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刊物信息

期刊名称:药物分析杂志
主管单位:中国科学技术协会
主办单位:中国药学会
承办:中国食品药品检定研究院
主编:金少鸿
地址:北京天坛西里2号
邮政编码:100050
电话:010-67012819,67058427
电子邮箱:ywfx@nifdc.org.cn
国际标准刊号:ISSN 0254-1793
国内统一刊号:CN 11-2224/R
邮发代号:2-237
 

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HPLC-FLD法测定欧前胡素对肿瘤耐药细胞悬液中罗丹明123含量的影响

Effect of imperatorin on the content of Rho 123 in the drug-resistant cell suspension of tumor by HPLC-FLD

分类号:R917
出版年·卷·期(页码):2019,39 (9):1567-1573
DOI: 10.16155/j.0254-1793.2017.01.01
-----摘要:-------------------------------------------------------------------------------------------

目的:建立高效液相色谱-荧光检测法(HPLC-FLD)测定肿瘤耐药细胞中罗丹明123(Rho 123)的浓度,并采用建立的检测方法考察欧前胡素对Rho 123含量的影响。方法:用乙腈沉淀法进行蛋白沉淀。色谱柱为Phenomenex-C18(250 mm×4.6 mm,5 μm),柱温为40℃,流动相为乙腈-1%三乙胺(磷酸调至pH为3.0)(28∶72),流速为1 mL·min-1,激发波长485 nm,发射波长546 nm,进样量为20 μL。结果:Rho 123标准曲线为Y=1.199 3X-4.003 6(R2=0.999 7),其质量浓度在9.375~600 ng·mL-1范围内线性良好,定量下限为1.172 ng·mL-1,日内与日间精密度均小于10%,绝对回收率为70.6%~80.6%,相对回收率为91.0%~106.7%,长期、短期以及反复冻融稳定性良好。2、5、10 μg·mL-1欧前胡素组的A2780/Taxol与K562/ADR细胞内的蓄积倍数分别为2.79、3.53、4.18与2.18、2.36、2.39。结论:本方法专属性强,灵敏度高,简单可靠,适用于检测肿瘤耐药细胞悬液中Rho 123的浓度,可用于细胞中P-糖蛋白(P-glycoprotein,P-gp)的活性和功能评价。欧前胡素对肿瘤耐药细胞中P-gp介导的Rho 123外排具有抑制作用。 

-----英文摘要:---------------------------------------------------------------------------------------

Objective:To establish a high performance liquid chromatography-fluorescence method for determining the concentration of Rho 123 in tumor multidrug resistance cell lines,and investigating the effect of imperatorin on Rho 123 content in cell. Method:The Rho 123 were extracted from cell lysis with protein precipitation by acetonitrile solution and supernatant separation was performed on Phenomenex-C18(250 mm×4.6 mm,5 μm)with temperature at 40℃. The elution with the mobile phase consisting of aqueous solution (1% triethylamine, adjusted to pH 4.0 with phosphoric acid) and acetonitrile at a flow rate of 1.0 mL·min-1 were applied for the chromatographic separation and the injection volume was 20 μL. The excitation wavelength was 485 nm,and the emission wavelength was 546 nm. Results:Methodological experiment results showed that the standard curve was Y=1.199 3X-4.003 69(R2=0.999 7),and the linear relationship was good in the range of 9.375-600 ng·mL-1. The lowest limit of quantification (LLOQ) of Rho 123 was 1.172 ng·mL-1. The intra-day and inter-day RSDs were both less than 10%. The absolute recovery rate was between 70.6% and 80.6%,the extraction recovery was between 91.0% and 106.7%. Rho 123 was stable in long-term,short-term and repeated freeze-thaw conditions. The accumulation ratios of A2780/Taxol and K562/ADR cells in the 2,5,and 10 μg·mL-1 imperatorin groups were 2.79,3.53,4.18,and 2.18,2.36,and 2.39,respectively. Conclusion:The method has strong specificity,high sensitive,simple and reliable,and is suitable for detecting the concentration of Rho 123 in cell suspension. It can provide a rapid and convenient method for evaluating the activity and function of P-glycoprotein(P-gp) in cells. Imperatorin can inhibit the excretion of Rho 123 which mediated by P-gp. 

-----参考文献:---------------------------------------------------------------------------------------

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