Ethambutol和Melamine的毛細管電泳分析方法開發

Abstract

本論文研究目的在建立具高靈敏度的毛細管電泳法以分析樣品中之微量成分。毛細管電泳具有高選擇性、高分離效率、分析時間短及所需樣品和溶劑量少等的優點，因此在近年被廣泛應用於各個領域之分析上。 本論文包含兩部分，第一部分為毛細管電泳連接雷射誘導激發螢光偵測器 (capillary electrophoresis-laser induced fluorescence)分析血漿中ethambutol的含量。先利用固相萃取方法 (solid phase extraction, SPE)移除血液樣品中的干擾物並濃縮分析物，再使用6-carboxyfluorescein succinimidyl ester (CFSE)與不具螢光性質的ethambutol進行管柱前衍生化。分析方法開發過程針對緩衝溶液酸鹼值、界面活性劑濃度、有機溶媒添加量、施加電壓及注射時間等參數進行探討，最適化條件為：毛細管內徑為75 μm，偵測長度為63 cm，所搭配之LIF激發波長和發射波長分別為488 nm和520 nm；緩衝液為50 mM 四硼酸鈉，10 mM SDS所組成，pH 9.0；施加電壓為15 kV；注射時間為8秒 (hydrodynamic mode, 50 mbar)，ethambutol可於13.5分鐘內完成分析，此條件可有效地將ethambutol與血液中內生性物質分離。本分析方法的精密度於峰面積，在一日內重複性及異日間再現性之RSD值分別為2.97 %及6.09 %；準確度由添加0.5、2.5及7.5 μg/mL ethambutol之血液樣品進行測試，所獲得之回收百分率分別為110.74 ± 1.04 %、97.33 ± 1.61 %和102.73 ± 1.45 %；偵測極限 (S/N= 3)為0.055 μg/mL。本研究並將此最適化分析條件應用於分析肺結核病人血漿，結果證實所開發之方法可以有效的定量病患血中ethambutol之濃度。 第二部分則是開發毛細管電泳法結合掃集法線上濃縮技術，以定量不同食物基質中的三聚氰胺。首先透過陽離子交換固相萃取法，移除部份食物基質中的干擾物，再利用掃集法 (sweeping)提高檢測靈敏度。本研究使用中心混成設計法進行最適化分析條件找尋，所得之最適化條件為：緩衝液為45 mM H3PO4，175 mM SDS，15 % MeOH所組成，pH值為2.15；施加電壓為– 22 kV；注射時間為5分鐘 (hydrodynamic mode, 50 mbar)；偵測波長為218 nm；溫度為25 ℃，最適化條件可於13分鐘內使三聚氰胺與食物基質中其他成分達基線分離，此條件於三聚氰胺標準品之偵測極限 (S/N= 3)為5 ng/mL。最後將確效之條件應用於多種食物樣品和實際含有三聚氰胺之樣本分析，準確度介於92.83 ± 3.32 - 108.29 ± 1.71%。本分析方法可提供一快速、準確且靈敏之三聚氰胺定量方法，且可廣泛應用於各種食物檢測，以確保食物之使用安全。

Due to its high selectivity, high resolution, short analytical time, and low sample and reagent consumption, capillary electrophoresis has become a useful tool in many analytical fields in recent years. This study aimed to establish high sensitive capillary electrophoresis methods to analyze the trace amounts of analytes. The thesis is composed of two parts. In the first part, we used capillary electrophoretic – laser induced fluorescence (CE-LIF) detection for the determination of ethambutol in patient plasma. Solid-phase extraction with Oasis HLB cartridge was used to remove the interference in plasma samples and concentrate the analyte. Owing to lack of intrinsic fluorescence characteristic of ethambutol, pre-capillary derivatization with 6-carboxyfluorescein succinimidyl ester (CFSE) was required for LIF detection. The derivatized product was then analyzed by micellar electrokinetic chromatography (MEKC). By using 50 mM sodium tetraborate (pH 9.0) containing 10 mM SDS as background electrolyte (BGE), ethambutol could be baseline separated with endogenous plasma components within 13.5 min. The capillary inner diameter was 75 μm, detection length was 63 cm, and the LIF excitation and emission wavelength were 488 nm and 520 nm, respectively. The limit of detection (LOD) (S/N= 3) of ethambutol was 0.055 μg/mL. 2’,7’-dichlorofluorescein was used as internal standard for the quantitative analysis. The run-to-run repeatability (n = 3) and intermediate precision (n = 3) of peak area ratios were less than 2.97 % and 6.09 % relative standard deviation (RSD), respectively. The accuracy was tested by spiking 0.5, 2.5 and 7.5 μg/mL of ethambutol into blank human plasma, and the recoveries were 110.74 ± 1.04 %, 97.33 ± 1.61 % and 102.73 ± 1.45 %, respectively. The developed method has been applied to analyze several plasma samples of tuberculosis patients. The result shows that the developed CE-LIF method is sensitive, efficient and accurate for the determination of plasma ethambutol concentration in tuberculous patients. The second part of this thesis developed a new approach for the determination of melamine in dietary products by sweeping-MEKC. We used cation-exchange solid-phase extraction to clean-up the ingredients in dietary products, and applied sweeping-micellar electrokinetic chromatography (sweeping-MEKC) for melamine determination. In order to have the best resolution, sweeping efficiency and separation speed, we used central composite designs (CCD) to search for the optimum sweeping-MEKC conditions. The optimum conditions used 45 mM H3PO4 containing 175 mM SDS with the addition of 15 % MeOH as BGE (pH 2.15), and 65 mM H3PO4 as sample solution. The applied voltage was -22 kV and the injection time was 5 min (hydrodynamic mode, 50 mbar). Under optimum sweeping-MEKC conditions, melamine content could be determined within 13 minutes, and the limit of detection (S/N= 3) was 5 ng/mL. The developed approach was validated by spiking melamine in various dietary products including milk, gluten, chicken feed and cookies. Run-to-run repeatability (n= 3) and intermediate precision (n= 3) of peak area were less than 5.65 % RSD. The accuracy were between 92.83 ± 3.32 % - 108.29 ± 1.71%. The developed approach was applied to analyze melamine contaminated milk powder and baking mix provided by the Joint research centre of the European Commission. The result reveals that the present sweeping-MEKC approach is sensitive, efficient and accurate for the determination of melamine in various dietary products.