博士 / 高雄醫學大學 / 藥學系博士班 / 105 / 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.
| Identifer | oai:union.ndltd.org:TW/105KMC05551005 |
| Date | January 2017 |
| Creators | Hung-Ju Lin, 林宏儒 |
| Contributors | Shou-Mei Wu, 吳秀梅 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 128 |
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