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標題: | 第一部份:開發固相萃取及高效液相層析的方法以定量生物樣品中的 aripiprazole及其代謝物dehydroaripiprazole
第二部份:以毛細管電泳及超高壓液相層析儀建立大黃的指紋圖譜 Part I: Development of a solid phase extraction coupled with high performance liquid chromatography method for the determination of aripiprazole and dehydroaripiprazole in biological fluid Part II: Fingerprint analysis of rhubarb by capillary electrophoresis and ultra-high pressure liquid chromatography |
作者: | Chun-Ting Kuo 郭俊廷 |
指導教授: | 郭錦樺 |
關鍵字: | Aripiprazole,Dehydroaripiprazole,高效液相層析,固相萃取,層析指紋圖譜,化學計量法,毛細管電泳,超效液相層析,大黃,主成分分析,去氧核醣核酸, Aripiprazole,Dehydroaripiprazole,HPLC,SPE,serum,Chromatographic fingerprint,Chemometric,Capillary electrophoresis,UPLC,Rhubarb,PCA,DNA, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | 第一部份:開發固相萃取及高效液相層析的方法以定量生物樣品中的 aripiprazole及其代謝物dehydroaripiprazole
Aripiprazole是第一個多巴胺部分促效劑專用來治療精神分裂症,dehydroaripiprazole是其主要代謝物。人體血液及尿液中aripiprazole 及dehydroaripiprazole的定性及定量研究是藉由固相萃取(SPE)及高效液相層析(HPLC)來分析。這分析方法包含下列步驟:1)利用酸鹼溶液進行前處理以去蛋白質,2)利用Oasis HLB 管柱進行固相萃取以達到清洗及濃縮樣品,3)高效液相層析分析,樣品在前處理的回收率高達88.20 - 99.83 %。高效液相層析的最佳化條件是使用 C18 X Terra®管柱,dipotassium hydrogen phosphate緩衝液,酸鹼值8.35,和乙睛以40 : 60 v/v的比例混和形成移動相,以流速每分鐘1.0 mL沖提,aripiprazole及dehydroaripiprazole的血中濃度可在五分鐘內測定。此方法波峰面積一日內重複性的相對標準偏差低於0.11 % (n = 4),而日與日間中間精密度(inter-day)的相對標準偏差低於5.16 % (n = 3)。此方法在aripiprazole濃度範圍50 - 1000 ppb及dehydroaripiprazole 濃度範圍50 - 800 ppb之間具有良好線性(R = 0.999)。本研究最後將已確效的方法分析正在接受aripiprazole治療之精神病病患的血清及尿液。本研究所開發的固相萃取-高效液相層析條件可以在藥物監控及臨床研究上正確定量病人血液及尿液中aripiprazole及dehydroaripiprazole的濃度。 第二部分:以毛細管電泳及超高壓液相層析儀建立大黃的指紋圖譜 本研究以毛細管電泳(CE)及超高壓液相層析法(UHPLC; Waters UPLC 系統)建立大黃的指紋圖譜、並配合化學計量法(Chemometric)鑑別大黃之品種。此研究分析的樣品為大黃的兩個不同種分別為藥用大黃(Rheum officinale)及唐古特大黃(Rheum tanguticum),分析方法開發選取兩者所共同含有的十種成分:aloe-emodine, (+) cathechin, chrysophanol, emodine, (-) epicathechine gallate, gallic acid, physcion, rhein, sennoside A and sennoside B進行條件最適化。毛細管電泳系統之最適化分析條件是利用膠束電動層析法(micellar electrokinetic chromatography; MEKC)模式,其緩衝液為:30 mM sodium tetraborate / sodium dihydrogen phosphate monohydrate, 30 mM sodium deoxycholic acid (SDC), pH 8.6及26 % acetonitrile (v/v)。超效液相層析之最適化分析條件使用Waters® Acquity UPLC BEH C18 column 的管柱,移動相組成為0.05 % 磷酸緩衝溶液(溶液A)及acetonitrile (溶液B),由梯度模式下進行沖提,梯度模式為(溶液A : 溶液B):0 min, 90 : 10; 25 min, 79 : 21; 35 min, 67 : 33; 40 min, 35 : 65; 45 min, 35 : 65)。兩分析方法的偵測器波長均設定於254 nm,CE與UPLC的分析時間分別為21分鐘及45分鐘。兩方法分別使用樣品中十個標準品的滯留時間計算出再現性的相對標準偏差,CE的低於0.668 % (n = 3)而UPLC的低於0.144 % (n = 3)。精準度(inter-day)的相對標準偏差CE的低於3.105 % (n = 3) 而UPLC的低於1.437 % (n = 3)。由於CE的時間再現性較差且基線漂移嚴重,所建立的指紋圖譜須經由基線校正及peak alignment後,再進行相似度比對,UPLC指紋圖譜再現性佳,可將圖譜直接使用化學計量法進行分析。經由主成分分析法(PCA)分析可得到成功的分群結果。對於指紋圖譜差異較大的樣品經由去氧核醣核酸(DNA)的序列比對,確認其為雜交之品種。本研究所建立的指紋圖譜可應用於藥用大黃及唐古特大黃的品質管制。 Part I: Development of a solid phase extraction coupled with high performance liquid chromatography method for the determination of aripiprazole and dehydroaripiprazole in biological fluid Aripiprazole is the first drug with dopamine partial agonist effect for schizophrenia. Dehydroaripiprazole is its major metabolite. The determination and validation of aripiprazole and dehydroaripiprazole in human serum and urine were performed by a combination of solid phase extraction (SPE) and high performance liquid chromatography (HPLC) in this study. The method includes the following steps: 1) pre-treatment of acid-base solutions for deproteination, 2) application of SPE using an Oasis HLB cartridge for cleaning-up and concentration of the samples, 3) HPLC analysis. The recovery of sample pretreatment step was relatively high with recovery rate of 88.20 - 99.83 %. The optimized HPLC conditions were using a C18 X Terra® column, with an isocratic elution consisted of dipotassium phosphate buffer, pH 8.35, and acetonitrile (40 : 60 v/v) at a flow rate of 1.0 mL/min. The concentration of aripiprazole and dehydroaripiprazole could be determined within 5 minutes. The relative standard deviation (RSD) of the peak area for method repeatability (n = 4) and intermediate precision (inter-day, n = 3) were lower than 0.11 % and 5.16 %, respectively. The calibration curves revealed the method that was linear with concentration range between 50 - 1000 ppb for aripiprazole and 50 - 800 ppb for dehydroaripiprazole. Finally, the validated method was successfully applied to analyze serum and urine samples collected from patients receiving the aripiprazole treatment. The developed method can be used to quantitative determination of aripiprazole and dehydroaripiprazole concentration in patients’ serum and urine for therapeutic monitoring and clinical research. Part II: Fingerprint analysis of rhubarb by capillary electrophoresis and ultra-high pressure liquid chromatography This study used capillary electrophoresis (CE) and ultra performance liquid chromatographic (UPLC) method for chromatographic fingerprint analysis of rhubarb. With the application of chemometric approach, chromatographic fingerprint could be used for species differentiation. Ten common constituents in rhubarb, including aloe-emodine, (+)catechin, chrysophanol, emodine, (-)epicatechin gallate, gallic acid, physcion, rhein, sennoside A and sennoside B, were selected for analytical method development. The optimum micellar electrokinetic chromatography (MEKC) conditions were as followed: 30 mM sodium tetraborate / sodium dihydrogen phosphate monohydrate, 30 mM sodium deoxycholate (SDC), pH 8.6 with 26 % acetonitrile (v/v) as background electrolyte. The optimum condition of UPLC method used a Waters Acquity UPLC BEH C18 column for the separation. The mobile phase was composed of 0.05 % phosphate solution (solution A) and acetonitrile (solution B). The gradient profile was ( solution A: solution B): 0 min, 90 : 10; 25 min, 79 : 21; 35 min, 67 : 33; 40 min, 35 : 65; 45, min 35 : 65. The detector wavelength was set at 254 nm for both methods, and the total analytical time was 21 min for CE and 45 min for UPLC. Sixteen samples of Rheum officinale and Rheum tanguticum collected from various sources were analyzed by optimum analytical conditions. Chromatographic fingerprints of CE were subjected to peak alignment and baseline correction for further similarity test. On the other side, analytical results of UPLC show high precision with flat baseline. Chromatographic fingerprints of UPLC were directly used for Principal component analysis (PCA) and similarity test. PCA shows the chromatographic fingerprints of the two species could be successfully classified. The sample showing the least correlation with the representative chromatographic fingerprint was studied for its DNA sequences. DNA analysis demonstrated the sample to be a hybrid rhizome. The developed CE and UPLC chromatographic fingerprint methods could be applied for the quality control of rhubarb. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45629 |
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