Immobilized human serum albumin: Liquid chromatography/mass spectrometry as a method of determining drug–protein binding

Abstract

A liquid chromatography/mass spectrometry methodology is described that enables the simultaneous estimation of the individual protein-binding affinities of a mixture of compounds. This approach has proved to be robust and enables a series of molecules to be ranked according to their protein-binding affinities within 20 minutes.

Application of High Performance Liquid Chromatography for the Measurements of Lipophilicity and Biomimetic Binding Properties in Drug Discovery

“…The best approach is to measure at pH 7.4, the pH of the plasma, and use isocratic conditions preferably without using an organic modifier in the mobile phase. As it was found by Tiller et al [44], the k/(k+1) proportion derived from the retention factor multiplied by 100 gave very close values to the reported plasma protein binding % for a variety of drug molecules even when injected together in a mixture and the chromatographic peaks were identified by MS. Valko et al [46] applied a fast iso-propanol gradient during the retention time measurements in order to reduce the analysis time for strongly bound compounds. The obtained correlation of the calibrated logarithmic retention time values showed acceptable correlation to the plasma protein binding data in the literature.…”

“…Several authors presented good correlations between the logarithmic retention factors measured on the HSA stationary phase and plasma protein binding of drugs obtained by equilibrium dialysis or ultra-filtration methods [43][44][45][46]. The principles of using immobilised target bio-polymers to measure drug affinity by HPLC have been reviewed by Bertucci et al [47].…”

Fast Gradient HPLC Method to Determine Compounds Binding to Human Serum Albumin. Relationships with Octanol/Water and Immobilized Artificial Membrane Lipophilicity

“…Although our HPLC based method measures only the HSA binding ability, it can aid compound selection at the early discovery phase. Previously published isocratic methods [13][14][15] can be time consuming to elute strongly bound compounds. Also, when the logK values are used the reproducibility of the data are dependent on the column to column reproducibility, while the calibration in our method takes care of the slight variations in the columns or HPLC systems.…”

“…[13][14][15] These methods are based on the assumption that the chemically bonded HSA retains the binding specificity and conformational mobility of the native HSA. Although it is assumed that the major binding sites of the HSA are intact, there are several nonspecific binding sites and other pharmacologically irrelevant interactions including the silica support that might contribute to the compound retention.…”

NMR in Pharmacokinetic and Pharmacodynamic Profiling

“…However, the throughput of these assays is limited because of the need to directly detect the free compound, typically through HPLC or radiometric detection. As a result, alternative methods that make use of chromatography, [29] mass spectrometry, [30] microcalorimetry, [31] and fluorescence spectroscopy [32,33] have been proposed for measuring the affinity constant of a compound for HSA. NMR has also been used to monitor compound binding to albumin, with the earliest report being from Oleg Jardetzky's lab in the study of penicillin binding to albumin.…”