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Dear all,
The principle upon which protein binding acts is
hydrophobic-hydrophobic interactions. When we add organic solvent like
acetonitrile/methanol to plasma, the aqueous layer that is acting as a
shield between plasma proteins gets dissolved into acetonitrile and
the protein part of plasma will crash/precipitate. My doubt is how the
analyte gets extracted into organic solvent. My assumption is if a
drug is having considerable protein binding it will bind to some amino
acid moiety of the protein. So how the drugs gets extracted into
organic solvent. Kindly clarify my doubt?
Thanks in advance
With Regards,
Veeravalli Vijaya Bhaskar,
Research Associate,
Aurigene Discovery Technologies Limited,
Bioanalytical Division,
Bangalore,
Email: vijay_b.-a-.aurigene.com
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Hi Vijay,
My two cents:
The protein precipitates not only due to a "salting out" phenomenon
when you add the organic solvent, but also due to protein denaturation
(the three dimensional conformation of the protein is destroyed). If
the drug is binding based on hydrophobic-hydrophobic interactions or
even hydrogen bonding, I think that the drug will released from it
association with the protein. That is not to say that adsorption on
the surface of the denatured protein is not taking place. Accordingly,
if your drug is not very heat-labile, some analysts would go one step
further and heat the mixture (organic, aqueous, protein and drug along
with everything else in plasma) up to 40 degree for a few minutes to
make sure even adsorption is not causing the drug to be kept away from
the extracting solvent).
I hope this helps,
Murad Melhem, PhD
Assistant Director PK/PD
Cognigen Corporation
Buffalo, NY
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The following message was posted to: PharmPK
Hello again,
With final exams over, I am glad to help.
Do not view binding, partitioning and distribution, as some kind of
binary
static system; its graded (from a global point of view), with
everything in
a dynamic state of equilibrium. The proteins precipitate in the
presence of
ACN (primarily) due to an unraveling of its tertiary structure,
exposing a
rather hydrophobic core (tried to gather what you meant by "aqueous
shield"
in terms of a hydrophobic effect, but its not clear). That said, drugs
that
were in love with some nonspecific electrostatic binding sites, may
stay, or
may leave for the potentially more favorable microenvironment of your
water-miscible ACN/MeOH mix. It sounds as if you are hypothesizing an
irreversible association on the protein, much like a covalent bond,
but the
binding is on and off, and some drug is likely still attached to your
protein, even though you can recover it in your organic solvent.
Everything depends on the nature of the analyte, and can be understood
further via an exposition on free energy of binding, or rather the
magnitude
of a change in free energy.
Kind regards,
SHAWN SPENCER, PhD.
Assistant Professor of Biopharmaceutics
Dyson Bldg., Rm 227
College of Pharmacy and Pharmaceutical Sciences Florida A&M University
Tallahassee, FL 32307 shawn.spencer.aaa.famu.edu
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