Hybridization LC-MS for Bioanalysis of Oligonucleotides

Oligonucleotide therapeutics, such as siRNAs and ASOs, have emerged as a promising modality to target rare and intractable diseases in the past decade. Chromatographic analysis of oligonucleotides provides higher specificity that enables discrimination of truncation metabolites and is a common alternative to hybridization-based ELISA (hELISA). Recently, hybridization has been coupled with chromatographic separation in the form of hybridization capture followed by liquid chromatography with mass spectrometry (LC-MS). However, the best practices for these hybridization-based chromatographic methods are still under debate. In this rapid fire talk, I will discuss the insights gained from a case study where we developed a hybridization capture followed by LC-MS as an alternative to an existing solid phase extraction (Clarity OTX) method. We evaluated two types of probes types: locked nucleic acids (LNA) and peptide nucleic acids (PNA), which are synthetic analogs of DNA that have higher affinity and stability than standard oligonucleotides. We tested both short (14 nucleotides) and long (20 nucleotides) versions of these probes. We found that the hybridization methods had some advantages over the LC-MS methods, such as lower sample volume requirement, higher recovery, and lower matrix effect. However, we also encountered some challenges with the hybridization method, such as apparent non-specific binding and poor recovery.