%0 Journal Article
%T Critical Evaluation of Acetylcholine Determination in Rat Brain Microdialysates using Ion-Pair Liquid Chromatography with Amperometric Detection
%A Dimitri De Bundel
%A Sophie Sarre
%A Ann Van Eeckhaut
%A Ilse Smolders
%J Sensors
%D 2008
%I MDPI AG
%X Liquid chromatography with amperometric detection remains the most widely used method for acetylcholine quantification in microdialysis samples. Separation of acetylcholine from choline and other matrix components on a microbore chromatographic column (1 mm internal diameter), conversion of acetylcholine in an immobilized enzyme reactor and detection of the produced hydrogen peroxide on a horseradish peroxidase redox polymer coated glassy carbon electrode, achieves sufficient sensitivity for acetylcholine quantification in rat brain microdialysates. However, a thourough validation within the concentration range required for this application has not been carried out before. Furthermore, a rapid degradation of the chromatographic columns and enzyme systems have been reported. In the present study an ion-pair liquid chromatography assay with amperometric detection was validated and its long-term stability evaluated. Working at pH 6.5 dramatically increased chromatographic stability without a loss in sensitivity compared to higher pH values. The lower limit of quantification of the method was 0.3 nM. At this concentration the repeatability was 15.7%, the inter-day precision 8.7% and the accuracy 103.6%. The chromatographic column was stable over 4 months, the immobilized enzyme reactor up to 2-3 months and the enzyme coating of the amperometric detector up to 1-2 months. The concentration of acetylcholine in 30     l microdialysates obtained under basal conditions from the hippocampus of freely moving rats was 0.40    ¡À 0.12 nM (mean    ¡À SD, n = 30). The present method is therefore suitable for acetylcholine determination in rat brain microdialysates.
%K Acetylcholine
%K microdialysis
%K liquid chromatography
%K amperometric detection
%U http://www.mdpi.com/1424-8220/8/8/5171/