BIOANALYTICAL QUANTIFICATION OF A LIPID CONJUGATED ANTI-SENSE OLIGONUCLEOTIDE ON A HRMS SYSTEM

Importance of the Topic

Oligonucleotide therapeutics have emerged as a transformative modality in drug development, offering precise modulation of gene expression with high target specificity and minimal off-target effects. As these therapies progress through discovery and development, robust bioanalytical workflows are essential to quantify drug levels in biological matrices, support pharmacokinetic and ADME studies, and ensure regulatory compliance. High-resolution mass spectrometry combined with standardized sample preparation platforms can streamline analysis of a broad range of oligonucleotides, reducing method development time and enhancing data quality.

Study Objectives and Overview

This work presents a streamlined workflow for the extraction and quantification of a lipid-conjugated antisense oligonucleotide (ASO) from rat plasma. Key goals include:

• Evaluating the performance of the OligoWorks SPE Microplate Kit for sample cleanup and recovery.
• Optimizing solid-phase extraction parameters to improve analyte yield and minimize matrix effects.
• Implementing an untargeted LC-HRMS acquisition method on the Xevo G3 QToF to enable sensitive and accurate quantification across a wide concentration range.

Methodology and Instrumentation

An optimized solid-phase extraction (SPE) protocol was developed using a mixed-mode OligoWorks WAX sorbent in a 96-well format. Critical steps included adjusting the organic content of the second wash solution to balance recovery of the lipid-conjugated ASO and the internal standard.

• Sample preparation: OligoWorks SPE Microplate Kit, rat plasma spiked with lipid-ASO, protocol adaptation from the Care & Use Manual.
• Chromatography: ACQUITY Premier Oligonucleotide BEH C18 column (2.1×50 mm, 1.7 µm), 55 °C, mobile phases containing hexafluoro-2-propanol and diisopropylethylamine.
• Mass spectrometry: Waters Xevo G3 QToF MS, ESI negative mode, full-scan m/z 50–2000, data captured in UNIFI software.
• Data processing: Evaluation of extracted ion chromatogram (XIC) windows (0.7–0.01 Da) to maximize signal-to-noise, with 0.025 Da chosen for routine quantification.

Main Results and Discussion

The optimized SPE conditions yielded recoveries of approximately 80% for the lipid-ASO and 60% for the internal standard, with matrix effects below 15%. Tightening the XIC window effectively reduced background interference, enhancing assay selectivity without compromising analyte signal.

• A linear calibration curve was achieved over 1–1000 pmol/mL (r2 = 0.991) using 1/x weighting.
• Accuracy across calibration points ranged from 87.8% to 114.2%, and precision (%CV) was 1.29%–10.61%.
• Quality control samples at low, mid, and high concentrations demonstrated mean accuracies of 98.2%, 113.2%, and 106.2%, with %CV below 4%.

Benefits and Practical Applications

This generic workflow offers several advantages for bioanalytical laboratories:

• Standardized SPE protocol reduces individual method development efforts and ensures reproducible recoveries across diverse oligonucleotide chemistries.
• Untargeted HRMS acquisition facilitates simultaneous characterization and quantification without custom transitions, accelerating project timelines.
• High resolving power enables retrospective data mining for metabolites and structural variants, supporting downstream ADME and safety studies.

Future Trends and Applications

Looking ahead, coupling standardized sample preparation with advanced HRMS platforms can further improve throughput and data richness. Emerging mass analyzers with enhanced sensitivity and resolution, along with automated data analytics, will enable comprehensive profiling of oligonucleotide therapeutics and their metabolites in a single run. Integration with microfluidic sample handling and artificial intelligence–driven interpretation will continue to streamline bioanalysis in discovery and clinical settings.

Conclusion

The combination of the OligoWorks SPE Microplate Kit and a generic LC-HRMS method on the Xevo G3 QToF provides a robust, sensitive, and high-throughput workflow for quantifying lipid-conjugated ASOs in plasma. This approach meets regulatory guidelines for bioanalytical method validation and supports broad application across therapeutic oligonucleotide programs.

Reference

1. OligoWorks SPE Kits and Components—Care and Use Manual
2. Quantification of a Lipid Conjugated Antisense Oligonucleotide Extracted From Rat Plasma Using the OligoWorks SPE Microplate Kit on a HRMS System
3. An Automated, Standardized, Kit-Based Sample Preparation Workflow for Bioanalytical Quantification of Therapeutic Oligonucleotides
4. Development of a Standardized, Kit-Based Approach for Selective and Reproducible Sample Preparation and Extraction for Therapeutic Oligonucleotides from Biological Matrices