荧光探针法测定血浆中硫代寡核苷酸PS20的方法初探  

Preliminary research on fluorescent probe method for quantitation of phosphorothioate oligonucleotide PS20 in plasma

作　　者：尚明美[1] 刘秀文[1] 汤仲明[1] 陈惠鹏[2] 孙效[1] 欧伦[1] 宋海峰[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《中国药理学与毒理学杂志》2005年第6期458-461,共4页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金项目(39870878);国家高技术研究发展计划资助项目(2003AA2Z347B)~~

摘　　要：目的为解决快速定量测定血浆中硫代寡核苷酸的问题.方法以一个全程硫代修饰的寡核苷酸,PS20为模型序列,用去离子水溶解PS20标准品,通过核酸蛋白分析仪定量,采用猕猴血浆稀释得到7个浓度梯度的血浆样品,再与稀释200倍的Oligreen荧光素Tris-EDTA缓冲液等体积混合.采用荧光高效液相色谱检测含不同浓度PS20的血浆样品.结果采用20 μL体系,所测血浆样品的积分面积和PS20浓度在50～2500 μg·L-1范围内呈良好线性关系,线性相关系数均大于0.997.对方法学的确证表明,荧光素法的线性范围、灵敏度、特异性、精密度和准确度均满足生物分析方法的要求,已经采用该方法成功的测定了PS20在猕猴血浆中的浓度,可以准确定量.结论荧光探针法可以快速定量血浆中硫代寡核苷酸的浓度,满足药代动力学研究的要求.AIM To solve the problem for rapid quantitation of the phosphorothioate oligodeoxynucleotides in plasma. METHODS Phosphorothioate oligodeoxynucleotide PS20 was dissolved in de-ionized water and quantitated by DU640 Nucleic Acid and Protein Analyzer at wavelength 260 nm. The aqueous solution of PS20 was diluted by blank plasma of rhesus monkey to make 7 density gradients, then mixed with OliGreen probe solution diluted at 1 ： 200 by Tris-EDTA buffer at ratio 1：1. Fluorescence high performance liquid chromatography was used to detect the change for signal of different plasma samples with different density of PS20. RESULTS For a 20 μL sample, a high degree of linearity between logarithm of integration area and the logarithm of concentration of PS20 in plasma was found in the range of 50 - 2500μg·L^-1 with the correlation coefficient more than 0. 997. The validation of the methods indicated that the sensitivity, specificity, precision, accuracy and the linear range met the requirements for bio-analytical methods. The method was successfully used to determine the concentration of an anti-cancer drug phosphorothioate oligonucleotide PS20 in rhesus monkey plasma. CONCLUSION The fluorescent probe combined FH- PLC method could be used to quantitation of phosphorothioate oligonucleotide in plasma, and the main parameters of the method met the requirements for pharmacokinetic study.

关 键 词：寡核苷酸类 反义 色谱法 高效液相 

分 类 号：R969[医药卫生—药理学]