| Pharmacokinetics is the study of the time course of a drug within the body and incorporates the processes of absorption, distribution, metabolism and excretion. To better interpret the effect and toxicity of the drug, an analytical method should be established to determine the drug and its metabolites quantitatively, which help us to know about their mechanisms of action and their pharmacokinetic properties during the initial state of drug development and in clinical therapy. With the improvement of combinatorial chemistry and separation and purification of natural products, a number of candidate compounds appeared and required pharmacokinetic analysis. Investigators have shown increasing interest in developing and optimizing high-throughput analytical methods with more sensitivity,accuracy and lower consumption for quantitating small molecule drugs, metabolites, and other xenobiotic biomolecules in biological matrices. Based on fast HPLC using a common silica column, and sample preparation in 96-well format, high-throughput bio-analysis method with accuracy and sensitivity was established and utilized in pharmacokinetic studies of curcumine.The thesis is composed of four chapters.In the first chapter, the application and development of high-throughput bio-analysis method in pharmacokinetic analysis were introduced, also, the methods reported to measure curcumine in vivo or in vitro were presented, finally, to sum all, we would show some opinions on the development hotspots and the envedents, accroding to which, we complete our experiments.In the second chapter, we established a fast and sensitive LC/MS/MS method to measure curcumine liposome in rat plasma. After a simple step of protein precipitation using acetonitrile containing the internal standard (IS), resveratrol, plasma samples were analyzed by LC/MS/MS. Curcumin and the IS were separated on a Ultimate XB C-18 analytical column (4.6mm×50mm, 5μm) using methanol-0.01 % formic acid (80:20, v/v) as mobile phase at a flow rate of 0.3mL/min. The electrospray ion source was run in a positive ionization mode. The samples were analyzed via selected reaction monitoring (SRM). The monitoring ions were set as m/z 367-149 for curcumine and m/z 227-143 for its IS. The collision energies were 21.5 eV and 16 eV, respectively.In the third chapter, a fast and convenient HPLC-UV method mainly basing on aptimizing common RP columns was established to ditermine curcumine in rat plasma. The influence of different parameters (internal standard, pH, the percentage of organic phase in mobile phase, column, exaction solution) was systematically studied. After a simple step of protein precipitation in 96-well format using acetonitrile containing the internal standard (IS), emodin, plasma samples were analyzed by reverse phase HPLC. Curcumin and the IS emodin were separated on a Diamonsil C18 analytical column (4.6mm×100mm, 5μm) using acetonitrile-5 % acetic acid (75:25, v/v) as mobile phase at a flow rate of 1.0 mL/min. The wavelength of the detection was at 420 nm. The injection volume was 50μL and the analysis time was 3 min per sample.In the fourth chapter, HPLC-FLD determination was developed for measuring curcumin in SD rat plasma. According to the defects of above methods, we do some optimizings, and finally, curcumin and the IS emodin were separated on a Diamonsil C18 analytical column (4.6mm×100mm, 5μm) using acetonitrile-3 % acetic acid (80:20, v/v) as mobile phase at a flow rate of 1.0 mL/min. Theλex and theλem were at 423 and 536nm, respectively. The injection volume was 50μL. Although protein precipitation in 96-well format is simple, this extraction procedure can damage the columns, for we use liquid liquid extraction in 96-well format, which can reduce the consumption of materials and improve the sensitivity of our experiment. |
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