毛细管凝胶电泳法测定硫代寡核苷酸药物癌泰得的含量

Determination of Antisense Phosphorothioate Oligonucleotide Drug Cantide by Capillary Gel Electrophoresis

癌泰得为20碱基的抑制端粒酶催化亚基hTERT的硫代反义寡核苷酸,体内、体外抗肿瘤活性评价显示其有较好的抗肿瘤活性。采用固相合成仪分别制备了癌泰得及与其碱基百分组成基本相同的硫代寡核苷酸内标,并使用制备型阴离子交换色谱和反相高效液相色谱进行纯化。采用毛细管凝胶电泳仪和单链DNA分离试剂盒测定了癌泰得的含量。所用毛细管规格为内径100μm,总长31cm,有效长度20cm;电动进样,进样电压-10kV,进样时间1s;分离电压-12 4kV;柱体温度40℃;样品储存温度为30℃;缓冲溶液为pH8 5的Tris 硼酸盐 7mol/L尿素缓冲液;紫外检测,波长为254nm。结果表明,在12 5～800mg/L时呈现良好的线性关系(r=0 99997);最低检测限为0 220mg/L;日内、日间的相对标准偏差(RSD)分别为0 449%～1 89%和1 01%～1 48%;低、中、高3种浓度的平均回收率为96 95%,RSD为6.88%。说明毛细管凝胶电泳内标法用于反义硫代寡核苷酸的含量测定结果准确、可靠。

Cantide is a 20-mer antisense phosphorothioate oligonucleotide that inhibits telomerase catalytic subunit hTERT. A capillary gel electrophoresis (CGE) method with internal standard was used for the determination of Cantide. Cantide and the phosphorothioate internal standard were prepared on a solid-phase DNA synthesizer and purified by preparative strong anion-exchange chromatography and reversed-phase high performance liquid chromatography. Cantide and the internal standard had approximately equal percentage of base composition. Cantide was determined with capillary electrophoresis instrument and ssDNA kit. The size of the capillary column was 31 cm×100 μm i.d. with an effective length of 20 cm. Samples were electrokinetically injected using -10 kV voltage for a duration of 1 s. The column temperature and sample storage temperature was 40 ℃ and 30 ℃, respectively. The detection wavelength was 254 nm.The running buffer was a mixture of 7 (mol/L) urea-tris-boric acid(pH 8.5). The calibration curve was linear in the range of 12.5-800(mg/L) with correlation coefficient of (0.999 97). The limit of detection of Cantide was 0.220 (mg/L). Intra-day and inter-day relative standard deviations (RSDs) for Cantide were 0.449%-1.89%, and 1.01%-1.48% , respectively. The average recovery was 96.95% with RSD of 6.88%. The assay validation study and the characterization of CGE revealed that the method can be used in quantitative analysis of Cantide for pharmacokinetic characterization, and the results are accurate and reproducible.