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 ultrafilt ration assays of ligand binding to human serum albumin, reducing the volume of the ultrafiltration chamber and combining pulsed ultrafiltration with h igh performance liquid chromatography-atmospheric pressure chemical ionizat ion mass spectrometry (LC-MS). Affinity constants for binding of ligands to human serum albumin were determined using pulsed ultrafiltration with ultr aviolet absorbance detection. The first affinity constants (K-a1) were meas ured for the binding of dansylsarcosine, dansylamide, 7-anilinocoumarin-4-a cetic acid and warfarin, and were determined to be 1.8 x 10(5), 5 x 10(4), 8 x 10(4,) and 2.0 x 105 M-1, respectively. The throughput of pulsed ultraf iltration 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 an d rank ordering of ligand mixtures for binding to serum albumin. For exampl e, the throughput of these pulsed ultrafiltration measurements was tripled by analyzing three ligands as a mixture.