SIMULTANEOUS DETERMINATION OF ACETYLCHOLINE, CHOLINE AND PHYSOSTIGMINE IN MICRODIALYSIS SAMPLES FROM RAT HIPPOCAMPUS BY MICROBORE LIQUID-CHROMATOGRAPHY ELECTROCHEMISTRY ON PEROXIDASE REDOX POLYMER-COATED ELECTRODES

Microbore column liquid chromatography with post-column immobilized en zyme reactor (IMER) and electrochemical detection on redox polymer coa ted electrodes was used for detection of acetylcholine (ACh) and choli ne (Ch) in microdialysis samples. The sensitivity of the coated electr odes, decreased gradually by about 10%/day, with highest reduction of 30% within the first 16 h of use. A number of choline derivatives were tested as possible internal standards, of those acetylethylhomocholin e (AEHCh) and butyrylcholine (BCh) were found the most suitable candid ates since they both provided high enzymatic conversion in the IMER. P hysostigmine produced a negative peak, possibly reflecting oxidation o f eseroline-a decarbamoylated product of reversible reaction of physos tigmine with immobilized acetylcholine esterase. The probes, implanted in the ventral hippocampi of awake rats were perfused at a flow-rate of 1.25 mu l/min with Ringer solution containing 10 mu M physostigmine or with artificial cerebrospinal fluid only. The concentrations of AC h in 10-mu l samples at basal conditions were between 0.9-2.5 nM, wher eas in the presence of physostigmine the ACh levels raised to 41-48 nM . Physostigmine concentration was reduced to 8.8 mu M, indicating its in vivo delivery of about 12%. The coefficients of variation were redu ced from 7.4% for external standard method after every sixth sample to 5.8% and 5.9% for internal standardization with AEHCh and BCh, respec tively. The latter method shortened the total analysis time by about 1 5%, thus being especially suitable for continuous long-lasting off-lin e or on-line monitoring. Additionally, other endogenous cholines such as butyrylcholine or synthetic choline derivatives could be detected b y the present method. (C) 1998 Elsevier Science B.V. All rights reserv ed.