Abstract: A method using anion exchange high performance liquid chromatography (AX-HPLC) and capillary gel electrophoresis (CGE) was established for the analysis of the related substances in phosphorothioate antisense oligonucleotide FT19 for quality control. The FT19 and its analogs of partial phosphodiester compounds (P=O)1 as well as deletion sequences (n-1) were analyzed. AX-HPLC with the DNA Pac PA-100 column (4 mm×250 mm) was used for the separation of phosphorothioate modifications. The size of the capillary column used in the CGE was 31 cm × 100 μm with an effective length of 20 cm. It was found that full phosphorothioate and partial phosphodiester oligonucleotides of the same length were successfully separated, and the deletion sequences (n-1) and the full-length sequence n were successfully separated in CGE. The results demonstrated that the related substances of phosphorothioate oligonucleotides can be well analyzed by AX-HPLC and CGE.

Key words: capillary gel electrophoresis (CGE), phosphorothioate oligonucleotide, related substances , anion exchange high performance liquid chromatography (AX-HPLC)