Determination of Acetylcholine and Choline in Rat Brain Tissue by Liquid Chromatography/Electrochemistry Using an Immobilized Enzyme Post Column Reactor

Following separation by conventional LC, acetylcholine and choline are converted to hydrogen peroxide in a packed bed reactor consisting of covalently bound acetylcholinesterase and choline oxidase. Hydrogen peroxide, the ultimate enzymatic product, is detected amperometrically at a platinum electrode thin-layer cell. This method is simple and highly sensitive to the detection of acetylcholine and choline, with detection limits of about 100 femtomoles. The immobilized enzyme columns were stable for at least 60 days and conserved precious and expensive supplies of enzyme relative to continuous addition schemes. The apparatus is generally applicable to other enzymatic reactions which yield electroactive products.