- 學位論文
多重濫用藥物毛髪檢驗之質譜研究
Hair analysis for multiple drugs of abuse by mass spectrometry
摘要
濫用藥物檢驗可以了解受檢者是否使用濫用藥物。然而，常見的尿液檢驗只有數日檢驗視窗。毛髮檢驗，有別於其他的生物檢體，具有長時間之檢驗視窗(數月~數年)，可完整記錄受檢者的用藥歷程。目前國內對濫用藥物毛髮檢驗的研究並不多，因此，本研究目的希望能針對國內外之濫用藥物，發展靈敏且可信賴之毛髮檢驗。 近年來，多重藥物濫用者有越來越多的趨勢，加上毛髮檢體量遠低於尿液，本研究首先針對國內4類常見濫用藥物及其代謝物，包括安非他命、甲基安非他命、MDMA、MDA、MDEA、嗎啡、可待因、6-乙醯嗎啡、K它命及Norketamine，成功地發展氣相層析質譜分析(GC/EI-MS)之毛髮檢測方法，不但大幅增加濫用藥物檢測廣度與分析的效率，而且減少了檢驗所需要的毛髮量。而為了解決一般需隔夜浸泡，才能溫和的自毛髮中取出藥物之耗時問題，本研究也成功地發展毛髮剝皮方法，將浸泡時間縮短為1小時，有效增進濫用藥物之毛髮檢驗效率。 接著，為了建立更多類濫用藥物與其代謝物的GC/EI-MS毛髮分析方法，研究併入了大麻、古柯鹼、配西汀、特拉嗎竇、丁基原啡因、美沙酮、Naloxone、2C-B、LSD等藥物及其代謝物。由於需要分析的藥物與其代謝物種類繁多，因此依照藥物結構與性質，分成醯化與矽化兩大衍生化系統，結果發現，在「系統A」有13種濫用藥物能以HFBA/EA/HFPOH進行醯化反應；而在「系統B」有14種濫用藥物能以BSTFA進行矽化反應。而為了提升微量毛髮檢體上之靈敏度，研究由以往的GC/EI-MS質譜方法提升至氣相層析/化學游離法質譜儀(GC/CI-MS)，分別以正離子化學游離化(GC/PCI-MS) 和負離子化學游離化(GC/NCI-MS)兩種方法進行分析，結果發現，使用GC/PCI-MS可分析13種以BSTFA衍生化之濫用藥物。而使用GC/NCI-MS可分析11種HFBA衍生化之濫用藥物。此外，由於GC/NCI-MS對K他命之高靈敏度，可靈敏並準確的訂出NK/K比例，進一步區分出毛髮中之藥物來自主動吸食K他命或外部汙染。而THC-COOH在毛髮中相當微量，為了提升其靈敏度，嘗試了醯化及烷化兩種衍生化方法，以GC/NCI-MS分析。結果發現，經PFPA/HFPOH衍生化後的THC-COOH其靈敏度最佳，可達到2.5 pg/mg hair。 由於液相質譜方法之樣品前處理步驟較為單純，具有高分析通量之特性，因此本研究發展濫用藥物之LC/MS/MS 方法與引入charge modified之LC/MS/MS方法。利用流動注入法注入藥物標準品，首先得到各藥物之全掃描圖譜與產物圖譜，並評估ESI與APCI對各藥物靈敏度，結果發現LC/MS/MS方法大多高於GC/MS方法。另外，針對感度較差的含酚類官能基濫用藥物，如嗎啡、6-AM、THC及THC-COOH，則使用dansyl chloride快速衍生化法加強其靈敏度，結果發現經由APCI游離化，在API 3000儀器下，嗎啡及6-AM可達0.1 pg/mg hair，而THC及THC-COOH可達1 pg/mg hair。
並列摘要
Drug testing provides objective information regarding an individual’s use of or exposure to illicit drugs. Traditionally, drug testing has used urine testing to monitor drug use. However, urine testing only provides a short-term historical record of drug exposure. Hair differs from other biological specimens due to its long detection window (months to years), enabling retrospective investigation of chronic and past consumption. In Taiwan, there are fewer researches about hair testing for drugs of abuse. The purpose of this study is to develop sensitive and trustworthy hair testing method for the determination drugs of abuse. Recently, the multiple-drug cocktails had become the worldwide trend. In addition, the amount of hair sample size is much lower than in urine. Therefore, a reliable gas chromatography/mass spectrometry (GC/MS) hair testing that we have developed can be successfully used for the simultaneous determination of drugs of common abuse in Asia, including amphetamines (amphetamine, AP; methamphetamine, MA; methylenedioxy amphetamine, MDA; methylenedioxy methamphetamine, MDMA; methylenedioxy ethylamphetamine, MDEA), ketamine (ketamine, K; norketamine, NK), and opiates (morphine, MOR; codeine, COD; 6-acetylmorphine, 6-AM). The simultaneous quantitative determination of several compound types is not only increases the ability and extent of drug testing but also decreases the amount of sample size. Then, a hair stripped method was developed for improving the time consuming of sample digestion/extraction pretreatment procedures. The analyses are conducted in less than 1 hr with minimal sample pretreatment. Then, we developed the GC/EI-MS hair testing methods of the more drugs such as cannabis, cocaine, pethidine, tramadol, buprenorphine (BUP), methadone, naloxone, 4-Bromo-2,5-dimethoxyphenethylamine (2C-B) and Lysergic acid diethylamide (LSD) in order to develop the more comprehensive hair testing methods. The “System Hair Testing” was according to the structure of drugs. The「system A」with HFBA/EA/HFPOH derivatization can be successfully used for the simultaneous determination of drugs of abuse, including AP, MA, MDMA, MDA, MDEA, K, NK, MOR, COD, 6-AM, 2C-B, cocaine (COC), Benzoylecgonine (BZE). The「system B」with BSTFA derivatization can be successfully used for the simultaneous determination of drugs of abuse, including COC, BZE, MOR, COD, 6-AM, pethidine, tramadol, BUP, Norbuprenorphine (NorBUP), methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP), naloxone and LSD. Drugs concentration in hair sample is much lower than in urine, so the hair sample calls for more sensitive analytical methods. Therefore, we have developed the Gas chromatography/chemical ionization mass spectrometry (GC/CI-MS) hair testing methods of the drugs of abuse. The「GC/PCI-MS」with BSTFA derivatization can be successfully used for the simultaneous determination of drugs of abuse, including COC, BZE, MOR, COD, 6-AM, pethidine, tramadol, BUP, NorBUP, methadone, EDDP, EMDP and naloxone. The 「GC/NCI-MS」with HFBA derivatization can be successfully used for the simultaneous determination of drugs of abuse, including AP, MA, MDMA, MDA, MDEA, K, NK, MOR, COD, 6-AM, 2C-B. The sensitivity of K and NK can be dramatically enhanced after GC/NCI-MS analysis, especially for NK. As a result, metabolite-to-drug ratio was useful to evaluate passive contamination from active K consumption. The low amount of THC-COOH incorporated in the hair and the complexity of the hair matrix, a very sensitive and specific technique is required for accurate quantification of this metabolite. In order to choose a sensitive derivatization reagent for THC-COOH, acylation and alkylation were evaluated. The LOD values obtained were 2.5 pg/mg after PFPA/HFPOH derivatization in GC/NCI-MS. The liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods which do not require derivatization or extensive sample clean-up procedures. Therefore, LC/MS/MS method and charge modified LC/MS/MS method were developed for drugs of abuse. All drugs were injected into tandem mass spectrometers; full scan spectrum and product ion scan spectrum were acquired. The sensitivity of LC/MS/MS seems better than GC/MS. The dansyl chloride has been used in order to enhance signal intensity of the phenolic compounds. After fast dansyl chloride derivatization, the derivatives were injected into tandem mass spectrometers. The LOD of opiates (MOR and 6-AM) and cannabis (THC and THC-COOH) obtained were 0.1 pg/mg and 1 pg/mg in API 3000 with APCI positive mode.