New Review Highlights HILIC Evolution for Oligonucleotide Analysis

In a recent review article published by scientists from the University of Georgia, in Athens, Georgia, Michael G. Bartlett looked at how hydrophilic interaction liquid chromatography (HILIC) has evolved regarding oligonucleotide analysis. His findings were published in the Journal of Chromatography A (1).

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Oligonucleotides are short DNA or RNA molecules, either single- or double-stranded, which include antisense oligonucleotides, RNA interference, and aptamer RNAs (2). These molecules, which are used to modulate gene and protein expression, have become a widely used class of molecules in the biomedical sciences. They act as reagents in many biological assays, disease biomarkers, and are seeing increased use as therapeutic agents. This has led to an increasing focus on continuously improving quality control and bioanalytical assays for these molecules. These molecules, which have seen increasing commercial use, have led to a shift away from classical biological assays, such hybridization immunoassay and polymerase chain reactions (PCR), and towards chromatographic methods with greater specificity.

Read more: Systematic Evaluation of HILIC Stationary Phases for MS Characterization of Oligonucleotides

One popular technique used to separate oligonucleotides is hydrophilic interaction liquid chromatography (HILIC). First recognized as a form of chromatography in 1990, HILIC is a high-performance liquid chromatography (HPLC) technique meant for separating polar and hydrophilic compounds (3). The technique is a normal-phase type of separation, though it uses reversed-phase type eluents. HILIC provides both a column with a hydrophilic stationary phase and an eluent with water, buffer, and a high concentration of a water-miscible organic solvent.

The main retention mechanism in HILIC is partitioning into a surface-absorbed water layer at the interface of the stationary phase. The rise of popularity in HILIC use has correlated with increasing oligonucleotide success as therapeutic treatments and reagents in biomedical research. As more scientists routinely analyze oligonucleotides, in addition to small molecules, peptides, and proteins using the same analytical instruments, traditional types of analyses, such as ion-pair reversed-phase chromatography, become more difficult to use.

In this review article, the scientists highlighted recent advancements in HILIC separation of oligonucleotides, with specific focus on underlying mechanisms of action. Due to higher water levels being present in the mobile phase, oligonucleotide HILIC occurs with unique mobile phase compositions compared to traditional HILIC. Compared to other types of chromatography, non-specific binding is a greater challenge for HILIC; this is likely due to its optimal pH being between 5 and 8, thereby causing greater metal surface charging. Regarding sensitivity, HILIC combined with mass spectrometry has improved, though it requires attention to alkali metal adduction. Increased water content in the mobile phase can also likely prevent great improvements in desolvation as is typically observed in HILIC.

While HILIC has grown significantly regarding performance, there are still challenges with using this approach, the scientists wrote. For example, there have been many studies on diastereomer resolution in ion-pair reversed-phase liquid chromatography (IPRP-LC), with most studies showing that most ion-pairing agents can be used to resolve diastereomers. However, some show limited abilities to resolve them, instead proving beneficial methods that only want to measure total oligonucleotide contents as they will narrow chromatographic peak shape, thus making it easier to observe impurities. Limited HILIC-based studies that use the technique to study diasteromer resolution show that they can also be resolved using this approach, having similar results to IPRP-LC. Future research into improving HILIC will focus on improving the performance of buffering systems, pH, common chemistries, and integrating microflow liquid chromatography.

References

(1) Bartlett, M. G. Current State of Hydrophilic Interaction Liquid Chromatography of Oligonucleotides. J. Chromatogr. A 2024, 1736, 465378. DOI: 10.1016/j.chroma.2024.465378

(2) Oligonucleotide. ScienceDirect 2013. https://www.sciencedirect.com/topics/neuroscience/oligonucleotide (accessed 2024-10-16)

(3) Hydrophilic Interaction Liquid Chromatography. MilliporeSigma 2024. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/analytical-chemistry/small-molecule-hplc/hilic (accessed 2024-10-21)