Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs

Abstract

In this study, two capillary electrophoresis-based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%.Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l-TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.

Sensitivity enhancement of capillary electrophoresis‐frontal analysis‐based method for characterization of drug‐protein interactions using on‐line sample preconcentration

“…The uncoated capillary was used throughout the whole study for simplification. In view of this fact, several washing procedures between the runs were tested, and finally, the washing step published by Mlčochová et al was adopted and slightly modified (see below) [35]. Further, the optimization of the volume of injected interaction mixture given by injection time and pressure was performed.…”

“…Further, the optimization of the volume of injected interaction mixture given by injection time and pressure was performed. Taking into account previous experience with CE-FA [4,7,10,35,36] and recommendations from the literature [5,37], hydrodynamic injection was used. Then, the HSA concentration used for the interaction study was optimized since the change in plateau heights between the drug without protein and the drug with protein is important for evaluating results.…”

Recent developments in capillary and microchip electroseparations of peptides (2021–mid‐2023)

“…The first one is based on the separation of the interacting species and the determination of their equilibrium concentration. It includes the Hummel–Dreyer method [149], the vacancy peak method [150], frontal analysis [151, 152], continuous frontal analysis [153, 154], and kinetic CE [155, 156]. The second class is based on the detection of a specific physicochemical property of the complexed analyte or its binding partner; mostly, it is the change of analyte effective mobility in the mobility shift ACE [151, 157] or change of migration times of analyte in the partial‐filling ACE [158–161].…”

Study of molecular interactions by nonequilibrium capillary electrophoresis of equilibrium mixtures: Originations, developments, and applications

“…ACE is a broad term that refers to the separation of substances that participate in specific or nonspecific affinity interactions during electrophoresis by CE [30]. Popular ACE methods include partial-filling ACE [31], mobility-shift ACE [32], the Hummel-Dreyer method [33], the vacancy peak method [34], vacancy-affinity CE [35], and frontal analysis CE [36]. the on-and off-rate constants, respectively) of protein-DNA interactions [38], and temperature in CE [39].…”