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

Significance of the topic

Reliable quality control of synthetic oligonucleotides is essential for applications in molecular diagnostics, therapeutics development, and research laboratories. As demand for custom DNA arrays grows, methods that combine high throughput, minimal sample preparation, and confident identification of intact sequences become increasingly valuable.

Objectives and study overview

This work demonstrates a step-by-step workflow for intact mass analysis, deconvolution, and reporting of oligonucleotide synthesis quality using a single quadrupole mass spectrometer. The goal is to enable rapid pass/fail confirmation of sequences ranging from 10 to 60 nucleotides without post-synthesis purification.

Methodology and instrumentation

Samples collected directly from a DNA synthesizer in 96-well plate format were injected neat onto a reversed-phase ion-pairing LC column. Chromatographic separation was performed on a Thermo Scientific DNAPac RP column using a Vanquish Flex UHPLC system with UV detection. The mobile phase incorporated hexafluoro-2-propanol (HFIP) and triethylamine to facilitate negative-mode electrospray ionization. Mass spectra were acquired on a Thermo Scientific ISQ EM single quadrupole instrument under negative polarity. Key source parameters (vaporizer and transfer tube temperatures, sheath and auxiliary gas pressures, spray voltage) were systematically tuned to maximize the most abundant charge state. HFIP concentration was optimized between 0.01 and 2 percent to balance signal intensity and adduct formation.

Used instrumentation

• Thermo Scientific Vanquish Flex Binary UHPLC System with Variable Wavelength Detector
• Thermo Scientific DNAPac RP column (2.1 by 50 mm, 4 µm)
• Thermo Scientific ISQ EM Single Quadrupole Mass Spectrometer
• Chromeleon 7.3 Chromatography Data System with Intact Protein Deconvolution module

Key results and discussion

Optimal MS source settings (vaporizer 350 °C, transfer tube 350 °C, sheath gas 75 psig, auxiliary gas 7.5 psig, spray voltage -3000 V) provided strong signal for the highest charge states. An HFIP concentration of 0.1 % delivered the best compromise between maximum spectral intensity and minimal adduct formation, reducing HFIP usage by a factor of twenty compared to the industry standard 2 %. Automated deconvolution parameters yielded clear pass/fail assessments against expected masses, even in a fully unattended sequence report.

Benefits and practical applications of the method

• No desalting or sample cleanup required—direct injection from synthesis plate
• Significant cost savings in HFIP consumption and reduced reagent waste
• Reliable intact mass confirmation for oligonucleotides from 10 to 60 bases
• User-friendly pass/fail reporting integrated into the chromatography data system

Future trends and potential applications

Emerging opportunities include coupling this workflow with high-resolution mass analyzers for improved sequence verification, integrating machine learning algorithms for enhanced deconvolution accuracy, implementing real-time monitoring of synthesis in automated platforms, and extending the approach to chemically modified or longer oligonucleotides for therapeutic applications.

Conclusion

This study delivers a robust, cost-effective LC-MS workflow for high-throughput quality control of synthetic oligonucleotides. By combining optimized ion-pairing chromatography, targeted source tuning, and automated deconvolution, laboratories can achieve rapid, reliable sequence confirmation with minimal sample handling.