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Hello,
Let me start this by saying I have no training in pharmacology, so
forgive the naivety of my question. I am interested in detecting a
novel drug in mouse plasma by HPLC. I think it is important to know
whether I am detecting free or protein bound drug or both. I know
there are various means of precipitating plasma proteins. Can someone
explain to me the various options and how and why they differ,
especially with regards to whether I am going to be detecting total or
only unbound drug? I know this is likely to be a rather complex
answer, so if someone has a good reference or text that they could
point me to instead, that would be great.
Thanks so much in advance,
Noelle Williams
Noelle S. Williams, Ph.D.
Research Assistant Professor
Department of Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Boulevard
Dallas, Texas 75390-9152
phone: 214-648-0348
fax: 214-648-3346
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The following message was posted to: PharmPK
Hello Dr. Williams,
It is considered that the commanly employed techniques for plasma sample
preparation, such as sample clean up by precipitation (using dilute
perchloric
acid or acidified methanol)or extraction using liquid liquid or solid
phase
extraction results in measurement of combined (bound + free) form of
the drug
in plasma.
Hope it helps
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Generally, after L-L extraction, SPE or plasma precipitation you will
measure the total amount of drug (the drug is bound to proteins by
physico-chemical interaction, easy to disrupt)
The free and bond to protein drug ratio can be determined by dialysis.
For precipitation:
4 volplasma +1 volsol HClO4 12% in water -----check recovery and
stability !!
if recovery is low (acidic compounds), try
2 volplasma +1 volsol HClO4 12% inACN or MET, let stand 15 min
after precipitation !!
or
1 vol plasma + 3 vol ACN or MET
Vlase Laurian
Vlase Laurian Univ. Med. & Pharmacy "I. Hatieganu" Dept. of
Pharmaceutical Technology and Biopharmaceutics Cluj-Napoca Romania
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To determine free (unbound) drug concentrations, dialysis membrane
(using centrifuge) is the most convenient method. Basically, you
separate the free drug from the protein-bound drug using the membrane.
You can get the ready-to-be-used centrifuge tubes from Millipore or
other commercial source. The trick is some drug will stick to the
membrane and complicate the analysis. You can disgard the initial
filtrate and collect later fractions.
To determine total drug conc., there are several methods (LLE, protein
precipiation using ACN or TCA, SPE), however, make sure you determine
the extraction recovery using spiked samples and compare the different
extraction methods. A higher recovery (>70%) will minimize analytical
errors and improve reproducibility.
Regards,
D. Song, Ph.D.
PK/PD Online
www.pkpdonline.com
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)