Chad Christianson (Alturas Analytics)
The analysis of oligonucleotides by HPLC–MS/MS is a technique that hasn’t been widely adopted but is an emerging technology due to the limitations of the current methods being utilized. ELISA, qPCR and HPLC–FL analysis lack selectivity, dynamic range or the capability to analyze oligonucleotides smaller than 20 nucleotides. Additionally, the time and cost to develop these methods are typically much greater than the HPLC–MS/MS approach. HPLC–MS/MS analysis provides better selectivity, increased dynamic range and the flexibility to quickly analyze a vast array of oligonucleotides from biological matrices.
Zamas Lam (QPS)
Traditional methodologies that are still being used are hybridization ligand binding assays, hybridization LC–fluorescence assay and LC–MS/MS assays using MRM. The newer methodologies are LC–HRMS using full scan or high-resolution MRM and hybridization LC–MS/MS.
Ken Cook (Thermo Fisher Scientific)
There has been a dramatic increase in the analytical requirements for oligonucleotide therapeutics following the success of the mRNA vaccines. Very recent developments in methodology to support this include sample preparation and software for direct sequence analysis of mRNA and smaller oligonucleotide therapeutics such as siRNA by LC–HRMS. There has never been any direct method adopted to verify the sequence of these oligonucleotide drug products before this year. Complete workflows for the sequence analysis of siRNA and mRNA are now available alongside accurate intact mass analysis. This is truly a game changer in the analysis of oligonucleotide therapeutics.
Alexey Wolfson (Advirna)
With a recent explosion in the development of oligonucleotide therapeutics – especially various oligonucleotide conjugates for targeted delivery – the availability of reliable, high-throughput and easy-to-implement methods for quantitative detection of oligonucleotides in tissues are increasingly in great demand. Their development will certainly significantly facilitate biodistribution studies.
The opinions expressed in this feature are those of the authors and do not necessarily reflect the views of Bioanalysis Zone or Future Science Group.