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 enzyme reactor (IMER) and electrochemical detection on redox polymer coated electrodes was used for detection of acetylcholine (ACh) and choline (Ch) in microdialysis samples. The sensitivity of the coated electrodes, 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 acetylethylhomocholine (AEHCh) and butyrylcholine (BCh) were found the most suitable candidates since they both provided high enzymatic conversion in the IMER. Physostigmine produced a negative peak, possibly reflecting oxidation of eseroline--a decarbamoylated product of reversible reaction of physostigmine with immobilized acetylcholine esterase. The probes, implanted in the ventral hippocampi of awake rats were perfused at a flow-rate of 1.25 microl/min with Ringer solution containing 10 microM physostigmine or with artificial cerebrospinal fluid only. The concentrations of ACh in 10-microl samples at basal conditions were between 0.9-2.5 nM, whereas in the presence of physostigmine the ACh levels raised to 41-48 nM. Physostigmine concentration was reduced to 8.8 microM, indicating its in vivo delivery of about 12%. The coefficients of variation were reduced from 7.4% for external standard method after every sixth sample to 5.8% and 5.9% for internal standardization with AEHCh and BCh, respectively. The latter method shortened the total analysis time by about 15%, thus being especially suitable for continuous long-lasting off-line or on-line monitoring. Additionally, other endogenous cholines such as butyrylcholine or synthetic choline derivatives could be detected by the present method.