脑微透析液中多巴胺及其代谢物的监测：结合冷冻干燥与液相色谱-串联质谱法的方法。

Monitoring of dopamine and its metabolites in brain microdialysates: method combining freeze-drying with liquid chromatography-tandem mass spectrometry.

机构信息

Institute of Chemical Technology, Technicka 5, Prague 6, Czech Republic.

出版信息

J Chromatogr A. 2011 May 27;1218(21):3382-91. doi: 10.1016/j.chroma.2011.02.006. Epub 2011 Feb 26.

DOI:10.1016/j.chroma.2011.02.006

Abstract

A sensitive assay method was developed for a parallel, rapid and precise determination of dopamine and its metabolites, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid, from brain microdialysates. The method consisted of a pre-treatment step, freeze-drying (lyophilization), to concentrate dopamine and its metabolites from the microdialysates, and a detection step using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the reaction monitoring mode was selected for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized by the following parameters: the precision of the developed method was determined as ≥88.6% for dopamine, ≥89.9% for homovanillic acid, ≥86.1% for 3-methoxytyramine and ≥88.1% for 3,4-dihydroxyphenylacetic acid; the mean accuracy was determined as ≥88.2% for dopamine, ≥88.3% for homovanillic acid, ≥85.9% for 3-methoxytyramine and ≥88.6% for 3,4-dihydroxyphenylacetic acid. The developed method was compared to (1) other combinations of pre-treatment methods (solid phase extraction and nitrogen stripping) with LC-MS and (2) another detection method, liquid chromatography, with electrochemical detection. The novel developed method using combination of lyophilization with LC-ESI-MS/MS was tested on real samples obtained from the nucleus accumbens of rat pups after an acute methamphetamine administration. It was proven that the developed assay could be applied to both a simultaneous analysis of all four substrates (dopamine, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid) in microdialysis samples acquired from the rat brain and the monitoring of their slight concentration changes on a picogram level over time following methamphetamine stimulus.

摘要

建立了一种灵敏的分析方法，用于平行、快速和精确地测定脑微透析液中的多巴胺及其代谢产物高香草酸、3-甲氧基酪胺和 3,4-二羟基苯乙酸。该方法包括预处理步骤（冻干），从微透析液中浓缩多巴胺及其代谢产物，以及检测步骤，使用液相色谱-电喷雾串联质谱法（LC-ESI-MS/MS）。特别是，选择反应监测模式是因为其具有极高的选择性，而稳定同位素稀释法则是因为其具有很高的定量精度。所建立的方法具有以下参数特征：该方法的精密度（以多巴胺计）≥88.6%，（以高香草酸计）≥89.9%，（以 3-甲氧基酪胺计）≥86.1%，（以 3,4-二羟基苯乙酸计）≥88.1%；平均准确度（以多巴胺计）≥88.2%，（以高香草酸计）≥88.3%，（以 3-甲氧基酪胺计）≥85.9%，（以 3,4-二羟基苯乙酸计）≥88.6%。所建立的方法与（1）其他预处理方法（固相萃取和氮气吹扫）与 LC-MS 联用和（2）另一种检测方法（液相色谱与电化学检测）进行了比较。该新方法使用冻干与 LC-ESI-MS/MS 联用，用于检测急性 methamphetamine 给药后大鼠伏隔核中获得的真实样本。结果证明，该检测方法可同时分析微透析样本中四种基质（多巴胺、高香草酸、3-甲氧基酪胺和 3,4-二羟基苯乙酸），并能监测 methamphetamine 刺激后其浓度在皮克水平上的轻微变化。