Analysis of deamination impurity of PS-ONs by chemical derivatization of the enzymatic digest and LC-MS/MS

The presence of deamination impurities in phosphorothioate oligonucleotides (PS-ONs) presents a significant challenge to quality control, as their detection is complicated by minimal mass differences from the parent molecule and difficulties in chromatographic separation. Herein, a bottom-up strategy based on enzymatic digestion, chemical derivatization, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was proposed for the sensitive qualitative and quantitative analysis of trace cytosine deamination in PS-ONs. Fomivirsen (FMVS) and its deamination impurity were used as models. The method involved digestion with S1 nuclease, followed by derivatization of the 5’-phosphorothioate in digests with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) to enhance chromatographic retention and mass spectrometric response. This approach effectively eliminated the need for ion-pairing (IP) reagents. The mass shift upon deamination was amplified in the smaller digested fragments, and a unique isotopic pattern in the derivatized products, specifically, the decrease of the first isotopic peak and the increase of the second isotopic peak was exploited for quantification. The method demonstrated excellent linearity over a weight ratio of deamination product to prototype range from 0.05:1 to 0.9:1. Satisfactory accuracy (80–120%) and precision (RSD < 15%) were achieved. Furthermore, the precise deamination site (C8) within a sample containing 10% deamination impurities was successfully identified. Thus, this method provides a powerful tool for profiling deamination impurities in PS-ONs, with potential applications in quality control, stability studies, and the characterization of other nucleic acid modifications.