Determination of Antisense Phosphorothioate Oligonucleotide Drug Cantide by Capillary Gel Electrophoresis
毛细管凝胶电泳法测定硫代寡核苷酸药物癌泰得的含量

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 x 100 microm 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 degrees C and 30 degrees C, 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 0.99997. 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.