采用 LC-MS/MS 法测定果蝇体内多巴胺及其代谢产物评价 PTUPB 的抗帕金森活性。
Evaluation of antiparkinson activity of PTUPB by measuring dopamine and its metabolites in Drosophila melanogaster: LC-MS/MS method development.
机构信息
Department of Pharmacology, JSS College of Pharmacy (A Constituent College of Jagadguru Sri Shivarathreeshwara University, Mysuru), Ootacamund, Tamilnadu, India.
Department of Pharmacology, JSS College of Pharmacy (A Constituent College of Jagadguru Sri Shivarathreeshwara University, Mysuru), Ootacamund, Tamilnadu, India.
出版信息
J Pharm Biomed Anal. 2018 Feb 5;149:457-464. doi: 10.1016/j.jpba.2017.11.043. Epub 2017 Nov 16.
Soluble epoxide hydrolase (sEH) inhibition is reported to elevate endogenous epoxyeicosatrienoic acids (EET's), which are known to play an important role in neuroprotection by inhibiting oxidative stress and neuroinflammation. In the present study, PTUPB, a dual inhibitor of sEH and COX-2, has been tested for its antiparkinson activity against rotenone (ROT) induced neurodegeneration in Drosophila model of Parkinson's disease (PD). To determine the efficacy and brain bioavailability of PTUPB a simple, rapid and sensitive LC-MS/MS method was developed and validated for the estimation of PTUPB (Method-I), dopamine (DA) and its metabolites (Method-II) in fly head. Mass spectrometric acquisitions of analytes signals were performed in positive and negative electron spray ionization MRM mode by monitoring the daughter ions. The isocratic elution using formic acid (0.1% v/v) and acetonitrile (20:80v/v) (for method I), and acetic acid (0.1% v/v) and methanol (for method II) on Jones C was carried out to achieve the separation. The results of brain PTUPB, DA and its metabolites estimation shows a dose dependent increase in PTUPB concentration and a dose dependent prevention of ROT induced changes in DA and its metabolites levels (p<0.05), indicating a significant neuroprotection activity of PTUPB. In the present study, we have successfully developed and validated LC-MS/MS methods to identify and quantify PTUPB, DA and its metabolites using a UFLC-ESI-QqQ mass spectrometer for the screening of neuroprotective agents in Drosophila Melanogaster.
可溶性环氧化物水解酶（sEH）抑制被报道可提高内源性环氧二十碳三烯酸（EETs）的水平，已知其通过抑制氧化应激和神经炎症在神经保护中发挥重要作用。在本研究中，PTUPB，一种 sEH 和 COX-2 的双重抑制剂，已在帕金森病（PD）的果蝇模型中针对鱼藤酮（ROT）诱导的神经退行性变测试其抗帕金森活性。为了确定 PTUPB 的疗效和脑生物利用度，开发并验证了一种简单、快速和灵敏的 LC-MS/MS 方法，用于测定 PTUPB（方法 I）、多巴胺（DA）及其代谢物（方法 II）在果蝇头部的含量。通过监测子离子，在正、负离子喷雾电离 MRM 模式下对分析物信号进行质谱采集。使用甲酸（0.1%v/v）和乙腈（20:80v/v）（用于方法 I）和乙酸（0.1%v/v）和甲醇（用于方法 II）进行等度洗脱，以实现分离。大脑中 PTUPB、DA 及其代谢物的测定结果表明，PTUPB 浓度呈剂量依赖性增加，并且可预防 ROT 诱导的 DA 及其代谢物水平的变化（p<0.05），表明 PTUPB 具有显著的神经保护活性。在本研究中，我们成功开发并验证了 LC-MS/MS 方法，使用 UFLC-ESI-QqQ 质谱仪鉴定和定量 PTUPB、DA 及其代谢物，用于筛选黑腹果蝇中的神经保护剂。
Zotero 插件