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研究生:劉毓芬
研究生(外文):Yu-fen Liu
論文名稱:以液相層析電噴灑游離法離子阱質譜儀建立濫用藥物在尿液樣本之廣篩方法
論文名稱(外文):Screening Procedures for Drugs of Abuse in Urine Samples with Liquid Chromatography-Electrospray Ionization Ion Trap Mass Spectrometry
指導教授:邱鐵雄邱鐵雄引用關係賴滄海賴滄海引用關係
指導教授(外文):TED-H ChiuChang-H Lai
學位類別:碩士
校院名稱:慈濟大學
系所名稱:藥理暨毒理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:185
中文關鍵詞:電噴灑游離法液相層析質譜儀篩檢藥物濫用
外文關鍵詞:Electrospray IonizationLC/MSScreeningDrugs of Abuse
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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
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