[Determination of acetylcholine and choline in microdialysates from rat brain by high performance liquid chromatography with electrochemical detection combined with a post-column immobilized enzyme reactor].

In the present work, acetylcholine(ACh) and choline(Ch) in the microdialysates from three brain areas of anesthetized rats and from hippocampus and frontal cortex of freely moving rats were simultaneously measured by high performance liquid chromatography(HPLC) with electrochemical detection combined with a post-column immobilized enzyme reactor(IMER). This assay was based on the separation of ACh and Ch on a polymer gel column followed by passage of the effluent through an IMER, on which the separated ACh and Ch reacted respectively to give each stoichiometric yield of hydrogen peroxide, which was detected electrochemically at a platinum electrode (potential + 0.5 V versus Ag/AgCl). The tip of concentric dialysis probe was made of the semipermeable dialysis membrane of 0.22 mm in outside diameter, and the effective length inserted into rat brain was 3.0 mm. The probe was perfused at a rate of 1 microL/min with Ringer's solution which contained 10 mumol/L (for anesthetized rats) or 1 mumol/L (for freely moving rats) neostigmine, a reversible cholinesterase inhibitor, to elevate ACh level in microdialysate. Before the experiment, the recovery of the probe in vitro was measured at room temperature, and the position of the probe was checked by histological procedure at the end of the experiment. In the range of 0.2-100 mumol/L, the relation between the amounts and the peak areas was linear (r = 0.9988 for ACh and r = 0.9985 for Ch). The detection limit for ACh and Ch, at a S/N ratio of two, was found to be 50 fmol per injection. The probe recoveries(%) for ACh and for Ch were 23.2 +/- 1.4 and 34.3 +/- 3.2(mean +/- SD) respectively. The basal levels of ACh in the microdialysates from striatum and frontal cortex of anesthetized rats as well as from hippocampus and frontal cortex of freely moving rats were 212 +/- 28 and 22 +/- 4 as well as 26 +/- 4 and 83 +/- 7(nmol/L, mean +/- SD, not corrected according to probe recovery) respectively. The perfusion of high concentration K+ (100 mmol/L) through the dialysis probe induced a large increase of ACh in the microdialysates. The critical points for HPLC analysis combined with IMER were briefly discussed.