以固相萃取法結合線上衍生GC-MS檢測人類尿液中酚類內分泌干擾物

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鍾雙鴻(Shuang-Hung Chung) 查詢紙本館藏

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論文名稱

以固相萃取法結合線上衍生GC-MS檢測人類尿液中酚類內分泌干擾物

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摘要(中)

酚類內分泌干擾物 (endrocrine disrupters, EDs) 主要以雙酚A (Bisphenol A, BPA) 及烷基酚 (Alkylphenols, APs) 中壬基酚 (4-Nonylpheols, 4-NPs) 及辛基酚 (4-t-Octylphenol, 4-t-OP) 最為常見。烷基酚前驅物中烷基酚聚乙氧基醇纇 (Alkylphenol polyethoxylates, APnEOs) 及雙酚 A 廣泛應用於家庭及工業產品，於使用後經降解釋放至環境中。人體藉由攝取及接觸而遭受暴露，且已被證實干擾生物體的內分泌系統。故針對人類尿液進行暴露之研究。
　　本研究使用固相萃取法 (Solid Phase Extraction，簡稱SPE) 搭配線上 (on-line) 衍生GC-MS 檢測尿液中酚類內分泌干擾物，並利用實驗統計設計及變異數分析做最佳化的探討。最佳化實驗步驟為1 mL尿液經由酵素水解後，使用Agilent bond Elut PH固相吸附材料50 mg、流速0.5 mL/min、以2 mL 10 %甲醇 (水) 清洗及以1 mL 30 % 二氯甲烷 (異丙醇) 沖提後，吹氮回溶並添加10 mM TBA-OH衍生化試劑利用線上衍生裝置導入GC注射埠中，以250 oC及4 min進行衍生。
　　本實驗4-t-OP、4-NPs及BPA線性範圍0.3、0.5及0.1-100 ng/mL，回歸係數皆高於0.9998，偵測極限 (LOD) 範圍0.02-0.1 ng/mL間，精密度 (precision) 以相對標準差 (RSD) 表示，皆小於15 %，準確度 (accuracy) 以回收率表示，在76至109 %間，顯示此方法穩定且具有良好的再現性。在尿液檢驗上成功檢測173組樣本，檢測出尿液總濃度介於未檢出 (n.d) 至19.4 ng/mL (mean 4.7 ng/mL)，檢出率達60 %以上，估算人體每日暴露量 (estimated daily intakes, EDI) 為5.1-559.4 ng/kg-bw/day (mean 140.1 ng/kg-bw/day) 其中又以年齡層30-39歲暴露較為嚴重。

摘要(英)

The most common phenolic endocrine disruptors (EDs) are bisphenol A (BPA), 4-nonylphenols (4-NPs) and 4-t-octylphenol (4-t-OP). A precursor of alkylphenols and bisphenol A is widely used in household and industrial products. Because the use of these products release phenolic EDs into the environment, the human body could be exposed to phenolic EDs, usually via ingestion of contaminated food.
In this study, a solid-phase extraction (SPE) method coupled with on-line derivatization and gas chromatography-mass spectrometry (GC-MS) was developed and was able to detect phenolic EDs in urine. For optimal, the best experiment conditions were: after hydrolysis of the urine sample and elution with 3:7 dichloromethane/ isopropanol for SPE (Agilent bond Elut PH 50 mg, 1mL), the eluent was dried under nitrogen and reconstituted with TBA-OH to derivatize the analytes. The derivatized sample was analyzed by GC/MS.
In this study, the linearity of three analytes (4-t-OP, 4-NPs and BPA) ranged 0.3, 0.5 and 0.1 to 100 ng/mL, respectively. The regression coefficients (r2) exceeded 0.995, and the limit of detection (LOD) ranged from 0.02 to 0.1 ng/mL. The method had good accuracy (recovery 76 - 109 %) and precision (RSD < 15 %). Successfully detected analytes in 173 samples in human urine. The maximum amount detected in the real samples was 19.4 ng/mL (mean value 4.7 ng/mL). The detection rate exceeded 60 %. The estimated daily intakes ranged from 5.1 to 559.4 ng/kg-bw/day and the exposure to phenolic EDs were more significant between age 30-39.

關鍵字(中)

★ 酚類內分泌干擾物
★ 氣相層析質譜儀
★ 尿液
★ 固相萃取法
★ 估計每日攝入量

關鍵字(英)

★ phenolic endrocrine disrupter
★ GC-MS
★ urine
★ SPE
★ EDI

論文目次

摘要 I
Abstract III
謝誌 V
目錄 VII
圖目錄 XIII
表目錄 XV
第一章　前言 1
1-1　研究起源 1
1-2 研究目標 4
第二章　文獻回顧 7
2-1 環境荷爾蒙 7
2-1-1　環境荷爾蒙簡介 7
2-1-2　環境荷爾蒙作用機制 8
2-1-3　酚類內分泌干擾物 10
2-1-3-1　相關性質 10
2-1-3-2　雙酚Ａ (Bisphenol A) 11
2-1-3-3　烷基酚 (alkylphenols, APs) 12
2-1-3-4　酚類內分泌干擾物危害 14
2-1-4　相關檢測 (研究) 文獻 22
2-1-5　人體尿液含量及暴露情形 25
2-2　樣品前處理-固相萃取法 32
2-2-1　固相萃取法優點 32
2-2-2 固相萃取法步驟及選用 34
2-3　氣相層析質譜儀 37
2-3-1　離子阱質譜儀 39
2-4　衍生化技術 40
2-4-1　離子對試劑 41
2-4-2　離子對衍生化原理 42
2-4-3　線上衍生化技術 43
2-5　β-D-glucosidase及Steroid sulfatase 酵素水解 44
2-6　實驗設計 46
2-6-1 實驗設計基本原理 46
2-6-2 反應曲面法 (Response surface methodology, RSM) 47
2-6-3　Box-Behnken設計 48
2-6-4　迴歸模型 50
2-7　線性回歸 52
2-7-1　Mandel test 52
2-7-2　The Lack-of-Fit by ANOVA 54
2-8　基質效應 (Matrix effect) 56
第三章實驗步驟與樣品分析 59
3-1　實驗藥品與設備 59
3-1-1　實驗藥品 59
3-1-2　儀器設備 61
3-2 實驗步驟 62
3-2-1　標準品配製 62
3-2-2　標準品的測定 63
3-2-3　固相萃取及線上衍生化步驟 67
3-2-4　真實樣品之酵素水解與萃取淨化步驟 68
3-3樣品採集 69
第四章結果與討論 71
4-1　氣相層析質譜儀對酚類內分泌干擾物的測定 71
4-1-1衍生化效率之探討 71
4-1-2　烷基化待測物分析 73
4-1-3　烷基化待測物質譜圖 74
4-1-4　檢量線及偵測極限 75
4-2　固相萃取法最佳化條件探討 76
4-2-1　固相萃取吸附劑選擇 76
4-2-2　沖提溶劑及清洗溶劑比例之選擇 77
4-2-3　固相萃取法實驗設計最佳化 80
4-3　Mandel test & Lack of fit 85
4-3-1　Mandel’s fitting test 85
4-3-2　Lack-of-fit 86
4-4　尿液樣品酵素水解探討 87
4-5　真實樣品之檢測 89
4-5-1　人體尿液檢測 89
4-5-2　人類每日暴露量 92
4-5-3　方法確效 95
4-5-4　基質效應檢視 96
第五章　結論 99
第六章　參考文獻 101

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指導教授

丁望賢(Wang-Hsien Ding)

審核日期

2015-6-26

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