Sensitive Bioanalysis of Antisense Oligonucleotides through Mitigation of Non‐specific Binding and Improved LC‐MS/MS Performance

Significance of the Topic

Antisense oligonucleotides (ASOs) are a rapidly expanding class of therapeutic agents that modulate gene expression. Accurate quantitation of ASOs in biological matrices is critical for pharmacokinetic profiling, safety evaluation and dosage optimization. However, their negative charge, tendency for non-specific adsorption and low ionization efficiency in negative electrospray mode pose significant analytical challenges.

Objectives and Study Overview

This work sought to develop and validate a highly sensitive, reproducible LC-MS/MS assay for quantifying a panel of seven ASOs in human plasma at sub-nanogram per milliliter levels. The selected oligonucleotides varied in length (18–33 nucleotides), backbone chemistry (phosphorothioate), sugar modifications (2′-MOE, 2′-OMe) and included a GalNAc-conjugated species to represent clinically relevant diversity.

Methodology and Instrumentation

Sample preparation employed liquid-liquid extraction with phenol:chloroform:isoamyl alcohol followed by chloroform wash and reconstitution in EDTA. GEM91 served as an internal standard. Chromatographic separation used a Waters ACQUITY Premier UPLC system with MaxPeak HPS plate, OST standards and a 1.7 µm C18 column (2.1 × 50 mm) at 50 °C. Mobile phases consisted of 100 mM HFIP and 15 mM DIPEA in water (A) and 80 % acetonitrile (B). The Xevo TQ Absolute tandem mass spectrometer operated in negative electrospray mode with a 0.5 mL/min flow and 15 µL injection volume.

Main Results and Discussion

The method achieved a lower limit of quantification of 0.1 ng/mL (0.2 ng/mL for the GalNAc-conjugate) across a calibration range of 0.1–1000 ng/mL, with regression coefficients (r2) exceeding 0.99 using 1/x2 weighting. Calibration standards and quality controls met acceptance criteria (±15 % accuracy, ±20 % at LLOQ) over three runs, demonstrating accuracy between 89–108 % and precision (RSD) below 15 %. MaxPeak HPS technology effectively minimized non-specific binding and metal adsorption, improving signal stability. Representative chromatograms confirmed clear peak definition at the lowest concentrations.

Benefits and Practical Applications

• The protocol supports sub-ng/mL sensitivity critical for early-phase pharmacokinetic studies.
• Wide dynamic range accommodates low and high concentration samples without dilution.
• Reduced non-specific adsorption enhances reproducibility in negative ion mode.
• Applicable to diverse ASO chemistries including conjugated and heavily modified sequences.

Future Trends and Opportunities

Integration of hybridization-based extraction could further improve specificity. Advances in ionization enhancers may boost sensitivity for challenging analytes. Automation and microsampling will streamline high-throughput workflows. Extending the approach to metabolite profiling and stability studies will broaden its impact in oligonucleotide drug development.

Conclusion

The combination of optimized sample preparation, MaxPeak HPS-enabled UPLC separation and Xevo TQ Absolute detection delivers a robust, sensitive and reproducible assay for antisense oligonucleotides in human plasma, meeting stringent bioanalytical requirements for modern drug discovery and development.

References

• Suma Veeramachineni, Mark Wrona, Sensitive LC-MS/MS Bioanalytical Quantitation of Antisense Oligonucleotides, Waters™ Application Notes, 720007574, March 2022.
• Jennifer M. Nguyen et al., Assessing the impact of nonspecific binding on oligonucleotide bioanalysis, Future Science, Bioanalysis, Vol. 13, No. 16.
• Guilherme J. Guimaraes et al., Impact of Nonspecific Adsorption to Metal Surfaces in Ion Pair-RP LC-MS Impurity Analysis of Oligonucleotides, Journal of Pharmaceutical and Biomedical Analysis, 208, 114439, 20 January 2022.