本研究以探討微透析技術在大鼠腦中紋狀體區進行取樣，以利用高效能液相層析-電化學偵測器同時偵測神經傳遞物質及其代謝物含量之變化。其微透析管總長為14mm，膜長為4mm，透析膜材質為poly-arylethersulfone (PAES)，可通透之最大分子量為2000 Daltons，本實驗在高效能液相層析-電化學偵測器條件使用包含C18 ODS-3μm 100 mm × 4.6 mm之層析管柱，移動相為91:9(v/v)之0.05M磷酸緩衝液(包含0.2mM sodium octyl sulfonate及0.05mM Na2EDTA，以磷酸調至pH=3)與甲醇，流速為1 ml/min，樣品注入量為20μl，電化學偵測器主要以石墨工作電極，銀/氯化銀為參考電極，電位設定在+0.6伏特。
本研究順利開發一個簡單高效能液相層析-電化學偵測器之方法可同時偵測單胺類神經傳遞物質及其代謝物，其最低偵測極限範圍為0.3-127nM，線性迴歸曲線之r2在0.99以上，而利用此分析方法結合微透析技術，觀察大鼠在急性、慢性嗎啡注射之不同狀態下腦部之紋狀體核區神經傳遞物質及其代謝物濃度之變化情形。 This study combined microdialysis sampling with high performance liquid chromatography-electrochemical detection (HPLC-ECD) for the determination of the monoamine neurotransmitters and metabolite levels in the rat corpus striatum.
The material of probe membrane (length 4mm) was poly- arylethersulfone (PAES) and the cut-offs of these membranes were 20,000 Daltons. Separation of the monoamine neurotransmitters was obtained by a C18 ODS-3μm 100 mm × 4.6 mm column at a mobile phase consisting of 9% methanol in buffer (0.2mM sodium octyl sulfonate (SOS) and 0.05mM ethylene diamine tetraacetic acid disodium salt (EDTA Na2) with phosphoric acid to pH of 3), operated at a flow rate of 1ml/min and a inject volume of 20μl .A potential seep of +0.6V was applied across the working and reference electrodes of the detector.
A Simple high-performance liquid chromatography with electrochemical detection using microdialysis was developed for simultaneous determination of monoamine neurotransmitters and metabolite levels in the rat corpus striatum. The detection limits of these compounds were 0.3~127nM, the correlation coefficients of regression were more than 0.99.
The method has been successfully applied to measure the neurotransmitters and their metabolite levels in the rat corpus striatum by morphine injection in acute and chronic morphine treatment.
