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Abstract：
The aim of the presented work was to develop a highly sensitive, accurate and rapid analytical method for the determination of concentration levels of tryptophan and its metabolites of kynurenine catabolic pathway, as well as neurotransmitters and their metabolites in complex biological matrices (brain tissue and blood plasma). The developed analytical method consists of analytes separation from the biological matrices by protein precipitation (blood plasma) or solvent extraction (brain tissue), derivatization of the analytes and their detection by high-performance liquid chromatography combined with mass spectrometry. Individual steps of the whole process were optimized and the method was validated in the terms of selectivity, linearity (R2≥0.980), precision (RSD ≤ 13.3%), recovery (≥82.0%), limit of detection (1.8 ng/mL of blood plasma, 2.2 pg/mg of brain tissue) and limit of quantification (2.5 ng/mL of blood plasma, 2.8 pg/mg of brain tissue). The method was subsequently verified by an animal study, where the concentration levels of the analytes in biological matrices (blood plasma and brain tissue) of T. gondii - infected rats and control animals were compared. All the data obtained from the animal study were statistically evaluated. Increased concentration levels of kynurenine catabolic pathway metabolites (e.g. kynurenine, 3-hydroxykynurenine, quinolinic acid) were observed in the case of T. gondii - infected rats in contrast to the control group. The opposite effect was determined in the case of serotonin and its metabolite 5-hydroxyindoleacetic acid, where higher concentration levels were found in blood plasma of healthy subjects. Finally, Principal Component Analysis (PCA) was utilized for a score plot formation. PCA score plots have demonstrated the similarities of individuals within each group and the differences among the groups.
摘要：
提出的工作的目的是开发一种高度敏感的，犬尿氨酸分解代谢途径中色氨酸及其代谢产物浓度水平的准确快速测定分析方法,以及复杂的生物基质（脑组织和血浆）中的神经递质及其代谢产物。所开发的分析方法包括通过蛋白质沉淀（血浆）或溶剂萃取（脑组织）从生物基质中分离分析物，分析物的衍生化及其通过高效液相色谱与质谱联用的检测。对整个过程的各个步骤进行了优化，并在选择性方面对该方法进行了验证。线性度（R2≥0.980），精度(RSD≤13.3%),回收率（≥82.0%），检测限(1.8ng/mL血浆，2.2pg/mg的脑组织）和定量限（2.5ng/mL的血浆，2.8pg/mg的脑组织）。该方法随后通过动物研究进行了验证，其中比较了弓形虫感染的大鼠和对照动物的生物基质（血浆和脑组织）中分析物的浓度水平。对从动物研究中获得的所有数据进行统计学评估。犬尿氨酸分解代谢途径代谢物的浓度水平增加（例如犬尿氨酸，3-羟基犬尿氨酸，与对照组相比，在弓形虫感染的大鼠中观察到喹啉酸）。在5-羟色胺及其代谢物5-羟基吲哚乙酸的情况下，在健康受试者的血浆中发现较高的浓度水平。最后,主成分分析(PCA)用于得分图形成。PCA得分图证明了每个组内个体的相似性以及组间的差异。
