臺灣博碩士論文加值系統

本實驗使用界面活性劑輔助分散式液液微萃取法並結合線上濃縮技術-聚合物堆積法 ( SA-DLLME-Polymer stacking )，搭配毛細管電泳儀 ( CE – UV ) 是一種簡單且具有高靈敏度的分析方法。將此方法用來偵測人體尿液及血清中之抗抑鬱藥物Mirtazapine及其代謝物Desmethyl mirtazapine 、8-Hydroxy mirtazapine之掌性異構物。並使用含有0.9 % [Poly (diallyldimethylammonium chloride)] ( PDDAC )及20 mM Dimethyl-β-cyclodextrin之10 mM 磷酸緩衝溶液 ( pH 3.0 ) 作為分離的電泳緩衝溶液。因高分子聚合物PDDAC具有高度黏性，可有效輔助堆積效果，使偵測靈敏度大幅提高。在最佳化條件下，可獲得2725 – 3878倍的濃縮倍率，線性範圍1.2 – 150.0 nM，最低偵測極限 ( LODs ) 範圍為0.3 – 0.5 nM。在真實樣品部分，已成功應用於人體尿液及血清中，尿液線性範圍1.5 – 150.0 nM，LOD範圍為0.3 – 0.5 nM，相對回收率89.0 – 116.5 %；血清線性範圍為5.0 – 250.0 nM，LOD範圍為1.3 – 2.0 nM，相對回收率88.5 – 112.8 %。本實驗所開發的方法，具有高濃縮倍率、良好的準確度與精密度等優點，可成功應用於測定尿液及血清中的藥物。

Surfactant-assisted dispersive liquid-liquid microextraction combined with on-line concentration - polymer stacking ( SA-DLLME-Polymer stacking ) in capillary electrophoresis was determined to be a simple and sensitive analytical method for detecting R,S-Mirtazapine and its metabolite R,S-Desmethyl mirtazapine, R,S-8-Hydroxy mirtazapine in human urine and serum. The electrophoretic analyses were carried out in 10 mM Phosphate buffer solution at pH 3.0 containing 0.9 % [Poly (diallyldimethylammonium chloride)] ( PDDAC ) and 20 mM Dimethyl-β-cyclodextrin.Because PDDAC exhibits viscosity, the effect of the polymer stacking and detection sensitivity could to substantially improved. The enrichment factors obtained under optimized conditions ranged from 2725 to 3879. The proposed method for detecting R,S-Mirtazapine and its metabolite provided strong linearity that ranged from 1.5 to 1500.0 nM, the limits of detection (LODs) ranged from 0.3 to 0.5 nM. In the real sample, the recovery of analytes for the urine sample at spiking levels of 1.5-150.0 nM and the serum sample at spiking levels of 5.0 – 250.0 nM were in the ranges of 89.0 – 116.5 % and 88.5 – 112.8 %, respectively. With the advantages of high enrichment factors and high accuracy and precision, the developed method was successfully applied to determine drugs in urine and serum.

目錄
摘要 I
ABSTRACT Ⅱ
目錄 III
圖目錄 V
表目錄 Ⅶ
第一章、緒論 1
一、抗抑鬱藥簡介 1
1.1分析物簡介 2
二、藥物分析方法 4
三、聚二甲基二烯丙基氯化銨 ( PDDAC ) 11
四、毛細管電泳簡介 12
4.1 毛細管電泳的分離原理 12
4.2 毛細管電泳的分離模式 13
五、使用之線上濃縮方法簡介 17
5.1 聚合物堆積法 ( Polymer stacking ) 17
六、使用之萃取方法簡介 19
6.1 界面活性劑輔助分散式液液微萃取法 ( Surfactant Assisted Dispersive Liquid-Liquid Microextraction, SA-DLLME ) 20
七、環糊精簡介 23
八、研究動機與目的 26
第二章、實驗 27
一、儀器設備 27
二、藥品 29
三、實驗方法 33
3.1 CE-UV偵測系統 33
3.2 儲存溶液的配製 34
3.3 分析物樣品與緩衝溶液的配製 35
3.4 毛細管電泳處理與進樣方式 36
3.5 界面活性劑輔助分散式液液微萃取法步驟 36
3.6 尿液樣品直接分析 37
3.7 尿液樣品經界面活性劑輔助分散式液液微萃取法後分析 37
3.8 血清樣品直接分析 37
3.9 血清樣品經界面活性劑輔助分散式液液微萃取後分析 38
第三章、結果與討論 39
一、毛細管電泳分離條件的探討 39
1.1 緩衝溶液種類的探討 40
1.2 環糊精種類及濃度對於分離的影響 42
1.3 緩衝溶液離子強度對於分離的影響 52
1.4 緩衝溶液pH值對於分離的影響 55
1.5 PDDAC含量對於堆積的影響 57
二、界面活性劑輔助分散式液液微萃取法的探討 60
2.1萃取劑的種類 62
2.2萃取劑的體積 65
2.3界面活性劑的種類 67
2.4溶液pH值 71
2.5萃取時間 73
2.6鹽類的添加 75
2.7 界面活性劑輔助分散式液液微萃取法之最佳化條件 77
三、線上濃縮技術 79
3.1 進樣時間探討 79
四、結合界面活性劑輔助分散式液液微萃取法與線上濃縮技術 83
五、方法確效 89
5.1 標準樣品直接分析 89
5.2 標準樣品經線上濃縮 ( Polymer stacking ) 堆積後分析 90
5.3 標準樣品經界面活性劑輔助分散式液液微萃取 ( SA-DLLME ) 後分析 90
5.4 標準樣品經界面活性劑輔助分散式液液微萃取與線上濃縮後 ( SA-DLLME-Polymer stacking ) 分析 91
5.5 標準樣品的靈敏度與濃縮倍率 92
六、生物真實樣品的應用 (尿液、血清) 99
6.1 尿液樣品的分析 99
6.2 尿液樣品之定量、靈敏度、精密度與準確度 102
6.3 血清樣品的分析 108
6.4 血清樣品之定量、靈敏度、精密度與準確度 111
第四章、結論 117
參考文獻 118



圖目錄
圖 1、PDDAC結構 11
圖 2、毛細管區帶電泳法示意圖 14
圖 3、微胞電動層析法示意圖 16
圖 4、聚合物堆積法 18
圖 5、界面活性劑輔助分散式液液微萃取法 ( SA-DLLME ) 示意圖 22
圖 6、環糊精的外觀 24
圖 7、環糊精的結構示意圖 24
圖 9、緩衝溶液種類的探討 41
圖 10、緩衝溶液中添加不同濃度之DM-Β-CD 進行探討 44
圖 11、緩衝溶液中添加不同濃度之HP-Β-CD 進行探討 45
圖 12、緩衝溶液中添加不同濃度之CM-Β-CD 進行探討 47
圖 13、緩衝溶液中添加不同濃度之TM-Β-CD 進行探討 48
圖 14、緩衝溶液中添加不同濃度之Β-CD 進行探討 49
圖 15、不同種類及濃度之環糊精最佳分離圖 51
圖 16、緩衝溶液離子強度的探討 53
圖 17、緩衝溶液PH值的探討 56
圖 18、PDDAC含量的探討 58
圖 19、萃取劑的種類 64
圖 20、萃取劑的體積 66
圖 21、不同種類界面活性劑之濃度差異 69
圖 22、界面活性劑的種類 70
圖 23、溶液PH值 72
圖 24、萃取時間 74
圖 25、鹽類的添加 76
圖 26、界面活性劑輔助分散式液液微萃取法之最佳化標準圖 78
圖 27、毛細管進樣時間與體積變化量 81
圖 28、分析物訊號高度 82
圖 29、分析物訊號面積 82
圖 30、不同濃縮方式之示意圖 84
圖 31、界面活性劑輔助分散式液液微萃取法結合線上濃縮技術 86
圖 32、萃取後並結合線上濃縮之分析物訊號高度 87
圖 33、萃取後並結合線上濃縮之分析物訊號面積 87
圖 34、標準樣品經SA-DLLME-POLYMER STACKING所能偵測之最低濃度 88
圖 35、尿液樣品直接分析與添加分析物後經SA-DLLME-POLYMER
STACKING之對照圖 101
圖 36、尿液樣品經SA-DLLME-POLYMER STACKING所能偵測之最低濃度
105
圖 37、血清樣品直接分析與添加分析物後經SA-DLLME-POLYMER
STACKING之對照圖 110
圖 38、血清樣品經SA-DLLME-POLYMER STACKING所能偵測之最低濃度
114



表 1、分析物及內標的化學結構及其物性 3
表 2、使用不同偵測儀器與樣品前處理應用於MRT、DMR、8-OHM藥物的分
析 6
表 3、不同種類的界面活性劑之CMC及HLB 22
表 4、環糊精的性質 25
表 5、不同緩衝溶液之理論板數 40
表 6、不同濃度DM-Β-CD之理論板數 43
表 7、不同種類及濃度之環糊精對分析物之分離解析度 50
表 8、不同種類及濃度之環糊精對分析物之理論板數 50
表 9、不同離子強度對分析物之分離解析度及理論板數 54
表 10、不同含量的PDDAC之分離解析度及理論板數 59
表 11、電泳分離之條件最佳化 59
表 12、萃取劑的性質 63
表 13、界面活性劑輔助分散式液液微萃取法之最佳化條件 77
表 14、標準樣品直接分析 93
表 15、標準樣品經線上濃縮 ( POLYMER STACKING ) 堆積後分析 94
表 16、標準樣品經界面活性劑輔助分散式液液微萃取 ( SA-DLLME ) 後分析
95
表 17、標準樣品經界面活性劑輔助分散式液液微萃取與線上濃縮後 ( SA-
DLLME-POLYMER STACKING ) 分析 96
表 18、比較標準樣品經不同分析方法之偵測極限值 97
表 19、比較標準樣品經不同分析方法之濃縮倍率 98
表 20、尿液樣品經界面活性劑輔助分散式液液微萃取與線上濃縮後 ( SA-
DLLME-POLYMER STACKING ) 分析 104
表 21、尿液樣品經添加後之回收率、精密度準確度 106
表 22、血清樣品經界面活性劑輔助分散式液液微萃取與線上濃縮後 ( SA-
DLLME-POLYMER STACKING ) 分析 113
表 23、血清樣品經添加後之回收率、精密度準確度 115

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