毛細管電泳法對阿茲海默症患者服用乙醯膽鹼酯酶抑制劑及其活性胺基酸之微量分析

Trace analysis of AChEIs and active amino acids in patients with Alzheimer’s disease by capillary electrophoresis

謝雅蕙(Ya-Hui Hsieh)

指導教授 ： 陳素惠

高雄醫學大學/藥學院/藥學研究所/博士(2011年)

摘要

本論文為利用毛細管電泳法(capillary electrophoresis; CE)分別對藥物及胺基酸發展微量的分析方法，主要的研究包括人體血漿中阿茲海默症用藥-乙醯膽鹼酯酶抑制劑(acetylcholinesterase inhibitors；AChEIs)的分析，以及興奮性胺基酸(excitatory amino acids；EAAs) aspartate與glutamate其光學異構物於人體血漿及腦脊髓液的分析。這兩個研究分別應用在阿茲海默症患者服用AChEIs後體內濃度測定以及患者體內EAAs濃度測定。其成果摘要如下：

甲、微胞電動層析法對血漿中AChEIs藥品的分析，並應用於阿茲海默症患者的用藥監測
本研究建立一個簡單且具感度的微胞電動層析法(micellar electrokinetic chromatography；MEKC)配合紫外光偵測，同時分析血漿中AChEIs的藥物galantamine、rivastigmine及rivastigmine代謝物NAP 226-90濃度。分析前，以液相萃取法(liquid-liquid extraction；LLE)作為樣品前處理，使用diethyl ether萃取後再進行MEKC的分析定量。定量分析使用熔矽毛細管全長為30.2 cm、內徑50.0 μm (有效長度20.0 cm)，在25℃之下，待測物可於10分鐘內達到有效的分離，分離所用的緩衝溶液為25 mM Tris (pH 5.0) 內含160 mM sodium octanesulfonate (SOS)、20% ACN及0.01% polyvinyl pyrrolidone (PVP)。此分析方法劇良好的感度，galantamine、rivastigmine及NAP 226-90的偵測極限分別為0.25、0.125及0.125 ng/ mL；此外，此方法也具良好的精密度與準確度。本研究應用於評估rivastigmine及其代謝物NAP 226-90的藥物血中濃度與時間的變化，也應用在11位阿茲海默症患者，口服Reminyl® (8 mg galantamine/ capsule)或Exelon® (3 mg rivastigmine/ capsule)後，監測其血漿中galantamine或rivastigmine及rivastigmine代謝物NAP 226-90的濃度。

乙、毛細管電泳法對生物檢品中興奮性胺基酸aspartate及glutamate光學異構物的同時分析，並應用於探討興奮性胺基酸的濃度與阿茲海默症之間的關係
本研究建立了簡單的區帶電泳法(capillary zone electrophoresis；CZE) 搭配雷射誘導螢光 (laser-induced fluorescence; LIF) 偵測器，分析aspartate (Asp)及glutamate (Glu)之光學異構物，並測定其在阿茲海默症病患的血漿與腦脊髓液中的含量。分析前，樣品先以離心過濾裝置(分子量3000)去除大分子的蛋白質，再將溶於DMSO之10 mM 6-carboxyfluorescein N-hydroxysuccinimide ester，以體積比5：1 (濾液：6-carboxyfluorescein N-hydroxysuccinimide ester) 的比例進行衍生反應，在室溫下以超音波震盪2小時。衍生後以水稀釋100倍再注入毛細管電泳儀進行分析，以0.5 psi注入5秒，分析的緩衝溶液為borate buffer 50 mM (pH 9.0)內含γ-CD 6 mM及0.1% PVP，分離電壓20 kV。本研究將應用於26位阿茲海默症患者，測定阿茲海默症患者體內D/L Asp及D/L Glu的濃度，並對照其臨床失智評估量表及簡易智能量表的指數，討論患者體內EAAs光學異構物濃度與疾病之相關性。研究結果顯示，病患血漿中的L-Asp濃度與臨床失智評估量表的指數據有中度負相關。

關鍵字

阿茲海默症 ； 毛細管電泳 ； 乙醯膽鹼酯酶抑制劑 ； 興奮性胺基酸

並列摘要

In this thesis, analytical methods were developed for the trace determination of drugs and amino acids by capillary electrophoresis (CE). The major studies are focused on the determination of acetylcholonesterase inhibitors (AChEIs) in plasma and chiral analysis of excitatory amino acids (EAAs), aspartate and glutamate, in human plasma and CSF. The two studies were applied in the evaluation of AChEIs concentrations and chiral excitatory amino acids concentrations in patients with Alzheimer’s disease, respectively. The summaries were listed as follows：

A. Simultaneous determination of galantamine, rivastigmine and NAP 226-90 in plasma by MEKC and its application in Alzheimer’s disease
A simple and sensitive MEKC with UV detection was developed for the simultaneous determination of AChEIs, including galantamine, rivastigmine and major metabolite NAP 226-90 in plasma. A sample pretreatment by liquid–liquid extraction with diethylether and subsequent quantification by MEKC was used. The optimum separation for these analytes was achieved in less than 10 min at 25℃ with a fused-silica capillary of 30.2 cm× 50 μm id (effective length 20 cm) and the run buffer was consisting of 25 mM Tris buffer (pH 5.0) with 160 mM sodium octanesulfonate, 20% ACN and 0.01% plyvinylpyrrolidone. In this method, the LODs of galantamine, rivastigmine and NAP 226-90 were 0.25, 0.125 and 0.125 ng/ mL, respectively. Besides, this MEKC method possessed good precision and accuracy. This method was applied in the drug concentration–time profile study of rivastigmine and its metabolite, NAP 226-90, and also applied in monitoring galantamine or rivastigmine and its metabolite NAP 226-90 in 11 Alzheimer’s disease patients’ plasma after oral administration of the commercial products Reminyl® (8 mg galantamine/capsule) or Exelon® (3 mg rivastigmine capsule), respectively.

B. Chiral analysis of aspartate and glutamate in biological samples by capillary electrophoresis and application in investigating the relationship between EAAs concentrations and Alzheimer’s disease
A simple CD-mediated CZE method equipped with laser-induced fluorescence (LIF) detector was developed in this study for chiral analysis of excitatory amino acids (EAAs), aspartate and glutamate, and to determine the EAAs concentrations in plasma and CSF of patients with Alzheimer’s disease. Before analysis, plasma and CSF samples were pretreated with centrifugal filter devices for removing proteins with high molecular weight (molecular weight cut off 3000) and then derivatized with 10 mM 6-carboxyfluorescein N-hydroxysuccinimide ester/ DMSO. The ratio of filtrate to the derivatizing reagent is 5 (v/v= 5：1). Mixed samples were sonicated for 2 h at 25℃ for chemical derivatization. After the derivatization reaction, reacted samples were diluted 100-fold with water and then hydrodynamically injected into CE instrument (0.5 psi for 5 s). The separation buffer was consisting of borate buffer 50 mM (pH 9.0) with 6 mM γ-CD and 0.1% PVP. The separation voltage was set at 20 kV. This method was applied in determining the EAAs concentrations of 26 patients with Alzheimer’s disease. We compared the EAAs concentrations and CDR-SB or MMSE and then discussed the relationship between EAAs concentrations and Alzheimer’s disease. From the results, there’s a moderately negative correlation between L-Asp concentration in plasma and CDR-SB values.

並列關鍵字

Alzheimer's disease ； Capillary electrophoresis ； Acetylcholinesterase inhibotors ； Excitatory amino acids

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