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Critical Evaluation of Acetylcholine Determination in Rat Brain Microdialysates using Ion-Pair Liquid Chromatography with Amperometric Detection
Experimental Neuropharmacology Research Group, Department of Pharmaceutical Chemistry and Drug Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium
* Author to whom correspondence should be addressed.
Received: 1 July 2008; in revised form: 24 August 2008 / Accepted: 25 August 2008 / Published: 28 August 2008
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.
Keywords: Acetylcholine; microdialysis; liquid chromatography; amperometric detection
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De Bundel, D.; Sarre, S.; Van Eeckhaut, A.; Smolders, I.; Michotte, Y. Critical Evaluation of Acetylcholine Determination in Rat Brain Microdialysates using Ion-Pair Liquid Chromatography with Amperometric Detection. Sensors 2008, 8, 5171-5185.
De Bundel D, Sarre S, Van Eeckhaut A, Smolders I, Michotte Y. Critical Evaluation of Acetylcholine Determination in Rat Brain Microdialysates using Ion-Pair Liquid Chromatography with Amperometric Detection. Sensors. 2008; 8(8):5171-5185.
De Bundel, Dimitri; Sarre, Sophie; Van Eeckhaut, Ann; Smolders, Ilse; Michotte, Yvette. 2008. "Critical Evaluation of Acetylcholine Determination in Rat Brain Microdialysates using Ion-Pair Liquid Chromatography with Amperometric Detection." Sensors 8, no. 8: 5171-5185.