Poster abstracts

Poster number 149 submitted by Scott Abernathy

Investigating the effectiveness of HILIC-type column chemistries for oligonucleotides

Scott Abernathy (Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati), Patrick A. Limbach (Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati)

Analyzing DNA and RNA has become increasingly important as more is learned about the transcriptome and more RNA-based drugs are being produced in an attempt combat various diseases. Traditionally, liquid chromatography – mass spectrometry (LC-MS) analysis of nucleic acids is done by digestion of the sample into nucleotides and oligonucleotides and then building the sequence from the bottom-up. The gold standard for oligonucleotide chromatography has been ion pairing reverse phase liquid chromatography (IP-RP-LC). IP-RP-LC utilizes mobile phase additives that aid the chromatographic retention and gas-phase ionization of oligonucleotides. While this method has been very effective, ion pairing reagents are inconvenient to work with and difficult to eliminate from HPLC instruments. Additionally, ion-pairing reagents can lead to increased background noise in mass spectrometry when trying to change assays or modes. Thus, most oligonucleotide assays can require dedicated instruments, which is not practical and can be very costly. Recently, hydrophilic interaction liquid chromatography (HILIC) for oligonucleotide separation during LC-MS analysis has been shown to provide competitive chromatographic and mass spectrometric figures of merit. Currently, assays have been reported for a diol and amide functionalized column. The focus of this project is to further understand the retention mechanism as well as the limitations of HILIC-type columns for oligonucleotide LC-MS assays.

Keywords: HILIC, Oligonucleotides