毛細管電泳法對生物檢體中濫用藥物之檢測研究

Study on analysis of abused drugs in biological samples by capillary electrophoresis

林宜慧(Yi-Hui Lin)

指導教授 ： 吳秀梅

高雄醫學大學/藥學院/藥學研究所/博士(2008年)

摘要

藥物濫用常與犯罪相關並且衍生出許多社會問題，它不僅在台灣，也在日本、韓國和東南亞等國家引起嚴重的問題。本論文之目的為開發常見濫用藥物的分析方法，由建立毛細管電泳線上樣品堆積技術來提高感度，並應用於個案藥物濫用之確定及戒斷追蹤。尿液及毛髮為利用非侵入性方法即可採集之檢品，適用於法醫鑑識且各具意義，尿液檢品可監測藥癮者近期之藥物服用品項、劑量及代謝情形，而毛髮檢品則可長期保存受檢者之吸毒歷程，因此本研究針對此二類檢體進行分析。研究成果如下：
本研究應用選擇性大量陽離子取樣搭配掃集式微胞電動層析法(cation-selective exhaustive injection - sweeping - micellar electrokinetic chromatography, CSEI-Sweep-MEKC) 對尿液檢品中甲基安非他命(methamphetamine, MA)、嗎啡(morphine, M)、可待因(codeine, C)和愷他命(ketamine, K)進行分析研究。本法在不需尿液樣品前處理之情況下可直接進行，於有效長度40 cm之熔矽毛細管(內徑50 ?慆)，先通入分離用緩衝液磷酸鹽溶液(50 mM, pH 2.5)內含30%甲醇，接著通入一較高導電度之緩衝液磷酸鹽溶液(100 mM, 6.9 kPa, 99.9 s)，之後以 10 kV電動取樣 500秒，再用-20 kV電壓，通以掃集用緩衝液磷酸鹽溶液(25 mM, pH 2.5)內含20%甲醇及100 mM SDS，將待測物堆積濃縮並分離，於200 nm偵測之。分析方法經確效後，M和C的定量範圍是150 - 3000 ng/mL，MA為250 - 5000 ng/mL，K為50 - 1000 ng/mL，其相關係數皆大於0.9982。各待測物之偵測極限(S/N = 3，10 kV電動取樣 500秒)，M及C為15 ng/mL，MA和K則為5 ng/mL，以MA比較本法及毛細管區帶電泳法，本法約可提高感度六千倍。本法應用於28個實際藥癮者尿液檢品分析，其濫用藥物鑑定結果與常用之免疫分析法及氣相層析質譜法(GC-MS)相符。
另將此CSEI-Sweep-MEKC法推展至毛髮檢品中MA、M、C和K之分析研究。毛髮樣品先經簡單清洗及萃取步驟後，再以此CE法進行分析；本法經確效驗證，所用定量範圍是每毫克頭髮中MA和K為0.15 - 80 ng/mg，M為0.5 - 50 ng/mg，C為0.3 - 30 ng/mg，其相關係數皆大於0.999。各待測物之偵測極限(S/N = 3，10 kV電動取樣 600秒)，於每毫克頭髮中M為200 pg，C為100 pg，MA和K則為50 pg。本法應用於4個實際藥癮者頭髮檢品分析，其濫用藥物鑑定結果與液相層析質譜法(LC-MS)分析結果相符。
建立管制藥品及其代謝物之分析技術，可明確驗證藥癮者使用之藥物品項，並可探討相關之毒物動力學。本研究亦應用CSEI-Sweep-MEKC法對尿液檢品中M及其四種代謝物 morphine-3-glucuronide (M3G)、morphine-6-glucuronide(M6G)、codeine (C) 和normorphine (NM)進行分析研究。樣品經固相萃取後進行分析。於有效長度40 cm之熔矽毛細管(內徑 50 ?慆)，先通入分離用緩衝液磷酸鹽溶液(75 mM, pH 2.5)內含30%甲醇，接著通入一較高導電度之緩衝液磷酸鹽溶液(120 mM, 6.9 kPa, 99.9 s)，之後以 10 kV電動取樣 600秒，再用-20 kV電壓，通以掃集用緩衝液磷酸鹽溶液(25 mM, pH 2.5)內含22%甲醇及100 mM SDS，將待測物堆積濃縮並分離，於200 nm偵測之。分析方法經確效後，M、C和NM的定量範圍是30 - 3000 ng/mL，M6G為100 - 2000 ng/mL，M3G為80 - 3200 ng/mL，其相關係數皆大於0.998。各待測物之偵測極限(S/N = 5，10 kV電動取樣 600秒)，M、NM及C為10 ng/mL，M6G為35 ng/mL， M3G則為25 ng/mL。本法應用於5個實際藥癮者尿液樣品分析，濫用藥物鑑定結果與液相層析串聯質譜法(LC-MS-MS)分析結果相符。
本研究可應用於法庭分析中濫用藥物之監測及確認，因準確性高，可大幅降低由免疫分析所造成之偽陽性判定，減少後續質譜鑑定相關工作之成本，並可應用於M及其代謝物之毒物動力學等相關研究。

關鍵字

毛細管電泳法 ； 濫用藥物 ； 尿液 ； 毛髮

並列摘要

Drug abuse is always associated with crimes, trafficking and mob, which generate many social issues. It causes serious problems not only in Taiwan but also in many countries. The objective of this study is to establish an analytical method for monitoring the presence of trace amounts of illicit drugs in biological samples by capillary electrophoresis. Urine and hair belongs to the realm of non-invasive samples. Urine is most commonly used, as it provides direct evidence for the short-term use of illicit drugs. Hair samples, on the other hand, can provide a longer retrospective period (months to years) for drug abuse. Therefore, this research carried on the analysis of these two kinds of test samples. Several works have been done, including:
A cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-Sweep-MEKC) method was directly used to test some abuse drugs in human urine, including morphine (M), codeine (C), ketamine (K) and methamphetamine (MA). Due to its high sepecificity to analytes and none interference from urine matrix, it was unnecessary to have sample pretreatments. First, phosphate buffer (50 mM, pH 2.5) containing 30% methanol was filled into an uncoated fused silica capillary (40 cm, 50 μm I.D.), and then high conductivity buffer (100 mM phosphate, 6.9 kPa for 99.9 s) was followed. Electrokinetic injection (10 kV, 500 s) was used to load samples and to enhance sensitivity. The stacking step and separation were performed at -20 kV and 200 nm using phosphate buffer (25 mM, pH 2.5) containing 20% methanol and 100 mM sodium dodecyl sulfate. Using CSEI-Sweep-MEKC, the analytes could be simultaneously analyzed and have a detection limit down to ppb level. During method validation, calibration plots were linear (r > 0.9982) over a range of 150 - 3000 ng/mL for M and C, 250 - 5000 ng/mL for MA, and 50 - 1000 ng/mL for K. The limits of detection (LODs, S/N = 3, sampling 500 s at 10 kV) were 15 ng/mL for M and C, and 5 ng/mL for MA and K. Comparing with capillary zone electrophoresis, the results indicated that this stacking CE method could increase about 6000-fold sensitivity for analysis of MA. Our method was applied for analysis of 28 real urine samples. The results showed good coincidence with immunoassay and GC-MS.
Furthermore, this CSEI-Sweep-MEKC method was extended to analyze abuse drugs in addicts’ hair. After pretreatment of hair sample, this method was used to test for the presence of abused drugs in human hair. These drugs include M, C, K and MA. Using this method, the analytes could be simultaneously analyzed and have a detection limit down to the level of picogram per mg hair (ppt level). During method validation, calibration plots were linear (r > 0.999) over a range of 0.15 - 80 ng/mg hair for MA and K, 0.3 - 30 ng/mg hair for C, and 0.5 - 50 ng/mg hair for M. The LODs (S/N = 3, sampling 600 s at 10 kV) were 50 pg/mg hair for MA and K, 100 pg/mg hair for C, and 200 pg/mg hair for M. Our method was applied for analysis of real hair samples taken from addicts. The addicts’ specimens were also analyzed by LC-MS, and showed good coincidence of those results.
It is important to establish a simultaneous analytic method for abuse drug and its metabolites, which could provide systemic confirmation data and used for investigation of toxicokinetics study. After solid-phase extraction, CSEI-Sweep-MEKC method was used to examine M and its four metabolites in human urine, including M, C, normorphine (NM), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). First, phosphate buffer (75 mM, pH 2.5) containing 30% methanol was filled into an uncoated fused silica capillary (40 cm, 50 μm I.D.), and then high conductivity buffer (120 mM phosphate, 10.3 kPa for 99.9 s) was followed. The sample was loaded by electrokinetic injection (10 kV, 600 s) after solid-phase extraction. The stacking step and separation were performed at -20 kV and detected at 200 nm using phosphate buffer (25 mM, pH 2.5) containing 22% methanol and 100 mM sodium dodecyl sulfate. During method validation, calibration plots were linear (r > 0.998) over a range of 30 - 3000 ng/mL for M, NM and C, 100 - 2000 ng/mL for M6G, and 80 - 3200 ng/mL for M3G. The LODs (S/N = 5, sampling 600 s at 10 kV) were 10 ng/mL for M, NM and C, 35 ng/mL for M6G and 25 ng/mL for M3G. Our method was applied for analysis of 5 real urine samples from addicts. The specimens were also analyzed by LC-MS-MS, and showed good coincidence of results.
This study was feasible for detection and confirming abused drugs in forensic analysis. Because of its high accuracy, it can greatly reduce the false positive results produced by immunoassay, and drastically decrease the costs of the following confirmation test by GC-MS. It was also applicable for the toxicokinetics investigations of M and its metabolites.

並列關鍵字

capillary electrophoresis ； abused drugs ； urine ； hair

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