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

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。 

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. 

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