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研究生：	官大為 Kuan, Da-Wei
論文名稱：	開發以高效能液相層析儀/紫外光偵測器同步測定環境水體中與生物體尿液中多種胺基甲酸鹽及其代謝物之分析方法 The development of an analytical method to simultaneously determine multiple carbamates and their indicative metabolites in environmental surface water and urine specimen with high performance liquid chromatography / ultraviolet detector
指導教授：	桂椿雄 Kuei, Chun-Hsiung 張火炎 Chang, Ho-Yuan
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2003
畢業學年度：	91
語文別：	中文
論文頁數：	75
中文關鍵詞：	加保利、固相萃取、自由型態、高效能液相層析、尿液、安丹、環境水體、胺基甲酸鹽、加保扶、代謝物
外文關鍵詞：	carbaryl, free form, HPLC, urine, surface water, solid phase extraction, metabolites, propoxur, carbofuran, carbamates
相關次數：	點閱：102 下載：3
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　　本研究利用高效能液相層析儀/紫外光偵測器(簡稱HPLC/UV)開發一種分析方法，得簡易又快速的前處理即可同步分析環境中水體及生物尿液樣本中加保利、加保扶、安丹等三種胺基甲酸鹽本體及1-naphthol、3-hydroxy carbofuran、3-keto carbofuran、2-isopropoxy phenol等四種代謝物。

　　本研究初步將七種胺基甲酸鹽及其代謝物之標準品溶入純乙腈溶劑中發現在HPLC/UV之分析圖譜因帯寬及不對稱而無法定性定量。將溶劑置換成高極性之溶劑後分析圖譜之表現即大量提升，可定性定量且可大量樣品注射，此乃本研究推論因分析物於分析過程中聚焦效應造成的。此技術直接應用於受污染河川水體中，發現經簡單過濾後，本方法偵測下限可達5μg/L再現性為94%以上，可適用一般環境水體之胺基甲酸鹽農藥殘留之檢驗。

　　在生物尿液樣本，本研究以Wistar鼠灌食胺基甲酸鹽本體前後之尿液測試否有胺基甲酸鹽本體及其主要代謝物之自由型態(free form)。以口服半致死量之1/10濃度進行灌食實驗發現七種胺基甲酸鹽及其代謝物皆能在鼠尿中發現自由形態，經以C18逆相固相萃取及吹氮濃縮之前處理進行HPLC/UV分析後，本研究發現可有效降低尿液中之基值干擾且回收率可達79%以上，CV%為12%以下。由動物實驗可知，應用本分析方法可成功將暴露前後之尿中胺基甲酸鹽及其代謝物清楚的區別，並可將暴露後之胺基甲酸鹽及其代謝物予以定量。因此本研究結論為本方法應可廣泛應用於環境水體中與生物尿液中之三種胺基甲酸鹽及其四種代謝物予以定性暨定量。

The aim of this study was to develop an analytical method to simply and rapidly determine three parent compounds of carbamates (carbaryl, carbofuran and propoxur) and their corresponding indicative metabolites (1-naphthol, 3-hydroxy carbofuran, 3-keto carbofuran and 2-isopropoxy phenol) in environmental surface water and urine specimen simultaneously by using high performance liquid chromatography / ultraviolet detector (HPLC/UV).

Seven compounds of interest prepared in pure acetonitrile in the beginning of the experiment were found to fail in neither qualitative nor quantitative determination due to broaden peaks and imbalanced peaks in HPLC/UV chromatogram. After changing from the less polar solvent to the highly polar solvent (H2O), the chromatographic performance was found to be significantly improved to accommodate the need of qualitative and quantitative determination. Moreover, it can also allow larger volume injection to improve the method of the sensitivity. Using this technology, we found with simple filtration this method was able to quantitatively determine the amount of parent compounds and their indicative metabolites of carbamates of interest in the water collected from a polluted river. The limit of the detection and the reproducibility for the environmental surface water was 5μg/L and >94%, respectively.

For the biological urine specimen, this study exposed the Wistar rats to three carbamates of interest to explore whether their existence in free form could be detected in rats’ urine prior to and posterior to the exposure. The rats were orally administered to a tenth of the 50th lethal dose and we found all seven free-form carbamate compounds of interest can be detected in rats’ urine. Using reverse-phase solid-phase extraction with C18 and nitrogen blow enrichment procedure along with HPLC/UV analysis, we found this sample preparation procedure was able to significantly reduce the matrix effect of the urine samples and yielded the recovery of more than 79% with CV% less than 12%. Based on the affirmative results in the animal experiments, this method was proved to be able to make clear distinction in the urine collected prior to and posterior to the exposure for seven parent compounds and their indicative metabolites of carbamates. We, therefore, concluded that the methods established by this study should be able to be applied successfully in the determination of the three parent compounds and their indicative metabolites of carbamates of interest qualitatively and quantitatively for environmental surface water and biological urine samples.

中文摘要………………………………………………………………Ⅰ
英文摘要………………………………………………………………Ⅲ
目錄……………………………………………………………………Ⅴ
表目錄…………………………………………………………………Ⅷ
圖目錄………………………………………………………………ⅩⅠ


第一章　緒論……………………………………………………………1
　　1-1 前言……………………………………………………………1
　　1-2 胺基甲酸鹽之簡介……………………………………………5

第二章　文獻回顧
　　2-1 胺基甲酸鹽的特性……………………………………………11
　　　　2-1-1 尿液方面………………………………………………11
　　2-2 比較不同的分析方法…………………………………………16
　　　　2-2-1 環境方面………………………………………………16
　　　　2-2-2 尿液方面………………………………………………19

第三章　研究方向………………………………………………………23
　　3-1 研究目標………………………………………………………23
　　3-2 研究內容………………………………………………………24

第四章　實驗部分
　　4-1 建立分析方法…………………………………………………26
　　　　4-1-1 藥品及儀器……………………………………………26
　　　　4-1-2 HPLC的簡介及原理……………………………………28
　　　　4-1-3 分析方法開發…………………………………………30
　　4-2 前處理純化……………………………………………………34
　　　　4-2-1 儀器及藥品……………………………………………34
　　　　4-2-2 前處理純化……………………………………………34
　　　　4-2-3 樣品添加………………………………………………36
　　4-3 分析真實樣品…………………………………………………37
　　　　4-3-1 應用在環境水樣………………………………………37
　　　　4-3-2 應用在尿液樣本………………………………………38

第五章　結果及討論
　　5-1 建立分析方法…………………………………………………46
　　　　5-1-1 分析物最大吸收波長…………………………………46
　　　　5-1-2 Focus 效應……………………………………………47
　　　　5-1-3 定性、定量及偵測下限………………………………53
　　5-2 環境水樣………………………………………………………55
　　　　5-2-1 建立檢量線……………………………………………55
　　　　5-2-2 方法偵測下限（MDL）……………………………… 56
　　　　5-2-3 環境真實樣本…………………………………………56
　　　　5-2-4 總結……………………………………………………57
　　5-3 尿液樣本………………………………………………………59
　　　　5-3-1 建立檢量線……………………………………………59
　　　　5-3-2 添加水樣………………………………………………59
　　　　5-3-3 尿液添加………………………………………………61
　　　　5-3-4 動物實驗………………………………………………63

第六章　結論……………………………………………………………70

第七章  參考文獻………………………………………………………72


表目錄

表1.1  胺基甲酸鹽種類及化學通式之相關性………………………9
表1.2  九種胺基甲酸鹽類農藥物理化學性質…………………… 10
表2.1.1  Off-line SPE of Carbamates for Water
……………………………………………  17
表2.1.2  On-line SPE of Carbamates and Metabolites for Water……………………………………18
表2.2.1  正常人體的尿液物理特性………………… 20
表2.2.2  尿液成分:成年男性，尿液體積1~2公升… 20
表4.1.1  實驗中所使用到的藥品…………………… 26
表4.1.2  實驗中所使用的儀器……………………… 27
表4.1.3  本研究之三種胺基甲酸鹽本體與四種代謝物之結構與分子式…………………………… 31
表4.1.4  HPLC/UV之分析操作條件及動相Gradient條件…………………………………………… 33
表4.2.1  前處理所需藥品、溶劑及儀器………………34
表4.3.1  動物實驗所需藥品及器材………………… 43
表4.3.2  胺基甲酸鹽農藥之LD50……………………44
表4.3.3  胺基甲酸鹽之Wistar鼠灌食尿液收集體積(mL)與時間表……………………………… 45
表5.1.1  七種胺基甲酸鹽之最大吸收波長………… 46
表5.1.2  添加七種胺基甲酸鹽及其代謝物10μg/L之滯留時間(SD)、peak area(CV%)(注射量100μL)…………………………………………54
表5.1.3  七種胺基甲酸鹽及其代謝物最佳分析條件及IDL………………………………………… 54
表5.2.1  七種胺基甲酸鹽及其代謝物之MDL……… 56
表5.2.2  研究開發之方法與其它之方法的比較
………………………………………………58
表5.3.1  以二次去離子水添加七種胺基甲酸鹽及代謝物20 μg/L、100 μg/L、400μg/L於水中的回收率(平均CV%)………………………59
表5.3.2  以空白尿液添加七種胺基甲酸鹽及代謝物之不同濃度20μg/L、100μg/L、400μg/L的回收率(平均CV%)…………………………62
表5.3.3  Wistar所需灌食三種胺基甲酸鹽本體的劑量表……………………………………………63
表5.3.4  Wistar鼠口服農藥實驗之暴露前後的最高濃度及時間……………………………………65


圖目錄
圖2.1.1  代表胺基甲酸鹽代謝途徑的通式…………12
圖 3.1  研究架構……………………………………………………25
圖 4.1.1   HPLC/UV儀器示意圖……………………………………27
圖4.2.1 前處理（ＳＰＥ）步驟………………………35
圖4.3.2-1  carbaryl在動物之代謝途徑圖…………40
圖4.3.2-2  carbofuran在動物之代謝途徑圖………41
圖4.3.2-3  propoxur在動物之代謝途徑圖…………42
圖5.1.1-1  濃度:1ppm(100%CH3CN),注射量:100μL
          …………………………………………… 50 
圖5.1.1-2  濃度:1ppm(CH3CN:H2O=10%:90%),注射量:100μL……………………………… 50
圖5.1.1-3  100%CH3CN與CH3CN:H2O=10%:90%之疊圖…
………………………………………… 51
圖5.1.2-2  樣品溶液組成CH3CN:H2O=1:9，七種胺基甲酸鹽及代謝物之標準層析圖……………52
圖5.2.1    七種胺基甲酸鹽分析物之環境水樣檢量線…………………………………………55

圖 5.2.2  環境水體空白圖譜…………………………56
圖 5.2.3  添加七種胺基甲酸鹽之分析物50μg/L於環境水體中…………………………………… 57
圖5.2.4   添加七種胺基甲酸鹽之分析物500μg/L於環境水體中…………………………………………… 57
圖 5.3.1  七種胺基甲酸鹽及代謝物之檢量線高(右)低(左)濃度配置圖…………………………… 60
圖5.3.2.1  Wistar大鼠灌食carbaryl後，所代謝成 1-naphthol之時間vs濃度累積百分率圖………………………………………… 67
圖5.3.2.2  Wistar大鼠灌食propoxur後，尿液殘留propoxur本體之時間vs濃度累積百分率圖………………………………………… 67
圖5.3.2.3  Wistar大鼠灌食propoxur後，所代謝成2-isopropoxy phenol之時間vs濃度累積百分率…………………………………… 67
圖5.3.2.4  wistar鼠尿液中含有propoxur及2-isopropoxy phenol與總量之時間vs濃度累積百分率………………………68
                                    

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