Evaluation of Association Constants between Drug Enantiomers and Human α₁-Acid Glycoprotein by Applying a Partial-Filling Technique in Affinity Capillary Electrophoresis

Abstract

The principles for evaluation of conditional association constants between drug enantiomers and proteins, exemplified here by alpha 1-acid glycoprotein (AGP), using capillary zone electrophoresis employing a partial filling technique, is presented. In the partial filling technique only the first part of the capillary is filled with the selector, and this selector zone (plug) length can be varied by introducing the selector solution at different times at constant pressure. An important feature of the technique is the low consumption of selector solution in this study only 40-290 nL is used per run, of special importance when the availability of the selector is limited, and also in case it is expensive. Conditions are chosen so that the protein has a net negative charge and migrates toward the anode, while the analytes migrate toward the detector at the cathodic side. The resolution is linearly related to the effective plug length, as shown in separations of the enantiomers of disopyramide and remoxipride. The effective plug length can be calculated, which forms the basis to apply this technique for determinations of association constants. The association between the enantiomers of the solutes and AGP varied with increasing temperature, as shown by determined association constants. It was found that the association between the enantiomers and AGP was strongest at 25 degrees C and decreased at both lower and higher temperatures. This unexpected finding may indicate conformational changes of the protein with temperature variations.

Highly Sensitive Methods Using Liquid Chromatography and Capillary Electrophoresis for Quantitative Analysis of Glycoprotein Glycans

“…Previously lectin ACE have been reported to demonstrate the usefulness in the determination of the specific structure of oligosaccharides, but the concentration, components and pH of the electrophoresis buffers have limited to sustain activity of the lectin used, and the usage of protein-containing buffers can complicate analyses due to adsorption of proteins to the capillary, vials and electrodes. A partial-filling ACE [23] can be used to overcome these situations. Fig.…”

Enantiomeric Separation of Basic Drugs with Partially Filled Serum Albumin as Chiral Selector in Capillary Electrophoresis

“…The apparent mobility of drug in the presence of protein (µD,P) can be evaluated according to the following equation: 39,40 µD,P = X µ*DP + X µ*D + (1 -X)µD (1) where [ ] represents the molar concentration of each species in the solution. µ*D, µ*DP and µD are the mobility of free drug, drug-protein complex in the protein zone and that of free drug in the buffer zone, respectively.…”

Determination of association constants between enantiomers of orciprenaline and methyl-β-cyclodextrin as chiral selector by capillary zone electrophoresis using a partial filling technique

“…In a recent paper [1], the principles for determination of association constants between enantiomers of some basic drugs and a 1 -acid glycoprotein (AGP) as chiral selector by a partial filling technique (PFT) were described. PFT is a new separation mode initially employed for enantioseparations using a UV-absorbing chiral selector [2].…”

“…Increasing the plug length results in a reduction of the solute mobility, due to the longer interaction time between the selector and analyte. The degree of reduction is proportional to the affinity between the selector and the solute, and allows for determination of association constants [1]. It has also been shown that the enantioresolution is affected by the concentration of the chiral selector [17±19].…”