Chromatography Forum

HI,

We all starting to use protein precipitation plate instead of centrifugation for bioanalytical work. Is there a known best way to use it? I know the plasma is usually added last but I am still getting high ion suppression and not good reproducibility. Any help is welcome.

What is the plasma sample volume and ratio of ACN or MeOH to Plasma that you are using. Additionally what is the retention time of your analyte and column dimensions/mobile phase comp.

The plasma/ACN is 1:3 and the molecule can trap easily in the 2mm C18 RP. I am just anot getting consistent results even from the internal standard

2mm by what length? What gradient?

I'm assuming that your using a large (>100ul) volume of plasma if its a 3:1 ratio maybe, if sensitivity allows, decrease plasma volumes and increase the acetonitrile volume or find a different I.S. assuming your not using an labelled I.S. or a derivative with high structural similarity? Alternatively if your using ESI switch to APCI as matrix effects are less severe with this ionization mode. Finally, if you try or have tried this use the merkl (I think thats the right spelling and gives credit to the group whom developed it) test to see where your anlalyte/I.S. sits in relation to the suppression zone of your injection volume and fiddle with your chromatography if possible. Hope this helps.

Protein precipitation does give you ion suppression, and it has little to do with insufficient precipitation of the proteins. It has to do with ion suppression due to a large number of coeluting compound, that actually may vary from sample to sample.

To get around ion suppression, you have several choices. You may be able to get around it by using another LC/MS method (e.g. HILIC, if suitable). You may use solid-phase extraction or liquid-liquid extraction. Among solid phase extraction, you have several methods available. A simple reversed-phase method, a more complex pH-solvent reversed-phase method, and a simple reversed-phase ion-exchange method on a mixed-mode sorbent. I have listed them in the order of increasing sample cleanliness, i.e. less ion suppression.

I can send you an article on this.

fishin' addict

could you explain what you mean by 'use the merkl' ?


regards Bert

I'm just referring or attempting to refer (most likely without accuracy) to the ion-suppresion experiment where analyte in appropriate solution is post-column infused with a syringe pump into a mixing-T while matrix blanks are injected and the resulting matrix induced suppression effect evidenced on the resulting chromatogram. The name may not be exact, but with respect to the party that I believe may have first published this test

I call them "ion suppressograms".

Okay I get the point I went to far on the citation I'll try to quote MG from Now on with ion suppressograms. Nice one