论文部分内容阅读
目的:建立U87肿瘤细胞中阿霉素的高效液相色谱荧光检测方法,为进一步研究化疗药对肿瘤细胞功能和活性的影响及逆转耐药研究提供方法学基础。方法:U87细胞以1×106·mL-1密度与不同浓度阿霉素孵育24h为药物处理条件。将细胞分为空白对照组和加药组,采用HPLC法测定细胞内外阿霉素的含量。色谱柱为Inertsil ODS-3(250mm×4.6mm,5μm);流动相为甲醇-乙腈-0.01mol·L-1磷酸二氢铵(40∶15∶45)(以0.1%冰醋酸调整pH至3.52);柱温35℃,流速1.4mL·min-1,荧光检测器检测波长λex=495nm,λem=560nm,以盐酸柔红霉素为内标。结果:细胞内阿霉素在0.025～7.5mg·L-1范围内线性关系好,r=0.9999;细胞外阿霉素在0.1～10.0mg·L-1范围内线性关系好,r=0.9999。细胞内外液阿霉素的检测限均为0.005mg·L-1,细胞外液的定量限为0.1mg·L-1。日内、日间RSD均小于10%,样品稳定性好。结论:采用HPLC荧光法测定U87细胞内外液中阿霉素的含量,方法简单、准确、线形范围宽、灵敏度高、精密度好,可用于对培养的肿瘤细胞内外化疗药物浓度的动态变化规律研究。
OBJECTIVE: To establish a method for the determination of doxorubicin in U87 tumor cells by fluorescence detection, and provide a methodological basis for further study on the effects of chemotherapeutics on the function and activity of tumor cells and the study of drug resistance reversal. Methods: U87 cells were incubated with different concentrations of doxorubicin at a density of 1 × 106 · mL-1 for 24h. The cells were divided into blank control group and dosing group. The contents of intracellular adriamycin and doxorubicin were determined by HPLC. The column was Inertsil ODS-3 (250 mm × 4.6 mm, 5 μm); the mobile phase consisted of methanol-acetonitrile-0.01 mol·L -1 ammonium dihydrogen phosphate (40:15:45) (pH was adjusted to 3.52 with 0.1% glacial acetic acid ). The column temperature was 35 ℃ and the flow rate was 1.4 mL · min-1. The detection wavelength λex = 495nm and λem = 560nm were detected by fluorescence detector with daunorubicin hydrochloride as internal standard. Results: The linearity of doxorubicin in the range of 0.025 ~ 7.5 mg · L-1 was good (r = 0.9999). The linearity of adriamycin in the range of 0.1-10.0 mg · L-1 was good (r = 0.9999). The detection limits of intracellular and extracellular doxorubicin were both 0.005 mg · L-1, and the limit of quantitation of extracellular fluid was 0.1 mg · L-1. Days, day RSD were less than 10%, good sample stability. Conclusion: HPLC method was used to determine the content of doxorubicin in U87 cells. The method is simple, accurate, wide range of linearity, high sensitivity and precision, and can be used to study the dynamic changes of drug concentration in vitro and in vivo. .