Plasma protein binding plays a critical role in drug therapy, being a key part in the characterization of any compound. Among other methods, this process is largely studied by ultrafiltration based on its advantages. However, the method also has some limitations that could negatively influence the experimental results. The aim of this study was to underline key aspects regarding the limitations of the ultrafiltration method, and the potential ways to overcome them. The main limitations are given by the non-specific binding of the substances, the effect of the volume ratio obtained, and the need of a rigorous control of the experimental conditions, especially pH and temperature. This review presents a variety of methods that can hypothetically reduce the limitations, and concludes that ultrafiltration remains a reliable method for the study of protein binding. However, the methodology of the study should be carefully chosen.

The aim of the present study was to assess the suitability of different Amicon Ultra and Centrifree ultrafiltration devices for the study of the plasma protein binding process in the case of carvedilol, a highly protein-bound and lipophilic beta-blocking agent. Samples at different levels of concentration were prepared in both proteic and non-proteic matrices (human plasma, 5% human serum albumin solution and saline solution) and subjected to the classical ultrafiltration method using the different devices considered. Furthermore, an attempt to apply a previously described modified ultrafiltration method was also made. The analysis and quantification was achieved using a validated LC-MS/MS method. For the Centrifree devices, the determined unbound fractions of carvedilol and the corresponding binding degree were in accordance to literature data, while for the Amicon Ultra devices a great degree of carvedilol adsorbtion to the sample reservoir was observed, the analyte not being detected in the ultrafiltrate samples. Thus, it was further demonstrated that the type of ultrafiltration device used has a significant influence on the outcome of a plasma protein binding study. In the case of carvedilol, the evaluation of the protein binding interaction could be achieved using the Centrifree ultrafiltration devices, but not the Amicon Ultra devices.