| Nucleotides play significant roles in the cell, especially nucleoside triphosphate, which includes ribonucleoside triphosphates ( NTP ) and deoxyribonucleoside triphosphates (dNTP). NTP are activated precursors for RNA synthesis, while dNTP are for DNA synthesis. Moreover, nucleoside triphosphate takes part in many important biochemical processes, such as ATP in tricarboxylic acid cycle. Because of the polarity, this kind of compounds was hardly separated by routine RPLC, and mainly separated by ion-pair RPLC method. In previous IP-RPLC method, some analytes could not be separated thoroughly, such as GTP and UTP. In this thesis, we firstly optimized the previous separation method, and did some further research in separation of these 8 analytes by other analytical methods that were different from IP-RPLC in mechanism. Then from the aspect of metabolic profiling analysis, we invested the metabolism between nomal tumor cell and tumor cell administeated by different drugs.In the very beginning, we grope and consummate the condition for transfer of culture, cryopreservation and resuscitation of HepG2 cell line. Then we draw the growth curve and observed the appearance of the cells in exponential phase of growth. Followed by, we invested inhibition of 3 common-used drugs, against HepG2 cell line. According to the inhibition effect, Vinristine was considered to be better one as a positive control in drug screening experiment.We selected tetrabutylammonium hydroxide as an ion pair agent, and optimized category and concentration of organic phase, pH value, and UV wave for detection, flow rate, and column temperature. HPLC analysis was performed on an Agilent 1100 series HPLC system. The UV detector was set at 260 nm. The analytical column was an Agilent Eclipse plus C18 column (250×4.6 mm, 5.0μm). The mobile phase was as follows: A–B (40:60) at 0 min→(10:90) at 50 min→(10:90) at 75 min. Solvent A contained 10mM tetrabutylammonium hydroxide, 10mM KH2PO4, and was adjusted to pH 7.0 with H3PO4. Solvent B consisted 5.6mM tetrabutylammonium hydroxide, 50mM KH2PO4 and 30% MeOH, and was neutralized to pH 7.0 with 1 M NaOH. The chromatographic system provided good separation of the compound without interfering peaks from other substance. The accuracy, precision, sensitivity, specificity and linearity of method well satisfied the requirements.The separation of 8 analytes was carried on by capillary zone electrophoresis. We optimized category and concentration of buffer salt, pH value, temperature, voltage, and organic moderator, length of capillary and inner diameter of capillary. In the optimized method, the total lengths of untreated fused silica capillary were 49 cm (40.5cm to detection window). The inner diameter was 50.0μm. Before each run, the capillary was rinsed for 5 min with 1M NaOH and 5 min with running buffer. Mixed standards were introduced from inlet side at 50mbar for 3 s. The separations were performed under a constant voltage of 18 kV and detection visualized at 260 nm. The temperature was set at 15℃. In this experiment, the CE separation method was only applied for mixed standards.HILIC method for separating these 8 analytes was invested, too. An amino column was selected as the analytical column, and a mass spectrum with SIM monitor was applied as the detector. We optimized category, concentration of the buffer salt, pH value of mobile phase and Mass parameters. The method could be used to separate mixed standards. But because of its intrinsic shortcoming, HILIC was not a developed technique, and need to be further research and developed.At last, from the aspect of metabolic profiling analysis, we designed experiment as follows: the 3 drugs mentioned above were set as irritation to HepG2 cell line. After administering 3 drugs, cells were extracted respectively. The metabolic variation between normal tumor cells and cells administered drugs was investigated.
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