Sensitive method for HPLC determination of acetylcholine, choline and their analogues using fluorometric detection.
A method is described for reversed-phase HPLC separation of acetylcholine and choline and of their homologues in tissue extracts or perfusion fluids, combined with postcolumn enzymatic derivatization and fluorometric quantification. The separation occurs on a polymeric resin derivatized with hydrophobic moiety and the mobile phase consists of Na2HPO4, 3-(p-hydroxyphenyl)propionic acid and sodium dodecylsulphate; postcolumn enzyme reactor contains immobilized acetylcholinesterase, choline oxidase, and peroxidase. The limits of detection are 1 pmol choline and 3 pmol acetylcholine per sample. The method is free of interferences encountered with electrochemical detection and well suited for non-attended automatic operation.
Rícný
J
J
Institute of Physiology, Czechoslovak Academy of Sciences, Prague.
Tucek
S
S
Vins
I
I
D000109:Acetylcholine / Q000031:analogs & derivatives / Q000032:analysis*; D000818:Animals; D001923:Brain Chemistry; D002794:Choline / Q000031:analogs & derivatives / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry; D004800:Enzymes, Immobilized; D006863:Hydrogen-Ion Concentration; D051381:Rats; D013050:Spectrometry, Fluorescence
D004800:Enzymes, Immobilized; D002794:Choline; D000109:Acetylcholine