臺灣博碩士論文加值系統

呋喃甲醛(furfural)被廣泛應用在化學石油工業上且為已知的食物性致突變物質，同時具有基因毒性，其暴露途徑包含吸入、皮膚接觸及食入。為了保護大眾的健康，降低勞工作業場所或一般環境下的致癌風險及基因毒性的影響，進行呋喃甲醛的暴露評估有其必要性。因此，本研究希望利用固相微萃取 (solid-phase microextraction, SPME) 與氣相層析/質譜儀（GC/MS）之結合，建立呋喃甲醛尿中代謝物與肌酸酐的分析技術，以發展方便、敏感且正確的生物偵測方法，進而提供呋喃甲醛暴露評估工具。
首先製備濃度為0.1∼3倍furfural BEIs (Biological Exposure Indices)的添加樣本，再利用固相微萃取技術進行頂空吸附，並以GC/MS分析。萃取過程中，除了測試包括: PA、PDMS、PDMS/DVB、CAR/PDMS、CW/DVB等纖維的吸附效果外，其他如:溫度、攪拌速度、離子及含鹽量等可能影響纖維吸附量的因素，亦都是研究中探討的重點。肌酸酐的分析亦利用已知濃度的添加樣本進行測試，但進行分析之前，已吸附肌酸酐的SPME纖維需先以無水三氟醋酸（trifluoroacetic anhydride, TFAA）頂空裹附，經與肌酸酐反應生成衍生物(trifluoroacetamide)後，再以GC/MS進行分析。
試驗結果顯示使用65m PDMS/DVB纖維最符合研究所需。關於肌酸酐的分析，可直接將纖維插入水樣本中，於室溫環境下配合1200 rpm的電磁攪拌速度，讓纖維在樣本中停留60sec，接著取出纖維並插入另一個置有100L TFAA的4mL vial中並停留30sec，再取出纖維另插入一個空的4mL vial中並等待10min後，以GC/MS在注射口溫度230C下進行分析(m/z=95、112)。至於呋喃甲酸的偵測，亦可使用 PDMS/DVB纖維在室溫下直接插入樣本並停留60sec進行吸附後(轉速 1200 rpm)，以GC/MS分析(m/z=95、112)。當纖維在注射口溫度230 C停留5min，其脫附效率>95%。本研究的呋喃甲酸的檢量線範圍: 0.03∼0.89 g/L (R2=0.996)，約等於0.1 ~ 3倍的BEIs值，分析準確度在±10％以內。分析變異係數CVa％為2.40％。方法偵測極限為19.42 μg，儀器偵測極限為0.26μg；肌酸酐的檢量線範圍: 0.32∼3.12 g/L (R2>0.995)，為有效尿液的肌酸酐濃度範圍。分析變異係數CVa％為3.5％。方法偵測極限為173.63μg，儀器偵測極限為0.26μg。不同條件對萃取呋喃甲酸的影響發現添加氯化鈉及亞硫酸鈉兩種鹽類及加溫、攪拌樣本均會增加纖維吸附呋喃甲酸的量；肌酸酐部分則發現添加氯化鈉、硫酸鈉及亞硫酸鈉會降低纖維萃取肌酸酐衍生物的量。
本研究發現固相微萃取技術可應用於尿液中呋喃甲酸及肌酸酐之分析。與現有方法相比較，本研究能有效縮短樣本前處理及分析時間並減少溶劑的使用，且與肌酸酐現有分析方法的平行比對具有一致性；然而在不更換衍生試劑下，本研究的方法並無法直接進行兩者的同步分析。可能的改善方式是先分析樣本中的呋喃甲酸濃度，再利用呋喃甲酸與肌酸酐混合經衍生後的檢量線來推算肌酸酐的濃度。

Furfural is widely used in petrochemical industry, and is a known dietary mutagens. The exposure routes of furfural include inhalation, skin contact and ingestion. Furfural is an irritant of the eyes, mucous membranes, and skin. Besides, furfural is a confirmed animal carcinogen. To perform the exposure assessment of furfural, biological monitoring is a better tool since inhalation is not the only route of exposure. However, there are drawbacks of the current methods. In recent years, a new analytical technique called solid-phase microextraction (SPME) has been developed which is favorable in terms of solvent-free and convenience. Therefore the purpose of this study was to develop a new analytical method for furoic acid and creatinine in urine based on SPME, to provide a better tool for the exposure assessment of furfural.
Known concentrations of furoic acid equal 0.1∼ 3 times BEIs (Biological Exposure Indices) of furfural were prepared in spiked urine samples. Headspace adsorptions were performed followed by the GC/MS analysis. Including 85m PA, PDMS, PDMS/DVB, CAR/PDMS, and CW/DVB were tested for the abilities of adsorption. Besides, temperatures, speeds of magnetic stirring, and pH values were all the factors that needed to be investigated. For the analysis of creatinine, known concentrations of spiked urine samples were also prepared. However, trifluoroacetic anhydride (TFAA) must be loaded onto the fiber after the adsorption of creatinine. The derivatives from the reaction of TFAA and creatinine were then analyzed by GC/MS.
The PDMS/DVB was selected for this research. For the analysis of creatinine, the SPME fiber was first directly immersed into a spiked water sample for 60 sec with 1200 rpm magnetic stirring. The fiber was then transferred to another 4 mL vial (which was filled with 100 L of TFAA) and stood for 30sec. Afterward the fiber was transferred to another blank 4 mL vial again and stood for 10 min before the analysis by GC/MS (DB-WAX column, injector temp.=230C, m/z=95,112). The detection of furoic acid was also performed by the adsorption of PDMS/DVB with direct urine sample immersed for 30sec (1200 rpm) followed by the GC/MS analysis. The desorption efficiency > 95% when the SPME fiber was stayed in the injector for 5 min (230C). The concentrations of the spiked furoic acid standards ranged from 0.03~0.89 g/L (R2=0.996) which was equal to 0.1~3 times BEIs. And the concentrations of the spiked creatinine standards ranged from 0.32∼3.12 g/L (R2>0.995). Besides, increasing temperatures, using magnetic stirring, and adding salts(sodium chloride、sodium sulfite) will increase the adsorption efficiency of fiber for the analysis of furoic acid. On the other hand , the adding of salts( sodium chloride、sodium sulfite and sodium sulfate) will decrease the adsorption efficiency of fiber for the analysis of creatinine.
The current SPME method has the advantages of solvent-free and easy-operation. The analysis of furoic acid and creatinine in urine by SPME were found to be possible. Besides, parallel comparisons for the analysis of creatinine between the SPME method and Jeffe’s method also showed consistency (R2=0.988).However , the analysis of creatinine and furoic acid simultamously was not possible due to theproblem of poor co-elution.

摘要 Ⅰ
Abstract Ⅱ
目錄 Ⅲ
表目錄 Ⅳ
圖目錄 Ⅴ
目次 頁次
第一章 研究概述
第一節 研究動機 1
第二節 研究目的 3
第三節 研究架構 4
第二章 文獻探討
第一節 醛類---呋喃甲醛 5
2-1.1 理化特性 5
2-1.2 暴露來源與用途 7
2-1.3 體內吸收、分佈、代謝及排除 9
2-1.3.1 動物試驗 9
2-1.3.2 人體暴露資料 11
2-1.5 健康危害特性 13
2-1.5.1基因毒性研究 13
2-1.5.2急毒性研究 15
2-1.6 暴露限制 15
2-1.7 偵測與分析 15
2-1.7.1空氣樣本採樣分析 15
2-1.7.2 生物偵測之尿液採樣 17
第二節 固相微萃取
2-2.1固相微萃取纖維及其選擇性 20
2-2.1.1 纖維的手動裝置（Fiber assemblies and holders） 20
2-2.2萃取方法的選擇 25
2-2.3固相微萃取平衡理論 27
2-2.4 萃取情形的最佳化 29
2-2.5 固相微萃取應用情形 30
第三節 氣相層析的衍生（Derivatives for chromatography）
2-3.1 衍生的原因 31
2-3.2 衍生試劑的選擇 31
2-3.3 SPME的衍生方式 32
2-3.4衍生反應 32
第四節 生物偵測（Biological Monitoring） 38
第三章 材料與方法
第一節 實驗器材 41
3-1.1 藥品試劑 41
3-1.2 儀器設備 41
第二節 儀器、玻璃器皿及試劑的使用 43
3-2.1 玻璃器皿清洗 43
3-2.2 分析儀器使用 43
3-2.3 試劑的配製 43
第三節分析方法 44
3-3.1 方法介紹及其原理 44
3-3.2樣本的前處理 44
3-3.2.1 纖維吸附方式 44
3-3.3 熱脫附的條件 45
3-3.4 建立檢量線 47
3-3.5 GC-MS分析條件 48
3-3.6分析方法驗證 49
3-3.6.1 平均相對誤差 49
3-3.6.5 纖維的脫附效率 50
第四章 結果與討論
第一節 纖維測試情形 51
第二節 呋喃甲酸的品保品管分析 51
4-2.1 呋喃甲酸之分析方法驗證 51
4-2.1.1 檢量線製作 51
4-2.1.2 平均相對誤差 54
4-2.1.3 分析變異係數CVa 54
4-2.1.4 偵測極限 54
4-2.1.5 脫附效率 54
第三節 肌酸酐的品保品管分析 59
4-3.1 肌酸酐之分析方法驗證 59
4-3.1.1 檢量線製作 59
4-3.1.2 分析變異係數CVa 59
4-3.1.3 偵測極限 61
4-3.1.4 脫附效率 61
4-3.3 與其他方法比對 63
第四節不同條件對萃取呋喃甲酸的影響 66
4-4.1含鹽量及不同離子對SPME萃取吸附量的影響 66
4-4.2 溫度對SPME萃取吸附量的影響 66
4-4.3 不同吸附時間對SPME萃取吸附量的影響 66
4-4.4 攪拌對SPME萃取吸附量的影響 67
第五節不同條件對萃取肌酸酐的影響 71
4-5.1 含鹽量及不同離子對SPME萃取吸附量的影響 71
第五章 結論與建議
第一節 討論 73
5-1.1呋喃甲酸（2-furoic acid） 73
5-1.2 肌酸酐(creatinine) 73
5-2.3 呋喃甲酸與肌酸酐同步分析的可行性與有待解決之處 75
第二節 結論 78
5-2.1 呋喃甲酸（2-furoic acid） 78
5-2.2 肌酸酐(creatinine) 78
參考文獻 79
表目錄
表2-1.1、呋喃甲醛之基本特性 6
表2-1.2、呋喃甲醛在工業上的使用(英國) 8
表2-1.3、呋喃甲醛之基因毒性研究 14
表2-1.4、呋喃甲醛之急毒性(動物試驗) 16
表2-1.5、呋喃甲酸與肌酸酐之基本特性 19
表2-2.1、市售SPME纖維規格 22
表2-2.2、市售SPME纖維種類 23
表2-3.1、衍生試劑選用指標 33
表2-4.1、物質的半衰期與採樣時間之選擇 40
表4-1.1、不同SPME纖維測試情形 52
表4-1.2、2-furoic acid分析之精密度（平均變異係數％） 55
表4-1.3、2-furoic acid分析之脫附效率
（不同濃度，樣本介質為水） 55
表4-1.4、2-furoic acid分析之脫附效率
（不同濃度，樣本介質為尿液） 57
表4-1.5、2-furoic acid分析之脫附效率
（不同脫附時間，樣本介質為水） 58
表4-1.6、2-furoic acid分析之脫附效率
（不同纖維，樣本介質為水） 57
表4-3.1、creatinine衍生物分析之脫附效率
（不同濃度，樣本介質為尿液，頂空萃取） 62
表4-3.2、creatinine衍生物分析之脫附效率
（不同濃度，樣本介質為尿液，直接萃取） 62
圖目錄
圖2-1.1a、Furfural在體內代謝情形(含動物) 10
圖2-1.1b、Furfural在人體代謝情形 12
圖2-2.1、SPME裝置設計圖 21
圖2-2.2、Fiber holder disassembles for fiber replacement (left) and fiber holder for manual sampling(right) 24
圖2-2.3、固相微萃取的萃取方式 26
圖2-3.1、利用固相微萃取（SPME）進行衍生的方式 34
圖2-3.2、肌酸酐與無水三氟醋酸（TFAA）衍生反應式及衍生物之質
譜圖 36
圖2-3.3、N-Methyl-N-[tert-butyldimethyl-silyl]trifluoroacetimide 結構 式 37
圖3-3.1、本研究之SPME採樣吸附系統 46
圖4-1.1、呋喃甲酸層析圖譜 53
圖4-1.2、呋喃甲酸檢量線 53
圖4-3.1、肌酸酐衍生物層析圖譜 60
圖4-3.2、肌酸酐衍生物檢量線 60
圖4-3.3、以Jeffe’s method量測尿中肌酸酐之實驗步驟 64
圖4-3.4、肌酸酐分析方法平行比對結果 65
圖4-4.1、添加不同鹽類及加溫對SPME萃取呋喃甲酸的影響 68
圖4-4.2、不同吸附時間對SPME萃取呋喃甲酸吸附量的影響 69
圖4-4.3、攪拌對SPME萃取呋喃甲酸的影響 70
圖4-5.1、添加不同鹽類對SPME萃取肌酸酐衍生物的影響 72
圖5-1.1、同步分析之採樣分析步驟 75
圖5-1.2、呋喃甲酸經TFAA衍生後之檢量線 76
圖5-1.3肌酸酐(呋喃甲酸濃度=353μg/mL)
經TFAA衍生之檢量線 76
圖5-1.4肌酸酐(呋喃甲酸濃度=1178.3μg/mL) 經TFAA衍生之檢量線 77
圖5-1.5肌酸酐(呋喃甲酸濃度=117.8μg/mL)
經TFAA衍生之檢量線 77

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