Plasma Protein Binding Assay2022-11-03T08:39:38-05:00


The goal of a clinical drug candidate is to reach its target in a sufficient amount to achieve the designed pharmacological outcomes. Once the drug is absorbed in the blood circulations via one of the administration routes, it may be bound to one of the main plasma proteins, such as albumin and beta globulin. This binding is non-specific, and many drugs may bind to the same protein because of the high concentration of the plasma proteins. Strong binding to the plasma proteins will minimize the drug’s free unbound concentration, and thus, higher drug concentration should be administered to achieve effective therapeutic levels. Therefore, determining the percentage of plasma protein binding affinity is an important step in the drug development process.

Visikol offers a panel of protein binding assays using state-of-the-art LC-MS/MS and technical expertise in the ADME field. We are using equilibrium dialysis, in which a compound is allowed to equilibrate between a plasma protein and a protein-free buffer, to determine the unbound percentage of the tested article.


MethodRapid equilibrium dialysis device (RED)
Plasma concentration10%, 50%, and 100% human plasma
Dialysis bufferPhosphate buffer saline (PBS)
Dialysis time4 hours
Test article concentration1-5 µM
Number of replicates2
Analysis methodLC-MS/MS
Data deliveryFraction unbound in 100% plasma recovery

Tested articles (1-5µM) in 100% human plasma are allowed to equilibrate with 10x PBS buffer at 37 oC in a rapid equilibrium dialysis device (Thermo Fisher Scientific). Following 4 hours of incubation, 50 µL of each compartment is taken and completed to a 100 µL volume with the opposite component. An internal standard solution (in acetonitrile) is added (400 µL) and then the samples are analyzed by LC-MS/MS. Data will be presented as a fraction of the unbound compounds to proteins (fu).


Warfarin, verapamil, and atenolol (1 µM in 100% human plasma solution) were individually equilibrated against 10x PBS at 37 oC for 4 hours. The concentrations of each compound were determined in the buffer and plasma champers, and the (fu) was calculated as follows:

% Free (fu) = (Concentration in the buffer chamber/Concentration in the plasma chamber) x 100

% Bound = 100-fu

As expected, both warfarin and verapamil showed high binding affinity to plasma proteins with fu values of 1.1% and 6.7%, respectively, while atenolol is not bound to plasma proteins (fu= 99%)

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