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Sensors 2008, 8(8), 5171-5185; doi:10.3390/s8085171

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 / Revised: 24 August 2008 / Accepted: 25 August 2008 / Published: 28 August 2008
(This article belongs to the Special Issue Amperometric Sensors and Techniques for Neurochemical Monitoring)
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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 Acetylcholine; microdialysis; liquid chromatography; amperometric detection
This is an open access article distributed under the Creative Commons Attribution License (CC BY) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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.

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