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[目的]建立高效液相色谱法定量分析大鼠血样和组织样本中苯肽胺酸的含量,为该药进一步研究提供参考依据。[方法]用高效液相色谱-紫外检测法对含有苯肽胺酸的空白样本进行检测并优化色谱条件使药物色谱峰与杂峰实现有效的分离。色谱柱为XB-NH_2柱,检测波长254 nm,柱温40℃,进样量10μL,流速1 mL/min,等度洗脱。血样和组织样本分别使用不同的流动相进行洗脱,血样洗脱的流动相中乙腈和水体积比为65∶35(水相含甲酸0.4%),动物组织样本洗脱的流动相中乙腈和水比例为80∶20(水相含甲酸0.2%)。按照设定的色谱条件进行精密度、重现性和稳定性的研究,再取空白血样和动物组织样本进行药物回收率的研究。[结果]日间及日内精密度RSD≤2%,重现性和稳定性RSD≤5%,所有样本回收率范围85.2%~97.5%。[结论]实验建立的高效液相色谱法检测生物样本中的苯肽胺酸稳定性和重现性均符合实验要求,可作为相关实验的参考方法。预试验结果显示,增加流动相的pH值和降低水相的pH值均能使保留时间延长。此外,研究结果显示pH值会对药物色谱峰的峰型有影响。
[Objective] To establish a HPLC method for the quantitative analysis of benzoic acid in rat blood samples and tissue samples, providing a reference for the further study of this drug. [Method] The blank samples containing peptidyl acid were detected by high performance liquid chromatography - ultraviolet detection method and the chromatographic conditions were optimized to achieve effective separation of the drug chromatographic peaks from the extracellular peaks. The column was XB-NH 2 column, the detection wavelength was 254 nm, the column temperature was 40 ℃, the injection volume was 10 μL, the flow rate was 1 mL / min and the isocratic elution was performed. Blood samples and tissue samples were eluted with different mobile phases, respectively. The mobile phase eluted from the blood sample consisted of a 65:35 volume ratio of acetonitrile to water (0.4% formic acid in the aqueous phase) and acetonitrile and The water ratio is 80:20 (aqueous phase contains 0.2% formic acid). According to the chromatographic conditions set for precision, reproducibility and stability of the study, then take blank blood samples and animal tissue samples for drug recovery studies. [Result] The RSD≤2%, RSD≤5% of reproducibility and stability during the day and the day, and the recoveries of all the samples ranged from 85.2% to 97.5%. [Conclusion] The established HPLC method for the determination of phenyl peptidase in biological samples was in accordance with the requirements of experiments and could be used as a reference method for related experiments. Pre-test results show that increasing the pH of the mobile phase and lowering the pH of the aqueous phase both prolong the retention time. In addition, the results show that the pH value of the drug peaks have an impact on the peak shape.