高效液相色谱法测定U87细胞内外液中的阿霉素含量  

HPLC fluorescence assay for determination of adriamycin concentration in U87 cells

作　　者：陈剑鸿[1] 夏培元[1] 王章阳[1] 吴南[2] 戴青[1] 孙凤军[1] 杨波[1] 向荣凤[1] 

机构地区：[1]第三军医大学西南医院药学部,重庆400038 [2]第三军医大学西南医院神经外科,重庆400038

出　　处：《中国医院药学杂志》2010年第5期353-356,共4页Chinese Journal of Hospital Pharmacy

基　　金：国家自然科学基金项目(编号:30873034);重庆市自然科学基金(编号:2007BB5094)

摘　　要：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 the HPLC with fluorescence detector method for determination of adriamycin （ADM） concentration in U87 cells, as the basis for further study the effect of chemotherapy agents on cancer cells and to reverse drug resistance of tumor cells. METHODS U87 at the density of 1 × 1 0^6 cells mL ^-1 were divided randomly into the groups as： control group and treatment groups treated with various concentration of ADM, the determination of ADM was performed after the cocultured with ADM for 24 h. HPLC fluorescence detector was used to determinate the concentration of ADM intra-and extra- cellular of U87 cells. The cells in each group were chromatographed on a reversed - phase column, Inertsil ODS-35 μm, 250 mm×4. 6 mm, mobile phase consisted of methyl alcohol （0. 01 mol·L^-1 ）, acetonitrile and ammonium dihydric phosphate （40： 15：45）, pH 3.52 and the flow rate of 1.4 mL·min^-1 The detective excitation and emission wavelengths were 495 nm and 561） nm respectively. The internal standard was daunorubicin. RESULTS A good linear relationship at the range of 0. 025 mg·L ^-1 to 7. 5 mg·L^-1 of ADM intracellular space was obtained, r= 0. 999 9, and a good linear relationship at the range of 0. 1 mg·L^-1 to 10 mg·L^-1 of ADM in extracellular space was obtained, r= 0. 999 8. The lowest quantity and testing concentration of intracellular and extracellular were 0.05 mg·L^-1 and 0. 1 mg·L^-1 respectively, the RSD of inter-day and within day were less than 10%. CONCLUSION The results showed that HPLC with fluorescence detector has been verified, it is accurate and sensitive in the determination of concentration of ADM in U87 cells, with low limit of determination, wide liner range, satisfied precision, and good mean recovery for cancer ceils dynamic status both of the intracellular and extraeellular concentrations of ADM, thus it could be a convenient and exact method for determination of chemotherapy agents in cultured cells.

关 键 词：高效液相色谱荧光分析 人恶性胶质瘤U87细胞 阿霉素 

分 类 号：R969[医药卫生—药理学]