臺灣博碩士論文加值系統

本研究主要是利用自動化的線上酵素水解(on-line enzyme hydrolysis) 系統，結合免疫親和管柱，使用大氣壓力化學游離質譜法(atmosphere pressure chemical ionization；APCI)，水解尿液中的Morphine-3-glucuronide(M3G)為嗎啡(morphine)，以改進了傳統酵素水解前處理的費時及使用固定化酵素(immobile enzyme)減少酵素使用量多的缺點，因為採取線上自動化分析，因此具有快速與自動化的優點。
另外水解過後，經由免疫親和管柱的吸附、脫附注入質譜後，經由APCI簡單的collision induced dissociation (CID)，M3G的水解率可以達到82％以上，而分析每一個樣品的時間只需21分鐘。而對於整個系統的穩定性，使用固定化的酵素，可以將酵素在4℃下保存10週。尿液樣品除了使用男性尿液添加嗎啡及M3G外，我們亦可在尿液樣品中偵測到偵測極限為100ppb的嗎啡。而尿液經過免疫親合管柱的萃取之後，可以發現並未受到干擾。在真實樣品上，與慈濟檢驗中心使用的法定方法判定陽性煙毒犯的結果上，並不會有太大的定量準確性受到影響。

An automatic on-line enzyme hydrolysis system, coupling with immunoaffinity reactor and the atmosphere pressure chemical ionization (APCI) mass spectrometry for the analysis of morphine in urine was studied. The time consuming enzyme hydrolysis using soluble β-D-glucuronidase was changed by immobilized reactor in this system to provide a time, enzyme saving, and automatic analysis.
The efficiency for morphine-3-glucuronide hydrolysis is at least 82% with 10 min hydrolysis operation. The immobilized enzyme reactor shows good stability after ten weeks. The limited of detection(LOD) for morphine in urine is 100ppb. It shows good correlation with this method and GC/MS (acid hydrolysis) method for urine sample analysis.

目錄
謝誌Ⅰ
摘要Ⅱ
英文摘要Ⅲ
目錄Ⅳ
圖目錄Ⅶ
表目錄Ⅸ
第一章、序論
1.1前言1
1.1.1毒品種類的區分:2
1.2嗎啡的介紹3
1.2.1 為什麼要偵測嗎啡3
1.2.2嗎啡的分析5
1.3水解的比較6
1.3.1酸水解6
1.3.2微波水解7
1.3.3酵素水解7
1.4免疫分析技術8
1.5質譜技術10
第二章 材料與方法
2.1實驗用藥品12
2.1.1抗體與酵素12
2.1.2試藥13
2.1.3抗體固定化試劑14
2.1.4自行添加標準品與真實樣品來源14
2.2實驗儀器14
2.3實驗步驟15
2.3.1抗體固定化方法15
2.3.1.1酸洗15
2.3.1.2矽烷化15
2.3.1.3抗體固定化16
2.3.1.4酵素固定化17
2.3.2 緩衝液(buffer)的配製17
2.3.3 載流液體(carrier solution)與脫附劑的配製17
2.3.4 酵素水解免疫吸附系統分析水樣中的嗎啡17
2.3.5 酵素水解免疫吸附系統分析尿液中的嗎啡18
2.3.6溫度對抗體與酵素的影響18
第三章、結果與討論
3.1大氣壓力化學游離法之條件選擇19
3.1.1 Corona voltage 19
3.1.2 Sample cone 的選擇19
3.1.3 HV lens 的選擇20
3.1.4 IE ramp的選擇20
3.1.5 Ion energy的選擇20
3.1.6 其他條件的選擇21
3.2酵素水解與抗體吸附條件的選擇21
3.2.1 水解時間21
3.2.2 水解的pH 22
3.2.3 水解的流速 22
3.2.4 水解溫度22
3.2.5 抗體脫附的elution time23
3.3酵素的量與酵素穩定的測試23
3.3.1酵素使用量的決定24
3.3.2 酵素使用24
3.4 嗎啡之檢量線25
3.5 真實尿液樣品的分析25
第四章、結論與展望27
第伍章、參考文獻28
圖目錄
圖1-1 嗎啡的化學結構3
圖1-2 嗎啡代謝圖4
圖1-3 免疫球蛋白G(IgG)簡示圖8
圖1-4抗體抗原結合圖9
圖1-5 APCI結構圖11
圖2-1 固定化機制流程16
圖2-2 酵素水解免疫吸附系統圖33
圖3-1 1ppm morphine 訊號圖34
圖3-2 corona的選擇35
圖3-3 cone電壓之選擇36
圖3-4 1ppm M3G 訊號圖37
圖3-5 HV lens 之選擇38
圖3-6 IE ramp之選擇39
圖3-7 Ion energy 之選擇40
圖3-8水解時間41
圖3-9水解pH42
圖3-10水解流速43
圖3-11水解溫度的效應44
圖3-12 morphine 脫附時間45
圖3-13 酵素水解週比較圖46
圖3-14嗎啡在質譜中的校正曲線47
圖3-15 M3G系統校正曲線48
圖3-16 M6G系統校正曲線49
圖3-17自行添加真實樣品系統校正曲線50
表目錄
表1-1 濫用藥物分類表2
表3-1 APCI質譜條件設定表51
表3-2 酵素水解unit 數的選擇52
表3-3 真實樣品的分析53
表3-4 真實煙毒犯樣品檢測54

1. 藥物濫用宣導教育防制手冊，行政院衛生署編印。
2. 防治嗎啡海洛因宣導教育手冊，行政院衛生署編印。
3. J. Huwyler, S. Rafer, E. Kuster, J. Drewe, Rapid and highly automated determination of morphine and morphine glucuronides in plasma by on-line solid-phase extraction and column liquid chromatography J. Chromatogr. B. 674 (1995) 57-63.
4.J. Beike, H. Kohler, B. Brinkmann, G. Blaschke, Immunoaffinity extraction of morphine, morphine-3-glucuronide and morphine-6-glucuronide from blood of heroin victims for simultaneous high-performance liquid chromatographic determination J. Chromatogr. 726 (1999) 111-119.
5. M. Freiermuth, J. C. Please, Determination of morphine and codeine in plasma by HPLC following solid phase extraction J. Pharm. Biomed Anal. 15 (1997) 759-764.
6. D.Bourquin, T. Lehmann, R. Hammig, M. Buhrer, R. Brenneisen High-performance liquid chromatographic monitoring of intravenously administered diacetylmorphine and morphine and their metabolites in human plasma J. Chromatogr. B. 694 (1997) 233-238
7. A. D. Nagy, L. Rivier, C. Giroud, M. Augsburger, P. Mangin, Method for quantification of morphine and its 3- and 6-glucuronides, codeine, codeine glucuronide and 6-monoacetylmorphine in human blood by liquid chromatography—electrospray mass spectrometry for routine analysis in forensic toxicology J. Chromatogz. A. 854 (1999) 109-118.17.
8.M. Blanchet, G. Bm, M. Guerret, M. Bromet-Petit, N. Bromet, Routine determination of morphine, morphine 3-β-d-glucuronide and morphine 6-β-d-glucuronide in human serum by liquid chromatography coupled to electrospray mass spectrometry J. Chromatog~.A. 854 (1999) 93-108.
9. N. Tyrefor, B. Hyllbrant, L. Ekman, M. Johansson, B. Langstrom, Determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human serum by solid-phase extraction and liquid chromatography-mass spectrometry with electrospray ionization J. Chromatogr. A. 729 (1996) 279-285.
10. M. J. Boguze, R. D. Maier, M. Ekman, S. Driessen, Determination of morphine and its 3- and 6-glucuronides, codeine, codeine-glucuronide and 6-monoacetylmorphine in body fluids by liquid chromatography atmospheric pressure chemical ionization mass spectrometry, J. Chromatogr. B. 703 (1997) 115-127.
11. G. Schanzle, S. Li, G. htfikus, U. Hofman, Rapid, highly sensitive method for the determination of morphine and its metabolites in body fluids by liquid chromatography—mass spectrometry J. Chromatogr..B. 721 (1999) 55-65.
12. R. Pacific, S. Pichlni, I. Altieri, A~Caronna, A. R. Passa, P. Zuccaro, High-performance liquid chromatographic-electrospray mass spectrometric determination of morphine and its 3- and 6-glucuronides: application to pharmacokinetic studies J. Chromatogr. B. 664 (1995) 329-334.
13. H. Keski-Hynnilä, Liquid Chromatography- and Capillary Electrophoresis-Mass Spectrometry in Glucuronide Analysis, permission of faculty of science. 2002 February 2nd.
14. B. Fryirs, M. Dawson, L. E. Mather, Highly sensitive gas chromatographic—mass spectrometric method for morphine determination in plasma that is suitable for pharmacokinetic studies J. Chromatogr. B. 693 (1997) 51-57.
15. Thomas A. Jennison, Elizabeth Wozniak, Gordon Nelson, and
Francis M. Urry, The Quantitative Conversion of Morphine 3-β-D Glucuronide to Morphine Using β-Glucuronidase Obtained from Patella vugata as Compared to Acid Hydrolysis J. Anal. Toxicol. 17, July/August (1993) 208-210.
16. Joan Combie, J.W. Blake, Thomas E.Nugent, and Thomas Tobin, Morphine Glucuronide Hydrolysis: Superior of β-glucuronidase from Patella vulgate Clin. Chem. 28 (1) (1982) 83-86.
17.濫用藥物尿液檢驗機構認可及管理相關規定，行政院衛生署編印。
18.濫用藥物檢驗手冊，慈濟醫學院編印。
19.紀柏亨、楊末雄、孫毓璋，為波消化之方法與應用，Chemistry, 56 (4) (1998) 264-284.
20.周靜怡、蘇宏基，序列注入線上微波水解免役質譜法分析尿液中之嗎啡與morphine-3-glucuronide，國立東華大學碩士論文，2002, July。
20. A. L. Ghindilis, P Atanasov, M. Wilkins & E. Wilkins, Immunosensors: electrochemical sensing and other engineering approaches” Biosensors
13 (1998) 113-131.
21. J. Cai and J. Henion, On-Line Immunoaffinity Extraction-Coupled Column Capillary Liquid Chromatography/Tandem Mass Spectrometry: Trace Analysis of LSD Analogs and Metabolites in Human Urine Anal. Chem. 68 (1996) 72-78.
22. 行政院國家科學委員會機密儀器發展中心，質譜分析數專輯， 1992。
23.J. Throck Watson, Ph.D. Introduction to Mass Spectrometry(third edition)，1997
24.林仁智，蘇宏基，自動化序列注入分析大氣壓力化學離子化與電灑質譜法偵測尿液中的嗎啡，國立東華大學碩士論文，2000，June。
25. P. Kintz*, V. Cn-imele, A. Tracqui, P. Mangin, Simultaneous determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine in human hair by gas chromatography-mass spectrometry
Journal of Chromatography B, 670 (1995) 162-166
26.Y. Nakahara, “Detection and diagnostic interpretation of amphetamines in hair Forensic Science International 70(1995) 135-153
27.C. Staub Analytical procedures for determination of opiates in hair: a review Forensic Science International 70 (1995) 111-123
28.P. Kintz, V. Cifimele, Interlaboratory comparison of quantitative determination of amphetamine and related compounds in hair samples Forensic Science International
84(1997) 151-156