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The following message was posted to: PharmPK
Hi,
Can somebody suggest a way to precipitate protein at
very high concentration?
The samples are from in-process and contain very high
preotein. We have to get rid of protein first.
Your help is highly appreciated.
Xiaodong
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The following message was posted to: PharmPK
Ice cold acetonitrile 4 volumes to one volume sample. Perchloric
acid also
works well, 1 part 0.4 M to 5 parts sample. Need to check the
stability of
your compounds to perchloric acid. Can ppt the perchloric with KOH if
needed.
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I think the best way will be ammonium sulfate precipitation or by any
other method of denaturation pptn
Swati
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The following message was posted to: PharmPK
Dear Xiaodong,
Have you tried searching the literature about protein precipitation
effectiveness?
"Optimization of protein precipitation based upon effectiveness of
protein removal and ionization effect in liquid chromatography-tandem
mass spectrometry." J Chromatogr B Analyt Technol Biomed Life Sci. 2003
Mar 5;785(2):263-75
Sincerely,
Rob ter Heine
--
Rob ter Heine, MSc, PharmD
Department of Pharmacology, Slotervaart Hospital
Amsterdam, The Netherlands
E: aprth.-at-.slz.nl
T: +31-20-5124737
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Hi Xiaodong,
I did seek for literature a few years ago and from my experiments -
acetonitrile, TCA and zinc sulfate were found to be optimal at
removing protein in their categories (>96, 92 and 91% protein
precipitation efficiency at a 2:1 ratio of precipitant to plasma,
respectively). Acetonitrile worked best for me due to it's
effectiveness in removing protein, minimal ionization effect in
positive ion TIS LC/MS/MS and decent chromatography obtained using RP
column.
I would say choose protein precipitants based not only upon their
effectiveness in protein removal but also on their compatibility with
the remainder of the assay. The literature reference mentioned by Rob
ter is a good one.
I hope this helps.
Anila
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The following message was posted to: PharmPK
Rob ter Heine,
I have this paper, but the problem is even I use 1:9
ratio of sample: ACN with 2.5% HCl, the precipitation
is not complete at very high protein concentration,
such as 150mg/mL.
Thanks for your info.
Xiaodong
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Ed,
Do you suggest use cold ACN alone or cold ACN with
Perchloric acid?
Thanks.
Xiaodong
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if you use acn with perchloric acid you will encounter a curious
phenomena
in the sample. sT Januarius' blood. It will start as a liquid then
gel. If
you shake it it will go back to liquid. Not good for autosamplers. Best
to use each independently/ need to centrifuge each at ~ 10xG FOR 5-10
MINUTES, 4C.
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The following message was posted to: PharmPK
Hi Xiaodong,
For a better protein precipitation always use ice cold
acetonitrile (1:2) or methanol(1:3) and keep tubes
after crashing on wet ice for 30 min or in -20*C
freezer. To get good results with methanol keep it in
-80*C freezer- Don't try this with acetonitrile it
would freeze. TCA would result in ion suppression, so
better avoid.
Hope this helps.
Jagdish
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The following message was posted to: PharmPK
Ed,
Thanks very much for your clarification. I really like
the idea using cold ACN.
Xiaodong
--
Jagdish.
After adding cold ACN and vortex, you put the tubes
into the refrigirator. Do you ever have solubility
issue for your analytes?
Thanks.
Xiaodong
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The following message was posted to: PharmPK
You may have problem if your drug is not having good
solubility in MeCN or methanol. Thanks for reminding
this.
Jagdish
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