Advances in chromatography in the study of drug-plasma protein interactions

After entering human blood circulation, small-molecule drugs interact extensively with various plasma proteins, such as human serum albumin and α₁-acid glycoprotein. These interactions profoundly affect the distribution of drugs in vivo and the binding of drugs to targets, thus affecting the efficacy of drugs. In-depth investigation of drug-plasma protein interactions is of great significance for the optimization of drug properties, the development of new drugs, risk assessment, and combination therapy of drugs. Therefore, it is essential to develop highly efficient, sensitive, and accurate methods for elucidating drug-plasma protein interactions. Chromatography is a powerful tool with high throughput, high separation performance, and high sensitivity in the characterization of drug-protein interactions. High-performance affinity chromatography (HPAC) and capillary electrophoresis (CE) have been widely utilized in this field. These methods include the determination of the effects of the posttranslational modification of proteins on binding and the competitive binding of multiple drugs. In addition, various chromatographic methods are used to obtain interaction information such as the binding constant, binding-site number, and dissociation rate constant. In this review, the common strategies and recent advances in HPAC and CE in the study of drug-plasma protein interactions are briefly reviewed. The immobilization methods of proteins, the principles and applications of frontal analysis, zonal elution, ultrafast affinity extraction, peak profiling, and peak decay analysis are discussed for HPAC and affinity capillary electrophoresis (ACE) and capillary electrophoresis frontal analysis (CE-FA) for CE. HPAC relies on the fixation of proteins on the surfaces of chromatographic stationary phases by covalent linking or physical adsorption, followed by obtaining the drug-protein interaction information through a variety of chromatographic methods. In the frontal chromatography analysis, mobile phases with different concentrations of drugs are passed through the HPAC column to obtain different breakthrough times. The process can determine the number of drug binding sites and the binding constant of each site in the affinity protein with high accuracy. The zonal elution method can detect the drug binding sites on proteins using site-specific probes to determine whether there is competition between drugs and probes. The sample consumption and analysis time of the zonal elution method are much less than those in frontal chromatography analysis. The ultrafast affinity extraction method can inject complex samples, such as serum, into affinity columns to determine the free drug components. It can measure the combination and dissociation constants of drug-protein interactions by changing the chromatography flow rate. Peak profiling and peak decay analyses are both effective methods for investigating the dissociation of drugs and proteins. In CE analysis, the drug and protein samples are dissolved in an electrophoresis buffer, and their interactions are measured during electrophoresis with high accuracy and low sample consumption. However, the adsorption of proteins on the capillary wall can compromise CE performance. Common CE methods in drug-protein interaction analysis are ACE and CE-FA. ACE is usually performed by changing the effective mobility of drugs via the addition of different concentrations of proteins. This method has been widely used, and several variant techniques have been developed recently. CE-FA involves the sampling of a drug premixed at a known concentration with a target protein. Compared with other CE methods, CE-FA exhibits the unique advantages of high throughput, automatic online analysis, and the ability to determine high-order drug-protein interactions. Finally, the shortcomings of current chromatography methods are summarized, and the application prospects and development direction of chromatography technology in the field of drug-plasma protein interaction research are discussed.