Critical Evaluation of Acetylcholine Determination in Rat Brain Microdialysates using Ion-Pair Liquid Chromatography with Amperometric DetectionReportar como inadecuado




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Experimental Neuropharmacology Research Group, Department of Pharmaceutical Chemistry and Drug Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium





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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. View Full-Text

Keywords: Acetylcholine; microdialysis; liquid chromatography; amperometric detection Acetylcholine; microdialysis; liquid chromatography; amperometric detection





Autor: Dimitri De Bundel, Sophie Sarre, Ann Van Eeckhaut, Ilse Smolders and Yvette Michotte *

Fuente: http://mdpi.com/



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