Determination of dopamine concentrations in brain extracellular fluid using microdialysis with short sampling intervals, analyzed by ultra high performance liquid chromatography tandem mass spectrometry

Abstract

IntroductionAn increase in striatal dopamine is considered essential for the rewarding and reinforcing effects of drugs of abuse. We have developed and validated an ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the analysis of dopamine in rat brain extracellular fluid (ECF) sampled with microdialysis. The method was applied to monitor changes in dopamine concentrations over time after an intravenous bolus injection of heroin. MethodsDopamine and dopamine-d3 were analyzed using a 2.1×100mm Aquity T3 column, 1.7μm particle size, with a formic acid and methanol gradient. The run time of the method was 2.5min including equilibration time. ResultsThe method had an LOQ of 0.15ng/mL, which equals 0.55pg on column. The calibration curves were linear in the tested area of 0.15 to 16ng/mL. Inter-assay coefficients of variation varied between 5–17%, with an accuracy expressed as bias of −10 to 5%. The intra-assay coefficients of variation varied between 9–15%, with an accuracy of −3–7%. DiscussionHeroin metabolism is very rapid. Sampling intervals of only 2min were thus required to obtain an adequate number of samples of dopamine analysis accompanying the concentration–time profile of opioids in the brain. Applying a flow of 2μL/min, 4μL of dialysate were sampled at 2min intervals, in 7μL internal standard. The injection volume onto the UPLC column was 10μL.Analyses of microdialysate samples from a rat given heroin i.v. showed that it was possible to measure baseline levels and rapid changes in dopamine concentrations with very short sampling periods.