An Ion-Pair Reversed Phase Liquid Chromatographic Method for Analysis of Related Substances in Phosphorothioate Antisense Oligonucleotide Flutide
An ion-pair reversed phase LC method(IP-RPLC) was developed for the analysis of related substances of a phosphorothioate antisense oligonucleotide flutide and routine quality control.The analytical result of IP-RPLC was compared with ion exchange chromatography(IEC) and capillary gel electrophoresis(CGE).The synthesized flutide and its analogs of monphosphodiester 5′(P=O)1 as well as deletion sequences 5′n-1 and 3′n-1 were analyzed.The optimized conditions of IP-RPLC were as follows: XTerra MS C18(250 mm × 4.6 mm,3.5μm) column;the mobile phase A was 142 mmol/L 1,1,1,3,3,3-hexafluoroisopropanol(HFIP)-36 mmol/L triethylamine acetate(TEA)-10% methanol(V/V),the mobile phase B was methanol;the gradient from 0 to 20% of solution B in 35 min,then back to 0% in 2 min,equilibrated 8 min;column temperature: 50 ℃;flow rate of mobile phase: 0.5 mL/min;detection wavelength: 260 nm.The result showed that under these conditions,flutide,deletion sequences 5′n-1 and 3′n-1 could be completely separated from each other and the resolution R of flutide and 5′(P=O)1 could reach to 0.94.The proposed IP-RPLC method has been applied for the related substances analysis and identification of crude and purified flutide samples,indicating that this method can be used for the quality control of phosphorothioate antisense oligonucleotide.