Do Ghost peaks matter in LC-MS(MS)?

Perhaps in the light of our new solvent filters with inbuilt ghost peak traps it is time to republish my article on how to eliminate ghost peaks. The new solvent filters make eliminating ghost peaks simpler than ever - https://www.shimadzu.co.uk/SolventFilter

Ghost Peaks in gradient methods have been the nemesis of the chromatographer using UV detection for many years, but those of us using LC-MS(MS) don’t need to worry about them – right? 

Often we LC-MS(MS) users believe that ghost peaks are not a concern to us due to the selectivity of our instrumentation, but is this a false assumption? Should the LC-MS(MS) user routinely consider ghost peaks; if we can’t see them – do they actually matter? Indeed, what about late eluting ghost peaks or even components that never actually elute from the column?

LC-MS(MS) users can be tempted to treat the LC column as little more than a filter or delay device for their LC-MS(MS). In doing so, they do the humble LC column a huge disservice – it is a fully integral component of your analytical system.

Before we consider the impact of the ghost peak, lets take a look at where ghost peaks come from.

Ghost peaks are also referred to as artefact peaks and occur in gradients, even when no sample is injected. The sources of ghost peaks are typically divided into three categories: those due to impurities other than the target substance, internal contamination of the LC instrument, and impurities in the mobile phase, as outlined below.

1. Impurities in instrument.
2. Impurities in sample.
3. Impurities in mobile phase.

Ghost Peaks Due to the Instrumentation

The most common reason for ghost peaks originating in the instrument is carryover attributable to the autosampler (or manual injector). When the needle is dipped into the vial to aspirate the sample, substances in the sample, become the source of carryover, as they can be adsorbed to the inside and outside surfaces of the needle. Those substances which are not eliminated even after needle rinsing are carried over to the next analysis, and can appear as ghost peaks. Since substances that exhibit particularly strong adsorption will appear even after several blank analyses (analysis in which only mobile phase is injected), it may be difficult to identify the autosampler as the source of the ghost peaks.

Ghost peaks due to the instrument are generally consistent and can be mitigated by use of advanced washing and a low carryover autosampler. Shimadzu autosamplers are specially engineered with patented technology in order to set the best in class when it comes to extremely low sample carryover.

Ghost Peaks due to the Sample

These are more commonly associated with UV detection as the additional specificity of LC-MS(MS) generally prevents different compounds interfering with each other, but this is not always the case. Since a ghost peak that originates in the sample does not appear in a blank injection, determining the source is (usually) relatively easy.

The scientist has a number of remedial approaches that may be applied:

• Develop the chromatography to resolve the interference and analyte of interest
• Devise a sample preparation procedure to eliminate the impurity.
• Use a different SRM/MRM

If the ghost peak is due to degradation of the sample, that degradation can be inhibited by using an autosampler with a vial cooling feature.

Ghost Peaks Due to Mobile Phase

Ghost peaks can also be generated from the mobile phase in various ways, including the following.

• Generation of organic substances in mobile phase due to prolonged use, or dissolution of organic substances from the air in the mobile phase
• Mobile phase contamination due to topping off existing mobile phase with new mobile phase over a long period of time instead of preparing a fresh bottle regularly
• Use of contaminated organic solvent and/or water to prepare mobile phase
• Use of contaminated mobile phase reagent
• Use of poor quality mobile phase components
• The presence of the analytes in the environment – such as perfluorinated compounds

Due to the high consumption of mobile phase and the relatively long periods of time it is used before being replaced, pre-treatment measures such as filtering that are typically used for samples are not practical for eliminating contaminants in mobile phase. Furthermore, mobile phase contaminants adhere to the inside of the instrument and tubing, small amounts are inevitably transported to the column along with the mobile phase during the solvent delivery process, and are likely to be detected as ghost peaks.

In reversed phase analysis using gradient elution, such substances that cause ghost peaks are retained in the column at the start of analysis, and are then eluted as the organic solvent ratio increases, making separation very difficult when they exhibit behaviour similar to that of the compound of interest.

Thus, it is difficult to specify the cause when ghost peaks are of mobile phase origin, and furthermore, it is difficult to address the problem by making changes to the method.

Invisible Ghost Peaks

One of the greatest strengths of LC-MS(MS) is its excellent specificity. Very often ghost peaks are ignored as they simply do not show themselves on the SRM/MRM. But is it safe to always ignore them? As part of your method development/validation you will be checking for matrix/ion suppression effects to ensure there are no routine ion suppression or enhancement effects coming from your samples or systems. But ghost peaks from the solvent can invisibly build up on the head of the column during column conditioning, during pauses in your run or if you have a long reconditioning phase; your next gradient will see these components elute – potentially influencing your results. 

A change in batch or supplier of solvent or issues with your pure water supply could result in impurities in your mobile phase that weren’t present during the validation. These build-up on the head of your column and, with the excellent specificity of LC-MS(MS), you remain unaware that these new components are now present and that you may be introducing a previously unknown matrix/ion suppression effect into your assay. The use of a Ghost Trap column will ensure that such components are prevented from impacting upon your results.

Utilising a Ghost Trap

Identifying the cause of ghost peaks in the mobile phase is difficult and drastic prevention measures are problematic; trapping of the causative substances is an effective, simple and low-cost means of suppressing the appearance of the ghost peaks.

The figure below depicts example Ghost Trap units that can be used to reduce or eliminate ghost peaks that are due to the mobile phase. Installing the Ghost Trap in the mobile phase flow line provides for adsorption of the substances responsible for the ghost peaks, especially when conducting gradient elution with reversed phase analysis. Ghost peaks are prevented from appearing because the mobile phase is sent to the column after removal of the problematic substances.

As a study a Ghost Trap was connected at various positions in the flow line according to the LC system configuration and the analysis application.

(a) Ghost Trap not used

(b) Ghost Trap positioned after solvent delivery pump A (aqueous solution pump)

(c) Ghost Trap positioned after solvent delivery pump B (organic solvent pump)

(d) Ghost Trap positioned before gradient mixer

(e) Ghost Trap positioned after gradient mixer

The figure below shows a comparison of the UV chromatographic results using the connection positions indicated in (a) to (e). The results clearly indicate that under these conditions, many ghost peaks originate in the aqueous flow line (most likely from the mobile phase rather than some contamination in pump A). It is also clear that there are slight differences in the ghost peak pattern when the Ghost Trap DS is connected before or after the gradient mixer, indicating that the presence of ghost peaks can also be due to contamination of the mixer. Since the Ghost Trap DS can also be connected downstream of the gradient mixer, more certain removal of ghost peaks can be expected, as shown in chromatogram (e).

The use of Ghost Traps improves data quality in particular when looking to quantify low level components – very often the raison d'être of LC-MS(MS). Quantitation of a peak that is being supplemented by an interference or carry-over is inherently flawed, the user needs to find a way of eliminating the influence of ghost peaks, the Ghost Trap column prevents these peaks from co-eluting with the peaks derived from the injection, thus protecting the validity of the result.

Effective for the quantitative analysis of impurities, the Ghost Trap DS/DS-HP effectively eliminates such ghost peaks to ensure highly accurate quantitation, even with very small target component peaks.

As a final thought, LC-MS(MS) users can sometimes neglect to give their LC column the recognition it deserves. When you are under pressure to get results issued, it is tempting to recondition the column ready for your next sample as soon as your last analyte of interest elutes. It is a good idea to always increase the organic component to a high concentration to ensure everything is flushed out of the column prior to injecting the next sample. Highly retained compounds that are not of interest can impact on later results. Unless the column is thoroughly flushed out, highly retained compounds will linger on the column; either never eluting, or eluting in later samples and potentially causing matrix/ion suppression effects that the analyst is unaware of.

These highly retained components can cause unexpected and unpredictable results, often a long time down the road; leading to difficulties as simple as a shortening of the column lifespan all the way through to placing a question mark over the validity of the results or even necessitating method redevelopment and validation.

For more information you visit: https://www.shimadzu.co.uk/mobile-phase-cleaner-hplc

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