Ultrafiltration in Serum Protein Binding Determinations

The ultrafiltration technique was evaluated theoretically and experimentally for use in clinical serum binding determinations. It is apparent from free energy considerations that the ultrafiltrate concentration approaches the true free concentration only as the pressure gradient causing flow reduces to zero. The theory presented accounts for the previously unexplained lower ultrafiltrate concentration observed at higher filtration pressures. Mathematical simulations of the molecular separation show that the ultrafiltrate concentration remains constant during filtration, and, thus, binding equilibria are not disturbed by this procedure, suggesting that an arbitrary restriction on the volume filtered is unnecessary. This finding greatly extends the value of the ultrafiltration technique in clinical binding determinations, especially for strongly bound, potent drugs where assays may be insufficiently sensitive to detect the extremely small free fractions reliably. These theoretical findings were verified experimentally by ultrafiltration of salicylate, ibuprofen, and carprofen in buffer, purified proteins, and whole serum.