title: Critical Evaluation of Acetylcholine Determination in Rat Brain Microdialysates using Ion-Pair Liquid Chromatography with Amperometric Detection.
abstract: 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 μl microdialysates obtained under basal conditions from the hippocampus of freely moving rats was 0.40 ± 0.12 nM (mean ± SD, n = 30). The present method is therefore suitable for acetylcholine determination in rat brain microdialysates.
authors.affiliation: Experimental Neuropharmacology Research Group, Department of Pharmaceutical Chemistry and Drug Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium. ddebunde@vub.ac.be.
authors.forename: Dimitri
authors.initials: D
authors.lastname: De Bundel
authors.affiliation: Experimental Neuropharmacology Research Group, Department of Pharmaceutical Chemistry and Drug Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium. sophie.sarre@vub.ac.be.
authors.forename: Sophie
authors.initials: S
authors.lastname: Sarre
authors.affiliation: Experimental Neuropharmacology Research Group, Department of Pharmaceutical Chemistry and Drug Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium. aveeckha@vub.ac.be.
authors.forename: Ann
authors.initials: A
authors.lastname: Van Eeckhaut
authors.affiliation: Experimental Neuropharmacology Research Group, Department of Pharmaceutical Chemistry and Drug Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium. ilse.smolders@vub.ac.be.
authors.forename: Ilse
authors.initials: I
authors.lastname: Smolders
authors.affiliation: Experimental Neuropharmacology Research Group, Department of Pharmaceutical Chemistry and Drug Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium. ymichot@vub.ac.be.
authors.forename: Yvette
authors.initials: Y
authors.lastname: Michotte
keywords: Acetylcholine; amperometric detection; liquid chromatography; microdialysis