以頂空固相微萃取法配合離子阱氣相層析質譜儀偵測尿液中的克倫特羅

簡易檢索 / 詳目顯示

回結果列表

研究生：	徐偉勛 Wei-Hsun Hsu
論文名稱：	以頂空固相微萃取法配合離子阱氣相層析質譜儀偵測尿液中的克倫特羅 Determination of Clenbuterol in Urine by Head-space Solid Phase Microextraction and Gas Chromatograph-Ion Trap Mass Spectrometer
指導教授：	黃賢達 Shang-Da Huang
口試委員:
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：	2004
畢業學年度：	92
語文別：	中文
論文頁數：	71
中文關鍵詞：	氣相層析質譜儀、衍生化、克倫特羅
外文關鍵詞：	GC-MS, Derivatives, Clenbuterol
相關次數：	點閱：1 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

本論文主要是利用頂空固相微萃取法配合氣相離子阱層析質譜儀（HSSPME-GC/ITMS）並使用衍生化法來分析人體尿液中的一種合成代謝劑：克倫特羅（Clenbuterol）。

固相微萃取法是近年來所使用的一種前處理技術，它可以一次做好採樣、萃取、濃縮以及樣品導入的步驟，讓我們在做前處理時能更方便以及快速。而本論文的目的就是在利用這種技術再配合氣相層析質譜儀分析人體尿液中克倫特羅的含量。這篇所探討的重心在各種影響萃取效率的變因做探討，包括；萃取所用的纖維種類、待測物的萃取溫度、萃取時間與冷卻系統、脫附溫度和脫附時間、溶液的攪拌速率與酸鹼度、樣品中的離子強度等八項。以求得最佳的實驗條件，再用這條件（纖維：PA、待測物萃取時間、溫度：90分鐘、90℃、待測物上端溫度5℃、衍生劑萃取時間、溫度：5分鐘、70℃、樣品攪拌速率：300rpm、樣品酸鹼值：12、離子強度：添加30％的氯化鈉、脫附時間、溫度：3分鐘、260℃）來應用在尿液中。

而本論文最後是使用PA纖維來做偵測。由實驗結果可以發現其偵測極限可達到ng/mL以下的層級，且在1~1000 ng/mL的濃度範圍下能擁有相當高的線性相關係數（0.9990）。而且精密度可以在4％以下。可證實此方法用於偵測克倫特羅的可行性。

In this thesis, we develop a method of using headspace solid-phase microextraction (HS-SPME) combined with gas chromatograph-ion trap mass spectrometer (GC-ITMS) to detect a β-agonist : clenbuterol.

We discuss and optimize eight parameters of extraction and desertion procedure, including types of fibers, extraction temperature, extraction time, desorption temperature, desorption time, stir rate, ionic strength, pH of sample and cooling system.

The method show good linearity over the concentration range 1–1000 ng/mL and gives detection limits below ng/mL level. Precision is also good (3.89%). Finally, the method is successfully applied to analyze clenbuterol in urine.

一、緒論..............................................1
1-1前言...............................................1
1-2合成代謝劑.........................................3
1-3固相微萃取法.......................................4
1-3-1固相微萃取裝置演進...............................4
1-3-2固相微萃取的實驗步驟.............................4
1-3-3固相微萃取的原理.................................4
1-3-4操作模式.........................................6
1-3-5固相微萃取的應用.................................7
1-4衍生化法...........................................9
1-5 Clenbuterol的檢測方法............................10
1-5-1酵素免疫連結吸附分析法..........................10
1-5-2高效能液相層析法................................10
1-5-3氣相層析法......................................11
1-6研究目的..........................................13
二、實驗部分.........................................14
2-1實驗藥品..........................................14
2-2實驗裝置..........................................14
2-3實驗儀器..........................................15
2-4標準溶液..........................................16
2-5真實樣品..........................................17
2-6實驗步驟..........................................17
三、結果與討論.......................................20
3-1各種變因之探討....................................20
3-1-1衍生反應........................................20
3-1-2纖維種類........................................20
3-1-3萃取時間........................................22
3-1-4攪拌速率........................................23
3-1-5脫附時間........................................24
3-1-6脫附溫度........................................25
3-1-7酸鹼值..........................................25
3-1-8離子強度........................................26
3-1-9萃取溫度與冷卻系統..............................28
3-1-10最佳化條件.....................................30
3-2線性、精密度及方法偵測極限........................31
3-3真實樣品的測試....................................32
四、結論.............................................33
五、參考文獻.........................................34

表一、Clenbuterol的性質..............................42
表二、HMDS的性質.....................................43
表三、固相微萃取纖維之調態與操作溫度條件.............44
表四、商業化固相微萃取纖維的比較.....................45
表五、吸附型與吸收型靜相纖維的比較...................46
表六、最佳化條件選擇結果.............................47
表七、線性係數、精密度及方法偵測極限.................48
表八、實驗結果與相關文獻的比較.......................49

圖一、Clenbuterol、HMDS、Clenbuterol-TMS的結購.......50
圖二、Pawliszyn實驗室最早設計的固相微萃取裝置圖......51
圖三、商業化的手動固相微萃取裝置圖...................52
圖四、固相微萃取裝置的操作流程圖.....................53
圖五、三種不同的固相微萃取操作模式...................54
圖六、直接注射1μl ,1 ppm的已衍生完的Clenbuterol-TMS溶液所得到的層析圖...........................................55
圖七、Clenbuterol-TMS 的質譜圖.......................56
圖八、Clenbuterol-TMS經EI撞擊後可能分裂的六離子......57
圖九、固相微萃取裝置架設圖...........................58
圖十、直接注射1μl ,2 ppm的標準Clenbuterol溶液所得到的層析圖...................................................59
圖十一、各種纖維之性質差異...........................60
圖十二、纖維種類與萃取效率之關係圖...................61
圖十三、萃取時間與萃取效率之關係圖...................62
圖十四、攪拌速率與萃取效率之關係圖...................63
圖十五、脫附時間與萃取效率之關係圖...................64
圖十六、脫附溫度與萃取效率之關係圖...................65
圖十七、酸鹼值與萃取效率之關係圖.....................66
圖十八、鹽類濃度與萃取效率之關係圖...................67
圖十九、萃取溫度與萃取效率之關係圖...................68
圖二十、冷卻系統的存在與否與萃取效率之關係圖.........69
圖二十一、以最佳化條件萃取真實尿液中所得到的層析圖...70
圖二十二、Clenbuterol-TMS 在標準樣品與真實人體尿液中質譜圖的比較.................................................71

                                

[1]Brambilla, G.; Cenci, T.; Franconi, F.; Galarini, R.; Macri, A.; Rodoni, F.; Strozzi, M.; Loizzo, A. “Clinical and pharmacological profile in a clenbuterol epidemic poisoning of contaminated beef meat in Italy” Toxicol. Lett., 2000, 114(1-3), 47-53.
[2]Clarkson, P.M.; Thompson, H.S. “Drugs and sport - Research findings and limitations” Sports Med., 1997, 24(6), 366-384.
[3]許美智，「運動員與禁藥」，一九九八年亞運會培訓教練研討會手冊，1998。
[4]許美智，「運動與禁藥」，台北合記圖書出版社，2000。
[5]詹德基，「運動禁藥之管制」，講義，2002。
[6]行政院衛生署動物用藥殘留標準，衛署食字第0900002580號公告修正，2001。
[7]Pawliszyn, J. Solid Phase Microextraction Theory and Practice , Wiley-VCH; New York, 1997.
[8]黃敬德，謝有容，「固相微萃取法」，科儀新知，1997，18(4)，59-67。
[9]Supelco, Solid Phase Microextraction Theory：Theory and Optimization of condiction, Bulletin 1998; Vol. 923, p1-5.
[10]黃賢達，「氣相層析及固相微萃取技術」，化學，1996，54，119-122。
[11]黃敬德，謝有容，「固相微萃取技術之原理及應用」，化學，1998，56，311-318。
[12]黃寶慧，李茂榮，「固相微量萃取法」，科儀新知，1996，18(2)，58-67。
[13]Zhang, Z. Y.; Pawliszyn, J. “Headspace Solid-Phase Microextraction” Anal. Chem., 1993, 65(14), 1843-1852.
[14]Arthur, C.L.; Killam, L.M.; Buchholz, K.D.; Pawliszyn, J.; Berg, J.R. “Automation and Optimization of Solid-Phase Microextraction” Anal. Chem., 1992, 64(17), 1960-1966.
[15]Buchholz, K.D.; Pawliszyn, J. “Optimization of Solid-Phase Microextraction Conditions for Determination of Phenols” Anal. Chem., 1994, 66(1), 160-167.
[16]Langenfeld, J.J.; Hawthorne, S.B.; Miller, D.J. “Optimizing split/splitless injection port parameters for solid-phase microextraction” J. Chromatogr. A., 1996, 740(1), 139-145.
[17]Doong, R.; Chang, S.; Sun, Y. “Solid-Phase Microextraction for Detemnining the Distribution of Sixteen US Environmental Protection Agency Polycyclic Aromatic Hydrocarbons in Water Samples” J. Chromatogr. A., 2000, 879(2), 177-188
[18]Goncalves, C.; Alpendurada, M.F. “Comparison of Three Different Poly(dimethylsiloxane)-Divinylbenzene Fibres for the Analysis of Pesticide Multiresidues in Water Samples: Structure and Efficiency” J. Chromatogr. A., 2002, 963(1-2), 19-26.
[19]Eisert, R.; Pawliszyn, J. “Automated In-Tube Solid-Phase Microextraction Coupled to High-Performance Liquid Chromatography” Anal. Chem., 1997, 69(16), 3140-3147.
[20]Hawthorne, S.B.; Grabanski, C.B.; Hageman, K.J.; Miller, D.J. “Simple Method for Estimating Polychlorinated Biphenyl Concentrations on Soils and Sediments Using Subcritical Water Extraction Coupled with Solid-Phase Microextraction” J. Chromatogr. A., 1998, 814(1-2), 151-160.
[21]Yang, Y.; Miller, D.J.; Hawthorne, S.B. “Solid-phase microextraction of polychlorinated biphenyls” J. Chromatogr. A., 1998, 800(2), 1866-1869.
[22]Gbatu, T.P.; Sutton, K.L.; Caruso, J.A. “Development of New SPME Fibers by Sol-Gel Technology for SPME-HPLC Determination of Organometals” Anal. Chim. Acta., 1999, 402(1-2), 67-79.
[23]Lock, C.M.; Chen, L.; Volmer, D.A. “Rapid Analysis of Tetracycline Antibiotics by Combined Solid Phase Microextraction/High Performance Liquid Chromatography/Mass Spectrometry” Rapid Commun. Mass. Spectrom., 1999, 13(17), 1744-1754.
[24]Lord, H.L.; Pawliszyn, J. “Method Optimization for the Analysis of Amphetamines in Urine by Solid-Phase Microextraction” Anal. Chem., 1997, 69(19), 3899-3906.
[25]Huang, M.K.; Liu, C.R.; Huang, S.D. “One Step and Highly Sensitive Headspace Solid-Phase Microextraction Sample Preparation Approach for the Analysis of Methamphetamine and Amphetamine in Human Urine” Analyst, 2002, 127(9), 1203-1206.
[26]Suzuki, T.; Tsuzurahara, K.; Murata, T.; Takeyama, S. “Quantitative-Determination of Trimetoquinol in Plasna as its Mixed Pentafluoropropionyl-Trimethylsilyl Derivative by Gas-Chromatography Mass-Spectrometry” Biomed. Mass Sp., 1982, 9(3), 94-98.
[27] Hoai, P.M.; Tsunoi, S.; Ike, M.; Kuratani, Y.; Kudou, K.; Viet, P.H.; Fujita, M.; Tanaka, M. ” Simultaneous Determination of Degradation Products of Nonylphenol Polyethoxylates and Their Halogenated Derivatives by Solid-Phase Extraction and Gas Chromatography-Tandem Mass Spectrometry After Trimethylsilylation” J. Chromatogr. A., 2003, 1020(2), 161-171.
[28] Purse, B.W.; Ballester, P.; Rebek, J. “Reactivity and Molecular Recognition: Amine Methylation by an Introverted Ester” J. Am. Chem. Soc., 2003, 125(48), 14682-14683.
[29]Ploum, M.E.; Haasnoot, W.; Paulussen, R.J.A.; Bruchem, G.D.; Hamers, A.R.M.; Schilt, R.; Huf, F.A. “Test Strip Enzyme Immunoassays And the Fast Screening of Nortestosterone and Clenbuterol Residues in Urine Samples at the Parts-Per-Billion Level” J. Chromatogr. Biomed. Appl., 1991, 564(2), 413-427.
[30]Qureshi, G.A.; Erisson, A. “Determination of Clenbuterol and Mabuterol in Equine Plasma by Ion-Pair Liquid-Chromatography with Electrochemical Detection - Chromatographic and Electrochemical Characteristics” J. Chromatogr., 1988, 441(1), 197-205.
[31]Haasnoot, W.; Ploum, M.E.; Paulussen, R.J.A.; Schilt, R.; Huf, F.A. “Rapid-Determination of Clenbuterol Residues in urine by High-Performance Liquid-Chromatography With Online Automated Sample Processing Using Immunoaffinity Chromatography” J. Chromatogr., 1990, 519(2), 323-335.
[32]Hooijerink, H.; Schilt, R.; Haasnoot, W.; Courtheijn, D.; “Determination of Clenbuterol in Urine of Calves by High-Performance Liquid-Chromatography with in Series Ultraviolet and Elevtrochemical Detection” J. Pharm. Biomed. Anal., 1991, 9(6), 485-492.
[33]Courtheyn, D.; Desaever, C.; Verhe, R. “High-Performance Liquid-Chromatographic Determination of Clenbuterol and Cimaterol Using Postcolumn Derivatizat” J. Chromatogr. Biomed. Appl., 1991, 564(2), 537-549.
[34]Aboul-Enein, H.Y.; Serignese, V. “Quantitative Determination of Clenbuterol Enantiomers in Human Plasma by High-Performance Liquid Chromatography Using the Macrocyclic Antibiotic Chiral Stationary Phase Teicoplanin” Biomed. Chromatogr., 1999, 13(8), 520-524.
[35]Diquet, B.; Doare, L.; Simon, P. “Determination of Clenbuterol in the High Nanogram Range in Plasma of Mice by High-Performance Liquid Chromatography With Amperometric Detection” J. Chromatogr., 1984, 336(2), 415-421.
[36]Guy, P.A.; Savoy, M.C.; Stadler, R.H. “Quantitative Analysis of Clenbuterol in Meat Products Using Liquid Chromatography-Electrospray Ionisation Tandem Mass Spectrometry” J. Chrmatogr. B., 1999, 736(1-2), 209-219.
[37]Dumasia, M.C.; Houghton, E. “Screenung and Confirmatory Analysis of Beta-Agonists, Beta-Antagonists and Their Metabolites in Horse Urine by Capillary Gas- Chromatography Mass-Spectrometry” J. Chrmatogr. Biomed. Appl., 1991, 564(2), 503-513.
[38] Vanginkel, L.A.; Stephany, R.W.; Vanrossum, H.J. “Development and Validation of a Multiresidue Method for Beta-Agonists in Biological Samples and Animal Feed” J. AOAC. Int., 1992, 75(3), 554-560.
[39]戴東發，華宏達，簡良潭，劉朝鑫，「台灣農業化學與食品科學」， 2000，38，42-50。
[40]Wilson, R.T.; Groneck, J.M.; Holland, K.P.; Henry, A.C.; “Determination of Clenbuterol in Cattle, Sheep, and Swine Tissues by Electron Ionization Gas-Chromatography Mass-Spectrometry” J. AOAC. Int., 1994, 77(4), 917-924.
[41]Pulce, C.; Lamaison, D.; Keck, G.; Bostvironnois, C.; Nicolas, J.; Descotes, J. “Collective Human Food Poisonings by Clenbuterol Residues in Veal Liver” Vet. Hum. Toxicol., 1991, 33(5), 480-481.
[42]Girault, J.; Fourtillan, J.B. “Determination of Clenbuterol in Bovine Plasma and Tissues by Gas-Chromatography Negative-Ion Chemical Ionization Mass-Spectrometry” J. Chromatogr., 1990, 518(1), 41-52.
[43]Engelmann, M.D.; Hinz, D.; Wenclawiak, B.W. “Solid-Phase Micro Extraction (SPME) and Headspace Derivatization of Clenbuterol Followed by GC-FID and GC-SIMMS Quantification” Anal. Bioanal. Chem., 2003, 375(5), 460-464.
[44]Musshoff, F.; Junker, H.P.; Lachenmeier, D.W.; Kroener, L.; Madea, B. “Fully Automated Determination of Amphetamines and Synthetic Designer Drugs in Hair Samples using Headspace Solid-Phase Microextraction and Gas Chromatography-Mass Spectrometry” J. Chromatogr. Sci., 2002, 40(6), 359-364.
[45]Kataoka, H.; Lord, H.L.; Pawliszyn, J. “Applications of Solid-Phase Microextraction in Food Analysis” J. Chromatogr. A., 2000, 800(1-2), 35-62.
[46]Leendert, A.G.; Stephany, R.W.; Rossum, H.J. “Development and Validation of a Multiresidue Method for β-agonists in Biological Samples and Animal Feed” J. AOAC. Int., 1992, 75(3), 554-560.
[47]Supelco, SPME application guide, 1999.
[48]Shirey, R.E.; Mindrup, R.F. “SPME-Adsorption verus Absorption: Which Fiber is Best for Your Application” 1-31 PTTTCON 2000.
[49]Thackston, E.L.; Wilson, D.J.; Hanson, J.S.; Miller, D.L. “Lead Removal with Adsorbing Colloid Flatation” J. Water. Pollut. Control. Fed., 1980, 52(2), 317-328.
[50]Horng, J.Y.; Huang, S.D. “Determination of The Semivolatile Compounds Nitrobenzene, Isophorone, 2,4-Dinitrotoluene and 2,6-Dinitrotoluene in Water Using Solid-Phase Microextraction with a Polydimethylsiloxane-Coated Fiber” J. Chromatogr. A., 1994, 678(2), 313-318.
[51]Pfaffl, M.W.; Reck, B.; Dreher, R.; Meyer, H.H.D. “Production of Clenbuterol, Diethylstilbestrol and Trenbolone Mass Standards in Lyophilised Bovine Urine” Anal. Chim. Acta., 2003, 483(1-2), 401-412.
[52] Van, Hout, M.W.J.; Niederlander, H.A.G.; de Zeeuw, R.A. de Jong, G.J. “Ion Suppression in the Determination of Clenbuterol in Urine by Solid-Phase Extraction Atmospheric Pressure Chemical Ionisation Ion-Trap Mass Spectrometry” Rapid Commun. Mass. Spectrom., 2003, 17(3), 245-250.
[53] Blomgren, A.; Berggren, C.; Holmberg, A.; Larsson, F.; Sellergren, B.; Ensing, K. “Extraction of Clenbuterol from Calf Urine Using a Molecularly Imprinted Polymer Followed by Quantitation by High-Performance Liquid Chromatography with UV Detection” J. Chromatogr. A., 2002, 975(1), 157-164.
[54]Van, Eenoo, P.; Delbeke, F.T.; Deprez, P. “Detection of Inhaled Clenbuterol in Horse Urine by GC/MS2” Biomed. Chromatogr., 2002, 16(7), 475-481.
[55]Guan, F.Y.; Uboh, C.E.; Soma, L.R.; Luo, Y.; Li, R.B.; Birks, E.K.; Teleis, D.; Rudy, J.A.; Tsang, D.S. “Quantification of Clenbuterol in Equine Plasma, Urine and Tissue by Liquid Chromatography Coupled On-Line with Quadrupole Time-Of-Flight Mass Spectrometry” Rapid Commun. Mass. Spectrom., 2002, 16(17), 1642-1651.
[56] Amendola, L.; Colamonici, C.; Rossi, F.; Botre, F. “Determination of Clenbuterol in Human Urine by GC-MS-MS-MS: Confirmation Analysis in Antidoping Control” J. Chromatogr. B. 2002, 773(1), 7-16.

全文公開日期本全文未授權公開 (校內網路)
全文公開日期本全文未授權公開 (校外網路)

簡易檢索 / 詳目顯示

相關論文