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

摘要

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 rea⁃gent, 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 car⁃ried out. The stable derivatives were obtained in aqueous acetonitrile ( pH 10�8 sodium carbon⁃ate⁃sodium bicarbonate buffer) at 37 ℃ for 3�0 min, and then were separated within 2�0 min using gradient elution. Linear range was 0�20-1 500�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.%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。