Investigation of Induced Peak Phenomenon in Capillary Electrophoresis with Electrochemiluminescence Detection

Abstract

Induced peak phenomenon in capillary zone electrophoresis with electrochemiluminescence detection for chiral separation of racemic phenylalanine mixture employing sulfated‐β‐cyclodextrin as chiral selector and acetonitrile as organic additive in the separation buffer was observed. Various experimental parameters influencing the intensity and the position of the induced peak were systematically investigated to find out the truth of the induced peak. Based on the experimental evidence, a reasonable mechanism involved in the formation of the induced peak was proposed. We found out the induced peak resulted from physical interactions between the components in the separation buffer and the injected sample during the electromigration process rather than chemical complexation interactions. Furthermore, suggestions to avoid the appearance of induced peak in capillary zone electrophoresis with electrochemiluminescence detection for chiral separations were presented.     

Enantioselective capillary electrophoresis for pharmacokinetic analysis of methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine in equines anesthetized with ketamine and isoflurane

An enantioselective assay for the determination of methadone and its main metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine in equine plasma based on capillary electrophoresis with highly sulfated γ‐cyclodextrin as chiral selector and electrokinetic analyte injection is described. The assay is based on liquid/liquid extraction of the analytes at alkaline pH from 0.1 mL plasma followed by electrokinetic sample injection of the analytes from the extract across a buffer plug without chiral selector. Separation occurs cationically at normal polarity in a pH 3 phosphate buffer containing 0.16% (w/v) of highly sulfated γ‐cyclodextrin. The developed assay is precise (intra‐ and interday RSD < 4% and < 7%, respectively), is capable to determine enantiomer levels of methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine in plasma down to 2.5 ng/mL, and was successfully applied to monitor enantiomer drug and metabolite levels in plasma of a pony that was anesthetized with racemic ketamine and isoflurane and received a bolus of racemic methadone and a bolus followed by constant rate infusion of racemic methadone. The data suggest that the assay is well suited for pharmacokinetic purposes.     

α-萘基缩水甘油醚对映体的毛细管区带电泳手性拆分

建立了毛细管区带电泳手性拆分α-萘基缩水甘油醚对映体的方法.考察了不同手性拆分试剂对手性选择性的影响,实验结果表明,20 mmol/L H3PO4-三乙醇胺(pH 2.5)、2%(w/V)HS-β-CD、毛细管温度20 ℃、运行电压-18 kV为最佳分离条件,在该分离条件下α-萘基缩水甘油醚对映体实现基线分离.方法简便、准确,可用于α-萘基缩水甘油醚的手性拆分和对映体过量值(ee,%)测定.     

Enantioseparation Of Aromatic Dipeptides Using Carboxymethyl-β-Cyclodextrin Polymer As Chiral Selector By Capillary Electrophoresis

ABSTRACT

Chiral separation of peptides is of interest because of the different biological activity of enantiomers. In this report, several underivatized dipeptides with benzene moieties were optically resolved by employing carboxymethyl-β-cyclodextrin polymer(CM-β-CD polymer) as chiral selector. The effects of different cyclodextrin types, selector concentration, buffer pH, and organic additive were examined. Selector concentration and buffer pH played significant roles in resolution. Enantioseparation was found to be negatively influenced by adding the organic additive into running buffer and even completely lost at the organic additive content of 16%. It was also noted that the dipeptides with short chain in the vicinity of the second chiral carbon atom showed better chiral resolution by using CM-β-CD polymer than by using either carboxyethyl-β-CD or succinylated-β-CD. Simultaneous chiral separation of a mixture of DL-Ala-DL-Phe and DL-Leu-DL-Phe was also obtained using 27 mg/ml CM-β-CD polymer in the running buffer at pH5.12.     

Optimization of a capillary electrophoresis method to determine the chiral purity of a drug

Capillary electrophoresis (CE) using hydroxypropyl-β-cyclodextrin (HP-β-CD) in the separation buffer was investigated to determine the overall chiral purity of a drug containing a single stereogenic center. The effects of primary factors —pH, buffer components, buffer concentration, cyclodextrin concentration and sample amount (concentration and injection volume) — on the resolution of the enantiomers were investigated. Secondary factors such as the HP-β -CD source, lot and degree of substitution that were expected to affect the robustness of the assay were investigated also. The linearity, quantitation limit for the trace enantiomer and the precision of the measurements were determined. This study shows that understanding and optimizing the assay conditions leads to a chiral CE separation that is comparable to that obtained by chiral HPLC. However, chiral CE separations achieved with buffer additives have the advantages of shorter run times, higher numbers of theoretical plates (greater resolution), smaller amounts of chiral additive (less cost) and greater ruggedness (separation virtually independent of column properties unlike HPLC).     

Capillary electrophoresis and molecular modeling of the chiral separation of aromatic amino acids using α/β-cyclodextrin and 18-crown-6

In this work, chiral separation of enantiomers of three amino acids was achieved using capillary electrophoresis technique with α-cyclodextrin (αCD) as a running buffer additive. Only tryptophan has exhibited baseline separation in the presence of αCD, while the enantiomers of the other two amino acids, phenylalanine and tyrosine, were only partially separated. The addition of 18-crown-6 (18C6) as a second additive imparted only slight improvement to the separation of all enantiomers. On the other hand, all three racemic amino acid mixtures demonstrated no indication of separation when the larger cavity cyclodextrin members, β- and γCD, are used as running buffer chiral additives. However, remarkable improvements in the separation of the enantiomers of phenylalanine and tyrosine were obtained when 18C6 is used together with βCD as a running buffer additive. Surprisingly, tryptophan enantiomers were not separated by the dual additive system of cyclodextrin and crown ether. Using electrospray ionization mass spectrometry (ESI-MS), all amino acids were found to form stable binary complexes with individual hosts as well as ternary compounds involving the crown ether and the cyclodextrin. Furthermore, we used molecular dynamics (MD) simulations to build a clear picture about the interaction between the guest and the hosts. Most of these complexes remained stable throughout the simulation times, and the molecular dynamics study allowed better understanding of these supramolecular assemblies.     

Use of various β‐cyclodextrin derivatives as chiral selectors for the enantiomeric separation of ofloxacin and its five related substances by capillary electrophoresis

A capillary electrophoretic method for the enantioseparation of ofloxacin and its five related substances (potential impurities, indicated as impurities B–F) was developed using β‐cyclodextrin derivatives as chiral selectors. To our knowledge, there are no previous studies about using capillary electrophoresis for the separation of impurities B–D. Six β‐cyclodextrin derivatives including cationic (piperidine‐ and cyclohexylamine‐), neutral (dimethyl‐ and hydroxypropyl‐), and anionic (carboxymethyl‐ and sulfated‐) β‐cyclodextrin derivatives were tested and operational parameters such as buffer pH and concentration of β‐cyclodextrin derivatives were investigated. The best resolutions were all obtained with anionic β‐cyclodextrin derivatives: ofloxacin, impurities C–F could be best resolved with carboxymethyl‐β‐cyclodextrin at satisfactory resolutions of 8.27, 9.98, 5.92, 8.49 and 6.78, respectively, while for impurity B, a particularly impressive resolution value, up to 21.38, was observed using sulfated‐β‐cyclodextrin. The enhancement of enantioseparation observed for the tested analytes using anionic β‐cyclodextrin derivatives might be due to some favorable interaction between selectors and analytes. Given the fact that the selection of chiral selector depends on the structures of analytes, with the help of structural similarities and differences of the analytes, the structure–separation relationship was further discussed.     

Dynamic high-resolution computer simulation of electrophoretic enantiomer separations with neutral cyclodextrins as chiral selectors

GENTRANS, a comprehensive one-dimensional dynamic simulator for electrophoretic separations and transport, was extended for handling electrokinetic chiral separations with a neutral ligand. The code can be employed to study the 1:1 interaction of monovalent weak and strong acids and bases with a single monovalent weak or strong acid or base additive, including a neutral cyclodextrin, under real experimental conditions. It is a tool to investigate the dynamics of chiral separations and to provide insight into the buffer systems used in chiral capillary zone electrophoresis (CZE) and chiral isotachophoresis. Analyte stacking across conductivity and buffer additive gradients, changes of additive concentration, buffer component concentration, pH, and conductivity across migrating sample zones and peaks, and the formation and migration of system peaks can thereby be investigated in a hitherto inaccessible way. For model systems with charged weak bases and neutral modified β-cyclodextrins at acidic pH, for which complexation constants, ionic mobilities, and mobilities of selector-analyte complexes have been determined by CZE, simulated and experimentally determined electropherograms and isotachopherograms are shown to be in good agreement. Simulation data reveal that CZE separations of cationic enantiomers performed in phosphate buffers at low pH occur behind a fast cationic migrating system peak that has a small impact on the buffer composition under which enantiomeric separation takes place.     

Enantioseparation of dihydrofurocoumarin derivatives by various separation modes of capillary electrophoresis

Chiral dihydrofurocoumarin compounds are currently the focus of industrial and pharmacological research. These derivatives have been shown to possess many physiological properties that could be medically beneficial. This work proposes four different chiral separation methods using capillary electrophoresis and micellar capillary electrophoresis (MCE). Several different cyclodextrin chiral selectors were examined to evaluate their effectiveness in the enantioseparation of dihydrofurocoumarins. In addition, the effects of the chiral selector concentration, the presence of an organic modifier, run buffer pH, and in two cases, the ratio between the chiral selector and an additional charged pseudophase were investigated. Overall, the best separations for this class of chiral compounds were achieved using sulfated beta-cyclodextrins at low pH in the reversed polarity mode.     

环糊精毛细管区带电泳分离手性药物对映体

分别以2种天然环糊精(β、γ-环糊精)、2种常用的电中性环糊精衍生物(羟丙基-β-环糊精、二甲基-β-环糊精)和3种新型荷电环糊精衍生物(高取代磺酸基α、β、γ-环糊精)作为毛细管区带电泳手性添加剂,研究了环糊精的类型对6种手性药物对映体分离的影响.2种天然环糊精对所研究的手性药物均无手性识别能力,而环糊精经过衍生化后手性识别能力得到了很大的提髙,尤其是高取代磺酸基β-环糊精使6种手性药物均得到了基线分离.还考察了缓冲溶液的pH值和有机添加剂对手性分离的影响.     

Separations of antifungal compounds in capillary electrophoresis with two anionic cyclodextrins

CD‐CZE methods were developed for complete stereoisomeric separations of a series of six γ‐lactam analogues, of which some were neutral, or cationic depending on the background electrolyte nature. The tested cyclodextrin was the versatile sulfobutylether‐ β‐CD, used either in a phosphate buffer using capillaries dynamically coated with polyethylene oxide or in a borate buffer using uncoated capillaries. Long‐end and short‐end modes and concentration variations of chiral selectors allowed finding conditions of complete separation of four out of the six derivatives (i.e., 1 , 2 , 3, and 4 ) in short run times, confirming their broad range of applications. To separate the two last compounds, the highly sulfated‐ γ‐CD was examined as chiral selector in acidic phosphate conditions. The enantiomers of the γ‐lactam analogues 5 and 6 were baseline resolved with 5.5 and 4%, respectively as concentration in the buffer.     

Quantitative determination of clenbuterol, salbutamol and tulobuterol enantiomers by capillary electrophoresis

Enantiomers of clenbuterol, salbutamol and tulobuterol were directly separated and quantitated from a spiked sample by capillary electrophoresis (CE) using sulfated β-cyclodextrin (SCD) as chiral selector and phosphate as running buffer. The SCD and buffer concentration, pH and field strength were the parameters studied to optimize the separation. Optimal separation was obtained using 50 mM of phosphate monobasic at pH = 2.24, 0.25% (w/w) of sulfated cyclodextrin and a field strength of 10 kV, with 20 min total time analysis. Comparison between two different injection modes (hydrodynamic and electrokinetic) was made. In the hydrodynamic mode, repeatability (expressed as relative standard deviation, RSD) was less than 1.2% for migration times for all the analyte peaks and less than 2% for peak area percentages. With respect to reproducibility, RSD was less than 3.8% for migration time and less than 3% for peak area percentages. Calibration curves were set up for two different sample concentration ranges (1 to 10 μg mL^–1 and 160– 800 ng mL^–1, of each of the racemates studied). Although the electrokinetic injection mode for an aqueous sample appeared to suffer from some enantiodiscrimination, calibration curves were linear in the range between 1 and 10 ng mL^–1 with regression coefficients ranging from 0.9996 to 0.9952. As in the case of hydrodynamic injection, the method was tested with a spiked sample.     

Fast separation of warfarin and 7‐hydroxywarfarin enantiomers by cyclodextrin‐assisted capillary electrophoresis

The enantioseparation of warfarin and its main metabolite has been achieved using several cyclodextrin types and buffers at different pH, including conditions that have not been attempted so far. Methyl‐β‐cyclodextrin, highly sulfated‐β‐cyclodextrin and highly sulfated‐γ‐cyclodextrin were the most efficient chiral selectors. The pH range, within which particular cyclodextrins support chiral separation, has been approximately determined for the first time. By shortening the effective capillary length to 10 cm, the time of analysis has been vastly reduced <2 min. Hence, baseline separations of warfarin and 7‐hydroxywarfarin enantiomers have been achieved in times unreported for those species until now. The established conditions are promising for the further development of new highly selective and fast methods involving warfarin, its derivatives, as well as the same cyclodextrin types.     

Capillary electrophoresis separation of peptide diastereomers that contain methionine sulfoxide by dual cyclodextrin‐crown ether systems†

A dual‐selector system employing achiral crown ethers in combination with cyclodextrins has been developed for the separation of peptide diastereomers that contain methionine sulfoxide. The combinations of the crown ethers 15‐crown‐5, 18‐crown‐6, Kryptofix® 21 and Kryptofix® 22 and β‐cyclodextrin, carboxymethyl‐β‐cyclodextrin, and sulfated β‐cyclodextrin were screened at pH 2.5 and pH 8.0 using a 40/50.2 cm, 50 μm id fused‐silica capillary and a separation voltage of 25 kV. No diastereomer separation was observed in the sole presence of crown ethers, while only sulfated β‐cyclodextrin was able to resolve some peptide diastereomers at pH 8.0. Depending on the amino acid sequence of the peptide and the applied cyclodextrin, the addition of crown ethers, especially the Krpytofix® diaza‐crown ethers, resulted in significantly enhanced chiral recognition. Keeping one selector of the dual system constant, increasing concentrations of the second selector resulted in increased peak resolution and analyte migration time for peptide‐crown ether‐cyclodextrin combinations. The simultaneous diastereomer separation of three structurally related peptides was achieved using the dual selector system.     

Quantitative determination of clenbuterol, salbutamol and tulobuterol enantiomers by capillary electrophoresis

Enantiomers of clenbuterol, salbutamol and tulobuterol were directly separated and quantitated from a spiked sample by capillary electrophoresis (CE) using sulfaited beta-cyclodextrin (SCD) as chiral selector and phosphate as running buffer. The SCD and buffer concentration, pH and field strength were the parameters studied to optimize the separation. Optimal separation was obtained using 50 mM of phosphate monobasic at pH = 2.24, 0.25% (w/w) of sulfated cyclodextrin and a field strength of 10 kV, with 20 min total time analysis. Comparison between two different injection modes (hydrodynamic and electrokinetic) was made. In the hydrodynamic mode, repeatability (expressed as relative standard deviation, RSD) was less than 1.2% for migration times for all the analyte peaks and less than 2% for peak area percentages. With respect to reproducibility, RSD was less than 3.8% for migration time and less than 3% for peak area percentages. Calibration curves were set up for two different sample concentration ranges (1 to 10 microg mL(-1) and 160-800 ng mL(-1), of each of the racemates studied). Although the electrokinetic injection mode for an aqueous sample appeared to suffer from some enantiodiscrimination, calibration curves were linear in the range between 1 and 10 ng mL(-1) with regression coefficients ranging from 0.9996 to 0.9952. As in the case of hydrodynamic injection, the method was tested with a spiked sample.     

A comparison of phosphated and sulfated β-cyclodextrins as chiral selectors for capillary electrophoresis

The enantioseparation capabilities of three different functionalized β-cyclodextrins, two sulfated β-cyclodextrins with 4 and 15 nominal degrees of substitution and a phosphated β-cyclodextrin with 8 degrees of substitution, were compared. While anodic detection was used with both sulfated cyclodextrins, the phosphated cyclodextrin required cathodic detection suggesting either lower ionization of the phosphated cyclodextrin or generally lower affinity of the analytes for the phosphated cyclodextrin. The effects of several experimental parameters were evaluated with respect to enantioseparation. The degrees of substitution of the cyclodextrin, pH of the background electrolyte as well as the concentration of the functionalized β-cyclodextrin, each had a significant influence on the successful enantiomeric separation of the chiral drugs investigated.     

Migration order of dipeptide and tripeptide enantiomers in the presence of single isomer and randomly sulfated cyclodextrins as a function of pH

The present study was conducted in order to evaluate the cyclodextrin (CD)-mediated chiral separation of peptide enantiomers as uncharged analytes at pH 5.3 using randomly sulfated beta-cyclodextrin, heptakis-6-sulfato-beta-CD and heptakis-(2,3-diacetyl-6-sulfato)-beta-CD as chiral selectors. Although less effective compared to stronger acidic conditions, the CDs proved to be suitable chiral selectors for the present set of peptides at pH 5.3. The carrier ability of the negatively charged CDs upon reversal of the applied voltage may also be exploited leading to a reversal of the migration order. In addition, reversal of the enantiomer migration order upon increasing the buffer pH from 2.5 to 5.3 was also observed for Ala-Tyr in the presence of randomly sulfated beta-CD, for Ala-Phe, Ala-Tyr, Phe-Phe, Asp-PheNH(2) and Gly-Ala-Phe in the presence of heptakis-6-sulfato-beta-CD, and for Phe-Phe and Ala-Leu in the presence of heptakis-(2,3-diacetyl-6-sulfato)-beta-CD. The migration behavior could be explained on the basis of the complexation constants and the mobilities of the peptide-CD complexes. While a change in the affinity pattern of the CDs upon increasing the pH was observed for some peptides, complex mobility was the primary factor for other peptide-CD combinations affecting the enantiomer migration order at the two pH values studied.     

A comparison of phosphated and sulfated beta-cyclodextrins as chiral selectors for capillary electrophoresis

The enantioseparation capabilities of three different functionalized beta-cyclodextrins, two sulfated beta-cyclodextrins with 4 and 15 nominal degrees of substitution and a phosphated beta-cyclodextrin with 8 degrees of substitution, were compared. While anodic detection was used with both sulfated cyclodextrins, the phosphated cyclodextrin required cathodic detection suggesting either lower ionization of the phosphated cyclodextrin or generally lower affinity of the analytes for the phosphated cyclodextrin. The effects of several experimental parameters were evaluated with respect to enantioseparation. The degrees of substitution of the cyclodextrin, pH of the background electrolyte as well as the concentration of the functionalized beta-cyclodextrin, each had a significant influence on the successful enantiomeric separation of the chiral drugs investigated.     

Ultrashort partial-filling technique in capillary electrophoresis for infinite resolution of tramadol enantiomers and its metabolites with highly sulfated cyclodextrins

The possibility to enhance resolution to infinite value in chiral capillary electrophoresis is attained as soon as the apparent mobility of one enantiomer becomes opposite to the other. This could be achieved on the basis of the carrier ability of multiple charged chiral selectors such as highly sulfated cyclodextrin (HS-CD). With tramadol and its phase I metabolites selected as model compounds, the HS-gamma-CD was found to be the most appropriate chiral selector. The CD concentration was determined where one enantiomer still migrated as a cation while the other migrated in the opposite side. Besides the chiral selector concentration, secondary parameters such as buffer concentration appeared to be critical to reach infinite resolution. The latter was achieved with partial filling technique using ultrashort separation zones (a few mm). In order to better understand the interaction mechanism between the selected CD and the analytes, the classical affinity capillary electrophoresis method, although not fully satisfactory because of ionic strength variations within a series of mobility shift measurements, was applied to estimate complexation constants and complex mobilities. The results obtained point to the prevailing role of complex mobility differences in the enantioselectivity mechanism.     

Capillary electrophoretic enantioseparation of basic drugs using a new single‐isomer cyclodextrin derivative and theoretical study of the chiral recognition mechanism

A novel single‐isomer cyclodextrin derivative, heptakis {2,6‐di‐O‐[3‐(1,3‐dicarboxyl propylamino)‐2‐hydroxypropyl]}‐β‐cyclodextrin (glutamic acid‐β‐cyclodextrin) was synthesized and used as a chiral selector in capillary electrophoresis for the enantioseparation of 12 basic drugs, including terbutaline, clorprenaline, tulobuterol, clenbuterol, procaterol, carvedilol, econazole, miconazole, homatropine methyl bromide, brompheniramine, chlorpheniramine and pheniramine. The primary factors affecting separation efficiency, which include the background electrolyte pH, the concentration of glutamic acid‐β‐cyclodextrin and phosphate buffer concentration, were investigated. Satisfactory enantioseparations were obtained using an uncoated fused‐silica capillary of 50 cm (effective length 40 cm) × 50 μm id with 120 mM phosphate buffer (pH 2.5–4.0) containing 0.5–4.5 mM glutamic acid‐β‐cyclodextrin as background electrolyte. A voltage of 20 kV was applied and the capillary temperature was kept at 20°C. The results proved that glutamic acid‐β‐cyclodextrin was an effective chiral selector for studied 12 basic drugs. Moreover, the possible chiral recognition mechanism of brompheniramine, chlorpheniramine and pheniramine on glutamic acid‐β‐cyclodextrin was investigated using the semi‐empirical Parametric Method 3.