Ultra-sensitive quantitation of therapeutic oligonucleotide in human plasma by a high resolution accurate mass Orbitrap mass spectrometer

Significance of topic

Therapeutic oligonucleotides such as antisense oligonucleotides (ASOs) and siRNAs are gaining prominence for the treatment of rare and genetic diseases. Precise measurement of these molecules in biological matrices is critical for pharmacokinetic characterization, dose optimization, and regulatory compliance. High resolution accurate mass (HRAM) mass spectrometry offers enhanced specificity and sensitivity, enabling reliable quantitation of low-abundance oligonucleotide drugs in complex samples.

Objectives and overview of study

This work aimed to develop and validate an ion-pair reversed-phase liquid chromatography coupled to HRAM mass spectrometry (IPRP-LC-HRAM-MS) method for quantifying two FDA-approved ASO drugs—Fomivirsen and Nusinersen—in human plasma. Key goals included achieving a lower limit of quantitation (LLOQ) of 0.05 ng/mL, establishing linearity across a wide dynamic range, and implementing a fit-for-purpose compliance-ready reporting workflow.

Methodology and used instrumentation

• Sample preparation: Human plasma samples (200 µL) were extracted via phenol/chloroform/isoamyl alcohol liquid-liquid extraction, dried, and reconstituted in ion-pair reagent mix with an internal standard (IS).
• Chromatography: A Vanquish Horizon UHPLC system with a C18 column (2.1 × 50 mm, 2.6 µm) was used. A binary gradient of 15 mM DIPEA/25 mM HFIP in water (Solvent A) and in 80/20 acetonitrile/water (Solvent B) delivered an 8-minute separation at 0.25 mL/min and 50 °C.
• Mass spectrometry: A Thermo Scientific™ Orbitrap Exploris™ 480 operated in targeted MS2 (tMS2) negative-ion mode. Key source settings included 3,000 V spray voltage, 35 Arb sheath gas, and HCD fragmentation at 23%. Precursor-to-product transitions were defined for Fomivirsen, Nusinersen, and IS.
• Data processing and reporting: Chromeleon CDS software (v7.3.2) provided compliance-ready data review, integration, and calibration report generation.

Main results and discussion

The method achieved chromatographic baseline resolution of Fomivirsen, Nusinersen, and the IS with peak widths under 10 s. Calibration curves were linear from 0.05 to 50 ng/mL (weighted 1/X), with R2 > 0.998. At the LLOQ of 0.05 ng/mL, intra- and inter-day precision and accuracy were within ±15%. No interference or crosstalk was observed in extracted plasma samples for the selected transitions, confirming high specificity.

Benefits and practical applications

The developed IPRP-LC-HRAM-MS method delivers:

• Ultra-sensitive quantitation down to 0.05 ng/mL for key ASO therapeutics.
• Robust accuracy and precision across at least three orders of magnitude.
• A streamlined, compliance-ready software solution for regulated bioanalysis laboratories.
• Enhanced specificity through high-resolution mass detection, minimizing matrix interferences.

Future trends and opportunities

Emerging directions include multiplexed HRAM assays for panels of oligonucleotide therapeutics, automated sample preparation and data processing pipelines, integration of ion mobility for added separation, and extension to siRNA and other nucleic acid modalities. Advances in instrument sensitivity and informatics will further accelerate bioanalysis in clinical development.

Conclusion

An ultra-sensitive IPRP-LC-HRAM-MS method was successfully established on a Vanquish Horizon UHPLC coupled to an Orbitrap Exploris 480 for quantifying Fomivirsen and Nusinersen in human plasma. The approach meets stringent sensitivity, linearity, accuracy, and precision requirements and is supported by a compliance-ready reporting framework, facilitating its deployment in regulated pharmacokinetic studies.

Reference

Yang H, Du M. Ultra-sensitive quantitation of therapeutic oligonucleotide in human plasma by a high resolution accurate mass Orbitrap mass spectrometer. Thermo Fisher Scientific application note, 2024.