DEVELOPMENT OF A BIOANALYTICAL SPE LC-MS/MS ASSAY FOR OLIGONUCLEOTIDES

Significance of the Topic

Oligonucleotide therapeutics have emerged as a pivotal class of biomolecules, driving the need for highly sensitive and selective bioanalytical assays. Their unique size, charge and structural diversity pose significant challenges in sample preparation, chromatographic retention and mass spectrometric detection. Addressing these hurdles is essential for accurate pharmacokinetic profiling and quality control in both research and clinical settings.

Objectives and Study Overview

This work aims to develop and optimize a straightforward solid‐phase extraction (SPE) and LC‐MS/MS workflow for the quantification of oligonucleotide standards (15–35 mer) and a fully thioated 50‐mer (GEM91). The study evaluates reversed‐phase (RP) and mixed‐mode SPE protocols, sub‐2 µm RP C18 chromatography, and tandem quadrupole mass spectrometry to achieve high recovery, sensitivity and reproducibility.

Methodology and Instrumentation

The sample cleanup employed Waters Oasis HLB and mixed‐mode WAX µElution 96‐well plates. Key parameters—loading, washing and elution solvents—were systematically varied to maximize recovery and minimize matrix effects. Chromatographic separation used a prototype 2.1 × 50 mm sub‐2 µm RP C18 column with a 5 min gradient of hexafluoroisopropanol/hexylamine in water and methanol. Detection was carried out on a Waters ACQUITY UPLC I-Class PLUS coupled to a Xevo TQ-XS operating in negative electrospray MRM mode.

Main Results and Discussion

Recovery experiments showed that HLB SPE with HFIP/TEA wash and TEA/ACN elution yielded highest extraction efficiencies for 15–35 mer oligos, while WAX SPE performed best with ammonium acetate/MeOH wash and MeOH/TEA elution. System suitability checks demonstrated stable retention times and peak areas over 96 injections. Chromatographic performance improved on the prototype sub-2 µm column, increasing peak heights and overall sensitivity. The mixed-mode WAX protocol enabled linear and accurate quantification of GEM91 down to 25 ng/mL in plasma.

Benefits and Practical Applications

• The μElution SPE format eliminates evaporation steps, reducing losses from nonspecific binding.
• Optimized SPE protocols afford high recoveries across a range of oligonucleotide lengths.
• Fast UPLC gradient and robust MRM detection support high‐throughput bioanalysis.
• Achieved low limits of quantification suitable for preclinical and clinical PK studies.

Future Trends and Applications

Continued miniaturization of SPE devices and further refinement of stationary phases are expected to improve throughput and sensitivity. Integration with high‐resolution mass spectrometry and automation will support multiplexed oligonucleotide assays. Emerging ionization enhancers and novel chromatographic chemistries may further reduce matrix interferences and extend capabilities to modified oligonucleotides.

Conclusion

A streamlined SPE‐LC‐MS/MS method has been established for reliable quantification of both standard and thioated oligonucleotides. The combined use of RP and mixed‐mode SPE in μElution format, paired with sub-2 µm LC separation and sensitive MRM detection, delivers robust recoveries, low detection limits and high reproducibility, meeting the stringent demands of modern oligonucleotide bioanalysis.

Instrumentation Used

• Waters Oasis HLB and WAX μElution 96‐well SPE plates
• Waters ACQUITY UPLC I-Class PLUS (FTN)
• Prototype sub-2 µm, 2.1 × 50 mm RP C18 column
• Waters Xevo TQ-XS tandem quadrupole MS

References

• Lame M., Brennan K., Donegan M., Paxton T., Edwards I. Development of a Bioanalytical SPE LC-MS/MS Assay for Oligonucleotides. Waters Corporation Poster MKT20013, 2020.