High-throughput Mass Spectrometry Analysis of Synthetic Oligonucleotides

Significance of the topic

The rapid growth of synthetic oligonucleotide applications in research and therapeutics demands analytical methods that combine speed with reliable desalting and characterization. High-throughput mass spectrometry workflows address these needs by minimizing run times while maintaining data quality.

Study objectives and overview

This work compares two high-throughput approaches for oligonucleotide analysis: a Fast LC–Q-TOF method using an Agilent 1290 Infinity II system and a RapidFire 400–Q-TOF approach. Both workflows were optimized for 18-mer oligos and evaluated across DNA and RNA sequences from 18 to 100 nucleotides.

Methodology

Fast LC: Dual-needle, smart-overlap injections on a guard column deliver a 0.6-minute gradient and desalting at 1.75 mL/min, acquiring 10 spectra/s.

RapidFire: A 4 µL PLRP-S cartridge executes a five-state cycle (aspirate, wash, elute, re-equilibrate) in ~13 s, with MS data collected continuously and parsed postacquisition at 4 spectra/s.

Used instrumentation

• Agilent 1290 Infinity II Binary Pump with Dual-Needle Multisampler
• AdvanceBio Oligo UHPLC Guard Column (2.1 × 5 mm, 1.7 µm)
• Agilent RapidFire 400 high-throughput MS system with PLRP-S cartridge
• Agilent 6545 LC/Q-TOF Mass Spectrometer
• MassHunter Bioconfirm B07 software for deconvolution and analysis

Main results and discussion

• Throughput: RapidFire achieved ~15 s/sample (≈5 760/day); Fast LC required ~40 s/sample (≈2 160/day).
• Desalting efficiency: RapidFire reduced sodium and potassium adducts 2–3× more effectively than Fast LC over 18–100 mer lengths.
• Signal intensity: Fast LC produced lower overall peak intensity (25–80% relative) due to higher flow and narrower peaks but enabled partial chromatographic separation.
• Reproducibility: Both methods showed stable pump pressures and consistent retention profiles, with Fast LC retention times varying by ~7 s across oligo sizes and RapidFire eluting all sizes simultaneously.
• Impurity profiling: RapidFire deconvolution resolved low-abundance truncations, depurination/depyrimidation products, and cation adducts down to ~0.5% relative area.

Benefits and practical applications

RapidFire offers ultrahigh throughput for large-scale QC, purity assessment, and impurity profiling with minimal chromatographic method development. Fast LC provides added separation to simplify mixture analysis and can be tuned to balance resolution and speed.

Future trends and potential applications

Oligonucleotide therapeutics growth will drive integration of rapid desalting platforms with automated sample handling and real-time data processing. Advances in cartridge chemistries, accelerated UHPLC gradients, high-speed detectors, and AI-driven deconvolution promise even faster cycle times, improved separation, and deeper impurity insights.

Conclusion

Both RapidFire and Fast LC high-throughput MS methods deliver robust, reproducible results for synthetic oligonucleotides. RapidFire excels in speed and impurity detection, while Fast LC adds separation for complex samples. These complementary workflows meet the accelerating analytical demands of oligonucleotide development and QC.