Innovative ICP-MS/MS approach for the determination of phosphodiester to phosphorothioate ratio in phosphorothioate oligonucleotides

The phosphodiester to phosphorothioate ratio (P=O/P=S) is considered a Critical Quality Attribute of phosphorothioate oligonucleotides and must be accurately determined during characterisation studies. Traditionally assessed using ³¹P-NMR, this measurement requires specialised equipment and is not easily implemented in regulated (GxP) environments. Alternative techniques such as strong-anion exchange chromatography present limitations, particularly in the presence of impurities.

This application note introduces a novel ICP-MS/MS-based approach relying on the quantification of phosphorus and sulphur after sample digestion. This method provides an alternative analytical strategy with promising performance characteristics for routine use. The complete technical details and results are available in the full application note available for download.

ANALYTICAL HIGHLIGHTS

ICP-MS/MS enables simultaneous quantification of phosphorus and sulphur to determine the P=O/P=S ratio.

• Accuracy close to 100% across tested levels, with maximum relative bias of 3% at low S/P ratios
• Excellent precision, with %RSD values below 0.6% on calibration standards and below 1% on oligonucleotide samples
• Strong linearity demonstrated across S/P ratios from 0 to 1
• Results obtained within one day of analytical work using approximately 10 mg of sample

HOW THIS SUPPORT YOUR OLIGONUCLEOTIDE DEVELOPMENT PROGRAMME?

This ICP-MS/MS approach provides a robust and practical alternative to traditional techniques for assessing a key quality attribute of phosphorothioate oligonucleotides. It supports both development and quality control activities within regulated environments.

• Enables determination of a critical quality attribute with high accuracy and precision
• Provides an alternative to ³¹P-NMR that is more suitable for GxP environments
• Reduces analytical limitations linked to impurity interference observed with chromatographic methods
• Supports routine testing with rapid turnaround time (within one day)
• Requires limited sample quantity (~10 mg), facilitating integration into standard workflows
• Offers potential for future simultaneous quantification of oligonucleotides alongside ratio determination

This method contributes to improving analytical reliability and operational efficiency in oligonucleotide characterisation.