Revolutionizing Oligonucleotide Synthesis: Streamlined Process Monitoring Enters a New Era

Oligonucleotides have become indispensable tools in molecular biology, diagnostics, and therapeutic development. Synthetically produced oligonucleotides are becoming increasingly complex due to chemical modifications, increased length, and structural diversity, making the demand for analytical techniques that are rapid, accurate, and require minimal or no complicated sample preparation all the more urgent. While traditional analytical methods remain effective, they typically necessitate extensive sample purification or matrix optimization, rendering them time-consuming and less suitable for high-throughput screening or process monitoring.

To address these limitations, AIS OPSI’s open sampling port is coupled with CMS mass spectrometry, providing a powerful solution for the direct, rapid, and atmospheric-pressure analysis of oligonucleotides. This innovative technology combines a novel sampling interface with a mass spectrometer, enabling real-time analysis of samples even when the sample preparation volume is extremely small. The OPSI-CMS allows for straightforward direct injection from complex matrices—such as reaction mixtures, buffers, or crude synthesis products—thereby significantly simplifying the workflow for oligonucleotide identification.

I. Experimental Method

Experimental instruments: All experiments were conducted on the AIS OPSI-CMS integrated system.

Mobile phase ： A 95/5 methanol/water mixture containing 10 mM TEA and 10 mM HFIP. The flow rate is 0.15 ml/min.

Sample Information ： Two oligonucleotide samples with molecular weights of 14,744.6 and 6,993.6 were used as examples for method evaluation. The samples were dissolved in water.

Injection volume: Transfer 3 microliters of the sample to the OPSI sample port for analysis.

II. Experimental Results

Figure 1: Sample 1: Molecular weight = 14,744.6

Add 3 microliters of the solution to the OPSI interface. Within seconds, a mass spectrum spanning a mass range of 500 to 2000 Da can be obtained. The total ion current chromatogram is shown in Figure 1A, and the average mass spectrum is shown in Figure 1B. Using the automatic charge deconvolution function, the uncharged masses can be measured from the average mass spectrum with just two clicks. The uncharged mass spectrum is shown in Figure 1C; the measured mass is 14744.0, which closely matches the reported value of 14744.6.

Figure 2: Sample 2: Molecular weight = 6993.6

Add 3 microliters of the solution to the OPSI interface. Within seconds, mass spectra spanning a mass range of 500 to 2000 Da can be obtained. The total ion current chromatogram is shown in Figure 2A, and the average mass spectrum is shown in Figure 2B. Using the automatic charge deconvolution function, the measurement of the uncharged mass can be performed with just two clicks based on the average mass spectrum. The uncharged mass spectrum is shown in Figure 2C; the measured mass is 6993.0, which closely matches the reported value of 6993.6.

III. Experimental Conclusions

For oligonucleotide R&D and manufacturing, the OPSI-CMS technology is like equipping it with “real-time monitoring eyes.” It reduces what would otherwise take hours of analysis to just seconds, enabling truly instantaneous insights into the synthesis process and providing a powerful impetus for accelerating the development of oligonucleotide drugs and diagnostic reagents.

The AIS OPSI-CMS system addresses the challenges of traditional detection methods:

“Directly to the Core”: Directly sample and analyze from complex matrices such as reaction mixtures and crude samples. Omit the purification step 。
“Sub-second speed”: Requires only a tiny amount of sample, Within seconds You can then obtain the mass spectrum.
“High accuracy”: Cases show that the deviation between measurement results and theoretical values is minimal. < 0.2 Da , high accuracy.
“One-Click Deconvolution”: The software features a built-in automatic charge deconvolution function. Double-click The target molecular weight can be obtained easily, and the operation is extremely simple.

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