Department of Organic Technology, University of Chemistry and Technology Prague, Technická 5, 166 28, Prague 6, Czech Republic.
Department of Organic Technology, University of Chemistry and Technology Prague, Technická 5, 166 28, Prague 6, Czech Republic.
J Pharm Biomed Anal. 2019 Jun 5;170:139-152. doi: 10.1016/j.jpba.2019.03.039. Epub 2019 Mar 18.
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 (R≥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.
本工作旨在开发一种高灵敏度、准确和快速的分析方法，用于测定色氨酸及其代谢物犬尿氨酸分解代谢途径以及神经递质及其代谢物在复杂生物基质（脑组织和血浆）中的浓度水平。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，对分析物进行衍生化，然后通过高效液相色谱-质谱联用进行检测。对整个过程的各个步骤进行了优化，并在选择性、线性（R≥0.980）、精密度（RSD≤13.3%）、回收率（≥82.0%）、检测限（血浆 1.8ng/mL，脑组织 2.2pg/mg）和定量限（血浆 2.5ng/mL，脑组织 2.8pg/mg）方面对方法进行了验证。随后通过动物研究对方法进行了验证，比较了感染弓形虫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。对动物研究获得的所有数据进行了统计评估。与对照组相比，感染弓形虫的大鼠的犬尿氨酸分解代谢途径代谢物（如犬尿氨酸、3-羟基犬尿氨酸、喹啉酸）浓度升高。而在健康受试者的血浆中发现了更高浓度的血清素及其代谢物 5-羟色氨酸乙酸盐，其浓度水平相反。最后，利用主成分分析（PCA）进行得分图的形成。PCA 得分图显示了每个组内个体的相似性以及组间的差异。
