Chromatography Forum

Hi,


We are trying to transfer a method for some antibiotics from HPLC-ECD (which gave us some major troubles to equilibrate) to UPLC-UV.
When we go low enough for our wavelength and with the sensitivity of the UPLC we are able to detect impurities at 0.05%.

But now we have to validate specificity and in the EP method for the HPLC-ECD method we have a system suitability sample with example chromatogram which shows a few impurities (eg imp A, imp B and imp C).

How can we find and identify this impurites in our new UPLC method?
The system suitability is a mixture of imp A, B and C, so it's not possible to inject them pure.

Is there a way to tackle this problem?


Best regards

Ace

Hi Ace,

One possibility is to separately collect the A, B and C fractions, separating them on your current system and inject them onto the UPLC system (separately).
Potentially you’ll need to load more of the system suitability sample than you use to, under the isolation procedure. Or collect more fractions and evaporate some of the liquid/eluent.

Best Regards

Have all the Impurities the same amounts? If not, compare amounts.

Alex

Dear Alex,

although they have not the same amount, it's a bit tricky to compare the responses of ECD with UV. But it could be an indication.


Ace