Improved Oligonucleotide SPE-LC-MS Analysis Using MaxPeak High Performance Technology

Importance of the topic

The development of next-generation oligonucleotide therapies requires robust bioanalytical methods capable of high sensitivity and selectivity in complex matrices such as plasma and sera

Objectives and study overview

This work describes a unified SPE-LC-MS/MS method to extract, separate, detect, and quantify unmodified oligodeoxythymidines (15–35 mer) and the phosphorothioated antisense oligonucleotide GEM91

Methodology and sample preparation

A mixed-mode sample cleanup was implemented using Waters Oasis HLB and WAX μElution 96-well SPE plates

• Neat solutions: direct SPE on WAX μElution plates in 50 mM ammonium acetate, pH 5.5
• Plasma/sera: phenol-chloroform liquid-liquid extraction (LLE) followed by WAX SPE to disrupt protein binding and remove interferences

Chromatography was performed on ACQUITY PREMIER Oligonucleotide C18, 1.7 μm, 2.1 × 50 mm column, at 50 °C, 0.6 mL/min, using HFIP/hexylamine aqueous and methanolic mobile phases

Used instrumentation

ACQUITY UPLC I-Class PLUS with FTN and single column heater
Xevo TQ-XS tandem quadrupole mass spectrometer (ESI-, MRM mode)
MassLynx v4.2 and TargetLynx software for data acquisition and quantification

Main results and discussion

Application of MaxPeak HPS technology minimized metal interactions, greatly improving out-of-the-box analyte recovery and reducing column passivation

• SPE recoveries exceeded 80% for both OST standards and GEM91 after LLE-WAX cleanup
• LLOQs of 0.0025 nmol/mL for OST 15-mer and 50 ng/mL for GEM91 in neat and extracted samples
• LOD of 2.5 ng/mL for GEM91
• Stable retention times and peak areas over 96 injections confirmed system robustness

Benefits and practical applications

The streamlined SPE protocol with μElution format eliminated evaporation steps, minimizing adsorption losses and improving throughput
High sensitivity and selectivity support pharmacokinetic and bioavailability studies of oligonucleotide therapeutics in drug development and quality control

Future trends and opportunities

Integration of surface-passivated hardware will expand analysis to other phosphorous-rich biomolecules such as phosphopeptides and small organophosphates
Automation of LLE-SPE workflows and coupling with high-resolution MS will further enhance sensitivity, specificity, and sample throughput

Conclusion

The presented SPE-LC-MS/MS method leverages MaxPeak HPS technology and mixed-mode sample preparation to achieve high recovery, low limits of quantification, and robust performance for unmodified and phosphorothioated oligonucleotides

References

1. GEM91 (Trecovirsen) oligonucleotide structure image, accessed September 2020