poor chromatography

[Smk]

Hello everybody
I'm working on Clavulanic acid validation in plasma, using LC-MS/MS , I nternal standard used was ampicillin, mobile phase contain high percentage water about 90% and ACN10%
with C8 column
every thing is ok in terms of selectivity, sensetivity, precision and accuracy
the main problem is the poor peak shape of clav after short time period injection on the column(abou 3days) while very sharp peak for internal standard
I'm confused why this happened , knowing that extraction procedure was ppt with ACN and back extraction with DCM to water (final sample was water)

Thanks

[Poly Styrene]

I am a bit confused about your procedure. Are you performing stability validation, or what is the reason for repeat analysis over several days? More details and preferably a copy of your chromatograms, showing good and poor peak shapes would help us to help you. Both clavulanic acid and your internal standard have several ionizable groups so pH control and buffering could be important. What is your injection solvent? And I would not be terribly surprised if the stability of your analyte is pretty low, which could also explain your issues.

[Smk]

Actually I'm in validation phase, I prepare 2 fresh validation batchs in the same day, the last injected batch show poor peaks shape , and the reinjection for any other samples give also poor peaks
below 2 images for the sharp and poor peaks:

http://img4.imagetitan.com/img.php?imag ... rppeak.png
http://img4.imagetitan.com/img.php?imag ... orpeak.png

the injected solvent is water

[tkubowicz]

Hello

I'd check column first. It is possible that you're facing problem called "phase collapse" or "dewetting." (especially after few injections) Check other column that is able to handle high aqueous mobile phase.

Regards

Tomasz Kubowicz

[Smk]

Is it possible that the column problem affect one analyte without affect the other analyte