protein precipitation

[sonal]

In my earlier post on "HPLC of a basic compound" I mentioned about the direct injection columns. I was not getting good results with it due to all the constraints on the mobile phases that come along with it. Hence I switched to the protein precipitation method.

However, my concern with this method is that if the drug is 95% plasma protein bound, would precipitating it with ACN make any difference? Would the drug be lost with the proteins?

[HW Mueller]

That´s what you have to find out. The ACN will drastically change your protein (denature it) which could also change the binding of the drug. But, if the drug is not soluble in your final ACN/H2O mix it will stick to the protein or vessel anyway.

[sonal]

When I spike my plasma sample for the standard curve I get almost 100% recovery. Would that be the same for in-vivo case when I take the blood sample from an animal? I was wondering that in such a case since the drug has more chance to interact with the plasma proteins could that in any way affect the results?

[HW Mueller]

If you are worried about this spike the whole blood immediatly after withdrawing. Nevertheless, there is always the possibility that something gets lost, metabolized, or whatever. If the literature doesn´t give a hint one has to check what different isolation methods do.