Assays of ligand-human serum albumin binding using pulsed ultrafiltration and liquid chromatography-mass spectrometry.

Two approaches were utilized to increase the throughput of pulsed ultrafiltration assays of ligand binding to human serum albumin, reducing the volume of the ultrafiltration chamber and combining pulsed ultrafiltration with high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-MS). Affinity constants for binding of ligands to human serum albumin were determined using pulsed ultrafiltration with ultraviolet absorbance detection. The first affinity constants (Ka1) were measured for the binding of dansylsarcosine, dansylamide, 7-anilinocoumarin-4-acetic acid and warfarin, and were determined to be 1.8 x 105, 5 x 104, 8 x 104, and 2.0 x 105 M-1, respectively. The throughput of pulsed ultrafiltration analyses was tripled compared to previous pulsed ultrafiltration measurements by reducing the volume of the chamber. In addition, the use of LC-MS with pulsed ultrafiltration permitted the simultaneous comparison and rank ordering of ligand mixtures for binding to serum albumin. For example, the throughput of these pulsed ultrafiltration measurements was tripled by analyzing three ligands as a mixture.