Measurement of acetylcholine and choline in brain by HPLC with electrochemical detection.
A simple and rapid method for measuring acetylcholine and choline using high performance liquid chromatography (HPLC) with electrochemical detection is presented. Acetylcholine and choline were first separated using reverse-phase chromatography; acetylcholine was then hydrolyzed post-column to choline by acetylcholinesterase. Choline was oxidized enzymatically by choline oxidase to betaine and hydrogen peroxide, and the peroxide was detected electrochemically. Changes in methodology from previous procedures include a different mobile phase, controlled heating of chromatography column and post-column reaction coil, and a different extraction method for quaternary amines. The changes resulted in less inhibition of derivatizing enzymes by mobile phase, narrow and consistent elution of peaks, and a rapid and efficient extraction of quaternary amines. Measurement of acetylcholine and choline in brain tissue was found to be replicable, and the levels agreed with literature values.
D000109:Acetylcholine / Q000032:analysis*; D000110:Acetylcholinesterase / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001923:Brain Chemistry; D002794:Choline / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism; D004563:Electrochemistry; D051381:Rats; D011919:Rats, Inbred Strains
D001285:Atropine; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D000110:Acetylcholinesterase; D002794:Choline; D000109:Acetylcholine