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

Abstract

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.