臺灣博碩士論文加值系統

人造皮製造業勞工尿液中代謝物之固相微萃取氣相層析質譜分析方法研究
目的：二甲基甲醯胺(N,N-dimethylformamide ; DMF)為工業中常使用的溶劑，特別在於人造皮製造業中。目前對於DMF的所常用生物暴露指標為尿中的NMF(N,N-methylformamide)。本研究擬開發一個以頂空固相微萃取吸附的方式，再以氣相層析質譜儀來分析尿液中DMF代謝物。
方法：本研究以Carbowax-Divinylbenzene(CW/DVB)纖維作為吸附介質。因DMF代謝物揮發性低，故執行分析前以加熱的方式增加其頂空濃度，再以頂空法萃取尿液中DMF代謝物。完成採集後的固相微萃取纖維以氣相層析質譜儀分析。實驗室技術建立時，係將DMF代謝物標準品添加於健康非暴露者尿液中，製備標準溶液進行分析。本研究將探討的參數包括：尿液樣本之固相微萃取pH值、加入鹽類(NaCl、KNO3、NaNO3、(NH4)2SO4)、溫度、攪拌(包括攪拌時的轉速、攪拌時間)對尿液中DMF代謝物萃取效率的影響，以及氣相層析質譜儀器分析條件設定。
結果：
1.SPME方法為：將pH值為7±0.2的4 mL尿液樣本置於15 mL的樣本瓶中，添加0.8 g的氯化鈉混合均勻，在60 ℃、轉速為1000 rpm的條件下，同時用CW/DVB 纖維進行頂空固相微萃取25分鐘，最後在氣相層析質譜儀注射口以240 ℃熱脫附 8分鐘。
2.在此萃取分析條件下，所求得的丙酮檢量線濃度範圍介於0.1582-31.64 µg/mL，DMF檢量線濃度範圍介於0.944-188.8 µg/mL，NMF檢量線濃度範圍介於1.992-398.4 µg/mL，其相關係數(r)皆在0.995 以上。此外，丙酮的方法偵測極限（MDL）為0.0177 µg/mL，DMF的方法偵測極限為0.432 µg/mL；NMF的方法偵測極限為0.446 µg/mL。
3. 本分析方法發現，丙酮、DMF、NMF 分別在15.82、0.791 µg/mL；94.4、4.72 µg/mL；199.2、9.96 µg/mL之濃度值的尿液樣本密封儲存於-20℃冰箱內，在四週內的回收率皆大於86 %。
結論：以HS-SPME-GC-MS的方式來分析尿液樣本中DMF代謝物，可省去繁雜的前處理步驟，同時其具有免溶劑、快速、方法簡便、以及不受樣本基質干擾等優點。
關鍵字：二甲基甲醯胺、生物暴露指標、固相微萃取、頂空法

SPME-GC/MS method development for synthetic leather worker urinary metabolites
Objective：N,N-dimethylformamide (DMF) has been used as a solvent in many industries, especially in artificial leather industry. Urinary N,N-methylformamide (U-NMF) is commonly used as the biomarker of DMF exposure. The purpose of this study is to develop an analytical method for determining the DMF metabolism using solid phase microextraction followed by gas chromatography/mass spectrometry analysis.
Method：The Carbowax-Divinylbenzene(CW/DVB) fiber was tested to be the most suitable material for sample extraction. Due to the characteristic of low vaporization of DMF metabolism, and to increasing the DMF metabolism consistency, heating procedure was executed prior to the analysis. The pretreated procedure was then used to extract DMF metabolism in urine by headspace extraction. The SPME fiber was inserted into the injection port of GC/MS and analyzed. The standard solution was prepared by spiking DMF metabolism standard into the blank pooled urine of unexposed person. The optimum extraction parameters investigated were: pH, salt (NaCl, KNO3, NaNO3, (NH4)2SO4), temperature, stirring speed, extraction time, as well as measurement condition of GC/MS.
Results：
1.The SPME method was placing 0.8 g NaCl and 4mL urine sample of pH=7±0.2 in an 15mL vial, stirring at 1000 rpm 60 ℃stirring plant, CW/DVB fiber was used to perform headspace solid-phase microextraction for 25 min and thermal desorption 240 ℃for 8 min.
2. For the quality control calibration, the concentration ranges of Acetone, DMF and NMF in urine were 0.1582-31.64 µg/mL, 0.944-188.8 µg/mL, and 1.992-398.4 µg/mL. The correlation coefficients(r) were all above 0.995. The method detection limit（MDL）for Acetone, DMF and NMF were 0.0177 µg/mL, 0.432 µg/mL and 0.446 µg/mL, respectively.
3. In the storage stability tests at -20 ℃, we found the recovery rates for the urine spiked with Acetone at 15.82、0.791 µg/mL, with DMF at 94.4、4.72 µg/mL, and with NMF at 199.2、9.96 µg/mL were all above 86 % within 4 weeks.
Conclusion：The analytical method of DMF metabolism by HS-SPME-GC-MS can be reduced complicated pretreated procedure, as well as having the advantage of solvent-free, rapidity, simplicity, and avoid interfering with a complex matrix.
Key word：N,N-dimethylformamide, biomarker, solid phase microextraction, headspace

目 錄
第壹章 序論...............................13
第一節 研究背景...........................13
第二節 研究目的...........................15
第三節 研究內容...........................15
第貳章 文獻探討...........................16
第一節 前言...............................16
第二節 物化性及使用.......................17
(一) DMF..................................17
(二) Acetone..............................17
第三節 DMF代謝探討 .......................18
(一) DMF代謝途徑...................18
(二) DMF代謝物比例及半衰期.........18
第四節 DMF吸收途徑及健康影響..............22
(一) DMF吸收途徑探討......................22
(二) DMF對健康的危害......................23
第五節 DMF暴露標準值......................25
第六節 傳統萃取檢體中DMF NMF的方法........26
第七節 固相微萃取(Solid-Phase Microextraction).......27
(一) 固相微萃取裝置.......................27
(二) 直接固相微萃取.......................28
(三) 頂空固相微萃取.......................29
(四) 固相微萃取操作程序...................30
(五) 影響固相微萃取效率的因素.............30
(六) 固相微萃取法於生物檢體的應用.........34
第叁章 材料設備與方法....................43
第一節 儀器設備與試藥.....................43
(一) 試藥 ................................43
(二) 設備 ................................44
第二節 儀器參數條件.......................47
(一) GC條件...............................47
(二) MS條件...............................47
第三節 研究架構...........................49
(一)空白尿液收集..........................49
(二)頂空固相微萃取方法建立................49
(三) 分析品質管制.........................54
第肆章 結果與討論........................60
第一節 樣本前處理及分析條件...............60
第二節 頂空固相微萃取方法建立.............61
(一) 固相微萃取纖維的選擇.................61
(二) 萃取時間.............................61
(三) 樣本體積對吸附量的影響...............62
(四) 脫附溫度探討.........................62
(五) 脫附時間探討.........................63
(六) 攪拌速度對萃取的影響.................63
(七) 樣本溫度探討.........................63
(八) 樣本pH值探討.........................64
(九) 加入鹽種的影響.......................64
(十) 加鹽量探討...........................65
第三節 分析品質管制.......................66
(一) 檢量線建立...........................66
(二) 分析之精密度與準確度.................66
(三) 方法偵測極限(MDL)....................66
(四) 樣本儲存穩定性.......................67
第四節 討論...............................68
第伍章 結論與建議........................94
(一) 結論與建議...........................94
(二) 未來發展.............................85
參考文獻..................................96

參考文獻
1. Yang JS, Kim EA, Lee MY, Park IJ, Kang SK. Biological monitoring of occupational exposure to N,N-dimethylformamide — the effects of co-exposure to toluene or dermal exposure. Int Arch Occup Environ Health 2000;73:463-470
2. Chang HY, Shih TS, Cheng CC, Tsai CY, Lai JS, Wang VS. The effects of co-exposure to methyl ethyl ketone on the biological monitoring of occupational exposure to N,N-dimethylformamide. Int Arch Occup Environ Health 2003;76:121-128
3. American Conference of Governmental Industrial Hygienists (2001). Threshold limit values for chemical substances and physical agents and biological indices. ACGIH 2001
4. Deutsche Forschungsgemeinschaft (2000). Maximum concentrations at the workplace and biological tolerance values for working materials. DFG 2000
5. Käfferlein HU, Angerer J. Simultaneous determination of two human urinary metabolites of N,N-dimethylformamide using gas chromatography-thermionic sensitive detection with mass spectrometry confirmation, Journal of Chromatography B. 1999;734:285-298
6. 林育群.張火炎: 建立唾液中二甲基甲醯胺職業暴露之生物指標分析方法。碩士論文 2003
7. 陳俊璋.王文忻: 聚脲樹脂及合成皮製造工廠勞工二甲基甲醯胺暴露研究。碩士論文 2000
8. 黃敬德.謝有容：固相微萃取法。科儀新知 1997;18(4):59-67
9. 黃敬德.謝有容：固相微萃取技術之原理及其應用。Chemistry(The Chinese Chem.Soc.,Taipei) 1998;56(4):311-318
10. 李德仁：固相微萃取樣本前處理技術應用於揮發性或半揮發性有機物質之GC分析。科儀新知 1998;19(6):16-25
11. Ulrich S, Solid-phase microextraction in biomedical analysis. Journal of Chromatography A 2000;902:167-194
12. Pawliszyn J. Solid Phase Microextraction: Theory and Practice. 1997
13. 鄭蓉瑛：PU人造皮製造業空氣中有機溶劑採樣分析方法研究。勞工安全衛生研究季刊 2003;9(1):51-65
14. Amato G, Grasso E, Longo V, Gervasi PG. Oxidation of N,N-dimethylformamide and N,N-diethylformamide by human liver microsomes and human recombinant P450s. Toxicology Letters 2001;124:11-19
15. 汪禧年.張吳名任.黃麗如.陳育瑾.楊惠如.林君黛：二甲基甲醯胺(職業性)暴露生物偵測分析方法之探討及驗證。勞工安全衛生研究季刊 1997;5(4):79-93
16. N,N-dimethylformamide: chronic toxicity summary. Determination of Noncancer Chronic Reference Exposure Levels Batch 2A.2000
17. N,N-dimethylformamide 物質安全資料表
18. 丙酮物質安全資料表
19. Lareo AC, Perico A, Bavazzano P, Soave C, Perbellini L. Biological monitoring of workers exposed to N,N-dimethylformamide. I. Methods of analysis. Int Arch Occup Environ Health 1995;67:41—46
20. Schailteur V, Hoffmann De E, Buchet JP, Lauwerys R. Study on in vivo and in vitro metabolism of dimethylformamide in male and female rats. Toxicology 1984;29:221-234
21. Wrbitzky R, Angerer J. N, N-Dimethylformamide - influence of working conditions and skin penetration on the internal exposure of workers in synthetic textile production. Int Arch Occup Environ Health 1998;71:309-316
22. Kimmerle G, Eben A. Metabolism studies of N,N-dimethylformamide studies in persons, Int Arch Arbeitsmed 1975;34:127-136
23. Mráz J, Nohová H. Absorption, metabolism and elimination of N,N-dimethylformamide in humans. Int Arch Occup Environ Health 1992;64:85-92
24. 朱祐民.張火炎: 應用跟暴露方式探討人造皮工廠暴露環境下N,N-dimethylformamide(DMF)不同暴露途徑之生物偵測。碩士論文 2003
25. Lauwerys R. Al. Biological surveillance of workers exposed to dimethylformamide and the influence of skin oritection on its percutaneous absorption. Int Arch Occup Environ Health 1980;45:189-203
26. Brugnone F, Perbellini L, Gaffuri E. N,N-dimethylformamide concentration in environment and alveolar air in an artificial leather factory. British Journal of Industrial Medicine 1980;37:185-188
27. Maxfield M, Barnes JR, Azar A. Urinary excretion of metabolite following experimental human exposures to DMF or to DMAC. J Occup Med 1975;17:506-511
28. Mráz J, Nohová H. Percutaneous absorption of N,N-dimethylformamide in humans. Int Arch Occup Environ Health 1992;64:79-83
29. Nomiyama T, Nakashima H, Chen LL, Tanaka S, Miyauchi H, Yamauchi T, Sakurai H, Omae K. N,N-dimethylformamide: Significance of dermal absorption and adjustment method for urinary N-methylformamide concentration as a biological exposure item. Int Arch Occup Environ Health 2001;74:224—228
30. Stula EF, Krauss WC. Embroytoxicity in rats and rabbits from cutaneous application of amidtype solvents and subsituted ureas. Toxicology and Applied Pharmacology 1977;41:35-55
31. Hellwig J, Merkle J, Klimisch HJ, Jackh R. Studies on the prenatal toxicity of N,N-dimethylformamind in mice,rats and rabbit. Fd chem Toxicity 1991;29:193-201
32. Kelich SL, Mercieca MD, Pohland RC. Developmental toxicity of N-methylformamide administered by gavage to CD rats and New Zealand white rabbits. Fundamental and Applied Toxicology 1995;27:239-246
33. Cai SX, Huang MY, Xi LQ, Li YL, Qu JB, Kaiwai T, et al. Occupational dimethylformamide exposure. 3. Health effects of dimethylformamide after occupational exposure at low concentrations. Int Arch Occup Environ Health 1992;63:461-468
34. Scalilteur V, Lauwerys R. Dimethylformamide(DMF) hepatotoxicity. Toxicology 1987;43:231-238
35. Wrbitzky R. Liver function in workers exposed to N,N-dimethylformamide during the production of synthetic textiles. Int Arch Occup Environ Health 1999;72:19-25
36. Angerer J, Göen T, Krämer A, Käfferlein HU. N-methylcarbamoyl adducts at the N-terminal valine of globin in workers exposed to N,N-dimethylformamide. Arch Toxicol 1998;72:309-313
37. Lyle WH, TWM Spence, WM Mckinneley, Ducker K. Dimethylformamide and alcohol intolerance. British Journal of Industrial Medicine 1979;36:63-66
38. Supelco, SPME application Guide.1999
39. SUPELCO：2000 Chromatography Products for Analysis and Purification. Bellefonte, PA: Sigma-Aldrich Co., 2000
40. Lee MR, Yeh YC, Hsiang WS, Chen CC. Application of solid-phase microextraction and gas chromatography-mass spectrometry for the determination of chlorophenols in urine. Journal of Chromatography B 1998;707:91-97
41. Centini F, Masti A, Comparini B. Quantitative and qualitative analysis of MDMA, MDEA, MA and amphetamine in urine by head-space/solid phase micro-extration (SPME) and GC/MS. Forensic Science International 1996;83:161-166
42. Snow NH. Solid-phase micro-extration of drugs from biological matrices (Review). Journal of Chromatography A 2000;885:445-455
43. Augusto F, Valente ALP. Applications of solid-phase microextraction to chemical analysis of live biological samples. Trends in Analytical Chemistry 2002;21:428-438
44. Käfferlein HU, Göen T, Müller J, Wrbitzky R, Angerer J. Biological monitoring of workers exposed to N, N-dimethylformamide in the synthetic fiber industry. Int Arch Occup Environ Health 2000;73:113-120
45. Imbriani M, Maestri L, Marraccini P, Saretto G, Alessio A, Negri S, Ghittori S. Urinary determination of N-acetyl-S-(N-methylcarbamoyl)cysteine and N-methylformamide in workers exposed to N, N-dimethylformamide. Int Arch Occup Environ Health 2002;75:445-452
46. 黃德琪.林宜長：以頂空固相微萃取法分析勞工尿液中1,4-二氯苯與其代謝物2,5-二氯酚的含量。碩士論文 2002