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研究生： 林和靜
Ho-Ching Lin
論文名稱： 利用Microbore-HPLC-ICP-MS連線分析系統進行尿中硒物種的分析研究
Speciation of selenium in urine using microbore HPLC-ICP-MS
指導教授： 楊末雄
口試委員:
學位類別： 碩士
Master
系所名稱： 原子科學院 - 生醫工程與環境科學系
Department of Biomedical Engineering and Environmental Sciences
論文出版年： 2002
畢業學年度： 90
語文別： 中文
論文頁數： 87
中文關鍵詞： 硒 、HPLC-ICP-MS
外文關鍵詞： selenium, HPLC-ICP-MS
相關次數： 點閱：2  下載：0 
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    • 硒是人體必需的微量元素，攝取不足或過量，均可能會對人體造成危害。資料顯示，硒的營養價值、生物有效性、毒性和抗癌性等都與其物種息息相關。因此為進一步瞭解硒在人體內的作用機制，確有開發各種硒物種分析方法的迫切需求。
    本研究旨在發展人體尿液中硒物種的分析方法。根據各待測硒物種的特性，開發陰離子層析配合 ICP-MS （AC-ICP-MS）及逆相層析配合ICP-MS（RPLC-ICP-MS）等兩套連線系統，分別對尿液中的Se（IV），Se（VI）與SeMet，及SeEt，SeCys與TMSe+等物種進行分析。

    研究中，分別對AC-ICP-MS及RPLC-ICP-MS分析系統的層析分離的條件，及ICP-MS可能造成的干擾問題等進行探討，最後將所求得的最適化分析條件予以整合，並應用於人體尿液的分析。結果顯示，在AC-ICP-MS的分析系統中，對Se(IV)、Se(VI)及TMSe的偵測極限分別可低達0.18 ppb、0.20 ppb、及3.45 ppb；在RPLC-ICP-MS的分析系統中，對SeCys、SeMet及SeEt的偵測極限則可低達0.59 ppb、0.26 ppb及0.51 ppb。所建立之連線分析系統的效能( analytical performance )，經由適當的評估程序後，最後將其實際應用於人體攝食含硒營養品後，尿液中硒濃度變化趨勢的探討。

    Selenium is now well known as an essential element for biological systems but also as a potential toxicant. However, the nutrition, bioavailability, toxicity and cancer chemoprotective activity of selenium have been found to be species-dependent.
    The aim of this work is to develop analytical methods for determination of selenium species in human urine. Owing to a wide range difference in physical and chemical properties among the selenium species, two chromatographic methods based on different separation principles were employed to meet the requirement for the determination of various selenium species. Anion-exchange chromatography was used to separate selenite（Se(IV)）, selenate（Se(VI)）and trimethylselenonium ion（TMSe）, and reversed-phase chromatography to separate selenocysteine（SeCys）, selenomethionine（SeMet）and selenoethionine（SeEt）. To achieve the determination of ultratrace levels of selenium species in normal urine, a highly sensitive ICP-MS was used for selenium detection. By proper coupling of the respective chromatographic units and ICP-MS, two hyphenated systems consisting of AC-ICP-MS and RPLC-ICP-MS were established.

    To achieve favorable analytical performance of the on-line systems for selenium speciation, efforts were made to optimize the chromatographic parameters and to eliminate the spectroscopic interferences in ICP-MS determination. With the use of optimized analytical conditions, the detection limits as low as 0.18 ppb, 0.20 ppb and 3.45 ppb respectively for Se(IV), Se(VI) and TMSe, and 0.59 ppb, 0.26 ppb and 0.51 ppb respectively for SeCys, SeMet and SeEt were achieved. The established methods have been tested for its applicability to the analysis of urinary selenium species in healthy people. The daily change of selenium species concentration in urine samples taken from the tested individuals after uptaking selenium supplement was monitore

    第一章 前言……………………………………………………… 1 1.1硒元素物種分析的重要性…………………………………… 1 1.2物種的分析技術……………………………………………… 4 1.3研究的動機與目的…………………………………………… 9 第二章 儀器分析原理………………………………………….. 11 2.1高效液相層析…………………………………………….…… 11 2.2感應偶合電漿質譜儀分析法………………………………… 13 2.3 微同軸式霧化器……………………………………………… 24 第三章 實驗部分………………………………………………... 26 3.1儀器裝置……………………………………………………… 26 3.2試劑及用水…………………………………………………… 26 3.3 容器清洗……………………………………………………… 27 3.4層析流洗液的配置…………………………………………… 28 3.5 微分離管柱的使用及保存…………………………………… 28 3.6 ICP-MS最適化條件探討……………………………………… 29 3.7 實驗流程……………………………………………………… 30 第四章 結果與討論……………………………………………... 32 4.1層析分離條件之探討………………………………………… 32 4.2 ICP-MS最適化操作條件之探討……………………………… 39 4.3連線系統分析效能的評估…………………………………… 45 4.4 尿液樣品分析的可行性……………………………………… 47 第五章 結論……………………………………………………… 53 參考文獻………………………………………………………….. 54 表目錄 表1-1 ICP-MS測定時之各項主要質譜干擾…………………. 58 表2-1 RPLC常使用溶劑之種類………………………………. 59 表2-2 複合離子對待測元素所形成之質譜性干擾…………. 59 表4-1 利用陰離子層析對於硒物種的分析…………………. 60 表4-2 硒物種之化學型態及其酸解離常數…………………. 61 表4-3 氫氣流速的變化對78Se/82Se和 80Se/82Se同位素比值 的影響……………………………………………………….61 表4-4 Cone voltage的變化78Se/82Se和 80Se/82Se同位素比值的影響………………………………………………………. 62 表4-5 ICP-MS系統之操作參數…………………………….. 63 表4-6 AC-ICP-MS系統的分析特性………………………….. 64 表4-7 RPLC-ICP-MS系統的分析特性……………………….. 64 表4-8 文獻上利用HPLC配合不同儀器連線對硒的物種分析. 65 表4-9 本研究所建立之分析系統對水樣與尿液中硒物種分析所得的檢量線斜率………………………………….……. 66 表4-10 AC-ICP-MS與RPLC-ICP-MS對尿液標準參考樣品（NIST SRM 2670E）中硒物種的分析結果………….…. 66 表4-11 第一位受測者(A)尿中硒物種的分析結果…………… 67 圖目錄 圖1-1 硒在人體中的代謝途徑……………………………… 68 圖1-2 HPLC-ICP-MS在生物化學上的應用…………………… 69 圖2-1 典型高效液相色層分析儀的示意圖………………… 70 圖2-2 Micromass platform ICP-MS儀器構造圖…………… 71 圖2-3 ICP-MS之焰炬、氣流和感應磁場是意圖…………… 72 圖2-4 各元素在6680k之游離程度…………………………… 73 圖2-5 ICP-MS介面與六極柱碰撞腔結構圖………………… 74 圖2-6 離子偵測之原理與結構示意圖……………………… 75 圖3-1 Microbore HPLC-MCN-ICP-MS系統圖………………… 76 圖4-1 利用 microbore AC-ICPMS分析系統在不同流洗液組成之下進行硒物種分析……………………………………… 77 圖4-2 pH值與滯留時間之關係……………………………… 78 圖4-3 流洗液濃度與各硒物種滯留時間之關係…………… 78 圖4-4 流洗液流速的變化對硒物種滯留時間的影響……… 79 圖4-5 利用 microbore RPLC-ICPMS分析系統在不同流洗液組成之下進行硒物種分析…………………………………… 80 圖4-6 流洗液流速變化對硒物種滯留時間的影響………… 81 圖4-7 AC-ICP-MS分析系統中對樣品溶液中Se(IV)、TMSe及Se(VI)的分析…………………………………………… 82 圖4-8 RPLC-ICP-MS分析系統中對樣品溶液中SeCys、SeMet及SeEt的分析……………………………………………… 82 圖4-9 RF功率與訊號強度之關係…………………………… 83 圖4-10 霧化氣體流速與訊號強度之關係…………………… 83 圖4-11 氦氣流速與訊號強度之關係………………………… 84 圖4-12 氦氣流速與訊號強度之關係………………………… 84 圖4-13 氫氣流速與訊號強度之關係………………………… 85 圖4-14 Hexapole bias potential 與80Se訊號強度的關係 85 圖4-15 Cone voltage 對80Se訊號強度的關係……………… 86 圖4-16 利用AC-ICP-MS分析A尿液中硒物種所得的層析圖… 86 圖4-17 連續食用含硒的營養品的天數與人體代謝硒的濃度關係………………………………………………………….. 87 圖4-18 食用營養品前後尿中硒濃度的變化情況…………… 87

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