By Jim on Tuesday, November 11, 2003 - 02:10 pm:
I have one polar acidic compound which has very low plasma recovery (about 20%) even using the simpest protein precipitation (PPT) and filtration procedure. Where did the compound go? Does it stick to the wall or simply lost during the filtration process? Thanks.
By Anonymous on Thursday, November 13, 2003 - 03:31 am:
Which kind of filters do you use? Often filtration is a very critical process for recovery, even when using special filters for analytical purposes. Try a filter material that is known to be free of basic functions and is low adsorptive. For control you can try to elute putatively adsorbed analytes changing to harsher conditions like diluted acid (that certainly have to be compatible with the filter material).
Another problem could be strong adsorption to proteins under precipitation conditions. You can try to use different protocols like organic solvents (there are many different protocols), TCA, heat etc. With these procedures you can also try to elute adsorbed analytes from the pellet by washing procedures for control.
And there´s certainly the possibility that your compound is chemically unstable under the conditions used, so a little more information would be useful.
By Einar Pontén - SeQuant AB on Friday, November 14, 2003 - 01:33 pm:
Yes, I agree. Adsorbtion to the precipitate is quite likely and it constitute a "surface" just like the filter device.
By Jim on Friday, November 14, 2003 - 04:05 pm:
Can you give an example on how to minimize the adsorption to proteins under precipitation conditions? Thanks.
By Uwe Neue on Saturday, November 15, 2003 - 03:37 pm:
Jim,
How do you do the protein precipitation? In SPE, the addition of acid or base is used to eliminate the binding of drug to protein. I would assume that one can do the same thing with PPT, but I have no experience with PPT.
By HW Mueller on Monday, November 17, 2003 - 12:09 am:
This has been discussed before, in short: If your analyte is not soluble enough in H2O it will stick to the protein (maybe at a different place than originally) or to the vessel, the filter (as mentioned above)..., if protein precipitation is done by TCA or other materials (ammonium sulfate, etc.) which do not influence analyte solubility much. Either do the precipitation with MeOH or ACN, etc., or add these solvents (probably lesser amts) together with TCA....