Phosphodiester to phosphorothioate ratio (P=O/P=S) is a critical quality attribute of phosphorothioate oligonucleotides. The determination of this ratio is commonly performed by P-NMR, which requires specific equipment and cannot be easily applied in a regulated (GxP) environment. For the first time, we present in this application note the preliminary results obtained for the development of an ICP-MS/MS method to determine P=O/P=S ratio.
At Quality Assistance, we have developed an ICP-MS/MS method based on the quantification of P and S after microwave digestion to determine the P=O/P=S ratio in oligonucleotide samples. Preliminary results are very encouraging, with excellent precision and accuracy on reference standards and therapeutic oligonucleotide samples.
The method could be used in routine testing of oligonucleotides in a GxP environment, as an orthogonal method to P-NMR. In addition, the method is currently evaluated for the potential simultaneous absolute quantification of the tested oligonucleotide in place of the classical UV determination.