Ⅰ. 毛細管電泳法對藥物於生物檢品中及光學異構物之分析研究 Ⅱ. 高效能液相層析法對抗腫瘤藥物Mitomycin C與Epirubicin之同時分析研究

Ⅰ. The Analysis of Drug in Biosamples by Capillary Electrophoresis Ⅱ. Simultaneous Determination of Mitomycin C and Epirubicin by High Performance Liquid Chromatography

周育緯(Yu-Wei Chou)

指導教授 ： 陳素惠

高雄醫學大學/藥學院/藥學研究所/博士(2009年)

摘要

本論文主題一為利用毛細管電泳法之高分離能力、分析速度快，取樣少和低污染的優勢，配合線上紫外光檢測器，建立carbapenem類抗生素meropenem之微量分析方法，並應用在臨床生物檢品上；另亦建立第二代抗組織胺藥品cetirizine之光學異構物的分析方法及其在製劑和血漿檢品上的應用。主題二則是利用高效能液相層析法對抗腫瘤藥物mitomycin C和epirubicin於血漿檢品中同時分析，並應用在監測肝癌病患經導管化學藥物動脈栓塞療法(transcatheter arterial chemoembolization，TACE) 治療後，化療藥物自栓塞洩漏之血中濃度變化。所得成果摘錄如下：

Ⅰ. 毛細管電泳法對藥物於生物檢品中及光學異構物之分析研究

(一) 利用微胞電動層析法對血漿和腦脊髓液中meropenem進行定量分析並應用到細菌性腦膜炎病患檢品
本研究建立carbapenem類抗生素meropenem於生物檢體(包含血漿及腦脊髓液中)之分析方法，並應用於細菌性腦膜炎病患檢體之檢測。以C18固相萃取(solid-phase extraction)管柱進行血漿檢品前處理，而腦脊髓液檢品則直接注入，以cefotaxime作為內部標準品(IS)結合微胞電動層析法進行meropenem之定量分析。利用未修飾的毛細管(全長40.2 cm有效30 cm，內徑50 μm)，以40 mM Tris buffer (pH 8.0)添加200 mM sodium dodecyl sulfate (SDS)界面活性劑作為分離緩衝液系統，以紫外光檢測器在300 nm下偵測。對於影響meropenem在血漿和腦脊髓液檢品中分析的各項參數，包括pH值、Tris buffer濃度、界面活性劑SDS添加濃度，皆加以探討。本法可定量0.5-50 μg/mL範圍之meroepnem，偵測極限於血漿和腦脊髓液中分別為0.2和0.3 μg/mL (S/N=3)。此方法可應用於投予meropenem治療細菌性腦膜炎病人之血漿和腦脊髓液臨床檢品濃度檢測。

(二) 毛細管電泳法對cetirizine光學異構物分析研究與運用
本研究建立毛細管電泳法針對第二代抗組織胺類消旋異構物cetirizine，配合分離緩衝液系統中添加對掌選擇劑(chiral selectors)進行光學異構物的分析，並應用在製劑和血漿檢品之分析研究。以添加sulfated β-cyclodextrin (sulfated β-CD)作為對掌選擇劑(chiral selectors)於毛細管電泳分離緩衝溶媒系統，利用不具光學活性之cefazolin作為內部標準品(IS)，以直接注入方式，對cetirizine之光學異構物進行光學分析。討論可能會影響分離的條件，包含borate buffer的pH值和濃度，對掌選擇劑的種類和濃度。分離毛細管(全長40.2 cm，有效30 cm，內徑50 μm)，於borate buffer (5 mM，pH 8.7)加入對掌選擇劑1 ℅ sulfated β-CD (w/v)，配合分離電壓10 kV和紫外光檢測器200 nm下，對cetirizine進行光學異構物分離。利用最佳化分離條件下，完整的分離可在7分鐘內完成，對於levocetirizine可在1.0-50.0 μg/mL範圍內進行定量，偵測極限則為0.5 μg/mL (S/N=3)。並應用於levocetirizine市售製劑Xyzal®之含量分析和血漿檢品之異構物分離。

Ⅱ. 高效能液相層析法對抗腫瘤藥物mitomycin C與epirubicin之同時分析研究

本實驗建立高效能液相層析法配合移動相的梯度沖提方法針對抗腫瘤藥品mitomycin C和epirubicin血漿檢品中的同時分析。兩分析物利用C18層析分離管柱分離，配合10 mM磷酸鹽緩衝溶液( pH 3.0 )以及acetonitrile做梯度沖提、波長設定於365和480 nm下進行偵測。此方法可應用於監測肝癌病患經導管化學藥物動脈栓塞療法(transcatheter arterial chemoembolization，TACE) 治療後之血漿檢品，分析mitomycin C和epirubicin是否自栓塞洩漏出進入全身循環。

關鍵字

毛細管

並列摘要

The purpose of this work is direct to the development of simple and sensitivity methods for the analysis of drugs in biosamples and enantioseparation by capillary electrophoresis and simultaneous determination of mitomycin C and epirubicin by high performance liquid chromatography. The results are summarized as follow:

Ⅰ. The analysis of drug in biosamples by capillary electrophoresis.

(1) Quantification of meropenem in plasma and cerebrospinal fluid by micellar electrokinetic capillary chromatography and application in bacterial meningitis patients

A high-performance micellar electrokinetic capillary chromatography (MEKC) has been demonstrated for the determination of meropenem in human plasma and in cerebrospinal fluid (CSF) and application in meningitis patients after intravenous (IV) administration. Plasma sample was pretreated by means of solid-phase extraction (SPE) on C18 cartridge and CSF sample was by direct injection without SPE sample pretreatment, with subsequent quantitation by MEKC. The separation of meropenem was carried out in an untreated fused-silica capillary (40.2 cm × 50 ?慆 I.D., effective length 30 cm) and was performed at 25oC using a background electrolyte consisting of Tris buffer (40 mM, pH 8.0) solution with sodium dodecyl sulfate (SDS) as the running buffer and on-column detection at 300 nm. Several parameters affecting the separation and sensitivity of the drug were studied, including pH, the concentrations of Tris buffer and surfactant. Using cefotaxime as an internal standard (IS), the linear ranges of the method for the determination of meropenem in plasma and in CSF were all over 0.5-50 ?慊/mL; the detection limits (signal to noise ratio = 3) of meropenem in plasma and in CSF were 0.2 ?慊/mL and 0.3 ?慊/mL, respectively.

(2) Enantioseparation of cetirizine using sulfated-β-cyclodextrin- mediated capillary electrophoresis

A sulfated-β-cyclodextrin (sulfated-β-CD)-mediated capillary electrophoresis method is described for the enantioseparation of cetirizine using achiral cefazolin as an internal standard. The enantioseparation of the drug was performed in a borate buffer (5 mM, pH 8.7) with 1% sulfated β-CD (w/v) as a chiral selector at 10 kV. Several parameters affecting the separation were studied, including the pH and concentration of borate buffer and chiral selector. Under optimized conditions, a baseline separation of two enantiomers was achieved in less than 7 min. Using cefazolin as an internal standard (IS), the linear ranges of the method for the determination of levocetirizine were over 1.0-50.0 ?慊/mL; the detection limit (signal to noise ratio = 3) of levocetirizine was 0.5 ?慊/mL. The method allowed the enantioseparation of cetirizine in bulk sample, enantiomeric purity evaluation of levocetirizine (R-enantiomer) in pharmaceutical tablets (Xyzal??) and it was also found to be suitable for enantioseparation in human plasma.
Ⅱ. Simultaneous determination of mitomycin C and epirubicin by high performance liquid chromatography.

A simple liquid chromatographic method was described for simultaneous determination of mitomycin C and epirubicin in human plasma. The mitomycin C and epirubicin were separated on a reverse-phase C18 column with a mixed solvent of acetonitrile and phosphate buffer (10 mM, pH 3.0) as the gradient mobile phase system. The separated analytes were monitored with a UV-Vis detector (365 nm and 480 nm for mitomyci C and epirubicn, respectively). The linear ranges of the method for the determination of mitomycin C and epirubicin were 2-50 and 2-100 ng/mL. Application of the method to the analysis of mitomycin C and epirubicin in the plasma samples of the patients with transcatheter arterial chemoembolization treatment was proved feasible.

並列關鍵字

capillary electrophoresis ； high-performance liquid chromatography

參考文獻

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