Chromatographic and LC–MS Strategies for Therapeutic Oligonucleotide Characterization

Therapeutic oligonucleotides are a rapidly growing drug class, but their extensive chemical modifications, conjugates, and diastereomeric complexity create major analytical challenges. Robust chromatographic separations are essential to support liquid chromatography-mass spectrometry (LC–MS) workflows for identity testing, impurity profiling, stability assessment, and quality control. Key techniques include ion-pair reversed-phase LC (IP-RPLC), anion-exchange LC (AEX), and emerging hydrophilic interaction LC (HILIC), each offering complementary selectivity for resolving closely related species. Multidimensional LC approaches further enhance peak capacity for complex mixtures. Together, these chromatographic strategies enable comprehensive characterization of oligonucleotide therapeutics and support regulatory-compliant bioanalysis and development workflows.

LCGC International spoke to Sandy Al Bardawil and Ludivine Ferey, authors of a review article published in Analytical Chemistry¹ summarizing recent advances in chromatographic methods coupled with MS for the characterization of therapeutic oligonucleotides.

How does ion-pair reversed-phase liquid chromatography (IP-RPLC) enable retention of highly polar oligonucleotides, and what role do alkylamine ion-pairing agents play in this mechanism?

Oligonucleotides are highly polar, negatively charged molecules, which means they do not retain well in classical reversed-phase chromatography. IP-RPLC solves this by adding positively charged alkylamine reagents in the mobile phase. These alkylamines associate with the negatively charged phosphate backbone of oligonucleotides, forming transient ion-pairs. This interaction partially neutralizes the charge and makes the complex more hydrophobic. As a result, the oligonucleotide can interact with the C18 stationary phase through hydrophobic (lipophilic) interactions, mainly via the alkyl chains of the ion-pairing reagent. In simple terms, retention is driven first by ionic pairing, then by hydrophobic interactions. The strength of retention depends on both the oligonucleotide properties (length, sequence, modifications) and the hydrophobicity of the alkylamine used. More hydrophobic amines lead to stronger retention, but this often comes at the cost of reduced mass spectrometry (MS) sensitivity.

What are the key trade-offs between chromatographic resolution and MS sensitivity when selecting ion-pairing reagents for electrospray Ionization-compatible IP-RPLC methods?

There’s always a balance between chromatographic performance and MS sensitivity. Stronger, more hydrophobic ion-pairing reagents like tributylamine, generally improve retention and resolution of oligonucleotides, but they also increase signal suppression in electrospray ionization, leading to reduced MS sensitivity.

On the other hand, weaker and more volatile ion-pairing reagents can improve MS response but may compromise chromatographic selectivity and peak resolution. Interestingly, the buffering acid plays a more critical role in MS compatibility than the ion-pairing reagent itself. That is why alkylamine/hexafluoro-2-propanol (HFIP)-based mobile phases are often used: HFIP helps improve desolvation and charge-state distribution, which benefits MS sensitivity. However, this comes with increased concerns regarding environmental impact and potential toxicity for operators, as well as system contamination issues. Overall, selecting an IP reagent is primarily about controlling retention and resolution, while MS compatibility is often more strongly influenced by the choice of buffering acid. Method development therefore requires finding the right balance between chromatographic selectivity and MS performance.

Why is hydrophilic interaction liquid chromatography (HILIC) considered orthogonal to IP-RPLC for oligonucleotide analysis, and how do their retention mechanisms fundamentally differ?

The two modes rely on fundamentally different retention mechanisms. In IP-RPLC, oligonucleotide retention is driven by the formation of hydrophobic ion-pair complexes that interaction a C18 hydrophobic stationary phase with ACN acting as the strong elution solvent. In contrast, HILIC, relies on partitioning into a water-rich layer on a polar stationary phase, along with hydrogen bonding and electrostatic interactions, where water is the strong solvent.

As a result, elution behavior can differ significantly between the two modes. Some species that coelute in IP-RPLC may be resolved in HILIC, and vice versa. For example, truncated or PO-related impurities that co-elute in IP-RPLC can often be resolved in HILIC due to its different selectivity. That complementary selectivity is particularly valuable for impurity profiling, as it improves confidence in both the detection and structural characterization of impurities.

In LC–MS analysis of oligonucleotides, how does adduct formation (e.g., Na⁺, K⁺, TEA⁺) impact spectral interpretation, and what strategies can be used to minimize these effects?

Adduct formation further complicates everything. In HILIC-MS, alkali metal adducts (Na⁺, K⁺, NH₄⁺) are particularly prevalent, while IP-RPLC-MS spectra often show adducts involving triethylamine (TEA⁺) and HFIP-related species. These adducts broaden charge-state distributions, complicate spectral interpretation and deconvolution, and ultimately reduce sensitivity.

Their formation is strongly influenced by solvent purity, glassware contamination, LC system memory effects, and ion-source conditions. To minimize these effects, we rely on high-purity solvents, bioinert LC systems, volatile buffers, andrigorous system cleaning protocols (for example, methane-sulfonic acid flushing). In addition, partial removal of TEA and HFIP adducts can be achieved by carefully tuning source parameters. This includes increasing in-source activation through higher gas flows, elevated source temperatures, or in source CID (collision-induced dissociation),which helps to reduce adduct stability. But it’s a balance — too much in-source activation can cause unwanted fragmentation. So, the goal is to suppress adducts without compromising molecular integrity.

What are the advantages and limitations of using high-resolution mass spectrometry (HRMS) compared to unit-resolution MS for impurity profiling of therapeutic oligonucleotides?

HRMS provides accurate mass measurements and can resolve closely related or isobaric impurities, which is critical for structural elucidation of impurities. This is particularly important because oligonucleotides produce highly complex spectra, with broad charge-state distributions, extensive adduct formation, and often low signal-to-noise ratios. In this context, HRMS systems also provide better sensitivity and resolving power for low-abundance impurities compared to unit-resolution MS.

In comparison, unit-resolution MS is suitable for routine identity confirmation but is limited for detailed impurity characterization. So, HRMS is more informative for comprehensive impurity profiling, but also more costly and less suited to routine QC applications. In contrast, unit-resolution MS remains widely used for routine identity confirmation due to its simplicity, robustness, and easier implementation. Ultimately, the choice depends on whether the objective is routine confirmation or detailed impurity characterization.

How does phosphorothioate (PS) backbone modification influence chromatographic behavior and peak shape in both IP-RPLC and HILIC?

Phosphorothioate (PS) modification significantly increases analytical complexity in both IP-RPLC and HILIC modes. A major challenge is stereochemistry: each PS linkage creates a chiral center, generating complex mixtures of diastereomers (e.g., up to 2¹⁹ for a 20-mer). This leads to peak broadening due to partial diastereomer separation, an effect seen in both chromatographic modes. In IP-RPLC, conditions such as the use of HFIP, hydrophobic ion-pairing systems and elevated temperatures help suppress diastereomer separation, improving peak shape and enhancing impurity resolution. Additionally, PS oligonucleotides are prone to oxidation, leading to the formation of phosphodiester (PO) impurities that must be effectively separated and monitored for QC. In IP-RPLC, PS oligonucleotides exhibit stronger retention than their PO counterparts due to the increased hydrophobicity imparted by sulfur. In contrast, in HILIC mode, PS oligonucleotides often display lower retention compared to PO analogues.

Explain how mobile phase pH affects both chromatographic retention and ionization efficiency in LC–MS analysis of oligonucleotides.

pH significantly affects both chromatographic performance and MS detection. In IP-RPLC, a pH of 8–9 provides an optimal compromise, ensuring efficient oligonucleotide and ion-pairing reagent ionization, increased retention, selectivity, and resolution. Lower pH (~7) increases nonspecific adsorption and ion suppression, while higher pH (~11) reduces alkylamine ionization, may induce aggregation, and shortens mobile phase lifetime.

In HILIC, pH has a smaller effect on retention but still influences electrostatic interactions, adsorption, and MS efficiency. As silanol groups ionize with increasing pH, electrostatic repulsion reduces oligonucleotide retention, whereas low pH (<7) enhances adsorption on positively charged metal surfaces. HILIC analyses are typically performed at pH 5–7 to balance retention and adsorption, though slightly higher pH can improve ESI desolvation, increase MS sensitivity, and reduce adsorption for sensitive analytes or when using non-bioinert chromatographic hardware.

What are the key challenges in analyzing small interfering RNA (siRNA) duplexes using LC–MS, and how does column temperature influence duplex stability during separation?

siRNAs are particularly challenging to analyze because they are double-stranded and structurally dynamic. Column temperature plays a critical role in their chromatographic behavior. Below the melting temperature (T_m), the duplex remains intact, typically producing a single peak. Above the T_m, the strands may separate if the method is sufficiently selective, resulting in two peaks. Operating near the T_m can cause partial melting, leading to broad or distorted peaks.

It is therefore recommended to perform a temperature study to evaluate the impact of column temperature on siRNA peak profiles both above and below T_m. Combining this with an orthogonal T_m determination under mobile phase-like conditions, such as ultraviolet (UV) or differential scanning calorimeter(DSC) measurements, provides a more reliable prediction and control of duplex behavior during LC–MS analysis. While exact replication of chromatographic conditions is challenging, due to factors such as the aqueous layer in HILIC, the acetonitrile-rich environment in IP-RPLC, and sample dilution, these measurements still provide valuable predictive insight.

Precise temperature control is essential, depending on whether the goal is to assess duplex integrity under non-denaturing conditions or strand purity under denaturing conditions.

Other key factors affecting duplex stability include the presence of cations in the sample or mobile phase, which enhance stability, and the percentage and type of organic solvent, which lower T_m in a solvent-dependent manner (MeOH < EtOH < MeCN). The addition of HFIP has a modest effect, decreasing T_m.

How can multidimensional LC approaches (e.g., IP-RPLC × HILIC) improve impurity profiling, and what practical limitations restrict their routine use

Multidimensional LC approaches such as IP-RPLC x HILIC or anion exchange (AEX) x HILIC, can increase peak capacity by combining complementary separation mechanisms. For example, impurities unresolved in the first dimension can be further separated in the second. In addition, when HILIC is used as the second dimension following AEX, it can serve as an efficient desalting step prior to MS detection, improving sensitivity.Overall, these approaches can enhance impurity profiling by enabling the detection of low-level species that may co-elute or remain unresolved in single-dimension methods.

However, the practical benefit of orthogonality is somewhat limited for oligonucleotides. In IP-RPLC, HILIC, and AEX, oligonucleotide retention is primarily governed by chain length, which reduces the true complementarity of these modes. Moreover, multidimensional LC comes with increased system complexity, including more sophisticated instrumentation and workflows (e.g., column switching or heart-cutting setups), longer and more demanding method development, and additional challenges in data acquisition and processing.Other limitations include reduced robustness, lower throughput, and difficulties in standardization across laboratories, which restrict routine implementation in QC environments where simplicity, speed, and reproducibility are essential.

In tandem mass spectrometry (MS/MS) of oligonucleotides, what factors influence fragmentation patterns and sequence coverage, and why is method optimization critical?

MS/MS is essential for oligonucleotide characterization, enabling sequence confirmation, impurity identification, and degradation analysis. Fragmentation, typically induced by collision-Induced dissociation (CID) or higher-energy collisional dissociation (HCD), depends strongly on precursor charge state, sequence length, chemical modifications, and collision energy.For example, phosphorothioate (PS) linkages and sugar modifications can alter fragmentation pathways, leading to preferential neutral and cleavage sites.Charge state selection is critical because it strongly influences sequence coverage. Without careful optimization, spectra may exhibit incomplete sequence information, insufficient fragmentation, or excessive spectral complexity. Targeted precursor selection, charge-state control, and fine-tuning of collision energies are therefore essential to obtain reproducible and structurally meaningful spectra.Challenges remain due to congested spectra with multiple charge states, metal adducts, and modification-dependent fragmentation, often requiring manual validation. These factors highlight the need for optimized acquisition strategies and specialized bioinformatics tools for therapeutic oligonucleotides.

References

1. Al Bardawil, S.; Barthélémy, P.; Ferey, L. Advances in Analysis of Therapeutic Oligonucleotides with Chromatography Coupled to Mass Spectrometry. Anal Chem. 2026, 98 (15),10895-10912. DOI: 10.1021/acs.analchem.5c06656