Development and validation of a method for routine base composition analysis of phosphorothioate oligonucleotides.

Abstract A method for routine base composition determination of phosphorothioate oligonucleotides containing as many as 21 nucleobases has been developed and systematically evaluated in terms of factors contributing to assay precision and accuracy. Phosphorothioate internucleotide linkages were oxidized with a mixture of tetrahydrofuran—water—methylimidazole (16:4:1, v/v/v) which has been shown to be 97.3% effective. This step was followed by enzymolysis and HPLC quantitation of individual nucleobases. RSD for inter-day base composition analysis ranged from 1.1 to 1.3%, and inter-lot variation was 0.6–2.0%. Accuracy of the determined nucleobase ratio was independently confirmed through sequencing of the oxidized oligomer by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS).