應用毛細管電泳及銅奈米團簇分析乙型海洋性貧血患者血中排鐵劑deferoxamine、deferiprone 及deferasirox 之濃度

Analysis deferoxamine, deferiprone, and deferasirox in β-thalassemia patients’ plasma by capillary electrophoresis and copper nanoclusters

林宏儒(Hung-Ju Lin)

指導教授 ： 吳秀梅

高雄醫學大學/藥學院/藥學系博士班/博士(2017年)

摘要

乙型海洋性貧血(β-thalassemia)屬於一種遺傳型溶血性慢性疾病，盛行於地中海、東南亞等沿海國家，台灣為盛行國家之一。治療方式主要依賴長期輸血，因而容易造成體內鐵含量過多，故需使用排鐵劑
(iron-chelating agents)排除過多的鐵。目前臨床上主要使用的三種排鐵劑為deferoxamine (DFO)、deferiprone (DFR)及 deferasirox (DFX)。本研究主要利用毛細管電泳(capillary electrophoresis, CE)，針對服用 DFO、DFR或DFX 的乙型海洋性貧血患者來監測其藥物血中濃度。另外，也設計銅奈米團簇(copper nanoclusters, CuNCs)搭配螢光分光光譜儀，來監測患者血中DFX濃度。本論文含以下三個部分。
第一部分，利用電場放大堆積法(field-amplified sample injection, FASI)結合線上掃集技術(sweeping)，以微胞電動層析法(micellar electrokinetic chromatography, MEKC)分離血漿檢體中的 DFO 及 DFR。最適化的分離條件為：背景電解質100 mM NaH2PO4 (pH 6.6)，內含50 mM三乙醇胺(triethanolamine, TEA)；分離緩衝液100 mM NaH2PO4 (pH 3.0)，內含150 mM十二烷基磺酸鈉(sodium dodecyl sulfate, SDS)；樣品以正向電壓10 kV 注入180 秒；毛細管有效長度為 40 公分；使用 UV 偵測器，波長為200 nm。DFO 及 DFR 於本分析方法的偵測極限(limit of detection, LOD) (S/N=3)分別為200 ng/mL及25 ng/mL，並已成功應用於監測重度乙型海洋性貧血患者血中 DFO 及 DFR 的濃度。
第二部分，亦利用電場放大堆積法(field-amplified sample stacking, FASS)搭配掃集，以 MEKC 之分離技術來分析血漿中 DFX 之含量。樣品注入採短端進樣。DFX 進到毛細管時，先被微胞進行掃集，然後因電場差異進行二次堆積。最適化分離條件為 40 mM Na2HPO4 (pH 4.5)，含100 mM SDS及20%甲醇；毛細管有效長度為10公分；樣品以5 psi注入15秒；偵測波長254 nm。本分析方法的LOD為300 ng/mL，已應用於臨床分析病人血中之DFX濃度。
第三部分，利用聚合物poly(allylamine hydrochloride) (PAH)包覆copper nanoclusters (CuNCs)用於快速測定血漿中DFX之濃度。CuNCs由1.0 M抗壞血酸(L-ascorbic acid, LAA)、0.1 M硫酸銅(CuSO4)及10% PAH，在60℃反應2小時。螢光最大激發及放射波長為λex/λem:360/450 nm。PAH-CuNCs平均粒徑為2.3±0.5 nm；經X射線光電子能譜儀(x-ray photoelectron spectroscopy)及基質輔助雷射脫附游離法(matrix-assisted laser desorption ionization)搭配質譜儀(Mass spectrometry)知PAH-CuNCs六個銅原子組成。PAH-CuNCs 與 DFX 的反應需時五分鐘，即可測定血漿中DFX濃度；專一性測試發現不會被數種金屬離子及胺基酸干擾。本分析方法的檢量線為Y=0.0173X+0.0166，線性範圍為1-100 μg/mL，LOD為0.5 μg/mL。本方法亦已應用於測定真實病人血中之DFX濃度。

關鍵字

毛細管電泳 ； 排鐵劑 ； 銅奈米團簇 ； 海洋性貧血

並列摘要

β-Thalassemia is a hereditary hemolytic disease and prevailing in Mediterranean, Southeast Asia, etc. Taiwan is one of the prevailing area. Long-term transfusion is the major therapy for β-thalassemia patients, but also results in iron overload. There are some iron-chelating agents, including deferoxamine (DFO), deferiprone (DFR), and deferasirox (DFX) to be used to remove the excess iron. This study utilized capillary electrophoresis (CE) to monitor the concentration of DFO, DFR, and DFX in β-thalassemia patients’ plasma. In addition, copper nanocluster (CuNCs) coupled with fluorescence spectrophotometer was also designed to measure the concentration of DFX. This dissertation could be divided into three parts.
Field-amplified sample injection (FASI) combined with sweeping and micellar electrokinetic chromatography (MEKC) was used to separate DFO and DFR in plasma. The optimal conditions were as following. Background eletrolyte was composed of phosphate buffer (100 mM) which contained 50 mM triethanolamine (TEA) and the pH value was 6.6. Separation buffer was phosphate buffer (100 mM, pH 3.0) containing 150 mM SDS. The plasma samples were electrokinetically injected into capillary at normal polarity 10 kV for 180 s. The effective length of capillary was 40 cm. UV detector was set at 200 nm to monitor the signals. The LODs (S/N = 3) were 200 ng/mL for DFO, and 25 ng/mL for DFR. This method was applied for clinical applications of β-thalassemia major patients.
Another method was developed by using field-amplified sample stacking (FASS) coupled with sweeping and MEKC. Short-end injection was utilized to inject sample and shortened the analysis time. Due to the uncharged property, DFX will be swept by micelle firstly, then stacking by the electric field difference. The optimal conditions were 40 mM Na2HPO4 (pH 4.5) containing 100 mM SDS and 20% methanol as separation buffer and background electrolyte. The effective length of uncoated fused-silica capillary was 10 cm. Hydrodynamic injection mode at 5 psi for 15 seconds. The detection wavelength was set at 254 nm. The LOD of this method for DFX was 300 ng/mL. This method was applied for determination of DFX in β-thalassemia major patients.
Finally, poly(allyamine hydrochloride) (PAH) was used to cap the copper nanoclusters (CuNCs) for rapid determination of DFX in plasma. L-ascorbic acid (LAA) was used to reduce copper sulfate (CuSO4) to CuNCs and protected by PAH to prevent oxidation and improve stability. The optimal reaction conditions were 0.1 M CuSO4 mixed with 1.0 M LAA and 10% PAH, then reacted at 60℃ for 2 hours. PAH-CuNCs has highly water solubility and excellent fluorescence stability. The excitation and emission wavelength of PAH-CuNCs were 360 and 450 nm, respectively. The average particle size of PAH-CuNCs was 2.3±0.5 nm. The composition of PAH-CuNCs was constituted with six cupper atoms and identified by X-ray photoelectron spectroscopy and matrix-assisted laser desorption ionization–mass spectrometry. It was only needed five minutes for DFX reacting with PAH-CuNCs. The specificity was good that there is no interference which from various metal ions and amino acids. The calibration curve of DFX was Y= 0.0173X + 0.0166 in the range of 1-100 μg/mL. LOD was 0.5 μg/mL. This method was applied for determination of DFX in β-thalassemia major patients.

並列關鍵字

capillary electrophoresis ； iron-chelating agents ； copper nanoclusters ； thalassemia

參考文獻

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