建立高感度同時分析老年人常用藥物及乳癌治療藥物之毛細管電泳法並應用於人體血漿檢體測定

Establish High Sensitive Capillary Electrophoresis Methods for Simultaneous Determination of Medicines in the Elderly Population and Patients with Breast Cancer in Human Plasma

魏劭芸(Shao-Yun Wei)

指導教授 ： 陳素惠

高雄醫學大學/藥學院/藥學研究所/博士(2012年)

摘要

此論文利用高效能毛細管電泳法(capillary electrophoresis, CE)針對老年人常用藥物與乳癌賀爾蒙治療藥物發展出簡單且高感度的微量分析方式並應用於人體血漿。主要研究包含以電壓取樣配合高感度之線上濃縮技術建立阿茲海默症、高血壓、高血脂及糖尿病常用治療藥物之同時分析，並利用大體積進樣之線上濃縮技術建立乳癌賀爾蒙治療藥物anastrozole, letrozole及tamoxifen之微量分析。主要研究成果摘錄如下：

A.建立高感度同時分析老年人常用藥物之毛細管電泳法並應用於人體血漿檢體測定
1.利用直接電壓取樣之毛細管電泳法針對第二型糖尿病治療藥物metformin之微量分析並與高壓液相層析法進行比較
本研究利用直接電壓取樣之毛細管電泳法對metformin建立簡單、快速及高感度之分析方式。實驗過程中不需任何樣品前處理，有效縮短整體分析所耗費的時間及試藥，達到偵測藥物治療濃度的目標，並與傳統高壓液相層析法進行比較，突顯出毛細管電泳法之優勢。此分析方法條件為：25 mM Tris緩衝溶液(pH 4)配合電壓取樣。此方法應用於監測病患的血漿濃度及計算健康受試者服藥後的藥物動力學參數。

2.利用電場加大樣品進樣法搭配sweeping之毛細管電泳法針對酸性藥物atorvastatin、irbesartan與rosuvastatin及鹼性藥物amlodipine與donepezil之同時微量分析
本方法以高感度線上濃縮技術之毛細管電泳法同時分析多個老年人常用藥物，同時包含酸性與鹼性藥物，並應用於人體血漿檢品之微量分析。前處理採用液相萃取法(liquid-liquid extraction, LLE)，分析方法先以平衡用緩衝溶液50 mM Tris緩衝液內含41%甲醇及0.1% poly(ethylene oxide) (pH 3)充填整個毛細管，以電壓取樣後，再利用分離用緩衝液25 mM Tris緩衝溶液內含60 mM sodium dodecyl sulfate (SDS) (pH 3)配合負電壓進行分離，利用微胞(micelles)產生sweeping的方式進行樣品線上濃縮且達到有效分離的目的。此分析方式應用於amlodipine、donepezil、atorvastatin、irbesartan與rosuvastatin市售錠劑之含量分析及病人血漿檢品之監測。

B.毛細管電泳法對人體血漿中乳癌賀爾蒙治療藥物anastrozole、letrozole及tamoxifen之微量分析研究
本實驗以大體積進樣的線上濃縮方式針對乳癌患者的賀爾蒙輔助藥物anastrozole、letrozole及tamoxifen之同時微量分析。此方法利用LLE進行樣品前處理，分析條件為25 mM磷酸緩衝液(pH 2.5)內含30%甲醇及50 mM SDS，以壓力進行大體積取樣後，配合負電壓同時產生樣品濃縮與分離的分析方式。此研究應用於乳癌患者血漿檢品中anastrozole、letrozole及tamoxifen之濃度監測。

關鍵字

Metformin ； Amlodipine ； Atorvastatin ； Anastrozole ； Donepezil ； Irbesartan ； Letrozole ； Rosuvastatin ； Tamoxifen ； 糖尿病 ； 高血壓 ； 高血脂 ； 阿茲海默症 ； 乳癌 ； 毛細管電泳 ； 線上濃縮 ； 藥物濃度監控

並列摘要

The purpose of this work is directed to develop the simple and sensitive methods for determination of medicines in the elderly population and in patients with breast cancer by capillary electrophoresis (CE). The main studies include on-line sample stacking coupled with electrokinetic injection for trace analysis of medicines for Alzheimer’s disease, hypertension, hyperlipidemia and diabetes mellitus and establishment of simultaneous analysis for breast cancer drugs, including anastrozole, letrozole and tamoxifen, by CE. The results are summarized as follow:

A.Establish high sensitive capillary electrophoresis methods for the simultaneous determination of medicines in the elderly population in human plasma

1.CE with direct sample injection for the determination of metformin in plasma for type 2 diabetic mellitus: An adequate alternative to high performance liquid chromatography (HPLC)
A simple, fast and sensitive capillary electrophoresis method combined with direct sample injection has been established for the determination of metformin. It is effective for shortness of total analysis time and reagents due to no sample pretreatment before sample injection and application for monitoring therapeutic concentration. CE was compared with the results obtained by a validated HPLC method. The analysis of metformin was performed in 25 mM Tris buffer (pH 4) with electrokinetic injection enabling application for monitoring plasma concentration of patients and assessment pharmacokinetic and clinical studies for a healthy volunteer.

2.Sample stacking by field-amplified sample injection (FASI) and sweeping for simultaneous analysis of acidic and basic components in clinic application
In this study, on-line sample concentration method was used to analyze acidic and basic components of medicines for the elderly group in a single run and also successfully applied to monitor the tested drugs in plasma. All plasma was pretreated by liquid-liquid extraction (LLE) before injection. The capillary was conditioned with rinse buffer (50 mM Tris buffer with 41% MeOH and 0.1% PEO, pH 3) and sample loading was achieved by electrokinetic injection. Then, the analytes were focused and separated by sweeping in the separation buffer (25 mM Tris buffer and 60 mM SDS, pH 3) under reverse polarity. This analysis method was determined concentrations of amlodipine, donepezil, atorvastatin, irbesartan and rosuvastatin in the commercial tablets and patients’ plasma.

B.Large volume sample stacking with sweeping of anastrozole, letrozole and tamoxifen in patients with breast cancer by capillary electrophoresis
The on-line sample stacking was developed for simultaneous determination of anastrozole, letrozole and tamoxifen in human plasma, which coupled large-volume sample stacking (LVSS) with sweeping. A LLE with hexane-toluene (80:20, v/v) was necessary as sample pretreatment. Baseline separation of the analytes was obtained on 25 mM phosphate buffer (pH 2.5) with 50 mM SDS and 30% MeOH as background electrolytes. After large volume sample injection, the voltage was performed at -15 kV for sample concentration and separation. Trace determinations of anastrozole, letrozole and tamoxifen in patients with breast cancer after oral administration the analytes were investigated.

並列關鍵字

Metformin ； Amlodipine ； Atorvastatin ； Anastrozole ； Donepezil ； Irbesartan ； Letrozole ； Rosuvastatin ； Tamoxifen ； Diabetes mellitus ； Hypertension ； Hyperlipidemia ； Alzheimer’s disease ； Breast cancer ； Capillary electrophoresis ； On-line stacking ； Drug montoring

參考文獻

01.Hines L.E., Murphy J.E., Potentially harmful drug-drug interactions in the elderly: a review, Am. J. Geriatr. Pharmacother. 2011, 9, 364-377.

02.Hastings S.N., Schmader K.E., Sloane R.J., Weinberger M., Pieper C.F., Goldberg K.C., Oddone E.Z., Quality of pharmacotherapy and outcomes for older veterans discharged from the emergency department, J. Am. Geriatr. Soc. 2008, 56, 875-880.

03.Qato D.M., Alexander G.C., Conti R.M., Johnson M., Schumm P., Lindau S.T., Use of prescription and over-the-counter medications and dietary supplements among older adults in the United States, JAMA. 2008, 300, 2867-2878.

04.Shargel L., Yu A.B.C., Applied Biopharmaceutics and Pharmacokinetics 3th Edition, Simon & Schuster Pte Ltd., Singapore, 1994.

05. Klotz U., Pharmacokinetics and drug metabolism in the elderly, Drug Metab. Rev. 2009, 41, 67-76.