Metabolite ID and relative quantification of oligonucleotides in plasma

Identify, quantify, and confirm the structure of oligonucleotide metabolites using the ZenoTOF 7600 system and Molecule Profiler software from SCIEX

Remco van Soest, Kerstin Pohl, Todd Stawicki and Elliott Jones


This technical note describes the identification, relative quantification and structural confirmation of the chain-shortened metabolites of a phosphorothioated oligonucleotide. Relative quantification was achieved at levels as low as 0.1% (w/w), while sequence coverage was realized at levels down to 1%.

Oligonucleotide therapeutics and gene therapies are rapidly gaining attention as their potency improves and delivery challenges are addressed. Modalities such as antisense oligonucleotides (ASOs) are becoming more important due to their high specificity and ability to reach formerly untreatable targets. To ensure safe drugs, methods for the identification and characterization of the full length product (FLP) and its metabolites are critical. High resolution mass spectrometry (HRMS) can be used for the identification of potential metabolites, by comparing the measured accurate masses and isotope patterns with those calculated. However, there is a lack of powerful yet intuitive processing software, and manual interpretation is cumbersome and time consuming. Furthermore, structural confirmation leveraging MS/MS adds an additional level of complexity.

Using the Molecule Profiler software to overcome these challenges, this technical note shows the identification and relative quantification of the 5’ and 3’ (n-1, 2 and 3) metabolites of an ASO spiked into a rat plasma extract, in the presence of the FLP, at levels between 0.1% and 10% (w/w) of the FLP. The software can perform relative quantification based on TOF MS and assign fragment ions of the potential metabolites to confirm their structures, facilitating metabolism studies of drugs in development. 

Key features of Molecule Profiler software for oligonucleotide metabolite analysis

  • Excellent quality and high mass accuracies for MS and MS/MS data allow for confident assignment of oligonucleotide FLP and metabolites in Molecule Profiler software
  • Straightforward relative quantification based on TOF MS can be achieved by grouping of charge states or, alternatively, UV data can be leveraged for quantification
  • Significant improvement of identification of low abundant metabolites by improved S/N of MS/MS spectra using the Zeno trap of the ZenoTOF 7600 system

Figure 1. Workflow representation for relative quantification and structural confirmation of metabolites using the Molecule Profiler software.