| Capillary electrophoresis (CE) is a relatively new separation technique compared to the well-known chromatographic procedures, such as HPLC and gas chromatography separation of a complex array of large and small molecules, including maromolecules proteins. Sample introduction is accomplished by immersing the end of the capillary into a sample vial and applying pressure, vacuum or voltage. High electric field strengths are used to separate molecules based on differences in charge, size and hydrophobicity. Depending on the types of capillary and electrolytes used, the technology of CE can be segmented into several separation techniques, the simplest form of CE is capillary zone electrophoresis(CZE). Fundamental to CZE are homogeneity of the buffer solution and constant field strength throughout the length of the capillary. The analytes are prepared by the differences in their charge-to-mass ratio.Methotrexate (MTX) is the main object of this experiment. It is extensively used in clinical as an antitumor drug through anti-metabolism path, nature MTX enantiomers have L and D two forms. In this essay, we establish two methods to evaluate the drug and separate the enantiomers by high performance capillary electrophoresis (HPCE). Subsequently, we invest the MTX enantiomers' antitumor effect by using the two methods. There are three main parts in the thesis.Part I Establishment and application of a method for determining MTX in serum by using HPCE.At first, the electrophoresis conditions were systemly studied which included pH value, run buffer, capillary temperature and sample treatment, i.e. The capillary is an naked silicon capillary, 60cM in full length and efficient length is 50.5cM,having a detection window at there, and id is 75μM. The run buffer is pH7.3, 75mM phosphate. The separation voltage is 25Kv, and the capillary temperature is at 25 ℃. MTX has ultraviolet absorbance at 306 run, so we utilize PDA detector to determine its concentration in fluid. Finally, MTX was achieved within 10 min. Also, the evaluationof methodology was made. The linearity was form l.lumol/L tollOO.Oumol/L.The minimal detection limit was 0.55umol/L.The recovery of added MTX was from 88.2%to 98.2% over the range 22-110umol/L.Wthin-run precision was 4.2%, and between-run precision was 5.4%. HPCE is an effect method in clinical and scientific research for its advantages such as rapidity, simplicity and accurate in determination of methotrexate in serum and separation.Part II Establishment and application of a method for separation MTX enantiomers by using HPCE.Pharmaceuticals with asymmetric carbons that exist as enantiomers provide a significant challenge. Stereoisomers are physically and chemically identical. Chiral environment is required for their separation. The capillary provides an ideal format for creating a chiral environment, as chiral reagents in solution are easily introduced by the simple application of pressure.By using capillary zone electrophoresis with nature cyclodextrin , we optimize various factors such as concentration of buffer, cyclodextrin, modifier, pH, voltage and temperature .Finally ,The methotrexate enantiomers was achieved baseline separation in 25 minutes with resolution factor of 1.51 by using 25mmol/L hydroxypropyl- P -cyclodextrin as the chiral selector, and 10% isopropylalcohol, 80mmol/L phosphate buffer (pH 7.4) as running buffer .The separation voltage was 18 kV and the temperature was 25 °C .High performance capillary zone electrophoresis is a simple, rapid and economic method for the chiral resolution of methotrexate, and it can be used to determine the purity and quality of drug.Part III The research of different bioactivity by methotrexate enantiomers in antitumor.To investigate the biological difference of enantiomers of methotrexate (L-(+)-MTX and D-(-)-MTX) against cancer cells by vein approach in vitro. The A549 pulmonary carcinoma cell line were cultured at various MTX enantiomers level, After 72...
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