Method Development of Enantiomer Separations by Affinity Capillary Electrophoresis,Cyclodextrin Electrokinetic Chromatography and Capillary Electrophoresis-Mass Spectrometry

 Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection     

色谱技术在药物-血浆蛋白相互作用研究中的应用进展

小分子药物进入人体血液循环系统后与人血清白蛋白(HSA)、α1-酸性糖蛋白(AGP)等血浆蛋白存在广泛的相互作用,这些相互作用深刻影响药物在体内的分布及其与靶标蛋白的结合,进而影响药物效应的发挥.深入探究药物与血浆蛋白间的相互作用对于候选药物的成药性优化、新药研发、联合用药的风险评控等意义重大.而发展高效、灵敏、准确的...     

Cation‐enhanced capillary electrophoresis separation of atropoisomer anions

CE was used to study the separation of the atropoisomers of four phosphoric acids and two sulfonic acids and the enantiomers of two phosphoric acids. All solutes are in their anionic forms in aqueous electrolytes. The chiral additives were two hydroxypropyl cyclodextrins (CDs) and cyclofructan 6 (CF6). The CDs were able to separate four solutes and the CF6 additive could separate only one: 1,1′‐binaphthyl‐2,2′‐diyl hydrogenphosphate (BHP). Since CF6 is able to bind with cations, nitrate of alkaline metals, Ba²⁺, and Pb²⁺ were added, greatly improving the BHP separation at the expense of longer migration times. There seems to be a link between CF6–cation‐binding constants and BHP resolution factors. Cation additions were also performed with CD selectors that are less prone to form complexes with cations. Significant improvements of enantiomer or atropoisomer separations were observed also associated with longer migration times. It is speculated that the anionic solutes associate with the added cations forming larger entities better differentiated by CDs.     

Chiral resolution of melatoninergic ligands by EKC using highly sulfated CDs

EKC methods for the enantiomeric resolutions of melatoninergic ligands were developed using anionic CDs (highly S-alpha-CD, highly S-beta-CD, and highly S-gamma-CD) as chiral selectors at acidic pH 2.5. The optimization of the various operational parameters (nature and concentration of the CD, phosphate buffer concentration, addition of organic modifiers in the BGE, and temperature) allows baseline enantioresolutions (superior to 2) in short analysis times (inferior to 7 min) for all studied analytes. Some analytical characteristics of the optimal method were then studied for each analyte: repeatability, linearity, and LOD and LOQ. Lastly, determination of the apparent binding constants for the 18 complexes formed between the six analytes and the three CDs led us to rationalize the complexation mechanisms.     

Fast enantiomeric separation of basis drugs by electrokinetic chromatography. Application to the quantitation of terbutaline in a pharmaceutical preparation

Electrokinetic chromatography (EKC) using micelles of bile salts alone or mixed with sodium dodecyl sulfate (SDS) and neutral, anionic, or cationic cyclodextrins (CDs) in the separation buffer has been employed in order to achieve fast enantiomeric separation of basic drugs. A study of the enantiomeric separation ability of these chiral selectors concerning four basic drugs (epinephrine, terbutaline, clenbuterol, and salbutamol) has been carried out under different experimental conditions. The best chiral selectors to perform the enantiomeric separation of these drugs were neutral beta-CD derivatives, specifically permethylated beta-CD PM-beta-CD. The effect of the PM-beta-CD concentration, temperature, and applied voltage on the enantiomeric resolution of the basic drugs was investigated. The use of a 25 mM ammonium acetate buffer (pH 5.0), 30 mM in PM-beta-CD together with an applied voltage of 20 kV and a temperature of 15 degrees C enabled the individual and fast enantiomeric separation of epinephrine, norepinephrine, terbutaline, clenbuterol, and salbutamol each one into its two enantiomers in less than 3 min. The EKC method was validated (precision and accuracy) to quantitate terbutaline in a pharmaceutical preparation, obtaining a limit of detection of 4 microg/mL.     

Separation of neutral and basic enantiomers by cyclodextrin electrokinetic chromatography using anionic cyclodextrin derivatives as chiral pseudo-stationary phases

Separations of neutral and basic racemates were performed using five different anionic cyclodextrin (CD) derivatives as chiral selectors, viz. carboxymethylated β-CD, β-CD phosphate sodium salt, sulfobutyl ether β-CD sodium salt, carboxymethylated γ-CD, and γ-CD phosphate sodium salt. For the separation of neutral racemates, an untreated fused silica capillary was employed and various neutral racemates were successfully separated. Since the pH of the buffer affected the electroosmotic flow (EOF), the resolution was improved by changing the buffer pH. A polyacrylamide coated capillary was employed for the separation of basic racemates to suppress EOF and to prevent adsorption of cationic analyte on the capillary surface. By choosing an appropriate type and concentration of anionic CD, about 40 basic racemates were successfully separated. Some rough binding constants of basic analytes with an anionic β-CD were measured to discuss the optimum concentration of the CD. The migration direction was dependent on the binding constants and the concentration of the CD. The analyte strongly bound to the anionic CD migrated towards the anode but the weakly bound one moved towards the cathode. Anionic γ-CDs were also very useful for the separation of basic enantiomers. Five neutral CDs were employed as chiral selectors to compare selectivity between charged and neutral CDs, and eleven racemates could only be resolved using anionic CDs. The separation of some basic racemates in human plasma was also described. The direct injection of plasma samples was possible for some enantiomers that did not interact strongly with plasma proteins.     

Cyclodextrins in capillary electrophoresis enantioseparations--recent developments and applications

Capillary EKC has been established as a versatile and robust CE method for the separation of enantiomers. Within the chiral selectors added to the BGE CDs continue as the most widely used selectors due to their structural variety and commercial availability. This is reflected in the large number of practical applications of CDs to analytical enantioseparations that have been reported between January 2006 and January 2008, the period of time covered by this review. Most of these applications cover aspects of life sciences such as drug analysis, bioanalysis, environmental analysis, or food analysis. Moreover, new CD derivatives have been developed in an attempt to achieve altered enantioselectivities and to further broaden the application range. Finally, efforts will be summarized that aim at an understanding of the molecular level of the chiral recognition between CDs and the analytes.     

System Peaks of Cyclodextrins in Capillary Electrophoresis

Using a running buffer containing cyclodextrins (CDs) and 2‐[N‐cyclohexylamino]‐ethanesulfonic acid (CHES), positive system peaks were observed in the analysis of a ganglioside mixture by CE‐UV. These system peaks were related to CDs in the running buffer because these peaks were also detected when a plug of solution devoid of CDs but having the same CHES concentration and pH as the running buffer was injected. Neutral CDs were separated owing to the formation of inclusion complexes with the anionic CHES ion. One possible explanation for the positive system peaks is that the anionic CD‐CHES inclusion complex is displaced by co‐ions with higher UV absorptivity.     

Recent innovations in the use of charged cyclodextrins in capillary electrophoresis for chiral separations in pharmaceutical analysis

A review is presented on the use of charged cyclodextrins (CDs) as chiral selectors in capillary electrophoresis (CE) for the separation of analytes in pharmaceutical analysis. An overview is given of theoretical models that have been developed for a better prediction of the enantiomeric resolution and for a better understanding of the separation mechanism. Several types of charged CDs have been used in chiral capillary electrophoretic separation (anionic, cationic, and amphoteric CDs). Especially the anionic CDs seem to be valuable due to the fact that many pharmaceutically interesting compounds can easily be protonated (e.g., amine groups). For that reason several anionic CDs are now commercially available. Cationic and amphoteric CDs are less common in chiral analysis and only a few are commercially available. Attention is paid to the most common synthesis routes and the characterization of the CDs used in chiral capillary electrophoretic separations. The degree of substitution in the synthesized CDs may vary from one manufacturer to another or even from batch to batch, which may have a detrimental effect on the reproducibility and ruggedness of the separation system. In Sections 4, 5, and 6 the applications of anionic, cationic, and amphoteric CDs for the chiral separation in CE are described. Many interesting examples are shown and the influence of important parameters on the enantioselectivity is discussed.     

Capillary electrophoresis-based analysis of phospholipid and glycosaminoglycan binding by human beta2-glycoprotein I

Human beta2-glycoprotein I (beta2gpI) is a phospholipid and heparin binding plasma glycoprotein involved in autoimmune diseases characterized by blood clotting disturbances (thrombosis) together with the occurrence of autoantibodies against beta2gpI. With the final goal of assessing autoantibody influence on binding interactions of beta2gpI we have studied the development of capillary electrophoresis (CE)-based assays for interactions of negatively charged ligands with beta2gpI. In the development of suitable conditions for analysis at neutral pH of this basic protein (pI about 8) we found the pH hysteresis behavior of fused silica surfaces useful since the protonated surface after an acid pre-wash counteracted protein adsorption efficiently in contrast to more laborious procedures including acrylamide/dimethylacrylamide coatings that did not permit analysis of this particular protein. This simple approach made estimates of heparin-beta2gpI interactions possible and the principle was shown also to work for detection of betagpI binding to anionic phospholipids. Utilizing the pH hysteresis effect may be a simple solution to the adsorption problems often encountered in analyses of proteins by CE.     

CE in anticancer metallodrug research--an update

With the current demographic development and the knowledge that the probability to be diagnosed with cancer increases with age, the search for new treatment options in cancer chemotherapy is of utmost importance for the society. Capillary electrophoretic methods have been applied in the last few years for studying the properties of metal-based drugs and drug candidates. Especially, the elucidation of the mode of action of such compounds could contribute significantly to design new drugs for overcoming the threat of cancer. This review article highlights the developments in metallodrug research applying CE during the last 4 years and follows a review from 2003 (Hartinger, C. G., Timerbaev, A. R., Keppler, B. K., Electrophoresis 2003, 24, 2023-2037). Most importantly the broadening of application areas of CE must be noted: especially the binding studies of metal complexes toward proteins (including the determination of association and rate constants), following redox reactions of metal complexes and their influence on the reactivity toward biotargets, etc. are important development areas of the last few years. In parallel with these new applications goes the usage of new or modified separation methods including microemulsion EKC or ACE, or the advantageous use of equipping the CE system with mass spectrometric detectors such as inductively coupled plasma (ICP) or ESI mass spectrometers (MS) for determining the degree of metallation of a protein or characterizing the adducts. Finally, upcoming requirements for expanding the method's application area are discussed including studies on new targets in the cell, analyzing real-world samples, methodological development, and contributions to improve the design of new anticancer agents.     

Analysis of drug interactions with serum proteins and related binding agents by affinity capillary electrophoresis: A review

Biomolecules such as serum proteins can interact with drugs in the body and influence their pharmaceutical effects. Specific and precise methods that analyze these interactions are critical for drug development or monitoring and for diagnostic purposes. Affinity capillary electrophoresis (ACE) is one technique that can be used to examine the binding between drugs and serum proteins, or other agents found in serum or blood. This article will review the basic principles of ACE, along with related affinity-based capillary electrophoresis (CE) methods, and examine recent developments that have occurred in this field as related to the characterization of drug–protein interactions. An overview will be given of the various formats that can be used in ACE and CE for such work, including the relative advantages or weaknesses of each approach. Various applications of ACE and affinity-based CE methods for the analysis of drug interactions with serum proteins and other binding agents will also be presented. Applications of ACE and related techniques that will be discussed include drug interaction studies with serum agents, chiral drug separations employing serum proteins, and the use of CE in hybrid methods to characterize drug binding with serum proteins.     

Characterization of basic drug-human serum protein interactions by capillary electrophoresis

Drug-protein interactions are determining factors in the therapeutic, pharmacodynamic and toxicological drug properties. The affinity of drugs towards plasmatic proteins is apparently well established in bibliography. Albumin (HSA) especially binds neutral and negatively charged compounds; alpha(1)-acid glycoprotein (AGP) binds many cationic drugs, lipoproteins bind to nonionic and lipophilic drugs and some anionic drugs while globulins interact inappreciably with the majority of drugs. In this paper, the characterization of the interaction between cationic drugs, beta-blockers and phenotiazines towards HSA, AGP, and both HSA + AGP mixtures of proteins under physiological conditions by CE-frontal analysis is presented. Furthermore, the binding of these drugs to all plasmatic proteins is evaluated by using ultrafiltration and CE. The results indicate that the hydrophobic character of compounds seems to be the key factor on the interaction between cationic drugs towards proteins. In fact, hydrophobic basic drugs bind in great extension to HSA, while hydrophilic basic drugs present low interactions with proteins and bind especially to AGP.     

Comparison of three methods for analyzing loureirin B and human serum albumin interaction using capillary electrophoresis

Loureirin B (LB), a bioactive drug, is widely used in the treatment of biological diseases. However, due to its poor solution in water, it is important to find the approach which helps LB to specific biological targets. As the most abundant protein in plasma, HSA plays the role of a carrier of numerous drug ligand. Thus, the interaction between LB and HSA was explored by ACE, CE frontal analysis, and pressure‐mediated ACE under simulated physiological conditions (pH 7.4). The binding constants were calculated as 13.14 × 10⁴ L/mol, 7.00 × 10⁴ L/mol, and 2.78 × 10⁴ L/mol for each method, respectively. At the same time, the binding site number (n = 1.429) could be only calculated by the CE frontal analysis method. Furthermore, good experimental repeatability was obtained by pressure‐mediated ACE with RSDs for retention times and peak areas within 2.149 and 1.228, respectively.     

Comparison of cyclodextrin-barbiturate noncovalent complexes using electrospray ionization mass spectrometry and capillary electrophoresis

Various noncovalent complexes between native and derivatized cyclodextrins (CDs) and barbiturates were studied using capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS). This paper involves the study of four aspects of CD-barbiturate noncovalent inclusion complexes. The first study focused on determining the formation of CD-barbiturate inclusion complexes in ESI-MS. This determination was accomplished by the comparison of migration data from CE with ESI-MS inclusion complex peak abundances, which were found to be complementary. The second study found the possibility of predicting native beta-CD mediated CE elution orders for barbiturates using data from ESI-MS. A third study focused on the formation of barbiturate inclusion complexes with derivatized beta-CD and gamma-CD. As part of this study, the effect of the extent of side chain substitution on native CD complexation behavior was investigated. The results indicated that the number of side chains on the CD does not affect the formation of barbiturate complexes with the hydrophobic CD cavity. Finally, a comparison of the hydroxypropyl-beta-CD-barbiturate and hydroxypropyl-gamma-CD-barbiturate complexes in CE and ESI-MS was made to study the relationship between strength of drug-CD binding and enantioresolution. The results from the above studies indicated that the gas phase and the solution state complexes showed comparable behavior indicating that similar interactions played a role in stabilizing these complexes. While it was possible to use the ESI-MS data to determine drug binding to the CDs, it was not possible to predict whether a separation of the enantiomers of a chiral barbiturate would occur. However, the ESI-MS data could be used to eliminate certain CDs from consideration as chiral selectors.     

Dispersive micro‐solid‐phase extraction combined with online preconcentration by capillary electrophoresis for the determination of glycopyrrolate stereoisomers in rat plasma

A simple and sensitive analytical method for four isomers of glycopyrrolate in rat plasma was developed using cation‐selective exhaustive injection‐sweeping cyclodextrin‐modified electrokinetic chromatography (CSEI‐Sweeping‐CDEKC) for online enrichment combined with dispersive micro‐solid‐phase extraction pretreatment. The CSEI‐Sweeping‐CDEKC was conducted on an uncoated fused silica capillary (40.2 cm × 75 μm) with an applied voltage of –20 kV. The electrophoretic analysis was carried out in 30 mM phosphate solution at pH 2.0 containing 20 mg/mL sulfated‐β‐cyclodextrin and 5% acetonitrile. Under these optimized conditions, the detection limit for racemic glycopyrrolate was found to be 2.0 ng/mL and this method could increase 495‐fold detection sensitivity compared with the traditional injection method. Additionally, the parameters that affected the extraction efficiency of dispersive micro‐solid‐phase extraction were also examined systematically. The glycopyrrolate isomers in rat plasma samples as low as 0.0625 μg/mL were able to be separated and detected by capillary electrophoresis with the aid of CSEI‐sweeping. The findings of this study show that the dispersive micro‐solid‐phase extraction pretreatment coupled with CSEI‐Sweeping‐CDEKC is a rapid and convenient method for analyzing glycopyrrolate isomers in rat plasma.     

Electrophoretic screening of ligands under suppressed EOF with an inert phospholipid coating

The applicability of dual injection CE for affinity selection of biopolymers that contain multiple binding sites is demonstrated. The efficient analysis of biomolecules such as carbohydrates and proteins, as well as pharmaceuticals by CE requires the reduction or elimination of nonspecific interactions with the capillary surface. Phospholipids are integral components of cell membranes and aqueous phospholipid liquid crystals adopt a bilayer structure on fused-silica. This phospholipid surface does not interact significantly with the following biomolecules: serum albumin, the 96-110 heparin binding domain of amyloid precursor protein (APP), polydisperse glycosaminoglycans, and variable chain-length oligosaccharides. Pharmaceuticals including five anionic nonsteroidal anti-inflammatory drugs, three cationic analgesics, and two cationic beta-blockers, also show minimal interaction with the surface. In addition, the use of a phospholipid coating suppresses EOF, which enables reversed-polarity separations, dual opposite injection CE, affinity screening via CE by dual opposite injection, and serial target-ligand injections.     

Enhancement of selectivity and resolution in the enantioseparation of uncharged compounds using mixtures of oppositely charged cyclodextrins in capillary electrophoresis

The enantiomeric separation of some nonsteroidal anti-inflammatory drugs (NSAIDs) was investigated in capillary electrophoresis (CE) using dual systems with mixtures of charged cyclodextrin (CD) derivatives. A significant enhancement of selectivity and resolution could be achieved in the enantioseparation of these analytes in their uncharged form by the simultaneous addition of two oppositely charged CD derivatives to the background electrolyte. The combination of the single-isomer cationic CD, permethyl-6-monoamino-6-monodeoxy-beta-CD (PMMAbetaCD) and the single-isomer polyanionic CD, heptakis-6-sulfato-beta-cyclodextrin (HSbetaCD) in a pH 2.5 phosphoric acid-triethanolamine buffer, was designed and employed for the enantioseparation of profens. The improvement in selectivity and resolution can be attributed to the fact that the two CDs, which lead to independent and enantioselective complexation with the analyte enantiomers, have not only opposite effects on the electrophoretic mobility of these compounds but also opposite affinity patterns towards the enantiomers of these compounds. Binding constants for these enantiomers with each CD were determined using linear regression approach, in order to be able to predict the effect of the concentrations of the two CDs on enantiomeric selectivity and resolution in such dual systems.     

Characterization of Interactions Between Fluoroquinolones and Human Serum Albumin by CE–Frontal Analysis

The binding of fluoroquinolones to the transport protein, human serum albumin (HSA), under simulated physiological conditions has been studied by capillary electrophoresis–frontal analysis (CE–FA). The binding of these drugs to human plasma was evaluated by using ultrafiltration and capillary electrophoresis. The free drug concentration [D]_f at each HSA concentration was determined by the plateau height in the electropherograms and the calibration lines. The binding constants of fluoroquinolones and HSA were estimated using nonlinear regression with origin 7.5 software. The fluoroquinolones were found to show low affinity toward HSA, with binding constants ranging from 1.73 × 10² to 5.40 × 10² M^?1. The percentages of protein binding (PB) for fluoroquinolones to HSA were between 8.6 and 22.2%, while the PB percentages for fluoroquinolones to human plasma were between 10.2 and 33.1%. It can be found that the PB percentages for fluoroquinolones to HSA are mostly lower than those for fluoroquinolones to human plasma. It suggests that HSA is the primary protein responsible for the binding of fluoroquinolones in human plasma. The thermodynamic parameters were obtained by CE–FA. The positive ?H and ?S values obtained by CE–FA showed that the binding reaction was an endothermic process, and the entropy drive the binding and hydrophobic interaction played major roles in the binding of fluoroquinolones to HSA.     

The Use of Dual Cyclodextrin Chiral Selector Systems in the Enantioseparation of Pharmaceuticals by Capillary Electrophoresis: An Overview

Cyclodextrin (CD) derivatives are the most efficient and frequently used chiral selectors (CSs) in capillary electrophoresis (CE). There are situations when the use of a single CD as CS is not enough to obtain efficient chiral discrimination of the enantiomers; in these cases, sometimes this problem can be resolved using a dual CD system. The use of dual CD systems can often dramatically enhance enantioseparation selectivity and can be applied for the separation of many analytes of pharmaceutical interest for which enantioseparation by CE with another CS systems can be problematic. Usually in a dual CD system an anionic CD is used together with a neutral one, but there are situations when the use of a cationic CD with a neutral one or the use of two neutral CDs or even two ionized CDs can be an efficient solution. In the current review we present general aspects of the use of dual CD systems in the analysis of pharmaceutical substances. Several examples of applications of the use of dual CD systems in the analysis of pharmaceuticals are selected and discussed. Theoretical aspects regarding the separation of enantiomers through simultaneous interaction with the two CSs are also explained. Finally, advantages, disadvantages, potential and new direction in this chiral analysis field are highlighted.