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  

LC-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

作　　者：苏媛 蔡思雨 时梦甜 王丹丹 于雨乐 严斌俊 SU Yuan;CAI Siyu;SHI Mengtian(School of Pharmaceutical Science,Zhejiang Chinese Medical University,Hangzhou(310053),China;Key Laboratory of Sys-tems Health Science of Zhejiang Province,School of Life Sciences,Hangzhou Institute for Advanced Study,University of Chinese Academy of Sciences)

机构地区：[1]浙江中医药大学药学院,杭州310053 [2]中国科学院大学杭州高等研究院,生命与健康科学学院,浙江省系统健康科学重点实验室

出　　处：《浙江中医药大学学报》2024年第7期862-871,共10页Journal of Zhejiang Chinese Medical University

基　　金：国家自然科学基金面上项目(82073956);浙江省基础公益研究计划项目(LGF19H280005)。

摘　　要：[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,5μm)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0.1~1000 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,与行为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。[Objective Objective]To establish a liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the determination of 5-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 Methods]The serum and hippocampus were taken and the concentrations of 5-HT and 5-HIAA were determined by LC-MS/MS after pretreatment of protein precipitation.Chromatographic separation was performed on Hypersil ODS(250 mm×4.6 mm,5μm)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 performed using 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 Results]5-HT and 5-HIAA had good linear relationship within the concentration range of 0.1~1000 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 of biological sample analysis.The degree of change in 5-HIAA/5-HT values in the Paeoniae Radix Alba group relative to the model group was 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 Conclusion]This LC-MS/MS method is simple and fast for operation,with accurate results,high sensitivity,good reproducibility and strong specificity which indicated that was suitable for the simultaneous determination of 5-HT and 5-HIAA in serum and hippocampus.It could be used for research and efficacy evaluation of neuroprotective drugs like Paeoniae Radix

关 键 词：液相色谱-串联质谱联用 5-羟色胺 5-羟基吲哚乙酸 小鼠血清 海马组织 白芍 抑郁症 药效评价 

分 类 号：R331[医药卫生—人体生理学]