臺灣博碩士論文加值系統

濫用藥物之種類因地區及時間而異，新興濫用藥物不斷出現，欲偵測個案是否使用濫用藥物，尿液是最常用的檢體。檢驗時通常分兩階段進行。先以免疫檢驗法進行篩檢，篩檢呈陽性的檢體再以氣相層析質譜儀(GC/MS)確認。
由於濫用藥物種類繁多，且不斷有新興的濫用藥物被使用，市售的免疫檢驗試劑無法跟上實際的需求，因此急需一可廣篩多種濫用藥物的方法。
本研究主要利用一高敏感度與高解析度之液相層析質譜儀為主要分析儀器，研發出可篩檢尿液中不同濫用藥物之方法。主要針對安非他命類、鴉片類、苯二氮平類、巴比妥酸鹽類、大麻以及古柯鹼等六大類濫用藥物進行分析，欲發展出一套同時分析此六大類藥物之方法。
本實驗主要將上述藥物種類在藉由酵素免疫分析(EIA)後之檢體，經過固相萃取後(Clean screen DAU column)，除巴比妥酸鹽類須保留methanol 之沖洗液外，其餘皆只保留CH2Cl2 : Isopropanol : Ammonia (78:20:2)之沖提液。沖提液吹乾後，溶於100 μL 之液相層析動相中(AmmoniumAcetate 10 mM : methanol 7:3,含0.05 %之 Trifluoroacetic acid, TFA)。以直接注入液相質譜儀(ESI-MS)之方式，不經層析管柱，先作一快速篩檢，利用移動相為AmmoniumAcetate 10 mM,含0.05 ％ TFA (5 ％) 與Methanol 含0.05 ％ TFA (95 ％)，流速：0.3 mL/min，注入量：10 μL；質譜儀之設定條件：掃瞄範圍m/z 100-500，Nebulizer gas N2，流速9 L/min，溫度350 ℃，Ion charge control (ICC control) : Target ion 30000，Max Accu Time : 300 ms，分析時間為 0.5 分鐘；除巴比妥酸鹽類以Negative ion mode 偵測外，其餘皆以positive ion mode 偵測。所有之檢體再利用GC/MS 作定性或定量之確認。
在以ESI-MS 篩檢結果上，安非他命類、鴉片類、苯二氮平類以及古柯鹼有其可用性，其中安非他命類、鴉片類與苯二氮平類分析之敏感度高，唯特異性低，故偽陽性機率偏高，但因有機會再作確認檢驗，因此仍有其可用性。而巴比妥酸鹽類與大麻較不適用於此方法做分析，因感度偏低。
因此，ESI-MS 能提供分析之時效性、簡單之流程，應用於快速篩檢尿液中不同種類之藥物。

The use of illicit drugs is a widespread phenomenon in today’s society. A two-step procedure was usually employed for routine drugs of abuse analysis in urine samples. Samples were screened with appropriate immunoassays, the presumptive positives were then confirmed with gas chromatography mass spectrometry (GC/MS). With the constant appearance of new drugs(designer drugs), the available immunoassays may not be able to detect the new drugs. It is thus desirable to have a universal assay that has the ability to identify different kind of drugs in urine samples.
Recently, high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) has become a powerful tool for quantitative confirmatory analysis of drugs of abuse and has begun to appear in the field of forensic toxicology. In this study, we evaluated several solid phase extraction columns and selected Clean Screen Column for further study. We developed and validated a sensitive and specific direct injection-electrospray tandem mass spectrometry method for the identification of Amphetamines, Opiates, Benzodiazepines, Cocaine, and respective metabolites in urine samples. A group of 870 urine samples were screened with Amphetamine, Opiates, THC, Cocaine metabolites, Benzodiazepines and Barbiturates immunoassay reagents. Parts of the samples were further analyzed with electrospray ionization-mass spectrometry (ESI-MS) screening, and GC-MS confirmation. Results obtained with these methods were compared to evaluate assay sensitivity and specificity. Extracted drugs were also analyzed with LC-ESI-MS. Elutes of CH2Cl2:Isopropanol:Ammonia (78:20:2) were saved for all the drugs except for the Barbiturates. The methanol and CH2Cl2:Isopropanol:Ammonia (78:20:2) elutes were saved for barbiturates. After evaporation to dryness, samples were dissolved in mobile phase and 10-μL was injected directly onto the ESI-MS system. The mobile phase was 5 % of 10 mM ammonium acetate pH 7.5 containing 0.05 % of trifluoroacetic acid (TFA) and 95 % of methanol containing 0.05 % of TFA, flow rate was set at 0.3 mL/min for all analysis. The MSD parameters were as follows : scan range m/z 100-500, drying gas temperature 350 ℃, nebulizer pressure 40 psi, positive ion mode was used to monitor all drugs, except barbiturates, which was monitored with negative ion mode. The quantitative confirmatory LC/MS parameters were as follows : Waters SymmetryShield RP18 (4.6 X 250 mm, 5μm) column was used, mobile phase was 70 % of 10 mM ammonium acetate pH 7.5 containing 0.05 % of TFA (A) and 30 % methanol containing 0.05 % of TFA (B), flow rate was set at 1 mL/min for all analysis in 17 min, the mobile phase gradient was set as follows : 3 min, (A) 70 %, (B) 30 %; 5-6 min, (A) 0 %, (B) 100 %; 7-15 min, (A) 70 %, (B) 30 %, post time : 2 min. The confirmation method were used for all drugs, except opiates.
Results of Amphetamines, Opiates, Benzodiazepines, Cocaine, and respective metabolites obtained with ESI-MS method, showed high sensitivity, low specificity and with many false positive samples as compared with GC/MS. Barbiturates and Cannabinoids can not be analyzed with this procedure because of the low sensitivity. LC-ESI-MS with SIM mode could be employed to increase sensitivity. The method was simple, time efficient, and applicable for the monitoring of many different kinds of illicit drugs.

目錄
壹、前言
1-1.背景介紹............................................1
1-1-1.毒品、藥物濫用回顧與分析..........................1
1-1-2.藥物總彙..........................................4
1-1-3.藥物各論..........................................4
1-2.分析方法及文獻回顧..................................27
1-3.本實驗研究方法介紹..................................33
1-4.研究目的............................................35
貳、實驗方法
2-1.藥物及化學溶劑......................................36
2-1-1.藥物之標準品......................................36
2-1-2.化學溶劑..........................................38
2-1-3.標準品與化學溶劑之配製............................38
2-2.檢體之來源..........................................41
2-3.萃取方法之選擇......................................42
2-4.儀器設備與參數設定..................................44
2-4-1.儀器設備..........................................44
2-4-2.液相層析質譜儀之參數設定..........................50
2-4-3.氣相層析質譜儀之參數設定..........................53
2-4-4.酵素免疫分析儀之（參數）設定......................57
2-5.敏感性(Sensitivity)和特異性(Specificity)實驗之評估..64
2-5-1.敏感性和特異性之實驗方法..........................64
2-5-2.敏感性和特異性之實驗步驟..........................65
2-5-3.敏感性和特異性實驗之計算..........................74
参、結果與討論
3-1.酵素免疫分析法之結果分析............................76
3-2.萃取方法之選擇......................................76
3-3.液相層析質譜儀之篩驗結果分析........................78
3-4.安非他命類之結果分析................................79
3-5.苯二氮平類之結果分析................................82
3-6.古柯鹼類之結果分析..................................83
3-7.巴比妥酸鹽類之結果分析..............................83
3-8.大麻類之結果分析....................................84
3-9.鴉片類之結果分析....................................86
3-10.本研究方法與其他研究方法之比較.....................88
肆、結論................................................90
參考資料...............................................159
附件...................................................165

1. 林杰樑. 臨床毒品藥物濫用學. 合記，1991.
2. 行政院衛生署管制藥品管理局，藥物濫用與防治，1997.
3. 行政院衛生署管制藥品管理局，92年台灣地區精神醫療院所通報藥物濫用使用藥物之種類分布統計表(http://www.nbcd.gov.tw/prop/prop_4-04.asp)
4. 行政院衛生署管制藥品管理局，87~94年台灣地區濫用藥物尿液檢驗統計表(http://www.nbcd.gov.tw/prop/prop_04-01_02-88-92.asp)
5. 李志恆. 物質濫用. 行政院衛生署管制藥品管理局，2003.
6. 詹舜安, 劉宗榮. 液相層析串聯式質譜儀之原理與應用. 科儀新知，2002；24：77-89.
7. 陳玫慧, 丁望賢. 以電灑游離質譜法研究環糊精非共價鍵錯合物之原理與應用. 科儀新知，2004；25：72-82.
8. Pfleger, K.; Maurer, H. H.; Weber, A. Mass spectral and GC data on drugs, posions, pesticides, pollutants and their metabolites; Verlag Chemie：Weinheim, Germany. 1992；pp 5-32.
9. McLafferty, F.W. Wiley Registry of Mass Spectral Data, 6th ed.; Wiley：New York. 1997.
10. Pfleger K, Maurer HH, Weber A. et al. Mass Spectral Library of Drugs, Poisons, Pesticides and their Metabolites. 3ed. Oalo Alto. CA：Angilent Technologies. 2000.
11. McFadden W.H., Schwartz H.L.and Evans S. J Chromatogr. 1976 ; 122:389.
12. Marquet P, Lachatre G. Liquid chromatography-mass spectrometry：potential in forensic and clinical toxicology. J Chromatogr B Biomed Sci Appl. 1999 Oct 15; 733(1-2): 93-118.
13. Van Bocxlaer JF, Clauwaert KM, Lambert WE, Deforce DL, Van den Eeckhout EG, Deleenheer AP. Liquid chromatography-mass spectrometry in forensic toxicology. Mass Spectrom Rev. 2000 Jul-Aug; 19(4): 165-214.
14. Maurer HH. Liquid chromatography- mass spectrometry in forensic and clinical toxicology. J Chromatogr B Biomed Sci Appl. 1998 Aug 21; 713(1): 3-25.
15. Xiaohua Chen, Jaap Wijsbeek, Jan Van Veen, Jan Piet Franke and Rokus A. De Zeeuw. Solid-phase extraction for the screening of acidic, neutral and basic drugs in plasma using a single-column procedure on Bond Elut Certify. J Chromatogr B Biomed Sci Appl. 1990 Feb 16; 529:161-166.
16. Xiaohua Chen, Jan Piet Franke, Jaap Wijsbeek and Rokus A. de Zeeuw. Study of lot-to-lot reproducibilities of Bond Elut Certify and Clean Screen DAU mixed-mode solid-phase extraction columns in the extraction of drugs from whole blood. J Chromatogr B Biomed Sci Appl. 1993 April 22; 617:147-151.
17. Saint-Marcoux F, Lachatre G, Marquet P. Evalution of an improved general unknown screening procedure using liquid chromatography-electrospray –mass spectrometry by comparison with gas chromatography and high-performance liquid-chromatography-diode array detection. J Am Soc Mass Spectrom. 2003 Jan; 14(1) : 14-22.
18. Helena K. Nordgren and Olof Beck. Multicomponent screening for drugs of abuse. Ther Drug Monit. 2004 Feb; 26(1):90-97.
19. Helena K. Nordgren, Per Holmgren, Paula Liljeberg, Nadja Eriksson and Olof Beck. Application of direct urine LC-MS-MS analysis for screening of novel substances in drug abusers. J Anal Toxicol. 2005 May-Jun; 29:1-6.
20. Statistics of positive rate of drugs of abuse in urine. www.nbcd.gov.tw Accessed August 12, 2004.
21. Nationwide trends, www.drugabuse.gov Accessed August 12, 2004.
22. Liu RH. Evaluation of common immunoassay kits for effective workplace drug testing. In “Handbook ofWorkplace Drug Testing”. Eds. Liu RH and Goldberger BA. AACC Press,Washington DC. 1995, pp 67-130.
23. Lua, AC, Hu AR, Lin BF, Yeh PC, Lin HR, Tseng YT. Evaluation of immunoassays for the determination of MDMAand cannabinoids in urine samples. J Food and Drug Analysis. 2003; 11:108-113.
24. Quinn PG. Kuhnert BR. Kaine CJ. Syracuse CD. Measurement of meperidine and normeperidine in human breast milk by selected ion monitoring. Biomed Environmental mass Spectrom. 1986; 13: 133-135.
25. Reid RW. Gerbeck CM. Detection of pentazocine and tripelennamine in urine. Clin Chem. 1981; 27: 10.
26. Van Nimmen NF, Poels KL, Veulemans HA. Highly sensitive GC-MS screening method for the determination of pictogram levels of fentanyl, sufentanil and alfentanil and their metabolites in urine of opioid exposed workers. J Chromatogr B Analyt Technol Biomed Life Sci. 2004; 804:375-387.
27. Goeringer KE, Logan BK, Christian GD. Identification of tramadol and its metabolites in blood from drug-related death and drug-impaired drivers. J Anal Toxicol. 1997; 21:529-537.
28. Tatsuno M. Nishikawa M. Katagi M. Tsuchihashi H. Simultaneous determination of illicit drugs in urine by liquid chromatography-mass spectrometry. J Anal Toxicol. 1996; 20:281-286.
29. Bogusz MJ. Liquid chromatography-mass spectrometry as a routine method in forensic sciences: a proof of maturity. J Chromatogr B Biomed Sci Applications. 2000; 748:3-19.
30. Maurer HH. Liquid chromatography-mass spectrometry in forensic and clinical toxicology. J Chromatogr B Biomed Sci Applications. 1998; 713:3-25.
31. Miki A. Tasuno M. Katagi M. Nishikawa M. Tsuchihashi H. Simultaneous determination of eleven benzodiazepine hypnotics and eleven relevant metabolites in urine by column-switching liquid chromatography-mass spectrometry. J Anal Toxicol. 2002; 26:87-93.
32. Zweingenbaum J. Henig K. Steinborner S.Wachs T. Henion J. High-throughput bioanalytical LC/MS/MS determination of benzodiazepines in human urine: 1000 samples 162 per 12 hours. Anal Chem. 1999; 71:2294-2300.
33. Tai SS.Welch MJ. Determination of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid in a urine-based standard reference material by isotope-dilution liquid chromatography-mass spectrometry with electrospray ionization. J Anal Toxicol. 2000; 24:385-389.
34. Moeller MR. Kraemer T. Drugs of abuse monitoring in blood for control of driving under the influence of drugs. Ther Drug Monit. 2002; 24:210-221.
35. Nordgren HK. Bodin K. Beck O. Chromatographic screening of zopiclone metabolites in urine using liquid chromatography and liquid chromatography-mass spectrometry techniques. Ther Drug Monit. 2002; 24:410-416.
36.Wey AB, ThormannW. Capillary electrophoresis and capillary electrophoresis ion trap mutiple-stage mass spectrometry for the differentiation and identification of oxycodone and its major metabolites in human urine. J chromatogr B Analyt Technol Biomed Life Sci. 2002; 770:191-205.
37. Dawson M, Fryirs B, Kelly T, Keegan J. Mather LE. A rapid and sensitive high-performance liquid chromatography-electrospray ionization triple quadrolple mass spectrometry method for the quantification of oxycodone in human plasma. J Chromatogr B. 2002; 40:40-4.
38. Lua AC and Lin HR. A rapid and sensitive screening procedure for ketamine in urine samples. The 18th International Conference of Clinical Chemistry and Laboratory Medicine. 2002; Kyoto, Japan.
39. Chace DH, Millington DS, Terada N, Kahler SG, Roe CR, Hofman LF. Rapid diagnosis of phenylketonuria by quantitative analysis for phenylalanine and tyrosine in neonatal blood spots by tandem mass spectrometry. Clin Chem. 1993; 39:66-71.
40. Chace DH, Hillman SL, Millington DS, Kahler SG, Roe CR, Naylor EW. Rapid diagnosis of maple syrup urine disease in blood spots from newborns by tandem mass spectrometry. Clin Chem. 1995; 41:62-8.
41. Liu RH, Gadzala DE. Extraction.Washington, DC: American Chemical Society, 1997:41-50.
42. 徐健民. 工作場所藥物檢驗彙編. 中央警察大學, 1997.
43. 黃寶慧, 李茂榮. 固相萃取技術. 化學, 1998; 56:319-26.
44. G. E. Platoff, Jr. and J. A. Gere. Solid phase extraction of abused drugs from urine. Forensic Sci Rev. 1991; 3:117-133.
45. 羅初英. 氣相層析質譜法的原理與應用. Chemistry (The Chinese Chem. Soc, Taiwan China). 1990 Mar; 48(1):pp 39-49.
46. Marion Villain, Marta Concheiro, Vincent Cirimele, Pascal Kintz. Screening method for benzodiazepines and hypnotics in hair at pg/mg level by liquid chromatography-mass spectrometry/mass spectrometry. J Chromatogr B. 2005 Feb; 825:72-78.
47. Yasushi Hori, Manami Fujisawa, Kenji Shimada, Yasuo Hirose, and Toshiharu Yoshioka. Method for screening and quantitative determination of serum levels of salicylic acid, acetaminophen, theophylline, phenobarbital, bromvalerylurea, pentobarbital, and amobarbital using liquid chromatography/electrospray mass spectrometry. Biol Pharm Bull. 2006; 29(1):7-13.
48. Helena Teixeira, Paula Proenca, Alain Verstraete, Francisco Corte-Real, Duarte Nuno Vieira. Analysis of △9-tetrahydrocannabinol in oral fluid samples using solid-phase extraction and high-performance liquid chromatography-electrospray ionization mass spectrometry. Forensic Sci Int. 2005 Apr; 150:205-211.
49. J. Eichhorst, M. Etter, J. Lepage, and D.C. Lehotay. Urinary screening for methylphenidate(Ritalin) abuse: a comparison of liquid chromatography-tandem mass spectrometry, gas chromatography-mass spectrometry, and immunoassay methods. Clin Biochem. 2004; 37:175-183.
50. Riet Dams, Constance M. Murphy, Willy E. Lambert and Marilyn A. Huestis. Urine drug testing for opioids, cocaine, and metabolites by direct injection liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2003; 17:1665-1670.