三口井類除草劑與啡噻口井類藥物在毛細管電泳法之分離與線上濃縮之研究

Abstract

本論文選擇環境污染物與藥物來探討偵測靈敏度的增強，著重小分子在毛細管電泳的線上濃縮技巧，主要分為三部份來探討線上濃縮的機制： 第一部份是討論環境污染物如硫甲基-s-三口井類除草劑(methylthio-s-triazine herbicides)在正向樣品濃縮模式的濃縮機制。添加陰離子界面活性劑如正十二烷基硫酸鈉(Sodium Dodecyl Sulfate, 簡稱SDS)於pH 9.0的硼酸鹽作為分離緩衝液，對其進行系統地研究。尋得理想的分離緩衝液條件為添加20 mM SDS於pH 9.0的20 mM硼酸鹽緩衝液，此時樣品基質組成為30 mM硼酸鹽緩衝液。當添加SDS濃度從10 ~ 150 mM時，分析物的吸收度顯現兩個最大值。分析物的濃縮效率與SDS的結合常數有關，在理想的電泳條件下，terbutryn可濃縮1000倍。當進樣時間為180秒且用UV偵測器偵測時，simetryn、ametryn、prometryn 與terbutryn的偵測極限分別為61、25、11和2 ng/mL (S/N=3)。 第二部份是探討三口井類除草劑在掃集(sweeping)技巧的濃縮。當添加陰離子界面活性劑於pH 5.6的磷酸鹽緩衝液作為分離緩衝液，可基線分離四個三口井類除草劑的最大進樣時間為480秒，此時理想條件為樣品基質是50 mM磷酸鹽緩衝液；分離緩衝液組成為添加50 mM SDS和20 % MeOH於pH 5.6的50 mM磷酸鹽緩衝液。分析物在掃集模式的有效濃縮體積為正向樣品濃縮模式的2.67倍，乃因掃集模式同時進行樣品堆積與移除樣品基質，降低樣品基質對樣品堆積的干擾。當使用UV偵測器偵測時，simetryn、ametryn、prometryn與terbutryn的偵測極限分別為3.0、2.0、1.2和1.5 ng/mL (S/N=3)。 第三部份是以pH接合-掃集模式(pH junction-sweeping)來探討啡口賽口井類藥物(Phenothiazines)的對掌分離與濃縮，並以添加多種環糊精(包括sulfated β-cyclodextrin (S-β-CD)、β-CD、HP-β-CD、DM-β-CD和γ-CD)於pH 3.0的磷酸鹽做為分離緩衝液。結果顯示在使用單環糊精做為對掌選擇試劑時，γ-CD對四種啡口賽口井類對掌異構物有最佳的對掌分離，包括promethazine、ethopropazine、trimeprazine和thioridazine；然而分離緩衝液中同時使用多種環糊精(S-β-CD/β-CD/γ-CD)系統時，可有效濃縮與對掌分離長達1800秒注入時間的啡口賽口井類對掌異構物。此超長樣品注入體積約佔毛細管有效長度的98.9 %或98.9 cm，可增強當使用雙環糊精(S-β-CD/β-CD)對掌分離分析物thioridazine (5a)的弱吸收峰訊號。

Environmental pollutants and pharmaceutical compounds were selected for studying the enhancement of detection sensitivity. In this dissertation, on-line concentration of small molecules on capillary electrophoresis studied is consisting of three parts: In the first part, we focus on the stacking of environmental pollutants, such as methylthio-s-triazines herbicides, in normal stacking mode using sodium dodecyl sulfate (SDS) as an anionic surfactant and borate electrolyte at pH 9.0. The sample matrix used for concentration of neutral analytes is 30 mM borate electrolyte, and the optimal electrophoretic system for separation consists of 20 mM SDS and 20 mM borate buffer at pH 9.0. Interestingly, two maxima were observed in the plot of absorbance versus SDS concentration in the range 10-150 mM. Stacking efficiency of each individual analyte depends on its binding constant to SDS micelles, terbutryn can afford about 1000-fold enhancement under an optimal electrophoretic system. With this stacking mode, the sample solution can be injected up to 180 s, and the limits of detection (S/N=3) of terbutryn, prometryn, ametryn and simetryn determined to be 61, 25, 11 and 2 ng / mL, respectively. In the second part, we focus on the stacking of s-triazines herbicides using sweeping technique. The separation buffer consisting of anionic surfactant and phosphate electrolyte at pH 5.6 was used. The injection time of 480 sec can be achieved for baseline separation of four s-triazines. The concentrations of phosphate buffer at 50 mM was used as the sample matrix, while separation buffer consists of 50 mM phosphate electrolyte and 50 mM SDS containing 20 % methanol at pH 5.6, sample matrix and separation buffer were optimized. With this sweeping mode, the sample solution can be injected up to 480 s, and the limits of detection (S/N=3) of terbutryn, prometryn, ametryn and simetryn determined to be 3.0, 2.0, 1.5 and 1.2 ng / mL, respectively, with UV detection. In the third part, the enantioseparation and stacking of phenothiazine enantiomers based on the pH junction-sweeping mechanism were investigated. Phenothiazine samples were dissolved in water at neutral pH, whereas phosphate buffer at pH 3.0 containing different type of cyclodextrins (CDs), such as randomly sulfated β-CD (S-β-CD), β-CD HP-β-CD, DM-β-CD and γ-CD, was used as separation buffer. γ-CD shows the best enantioresolution for four phenothiazines, including promethazine, ethopropazine, trimeprazine and thioridazine. Effective enantioseparation and stacking of phenothiazine enantiomers could be achieved with the injection time of 1800 sec, when multi-cyclodextrins, such as S-β-CD / β-CD / γ-CD was employed. Due to extremely large injection volume (a filling of 98.9 % effective length), the signal of thioridazine (5a) could be greatly enhanced.