How to deal with Sodium and Potassium Ions in Oligos Analysis by Mass Spectrometry

Metal ions such as sodium (Na) and potassium (K) are commonly found in biological samples. They can have a significant impact on the detection of biomolecules by mass spectrometry (MS) analysis. Herin, we will explore how these metal ions cause signal suppression during MS analysis of oligonucleotides and discuss strategies to mitigate this effect.

Mass spectrometry is a powerful analytical technique used for the identification and quantification of molecules in complex samples. It works by ionizing molecules and separating them based on their mass-to-charge (M/Z) ratio. However, the presence of metal ions in the sample can interfere with this process and lead to signal suppression, where the intensity of the analyte signal is reduced.

One of the main reasons why metal ions such as Na and K can cause signal suppression in MS is their high ionization efficiency compared to other analytes. When the sample is ionized in the mass spectrometer, these metal ions can compete with the analytes for ionization, leading to a decrease in the signal intensity of the analytes. This effect is particularly pronounced in electrospray ionization (ESI), a predominant ionization method for oligonucleotide analysis. Furthermore, since oligonucleotides are polyanionic molecules, they most often show up as multiple MS signals, corresponding to the different protonation states of the biomolecule. This implies that software needs to be used to help deconvolute the different signals into one atomic mass, and the presence of multiple Na/K adducts drastically reduces the sensitivity of deconvolution algorithms, leading to an even more significant signal reduction.

Metal ions can come from many sources, including from the sample itself, the water used in HPLC solvents, or the materials used in the HPLC system to which the MS is coupled. Only MS-grade water should be used to prepare solvents, and plastic glassware should be used for both solvent preparation and storage to minimize sodium leakage. Pre-soaking plastic components in isopropyl alcohol for 1h before use is recommended to remove any extractibles. In case the HPLC system becomes contaminated with Na/K, it can be useful to perform an acidic wash (10% Phosphoric Acid can be used for HPLC tubings and pumps

and 0.2% Acetic Acid for HPLC columns), as it has been shown to help remove metal ions (Birdsall et al, DOI:10.1002/rcm.7596). Metal ions coming from the samples can be more difficult to deal with, as removing them isn't always possible.

To reduce the effect of metal ions that cannot be eliminated from the sample on MS signal suppression, various strategies can be utilized. One option is to utilize chelating agents like ethylenediaminetetraacetic acid (EDTA) to bind metal ions in the sample before analysis. EDTA creates stable complexes with metal ions, hindering their interference with the ionization of analytes in the mass spectrometer. Another common choice is Medronic Acid, which functions similarly.

Another strategy is to optimize the MS conditions to minimize the ionization of metal ions. This can be achieved by adjusting the pH of the solvent or using selective ion monitoring (SIM) to only detect specific ions of interest while excluding metal ions from the analysis.

Furthermore, sample preparation techniques such as solid-phase extraction (SPE) can be used to remove metal ions from the sample before MS analysis. By selectively isolating analytes from metal ions, SPE can help reduce signal suppression and improve the sensitivity of MS detection. The C18-based stationary phase is one of the most commonly used media for SPE. In the context of oligonucleotide purification, SPE is often performed in an ion-pairing fashion, with triethylammonium counter ions helping increase the oligo's affinity for the C18 stationary phase. The excessive presence of salts in the sample can also be removed from oligos samples by using size exclusion columns (the columns should have a MW cutoff of at least 2x the molecular weight of the desired analyte).

In conclusion, metal ions such as Na and K can cause signal suppression in MS analysis due to their high ionization efficiency. By employing strategies such as chelating agents, optimization of MS conditions, and sample preparation techniques, the impact of metal ions on MS signal suppression can be minimized, leading to more accurate and reliable analytical results. In the event the HPLC system becomes contaminated with excess Na/K, acidic buffers can be used to help remove them. A good practice is to perform an acidic wash at the end of each day, to avoid the accumulation of ions over multiple injections and help wash the column thoroughly. Switching the polarity of the mass spectrometer (from negative to positive) during this washing step can also help to maintain sensitivity by a de-lensing effect.