Indirect detection of anti-acetylcholinesterase compounds in microcolumn liquid chromatography using packed bed reactor with immobilized human red blood cell acetylcholinesterase and choline oxidase.
The inhibiting compounds were separated by micro-column liquid chromatography in the mobile phase containing the natural substrate acetylcholine. A home-made packed bed microbioreactor system containing immobilized enzyme acetylcholinesterase (ACHE) in human red blood cell membrane and choline oxidase (CHO) from alcaligenes was used for the post-column conversion of acetylcholine to hydrogen peroxide which was detected by an electrochemical detector. The inhibition effect of the solutes caused a decrease in the acetylcholinesterase activity, a decrease in the formation of hydrogen peroxide and also a decrease in the response corresponding to the concentration of the solutes. The rate of the enzyme regeneration was also recorded. The micro-system was compared with a conventional LC system comprising commercially prepared enzyme reactor. The stability of the enzymes is at least 3 weeks at ambient temperature. The limit of detection depends on biological activity of inhibition and for galanthamine was 1 pmol.
Salamoun
J
J
Walther-Straub Institute of Pharmacology and Toxicology, Munich, Germany.
Remien
J
J
D000110:Acetylcholinesterase / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D002800:Cholinesterase Inhibitors / Q000032:analysis*; D002853:Chromatography, Liquid; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry
D002800:Cholinesterase Inhibitors; D004800:Enzymes, Immobilized; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D000110:Acetylcholinesterase