反相离子对高效液相色谱法分析硫代反义寡核苷酸CT102相关杂质

Analysis on related impurities of antisense thio-oligonucleotide drug CT102 by ion-pair reversed phase high-performance liquid chromatography

目的:建立硫代反义寡核苷酸CT102及其相关杂质的反相离子对高效液相色谱(IP-RP-HPLC)分析方法。方法:对可能存在的3'n-1、5'n-1序列、5'(P=S/O)不完全硫代序列与CT102全长序列(n)的分离情况进行了考察,采用的色谱柱为XBridge OST C18 Column(50 mm×4.6 mm,2.5μm);流动相A为100 mmol.L-1 HFIP/31.6 mmol.L-1 TEA,10%甲醇(V/V));流动相B为100 mmol.L-1HFIP/31.6 mmol.L-1TEA,20%甲醇,洗脱梯度为0～12 min,17%→43%B;柱温为55℃;流速为0.4 mL.min-1;检测波长为260 nm。结果:在优化的条件下,合成过程中的主要杂质5'n-1、3'n-1以及5'(P=S/O)均能够实现与全长全硫代序列n基线分离。该法用于含量测定,CT102在50-600μg·mL-1浓度范围内线性良好,r=0.9999,检出限为2.5μg·mL-1,精密度与重现性良好,RSD小于2%,平均回收率为100.1%,(RSD=1.7%,n=9)。结论:该方法可应用于CT102原料药的纯度检测和含量测定,为该药物的深入研究提供保证。

Objective： To develop an ion-pair reversed phase high-performance liquid chromatography method（IP-RP-LC） for the analysis of an antisense thio-oligonucleotide drug CT102 and its related impurities.Methods： The full-length sequence of CT102 as well as the possibly existing sequences of 3＇n-1 and 5＇n-1 and the truncated thio-sequence of 5＇（P = S / O） were investigated to study their separation conditions.HPLC separation was performed on a XBridge OST C18 Column（50 mm × 4.6 mm,2.5 μm）;the mobile phase A and B were 100 mmol.L-1 HFIP /31.6 mmol.L-1 TEA-10% methanol（v / v） and 100 mmol.L-1 HFIP /31.6 mmol.L-1 TEA-20% methanol（v / v） for gradient elution with 17%→43% of solution B at 0-12 min;the column temperature was 55 oC,the flow rate 0.4 mL.min-1,the detection wavelength 260 nm.Results： Under the optimal condition,the main impurities,5＇n-1 and 3＇n-1together with 5＇P = S / O could be completely separated from the full-length complete thio-sequence n baseline of CT102.By this method for content detection,a good linear relationship was obtained in the CT102 concentration range of 50-600 μg.mL-1（r =0.9999）,the limits of detection was 2.5 μg.mL-1with satisfactory precision and reproducibility.RSD was less than 2%,and the average recovery was 100.1%（RSD = 1.7%,n = 9）.Conclusion： The proposed IP-RP-HPLC method can be applied for the purity analysis and assay,providing foundation for further studies.