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I have seen some papers talking about using acetonitrile to extract
small molecules from the cells and precipitate the protein at the same
time. But I'm not sure if acetonitrile can be used to break the cells
or not. If not, what's the mechanism to use acetonitrile to extract
small molecules out of the cell
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The following message was posted to: PharmPK
You can break the cells with a mixture of methanol or acetonitrile
with or without a buffer (i.e.: tris buffer). You should choose a
percentage of methanol or acetonitrile suitable for analyte solubility.
Hope this help
Henri BENECH
CEA
Service de Pharmacologie et d'Immunoanalyse
Institut de Biologie et de Technologies de Saclay
France
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I know that methanol can be used to extract the drug out of cell (by
breaking the cell wall) without causing significant protein
precipitation. Its a volume dependent effect. I hope that acetonitrile
must be acting in a similar way.
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The following message was posted to: PharmPK
My two cents on the topic:
Cells can be lysed in almost any non-physiological medium (for example
perchloric acid, distilled water, 50% MeOH in water, 100% acetonitrile
and so on....) or by mechanical rupture (sonication, freeze-thaw
cycles, etc). However, for extraction purposes you need a medium in
which your analytes dissolves and which doesn't interfere with your
analytical method.There is no gold standard and there's only one way
to find which medium suits your analyte: try different methods and
mediums. Start with an extraction medium that is known to dissolve
your analyte.
Cheers,
Rob ter Heine
--
Rob ter Heine, MSc, PharmD
Department of Pharmacology, Slotervaart Hospital
Amsterdam, The Netherlands
E: rob.terheine.-a-.slz.nl
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Rob has provided an excellent summary of the different options
available: one has to perform a comparative evaluation of the
different methods. The goal is to achieve a high recovery of the drug
following extraction of the biological sample, while simultaneously
minimizing on cellular material, which can interfere with the assay of
the drug.
Simply spike the cells with the drug at a representative concentration
and split cellular material into different replicate volumes in test
tubes. Evaluate the different procedures using these samples.
For example once I worked on the assay of a drug in whole blood using
acetonitrile with ultrasonics followed by centrifugation.
Subsequently a colleague found that 6% perchoric acid provided the
same drug recovery but the sample was much "cleaner" when
chromatograms were compared.
Hope this helps;
Angus McLean
8125 Langport Terrace,
Gaithersburg,
MD 20877
* E-mail angusmdmclean.-at-.aol.com
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