Improved Method for Determination of Acetylcholine, Choline, and Other Biogenic Amines in a Single Brain Tissue Sample Using High Performance Liquid Chromatography and Electrochemical Detection

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Abstract
A simple method for determination of ACh, Ch, NA, DA, 5-HT and their related metabolites on the same brain tissue sample was developed by HPLC-ED. The electrochemical detection system is equipped with a platinum electrode for ACh and Ch detection, or a glassy carbon electrode for CA and 5-HT detection. ACh and Ch can be separated with bonded silica or polystyrene reverse phase columns, using a pH 7 mobile phase. They are converted to H2O2 by the passage of the effluent through an in line post column reactor with covalently bonded ACh esterase and Ch oxidase. This step ensures sensitivity, reliability and enzyme economy. Tissue preparation consists of formic acid/acetone extraction and purification by tetraphenyl boron exchange with high reproductible recoveries. The time necessary for the whole procedure is short, making it well adapted to large series. CA, 5-HT and related metabolites can be simply analysed on an aliquot of the tissue extract.

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