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  • 陈磊,杜毅,李盛建,周瑾,蔡溱.超高效液相色谱-串联质谱法同时测定抑郁大鼠脑脊液中5-羟色胺和5-羟吲哚乙酸[J].第二军医大学学报,2018,39(12):1354-1359    [点击复制]
  • CHEN Lei,DU Yi,LI Cheng-jian,ZHOU Jin,CAI Zhen.Simultaneous determination of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in cerebrospinal fluid of depressive rats by ultra high performance liquid chromatography-tandem quadrupole mass spectrometry[J].Acad J Sec Mil Med Univ,2018,39(12):1354-1359   [点击复制]
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超高效液相色谱-串联质谱法同时测定抑郁大鼠脑脊液中5-羟色胺和5-羟吲哚乙酸
陈磊1,杜毅2,李盛建3,周瑾3,蔡溱1*
0
(1. 海军军医大学(第二军医大学)长海医院药材科, 上海 200433;
2. 上海交通大学医学院附属新华医院药学部, 上海 200092;
3. 海军军医大学(第二军医大学)东方肝胆外科医院药材科, 上海 200438
*通信作者)
摘要:
目的 建立超高效液相色谱-串联质谱(UPLC-MS/MS)法,同时检测抑郁模型大鼠脑脊液中5-羟色胺及其代谢产物5-羟吲哚乙酸(5-HIAA)的水平,并观察氟西汀治疗后抑郁模型大鼠脑脊液中5-羟色胺和5-HIAA的含量变化。方法 将24只SD大鼠随机分为对照组、模型组和氟西汀治疗组,每组8只。造模第35天,待行为学测试后取大鼠脑脊液,用蛋白沉淀法进行前处理,然后采用UPLC-MS/MS测定5-羟色胺和5-HIAA的含量。色谱柱为ACE C18-PFP柱(4.6 mm×150 mm,3 μm),流动相为乙腈-0.2%甲酸水,梯度洗脱,流速为0.4 mL/min,柱温为25℃,质谱检测采用动态多反应监测(DMRM)模式。结果 大鼠脑脊液中5-羟色胺和5-HIAA分别在0.10~100.00 ng/mL和1.00~1 000.00 ng/mL浓度范围内线性关系良好(r>0.999),日内和日间精密度均<15.00%,提取回收率为86.86%~98.49%,基质效应为89.80%~95.25%,样品稳定性良好。与对照组比较,模型组大鼠脑脊液中5-羟色胺的含量降低(P<0.01),5-HIAA含量上升(P<0.01);与模型组比较,氟西汀组大鼠脑脊液中5-羟色胺含量上升(P<0.01),5-HIAA含量降低(P<0.01)。结论 本实验所建立的方法操作简便,结果准确,灵敏度高,重现性好,适用于检测抑郁大鼠脑脊液中5-羟色胺和5-羟吲哚乙酸水平。
关键词:  超高效液相色谱法  串联质谱法  抑郁  脑脊液  5-羟色胺  5-羟吲哚乙酸
DOI:10.16781/j.0258-879x.2018.12.1354
投稿时间:2018-06-05修订日期:2018-07-09
基金项目:
Simultaneous determination of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in cerebrospinal fluid of depressive rats by ultra high performance liquid chromatography-tandem quadrupole mass spectrometry
CHEN Lei1,DU Yi2,LI Cheng-jian3,ZHOU Jin3,CAI Zhen1*
(1. Department of Pharmacy, Changhai Hospital, Navy Medical University(Second Military Medical University), Shanghai 200433, China;
2. Department of Pharmacy, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China;
3. Department of Pharmacy, Eastern Hepatobiliary Surgery Hospital, Navy Medical University(Second Military Medical University), Shanghai 200438, China
*Corresponding author)
Abstract:
Objective To establish an ultra high performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-MS/MS) method for simultaneous determination of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in cerebrospinal fluid of depressive rats, and to investigate the concentrations of 5-HT and 5-HIAA in cerebrospinal fluid of depression rats and fluoxetine treated rats. Methods Twenty-four SD rats were randomly divided into control group, model group and fluoxetine group, with 8 rats in each group. At 30 d of model building, the cerebrospinal fluids were taken after the test of behaviors, and the concentrations of 5-HT and 5-HIAA were determined by UPLC-MS/MS after pretreatment of protein precipitation. Chromatographic column was ACE C18-PFP column (4.6 mm×150 mm, 3 μm), mobile phase was acetonitrile and 0.2% formic acid-water solution, flow rate was 0.4 mL/min, and the column temperature was 25℃. The mass spectrometry detection was performed using dynamic multiple reaction monitoring (DMRM). Results 5-HT and 5-HIAA in cerebrospinal fluid of depression rats had good linear relationship within the concentration range of 0.10-100.00 ng/mL and 1.00-1 000.00 ng/mL, respectively (r>0.999). Intra-and inter-day precision was less than 15.00%; the recoveries ranged from 86.86% to 98.49%; and the matrix effects ranged from 89.80% to 95.25%. The samples remained stable during analysis. Compared with the control group, the concentration of 5-HT in cerebrospinal fluid of rats in the model group was significantly decreased (P<0.01), and the concentration of 5-HIAA was significantly increased (P<0.01). Compared with the model group, the concentration of 5-HT in cerebrospinal fluid of rats in the fluoxetine group was significantly increased (P<0.01), and the concentration of 5-HIAA was significantly decreased (P<0.01). Conclusion The UPLC-MS/MS method is simple, accurate, sensitive, reproducible and suitable for simultaneous determination of 5-HT and 5-HIAA in cerebrospinal fluid of depression rats.
Key words:  ultra high performance liquid chromatography  tandem quadrupole mass spectrometry  depression  cerebrospinal fluid  5-hydroxytryptamine  5-hydroxyindoleacetic acid