LC/MS bioanalysis of oligonucleotides has had its historical challenges in all areas of the workflow including extraction, liquid chromatography, and mass spectrometry detection. Among the primary pain points for the extraction of oligonucleotides is poor recovery from nonspecific binding or poor extraction efficiency using the most common extraction approaches. Recent publications of a more specific biotinylated probe hybridization approach have addressed these challenges, however, there hasn’t been much presented on the specific advantages for different probe types that can be used for this hybridization work. Although there has been a focus on DNA, LNA, and PNA probe design with research to demonstrate the specific attributes each offers, there has been limited discussion on the overall impact on recovery and interference from these different probes. Biotinylated probe design has been focused on limiting self-hybridization of the probe while maintaining a complimentary sequence with a sufficiently high score to out-compete any interferences from matrix or from the sense strand in siRNA modalities while keeping the melting temperature (Tm) of the hybridized duplex low enough to ensure recovery from the streptavidin beads. Recently, while developing an assay for a siRNA complex using the LNA approach, we observed interferences from the probe to the antisense strand when it was analyzed by mass spectrometry. We modified the melting temperature to release the streptavidin/biotinylated hybridized to avoid releasing most of the biotinylated probe/antisense complex, but the limited release still had enough of the probe in the final extracts to cause interferences in the LLOQ samples. Modifying LC conditions helped to resolve the interference, but those changes were not entirely successful due to peak shape issues with the internal standard. After reviewing the probe design, we implemented an alternate PNA probe with the intention that any residual PNA probe would be easier to resolve chromatographically.
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