LC-MS analysis of RNA through hydrogen bonding of alkylamines

Nucleic acid oligonucleotides represent a promising therapy for treating disease. Production of these biopolymers happens synthetically which can produce impurities concomitant with the desired product. The use of liquid chromatography coupled to high resolution mass spectrometry is a robust analytical technique for determination of sample purity. Traditionally, ion pairing reagents are used to facilitate chromatography separation before sampling with the mass spectrometer. The choice of reagent for analysis can impact the analysis as the length of the oligonucleotide increases. This is in part due to the thermodynamics of transitioning the oligonucleotide from the liquid phase to the gas phase. Here we show that the use of a hydrogen bond donating alkylamine assists with overcoming the energy barrier of phase transition by bonding to the nucleobase as well as the phosphodiester backbone. By increasing the relative hydrophobicity of the oligonucleotide, the transition to the gas phase is increased producing a cleaner mass spectrum. Furthermore, we show that by using a hydrogen bond donating alkylamine, we can remove the fluoroalcohol reagent commonly used in ion pairing mass spectrometry of nucleic acids.