臺灣博碩士論文加值系統

以往因受限於分析技術的不足，文獻中鮮少有報導能直接測得活體動物體內砷物種動態的變化情形，而長久以來，砷物種在活體動物體內的轉換代謝機轉，又一直是生物醫學界亟欲窺探的重要資訊。為達到現場（in-situ）進行活體動物體內（in-vivo）連續（continuous）監測砷物種動態（dynamic）變化的目的，本研究已成功地建立二套微型化（Miniaturized）microdialysis-microbore HPLC-ICP-MS與microdialysis-microbore HPLC-HG-ICP-MS之線上（on-line）分析技術，並實際針對活體動物體內三氧化二砷代謝過程中，砷物種的動態（dynamic）變化趨勢提供了直接的證據。
為開發適合上述連續監測活體動物體內砷物種動態變化的分析系統，本研究依層析、微透析及ICP-MS儀器測定等三大部分分別進行最佳化操作條件的探討。在完成各關鍵操作參數的最佳化後，本研究即針對所建立之連線系統的分析效能進行探討，實驗結果顯示，對本研究欲測定四個砷物種As(III)、DMA、MMA及As(V)而言，最低可達到的偵測極限分別為0.37, 0.56, 1.42 及 1.88 ng/mL，測定時間的解析度亦可縮短至30分鐘，在長時間的監測過程中，所得到之分析結果的穩定度（RSD值）則均可維持在10 %以內。
在確認利用所建立之連線分析系統，進行活體動物體內砷物種連續監測可行性的過程中，本研究係將微透析探針佈植到大白鼠的肝臟及膀胱內，並以腹腔注射的方式將As2O3注入大白鼠的體內，再進行大白鼠的肝臟及膀胱尿液中砷物種濃度變化的連續動態監測。結果顯示，當大白鼠接受到高劑量無機砷的注射後，其肝臟中及膀胱尿液中隨即出現大量的As(III)與DMA，且DMA出現的時間較As(III) 約慢了60 分鐘，此一結果與文獻中推測之無機砷代謝機制頗為吻合，顯示本研究所建立之連線分析技術確實已具備了現場(in-situ)，體內(in-vivo)及連續(continuous)監測活體動物體內砷物種動態變化的可行性。

The toxicological and biological roles of trace elements are reported highly dependent on their chemical forms and/or oxidation states. In the past decade, the understanding of the impact of various species of trace elements on biological and environmental systems has been remarkably advanced, due mainly to the significant improvement in the speciation techniques. In view of the need of pharmacokinetics studies, the metabolic and biotransportation information of arsenicals in target organs has been considered indispensable in adequately characterizing the effectiveness and impact of arsenic trioxide. Based on aforementioned requirements, an in vivo, in-situ and continuous analytical system was developed to monitor the dynamic variation of different arsenic metabolites in anesthetized rat.
In our study, two miniaturized hyphenation techniques, named microdialysis-micro HPLC-ICP-MS and microdialysis-micro HPLC-HG-ICP-MS, were developed. To extract analyte species from target organs, Ringer’s solution was used to perfuse arsenicals from target organs at flow rate of 0.5 mL/min. An on-line injector (CMA 160) equipped with a 10-mL sample loop and a hydride generator was used to interface microdialysis probe, microbore-LC column and ICP-MS. Under the optimized conditions, the detection limits obtained with the proposed method can be as low as 0.37, 0.56, 1.42 and 1.88 ng/mL for As(III), DMA, MMA and As(V), respectively. The stability of this method can be controlled down to approximately 10 % after continuous operation up to 360 min, permitting to continuously monitor the variation of analyte arsenic species in anesthetized rat after the administration of arsenic trioxide. Based on the achieved analytical results, an in vivo, in-situ and continuous monitoring technique for the investigation of dynamic variation of trace arsenicals in anesthetized rat has been performed by this study. On behalf of the novelty of our proposed method, further characterization of the detoxification and metabolic mechanism of arsenic in various organisms has become possible.

第一章 前言……………………………………………………. 1
1.1微小化分析系統的發展……………………………… 1
1.2物種分析的演進及其在生物醫學研究上的重要性…. 10
1.3生物樣品中微量元素連線分析方法…………………. 13
1.4生物體內砷物種分析的意義…………………………. 16
1.5研究目的及方法………………………………………. 20
第二章 儀器分析原理…………………………………………. 22
2.1感應耦合電漿質譜儀分析法（ICP-MS）…………… 22
2.2氫化物產生系統原理（Hydride generation）……….. 36
2.3微透析取樣法（Microdialysis sampling）……………… 36
2.4高效能液相色層分析法之分析原理（HPLC）……… 40
2.5陰離子交換層析法(Anion-exchange chromatography). 44
第三章 實驗部分………………………………………………. 47
3.1Microdialysis-microbore HPLC-ICP-MS連線分析系統及其應用於活體動物中砷物種的連續動態偵測.…. 47
3.1.1 Microbore HPLC-ICP-MS連線分析系統……….. 47
3.1.2微透析取樣系統（Microdialysis）………………. 51
3.1.3 Microdialysis-microbore HPLC-ICP-MS連線分析系統的建立……………………………………... 53
3.1.4 Microdialysis-microbore HPLC-ICP-MS連線分析系統應用於活體動物體內砷物種的連續動態偵測…………………………………………………... 54
3.1.5建立Microdialysis-microbore HPLC-HG-ICP-MS連線分析系統及其應用於活體動物體內砷物種的連續動態偵測……………………………………… 55
第四章 結果與討論……………………………………………. 57
4.1 Microdialysis-microbore HPLC-ICP-MS連線分析系統……………………………………………………… 57
4.1.1 ICP-MS儀器最適化操作條件的探討………….. 58
4.1.2 Microbore HPLC 最適分離條件的探討……….. 64
4.1.3微透析取樣系統的最佳操作參數探討…………. 72
4.1.4不同砷物種透析效率的探討…………………… 80
4.2 系統分析特性之探討………………………………… 82
4.3活體動物體內砷物種之連續動態之測定…………… 84
第五章 結論……………………………………………………. 92
第六章 參考文獻………………………………………………. 95

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