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The whys behind the dos and don’ts of oligonucleotide analysis

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We know LC-MS oligonucleotide analysis can have its share of challenges—challenges with sensitivity, challenges with adduct formation, and challenges with data analysis (to name a few). That’s why we’ve created an Oligonucleotide Analysis Knowledge Accelerator that includes an easy-to-read, one-page infographic with key dos and don’ts for successful oligonucleotide analysis. In this blog, we take a closer look at these dos and don’ts and explore the reasons why we believe they are keys to success. We discuss how following these simple rules can vastly improve your oligonucleotide characterization and quantification efficiency and success. 

The whys behind the dos

  • Do use columns with high pH and high temperature stability
    • Oligonucleotides are frequently separated using ion-pairing, reverse-phase chromatography for LC-MS applications. The most commonly used ion-pairing systems have a pH of around 9. In addition, separations are performed at temperatures at or above 60 °C which, when combined with the use of organic solvent, results in full melting of duplex species or self-complementarity. These conditions are very aggressive for conventional chromatographic media and can lead to poor performance and decreased column life. Using a column specifically designed for oligonucleotide applications helps ensure long column lifetime at optimal performance.
  • Do re-equilibrate columns with at least 5 column volumes
    • Because oligonucleotides are prone to high non-specific binding and adduct formation, extensive flushing between samples can greatly minimize carryover and contact with other impurities and adducts.
  • Do dedicate an LC for oligonucleotide analysis
    • Non-specific binding and adduct formation can severely degrade oligonucleotide assay sensitivity and data quality. Any small molecules (such as salts or proteins) that are exposed to the LC column, tubing, and system can potentially interfere with oligonucleotides and cause degradations in assay performance. For this reason, dedicated labware is also recommended.
  • Do optimize collision energy
    • A collision energy that is too high can fragment the oligonucleotide too much, leading to lower-mass fragment ions that are not specific to the oligonucleotide. The fragments could also be in a mass range that is crowded with background matrix ions. For better performance, the collision energy should be optimized to ensure a more diagnostic fragment ion in a less crowded mass range.
  • Do adopt a consistent cleaning protocol for your MS and LC to prevent adduct formation
    • This is particularly important for maintaining high sensitivity and reproducibility in quantitative studies where adduct formation with Na+ and K+ can spread the signal between multiple species and degrade overall assay consistency and performance. Preventing adduct formation is one of the most important rules for generating high quality oligonucleotide LC-MS data.
  • Do verify system performance with a system suitability standard
    • Using a known oligonucleotide standard for system performance testing can help to show whether the system is clean of interferences and adduct forming components before valuable sample is run.

The whys behind the don’ts

  • Don’t use bottled HPLC water
    • Na+ and K+ can readily form adducts with oligonucleotides. This can disperse the signal between different cation species and greatly reduce the overall MS sensitivity and reproducibility. Because bottled water can contain Na+ and K+, high-purity water, such as Milli-Q water, is best for reducing cation adducts.
  • Don’t use low-quality reagents
    • Low-quality reagents can affect ion-pairing capabilities, retention times, peak shapes, and adduct formation. They can lead to lower sensitivity, accuracy, and reproducibility.
  • Don’t use mobile phase for more than 1 day
    • The mobile phase additives, including your ion-pairing reagent (such as HFIP) and your buffering component (TEA, for example), are volatile and have limited aqueous solubility. This can lead to changes in concentrations over time.  Also, commonly used buffers like TEA are not strongly buffered at the desired pH, so they have the propensity to drift over time.
  • Don’t use a high MS source temperature
    • Oligonucleotides are susceptible to in-source fragmentation, particularly depurination.  Lowering the source temperature reduces the likelihood of causing undesirable in-source fragmentation. Preventing in-source fragmentation is another key rule for generating high-quality oligonucleotide LC-MS data.
  • Don’t use an MS scan range below m/z 400
    • Matrix effects and other interfering components are more abundant at lower m/z. By starting the scan range above m/z 400 and focusing only on the higher m/z oligonucleotide ions, these interferences can be minimized and sensitivity can be enhanced.
  • Don’t assume all charge states fragment the same way
    • Different precursor charge states can behave differently under MS/MS conditions. Some might fragment more easily than others. This can lead either to more “rich” MS/MS spectra, or to spectra that have too many low m/z fragments. It’s best to optimize on the precursor charge state that produces both a high intensity signal in MS mode and a strong, higher m/z fragment ion (in a clean portion of the spectrum) in MS/MS mode.

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