peak tailing and disappearing peak in LC/MS/MS

[cn-knutsch]

Hi, I've had some trouble with my LC/MS/MS system lately. Standard substances which have been analyzed with the same kind of column show great peak tailing. I tried to use new columns, cleaned the HPLC system, changed PEEK capillaries, cut the ends of PEEK capillaries (does anyone know a really good capillary cutter?) and and and... I did everything I could think of- without success. I'd appreciate it a lot if anyone had further hints for me.
Moreover, I've analyzed an unknown compound (pharmaceutical). I got pretty good peaks and they were stable for quite some time. Then all of a sudden another assay was run and the retention time was shifting in every run. Finally the peak diappeared. I prepared a new stock solution but still could not see any peak. According to the UV system the solution seems to be ok. I did a scan but the peak belonging to the right mass was extremely low and at the very end of the chromatogram. The bigger peak correlated to a different mass which did not make much sense. I tried to infuse it via the syringe pump and did the whole procedure to get a MS method. The MS method is ok (the signal is a little lower, though). However, I don't get a signal when using an HPLC column. I tried a new column, cleaned the whole system, prepared to eluents... but still there's no peak. I haven't tried a different column (different phase, different provider) yet. I'd be really greatful if someone could help me out and had some useful hints for me...
Thanks!!!

[Kostas Petritis]

Did you happen to change the injection solvent (what do you use anyways and what is your mobile phase and gradient)?

[cn-knutsch]

The compounds are analyzed in PBS (containing 1 to 2% DMSO) or after protein precipitation (diluted plasma plus ACN; 1:2). The eluents are: ACN and H2O, each with 0.2% formic acid. The standard gradient is:

95% H2O for 0.5 min.
linear gradient to 10% H2O in 2 minutes
hold for 0.5 min.
back to 5% H2O in 0.5 min.
total run time 6 and 7 min, respectively

I tried to different eluents (ammonium acetate, water plus acetic acid, methanol instead of ACN...) but it didn't help. These conditions worked pretty well for some years.

[Kostas Petritis]

I see several potential problems with your method.

1) If you really use 1:2 diluted plasma:ACN, your injection solvent is too strong and you should expect problems of the nature you described.

2) Your linear gradient is too steep and total re-equilibration time probably insufficient (extrapolating from the total run time given). Actually depending your system delay volumes the re-equilibration time might or not be sufficient. That might explain the differences when you either make differences in your current LC system or trying to transfer your method to "equivalent" LC systems.

I am not quite sure but you seem to imply that you are using a nanoLC system? If yes, then delay volumes might be even of a higher concerns and small things like by-passing or not the injection loop after sample loading can make a huge difference.

Bottom line, I would use a much lower eluting strength injection solvent and I would allow higher re-equilibration times as first steps for trouble shooting the problem.

[cn-knutsch]

Hi,

thanks for your input... but actually I doesn't make sense to me why all of a sudden the method described shouldn't work anymore... I took over the method my predecessor used for quite some years. We never really had trouble with it when running our usual assays. Of course, I expected the peaks to become broader when running the protein precipitated samples. But the broadening was still acceptable until a couple of weeks ago. As I mentioned before I tried to change the gradients but without much success... Besides for a couple of samples I don't get signal at all anymore when coupling the MS to the HPLC. It's a regular HPLC system (no nano-LC) and it has been working alright before all of sudden the peak intensity got worse and some other compounds can't be seen at all anymore... I'm just a little worried about the MS system. The vaccum has been about 2.7 x10^5 Pa and whenever it increased to 2.8 or 2.9 x 10^5 the signal intensity got worse. Right now the vaccum remains at 3.0 x 10^5 and even though we have cleaned it several times we can't get it too drop again. Might this be a reason for the missing peaks?

[cn-knutsch]

I forgot to mention: We can't even see the compound anymore in PBS (2% DMSO)... It's totally gone (we tried longer equilibration times before but weren't successful either)...

[sassman]

You should try to determine if the problem is with the MS or the LC system. If your compound responds to UV, you could try replacing the MS with a UV detector and run a high enough concentration to show up by UV. If you get reproducible and reasonable retention time and peak area, then your LC should be OK. For MS, you should try doing a mass and resolution calibration to see if that helps. Also make sure that none of the settings on the MS have been inadvertently changed.

[lc_k]

Dear cn-knutsch (or others!),

you posted a question a couple of years ago about a sudden unexcepcted problem with LC system (broad/missing chromatographic peak). I was wondering how things proceeded - what was the cause? I am now having a similar problem. Only the late eluting compounds are affected. A replacement of the column did not help nor did the cleaning operations I have carried out. Thank you for any advice!

[Ewelelek]

Dear cn-knutsch (or others!),

you posted a question a couple of years ago about a sudden unexcepcted problem with LC system (broad/missing chromatographic peak). I was wondering how things proceeded - what was the cause? I am now having a similar problem. Only the late eluting compounds are affected. A replacement of the column did not help nor did the cleaning operations I have carried out. Thank you for any advice!

Hi everyone,

I have a similar problem. I'm working with antibiotics in solid samples. I have been using the same method for some time, now the late eluting compounds are not present anymore.
What was the reason of your problems?