测定肿瘤细胞内外5-氟尿嘧啶含量的方法研究

Method Research for Determination of Intracellular and Extracellular 5-Fluorouracil

目的建立了高效液相色谱法测定A549细胞内外5-氟尿嘧啶含量的方法,为探索5-氟尿嘧啶的作用机理奠定基础。方法色谱柱采用Hypersil ODS柱(4 6 mm×250 mm,5μm);流动相为甲醇-水=2:98(V/V);流速:0.8 ml/min;柱温:30℃;检测波长:265 nm;进样量为5μl。结果细胞内液中5-氟尿嘧啶在0.1～50.0μg/ml范围内线性关系良好(r=0.999 6),平均回收率为97.65%,RSD为0.40%,日内和日间精密度分别为1.30%,1.04%;细胞外液中5-氟尿嘧啶在1.0～250.0 μg/ml范围内线性关系良好(r=0.999 9),平均回收率为102.34%,RSD为2.11%,日内和日间精密度分别为1.32%,1.06%。结论该色谱方法简便易行,准确度高,重现性好,可用于细胞内外5-氟尿嘧啶浓度的动态变化规律研究。

Objectlve A HPLC method for the determination of 5-FU in intracellular and extraeenular is established so as to understand more about antitumor mechamism of 5-FU. Methods The Hypersil ODS C18 column （250 mm × 4.6 mm,5 μm）was applied, and the parameters are as following： the mobile phase was methanolwater（2： 98, V/V） ; the flow rate was 0. 8 ml/min;the column temperature was 30℃ and the detection wavelength was 265 nm; the injection volume was 5 μl. Results The linear range of intracellular 5-FU was 0. 1 - 50. 0 μg/ml （ r = 0. 999 8） ;the average recovery was 97.65% ,and RSD was 0. 40% ;the precision of intra-day and inter-day was 1.30% and 1.04% respectively. And the linear range of extracellular 5-FU was 1.0 - 250. 0 μg/ml （ r = 0. 999 9 ）, the average recovery was 102.34%, RSD was 2. 11% ,the precision of intra-day and inter-day was 1.32% and 1.06%. Conclusion The method is simple,accurate and of good reproduc- ibility which can be used for the dynamic study of intracellular and extracellular 5-FU.