Oligonucleotide analysis by hydrophilic interaction liquid chromatography-mass spectrometry in the absence of ion-pair reagents

Abstract

Improving our understanding of nucleic acids, both in biological and synthetic applications, remains a bustling area of research for both academic and industrial laboratories. As nucleic acids research evolves, so must the analytical techniques used to characterize nucleic acids. One powerful analytical technique has been coupled liquid chromatography – tandem mass spectrometry (LC–MS/MS). To date, the most successful chromatographic mode has been ion-pairing reversed-phase liquid chromatography. Hydrophilic interaction liquid chromatography (HILIC), in the absence of ion-pair reagents, has been investigated here as an alternative chromatographic approach to the analysis of oligonucleotides. By combining a mobile phase system using commonly employed in liquid chromatography-mass spectrometry (LC–MS) - i.e., water, acetonitrile, and ammonium acetate - and a new, commercially available diol-based HILIC column, high chromatographic and mass spectrometric performance for a wide range of oligonucleotides is demonstrated. Particular applications of HILIC-MS for the analysis of deoxynucleic acid (DNA) oligomers, modified and unmodified oligoribonucleotides, and phosphorothioate DNA oligonucleotides are presented. Based on the LC–MS performance, this HILIC-based approach provides an attractive, sensitive and robust alternative to prior ion-pairing dependent methods with potential utility for both qualitative and quantitative analyses of oligonucleotides without compromising chromatographic or mass spectrometric performance.