Synthetic oligonucleotides, including small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs) and aptamers, continue to grow as a viable therapeutic modality. All of these classes utilize a serial synthesis for their production, which is prone to impurities that are multiplicative in nature. Many of these impurities can be isoelemental and thus almost impossible to fully characterize by a precursor ion only LC-MS workflow. Therefore, the need exists for a combined LC-MS and LC-MS/MS workflow to fully characterize the main product and impurities with high confidence. Unfortunately, the complexity of MS/MS spectra for oligonucleotides has made it difficult to develop an effective workflow solution including streamlined data reduction. Here, a novel ion trap (Zeno trap) is employed to achieve significantly improved MS/MS fragment ion coverage and oligonucleotide sequence coverage on a new LC-MS/MS QTOF system (ZenoTOF 7600 system, SCIEX). In addition, a highly automated software, which leverages MS, MS/MS and optical data, is utilized for impurity and biotransformation studies. A 2’-O-methoxyethyl phosphorothioated RNA (18-mer) and several n-X impurities at various concentrations were spiked into buffer. The samples were subjected to LC-MS/MS acquisition. The data were processed in the novel Molecule Profiler software against custom impurity libraries for oligonucleotides. MS1 results showed a very high level of agreement between intact isotope distributions and theoretical isotope models with excellent mass accuracies. Based on MS1 identification, a targeted MS/MS method was set up with and without the usage of the Zeno trap. Highly descriptive fragment ion spectra were generated. Matching data against in-silico information in the software allowed for detection and identification of spiked-in impurities down to a 0.1% spike. A significant enhancement of MS/MS data quality could be observed, resulting in higher coverage of consecutive residues when using the Zeno trap, due to increasing the duty cycle.