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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Oli-green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20μL体系,所测血浆样品的积分面积和PS20浓度在50~2500μg.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。
Objective To solve the problem of rapid and quantitative determination of thio-oligonucleotides in plasma. Methods A full-length thio-modified oligonucleotide, PS20, was used as a model sequence. PS20 standards were dissolved in deionized water and quantitated by a nucleic acid protein analyzer. Seven plasma samples were diluted with cynomolgus plasma to obtain seven plasma samples. 200-fold Oli-green Fluorescein Tris-EDTA buffer and the like. Plasma samples containing different concentrations of PS20 were detected by fluorescence high performance liquid chromatography. Results The 20μL system was used. The linear relationship between the integrated area of the measured plasma samples and the PS20 concentration in the range of 50 ~ 2500μg.L-1 was obtained. The linear correlation coefficients were all greater than 0.997. Confirmatory of the methodology shows that the linear range, sensitivity, specificity, precision and accuracy of the fluorescein method all meet the requirements of the bioanalysis method. The concentration of PS20 in the rhesus monkey plasma has been successfully determined by this method and can be accurately quantified . Conclusion Fluorescence probe method can rapidly quantify the concentration of thio-oligonucleotide in plasma and meet the requirements of pharmacokinetic studies.