Monitoring of dopamine and its metabolites in brain microdialysates: Method combining freeze-drying with liquid chromatography-tandem mass spectrometry
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 (Iyophilization), 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 stimulu