Hydrophilic-Phase Extraction: A New Avenue of Solid-Phase Extraction for Oligonucleotide Bioanalysis

A novel mode of bioanalytical solid-phase extraction (SPE) has been developed and used for a quantitative application involving an RNA oligonucleotide therapeutic candidate. Previously established protocols and media showed very little recovery, and so this new approach, based on hydrophilic interaction liquid chromatography (HILIC) with an aminopropyl-bonded phase on a silica support and providing an essentially uncharted avenue of selectivity, was explored. The procedure featured only one step prior to sample load, high-organic sorbent conditioning, two washes following the load that switched between low and high pH, followed by elution in an a highly eluotropic HILIC composition. The moniker of “hydrophilic-phase extraction” (HPE) was given to the procedure. The HPE was proven to be a critical part of a fully quantitative HILIC–high-resolution mass spectrometry (HRMS) method for the RNA oligonucleotide and has potential for general application.