Improve Sensitivity for Quantification of Antisense Oligonucleotides in Plasma Using Microflow LC MRM Methodology

Significance of the Topic

Oligonucleotide therapeutics are emerging as a powerful class of drugs, driving a growing need for sensitive bioanalytical methods to support pharmacokinetics and safety studies. Conventional mass spectrometry-based assays struggle to detect ultra-low concentrations in biological matrices. Combining advanced mass spectrometers with microflow chromatography bridges this gap, enabling quantification levels comparable to hybridization ELISA while maintaining method flexibility and robustness.

Objectives and Study Overview

This work aimed to develop and validate an ultra-sensitive LC-MS/MS workflow for the quantification of the antisense oligonucleotide fomivirsen in rat plasma. The study compared traditional high-flow LC-MS against a microflow LC-MS approach on the same QTRAP® 6500+ system to assess improvements in signal-to-noise ratio, calibration linearity, precision, and accuracy across a wide concentration range (0.1–1,000 ng/mL).

Methodology and Instrumentation Used

Sample Preparation:

• Rat plasma (100 µL) was processed using OTX Clarity SPE plates for protein removal and concentration.
• Eluates were dried under nitrogen and reconstituted in mobile phase containing HFIP, DIEA, and EDTA.
• Calibration standards prepared by spiking fomivirsen into extracted plasma (0.1–1,000 ng/mL).

LC-MS/MS Conditions:

• System: QTRAP 6500+ with OptiFlow™ Interface and M5 MicroLC for microflow (1–50 µL/min) or ExionLCTM high-flow setup.
• Chromatography: Comparison of high-flow vs. microflow separations using ion-pairing reagents HFIP and DIEA.
• Detection: On-column MRM transitions optimized for fomivirsen and internal standard.

Key Results and Discussion

Calibration and Sensitivity:

• Microflow LC-MS/MS achieved a lower limit of quantification of 0.1 ng/mL, compared to 1 ng/mL with high-flow.
• Average 5-fold improvement in signal-to-noise ratio using microflow conditions.

Precision and Accuracy:

• Across eight calibration points (0.1–1,000 ng/mL), intra- and inter-day CVs were <12%, and accuracy ranged from 94–108%.
• Recovery for the sample preparation protocol averaged ~80%.

Robustness:

• Reduced consumption of ion-pairing reagents enhanced long-term instrument stability.
• Minimal re-optimization required when switching between flow regimes.

Benefits and Practical Applications

• Enables quantification of antisense oligonucleotides at sub-nanogram per milliliter levels directly in plasma.
• Streamlines bioanalytical workflows by leveraging a single mass spectrometer platform for both high-flow and microflow analyses.
• Supports drug development programs by providing reliable PK data, with potential extension to other oligonucleotide modalities.

Future Trends and Opportunities

Ongoing advancements in ion source design, microfluidic chromatography, and multiplexed MRM methods will further lower detection limits and increase throughput. Integration with automated sample preparation and artificial intelligence–driven data processing can accelerate method development. Moreover, applying similar workflows to emerging modalities such as siRNA, mRNA, and CRISPR guide RNAs will broaden the impact of microflow LC-MS in pharmaceutical research.

Conclusion

The combination of the QTRAP 6500+ system with OptiFlow and M5 MicroLC delivers an ultra-sensitive, robust, and versatile platform for oligonucleotide quantification in complex matrices. The demonstrated 5-fold S/N improvement and sub-nanogram detection capability mark a significant advancement over conventional high-flow LC-MS assays, aligning mass spectrometry performance with hybridization-based techniques while retaining the benefits of MS specificity.

Reference

Jiang J., McCarthy S., Candish E., Xiong L. Improve Sensitivity for Quantification of Antisense Oligonucleotides in Plasma Using Microflow LC-MRM Methodology. SCIEX Application Note RUO-MKT-10-12584-A (2020).