High-throughput analysis of oligonucleotides using a single quadrupole mass spectrometer for quality control

Significance of the topic

High throughput quality control of synthetic oligonucleotides is essential for laboratories producing custom DNA arrays. Rapid intact mass determination helps ensure correct sequence synthesis, reduces reagent waste and supports automated workflows in research and industrial settings.

Objectives and Study Overview

This study demonstrates a complete step by step workflow for assessing oligonucleotide synthesis quality using a single quadrupole mass spectrometer. Key aims include optimization of chromatographic conditions, source parameters, spectral deconvolution and automated pass fail reporting.

Methodology

• Samples collected directly from DNA synthesizer in 96 well plates and injected without desalting or further preparation
• Reversed phase ion pairing chromatography on Thermo Scientific DNAPac RP column with gradient elution using HFIP and TEA mobile phases
• Screening HFIP concentrations from 0.01 to 2.0 % to maximize signal and minimize adduct formation
• Sequential tuning of source parameters: vaporizer temperature, transfer tube temperature, sheath gas and auxiliary gas pressures, spray voltage
• Data acquired and processed in Chromeleon CDS with intact protein deconvolution engine configured for oligonucleotide mass deconvolution

Used Instrumentation

• Vanquish Flex UHPLC system with DNAPac RP 2.1 x 50 mm column
• ISQ EM Single Quadrupole Mass Spectrometer with HESI source
• Chromeleon 7.3 Chromatography Data System with Intact Protein Deconvolution module

Main Results and Discussion

• Optimal HFIP concentration identified at 0.1 % gives highest peak area and lowest adduct formation, reducing solvent use by twenty fold
• Source settings of 350 °C vaporizer and transfer tube temperature, 75 psig sheath gas, 7.5 psig auxiliary gas and 3000 V spray voltage provided best spectral quality
• Intact mass deconvolution reliably detected oligonucleotides from 10 to 60 bases and provided clear pass fail assessment versus expected masses
• Chromatograms confirmed removal of residual reagents and clean single peak elution of target oligomers

Benefits and Practical Applications

• Substantial cost savings in HFIP consumption for daily high throughput workflows
• Elimination of sample cleanup steps accelerates turnaround time
• Automated deconvolution and customized injection variables enable real time pass fail reporting
• Applicable to quality control of diverse oligonucleotide lengths with minimal method adjustments

Future Trends and Potential Applications

Advances may include integration of higher resolution mass analyzers to resolve isobaric variants, further reduction of ion pairing agent usage, machine learning algorithms for spectral interpretation and fully automated end to end robotic synthesis to reporting platforms. Expanded applications could cover RNA aptamers, modified nucleic acids and emerging therapeutic oligonucleotides.

Conclusion

This work establishes a robust high throughput LC MS workflow for intact oligonucleotide quality control using a single quadrupole detector. Optimized chromatographic and source conditions along with automated deconvolution deliver rapid, cost effective and reliable pass fail results for synthesized DNA sequences.