(T1330-06-33) Ultra-High Throughput HILIC-Free Analysis of Oligonucleotides Using Acoustic Ejection Mass Spectrometry

Purpose: LC-MS methods for analyzing oligonucleotides (oligos) traditionally rely on ion-pairing reverse-phase chromatography. This approach typically ensures good separation and mass spectrometry (MS) response in negative mode. However, many ion-pairing reagents can contaminate the system, reducing the system's performance in positive mode. This presentation describes an ultra-high throughput method for characterizing an oligonucleotide using the Echo^® MS+ system with ZenoTOF 7600 system. The system utilizes acoustic energy to generate nanoliters of samples that are subsequently transferred into the MS for analysis. This contactless method enables analysis speeds upwards of 1 sample per second while providing robustness and high reproducibility sensitivity required for high-quality oligo characterization.

Methods: This presentation describes the use of Acoustic Ejection Mass Spectrometry (AEMS) for the analysis of intact oligonucleotides. The system uses an alternative approach to rapidly solve challenges traditional techniques face such as the dedication of a single system for ion-paring reagents for the confirmation of the molecular weight. MassPREP oligonucleotide standard (15-35 T) and 20mer ssDNA were purchased from Waters and diluted in DI H₂O. Larger length ssDNA standards (73, 125, and 175) were purchased from Integrated DNA Technologies and diluted in water. The samples were pipetted into 384 or 1536 well plates and acoustic energy is applied to the bottom of the well plate, causing a nanoliter droplet of the sample to be ejected. The Open Port Interface (OPI) is located above the well plate and captures the ejected droplet for dilution and transport by a carrier solvent (~400 µL/min) of the diluted sample to an ESI for ionization and delivery of diluted sample ions to a high-resolution mass spectrometer for analysis.

Results: This study explores important parameters (for example, carrier solvent composition, chemical additives, and varying matrices) that affect the data quality of high-resolution AEMS on a high-throughput scale for intact oligonucleotide analysis. Preliminary AEMS data demonstrate the rapid analysis of analytes, as a 384-well plate can be analyzed in 10-30 minutes (1-5 seconds/sample). In addition, the second half of this study focused on both small molecular weight oligonucleotides ~20 nt and characterization of the upper size limit ~175 nt. Poly T ladders were analyzed in one sample to show the complexity of an impure sample with mass accuracy under 20 ppm for the largest oligonucleotide. Preliminary AEMS data, without automation, show rapid analysis of compounds, measuring a 384-well plate in less than 10 min. Subsequent ejections result in baseline resolution between ejection peaks and no carry-over between measurements. Reproducibility was observed across a single plate and when measuring many plates in series, with an average of < 10% CV across ~300,000 replicate ejections. The addition of the liquid handler can increase workflow and sample throughput by enabling the preparation and scheduling of subsequent plates around the clock. By doing so, the number of samples analyzed drastically increases while still maintaining data quality, reproducibility, and system robustness.

Conclusion: For the first time, oligonucleotide analysis has been shown on an Acoustic Ejection Mass Spectrometer for oligonucleotides ranging from 20 -175 nt long with quality, reproducibility, and system versatility being key attributes of system performance for intact analysis. This system allows for swapping between positive and negative mode without having to dedicate an LC-MS system for HILIC and Ion paring reagents that can lead to potential contamination.

MassPREP oligonucleotide standard: poly-T ladder 50 nL ejection @ 4 pmol/µL without doing any chromatography