高效液相色谱法测定细胞核内多柔比星浓度

 目的建立高效液相色谱-荧光测定法测定K562/DOX(doxorubicin-resistant K562 cells,耐多柔比星K562细胞)细胞核多柔比星浓度。方法分离K562/DOX细胞核,多柔比星浓度测定采用Diamond C₁₈色谱柱,甲醇-水-醋酸(50∶50∶0.25)为流动相,流速1.0mL·min^-1,柱温35℃,荧光检测,激发波长497nm,发射波长555nm,以盐酸柔红霉素为内标。结果细胞核中多柔比星浓度在5.0～5000.0μg·L^-1内呈良好线性关系,r=0.9999。加样回收率为98.55%～100.84%(RSD1.77%～3.27%),日内和日间精密度均<15%,最低检测浓度为5.0μg·L^-1。结论该方法简单、方便,检测限低,精确度和回收率良好,可用于多药耐药肿瘤细胞内多柔比星浓度的胞内动力学研究。

Determination of Doxorubiciu Concentrations in MDR Cancer Cell Nuclei by HPLC

OBJECTIVE To develop a method using HPLC with fluorescence detector for determining the Doxorubicin concentrations in K562/DOX cell nuclei.METHODS Nuclei were isolated by modifying a method that employed isotonic conditions and detergent lysis. The mobile phase consisted of methanol-water-acetic acid (50∶50∶0.25). The flow rate of the mobile phase was set at 1.0 mL·min^-1, and the eluent was monitored at an excitation wavelength of 497 nm and an emission wavelength of 555 nm. Calibration curves were computed using the area ratio of doxorubicin to daunorubicin (the internal standard).RESULTS The linear calibration curve was observed in the concentration range of 5.0-1 000 μg·L^-1. The lower limit of quantification was 5.0 μg·L^-1. The spotting recoveries were 98.55%-100.84%. The inter-and intra-day precision (RSD) was below 15%. CONCLUSION The method was proved to be convenient,sensitive and rapid,and suitable for the determination of doxorubicin in nuclei.