Studying Mobile Phases for HILIC UV/MS Analysis of Oligonucleotide Therapeutics including an siRNA Duplex, a Lipid Conjugated ASO, and a CRISPR sgRNA

Significance of the Topic

Hydrophilic interaction liquid chromatography (HILIC) has emerged as a powerful alternative to ion-pair reversed-phase methods for the separation of therapeutic oligonucleotides. It supports sensitive and robust analysis of diastereomers, duplexes, and single strands while enabling direct mass spectrometric detection without ion-pairing additives.

Objectives and Study Overview

This study aimed to tune HILIC mobile phases for optimal separation of oligonucleotide standards, siRNA duplexes and a lipid-conjugated antisense oligonucleotide (ASO), and to demonstrate compatibility with UV and mass spectrometric detection. Key goals included systematic evaluation of ammonium salt additives, use of design of experiments, bracketed injection to mitigate breakthrough, and proof-of-concept LC-MS analysis of a CRISPR single guide RNA (sgRNA) and a lipid ASO.

Methodology

A three-factor design of experiments (DoE) explored the effects of gradient steepness, column temperature and ionic strength across ammonium acetate, formate and bicarbonate at 10–50 mM. A concave multistep gradient was optimized in DryLab software to achieve rapid separations. Sample introduction employed a bracketed injection scheme combining weak solvent plugs around aqueous sample to prevent peak splitting. Oligonucleotide standards included a dT ladder, ssDNA ladder, a lipid-conjugated ASO, and a duplex siRNA. Mobile phases comprised water/acetonitrile mixtures with controlled salt concentration. UV detection was performed at 260 nm and LC-MS analysis used a BioAccord system in full-scan mode (m/z 400–5000).

Instrument Used

• ACQUITY UPLC H-Class Bio System with Binary Solvent Manager and High-pH Kit
• ACQUITY TUV Detector (Titanium Flow Cell)
• BioAccord LC-MS System with full-scan MS detection
• GTxResolve Premier BEH Amide 300 Å 1.7 µm, 2.1×50 mm Column
• Empower Pro 3 and DryLab 4.4 software

Main Results and Discussion

Retention decreased at ionic strengths below 40 mM, with unacceptable peak splitting and breakthrough at 10–20 mM. Ammonium acetate and formate yielded similar retention and selectivity, whereas bicarbonate required a shifted gradient and showed slight differences in early peak behavior. Temperature played a critical role in tuning selectivity and controlling duplex stability: below 55 °C the siRNA duplex eluted as a single peak, began dissociating around 60 °C, and fully resolved into single strands at 65–70 °C. Bracketed injection effectively eliminated breakthrough, enabling up to 2 µL injections without distortion. In LC-MS mode, a 75 °C method with 100 mM ammonium acetate eluent yielded a clean sgRNA peak (molecular weight 32 276.97 Da from [M-11H]11– and [M-10H]10– ions). Analysis of a lipid-conjugated ASO using 25 mM ammonium acetate resolved isobaric diastereomers and detected a 419 Da impurity.

Benefits and Practical Applications

• Ion's pairing agent-free mobile phases enhance MS compatibility
• Adjustable native/denaturing modes support duplex and single-strand separation
• Systematic DoE accelerates method development
• Bracketed injection prevents solvent mismatch effects and peak splitting
• High resolution of diastereomers and impurities for therapeutic QC

Future Trends and Opportunities

Further elucidation of the HILIC retention mechanism for oligonucleotides, exploration of alternative co-solvents, and integration with advanced MS/MS fragmentation strategies offer pathways to deeper structural characterization. Automated multidimensional modelling and expanding to novel therapeutic modalities, such as mRNA vaccines and large guide RNAs, will broaden application scope.

Conclusion

HILIC affords a versatile, robust platform for the UV and MS analysis of oligonucleotide therapeutics. Systematic optimization of mobile phase parameters, bracketed injection, and high-temperature operation enable high-resolution separation of diastereomers, duplexes and single strands. Direct LC-MS compatibility streamlines mass measurement and purity assessment, making HILIC an attractive choice for research and quality control in oligonucleotide drug development.

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