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

Oct 1, 2023 | Biopharma, Blogs | 0 comments

We know that LC-MS oligonucleotide analysis can have its share of challenges—challenges with sensitivity, challenges with adduct formation and challenges with data analysis, to name just a few. That’s why this blog takes a closer look at the dos and don’ts of this type of analysis and explores some keys to success. It also explains why following these simple rules can vastly improve your oligonucleotide characterization and quantitation efficiency and success.


The dos (and the whys behind them)

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, separation is frequently performed at temperatures at or above 60°C. Combined with the use of organic solvent, high temperatures result in melting of duplex species and prevention of secondary structures based on complementarity bases. However, 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. Our recommendation is the Biozen Oligo from Phenomenex.

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 and column for oligonucleotide analysis. Non-specific binding and adduct formation can severely impact oligonucleotide assay sensitivity and data quality. 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. In addition, other types of analysis can be impaired by remaining ion-pairing agents, and therefore it is best practice to dedicate an LC and consumables to oligonucleotide analysis.

Do monitor spectra for undesired depurination. Two main factors contribute to the loss of a purine base, guanidine or adenosine, respectively, in the gas phase: the source temperature and voltages in the ion path. We recommend tuning the source temperature for best performance while keeping depurination to a minimum. For the radio frequency (RF) of the QJet ion guide, we recommend a value of XA1 = 180 V. Your SCIEX application specialist can help you with recommendations for your system.

Do optimize collision energy. For MS/MS experiments, the collision energy should be optimized for synthetic oligonucleotides. 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 for each analyte and charge state to help ensure more diagnostic fragment ions in a less crowded mass range. Please note: The most intense precursor charge state does not always result in the best-performing fragment ion. We recommend using a few charge states for method development, including the relevant matrix.

Do adopt a consistent cleaning protocol for your MS and LC to limit adduct formation. A cleaning protocol 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. Reach out to your SCIEX support specialist for recommendations on how to clean your LC and MS systems.

Do verify system performance with a system suitability test. 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 running valuable samples. We highly recommend including the LC in your system test to ensure your entire system is working well for oligonucleotide analysis.


The don’ts (and the whys behind them)

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. Keep an eye on your water quality: Na+ and K+ can readily form adducts with oligonucleotides. This can disperse the signal between different cation species and greatly reduce overall MS sensitivity and reproducibility. If you are using bottled water, be sure to use LC-MS grade water and be aware that suppliers and batches can differ in quality. Milli-Q water from a well-maintained system is a great way to reduce cation adducts without dependency on suppliers.

Don’t use mobile phase for more than a couple of days. Mobile phase additives—including ion-pairing reagents such as hexafluoroisopropanol (HFIP) and buffering components such as triethylamine (TEA)—are volatile and have limited aqueous solubility. This can lead to changes in concentration over time. In addition, commonly used buffers such as TEA are not strongly buffered at the desired pH, so they have the propensity to drift over time. To ensure consistency of results in terms of retention times, peak shape and intensities, prepare mobile phases accurately and freshly every 1–2 days.

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. Your SCIEX application specialist can help you with recommendations for your system.

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 for TOF MS scans or Q3 scans above m/z 400 and focusing 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 varying MS/MS conditions. Some might fragment more easily than others. This can lead to either more “rich” MS/MS spectra or spectra that have many unspecific, low m/z fragments. It’s best to optimize on a 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.


With the recommendations outlined here, and assistance from your SCIEX application specialist, you can be well positioned on the path to oligonucleotide analysis success. Also be sure to check out our latest solutions for oligonucleotide analysis, which can be a valuable tool on your journey.


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