線上濃縮毛細管電泳法對人體血漿中抗憂鬱藥fluoxetine及肉品中瘦肉精ractopamine之分析研究

On-line Stacking Capillary Electrophoresis for the Analysis of Fluoxetine in Plasma and Ractopamine in Meat

盧家加(Chia-Chia Lu)

指導教授 ： 吳秀梅

高雄醫學大學/藥學院/藥學研究所/博士(2012年)

摘要

本論文建立了兩種毛細管電泳分析法來監測生物檢體(血漿及肉品)中之微量殘留藥物。為達ppb之感度，本研究利用兩種線上濃縮技術：(一) 以電場放大電動取樣法(field-amplified sample injection)分析血漿中之fluoxetine(FL)及norfluoxetine(NFL)；（二）以大量注入陽離子及掃集微胞電動層析法(cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography, CSEI-sweep-MEKC)分析肉品中之ractopamine(RP)及dehydroxyractopamine(DRP)。

研究中利用化學計量統計實驗設計理論作為輔助，來完成實驗參數的最適化。在經由預試驗以建立基本分析條件後，部分因子設計法(fractional factorial design)可減少實驗次數，篩選出對實驗結果較有意義之因子，接著再以中心混成設計法(central composite design)來決定出最適化的實驗條件。

於第一篇研究中以電場放大電動取樣法分析血漿中抗憂鬱藥FL及其主要代謝物NFL。血漿檢體經由液相萃取後，回溶之樣品以電動取樣(–5 kV, 99.9 s)經毛細管之短端注入，如此可縮短分離時間至四分鐘內，達到快速分析之目的。分離緩衝液為磷酸鹽及三乙醇胺(pH 2.5)之混合溶液，並另外添加5%甲醇，使待測物於–10 kV電壓下可達基線分離。經方法確效後，其監測範圍為10-500 ng/mL，檢量線呈現良好的線性關係(r > 0.995)。此法之偵測極限為4 ng/mL (S/N = 3)，且成功的應用於定量三名真實血漿檢體，所獲得之血漿濃度與文獻中相符合。

第二篇研究以大量注入陽離子及掃集微胞電動層析法分析肉品中的RP及其同系物DRP。肉品經過均質及水解後，再以液相萃取法做前處理，接著再利用CSEI-sweep-MEKC的技術，其偵測極限可達5 ng/g (RP)及3 ng/g (DRP) (S/N = 3)。此線上濃縮法利用先注入一大段高導電性緩衝液，如此可進行較長時間的電動取樣，讓待測物堆積於此區帶中，再以正十二烷基硫酸鈉掃集，給予–25 kV電壓進行分離。此法經過確效，其檢量線範圍為10-300 ng/g，具有良好的線性關係(r > 0.994)。之後以市售培林試紙驗證本法測定含有RP之真實檢體，結果呈現陽性反應，證實此法確實可成功應用於分析傳統市場及超市中取得之肉品檢體，並定量其中之RP含量。

關鍵字

毛細管電泳 ； 線上堆積 ； 憂鬱症 ； 瘦肉精 ； 電場放大電動取樣法 ； 大量注入陽離子及掃集微胞電動層析法

並列摘要

In this research, two on-line sample stacking CE methods have been developed, field-amplified sample injection (FASI) and cation-selective exhaustive injection coupled with sweeping micellar electrokinetic chromatography (CSEI-sweep-MEKC), to analyze the residual drugs from complex, biological matrices, human plasma and porcine meat.

Both methods were established with the aid of chemometric experimental design to improve the efficiency and reduce the time and cost for finding the optimized parameters. Fractional factorial design (fFD) served the purpose to screen all potential factors with minimal number of runs and determined which variables were significant. Central composite design (CCD) was performed to reveal the optimized parameters for the analysis.

In the first work, antidepressant fluoxetine (FL) and its major metabolite norfluoxetine (NFL) were determined in plasma samples. After LLE, samples were electrokinetically injected (–5 kV, 99.9 s) into the capillary from the short end (detector end), which enabled a short analysis time of only 4 min. Good resolution of FL and NFL was achieved using a BGE formulation consisting of phosphate–triethanolamine at low pH, and a separation voltage of –10 kV. Five percent methanol was added as organic modifier to enhance selectivity and resolution. The linear range was between 10 and 500 ng/mL (r > 0.995), covering the expected plasma therapeutic ranges. The LOD in plasma were 4 ng/mL (S/N = 3), a value comparable to that obtained using LC-MS, showing the success of the online stacking technique. This method was also successfully validated in quantification and pharmacokinetic studies with three volunteer plasma samples, and could be applied to pharmacogenetic studies.

The second work was the porcine meat analysis for ractopamine (RP) and its homolog dehydroxyractopamine (DRP). Meat samples were homogenized and hydrolyzed prior to LLE. CSEI-sweep-MEKC enabled ppb level analysis, with LODs in meat to be 5 ng/g for RP and 3 ng/g for DRP (S/N = 3). A high conductivity buffer (HCB) zone was injected into the capillary, allowing the analytes to be electrokinetically injected for 12 min with a voltage of 9 kV. With 125 mM of SDS and 15-25% of MeOH in the separation and sweeping buffer, RP and DRP were well separated. The method was validated with a linear calibration curve of 10-300 ng/g (r > 0.994). Real samples obtained from meat stands and supermarkets were analyzed. One sample that contained RP was tested with commercial rapid test kit, and confirmed to be positive, showing that this developed method could be successfully applied to detect and quantify RP in real samples.

並列關鍵字

Fluoxetine ； Ractopamine ； Capillary electrophoresis ； On-line stacking ； Field-amplified sample injection ； Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography

參考文獻

參考文獻

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