离子对反相液相色谱法分析硫代寡核苷酸药物流感泰得的有关物质  

An Ion-Pair Reversed Phase Liquid Chromatographic Method for Analysis of Related Substances in Phosphorothioate Antisense Oligonucleotide Flutide

作　　者：王颖[1] 鲁丹丹[2] 张玉林[2] 高婵[2] 李树[2] 王升启[2] 

机构地区：[1]安徽医科大学,合肥230032 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《分析化学》2011年第12期1811-1816,共6页Chinese Journal of Analytical Chemistry

基　　金："重大新药创制"科技重大专项(No.2009ZX09503-021;2011ZX09102-009-09)基金资助

摘　　要：建立了离子对反相液相色谱(IP-RPLC)分析流感泰得(Flutide)有关物质的方法,并将其与其它两种常用方法(离子交换色谱(IEC)和毛细管凝胶电泳(CGE)法)的分析结果进行比较。合成了Flutide及其单核苷酸缩短序列5'n-1、3'n-1和单氧代序列5'(P=O)1,将它们作为已知杂质,在IP-RPLC上进行分离条件的优化,使用的分析柱为XTerra MS C18柱(250 mm×4.6 mm,3.5μm);流动相A为142 mmol/L HFIP-36 mmol/L TEA-10%甲醇(V/V),流动相B为甲醇;梯度洗脱;流速为0.5 mL/min;柱温为50℃;检测波长为260 nm。结果表明,在该条件下,Flutide能同时与这几种杂质进行分离,其中与缩短序列5'n-1和3'n-1可实现基线分离,而与5'(P=O)1之间的分离度也能达到0.94。所建立的IP-RPLC方法已成功应用于Flutide粗品和纯品中各类杂质的分析和鉴定。An ion-pair reversed phase LC method（IP-RPLC） was developed for the analysis of related substances of a phosphorothioate antisense oligonucleotide flutide and routine quality control.The analytical result of IP-RPLC was compared with ion exchange chromatography（IEC） and capillary gel electrophoresis（CGE）.The synthesized flutide and its analogs of monphosphodiester 5′（P=O）1 as well as deletion sequences 5′n-1 and 3′n-1 were analyzed.The optimized conditions of IP-RPLC were as follows： XTerra MS C18（250 mm × 4.6 mm,3.5μm） column;the mobile phase A was 142 mmol/L 1,1,1,3,3,3-hexafluoroisopropanol（HFIP）-36 mmol/L triethylamine acetate（TEA）-10% methanol（V/V）,the mobile phase B was methanol;the gradient from 0 to 20% of solution B in 35 min,then back to 0% in 2 min,equilibrated 8 min;column temperature： 50 ℃;flow rate of mobile phase： 0.5 mL/min;detection wavelength： 260 nm.The result showed that under these conditions,flutide,deletion sequences 5′n-1 and 3′n-1 could be completely separated from each other and the resolution R of flutide and 5′（P=O）1 could reach to 0.94.The proposed IP-RPLC method has been applied for the related substances analysis and identification of crude and purified flutide samples,indicating that this method can be used for the quality control of phosphorothioate antisense oligonucleotide.

关 键 词：离子对反相液相色谱 硫代反义寡核苷酸 流感泰得 质量控制 杂质 

分 类 号：R927.2[医药卫生—药学] O657.72[理学—分析化学]