Chen Jiahui, Hou Waner, Han Bo, Liu Guanghui, Gong Jin, Li Yemeng, Zhong Danmin, Liao Qiongfeng, Xie Zhiyong
School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, 510407, China.
School of Pharmacy, Shihezi University, Shihezi, 832000, China.
Anal Bioanal Chem. 2016 Apr;408(10):2527-42. doi: 10.1007/s00216-016-9352-z. Epub 2016 Feb 12.
Amino acids, neurotransmitters, purines, and pyrimidines are bioactive molecules that play fundamental roles in maintaining various physiological functions. Their metabolism is closely related to the health, growth, development, reproduction, and homeostasis of organisms. Most recently, comprehensive measurements of these metabolites have shown their potential as innovative approaches in disease surveillance or drug intervention. However, simultaneous measurement of these metabolites presents great difficulties. Here, we report a novel quantitative method that uses hydrophilic interaction ultra-high-performance liquid chromatography-tandem mass spectrometry (HILIC-UPLC-MS/MS), which is highly selective, high throughput, and exhibits better chromatographic behavior than existing methods. The developed method enabled the rapid quantification of 37 metabolites, spanning amino acids, neurotransmitters, purines, and pyrimidines pathways, within 6.5 min. The compounds were separated on an ACQUITY UPLC® BEH Amide column. Serum and brain homogenate were extracted by protein precipitation. The intra- and interday precision of all of the analytes was less than 11.34 %, and the accuracy was between -11.74 and 11.51 % for all quality control (QC) levels. The extraction recoveries of serum ranged from 84.58 % to 116.43 % and those of brain samples from 80.80 % to 119.39 %, while the RSD was 14.61 % or less for all recoveries. This method was used to successfully characterize alterations in the rat brain and, in particular, their dynamics in serum. The following study was performed to simultaneously test global changes of these metabolites in a serotonin antagonist p-chlorophenylalanine (PCPA)-induced anxiety and insomnia rat model to understand the effect and mechanism of PCPA. Taken together, these results show that the method is able to simultaneously monitor a large panel of metabolites and that this protocol may represent a metabolomic method to diagnose toxicological and pathophysiological states.
氨基酸、神经递质、嘌呤和嘧啶是生物活性分子，在维持各种生理功能中发挥着重要作用。它们的代谢与生物体的健康、生长、发育、繁殖和体内平衡密切相关。最近，对这些代谢物的综合测量显示了它们作为疾病监测或药物干预创新方法的潜力。然而，同时测量这些代谢物存在很大困难。在此，我们报告一种新颖的定量方法，该方法使用亲水相互作用超高效液相色谱 - 串联质谱法（HILIC-UPLC-MS/MS），具有高选择性、高通量，且色谱行为比现有方法更好。所开发的方法能够在6.5分钟内快速定量37种代谢物，涵盖氨基酸、神经递质、嘌呤和嘧啶途径。这些化合物在ACQUITY UPLC® BEH酰胺柱上分离。血清和脑匀浆通过蛋白沉淀法提取。所有分析物的日内和日间精密度均小于11.34%，所有质量控制（QC）水平的准确度在-11.74%至11.51%之间。血清的提取回收率在84.58%至116.43%之间，脑样本的提取回收率在80.80%至119.39%之间，所有回收率的相对标准偏差（RSD）为14.61%或更低。该方法成功用于表征大鼠脑中的变化，特别是血清中的动态变化。接下来进行的研究是在5-羟色胺拮抗剂对氯苯丙氨酸（PCPA）诱导的焦虑和失眠大鼠模型中同时测试这些代谢物的整体变化，以了解PCPA的作用和机制。综上所述，这些结果表明该方法能够同时监测大量代谢物，并且该方案可能代表一种用于诊断毒理学和病理生理状态的代谢组学方法。
