Ultrafiltration

[chhubert]

Recently I use some ultrafiltration membrane filter for removing large protein molecules from my tissue extract for drug analysis. Some of polar drugs are found to have low recovery. Knowing that it needs some "passivation" for the membrane prior to filtration to avoid this. Any one has the experience of using it and how? Thanks.

[HW Mueller]

Addition of some methanol prevented the substances which I ultrafiltered from attaching to the protein or membrane.

[Keaka]

Your drugs obviously have some degree of protein binding to what is being separated by ultrafiltration if that is the case. Adding 6M Urea should hopefully disrupt the proteins themselves (and thus binding) prior to ultrafiltration.

The other option would just be to do a protein precipitation (methanol, acetonitrile) extraction.

[HW Mueller]

If chaotropes like urea don´t increase the solubility of the analyte in the aqu. phase you will still loose analyte.

[chhubert]

Thanks. I have tried methanol with no avail. Some says passivating the membrane with some non-ionic surfactants, e.g. Tween 80, PEG-800 etc. Have someone tried that before?

[HW Mueller]

Reading the original post again I wonder whether I understand it correctly. You have low recovery of polar substances only, nonpolar recover ok? That is the opposite of what one would expect. Did you dissolve the "drugs" in aqu. solutions, without protein, and ultrafilter, what is the recovery then? What exactly did you do with the MeOH? What is your membrane?

[chhubert]

I used Millipore Ultracel UF membrane (10000 MWCO). Basically I analysed polar drugs only in tissue extract. After ultrafiltrating the extract (containing methanol and acetonitrile), lots of polar drugs lost. I did perform similar test with drugs in 50% methanol with more or less the same results. Any suggestions?

[HW Mueller]

It is senseless to make any further suggestions unless I know how aqueous solutions (standards) of these drugs behave on ultrafiltration.

[Friederike]

Hi chhubert,
maybe the following publication would be interesting for you:
B.X.Mayer et al., Electrophoresis 2000, 21: 1558-1564

The authors faced a similar problem with the polar antimicrobial agent cefpirome with filter units from one manufacturer but not from another. They ascribed the problem to wetting materials on the filter membrane.

If you have no access to this paper, contact me by e-mail and I will send you the pdf.

[MaryCarson]

I suspect, as with any other filter, you will see differences from one drug to the next in recovery. Years ago I used Amicon ultrafilters to remove protein prior to analyzing tetracyclines in milk. The mild acid extraction was not sufficient to precipitate all protein. I did prewet/rinse the filters, partially to remove the protective glycerin, and partially to ensure constant pH through filtration. It worked fine for TCs, but I'm not sure I would have trusted it for fluoroquinolones or aminoglycosides (have not personally tried it, though).

I do not believe your problem is solely because of drug binding to proteins, though that might contribute. I do believe that membranes are not always completely inert to all compounds.

These extracts would be going onto a mass spec, right? I would think that would rule out use of most chaotropic agents, but again, I have never tried it.

[HW Mueller]

There is nothing mysterious about filters. There should be conditions for most compounds under which there is no adsorption. The proper conditions can easily be ascertained via standard solutions.
It appears that some people, in trying to determine absolute recovery, forget to wash the filter and protein retantate which has variable amounts of liquid that contains analyte.

[chhubert]

Thanks all for the replies. I did attempt to improve the recovery or to reduce the adsorption by soaking the membrane filter overnight with 5% SDS or PEG or sorbitol in water. Some analytes (e.g. coccidiostats) are still missing by passing standard solution (in ~50% methanol) thru it. It may be true that the regenerated cellulose membrane still contains some "active sites" that absorb polar drugs.

[HW Mueller]

Does MeOH increase the solubility of coccidiostats? What sort of compounds are these? Did you ever wash, extensively, the filters with analyte solvent before applying the analyte. Did you wash after applying the analyte?