Analysis of Synthetic DNA

Significance of the Topic

Synthetic oligonucleotides play a growing role in pharmaceuticals, diagnostics, and gene therapy. Reliable analytical methods are essential to confirm sequence integrity, assess purity, and detect process-related impurities. Reversed-phase liquid chromatography coupled with mass spectrometry (LC–MS) offers high sensitivity and specificity for oligonucleotide characterization, making it indispensable in quality control and research laboratories.

Objectives and Study Overview

This application report demonstrates a streamlined LC–MS workflow using the Shim-pack Scepter™ Claris C18-120 column and LCMS-2050 system for the separation and detection of synthetic DNA oligonucleotides. The study aims to optimize chromatographic conditions, evaluate mass spectrometric parameters in negative ion mode, and illustrate the method’s suitability for routine analysis of oligonucleotide pharmaceuticals.

Methodology and Instrumentation

The analytical method combines high-efficiency reversed-phase chromatography with electrospray ionization mass spectrometry. Key conditions include:

• Column: Shim-pack Scepter Claris C18-120 (100 mm × 2.1 mm, 1.9 µm particle size)
• Mobile Phases: A – 95.4 mM HFIP/7.1 mM TEA in water; B – same additives in methanol
• Gradient: 5% B initial, linear increase to 35% at 15 min, ramp to 80% at 16–17 min, return to 5% by 25 min
• Flow Rate: 0.3 mL/min; Column Temperature: 50 °C; Injection Volume: 2.33 µL (10 pmol)
• Detection: Photodiode array monitoring 200–400 nm; MS: ESI/APCI negative mode, m/z 600–2000

Main Results and Discussion

The method achieved sharp, well-resolved peaks for target oligonucleotides within a 25-minute runtime. The ion-pairing reagents HFIP and TEA enhanced retention and peak shape. Mass spectra displayed clean deprotonated molecular ions with minimal adduct formation. System reproducibility and sensitivity were sufficient for low-picomole quantitation, supporting accurate identification and purity assessment.

Benefits and Practical Applications

• High-resolution separation of closely related oligonucleotide sequences
• Robust detection in negative ion mode for improved signal-to-noise ratio
• Rapid method suitable for routine QC of therapeutic oligonucleotides
• Minimal sample preparation with direct injection of aqueous samples

Future Trends and Opportunities

Advances in ultra-high–pressure liquid chromatography and high-resolution mass spectrometry promise even faster, more detailed oligonucleotide profiling. Integration with automated sample handling and MS/MS fragmentation will enable deeper impurity characterization and sequence confirmation. Emerging ion-pair alternatives and microflow technologies may further reduce analysis time and solvent consumption.

Conclusion

This application demonstrates an effective LC–MS platform for synthetic DNA analysis, combining Shim-pack Scepter™ Claris C18 column efficiency with sensitive MS detection. The optimized method delivers rapid, reliable separation and characterization of oligonucleotides, meeting the demands of pharmaceutical development and quality control.

References

• Shimadzu Corporation. Application News 01-00662 (JP, ENG), 2024.
• Shim-pack Scepter Claris C18-120, Nexera™ XS inert, LCMS-2050 brochure, Shimadzu Corporation, First Edition: Sep. 2024.