进行在线固相萃取与 LC-MS/MS 联用。可以同时测量 15 种代谢物，3 种前体，8 种痕量胺和 4 种代谢物。根据国际验证指南对该方法进行了验证。评估了痕量胺的预分析稳定性。
该新方法成功地定量了血浆中的痕量胺、其前体和代谢物。使用仅 50µl 人血浆，我们能够实现 2-苯乙胺和 N-甲基苯乙胺的定量限为 0.2nmol/L 和 0.1nmol/L，而对酪胺和 N-甲基酪胺的定量限为 0.1nmol/L。所有分析物的批内和批间精密度均<15%。稳定性评估表明，某些痕量胺（如 2-苯乙胺和 N-甲基苯乙胺）易在血浆中发生酶降解。向血浆中添加单胺氧化酶抑制剂帕吉林可防止这种酶降解。
我们开发了一种新的 LC-MS/MS 方法，该方法 1）使用新的双衍生化技术，2）具有在线 SPE 自动化功能，3）比以前的方法使用更少的样本量，4）在同一样品中检测到更多的成分（八种痕量胺、三种前体和四种代谢物），具有高特异性和选择性。此外，添加 MAO A/B 抑制剂可防止降解并保证痕量胺更准确的定量。
