RP-HPLC法测定细胞内三磷酸阿糖胞苷浓度

Determination of Intracellular Ara-CTP Level by RP-HPLC

目的:建立一种简单、快速测定细胞内三磷酸阿糖胞苷浓度的反相高效液相色谱法。方法:自全血中分离单个核细胞,离心后进样检测。其中,色谱柱为SymmetryC18,流动相为0.1mol·L-1磷酸二氢钾缓冲液加0.01mol·L-1的四丁基硫酸氢铵(pH2.7),流速为1.0mL·min-1,紫外检测波长为278nm。结果:三磷酸阿糖胞苷检测浓度在0.35～9.12μg·mL-1范围内线性关系良好(r=0.9986),检测限为0.35μg·mL-1;低、中、高浓度的回收率、日内及日间精密度均符合生物样本测定方法学要求。结论:本法简便、快速、稳定、重现性好,适用于细胞内三磷酸阿糖胞苷浓度的监测。

OBJECTIVE： To establish a simple and rapid ion pair RP HPLC method for the determination of intracellular concentration of ara CTP. METHODS： Mononuclearcells were separated from the whole blood, centrifugated and injected for determination on Symmetry C18 column with the mobile phase consisted of 0.1 mol · L ^-1 KDP buffer solution and 0.01 mol · L ^-1 tetrabutylammonium hydrogen sulfate （pH = 2.7） at a flow rate of 1.0 mL · min ^-1 with the UV wavelength set at 278 nm. RESULTS： The linear range of ara - CTP was 0.35 - 9.12 μg · mL ^-1（ r = 0. 998 6） with its detection limit at 0.35 μg · mL ^-1. The recoveries of ara -CTP at low, middle and high concentrations, and the intra day and interday RSDs were all up to the standards. CONCLUSION： The established method is simple, rapid, stable, reproducible and applicable for the monitoring of intracellular concentration of ara -CTP.