Specific peak shape problem

[zokitano]

Dear colleagues,

Recently I got a specific peak shape problem during RP chromatography and using MS/MS (QqQ) detection. Tried couple of weeks to troubleshoot it without success. Any of your insights and comments are welcomed.

Firstly, about the hardware: Agilent 1100 coupled with API 4000 QTrap
Column: 3.0 mm ID, C18 column with an appropriate guard column
Mobile phase: methanol/THF/10mM formate buffer pH3 (isocratic elution)
Folw: 0.4mL/min, t(column) = 30 degrees
Ionization mode: ESI

The picture of the SRM chromatogram with the problematic peaks (designated with arrows and numbers):


All compounds are nitro-containing compounds (proprietary) with molecular weights below 250.
They all have similar backbone (same groups - arranged differently on the same backbone -> positional isomers) and the problematic peaks No. 2, 3 and 4 have additional OH (hydroxyl group), while the peak No. 1 has additional COOH (carboxyl group). They all show severe fronting and tailing, resulting in a very broadened low intensity peaks. Other peaks are symmetrical and do not show the same problem (as you can see from the picture).

So far: - I changed the column and its guard column with new ones (no reslut)
- I changed the tubing. From pump to the injector i have SS tubing, while from injector to column and from column to ESI source - PEEK tubing. Reduced the length of the pre and post column tubings (no result) as much as it is allowed.
- made PM, changed the injector rotor seal and pump seals. Made the leak and pressure tests afterward. Everything seems to work properly with all tests passed
- i did passivization of HPLC according to the Agilent procedure with nitric acid (because the peak No. 4 cannot be detected, which usually gives nice peak)
- increased the buffer ionic strength twice - the problem still persists.

Under the same conditions only on Thermo HPLC and MS the chromatography is nice, without fronting/tailing and sensitivity issues.
I ran out of ideas what to change next in order to improve the overall picture. I suspect that the problem is before the column, not after it, but maybe i am wrong. The MS was recently PM-ed (calibrated , cleaned and works normally)

Any ideas?

Thanks a million!

[zokitano]

Forgot to add that I ran only standards for now. The picture above shows standard chromatogram. They are prepared in the mobile phase. Even when I prepared them in weaker eluting injection solvent the problem is still here. The injection volume is 20 microliters.

Thanks in advance!

[yangz00g]

Based on your description, to me it is more likely due to sensitivity issue than chromatographic issue (background noise high for all the four compound).

Because you cannot simply transfer the MS parameters from API4000 to Thermo instrument, re-optimization of your API4000 parameters may help.

Good luck

[zokitano]

Based on your description, to me it is more likely due to sensitivity issue than chromatographic issue (background noise high for all the four compound).

Because you cannot simply transfer the MS parameters from API4000 to Thermo instrument, re-optimization of your API4000 parameters may help.

Good luck

Thank you for your response!
Actually the method was initially built on my instrument and transferred to the other lab (Thermo equipment). The MS/MS and ion source parameters were carefully optimized for the best sensitivity and specificity.

You're right about the higher background noise for all 4 compounds SRMs. Most of the compounds investigated share the same precursor or product ion in SRM (related compounds, isomers and homologs), but for the other compounds there isn't elevated background. That is why I suspect on the LC part. Also the compounds can be oxidized or reduced electrochemically and the background of ESI involves also electrochemistry. But why some compounds with additional polar group are more susceptible, than the other that show nice peak shapes?

Could be the problem due to an issue with the ion spray capillary on which the ion spray voltage is applied?

Thanks!

[Alp]

An interesting problem.

You said...
"Under the same conditions only on Thermo HPLC and MS the chromatography is nice, without fronting/tailing and sensitivity issues."
Do you mean using Thermo HPLC and Thermo MS? Or do you mean using Thermo HPLC and API 4000 in MS mode? It looks like from one of your follow up messages that you mean the former you get good peak shape for all compounds but on API 4000 and agilent HPLC something bad happens.

If you are able to get decent chromatography using the same column and the same mobile phase and the same standard composition, then chromatography is probably not your problem. It may be a hardware problem. Perhaps even an optimization problem on the API 4000.

Has the relative area of the peaks remained about the same? (eg ratio of the area of peak 1 vs peak 3)? Maybe the sensitivity is about the same but for some reason your peaks are getting smeared out due to sticking on some surface in the agilent/api 4000 set-up. This is my first impression from your chromatogram.

Even slight changes in position of groups can affect the fragility of the molecule. Maybe some of the energies (declustering potential) on your api4000 is too high and some of your compounds are fragmenting in the source.

I am curious, with a mobilephase of pH 3 I assume you are using positive ion mode ESI on the api 4000 (although not necessarily).
What is the ionization method on the thermo MS system?


Alp

[zokitano]

Thanks Alp for your valuable insight!

Just to clear some doubts: the Thermo equipment (Surveyor Plus LC and linear ion trap LXQ MS equipped with ESI source) is separate and belongs to our partner laboratory in Belgium.

Yes it seems that something is wrong with our equipment, although I regularly clean and take very good care of it. Even after passivization of all the capillaries and parts of the LC used, excluding the MS spray capillary, i still have the same problem. I want to try APCI for which i also optimized the conditions for my analytes and see if there is something wrong with my ESI capillary or ESI process itself? When I'll try it I will post the results.

The ratio of peak areas of peaks No 1 and 3 is the same more or less, having in mind the natural variability of the ESI process. I also thought that I have hardware problem, that's why I changed almost all the parts and capillaries and passivated the system.

The ESI response for all compounds was checked after careful optimization of the compound dependent parameters (declustering potential, collision energy, collision cell exit potential etc) using direct infusion-MS (syringe infusion). Using FIA-MS I optimized the ion source parameters (temperature of desolvation, capillary voltage, curtain gas, pressure of the drying and nebulizer gas...)

Actually we are using negative ESI with pH 3 buffer, because of the presence of the nitro group, the molecules' OH groups are more acidic and tend to lose proton and to give higher signal in negative than in positive mode.

Thanks!

[carls]

Perhaps an FIA experiment will shed some light on this.

If all compounds give similar peak profiles (i.e. widths) then the source can likely be ruled out.

I'm skeptical that a problem in the MS can produce such a wide peak. The only way this is possible is if the compounds are adsorbing onto something and relatively slowly desorbing. This could be happening in the spray capillary or the source housing but I cant remember ever seeing such a problem.

If the FIA experiment shows similar peak widths for the well-behaved and misbehaved compounds then the MS source (and HPLC system) can likely be ruled out. If the problematic compounds are MUCH wider (several minutes wide in your example) then perhaps the source needs to be cleaned or the spray tube replaced.

Also, dont forget about the grounding union. It too can become contaminated. Try bypassing it as well.

Please share the results of the FIA experiment.

[zokitano]

Thanks carls for your input!

I totally agree with your opinions. I am starting to thinking that maybe some contamination of the source or ion lenses could provoke this kind of problem.

Today I backed up this idea by applying APCI (Heated Nebulizer) instead of ESI (TurboIonSpray) source. Even with APCI I am getting the same peak shape problems in the chromatogram (chromatographic parameters and standard solutions are the same from my previous experiments with the ESI source). It is evident that the different ion formation processes (ESI vs. APCI) do not influence the peak shapes of my analytes. The final picture is the same.

I also by-passed the grounding union (as a possible place for contaminants buildup) and no change was observed in the final chromatogram. Still, the problem persists.

I did also FIA-APCI-MS and the peak profiles for the "problematic" analytes follows the general profile of all other analytes (with good chromatographic peak shapes).

I noticed these days one more thing. When the injection valve is turning from inject to load position and vice versa (during the injection of the samples) the whole injection valve assembly moves little bit in the direction of the turning rotor. I think that this assembly is loose and not firmly attached to the rest of the autosampler unit. Service engineer said that it is not a problem for the injection valve to be loose and didn't treat the "problem" during the PM, 2 days ago. Explained that the whole autosampler unit should be opened in order to tighten the screws that hold the injection valve assembly into the autosampler. Also checked for leaks and made pressure and leak tests and the injector seems to work properly.
Could this "problem" introduce the specific peak shape problems that I am observing?

Thanks to all for your valuable suggestions and ideas. I will keep informing you.

Best regards!

[Alp]

Zokitano,

Nitro groups, negative ion mode.....DOH!

As for the loose switch valve, if the chromatography is consistent (peak shapes always the same shape and intensity for multiple injections of the same sample) then it is likely that the valve itself is OK and only the mounting is a bit loose as you have said. However, this does not rule out some build-up may be in the valve. It should not hurt to disassemble it, clean it and put it back together. I would expect that this is usually done during a PM (which would necessitate properly srewing back the valve onto the unit, so why it is loose...?) and which may also require to replace the seals and stator during the PM, or maybe not. If the valve was completley PM'd then there might not be a need to clean it again. You might want to look at the log for the PM to see what was replaced.
You also might want to assess whether the valves in both systems are the same type ( PEEK or Stainless Steel) and what type of material the stators are made of.

Has the exact same column been used on the other system (that gives good peaks shape) or has only the exact same TYPE of column been used on the other system?

Alp

[zokitano]

Hi Alp,

The chromatography seems to be consistent and reproducible (nice peaks are always nice, bad peaks are always bad - also their intensity is reproducible),so that probably means that the loose injector assembly is not a major cause for the problem. The service engineer did the PM of the injector valve last week. BTW, the rotor seal was also replaced.

For now, because of this problem I am not using another diverter valve before the MS and after the column. I wanted to minimize the post-column extra volume to minimum and not to introduce another segments which could interfere with the chromatography.

The column that is used in the other lab is the same (same manufacturer, same stationary phase, same particle size, same pore size) with ours, only the difference is in the column internal diameter. We're using 3.0mm i.d column and they are using 2.1mm i.d.

Thanks for your help!

Regards

[carls]

Today I backed up this idea by applying APCI (Heated Nebulizer) instead of ESI (TurboIonSpray) source. Even with APCI I am getting the same peak shape problems in the chromatogram (chromatographic parameters and standard solutions are the same from my previous experiments with the ESI source). It is evident that the different ion formation processes (ESI vs. APCI) do not influence the peak shapes of my analytes. The final picture is the same.

I also by-passed the grounding union (as a possible place for contaminants buildup) and no change was observed in the final chromatogram. Still, the problem persists.

I did also FIA-APCI-MS and the peak profiles for the "problematic" analytes follows the general profile of all other analytes (with good chromatographic peak shapes).

Since the peak profiles all look similar in FIA (were all profiles normal, i.e. <~30-45s wide?) the source is clearing normally for all analytes. This rules out the source and the HPLC system (exc. the HPLC column) as the source of the broadening. This leaves the chromatographic column as the problem area. Remove the guard column and see if the chromatography improves any. If not, you'll need to try a different brand or type of C18 column. This problem appears to be due to undesirable (perhaps uncontrollable on this phase) secondary interactions. Are any of the problematic compounds chealators?

[zokitano]

Thank you Mr. Carl Sanchez!

The column is Atlantis T3, end-capped C18. I will check the chromatography without the guard column, although it is new as the analytical column.
The compounds have vicinal polar groups that could act as chelators, you're right about that. That is why I did complete passivization of the system when tried to solve the problem here. It is obvious that the method works on the same column (in the other lab), so I should find out what is the problem here.

I am trying several things at the moment, so I will come with more information.

Thanks for your help!

Best regards

[carls]

Unfortunately all columns and batches of sorbent are not "created equally." Sometimes youll get unlucky and come across a "special" batch of sorbent. Check to see if the batch/lot # of your column and the one that gives good chromatography are the same.

[Alp]

Carls is right.

Columns from different batches can perform differently.

And the column that works has NOT been tried on the system you are having the trouble with.
I have seen DEAD columns give good peak shape for some compounds, but put them on a new column and you have to start developing chromatography all over again!

I think you should try the column that works from the other lab on the equipment you are seeing the problem on.

Alp

[zokitano]

Unfortunately all columns and batches of sorbent are not "created equally." Sometimes youll get unlucky and come across a "special" batch of sorbent. Check to see if the batch/lot # of your column and the one that gives good chromatography are the same.

Just as information: I have two identical (by specifications) columns, the only difference is that one was used in the method development for the studied analytes and the second one is new. They belong to different batches. Before I started the LC-MS, I have used LC-UV and both columns gave me the same separation and very same retention times as well. The reproducibility is great.

I used the chromatographic parameters for LC-MS method (I am using it today) in the previous LC-UV analysis. In LC-UV chromatograms I could not see the excessive tailing or fronting of the peaks. Peaks were symmetrical and well separated. The concentration of the analytes injected in LC-UV was in the range of the low micrograms/mL.
In LC-MS I am working with ng/mL concentrations and some peak shape effects are more pronounced here?

Anyhow, to update the current knowledge about the problem:
- I cleaned extensively the whole MS interface: TurboIonSpray housing, TurboIonSpray probe, curtain plate, the interface (orifice), skimmer and Q0 region.
- I changed the ESI capillary with new one (the replaced was used regularly for more than a half year)

After these changes I start the analysis and the problem was still here (it is so reproducible that it goes on my nerves )

Then I removed the guard column and used only the analytical column, and the improvement is evident (and is reproducible):



Still there is some tailing/fronting for the problematic peaks, but at least now they are not so excessive. Probably there was something wrong with the new guard column or maybe the guard column holder (active site or something). I should confirm that when I'll get the new order of guard columns.

Anyway, for now, the problem is at least partially solved. I would like to try the chromatography with higher ionic strength (more than twice from that used) mobile phase to see if some secondary interactions are causing these "problematic" peaks to have visible tailing/fronting.

What do you think?

Thanks