高效液相色谱-柱后固定化酶反应器-电化学检测法分析大鼠脑微透析液中的乙酰胆碱和胆碱

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

建立了用高效液相色谱分离－柱后固定化酶反应器酶解－电化学检测器检测酶解最终产物Ｈ２Ｏ２的方法，分析了麻醉和自由活动大鼠脑微透析液中乙酰胆碱（ＡＣｈ）和胆碱（Ｃｈ）的含量。至少在０．２～１００μｍｏｌ／Ｌ范围内ＡＣｈ和Ｃｈ的浓度与其响应的线性关系良好，它们的检测极限都可达５０ｆｍｏｌ。对高效液相色谱结合固定化酶反应器的分析方法作了简要的讨论。

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+0.5V versus Ag/AgCl). The tip of concentric dialysis probe was made of the semipermeable dialysis membrane of 0.22mm in outside diameter, and the effective length inserted into rat brain was 3.0mm. The probe was perfused at a rate of 1μL/min with Ringer′s solution which contained 10μmol/L (for anesthetized rats) or 1μmol/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 100μmol/L, the relation between the amounts and the peak areas was linear (r= 0 998 8 for ACh and r=0.998 5 for Ch). The detection limit for ACh and Ch, at a S/N ratio of two, was found to be 50fmol 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 +(100mmol/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.