Quantification of a Lipid Conjugated Antisense Oligonucleotide (ASO) Extracted From Rat Plasma Using the OligoWorks™ SPE Microplate Kit on a HRMS System

Significance of the Topic

Oligonucleotide therapeutics are a growing class of drugs that target gene expression with high specificity and low toxicity. Quantifying lipid conjugated antisense oligonucleotides in plasma is critical for evaluating pharmacokinetics and supporting drug discovery. Robust sample preparation and high resolution mass spectrometry enable accurate bioanalysis under tight timelines in discovery laboratories.

Objectives and Study Overview

This study aimed to develop a standardized workflow for extracting a lipid conjugated antisense oligonucleotide from rat plasma using a microplate solid phase extraction kit and to demonstrate a generic high resolution mass spectrometry method for quantification. Emphasis was placed on minimal method development and high recovery across concentration ranges.

Methodology and Instrumentation

• Sample preparation employed proteinase K digestion and a mixed mode weak anion exchange reversed phase microplate kit for solid phase extraction
• Wash steps were optimized with ammonium acetate buffer at pH 5.5 and methanol water mixtures to maximize recovery
• Liquid chromatography used an ultraperformance system with an oligonucleotide C18 column at 55 degrees Celsius, mobile phases of HFIP and DIPEA in water and acetonitrile water, and a gradient from 25 to 75 percent organic in 3.25 minutes
• High resolution mass spectrometry was performed on a quadrupole time of flight instrument in negative electrospray ionization full scan mode with optimized desolvation conditions
• Data acquisition and processing used a unified software platform with narrow mass extraction windows

Key Findings and Discussion

• Initial recoveries reached approximately 80 percent for the analyte and 60 percent for the internal standard, with matrix effects below 15 percent
• Reducing methanol in the second wash improved analyte recovery but affected the internal standard, demonstrating the impact of organic composition on sorbent interactions
• Optimizing the liquid chromatography gradient centered the analyte peak and improved throughput
• Narrowing the extracted ion chromatogram window to 0.025 Da enhanced signal to noise without compromising peak area
• The method was linear from 1 to 1000 pmol per milliliter with a correlation coefficient of 0.991 and met accuracy and precision criteria from low to high quality control levels

Benefits and Practical Applications

• Standardized detergent free kit reduces development time and ensures reproducibility
• Generic full scan high resolution mass spectrometry can be applied to diverse oligonucleotides without additional tuning
• High throughput workflow supports rapid decision making in discovery bioanalysis
• Rich data sets enable retrospective investigation of metabolites and ADME profiles

Future Trends and Opportunities

Analytical workflows may evolve to include automation and microfluidic extraction formats, expanded support for complex oligonucleotide chemistries and modifications, and integration of artificial intelligence driven data analysis for pattern recognition and metabolite screening. High resolution mass spectrometry will continue to play a central role in untargeted discovery and quantitative studies.

Conclusion

A streamlined workflow combining a microplate solid phase extraction kit and generic high resolution mass spectrometry provides sensitive accurate quantification of lipid conjugated antisense oligonucleotides from plasma. This approach meets regulatory bioanalytical criteria and enhances productivity in discovery laboratories.

Reference

1. N Tanna M Trudeau Quantification of a Lipid Conjugated Antisense Oligonucleotide Extracted From Rat Plasma Using the OligoWorks SPE Microplate Kit on a HRMS System Application Note Waters Corporation February 2024