IMPROVED TISSUE HOMOGENIZATION AND SPE-BASED SAMPLE PREPARATIONS FOR THE QUANTITATIVE LC-MS ANALYSIS OF OLIGONUCLEOTIDE THERAPEUTICS

Importance of the Topic

The accurate quantitation of oligonucleotide therapeutics in tissue samples is vital for drug metabolism and pharmacokinetic studies. Modifications and conjugations on oligonucleotides pose challenges for extraction and reproducible analysis. Optimized sample preparation enhances sensitivity and throughput in bioanalysis.

Study Objectives and Overview

This work aimed to develop robust protocols for tissue homogenization and solid phase extraction based quantitation of antisense oligonucleotides including lipid conjugates. Four model oligonucleotides were spiked into bovine liver and porcine brain to evaluate recovery, matrix interference, and reproducibility.

Materials and Methods

The protocol integrates bead beating tissue homogenization followed by enzymatic digestion using high purity recombinant proteinase K. Organic solvents were tested to assist protein precipitation and analyte solubilization. A weak anion exchange microplate format SPE device was employed for selective adsorption, wash, and elution of oligonucleotides. Chromatography was performed on UHPLC using a BEH C18 low adsorption column (2.1 by 50 millimeters, 1.7 micrometer, 130 angstrom). Detection was achieved with a benchtop time of flight mass spectrometer and a triple quadrupole instrument in MRM mode. Method parameters were optimized to minimize matrix effects.

Results and Discussion

The optimized organic solvent assisted pretreatment enabled efficient recovery of unmodified and conjugated antisense oligonucleotides. The inclusion of a non ionic detergent alternative significantly improved the recovery of lipid linked sequences. The microplate SPE format achieved high concentration factors, permitting quantitative analysis from minimal tissue mass. Recoveries were independent of sample amount and exhibited minimal matrix interference. Data repeatability was demonstrated across multiple sorbent batches and with or without internal standard.

Instrumentation

• UHPLC system equipped with BEH C18 column
• Benchtop time of flight mass spectrometer
• Triple quadrupole mass spectrometer
• Proteinase K digestion system
• Weak anion exchange microplate SPE device

Practical Applications and Benefits

The protocol enables direct injection LC MS quantitation of antisense oligonucleotides from diverse tissue matrices with high accuracy and throughput. Eliminating drying and reconstitution steps reduces sample loss and preparation time. The method supports both full scan and targeted MRM analyses, facilitating DMPK, QA QC, and industrial bioanalysis applications.

Future Trends and Potential Applications

Advances may include automation of kit based workflows, integration with high resolution mass spectrometry, and expanded sorbent chemistries for modified nucleic acids. Multiplexed quantitation and predictive modeling in silico are emerging to streamline assay development for novel oligonucleotide therapeutics.

Conclusion

The developed tissue homogenization and SPE based sample preparation protocols deliver robust, reproducible, and efficient quantitation of antisense oligonucleotides in tissue. The streamlined workflow and minimal sample requirements enhance confidence in LC MS bioanalysis for therapeutic evaluation.

References

• Araya M et al 2023 Elements of Robust SPE Based Oligonucleotide Extraction 720008141
• Lee M Tanna N Trudeau M 2023 Development of a Standardized Kit Based Approach for Selective and Reproducible Sample Preparation and Extraction for Therapeutic Oligonucleotides from Biological Matrices 720008086
• Tanna N Trudeau M Lee M 2023 An Automated Standardized Kit Based Sample Preparation Workflow for Bioanalytical Quantification of Therapeutic Oligonucleotides 720008068
• Tanna N Trudeau M 2024 Quantification of a Lipid Conjugated Antisense Oligonucleotide Extracted From Rat Plasma Using the OligoWorks SPE Microplate Kit on a HRMS System 720008256
• Tran KV Trudeau M Lauber M 2018 High Oligonucleotide Recovery From Liver Tissue 720008270
• Waters Corporation 2024 OligoWorks Kits Application