血清与海马内 5-羟色胺和 5-羟基吲哚乙酸的 LC-MS/MS测定及其在白芍抗抑郁功效评价中的应用

[目的]建立一种用于测定血清和海马组织中 5-羟色胺(5-hydroxytryptamine，5-HT)和 5-羟基吲哚乙酸(5-hydroxyindole acetic acid，5-HIAA)含量的液相色谱与串联质谱联用(liquid chromatography-tandem mass spectrometry，LC-MS/MS)分析方法，并应用于中药抗抑郁功效的评价。[方法]取小鼠血清和海马组织，通过蛋白沉淀法进行样品前处理后，利用所建立的 LC-MS/MS分析方法测定 5-HT和 5-HIAA的含量。采用 Hypersil ODS(250 mm×4.6 mm，5 μm)色谱柱，柱温为 40 ℃，流动相为 0.1%甲酸水(含 2 mmol·L-1乙酸铵):乙腈，流速为 1 mL·min-1质谱检测选择多重反应监测(multiple reaction monitoring，MRM)模式。将 198只小鼠随机分为正常对照组、模型组、盐酸氟西汀组和，30个白芍提取物组，每组 6只。测定血清和海马中 5-HT和 5-HIAA含量，并进行悬尾实验与强迫游泳实验。[结果] 5-HT和 5-HIAA分别在 0.1～1 000 ng·mL-1和 1～500 ng·mL-1浓度范围内线性关系良好(r> 0.999)加样回收率和精密度均符合生物样品的分析要求。白芍提取物组 5-HIAA/5-HT值相对于模型组的变化程度，与行学实验指标变，化程度具有一定相关性，且变化程度显著高于行为学实验，组内相对标准偏差(relative standard deviation，RSD)更小。[结论]建立的 LC-MS/MS分析方法操作简便快捷、结果准确、灵敏度高、重现性好、专属性强，可同时测定血清、海马组织中 5-HT和 5-HIAA的含量，适用于白芍等具有神经保护功效药物的研究及药效评价。

C-MS/MS Determination of 5-Hydroxytryptamine and 5-Hydroxyindole Acetic Acid in Serum and Hippocampus and Its Ap. plication in the Evaluation of Antidepressant Efficacy of Paeoniae Radix Alba

[Objective] To establish a liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for the determination of5-hydroxytryptamine(5-HT) and 5-hydroxyindole acetic acid(5-HIAA) in mouse serum and hippocampus，applied to the evaluation of the antidepressant efficacy of traditional Chinese medicine. [Methods] The serum and hippocampus were taken and the concentrations of 5-HTand 5-HIAA were determined by LC-MS/MS after pretreatment of protein precipitation. Chromatographic separation was performed onHypersil ODS (250 mm×4.6 mm，5 μ m) at column temperature of 40 ℃. The mobile phase was 0.1% formic acid-water solution with 2 mmol·L-1 ammonium acetate and acetonitrile whereas flow rate was set at 1 mL·min-1. The mass spectrometry detection was performedusing multiple reaction monitoring(MRM). One hundred and ninety-eight mice were randomly divided into normal control group，model group，fluoxetine hydrochloride group and 30 groups of Paeoniae Radix Alba with 6 rats in each group. Tail suspension test and forced swimming test were carried out. [Results] 5-HT and 5-HIAA had good linear relationship within the concentration range of 0.1～ 1 000 ng · mL-1 and 1～500 ng·mL-1，respectively (r>0.999). Precision and recovery of this method were in line with the requirements ofbiological sample analysis. The degree of change in 5-HIAA/5-HT values in the Paeoniae Radix Alba group relative to the model groupwas correlated with the degree of change in indicators of the behavioral experiment，and the degree of change was significantly higher than that of the behavioral experiment，with a much smaller within-group relative standard deviation(RSD). [Conclusion] This LC-MS/MS method is simple and fast for operation，with accurate results，high sensitivity，good reproducibility and strong specificity which indicatedthat was suitable for the simultaneous determination of 5-HT and 5-HIAA in serum and hippocampus. It could be used for research andefficacy evaluation of neuroprotective drugs like Paeoniae Radix Alba.