臺灣博碩士論文加值系統

鑑於近年來國內使用海洛因成癮者不斷激增，其中不乏是共用針頭，卻又成傳染愛滋病的媒介，因此過去歐美曾以美沙冬 (Methadone)為戒癮藥物，但其毒性強對身體的傷害大，所以台灣從未開放；直到兩年前，美國核准以丁基原啡因(目前屬於第三級管制藥品)用於戒癮療法，主要是這種止痛藥成分雖同樣源於鴉片類，因脂溶性高,肌肉注射、舌下、或口服皆吸收良好且成癮性較低，副作用也較少，因此以丁基原啡因(Buprenorphine)作為海洛因成癮者戒癮療法的用藥。衛生署管制藥品管理局於94年底通過「毒品病患愛滋減害試辦計畫」，推動「替代療法」嘗試採取替代性戒癮藥物,例如美沙（Methadone）、丁基原啡因（Buprenorphine）以協助海洛因成癮患者之治療計畫,藉由門診醫療服務，提高有效戒癮治療的可行性,市立療養院最近完成了一份「海洛因成癮治療研究報告」，發現其治療成果與國外研究文獻接近，且未見其他明顯副作用，安全性令人滿意。
本論文以線上固相萃取結合液相層析/電灑譜儀檢測尿液中丁基原啡因(buprenorphine)及其代謝物、nor-buprenorphine (NBUP)、buprenorphine–glucuronide(BG) 、norbuprenorphine–glucuronide(NBG)之分析方法。
本研究先利用0.1%三氟酸使尿液中的蛋白質沉澱，注入HLB mixed mode 的線上固相萃取管柱，樣品再經由線上固相萃取減少基質的干擾，並利用二次質譜(MS/MS)進行定量檢測，因此不需繁瑣的化學衍生化法並可自動化，節省許多人力與時間。回收率可達96.6~100.9%，Bup、BG、NBG線性範圍約為0.5ng/ml~100ng/ml，NBUP線性範圍約為1.0ng/ml~100ng/ml，偵測極限約為0.25~0.50ng/ml 。

中文摘要
第一章 緒論 1
第一節 丁基原啡因類藥物之介紹 1
第二節 線上固相萃取結合液相層析/電灑/質譜檢測
尿液中丁基原啡因類藥物 6
第三節 液相層析/電灑/質譜儀的應用與發展 7
第四節 離子阱質譜儀 12

第二章 實驗部份 16
第一節 樣品及試劑 16
第二節 儀器及設備 16
第三節 實驗架構及步驟 21

第三章 結果與討論 35
第一節 離子阱質譜儀部分 35
(1) 質譜儀毛細管出口端電壓對訊號強度的影響 35
(2) 質譜儀斷裂電壓對訊號強度的影響 37
第二節 線上固相萃取連接液相層析管柱 53
2-1液相層析管柱條件之測試 53
2-2線上固相萃取管柱連接液相層析管柱條件
之測試 53
(1) 線上固相萃取管柱進樣液濃度之探討 53
(2) 線上固相萃取管柱沖洗液濃度之探討 57
(3) 線上固相萃取與液相層析管柱連接之相容性 57
(4) 注入的樣品體積探討 67
第三節 尿液基質效應探討 70
(1) 基質效應於尿液樣本之探討 70
(2) 靈敏度及偵測極限 75
第四節 線性關係及真實樣品 78
(1) 線性關係 78
(2) 精確度與準確度之測試 78
(3) 固相萃取回收率之測試 80
(4) 基質效應對線性的影響 85
(5) 丁基原啡因類藥物穩定性測試 85
(6) 真實尿液樣本之檢測 90
第五節 結論 94

第四章 參考文獻 95
附錄 110

1.R.D. Voyksner, “Atmospheric-Pressure Ionization LC/MS”, Environ. Technol. , 1994, 28, 118-127.
2.A.P. Bruins, T.R. Covry, J.D. Henion, “Ion Spray Interface for Combined Liquid Chromatography/Atmospheric Pressure Ionization Mass-Spectrometry”, Anal Chem., 1987, 59, 2642-2646.
3.M.H. Allen, B.I. Shushan, “Atmospheric-Pressure Ionization-Mass Spectrometry Detection for Liquid Chromatography and Capillary Electrophoresis”, LC-GC , 1993, 11, 112-118.
4.A.P. Bruins, “Mass Spectrometry with Ion Sources Operating at Atmospheric Pressure”, Mass Spectrom. Rev., 1991, 10, 53-77.
5.L. Bräutigam, J.U. Nefflen, G. Geisslinger, “Determination of etoricoxib in human plasma by liquid chromatography–tandem mass spectrometry with electrospray ionisation”, J. Chromatogr. B, 2003, 788, 309-315.
6.C.H. Oliveira, R.E. Barrientos-Astigarraga, E. Abib, G.D. Mendes, D. R. da Silva, G. de Nucci, “Lansoprazole quantification in human plasma by liquid chromatography–electrospray tandem mass spectrometry”, J. Chromatogr. B, 2003, 783, 453-459.
7.H. Yang, Y. Feng, Y. Luan, “Determination of Simvastatin in human plasma by liquid chromatography–mass spectrometry”, J. Chromatogr. B, 2003, 785, 369-375.
8.Y. Xu, J.L. Grem, “Liquid chromatography–mass spectrometry method for the analysis of the anti-cancer agent capecitabine and its nucleoside metabolites in human plasma”, J. Chromatogr. B, 2003, 783, 273-285.
9.C. M. Murphy ,M. A. Huestis, “Liquid chromatographic/electrospray ionization tandem mass spectrometric analysis for the quantification of buprenorphine, norbuprenorphine, buprenorphine-3-b-D-
glucuronide and norbuprenorphine-3-b-D-glucuronide in human
plasma”, J. Mass Spectrom, 2005, 40, 70-74.
10.A. Polettini, M. A. Huestis, “Simultaneous determination of buprenorphine, norbuprenorphine, and buprenorphine–glucuronide in plasma by liquid chromatography–tandem mass spectrometry”, J. Chromatogr. B, 2001, 754, 447-459.
11.A. Ceccato , R. Klinkenberg , Ph. Hubert , B. Streel , “ Sensitive determination of buprenorphine and its N-dealkylated metabolite norbuprenorphine in human plasma by liquid chromatography coupled to tandem mass spectrometry”, J. Pharm. Biomed. Anal. ,2003 ,32,619-631.
12.S. Pirnay, F. Herv′e, S. Bouchonnet ,B. Perrin , F. J. Baud, I. Ricordel , “Liquid chromatographic–electrospray ionization mass spectrometric quantitative analysis of buprenorphine, norbuprenorphine,nordiazepam and oxazepam in rat plasma ”, J. Pharm. Biomed. Anal. , 2006 ,41,1135-/1145.
13.D. Favretto, G. Frison, S. Vogliardi and S. D. Ferrara, “Potentials of ion trap collisional spectrometry for liquid chromatography/
electrospray ionization tandem mass spectrometry determination of buprenorphine and nor-buprenorphine in urine, blood and hair
samples”, Rapid Commun. Mass Spectrom.,2006, 20,1257-1265.
14.S. Gopal, T. Tzeng, A. Cowan, “Development and validation of a sensitive analytical method for the simultaneous determination of buprenorphine and norbuprenorphine in human plasma”, Eur.
J. Pharm. Biopharm,2001, 51,147-151.
15.S. Pirnay , S. Bouchonnet , F. Hervé , D. Libong ,N. Milan , P. Athis , F. Baudb, I. Ricordel, “Development and validation of a gas chromatography–mass spectrometry method for the simultaneous determination of buprenorphine,flunitrazepam and their metabolites in rat plasma: application to the pharmacokinetic study”, J. Chromatogr. B, 2004, 807, 335-342.
16.D. E. Moody, M. H. Slawson, E. C. Strain, J. D. Laycock,A. C.
Spanbauer,R. L. Foltz, “A Liquid Chromatographic-Electrospray Ionization-Tandem Mass Spectrometric Method for Determination
of Buprenorphine,Its Metabolite,norBuprenorphine, and a Coformulant,Naloxone,That Is Suitable for in Vivo and in Vitro Metabolism Studies”, Anal. Biochem ,2002; 306: 31.

17.S. D. McAleer , R. J. Mills, T. Polack, T. Hussain,P. E. Rolan, A. D. Gibbs, F. G.P. Mullins, Z. Hussein, “Pharmacokinetics of high-dose buprenorphine following single administration of sublingual tablet formulations in opioid naıvehealthy male volunteers under a naltrexone block”, Drug Alcohol Depend, 2003 ,72,75-/83.
18.http://www.nbcd.gov.tw/home/announce/list.aspx?id=2005
19.http://www.nbcd.gov.tw/admin/uploads/20061115050943312570555/951116%B7s%BBD%BDZ.pdf
20.W.M.A. Nissen, J.V.D. Greff., “Liquid Chromatography Mass Spectrometry. Principle and Application” Chromatographic Science. , MARCEL DEKKER, INC. New York. USA, 1992, 58, 235-239.
21.M. Yamashita, J.B. Fenn, “Electrospray Ion Source”, J. Phys. Chem. , 1984, 88, 4451-4471.
22.C.M. Whitehouse, R.N. Dreyer, M. Yamashita, J.B. Fenn, “Electrospray Interface for Liquid Chromatography and Mass Spectrometers”, Anal. Chem. , 1985, 57, 675-679.
23.M. Salairi, H. Kambara, “Chromatography/Atomspheric Pressure Ionization Mass Spectrometer”, Anal. Chem. , 1988, 60, 774-780.
24.M. Yamashita, J.B. Fenn, “Negative Ion Production with the Electrospray Ion Source”, J. Phys. Chem. , 1984, 88, 4671-4675.
25.C.M. Whitehouse, R.N. Dreyer, M. Yamashita, J.B. Fenn, “Electrospray interface for Liquid Chromatography and Mass Spectrometers”, Anal. Chem. , 1985, 57, 675-679.
26.M.G. Ikonomou, A.T. Blades, P. Kebarle. “Electrospray-Ion Spray: A Comparison of Mechanisms and Performance”, Anal. Chem. , 1991, 63, 1989-1998.
27.G. Hopfgartner, T. Wachs, K. Bean, J. Henion, “High-Flow Ion Spray Liquid Chromatography/Mass Spectrometry”, Anal. Chem. , 1993, 65, 439-446.
28.M.G. Ikonomou, A.T. Blades, P. Kebarle, “Electrospray-Ion Spray: Comparison of Mechanisms and Performance”, Anal. Chem. , 1991, 63, 1989-1998.
29.J. Sharma, M. Panico, J. Barber, H.R. Morris, “Purification and Determination of Intact Molecular-Mass by Electrospray-Ionization Mass-Spectrometry of the Photosystem-2 Reaction-Center Subunits”, J. Biol. Chem. , 1997, 272, 33153-33157.
30.Ion Trap Software 4.1 , “Agilent 1100 Series LC/MSD Trap Techiques and Operation - Course Number H8966A Volume 1”, Agilent Technologies, 2002, 62.
31.http://www.chm.bris.ac.uk/ms/theory/esi-ionisation.html
32.R. Miao, G.S. Yang, Y. Miao, Y.H. Mei, J. Hong, C.M. Zhao, L.G. Zhu, “Interactions of platinuim(II) complexes with sulfur-containing peptides studied by electrospray ionization mass spectrometry and tandem mass spectrometry”, Rapid Commun. Mass Spectrom. , 2005, 19, 1031-1040.
33.J.M. Zhang, J.S. Brodbelt, “Silver complexation and tandem mass spectrometry for differentiation of isomeric flavonoid diglycosides”, Anal. Chem. , 2005, 77, 1761-1770.
34.I.K. Chu, S.O. Siu, C.N.W. Lam, J.C.Y. Chan, C.F. Rodriquez, “Formation of molecular radical cations of aliphatic tripeptides from their complexes with Cu-II(12-crown-4)”, Rapid Commun. Mass Spectrom. , 2004, 18 , 1798-1802.
35.A. Kloepfer, J.B. Quintana, T. Reemtsma, “Operational options to reduce matrix effects in liquid chromatography-electrospray ionization-mass spectrometry analysis of aqueous environmental samples”, J. Chromatogr. A , 2005, 1067, 153-160.
36.J.Y. Tsai, P.B. Shevlin, “Structure of the Products of the Cyclooligomerization of Sulfur Dioxide with Diethyl Diallymalonate”, J. org. chem. , 1998, 63, 3230-3234.
37.S.J. Rowan, J.K.M. Shanders, “Macrocycles Derived from Cinchona Alkaloids – A Thermodynamic VS Kinetic Study”, J. org. chem. , 1998, 63, 1536-1546.
38.J.D. Williams, M. Flanagan, L. Lopez, S. Fischer, “Using accurate mass electrospray ionization–time-of-flight mass spectrometry with in-source collision-induced dissociation to sequence peptide mixtures”, J. Chromatogr. A , 2003, 1020, 11-26.
39.T. Keqi, J.S. Page, R.D. Smith, “Charge Competition and the Linear Dynamic Range of Detection in Electrospray Ionization Mass Spectrometry”, J. Am. Soc. Mass Spectrom. , 2004, 15, 1416-1423.
40.W.F. Smyth, “Electrospray ionisation mass spectrometric behaviour of selected drugs and their metabolites”, Anal. Chim. Acta , 2003, 492, 1-16.
41.W. Lam, R. Ramanathan, “In Electrospray Ionization Source Hydrogen/Deuterium Exchange LC-MS and LC-MS/MS for Characterization of Metabolites”, J. Am. Soc. Mass Spectrom. , 2002, 13, 345-353.
42.E.B. Kujawinski, M.A. Freitas,*, X. Zang, P.G. Hatcher, K.B. Green-Church, R. B. Jones, “The application of electrospray ionization mass spectrometry (ESI MS) to the structural characterization of natural organic matter”, Org. Geochem. , 2002, 33, 171-180.
43.E. Rosenberg , “The potential of organic (electrospray- and atmospheric pressure chemical ionisation) mass spectrometric techniques coupled to liquid-phase separation for speciation analysis”, J. Chromatogr. A , 2003, 1000, 841-889.
44.T.P. Knepper, “Mass spectrometric strategies for the analysis of polar industrial chemicals and their by-products in wastewater and surface water”, J. Chromatogr. A , 2002, 974, 111-121.
45.E. Dijkman, D. Mooibroek, R. Hoogerbrugge, E. Hogendoorn, J.V. Sancho, O. Pozo, F. Hernandez, “Study of matrix effects on the direct trace analysis of acidic pesticides in water using various liquid chromatographic modes coupled to tandem mass spectrometric detection”, J. Chromatogr. A , 2001, 926, 113-125.
46.C. Chevrin, J. Le Bras, F. Henin, J. Muzart, A. Pla-Quintana, A. Roglans, R. Pleixats, “Allylic substitution mediated by water and palladium: Unusual role of a palladium(II) catalyst and ESI-MS analysis”, Organometallics , 2004, 23, 4796-4799.
47.H.K. Nordgren, P. Holmgren, P. Liljeberg, N. Eriksson, O. Beck, “Application of direct urine LC-MS-MS analysis for screening of novel substances in drug abusers”, J. Anal. Toxicol. , 2005, 29, 234-239.
48.W.Y. Hua, E.R. Bennett, R.J. Letcher, “Triclosan in waste and surface waters from the upper Detroit River by liquid chromatography-electros
pray-tandem quadrupole mass spectrometry”, Environ. Int. , 2005, 31, 621-625.
49.R. Rodil, J.B. Quintana, T. Reemtsma, “Liquid chromatography
-tandem mass spectrometry determination of nonionic
organophosphorus flame retardants and plasticizers in wastewater samples”, Anal. Chem. , 2005, 17, 3083-3089.
50.S. Bhoopathy, B. Xin, S.E. Unger, H.T. Karnes, “A novel incubation direct injection LC/MS/MS technique for in vitro drug metabolism screening studies involving the CYP 2D6 and the CYP 3A4 isozymes”, J. Pharm. Biomed. Anal. , 2005, 37, 730-749.
51.F. Liang, L.J. Li, Z. Abliz, Y.C. Yang, J.G. Shi, “Structural characterization of steroidal saponins by electrospray ionization and fast-atom bombardment tandem mass spectrometry”, Rapid Commun. Mass Spectrom. , 2002, 16, 1168-1173.
52.S. Aggarwal, N.N. Ghosh, R. Aneja, H. Joshi, R. Chandra, “Mass spectral studies on aryl-substituted N-carbamoyl/N-thiocarbamoyl narcotine and related compounds”, Rapid Commun. Mass Spectrom. , 2002, 16, 923-928.
53.S.M. Sheehy, J. Szpunar, R. Morabito, P. Quevauviller, “The speciation of arsenic in biological tissues and the certification of reference materials for quality control”, Trends Anal. Chem. , 2003, 22, 191-209.
54.L. Bräutigam, J.U. Nefflen, G. Geisslinger, “Determination of etoricoxib in human plasma by liquid chromatography–tandem mass spectrometry with electrospray ionisation”, J. Chromatogr. B , 2003, 788, 309-315.
55.C.H. Oliveira, R.E. Barrientos-Astigarraga, E. Abib, G.D. Mendes, D.R. da Silva, G. de Nucci, “Lansoprazole quantification in human plasma by liquid chromatography–electrospray tandem mass spectrometry”, J. Chromatogr. B , 2003, 783, 453-459.
56.H. Yang, Y. Feng, Y. Luan, “Determination of Simvastatin in human plasma by liquid chromatography–mass spectrometry”, J. Chromatogr. B , 2003, 785, 369-375.
57.Y. Xu, J.L. Grem, “Liquid chromatography–mass spectrometry method for the analysis of the anti-cancer agent capecitabine and its nucleoside metabolites in human plasma”, J. Chromatogr. B , 2003, 783, 273-285.
58.A. Latorre, A. Rigol, S. Lacorte, D. Barceló, “Comparison of gas chromatography–mass spectrometry and liquid chromatography– mass spectrometry for the determination of fatty and resin acids in paper mill process waters”, J. Chromatogr. A , 2003, 991, 205-215.
59.S.E. Nielsen, B. Sandstrỏm, “Simultaneous determination of hydroxycinnamates and catechins in human urine samples by column switching liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry”, J. Chromatogr. B , 2003, 787, 369-379.
60.E. de Rijke, H. Zappey, F. Ariese, C. Gooijer, U.A.T. Brinkman, “Liquid chromatography with atmospheric pressure chemical ionization and electrospray ionization mass spectrometry of flavonoids with triple-quadrupole and ion-trap instruments”, J. Chromatogr. A , 2003, 984, 45-58.
61.R. Draisci, L. Palleschi, E. Ferretti, L. Lucentini, F.D. Quadri, “Confirmatory analysis of 17b-boldenone, 17a-boldenone and androsta-1,4-diene-3,17-dione in bovine urine by liquid chromatography–tandem mass spectrometry”, J. Chromatogr. B , 2003, 789, 219-226.
62.Y. Amet, F. Adas, F. Berthou, “High performance liquid chromatography of fatty acid metabolites Improvement of sensitivity by radiometric, fluorimetric and mass spectrometric methods”, Anal. Chim. Acta , 2002, 465, 193-198.
63.M. Meger, I. Meger-Kossien, A. Schuler-Metz, D. Janket, G. Scherer, “Simultaneous determination of nicotine and eight nicotine metabolites in urine of smokers using liquid chromatography– tandem mass spectrometry ”, J. Chromatogr. B , 2002, 778, 251-261.
64.何國榮, 張耀仁, “離子阱質譜儀”, 科儀新知, 1993, 14(5), 48-55.
65.http://www.chm.bris.ac.uk/ms/theory/qit-massspec.html
66.M. R. Fuh, T.Y. Wu, T. Y. Lin, “Determination of amphetamine and methamphetamine in urine by solid phase extraction and ion-pair liquid chromatography–electrospray–tandem mass spectrometry”, Talanta, 2006 ,68, 987–991.
67.M. R. Fuh, T.Y. Wu, “Determination of amphetamine, methamphetamine,3,4-methylenedioxyamphetamine, 3,4-
methylenedioxyethylamphetamine, and 3,4-
methylenedioxymethamphetamine in urine by online
solid-phase extraction and ion-pairing liquid
chromatography with detection by electrospray
tandem mass spectrometry”, Rapid Commun. Mass Spectrom. ,2005, 19, 775–780.


68.B.A. Sinnaeve, T.N. Decaestecker, P.G. Risha, J.P. Remon, C. Vervaet, J. F. Van Bocxlaer, “Liquid chromatographic-mass spectrometric assay for simultaneous pyrimethamine and sulfadoxine determination in human plasma samples”, J. Chromatogr. A , 2005, 1076, 97-106.
69.S. Bardin, W. Guo, J.L. Johnson , S. Khan, A. Ahmad, J.X. Duggan, J. Ayoub, I. Ahmad, “Liquid chromatographic-tandem mass spectrometric assay for the simultaneous quantification of Camptosar((R)) and its metabolite SN-38 in mouse plasma and tissues”, J. Chromatogr. A , 2005, 1073, 249-255.
70.J.M. Flaherty, P.D. Connolly, E.R. Decker, S.M. Kennedy,M.E. Ellefson, W.K. Reagen, B. Szostek, “Quantitative determination of perfluorooctanoic acid in serum and plasma by liquid chromatography tandem mass spectrometry”, J. chromatogr. B , 2005, 819,329-388.
71.T.S. Ho, J.L.E. Reubsaet, H.S. Anthonsen, S. Pedersen-Bjergaard, K.E. Rasmussen, “Liquid-phase microextraction based on carrier mediated transport combined with liquid chromatography-mass spectrometry - New concept for the determination of polar drugs in a single drop of human plasma”, J. Chromatogr. A , 2005, 1072, 29-36.
72.C. Polson, P. Sarkar, B. Incledon, V. Raguvaran, R. Grant, “Optimization of protein precipitation based upon effectiveness
of protein removal and ionization effect in liquid chrom
atography–tandem mass spectrometry”, J. Chromatogr. B , 2003, 263–275.
73.呂理霖,“線上固相萃取結合液相成層析質譜儀檢測尿液中嗎啡類藥物”， 東吳大學碩士論文，中華明國九十五年六月。
74.S. Souverain, S. Rudaz, J.L. Veuthey, “Matrix effect in LC-ESI-MS and LC-APCI-MS with off-line and on-line extraction procedures”, J. Chromatogr. A , 2004, 1058, 61–66.
75.R. King, R. Bonfiglio, C.F. Metzler, C.M. Stein, T. Olah, “Mechanisitic investigation of ionization suppression in electrospray ionization”, J. Am. Soc. Mass Spectrom. , 2000, 11, 942-950.