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Question regarding protien precipitation effeciency.
Is there a study out there that performed experimented on how much
organic reagent should be used to precipitate the proteins out of
plasma? My practice has been at least 3x organic to plasma but is
there a study out there that talks to this?
also,
when performing recovery studies the FDA guidance doc talks about
comparing response from analyte added to and extracted from a matrix,
to a pure authentic standard. in the next sentence is does talk about
extraction effiency but it doesn't read well. The question is, would
a correct recovery study be running your QC samples against samples
that you spiked AFTER you extracted blank plasma samples. for
example, extract blank plasma just like in your procedure, take the
supernatent out and then spike in your known analyte. run these
samples against each other.
any thoughts?
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The following message was posted to: PharmPK
Absolute recovery would compare material spiked in mobile phase or
buffer compared to sample matrix.
Usually the folks selling centricon tubes(AMICON) or empore protein
extraction disks (3M)willhave that info. Usually 3-3.5 volumes of ice
cold ACN works well. Some combine it with MeOH. Some use chloroform
(3 layers protein in the middle. PCA with KOH neutralization works well
but it is complicated.
Ed O'Connor, PhD
Technical Director, Immunoanalytical
Tandem Laboratories
115 Silvia Street
West Trenton, New Jersey
609-228-0243
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The following message was posted to: PharmPK
One paper that discussed various precipitants and ratios is Polson et
al. 2003 J. Chromatog. B, 785, 263-275.
Regards,
Adrian Sheldon, Ph.D.
Assoc. Dir., In Vitro ADMET and Immunochem.
Charles River Labs.
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)