Liquid chromatography - high resolution mass spectrometry analysis of intact ribonucleic acid through hydrogen...

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 with high resolution mass spectrometry is a robust analytical technique for determination of sample purity. Traditionally, ion pairing reagents are used to facilitate chromatographic separation before sampling with a mass spectrometer, termed ion-pair reversed phase liquid chromatography mass spectrometry (IPRP-LC-MS). The choice of reagents for IPRP-LC-MS can have an impact on the results as the length of the measured oligonucleotide increases. This is, in part, due to the thermodynamic energy barrier of transitioning the oligonucleotide from the liquid phase to the gas phase. In this work, we used the quantum chemistry software package Orca 6.0 to model the theoretical docking of a secondary amine to the nucleobase of a synthetic oligonucleotide through hydrogen bonding. We then validated these results using IPRP-LC coupled to high resolution accurate mass spectrometry (HRAM-MS). By increasing the relative hydrophobicity of the oligonucleotide, the transition to the gas phase is increased resulting in enhanced sensitivity. This in turn allowed for impurity analysis of guide RNAs at < 5 % relative abundance. Furthermore, we show that monoisotopic mass confirmation can be performed without the need for fluoroalcohol.