Development of a capillary electrophoresis method for the determination of the chiral purity of dextromethorphan by a dual selector system using quality by design methodology

Dextromethorphan is a centrally acting antitussive drug, while its enantiomer levomethorphan is an illicit drug with opioid analgesic effects. As capillary electrophoresis has been proven as an ideal technique for enantiomer analysis, the present study was conducted in order to develop a capillary electrophoresis‐based limit test for levomethorphan. The analytical target profile was defined as a method that should be able to determine levomethorphan with acceptable precision and accuracy at the 0.1 % level. From initial scouting experiments, a dual selector system consisting of sulfated β‐cyclodextrin and methyl‐α‐cyclodextrin was identified. The critical process parameters were evaluated in a fractional factorial resolution IV design followed by a central composite face‐centered design and Monte Carlo simulations for defining the design space of the method. The selected working conditions consisted of a 30/40.2 cm, 50 μm id fused‐silica capillary, 30 mM sodium phosphate buffer, pH 6.5, 16 mg/mL sulfated β‐cyclodextrin, and 14 mg/mL methyl‐α‐cyclodextrin at 20°C and 20 kV. The method was validated according to ICH guideline Q2(R1) and applied to the analysis of a capsule formulation. Furthermore, the apparent binding constants between the enantiomers and the cyclodextrins as well as complex mobilities were determined to understand the migration behavior of the analytes.     

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.     

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.     

A cationic β‐cyclodextrin as a dynamic coating for the separation of proteins in capillary electrophoresis

A cationic cyclodextrin was used as dynamic coating for the capillary electrophoresis of a model mixture of proteins (i.e., ubiquitin, α‐lactoglobulin, cytochrome‐c, and myoglobin) as positively charged species in a fused silica capillary. An interesting feature of the coating is that by simple adjustment of the concentration of cyclodextrin added into the background electrolyte, a neutral or positively charged surface, which was beneficial in preventing protein adsorption at the inner capillary wall surface, was obtained. This is the first demonstration of a dynamic coating that yielded a neutral surface for protein separations in capillary electrophoresis. Based on electro‐osmotic flow measurements, addition of 0.05 to 0.10 mg/mL quaternary β‐cyclodextrin in a low pH electrolyte resulted in a neutral or positive surface (undetectable to very slow anodic electro‐osmotic flow). The coating approach afforded the electrophoretic separation of the mixture of proteins at positive polarity with good repeatability and separation performance.     

Comparison of hydrodynamically closed two‐dimensional capillary electrophoresis coupled with ultraviolet detection and hydrodynamically open capillary electrophoresis hyphenated with mass spectrometry in the bioanalysis of varenicline

Two capillary electrophoresis methods for monitoring renally excreted varenicline, a highly effective drug prescribed for smoking cessation, in human urine were developed and compared. A method combining capillary electrophoresis with mass spectrometry was proposed for the fast analysis of varenicline (analysis time up to 7 min). Here, mass spectrometry was a prerequisite for achieving high sensitivity and selectivity of the analysis suitable for the quantification of a 15 ng/mL level of varenicline in un‐pretreated urine matrices. An alternative approach, two‐dimensional (column‐coupled) capillary electrophoresis with enhanced sample load capacity and ultraviolet detection, was proposed as a low‐cost alternative to capillary electrophoresis with mass spectrometry. The isotachophoresis on‐line sample treatment included simple elimination of the major matrix constituents and stacking of the sample in a large volume so that threefold lower quantitation limits could be easily achieved in comparison to the capillary electrophoresis with mass spectrometry. On the other hand, longer analysis time (ca. 4.5‐fold) and more complex electrolyte system in the coupled zone electrophoresis step (including two additives enhancing separation selectivity, i.e. isopropanol and cyclodextrin) were prerequisites for the complete separation of varenicline from the sample matrix. Anyway, both the developed methods were validated according to the Food and Drug Administration guidelines showing favorable performance parameters, suitable for their routine biomedical use.     

Capillary electrophoretic assay of human acetyl‐coenzyme A carboxylase 2

Human acetyl‐coenzyme A carboxylase 2 catalyzes the carboxylation of acetyl coenzyme A to form malonyl coenzyme A, along with the conversion of magnesium‐adenosine triphosphate complex to magnesium‐adenosine diphosphate complex. A simple off‐column capillary electrophoresis assay for human acetyl‐coenzyme A carboxylase 2 was developed based on the separation of magnesium‐adenosine triphosphate complex, magnesium‐adenosine diphosphate complex, acetyl coenzyme A and malonyl coenzyme A with detection by ultraviolet absorption at 256 nm. When Mg²⁺ was absent from the separation buffer, the zones due to magnesium‐adenosine triphosphate complex and magnesium‐adenosine diphosphate complex both split and migrated as two separate peaks. With Mg²⁺ added to the separation buffer, magnesium‐adenosine triphosphate complex and magnesium‐adenosine diphosphate complex produced single peaks, and the reproducibility of peak shape and area improved for human acetyl‐coenzyme A carboxylase 2 assay components. The final separation buffer used was 30.0 mM HEPES, 3.0 mM MgCl₂, 2.5 mM KHCO₃, and 2.5 mM potassium citrate at pH 7.50. The same buffer was used for the enzyme‐catalyzed reaction (off‐column). Inhibition of human acetyl‐coenzyme A carboxylase 2 by CP‐640186, a known inhibitor, was detected using the capillary electrophoresis assay.     

Surfactant‐assisted electromembrane extraction combined with cyclodextrin‐modified capillary electrophoresis for the separation and quantification of Tranylcypromine enantiomers in biological samples

Surfactant‐assisted electromembrane extraction coupled with cyclodextrin‐modified capillary electrophoresis was developed for the separation and determination of Tranylcypromine enantiomers in biological samples. This combination would provide a new strategy for selective and sensitive determination of target analytes. The addition of surfactant in the donor solution improved the analyte transport into the lumen of hollow fiber that resulted in an enhancement in the analytes migration into acceptor solution. Optimization of the variables, affecting proposed method, was carried out and best results were achieved with a 175 V potential as driving force of the electromembrane extraction, 2‐nitrophenyloctylether as the supported liquid membrane, donor solution containing 0.2 mM Triton X‐100 with pH 3 and 0.1 M HCl for acceptor solution. Then, the extract was analyzed using cyclodextrin‐modified capillary electrophoresis method for separation of Tranylcypromine enantiomers. The best results were obtained with a phosphate running buffer (100 mM, pH 2.0) containing 7% w/v hydroxypropyl‐α‐cyclodextrin. Under the optimum conditions, a low limit of detection (3.03 ng/mL), good linearity (R² > 0.9953), and relative standard deviations below 4.0% (n = 5) were obtained. Finally, this procedure was applied to determine the concentration of Tranylcypromine enantiomers in urine samples with satisfactory results.     

Chiral separation of lenalidomide by liquid chromatography on polysaccharide‐type stationary phases and by capillary electrophoresis using cyclodextrin selectors

Complementary techniques were applied for the investigation of the chiral recognition and enantiomeric resolution of lenalidomide using various cyclodextrins and polysaccharides as chiral selectors. The high‐performance liquid chromatography enantioseparation of the anticancer drug was achieved using polysaccharide‐type chiral stationary phases in polar organic mode. Elution order and absolute configuration were elucidated by combined circular dichroism spectroscopy and time‐dependent density functional theory calculations after the isolation of pure enantiomers. Chiral selector dependent and mobile‐phase dependent reversal of the enantiomer elution order was observed, and the nonracemic nature of the lenalidomide sample was also demonstrated. Eight anionic cyclodextrins were screened for their ability to discriminate between the uncharged enantiomers by using capillary electrophoresis. Only two derivatives presented chiral interactions, these cases being interpreted in terms of apparent stability constants and complex mobilities. The best results were delivered by sulfobutylether‐β‐cyclodextrin, where quasi‐equal stability constants were recorded and the enantiodiscrimination process was mainly driven by different mobilities of the transient diastereomeric complexes. The optimized high‐performance liquid chromatography (Chiralcel OJ column, pure ethanol with 0.6 mL/min flow rate, 40°C) and capillary electrophoresis methods (30 mM sulfobutylether‐β‐cyclodextrin, 30 mM phosphate pH 6.5, 12 kV applied voltage, 10°C) were validated for the determination of 0.1% (R)‐lenalidomide as a chiral impurity, which could be important if a racemic switch is achieved.     

Simultaneous detection of zinc dimethyldithiocarbamate and zinc ethylenebisdithiocarbamate in cabbage leaves by capillary electrophoresis with inductively coupled plasma mass spectrometry

We report a simple and highly sensitive method for the simultaneous detection of trace zinc dimethyldithiocarbamate and zinc ethylenebisdithiocarbamate by capillary electrophoresis with inductively coupled plasma mass spectrometry. Zinc dimethyldithiocarbamate and zinc ethylenebisdithiocarbamate were chelated with trans‐1,2‐diaminocyclohexane‐N,N,N′,N′‐tetraacetic acid to form a macromolecule complex. Then, these two compounds were separated by α‐cyclodextrin‐modified capillary electrophoresis within 12 min at a separation voltage of 15 kV and measured by inductively coupled plasma mass spectrometry. The developed method is sensitive with detection limit of 1.9 and 3.0 ng Zn/mL for zinc dimethyldithiocarbamate and zinc ethylenebisdithiocarbamate, respectively. By means of ultrasound‐assisted extraction methods, zinc dimethyldithiocarbamate and zinc ethylenebisdithiocarbamate spiked into cabbage leaves were successfully extracted and determined with a relative standard deviation (n  = 5) ≤ 6% and a recovery of 95–107%.     

Development and validation of a sensitive assay for the quantification of imatinib using LC/LC‐MS/MS in human whole blood and cell culture

We developed and validated a semi‐automated LC/LC‐MS/MS assay for the quantification of imatinib in human whole blood and leukemia cells. After protein precipitation, samples were injected into the HPLC system and trapped onto the enrichment column (flow 5 mL/min); extracts were back‐flushed onto the analytical column. Ion transitions [M + H]⁺ of imatinib (m/z = 494.3 → 394.3) and its internal standard trazodone (372.5 → 176.3) were monitored. The range of reliable response was 0.03–75 ng/mL. The inter‐day precisions were: 8.4% (0.03 ng/mL), 7.2% (0.1 ng/mL), 6.5% (1 ng/mL), 8.2% (10 ng/mL) and 4.3% (75 ng/mL) with no interference from ion suppression. Autosampler stability was 24 hs and samples were stable over three freeze–thaw cycles. This semi‐automated method is simple with only one manual step, uses a commercially available internal standard, and has proven to be robust in larger studies. Copyright © 2009 John Wiley & Sons, Ltd.     

A rapid and simple HPLC method for the determination of curcumin in rat plasma: assay development,validation and application to a pharmacokinetic study of curcumin liposome

This paper describes a sensitive, specific and rapid high‐performance liquid chromatography (HPLC) method for the determination of curcumin in rat plasma. After a simple step of protein precipitation in 96‐well format using acetonitrile containing the internal standard (IS), emodin, plasma samples were analyzed by reverse‐phase HPLC. Curcumin and the IS emodin were separated on a Diamonsil C₁₈ analytical column (4.6 × 100 mm, 5 µm) using acetonitrile–5% acetic acid (75:25, v/v) as mobile phase at a flow rate of 1.0 mL/min. The method was sensitive with a lower limit of quantitation of 1 ng/mL, with good linearity (r² ≥ 0.999) over the linear range 1–500 ng/mL. All the validation data, such as accuracy and precision, were within the required limits. A run time of 3.0 min for each sample made high‐throughput bioanalysis possible. The assay method was successfully applied to the study of the pharmacokinetics of curcumin liposome in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

A liposome-based assay for quantitation of detergents

The amount of enzyme released from liposomes exposed to detergents varies with the amount of detergent present. This fact makes it possible to quantitate the detergent. Multilamellar liposomes containing entrapped peroxidase were adsorbed to paper discs and then exposed to solutions containing detergents. The assay procedure proved useful for assaying detergents down to their critical micelle concentration (CMC), and for an induced leakage of ionic detergents also below their CMCs.     

Refolding of detergent‐denatured lysozyme using β‐cyclodextrin‐assisted ion exchange chromatography

Chromatography‐based protein refolding is widely used. Detergent is increasingly used for protein solubilization from inclusion bodies. Therefore, it is necessary to develop a refolding method for detergent‐denatured/solubilized proteins based on liquid chromatography. In the present work, sarkosyl‐denatured/dithiothreitol‐reduced lysozyme was used as a model, and a refolding method based on ion exchange chromatography, assisted by β‐cyclodextrin, was developed for refolding detergent‐denatured proteins. Many factors affecting the refolding, such as concentration of urea, concentration of β‐cyclodextrin, pH and flow rate of mobile phases, were investigated to optimize the refolding conditions for sarkosyl‐denatured lysozymes. The results showed that the sarkosyl‐denatured lysozyme could be successfully refolded using β‐cyclodextrin‐assisted ion exchange chromatography. Copyright © 2012 John Wiley & Sons, Ltd.     

Field‐amplified sample injection combined with CE for the enantiodetermination of cathinones in urine samples

This work presents a capillary electrophoresis methodology for the enantiodetermination of cathinones in urine employing a liquid–liquid extraction sample pretreatment. The cathinones were enantioseparated by adding a mixture of 8 mM 2‐hydroxypropyl β‐cyclodextrin and 5 mM β‐cyclodextrin to the background electrolyte, which consists of 70 mM of monosodium phosphate aqueous solution at pH 2.5. Field‐amplified sample injection was used as preconcentration strategy to improve the sensitivity. We studied various parameters that affect this stacking strategy, in particular, the sample solvent and its pH, the presence or absence of a low conductivity solvent plug introduced before the sample injection, the nature and volume of this plug, and the voltage and time of the electrokinetic injection of the sample. The optimum conditions were achieved by injecting a plug of isopropanol:H₂O 50/50 at 50 mbar for 5 s prior to the electrokinetic injection of the sample prepared in an aqueous solution of HCl 10^?6 M. The sensitivity enhancement factors were from 562 to 601 in terms of peak area and from 444 to 472 in terms of peak height. The method was validated by analyzing spiked urine samples, obtaining a linear range of 25 to 1000 ng/mL and limits of detection ranging from 15 to 45 ng/mL.     

Magnetic chitosan functionalized with β‐cyclodextrin as ultrasound‐assisted extraction adsorbents for the removal of methyl orange in wastewater coupled with high‐performance liquid chromatography

Three types of choline chloride based deep eutectic solvents were prepared and used to modify magnetic chitosan. The adsorption capacity of the three deep‐eutectic‐solvent‐modified magnetic chitosan/carboxymethyl‐β‐cyclodextrin for removing methyl orange from wastewater was examined. The different deep eutectic solvents were used to strengthen the adsorption capacity of magnetic chitosan. Deep‐eutectic‐solvent‐modified magnetic chitosan/carboxymethyl‐β‐cyclodextrin materials were characterized by Fourier transform infrared spectroscopy and Brunauer–Emmett–Teller surface area measurements. Among the three deep eutectic solvents, choline chloride/glycerol (1:2) modified magnetic chitosan/carboxymethyl‐β‐cyclodextrin showed the highest adsorption capacity to methyl orange. Therefore, choline chloride/glycerol (1:3, 1:4, 1:5, 1:6) deep eutectic solvents were prepared for the assay, and choline chloride/glycerol‐modified magnetic chitosan/carboxymethyl‐β‐cyclodextrin prepared with choline chloride/glycerol (1:3) (volume: 40 μg, contact time: 30 min, and pH: 6) had the best adsorption capacity over the concentration range of 10–200 μg/mL.     

Solid phase immunoradiometric assay for CA125 antigen levels in blood using monoclonal antibodies

We have developed a simple one step ‘sandwich’ immunoradiometric assay for CA125 using monoclonal antibodies directed against two different epitopes of the antigen. The detection antibody was radiolabeled with I-125 and the selected capture antibody was chemically coupled to magnetizable cellulose to form immobilized solid support. In the developed inclusive assay procedure, 200 μL of standard or sample was incubated with 100 μL of radiolabeled and capture antibody suspension for 18 h at room temperature with shaking. At the end of the incubation, the sandwich complex attached to solid phase is separated and counted for associated radioactivity. The analytical sensitivity for the developed assay procedure was observed to be 3.0 U/mL with an assay range up to 500 U/mL of CA125. The developed assay displayed acceptable precision; expressed in terms of percentage Coefficient of Variation (CV) estimated by repeated analyses of the quality control samples. Intra-assay CV was observed to be less than 5% whereas inter-assay CV was also less than 6%. The analytical recovery of the developed assay observed to be in the range of 88–107%. The clinical samples analyzed by the developed procedure showed a good correlation with that of a commercial kit (r = 0.99; y = 1.0052x − 38.942).     

Cyclodextrins in Polymer Synthesis: Photoinitiated Free‐Radical Polymerization of N‐Isopropylacrylamide in Water Initiated by a Methylated β‐Cyclodextrin/2‐Hydroxy‐2‐methyl‐1‐phenylpropan‐1‐one Host/Guest Complex

Methylated β‐cyclodextrin (Me‐β‐CD) was used to complex a free‐radical photoinitiator, 2‐hydroxy‐2‐methyl‐1‐phenylpropan‐1‐one ( 1 ), yielding the water‐soluble 1 : 1 host/guest complex 1 a . The structure of complex 1 a was verified by means of IR, UV/vis and ¹H NMR spectroscopy. The influence of Me‐β‐CD as the host on the photopolymerization kinetics of N‐isopropylacrylamide was studied. Compared to the photopolymerization carried out under nearly identical conditions but without cyclodextrin, an increase in the polymerization rate was registered in the presence of complex 1 a .     

Chiral separation of 12 pairs of enantiomers by capillary electrophoresis using heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector and the elucidation of the chiral recognition mechanism by computational methods

Chiral separation of 12 pairs of basic analyte enantiomers including oxybutynin, bambuterol, tradinterol, clenbuterol, clorprenaline, terbutaline, tulobuterol, citalopram, phencynonate, fexofenadine, salbutamol, and penehyclidine was conducted by capillary electrophoresis using a single‐isomer anionic β‐cyclodextrin derivative, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector. Parameters influencing separation were studied, including background electrolyte pH, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin concentration, buffer concentration, and separation voltage. A background electrolyte consisting of 50 mM Tris‐H₃PO₄ and 6 mM heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin at pH 2.5 was found to be highly efficient for the separation of most enantiomers, with other conditions of normal polarity mode at 10 kV, detection wavelength of 210 nm using hydrodynamic injection for 3 s. Under the optimal conditions, baseline resolution (>1.50) for 11 pairs of enantiomers and somewhat lower resolution for penehyclidine enantiomers (1.17) were generated. Moreover, the possible mechanism of separation of clenbuterol, oxybutynin, salbutamol, and penehyclidine was investigated using a computational modeling method.     

In situ self‐assembled reduced graphene oxide aerogel embedded with nickel oxide nanoparticles for the high‐efficiency separation of ovalbumin

A three‐dimensional reduced graphene oxide aerogel with embedded nickel oxide nanoparticles was prepared by a one‐step self‐assembly reaction in a short time. The nanoparticles could be captured into the interior of reduced graphene oxide network during the formation of the three‐dimensional architecture. The composite exhibited porosity, good biocompatibility, and abundant metal affinity binding sites. The aerogel was used to isolate ovalbumin selectively from egg white, and favorable adsorption was achieved at pH 3. An adsorption efficiency of 90.6% was obtained by using 1 mg of the composite for adsorbing 70 μg/mL of ovalbumin in 1.0 mL of sample solution, and afterwards a recovery of 90.7% was achieved by using an eluent of 1.0 mL Britton–Robinson buffer solution at pH 5. After the adsorption/desorption, ovalbumin showed no change in the conformation. The adsorption behavior of ovalbumin on the reduced graphene oxide composite well fitted to the Langmuir adsorption model, and a corresponding theoretical maximum adsorption capacity was 1695.2 mg/g. A sodium dodecyl sulfate polyacrylamide gel electrophoresis assay demonstrated that the aerogel could selectively isolate ovalbumin from chicken egg white.     

Influence of substituent position and cavity size of the regioisomers of monocarboxymethyl‐α‐, β‐, and γ‐cyclodextrins on the apparent stability constants of their complexes with both enantiomers of Tröger's base

Enantiomers of Tröger's base were separated by capillary electrophoresis using 2^I‐O‐, 3^I‐O‐, and 6^I‐O‐carboxymethyl‐α‐, β‐, and γ‐cyclodextrin and native α‐, β‐, and γ‐cyclodextrin as chiral additives at 0–12 mmol/L for β‐cyclodextrin and its derivatives and 0–50 mmol/L for α‐ and γ‐cyclodextrins and their derivatives in a background electrolyte composed of sodium phosphate buffer at 20 mmol/L concentration and pH 2.5. Apparent stability constants of all cyclodextrin–Tröger's base complexes were calculated based on capillary electrophoresis data. The obtained results showed that the position of the carboxymethyl group as well as the cavity size of the individual cyclodextrin significantly influences the apparent stability constants of cyclodextrin–Tröger's base complexes.