Sensitive Quantification of Therapeutic Oligonucleotides Extracted from Plasma

Importance of the Topic

Advances in antisense oligonucleotides (ASO) and siRNA therapies are unlocking new treatment options for previously undruggable targets. Sensitive and specific bioanalytical methods are essential to track pharmacokinetics, metabolism, impurities and ensure safety and efficacy in clinical and preclinical studies.

Objectives and Study Overview

This study aimed to develop and validate a robust, high-sensitivity LC-MS/MS assay for quantifying a fully phosphorothioated 20-mer ASO in human plasma. The method leverages the SCIEX QTRAP 6500+ system to achieve a low limit of quantification and broad dynamic range.

Materials and Methods

Sample Preparation

• Double liquid–liquid extraction using phenol:chloroform:isoamyl alcohol and chloroform
• Addition of Tris-HCl, EDTA and ammonium hydroxide for protein removal
• Drying and reconstitution in water with EDTA

Chromatography

• Agilent 1290 Infinity UHPLC with Phenomenex Oligo-XT column (2.1×50 mm, 2.6 μm)
• Mobile phases: 15 mM DIEA/100 mM HFIP in water and in methanol
• Gradient from 15% to 90% organic, 300 μL/min flow, column at 60 °C

Mass Spectrometry

• QTRAP 6500+ with IonDrive Turbo V source operated in negative mode
• Source gases and voltages optimized for minimal in-source degradation
• Information dependent acquisition using EMS to select precursors and EPI for product ion identification
• MRM transitions optimized for the −10 and −9 charge states

Data Processing

• Analyst and SCIEX OS-Q Software for method development and quantification
• Linear regression with 1/x2 weighting for calibration

Used Instrumentation

• SCIEX QTRAP 6500+ LC-MS/MS system with IonDrive QJet Ion Guide and linear ion trap functionality
• Agilent 1290 Infinity UHPLC
• Phenomenex Clarity Oligo-XT column

Main Results and Discussion

• Charge states from −4 to −11 were observed; the −10 and −9 charge states provided the highest signal for MRM selection.
• Enhanced product ion (EPI) scans delivered 6- to 15-fold improvements in sensitivity and superior resolution compared to standard product ion scans.
• Sodium adduct formation was limited (~2%), indicating effective system cleanliness.
• Three MRM transitions (716.6→392.1, 716.6→319.1, 716.6→733.3 for −10 state) were chosen following collision energy optimization.
• Calibration range of 0.2–1000 ng/mL achieved across 3.5 orders of magnitude (r = 0.995).
• LLOQ of 0.2 ng/mL demonstrated accuracy within ±15% and precision below 20%.

Benefits and Practical Applications

• Offers superior specificity over hybridization assays by distinguishing full-length oligonucleotide, metabolites and impurities
• Broad dynamic range accommodates low-level exposure studies and high-dose pharmacokinetics
• Rapid on-column optimization accelerates method development in support of drug discovery and preclinical research

Future Trends and Potential Applications

• Integration of ultra-high resolution MS and nano-flow chromatography for even lower LLOQs
• Implementation of automated sample prep and high-throughput workflows
• Expansion to multiplexed quantification of multiple oligonucleotides and their metabolites
• Application of machine learning for deeper spectral deconvolution and adduct prediction

Conclusion

The validated QTRAP 6500+ LC-MS/MS assay delivers a robust, sensitive and specific platform for ASO quantification in plasma, achieving sub-nanogram limits and a wide dynamic range. This approach supports the growing demand for precise bioanalysis of oligonucleotide therapeutics.

References

1. Iwamoto N et al. Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides. Nat Biotechnol. 2017;35(9):845-851.