Development of a hydrophilic interaction-based procedure for the extraction, separation, and determination of therapeutic oligonucleotides

Abstract

Background: For analyzing oligonucleotide impurities and metabolites in serum samples, where mass spectrometry detection is essential, hydrophilic interaction liquid chromatography utilizing fully mass spectrometry compatible mobile phases is the best choice. However, reliable analysis must be preceded by a reliable extraction method providing sufficient and repeatable recoveries. Thus, this work aimed to develop a simple and repeatable methodology for extracting and analyzing modified oligonucleotides differing in length (4mer-14mer), both under hydrophilic interaction conditions. Results: A comprehensive retention study examining the effects of mobile phase composition and temperature on retention and peak shape was conducted for the zwitterionic and amide columns. Based on the results, the amide column was chosen for further study, mainly because the zwitterionic column does not permit elution of longer oligonucleotides (>10mer). In addition, the amide column exhibits greater robustness (minor effects of tested parameters on chromatographic performance), which ensures higher repeatability. The optimization of mass spectrometry parameters was performed to obtain the highest possible sensitivity, which is well well-known issue because of adducts formation. A validated procedure was applied to analyze enriched serum extracts - a newly synthesized amide-based sorbent was used for the dispersive solid-phase extraction. The conditions for adsorption and desorption, which are driven by a weak ion-exchange mechanism and thus by pH change, were optimized to obtain the highest recoveries. Significance: A fully hydrophilic interaction-based procedure was utilized for the first time f extraction and analysis of a mixture of 5 modified oligonucleotides differing in length from enriched serum. This method proved to be simple, repeatable, and provides recoveries higher than 75% (RSD <5%).