Quantification of Large Oligonucleotides using High Resolution MS/MS on the TripleTOFTM 5600 System (SCIEX)

Authors: Thomas Knapman, Vicki Gallant and Martyn HemsleySCIEX_LOGO_BioZonesmall

Key Challenges of Oligonucleotide Bioanalytical Assay

  1. The bioanalysis of oligonucleotides as therapeutics requires sensitive, specific and robust analysis.
  2. Many ELISA-based oligonucleotide measurements do not accurately distinguish large metabolites from the full-length oligonucleotide of interest.
  3. ELISA- and UV-based measurements have limited dynamic range which complicates quantitative analysis of oligonucleotides in complex matrices.

Key Benefits of MRMHR Workflow for Oligonucleotide Bioanalytical Assay

  1. High sensitivity MS/MS enables the quantitative MRMHR workflow, providing high selectivity in biological matrices.
  2. High resolution, accurate mass MS/MS spectra enable qualitative verification of oligonucleotide sequences.
  3. The MRMHR workflow offers a dynamic range of two to three orders of magnitude.

Unique Features of MRMHR Workflow on TripleTOF™ 5600 System

  1. Summing of multiple ion transitions to increase both sensitivity and selectivity of quantitation.
  2. Accelerated method development times, since ion transitions can be selected post-acquisition to eliminate background interferences.
  3. High multiplexing due to high acquisition speeds (up to 100 spectra per second) for simultaneous quantitation of multiple species, including multiple oligonucleotide sequences and/or their metabolites.

Read the full application note here.

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