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I am intending to determine protein binding for a compound using an
filtration technique. What are some precautions that I should take to
get an accurate estimate of the free fraction?
[Binding to filtration device should be checked - db]
Hi Khalid,Back to the Top
The most important thing you have to consider in determining plasma
protein binding using ultrafiltration is not to disturb the equilibrium
Using ultrafiltration the filtration itself may influence your bound
versus unbound equilibrium, since you are filtering free compound from
the equilibrium. When you have compounds that bind moderatly to protein
this may lead to erroneous fraction unbound determinations. With
compounds that bind strongly to protein this effect may be negligible.
Separation times should be as short as possible.
These considerations are reviewed in Jenifer Wright et al., 1996.
Measurement and Analysis of Unbound Drug Concentrations, Clin.
Pharcokinet Jun 30 (6) 445-462. This paper I found very useful in
setting up a routine plasma protein binding assay.
I hope this information was useful to you,
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I hope you have taken the possible non-specific binding of the compound
the filter membrane into consideration. Depending on the solubility of
compound, you can run a preliminary study in PBS (Phosphate Buffer
against a control (usually in the solvent in which drug has high
This experiment would confirm the non-specific binding, if any. This
would avoid any misinterpretation of the results.
Hope this helps,
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