Improved Bioanalytical Extraction of Therapeutic Antisense Oligonucleotides, Including a Lipid Conjugated ASO From Liver and Brain Tissue Using the OligoWorks™ SPE Microplate Kit

Significance of the Topic

Oligonucleotide therapeutics are emerging tools for addressing genetic diseases, cancers, and viral infections through targeted modulation of gene expression. Accurate quantification of these molecules in tissues is essential for drug metabolism and pharmacokinetic studies, yet tissue complexity and chemical modifications of oligonucleotides present significant analytical challenges.

Objectives and Study Overview

This application note describes an optimized bioanalytical workflow using a proteinase K facilitated digestion and mixed-mode weak anion exchange SPE in a 96-well microplate format to achieve high recovery and reproducibility of phosphorothioated and lipid-conjugated antisense oligonucleotides from liver and brain tissues.

Methodology and Used Instrumentation

The sample preparation protocol comprises:

• Spiking 50 mg of tissue with oligonucleotide standards (GEM 91, GEM 132, lipid-conjugated ASO) in ammonium acetate buffer
• Solvent-assisted homogenization with acetonitrile, Tris-HCl, guanidine HCl, TCEP and RapiZyme proteinase K digestion
• Optional addition of 1–2.5 % NP-40 alternative surfactant for enhanced lipid-conjugated ASO recovery
• Mixed-mode WAX SPE using the OligoWorks SPE Microplate Kit with optimized wash and elution steps
• Direct injection of the MS-compatible eluate into LC-MS/MS without drying and reconstitution

Used instrumentation:

• UPLC system: ACQUITY UPLC H-Class PLUS with Quaternary Solvent Manager and Flow-Through Needle sample manager
• Column: ACQUITY Premier Oligonucleotide C18, 130 Å, 1.7 µm, 2.1×50 mm held at 55 °C
• Mobile phases: 1% HFIP/0.1% DIPEA in water and 0.75% HFIP/0.0375% DIPEA in 65% acetonitrile
• Mass spectrometer: Xevo TQ-Absolute triple quadrupole with electrospray ionization in MRM mode

Main Results and Discussion

Recoveries for GEM 91, GEM 132 and lipid-conjugated ASO exceeded 80% with standard deviations below 10% and calibration linearities above 0.98 in liver and brain matrices. The incorporation of NP-40 alternative surfactant raised lipid-conjugated ASO recovery to 100% in both tissue types. Matrix effects remained low, and no internal standard was required. The workflow supports high throughput analysis via a short LC gradient and direct SPE eluate injection.

Benefits and Practical Applications

• High and reproducible oligonucleotide recoveries from complex tissues (≥80%, SD ≤10%)
• Efficient removal of proteins, lipids and surfactants through mixed-mode SPE
• Concentrated, MS-compatible eluate suitable for direct LC-MS/MS injection
• Scalable 96-well format enabling high throughput drug metabolism and pharmacokinetic assays

Future Trends and Applications

Advances may include integration of isotopically labeled internal standards for even greater accuracy, automation of the SPE workflow, application to newer oligonucleotide modalities such as siRNA and gapmers, and exploration of alternative surfactants or enzymatic modules for challenging matrices.

Conclusion

The described kit-based workflow combining proteinase K digestion, optional NP-40 alternative surfactant, and mixed-mode SPE delivers robust, high-recovery extraction of therapeutic antisense oligonucleotides from liver and brain tissues. This streamlined approach supports sensitive, high-throughput LC-MS/MS quantification critical to drug discovery and clinical research.

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