色谱

色谱 ›› 2015, Vol. 33 ›› Issue (9): 981-987.DOI: 10.3724/SP.J.1123.2015.04049

• 研究论文 • 上一篇    下一篇

稳定同位素编码衍生-分散液液微萃取-超高效液相色谱-三重四极杆质谱检测大鼠脑微透析液中左旋多巴和多巴胺

亓伟梅1, 赵先恩2, 亓永1, 孙志伟2, 陈光2, 尤进茂2,3, 索有瑞3   

  1. 1. 莱芜职业技术学院化工教研室, 山东 莱芜 271100;
    2. 山东省生命有机分析重点实验室, 山东省高校绿色天然产物与医药中间体重点实验室, 曲阜师范大学化学与化工学院, 山东 曲阜 273165;
    3. 中国科学院西北高原生物研究所, 青海 西宁 810001
  • 收稿日期:2015-04-28 出版日期:2015-09-08 发布日期:2015-09-09
  • 通讯作者: 赵先恩, 尤进茂
  • 基金资助:

    国家自然科学基金项目(81303179).

Determination of L-dopa and dopamine in rat brain microdialysate by ultra high performance liquid chromatography-tandem mass spectrometry using stable isotope-coded derivatization coupled with dispersive liquid-liquid microextraction

QI Weimei1, ZHAO Xian-en2, QI Yong1, SUN Zhiwei2, CHEN Guang2, YOU Jinmao2,3, SUO Yourui3   

  1. 1. Chemical Department, Laiwu Vocational and Technical College, Laiwu 271100, China;
    2. Shandong Provincial Key Laboratory of Life-organic Analysis, Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China;
    3. Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China
  • Received:2015-04-28 Online:2015-09-08 Published:2015-09-09

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192

被引次数

4

摘要：

帕金森病(PD)大鼠脑微透析液中左旋多巴(L-DOPA)和多巴胺(DA)的高灵敏检测技术,是PD相关的临床医学及L-DOPA减毒增效协同药物筛选必不可少的手段。采用d0/d3-10-甲基-吖啶酮-2-磺酰氯(d0/d3-MASC)作为稳定同位素编码衍生试剂,联合超声波辅助-分散液液微萃取(UA-DLLME)技术,建立并验证了超高效液相色谱-串联质谱(UHPLC-MS/MS)快速检测L-DOPA和DA的分析方法。分别使用d0-MASC和d3-MASC衍生微透析液样品和混合对照品,将衍生溶液混合后采用UA-DLLME技术富集净化,继而进行UHPLC-MS/MS检测(多反应监测模式),以d3-MASC衍生物作为d0-MASC衍生物的内标物进行定量。实验表明,在乙腈/水(pH 10.8碳酸钠-碳酸氢钠缓冲液)溶液中37 ℃下反应3.0 min后衍生化反应完成,梯度洗脱条件下2.0 min可完成分离检测,线性范围为0.20~1500.0 nmol/L,相关系数大于0.994, L-DOPA和DA的检出限(S/N=3)分别为0.005和0.009 nmol/L。分析方法评价结果良好,与已报道方法相比在灵敏度、分析速度和抗基质干扰等方面具有优势,本方法已成功应用于测定中药方剂首乌方对PD大鼠脑微透析液中L-DOPA和DA浓度波动的影响。

关键词: 超高效液相色谱, 大鼠活体微透析, 帕金森病, 首乌方, 药物筛选, 质谱增敏分析

Abstract:

The sensitive detection method of levodopa (L-DOPA) and dopamine (DA) in rat brain microdialysate of Parkinson's disease (PD) is an essential tool for the clinical study and attenuated synergistic drug screening for L-DOPA from traditional Chinese medicines. Using d0/d3-10-methyl-acridone-2-sulfonyl chloride (d0/d3-MASC) as stable isotope derivatization reagent, a novel ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for L-DOPA and DA by stable isotope-coded derivatization coupled with ultrasonic-assisted dispersive liquid-liquid microextraction (UA-DLLME). d0-MASC (light) and d3-MASC (heavy) were used as derivatization reagents for microdialysate samples and standards, respectively. Mixtures of the two solutions were prepared by UA-DLLME for UHPLC-MS/MS analysis with multiple reaction monitoring (MRM) mode. With d3-MASC heavy derivatives as internal standards for corresponding light derivatives from samples, the stable isotope internal standard quantification for L-DOPA and DA was carried out. The stable derivatives were obtained in aqueous acetonitrile (pH 10.8 sodium carbonate-sodium bicarbonate buffer) at 37 ℃ for 3.0 min, and then were separated within 2.0 min using gradient elution. Linear range was 0.20-1500.0 nmol/L (R> 0.994). LODs were 0.005 and 0.009 nmol/L for DA and L-DOPA (S/N=3), respectively. This method was validated, and it showed obvious advantages in comparing with the reported methods in terms of sensitivity, analysis speed and anti-matrix interference. This method has been successfully applied to the study of effect of Shouwu Fang on L-DOPA and DA concentration fluctuations in PD rat brain microdialysate.

Key words: drug screening, mass spectrometry sensitization analysis, Parkinson's disease (PD), rat in vivo microdialysis, Showwu Fang, ultra high performance liquid chromatography (UHPLC)

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