A Sequential Ultrafiltration Method to Enhance the Accuracy and Throughput in Plasma Protein Binding Tests

Area of Science:

• Pharmacology
• Analytical Chemistry
• Biochemistry

Background:

• Plasma protein binding (PPB) is crucial for drug efficacy and disposition.
• Conventional ultrafiltration (UF) methods for PPB assessment are susceptible to non-specific binding (NSB) to devices, causing inaccurate results.
• Accurate PPB determination is essential for drug development and safety.

Purpose of the Study:

• To develop and validate a modified UF method, termed sequential UF, to mitigate NSB bias.
• To assess the simplicity, efficiency, and accuracy of the sequential UF method.
• To compare PPB results from sequential UF with a standard mass balance UF method.

Main Methods:

• A sequential UF method was developed, incorporating a 2-minute pre-UF phase to saturate NSB sites.
• The PPB of nine diverse compounds was measured using both sequential UF and conventional mass balance UF.
• Results were compared to evaluate the accuracy and reliability of the sequential UF approach.

Main Results:

• Sequential UF yielded PPB values highly consistent with the mass balance UF method (97.9%–113.8% fold difference, average 103.5%).
• No significant discrepancies were found between the methods for most compounds.
• Quercetin exhibited an unusually high PPB, noted as an exception.

Conclusions:

• Sequential UF effectively corrects for NSB in PPB assays.
• The sequential UF method offers a straightforward and efficient alternative to conventional techniques.
• This modified UF approach enhances the reliability of drug plasma protein binding measurements.