When it comes to LC-MS analysis one of the main contributors to inaccurate results, especially with biological sample analysis, is ion suppression.

So, what is ion suppression, how do you identify it and what steps can be taken to minimise its impact?

What is ion suppression?

In simple terms, ion suppression is the adverse effect on detector response of the analyte of interest due to reduced ionisation efficiency. This is often due to the presence of a competing or inhibiting species present in the sample matrix, which negatively impacts efficient ionisation.

LC-MS/MS often gives the false impression that there is no interfering species present, as no chromatographic impurities can be detected. If the interfering species isn’t isobaric, it can still suppress ionisation and have a negative impact sensitivity, precision and accuracy of analysis.

Although not fully understood completely, there are multiple theories around why electrospray ionisation (ESI) is more prone to ion suppression than atmospheric pressure chemical ionisation (APCI).

  • ESI involves a far more complex ionisation mechanism when compared to APCI, which means there are more factors involved which can lead to increased ion suppression.
  • The physical properties of droplets (such as surface tension and viscosity) when containing high concentrations of interfering compounds can lead to a reduction of solvent evaporation, which is heavily linked to ion suppression.
  • The presence of non-volatile species preventing ionisation of the analyte through co-precipitation or preventing the contraction of the droplet size to the point required for ion evaporation (critical radius of the droplet)

Identifying the problem

The issue with ion suppression is that it can, dependant on the method, be extremely hard to detect. Initial chromatograms during method development may looks very promising, even with substantial ion suppression! It’s often not until a project moves along that issues, such as reduced sensitivity and an increase in percent relative standard deviation (%RSD), become apparent. With those doubts raised, the method can no longer be considered reliable and cannot be validated!

A lot of time and money can be wasted attempting to troubleshoot the problem, such as replacing your analytical column, preparing a new mobile phase for further analysis, or replacing the tubing in your system. The solution is clearly to proactively prevent ion suppression, rather than to try and diagnose the issue once problems arise!

Negating ion suppression

Although not completely understood, there are a few strategies currently employed to minimise/remove ion suppression during analysis.

Ionisation Source Selection: As mentioned in the earlier section, APCI suffers from ion suppression less than ESI, so when there is no other way of impact minimisation, it is advisable to switch from ESI to APCI. If this isn’t possible (due to the analysis being undertaken), then it is advised to switch the ESI ionisation mode from positive to negative. As less compounds are ionisable in negative mode, there is a chance that the competing (ion-suppressing) compound is removed from the analysis. This is of course assuming that the analyte of interest can be ionised in negative mode, otherwise this strategy would be pointless!

Sample Preparation: An effective preparation protocol can greatly (if not completely) reduce the risk of ion suppression. Usually liquid-liquid extraction or solid-phase extraction are the most commonly employed, alongside derivatisation, in order to remove ion-suppressing species. This also has the added benefit of removing other interference, such as isobaric species!

Sample Concentration: By either reducing the volume of sample injected or diluting the sample, it is possible to reduce the number of interfering species present during analysis. The downside to this is that you also reduce the amount of the analyte of interest, so this strategy is not appropriate for trace analysis.

Ultimately the most appropriate technique and strategies that should be employed to reduce the impact of ion suppression is entirely dependent on the analysis you are running and should be determined on a case-by-case basis.

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