Quantification Of Large Oligonucleotides Using High Resolution MS/MS

By Thomas Knapman and Vicki Gallant, SCIEX and Martyn Hemsley, Covance

Quantitative analysis of synthetic oligonucleotides in biological matrices is an important aspect of pharmacokinetic (PK), toxicokinetic (TK) and metabolic pathway studies in drug development. With an increasing number of oligonucleotide based drugs in research pipelines, the acceleration of the drug development process by reducing the time spent on method development, and by performing simultaneous qualitative structural analysis with quantitative analysis are crucial advantages in any potential quantitation approach.

Current LC-MS approaches to oligonucleotide quantitation predominantly use multiple reaction monitoring (MRM), however the complex fragmentation pathways of oligonucleotide species coupled with the variability of matrix effects mean that it can be difficult to predict the sensitivity and selectivity of a given MRM transition without significant optimization. These effects limit the utility of low resolution quantitation methods both in terms of the achievable limits of quantitation and in sample throughput, particularly when quantifying large numbers of potential drug candidates of different sequences, and their metabolites.

We explore how to achieve sensitive, high-resolution analysis of large oligonucleotides, with the opportunity to perform both qualitative and quantitative analysis in a single run.