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Dear all (especially the analytical chemist ones)
Plasma concentrations of a compound are measured, using a solid phase
extraction method according to the following procedure:
0.5 mL of human plasma was added with I.S. and 0.4 mL of 10 mM
Ammonium acetate, mixture was passed through SPE column (polymeric
sorbent). Column was washed with 1 mL of 0.6% MeOH and CVT-3146 was
eluted with 1 mL 90% MeoH. 90% MeoH eluant was evaporated to dryness
and reconstituted with 20% MeOH before LC/MS/MS quantification.
My question is: does this process measures total or free fraction only?
Toufigh Gordi
Associate Director of Clinical Pharmacology
CV Therapeutics Inc.
3172 Porter Dr.
Palo Alto, CA 94304
Tel.: 650-384-8929
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Total, because it denatures the protein.
lourdes
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I think that the solid phase extraction column should have a higher
affinity for the drug that the protein binding site has. Also the
hydrophobic group of the solid phase column will tend to displace the
drug from the protein binding site: thus I would expect that the
total amount of drug ends up on the solid phase column. The eluent
then removes the drug from the solid phase column; hence you have a
value for total drug present per mL plasma.
Best Regards,
Angus McLean Ph.D,
8125 Langport Terrace,
Suite 100,
Gaithersburg,
MD 20877
tel 301-869-1009
fax 301-869-5737
BioPharm Global Inc.
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Toufigh,
I would say that the only way to be certain is to perform equilibrium
dialysis on the sample prior to SPE. This will give you the free
fraction. There are some higher throughput devices for determination
of protein binding, but equilibrium dialysis is usually the most
reliable.
There is a good chance that the methanol will disrupt the protein-
analyte interaction, but it may not be quantitative given the short
exposure time in the SPE cartridge.
Hope this helps.
HH
--
Howard Hendrickson, Ph.D.
Assistant Professor
University of Arkansas for Medical Sciences
College of Pharmacy
Department of Pharmaceutical Sciences #522
Little Rock, AR 72205
Office: 501-603-1547
Fax: 501-526-4618
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Dear Toufigh:
Depends on how you prepared your standard curve; If
you it was prepared the same way as your samples then
you are measuring total. Just for curiosity, any
particular reason to use ammonium acetate?
Rostam
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What's your absolute recovery like when using this method?
Regards,
Frederik Pruijn
--
Frederik B. Pruijn PhD MSc (Senior Research Fellow)
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland
New Zealand
Phone: +64-9-3737 599 x86939 or x86090
Fax: +64-9-3737 571
E-mail: f.pruijn.at.auckland.ac.nz
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The following message was posted to: PharmPK
What's your absolute recovery like when using this method?
Regards,
Frederik Pruijn
--
Frederik B. Pruijn PhD MSc (Senior Research Fellow)
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland
New Zealand
Phone: +64-9-3737 599 x86939 or x86090
Fax: +64-9-3737 571
E-mail: f.pruijn.-at-.auckland.ac.nz
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Measures the total fraction - no doubt about that.
Yati Chugh Ph.D
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Dear Toufigh,
Total drug. It is all a question of binding equilibria.
Even if the affinity of the SPE material is not quite as high as that
for the protein binding site, the SPE capacity should be much
greater. Therefore, as the plasma passes down the column the binding
equilibrium will shift as drug partitions from the protein to the SPE
material.
Obviously if the koff from protein is slow (unlikely but
theoretically possible) then not all drug may come off in the short
time of passage through the column and you will get incomplete
recovery. Also, if the binding to protein is very tight and there is
insufficient capacity on the SPE material, then the recovery will be
a function of the two equilibria of protein <-> free drug <-> SPE, as
well as transit time.
In theory this could be modelled if one knew the binding constants!
Best regards, Phil.
Modelling & Simulation
Novartis Pharmaceuticals AG
CH-4002 Basel
Switzerland
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DearToufigh,
To my experience, proteins will denature in this process and the free
drug partitions into the SPE cartridge. Thus, one will get the total
plasma concentration but not unbound drug.
Further, when a standard curve is prepared and used to intrapolate
the samples (following exactly the same procedure), you will always
get the total drug present irrespsctive of extent of binding and
extraction efficiency (extraction efficiency only helps in improving
the sensitivity) because you are considering the total spiked drug in
the X-axis of Std curve.
Best regards
Varma Manthena
Pharmaceutical R&D
Nicholas Piramal Research Center,
Mumbai, India.
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