利用實驗設計搭配毛細管電泳線上濃縮技術分析尿液中數種濫用藥品及部分主要代謝物

Experimental design and on-line stacking capillary electrophoresis for the determination of abused drugs and some major metabolites in urine

蕭郁慈(Yu-Tzu Hsiao)

指導教授 ： 吳秀梅

高雄醫學大學/藥學院/臨床藥學研究所/碩士(2007年)

摘要

本論文利用化學計量統計實驗設計理論（chemometric experimental design）建立毛細管電泳分析法，監測尿液中常見濫用藥品及其指標代謝物，包括codeine、morphine、ketamine、methamphetamine、alprazolam、α-hydroxyalprazolam、clonazepam、7-aminoclonazepam、diazepam、nordiazepam、oxazepam、flunitrazepam、7-aminoflunitrazepam及
N-demethylflunitrazepam。
最適化毛細管電泳條件的尋找是利用化學計量統計實驗設計理論採三個步驟進行，包含預試驗、二水準部分因子設計法及中心混成設計法。經由預試驗推測出本研究進行二水準部份因子設計法時，應考慮六項可能影響到實驗目標結果的參數，包括磷酸鹽緩衝液的濃度、pH值、掃集緩衝液中添加的界面活性劑濃度及有機修飾物的添加量、分離緩衝液中有機修飾物的添加量及溶解萃取產物之緩衝液濃度。由二水準部分因子設計法得到的分析結果，具有明顯影響的參數為磷酸鹽緩衝液濃度、pH值、掃集緩衝液中添加的界面活性劑濃度及分離緩衝液中有機修飾物的添加量，這四個參數再利用中心混成設計進行探討，以建立理想分析條件。
利用液相萃取法進行尿液樣品前處理後，再用逆向移動的微胞形成堆積及掃集的線上濃縮方式搭配紫外光偵測進行分析。毛細管電泳儀的條件如下，首先毛細管先注滿75 mM含30％甲醇的磷酸鹽緩衝液 (pH 2.5)，接著將待測檢品用壓力1 psi 取樣200秒注入毛細管內。檢品取樣完成後，將毛細管放置含有10％甲醇及65 mM正十二烷基硫酸鈉的掃集緩衝液中，給予-15 kV的電壓(陽極在檢測器端)進行分離。
經方法確效後，morphine的監測濃度範圍為0.15-3 μg/mL，codeine、methamphtetamine及ketamine皆為0.1-3 μg/mL，alprazolam及oxazeapm為0.1-1 μg/mL，其餘藥品皆為0.1-1.2 μg/mL。所有檢量線均呈現良好的線性關係 ( r≧0.992 )﹔評估同日間及異日間分析的精確度及準確性，其RSD和RE值皆小於14%，且偵測極限可達20-50 ng/mL。此方法也成功的應用於濫用藥品成癮者的尿液檢品，並經過GC-MS 或LC-ESI-MS的確認。

關鍵字

毛細管電泳 ； 濫用藥品 ； 尿液 ； 實驗設計

並列摘要

A capillary electrophoresis method was established for the determination of some common abused drugs in urine samples, including alprazolam, α-hydroxyalprazolam, clonazepam, 7-aminoclonazepam, diazepam, nordiazepam, flunitrazepam, 7-aminoflunitrazepam, norflunitrazepam, oxazepam, codeine, morphine, methamphetamine and ketamine . The method was developed using chemometric experimental design in a three-step procedure, including preliminary experiments, fractional factorial design (fFD) and central composite design (CCD). To find the significant variables, six factors were selected from the preliminary experiments, including the concentration and the pH of the phosphate buffer, the concentration of surfactant in the sweeping buffer, the percentage of organic solvent in the separation and the sweeping buffer, and the concentration of reconstitution solution. Then fFD was performed. Four out of these six factors were found to be siginificant, and were further investigated in the final step, CCD.
In our study, samples were pre-treated with liquid-liquid extraction, then analyzed by using stacking with reverse migrating micelles as the on-line concentration method. The optimized CE conditions were as follow: First, phosphate buffer (75 mM, pH 2.5) containing 30% methanol was filled into capillary, then pressure injection (1 psi, 200 s) was used to load the samples. Phosphate buffer (75 mM, pH 2.5) containing 10% methanol and 65 mM SDS was used as the sweeping buffer to separate the analytes at −15 kV.
During method validation, calibration plots were linear (r >0.992) over a range of 0.1-3 μg/mL for codeine, ketamine and methamphetamine and 0.15-3 μg/mL for morphine, 0.1-1 μg/mL for alprazolam and oxazepam, and 0.1-1.2 μg/mL for other analytes. During intra- and inter-day analysis, relative standard deviations (RSD) and relative errors (RE) were all less than 14%. The analytes could be simultaneously analyzed and have a detection limit down to 20-50 ng/mL.
The results showed good coincidence with GC–MS or LC-ESI-MS. This method was feasible for application to detect trace levels of abused drugs.

並列關鍵字

capillary electrophoresis ； aubsed drugs ； urine ； experimental design

參考文獻

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