Recent developments in the characterization of nucleic acids by liquid chromatography, capillary electrophoresis, ion mobility, and mass spectrometry (2010–2020)

Abstract

The development of new strategies for the analysis of nucleic acids has gained momentum due to the increased interest in using these biomolecules as drugs or drug targets. The application of new mass spectrometry ion activation techniques and the optimization of separation methods including liquid chromatography, capillary electrophoresis, and ion mobility have allowed more detailed characterization of nucleic acids and oligonucleotide therapeutics including confirmation of sequence, localization of modifications and interaction sites, and structural analysis as well as identification of failed sequences and degradation products. This review will cover tandem mass spectrometry methods as well as the recent developments in liquid chromatography, capillary electrophoresis, and ion mobility coupled to mass spectrometry for the analysis of nucleic acids and oligonucleotides.

Tandem mass spectrometric sequence characterization of synthetic thymidine‐rich oligonucleotides

“…For this work, hydrophilic interaction liquid chromatography (HILIC), which uses MS compatible mobile phases, was used as an alternative to more widely used ion-pair reversed-phase liquid chromatography (IPRP LC) to separate oligonucleotides prior to MS analysis. 6,7,11,[20][21][22] The absence of ion-pairing (IP) reagents during HILIC-MS analysis helps address some of the IP reagents-related technical hurdles, including The results demonstrated a good peak shape and an envelope of charge state distribution centered on the À4 charge state (Figure 1 inset), which was different from the wide charge state distributions commonly observed during IPRP LC-MS analysis of oligonucleotides. 20 Importantly, a predominant charge state offers better analytical sensitivity by consolidating the molecular ion count and is beneficial when using an extracted ion chromatogram for quantitation or when selecting a precursor ion in MS/MS.…”

“…Structural characterization of synthetic oligonucleotides, including any incorporated modifications, is essential for drug development, manufacturing processes, and quality control of final products. Tandem mass spectrometry (MS/MS) has been shown to provide rapid and reliable structural characterization and sequence determination of synthetic oligonucleotides 5–10. The mass spectrometry‐based technique has particular advantages in sequencing modified oligonucleotides, for which the classical enzyme‐based methods have faced great challenges due to the presence of modified structural elements.…”

Applications of capillary electrophoresis in the fields of environmental, pharmaceutical, clinical, and food analysis (2019–2021)

“…Owing to high selectivity and potential tolerance to matrix components, CE can be used to diagnose diseases by analyzing small ions [55], organic acid-base compounds [56], amino acids [57,58], peptides [59], proteins [60][61][62] and nucleic acids [63][64][65] in biological body fluids. As a result, CE has obtained a significant position in the field of clinical medical diagnosis [66,67].…”

Regulatory guidelines and preclinical tools to study the biodistribution of RNA therapeutics

“…For example, the European Public Assessment Reports of siRNA drugs Onpattro® and Givlaari® state that LC-MS/MS was used for quantification of siRNA in plasma and/or tissue samples [22] , [54] . The various MS modalities for detailed analysis of oligonucleotides beyond biodistribution have recently been extensively reviewed [78] , [79] and is beyond the scope of this review. In contrast to siRNA, mRNA molecules can exhibit a molecular weight into the MDa range.…”