Hi Justin,

What type of compounds are you studying? It is not unlikely that different precursor ions yield the same product ion(s), for example in the case of peptides with similar amino acid sequences/composition. If you observe the same precursor ion at various elution times, that could be due to the fact that you're separating isomers of the same compound on your chromatographic column.

The fact that compounds are not constantly eluting at the same retention time is a problem more frequently observed in chromatography and could be due to slightly different composition of solvents that were used, the column being not completely (re-)equilibrated prior to injection, and so on.

Hope this helps.

It sounds like you are describing carry over contamination in your HPLC system. This most commonly happens when you either fail to properly flush/wash your HPLC column properly (leading to a build up of material or fouling of the column support over time) OR have a maintenance issue to address such as a worn autoinjector rotary seal. Both types of problems result in sample peaks reappearing at random times.

Here is an article link to a paper that I wrote on the topic of carry over contamination in HPLC and LC/MS systems which may help you; "Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems".

The other possibility is that you have contaminated the MS source (or associated ion optics, nebulizer and so on). This can also result in the MS "seeing" the sample at random times as it breaks off and is re-detected. A thorough wash can be tried first using strong solvents to dissolve and flush away the material. If that does not work, then a full source cleaning is in order (a maintenance procedure).

http://hplctips.blogspot.com/2015/02/carry-over-carryover-contamination-in.html

Dear all, thank you for your responses. Actually on the topic of cleaning my column, what would be a good solvent system and for how long?

Justin: You did not provide us with any information about your HPLC method or LC column so it is not possible to provide specific recommendations to you. However, I will provide a few suggestions which I teach in my classes that may help you. (1) Contact the column manufacturer for their recommendations as to which solvent are SAFE to use on your specific column. Depending on the exact column type, some liquids can damage the column so check with them first. (2) After each analysis you want to wash the column down with a mixture of stronger solvents than found in your analysis method which will fully dissolve ALL of the materials you have been injecting into the system AND allow them time to fully elute off the column. Now that statement is also true for your actual HPLC method as you should have a method which fully dissolves everything and elutes off EVERYTHING in the same run. Ideally, your analysis method is one run and you have a separate method to flush and re-equilibrate the column with next. *Do not combine the analysis method with the flushing and re-equilibration method (always separate them). Well developed methods require only a simple wash, then re-equilibration step.

Example: If you are using a common C18 column and your method uses MeOH/DH20 mobile phase, then you would want to run a wash which incorporates some ACN (stronger than MeOH to elute peaks faster) as well as a higher than normal percentage of MeOH to wash the column with. You must also take into account pH and any additives used too. There is no "one" wash solution as it must be developed specifically for your exact method.

Dear Sir, thanks for your response. Actually I have been running this assay for quite some time already - like 6 months and this problem has been recurring.

Here are some information which you may find useful to help me troubleshoot the problem:

Column: Agilent ZORBAX Eclipse Plus C18 (3.0 mm x 150 mm x 5µm)

Solvent system for running my LC: 75% Methanol and 25% water (both with 0.1% formic acid)

Thank you.

Thank you for the additional info.

So as mentioned before, check your system for any maintenance issues or hardware problems as these can cause some of the problems that you are seeing.

It sounds like you are running an isocratic analysis ??? If so, that will be a problem if you are not following up with a wash method which utilizes a stronger solvent than your actual analysis does. If possible use a gradient analysis to avoid the issue, but if you must continue to use the isocratic analysis, then follow up with a wash method which starts off with 95% MeOH/5% Water (with formic) and see how that goes.

If this problem has been going on for some time, then it really should have been addressed much earlier. The quality of your data depends on a properly functioning instrument and method. It is possible that your entire LC/MS system may need a professional cleaning and servicing at this point.

Dear Sir, noted with thanks. Will conduct an extensive flushing of my column as soon as possible before doing any other assays. Thanks.

It sounds that your Solvent delivery module is slightly blocked. The inline solvent filter of the line inserted in water (with formic) suffer sometimes bock, you just need to clean this part, purge each line separately. Or, to make sure that this is the point, just use ONE delivery line contain the mobile system, and test using isocratic elution.

Dear Sir, thank you for your kind reply :)

Dear Justin

How are you preparing your samples?

The symptoms may be due to your sample solvent if the injection volume is fairly large.

What is the matrix of your samples? This could have a large number of contaminants that could influence the separation. Especially if the analyte you are looking for can be ionised under the acid conditions you are using.

More detail would help.

Dear Sir, thank you for assisting. My samples are extracted from cell lysates using 70% cyclohexane : 30% ethyl acetate as the liquid-liquid extraction solvent after 1h incubation of the cell lysate (containing the desired enzyme) with the substrate. The injection volume is kept between 5-10 microliter.

Has there been any papers using LC-MS/MS analysis with cell lysates after liquid-liquid extraction?

For your kind advice, please.

Thank you.

Warmest regards,

Justin

Dear Justin

I would suggest the bulk of LC-MS/MS methods follow a liquid liquid extraction process, so find cell lysate analysis and you will have some examples of what has been done.

I note that your extraction solvent is non-polar and your mobile phases are in fact fairly polar. 

I take it that you are drying your extract down completely as I would doubt the miscibility of the extract solvent in your mobile phase and this would cause all kinds of reproducibility problems in addition to split peaks as you describe.

You would also need to be sure the final sample solvent dissolves your analyte and is compatible with your starting mobile phase (if you are running a gradient which is not clear from the information given).

Dear Sir,

Thank you for the information. Would surely do an evaporation step to remove the liquid extraction solvent before reconstituting it with the mobile phase for better solubility.

Warmest regards,

Justin

Dear all,

I am facing some what similar situation. My HPLC shows a single peak so I presume a pure compound but on analysis with LC-MS I got 28 compounds but most of them having the similar RT. I will be thankful to you for any suggestion how to decide which is the major compound present in the sample. Is it the volume% on which we can decide the major compound on the sample. I will appreciate your advices.

Regards

Rath

It sounds like you probably failed to develop a proper HPLC method which retains, separates, resolves and elutes all of the compounds present in your sample. Perhaps they all eluted at the tzero? I see this often, even in published papers describing so-called "validated methods" where none of the samples are retained on the column and all of them appear to be "pure".

If you would do us the favor of sharing ALL of the HPLC method, peak and column details in your request (or starting a NEW question thread), then we may be able to provide you with a quick explanation.