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

[Arne]

Inject a standard of Clavulanic acid. Do you still get poor peak shape. If yes, your column is somehow affected by the analyses over time. If no, your injection solvent may be off.

Try cleaning the column with organic solvent and re-injecting the samples and standards. Do you get good performance now? If yes, you might want to add a solvent wash at the end of the method or require it to be done once a day manually in the SOP.

Best of Luck!

Arne

[James_Ball]

I have seen problems before when using high aqueous mobile phases on C8 columns. Usually if you wash the column with 100% methanol and let it sit overnight, then re-equilibrate with mobile phase before analysis it will return to normal. The C8 has a tendency to collapse with extended use of high aqueous mobile phase but can be regenerated easily.

[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