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

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,2.5 μm);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^-1 HFIP/31.6 mmol·L^-1 TEA,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50-600 μg·mL^-1 浓度范围内线性良好,r=0.9999,检出限为2.5 μg·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^-1 with 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.