Miękus Natalia, Konieczna Lucyna, Kowiański Przemysław, Moryś Janusz, Bączek Tomasz
Department of Animal and Human Physiology, University of Gdańsk, Wita Stwosza 59, 80-308Gdańsk, Poland.
Department of Pharmaceutical Chemistry, Medical University of Gdańsk, Gen. J. Hallera 107, 80-416Gdańsk, Poland.
Transl Neurosci. 2017 Aug 12;8:70-75. doi: 10.1515/tnsci-2017-0012. eCollection 2017.
Clinicians often rely on selected small molecular compounds from body fluids for the detection, screening or monitoring of numerous life-threatening diseases. Among others, important monoamines - biogenic amines (BAs) - and their metabolites serve as sensitive biomarkers to study the progression or even early detection of on-going brain pathologies or tumors of neuroendocrine origins. Undertaking the task to optimize a reliable method for the simultaneous analysis of the most relevant BAs in biological matrices is of utmost importance for scientists. Hydrophilic interaction liquid chromatography (HILIC) with mass spectrometry (MS) detection provides a specific and sensitive technique for the separation and assessment of several neurotransmitter concentrations in body fluids (blood, urine, tissues). The present study was focused on the optimization of a straightforward, sensitive and reliable method for the simultaneous analysis of the ten most important BAs and their acidic metabolites from homogenates of rat brain tissues by use of HILIC-MS. Here, we present the optimized experimental workflow in terms of sample preparation, buffer compositions, HILIC and MS settings and data analysis. The presented method is reliable, straightforward and sensitive. Our method permits the unbiased, qualitative and quantitative determination of several BAs and their metabolites simultaneously. The optimized method was applied to the analysis of rat brain tissue samples from healthy hemispheres or those with induced transient ischemic attack (TIA). The undertaken pilot study demonstrated that the proposed approach could be applied to reveal the perturbation in neurotransmitters concentration after TIA in rat brains.
临床医生常常依赖于从体液中选取的小分子化合物来检测、筛查或监测众多危及生命的疾病。其中，重要的单胺类物质——生物胺（BAs）及其代谢产物，可作为敏感的生物标志物，用于研究正在发生的脑部病变或神经内分泌源性肿瘤的进展情况，甚至早期检测。对于科学家而言，承担优化一种可靠方法以同时分析生物基质中最相关生物胺的任务至关重要。亲水作用液相色谱（HILIC）与质谱（MS）检测相结合，为分离和评估体液（血液、尿液、组织）中几种神经递质的浓度提供了一种特异且灵敏的技术。本研究聚焦于优化一种直接、灵敏且可靠的方法，通过HILIC-MS同时分析大鼠脑组织匀浆中十种最重要的生物胺及其酸性代谢产物。在此，我们从样品制备、缓冲液组成、HILIC和MS设置以及数据分析等方面展示优化后的实验流程。所呈现的方法可靠、直接且灵敏。我们的方法能够同时对几种生物胺及其代谢产物进行无偏倚的定性和定量测定。优化后的方法应用于分析来自健康半球或诱导短暂性脑缺血发作（TIA）的大鼠脑组织样本。所进行的初步研究表明，所提出的方法可用于揭示大鼠脑内TIA后神经递质浓度的扰动情况。
