Chromatography Forum

Hello all,

I try to transfer an HPLC method to our mass spectrometry department for the quantification of a penicillin.
I use 50mM of Ammonium Acetate + 5% of CH3CN at ph7 as mobile phase.
My colleague says that this method will not work in HPLC/ESI because there is too much ammonium adduct.
Is there a trick to decrease ammonium adduct ?
Is it really annoying if we perform a calibration curve befor each quantification ?

Thanks for your help,

Laurent

Hi,

Are you only quantifying penicillin? If so, it will be a lot easier to play around with the method. I would start by decreasing the salt concentration on machine or via an HPLC in your laboratory if you can detect the molecule by UV. If the k' value is reasonable as well as peak shape parameters, you should be fine. The problem with using that high of salt in the mobile phase is that it will 1) dirty the source quicker which will decrease sensitivity drastically over time and 2) form ammonium adducts with molecules in the sample instead of hydrogen adducts. For the second question, I always run a standard curve in the same run as my samples. If you don't, you have to run the samples with a quality check sample that is essentially a standard that falls within the range of an old standard curve you have performed. This is not an ideal scenario to pursue. In addition, you should certainly look into possible internal standards (heavy labeled penicillin or some similar molecule with a similar elution time.)

Hello all,

I try to transfer an HPLC method to our mass spectrometry department for the quantification of a penicillin.
I use 50mM of Ammonium Acetate + 5% of CH3CN at ph7 as mobile phase.
My colleague says that this method will not work in HPLC/ESI because there is too much ammonium adduct.
Is there a trick to decrease ammonium adduct ?
Is it really annoying if we perform a calibration curve befor each quantification ?

Thanks for your help,

Laurent

First off, 50 mM ammonium acetate is way higher than really necessary. LC-MS tends to have optimal signal enhancement in the range of 0-5 mM. A bit of suppression from additives is okay, because it helps even out the possible enhancement/suppression that can result from sample matrix. But above 5 mM you're really getting into strong suppression territory.

Now, it's actually quite common to quantify off the ammonium adduct for compounds that get a better signal on that channel. Around 10-20% of the pesticides I'm analyzing are best seen via the ammonium adduct. As long as you are controlling the amount of ammonium present, you will get consistent results. Is there a reason you would prefer not to use the adduct?

If neutral pH is not essential for chromatography or stability, you could use 0.05-0.1% formic or acetic acid as a modifier.