Sensitive quantitation of antisense oligonucleotides in plasma using high-resolution, accurate-mass (HRAM) mass spectrometry

Significance of Topic

Antisense oligonucleotides (ASOs) represent a rapidly growing class of therapeutic agents that modulate gene expression by sequence-specific binding to target mRNA. As more ASO drugs enter clinical use or advanced trials, reliable and sensitive bioanalytical methods are essential to characterize their pharmacokinetic and metabolic profiles in complex biological matrices such as human plasma.

Goals and Study Overview

The study aims to develop and validate a high-resolution, accurate-mass liquid chromatography-mass spectrometry (LC-HRAM-MS) workflow for trace quantitation of two FDA-approved ASOs—fomivirsen and nusinersen—in human plasma. The method integrates ion-pairing reversed-phase LC separation, targeted MS/MS acquisition, and compliance-ready data processing to achieve low detection limits, reproducibility, and regulatory suitability.

Methodology and Instrumentation

• Sample Preparation: One-step liquid-liquid extraction (LLE) using phenol/chloroform/isoamyl alcohol (25:24:1) stabilized with Tris-EDTA at pH 8.0, followed by drying and reconstitution with internal standard (IS).
• Chromatography: Thermo Scientific Vanquish Horizon UHPLC system with C18 column (2.1×50 mm, 2.6 µm), 8-min gradient of DIPEA/HFIP aqueous versus organic mobile phases, 20 µL injection volume, column temperature 50 °C.
• Mass Spectrometry: Thermo Scientific Orbitrap Exploris 120 operated in negative mode with targeted MS/MS (tMS2) at 60 000 resolution, HCD fragmentation, monitoring specific precursor-to-product ion transitions for each analyte and IS.
• Data Processing: Thermo Scientific Chromeleon CDS v7.3.2 for automated peak integration, calibration curve fitting (1/x weighting), and fit-for-purpose reporting in a cGLP-compliant environment.

Main Results and Discussion

• Lower limit of quantitation (LLOQ): 0.20 ng/mL for fomivirsen and 0.10 ng/mL for nusinersen in human plasma.
• Linearity: Calibration range 0.10–100 ng/mL, correlation coefficient (R2) ≥ 0.994 for both analytes.
• Precision and Accuracy: Five replicates per level showed ≤ 14.6% RSD and accuracy within ± 16% at low levels, improving at higher concentrations.
• Specificity: No detectable crosstalk or matrix interferences in extracted human plasma for selected MS/MS transitions.
• Chromatographic Performance: Baseline resolution of analytes and IS, peak widths < 10 s, total run time 8 min per injection.
• Regulatory Compliance: Automated audit trail of manual integrations and fit-for-purpose summary reports support regulated bioanalysis workflows.

Benefits and Practical Applications

• Enables sensitive and reproducible quantitation of ASO therapeutics in preclinical and clinical pharmacokinetic studies.
• Streamlines method development by targeting specific precursor and fragment ions against real sample matrices.
• Supports high throughput analysis with short run times and automated processing in a compliance-ready software environment.

Future Trends and Possibilities

• Expansion to multiplexed assays for simultaneously quantifying multiple oligonucleotide therapeutics and metabolites.
• Integration with microflow or nanoflow LC to further improve sensitivity and reduce sample volume requirements.
• Application of hybrid data acquisition strategies combining full-scan HRAM and targeted MS/MS for comprehensive metabolite profiling.
• Enhanced automation and AI-driven data review to accelerate turnaround in regulated bioanalysis laboratories.

Conclusion

The developed IPRP-LC-HRAM-MS method on the Vanquish Horizon UHPLC coupled with the Orbitrap Exploris 120 provides a robust, sensitive, and compliance-ready solution for quantifying fomivirsen and nusinersen in human plasma. It achieves low ng/mL detection limits, excellent linearity, and reproducible performance, supporting the growing demands of ASO therapeutic development.

Reference

1. Bennett CF, Baker BF, Pham N, Swayze E, Geary RS. Pharmacology of antisense drugs. Annu Rev Pharmacol Toxicol. 2017;57:81–105.
2. Dhuri K, Bechtold C, Quijano E, Pham H, Gupta A, Vikram A, Bahal R. Antisense oligonucleotides: an emerging area in drug discovery and development. J Clin Med. 2020;9(6):2004.
3. Patrinos G, Danielson P, Ansorge W. Molecular Diagnostics. 3rd ed. Elsevier; 2017.