Micellar Capillary Electrophoresis Research Articles

Application of micellar electrokinetic capillary chromatography for identification, quantitative detection and unfolding analysis of interleukin-12

To establish a micellar electrokinetic capillary chromatography-based method for identification and quantitative detection of interleukin-12 (IL-12) and analysis of its unfolding process. An uncoated fused-silica capillary (inner diameter 50 μm) with a total length of 48.5 cm (40 cm to the detector) was used for the experiment. The factors influencing the separation efficiency of IL-12 were analyzed, and a standard curve of IL-12 concentration was established. The mixture of IL-12 and anti-IL-12 antibody was incubated in a water bath at 38 ℃ for 40 min, and capillary electrophoresis was then performed under the same conditions. The results were compared with those of IL-12 and anti-IL-12 antibody to identify IL-12. IL-12 and dithiothreitol (DTT) were incubated at 60 ℃ in water bath for different lengths of times, and the unfolding process of IL-12 was analyzed based on electrophoresis results of IL-12 in different states. A micellar capillary electrophoresis on-line sweep method was established with 80 mmol/L borate (pH=9.3) containing 30 mmol/L sodium dodecyl sulfate (SDS) as the buffer solution. This system showed a good linear relationship between the peak area and the mass concentration of IL-12 with a linear correlation coefficient of 0.9991 within the linear range of 2 to 120 ng/L. As the incubation time of IL-12 and DTT prolonged, the disulfide bond of IL-12 gradually opened and resulted in distinct changes in the protein peak. This capillary electrophoresis-based method is simple and sensitive for IL-2 analysis and allows rapid detection of changes in IL-12 content in the setting of tumors and analysis of the possible causes.

Enantioseparation of amino acids by micellar capillary electrophoresis using binary chiral selectors and determination of D-glutamic acid and D-aspartic acid in rice wine

ABSTRACTA new chiral capillary electrophoresis (CE) analytical method for the determination of six pairs of amino acid enantiomer-derivatized precapillary with 9-fluorenylmethoxycarbonyl chloride (FMOC) was developed. CE separation parameters such as the pH of background electrolyte, the boric acid concentration, the addition of D-fructose and isopropanol, and the effect of new binary chiral selectors on the electropherograms were investigated. Precapillary and in-capillary derivatization was compared as well. The results showed that enantiomeric separations were obtained with enantio-resolution (Rs) ranged from 2.61 to 9.99 for the FMOC-derivatized amino acid enantiomers except alanine with Rs 1.06. Precapillary derivatization method provides overall better Rs for nearly all the targets except FMOC-aspartate with efficiency up to 1.309 × 106 plates and limit of detection (LOD) down to 2.5 μM. This method was successfully applied to analyze the concentration of the D/L-glutamic acid and D/L-aspartate in seven rice wine samples.

Determination of malachite green, crystal violet, brilliant green and methylene blue by micro-cloud-point extraction and nonlinear laser wave-mixing detection interfaced to micellar capillary electrophoresis

A laser wave-mixing CE detection method is reported for separation and detection of malachite green (MG), crystal violet (CV), brilliant green (BG), methylene blue (MB), and the leuco-metabolites of MG and CV residues in aquacultures.

Polydispersity and assembling phenomena of native and reactive dye-labelled nanocellulose

The assembling (self-association vs. aggregation or agglomeration) of highly polydispersed nanocelluloses (NCs) is a well-known but difficult to identify phenomena. In this research complementary analytical tools were applied for tracking and assessing this phenomena under different conditions (base vs. buffer) using both native and dye-labelled cellulose nanofibrils (CNFs) and cellulose nanocrystals. For this purpose, dichlorotriazine-type reactive blue 4 (RB4) dye was covalently (through alkyl-aryl ether bond) and regioselectively (to C6–OH groups) attached to the NC, being identified by ATR-FTIR and 13C CP/MAS solid-state NMR spectroscopies, and quantified by UV–Vis spectroscopy. The introduced RB4’s anthraquinone (i.e. hydrophobic) moieties evoked aggregation of those CNFs being quantified from the shifting and broadening of size-distribution profiles within differential light scattering analysis and their ζ-potential reduction. In addition, their high polydispersity profiles was assessed by qualitatively supported differential centrifugal sedimentation, nanoparticle tracking analysis, and transmission electron microscopy. A desorption of chemically non-bonded, yet highly substantive (i.e. partially or fully hydrolysed), dye from both types of NCs was also identified by capillary electrophoresis, which simultaneously excluded the non-labelled fractions, thus obtaining free dye-absent NC dispersions. Finally, labelling extent-triggered separation within the RB4-labelled CNFs was identified by applying the micellar capillary electrophoresis, thus confirming the conformational changes affecting NCs’ hydrodynamic (size) profiles.

Development of alternative methods for the determination of raloxifene hydrochloride in tablet dosage form

<p>Three methods are proposed for the quantitative determination of raloxifene hydrochloride in pharmaceutical dosage form: ultraviolet method (UV) high performance liquid chromatography (HPLC) and micellar capillary electrophoresis (MEKC). These methods were developed and validated and showed good linearity, precision and accuracy. Also they demonstrated to be specific and robust. The HPLC and MEKC methods were tested in regards to be stability indicating methods and they showed to have this attribute. The UV method used methanol as solvent and optimal wavelength at 284 nm, obeying Lambert-Beer law in these conditions. The chromatographic conditions for the HPLC method included: NST column C18 (250 x 4.6 mm x 5 µm), mobile phase water:acetonitrile:triethylamine (67:33:0,3 v/v), pH 3.5, flow rate 1.0 mL min<sup>-1</sup>, injection volume 20.0 µl, UV detection 287 nm and analysis temperature 30 °C. The MEKC method was performed on a fused-silica capillary (40 cm effective length x 50 µm i.d.) using as background electrolyte 35.0 mmol L<sup>-1</sup> borate buffer and 50.0 mmol L<sup>-1</sup> anionic detergent sodium dodecyl sulfate (SDS) at pH 8.8. The capillary temperature was 32°C, applied voltage 25 kV, UV detection at 280 nm and injection was perfomed at 45 mBar for 4 s, hydrodimanic mode. In this MEKC method, potassium diclofenac (200.0 µg mL<sup>-1)</sup> was used as internal standard. All these methods were statistically analyzed and demonstrated to be equivalent for quantitative analysis of RLX in tablets and were successfully applied for the determination of the drug.</p>

Development and Validation of a Micellar Capillary Electrophoresis Method for Determination of IFNβ-1b in Lyophilized Formulation of a Biosimilar Product.

Human interferons (IFNs) are key cytokines secreted by immune system. They have several effects such as antiviral and anti tumors activity, activating immune cells and healing of multiple sclerosis. The type IFNs present in humans are α ,β and Υ. IFN β is a polypeptide, normally produced by fibroblasts and seems to be more species-specific than IFN. Structural properties of IFNs are important for their biologic effects. There are a few analytical techniques for separation, identification and determination of IFNs in its formulations such as mass spectroscopy, RP-HPLC and capillary electrophoresis (CE). In this study we used Micellar Electrokinetic Chromatography (MEKC) as a unique mode of CE because of its capability to separate neutral as well as charged solutes. We used sodium tetraborate (Borax) as buffer without any modifier and sodium dodecyl sulfate (SDS) as surfactant. The optimum MECK running buffer consisted of Borate 50 Mm; SDS 20 mM pH =9.6. The validated method was used for determination of the IFN β-1b formulation which is manufactured in Iran. From 9 collected different batches, all of them had acceptable potency as claimed on their label with average 102.25 ±10.030 %. This is the first time that a MEKC method is introduced for quantification of IFN β-1b in its pharmaceutical dosage forms. The method is reliable safe, rapid and accurate.

Micellar capillary electrophoresis – Electrochemical detection of neurochemicals from Drosophila

The fruit fly is one of the most heavily studied model organisms for genetics research and has significantly contributed to the molecular, cellular, and evolutionary understandings of human behavior. Recent research in the analytical chemistry of the fruit fly has focused on developing methods to obtain highly sensitive chemical quantification information of Drosophila melanogaster, especially looking at the nervous system. We provide a brief overview of work in the area of CE of the fly head and brain.

Enantiomeric separation of chiral ruthenium(II) complexes using capillary electrophoresis

Capillary zone electrophoresis (CZE) and micellar capillary electrophoresis (MCE) were applied for the enantiomeric separation of nine mononuclear tris(diimine)ruthenium(II) complexes as well as the separation of all stereoisomers of a dinuclear tris(diimine)ruthenium(II) complex. Nine cyclodextrin (CD) based chiral selectors were examined as run buffer additives to evaluate their effectiveness in the enantiomeric separation of tris(diimine)ruthenium(II) complexes. Seven showed enantioselectivity. Sulfated gamma-cyclodextrin (SGC), with four baseline and three partial separations, was found to be the most useful chiral selector. In CZE mode, the derivatized gamma-CDs were more effective than beta-CDs while sulfated CDs work better than carboxymethyl CDs. In MCE mode, hydroxypropyl beta-CD separated the greatest number of tris(diimine) ruthenium(II) complexes. The effects of chiral selector concentration, run buffer pH and concentration, the concentration ratio between chiral selector and other factors were investigated.

Enantiomeric Separation of Isochromene Derivatives by Cyclodextrin-Modified Micellar Capillary Electrophoresis

The enantiomeric separations of highly hydrophobic isochromene derivatives were performed and optimized using cyclodextrin-modified micellar capillary electrophoresis. Hydroxypropyl-γ-cyclodextrin proved to be the most effective chiral selector for the enantioseparation of these analytes. The effects of cyclodextrin and sodium dodecyl sulfate concentration and organic modifier were examined in order to optimize the separation conditions. Addition of an organic solvent modifier to the run buffer served to increase the analyte' solubility and enhance the separation efficiency. A highly acidic pH was necessary to effectively suppress the electroosmotic flow when operating in the reverse polarity mode.

Determination of linezolid and its achiral impurities using sweeping preconcentration by micellar capillary electrophoresis

Linezolid is the first compound of a truly new class of antibiotics—the oxazolidinones. The elaborated method of capillary electrophoresis (CE) of linezolid separation from its achiral impurities was successfully performed using sweeping preconcentration, followed by UV absorption detection at 254 nm. The best results were obtained with 125 mM Tris buffer, pH 2.0, with addition of 20% (v/v) methanol as background electrolyte. Sodium dodecyl sulfate (150 mM) was added to the electrolyte in the inlet vial as the sweeping agent. The separation was carried out at negative polarity. Then, the optimized method was validated in terms of linearity, accuracy and precision. Sweeping preconcentration of linezolid provides detection limit at 0.05 μg/ml level. The evaluated CE method was applied in the analysis of medicinal product containing linezolid–linezolid solution for infusion.

Differentiation of opium and poppy straw using capillary electrophoresis and pattern recognition techniques

Opium samples from four different locations and poppy straw from different plant varieties have been assayed using micellar capillary electrophoresis incorporating a sweeping technique. Individual alkaloids (morphine, codeine, papaverine, noscapine, thebaine, oripavine, reticuline and narceine) were quantitatively determined in the different samples by a validated capillary electrophoresis method. Unsupervised pattern recognition of the opium samples and the poppy straw samples using hierarchical cluster analysis (HCA) and principal component analysis (PCA), showed distinct clusters. Supervised pattern recognition using soft independent modelling of class analogy (SIMCA) was performed to show individual groupings and allow unknown samples to be classified according to the models built using the CZE assay results.

Comparison between micellar liquid chromatography and capillary zone electrophoresis for the determination of hydrophobic basic drugs in pharmaceutical preparations

The determination of highly hydrophobic basic compounds by means of conventional reversed-phase liquid chromatographic methods has several drawbacks. Owing to the characteristics of micellar liquid chromatography (MLC) and capillary electrophoresis (CE), these techniques could be advantageous alternatives to reversed-phase chromatographic methods for the determination of these kinds of compounds. The objective of this study was to develop and compare MLC and CE methods for the determination of antipsychotic basic drugs (amitryptiline, haloperidol, perphenazine and thioridazine) in pharmaceutical preparations. The chromatographic determination of the analytes was performed on a Kromasil C(18) analytical column; the mobile phase was 0.04 m cetyltrimethylammonium bromide (CTAB), at pH 3, containing 5% 1-butanol, at a flow rate of 1 mL/min. The CE separation was performed in a fused-silica capillary with a 50 mm tris-(hydroxymethyl)-aminomethane buffer, pH 7, at an applied voltage of 20 kV, using barbital as internal stardard. The proposed methods are suitable for a reliable quantitation of these compounds in the commercial tablets and drops in terms of accuracy and precision and require a very simple pre-treatment of the samples. By comparing the performance characteristics and experimental details of the MLC and CE methods we conclude that CE seems to be slightly better than MLC in the determination of highly hydrophobic compounds in pharmaceuticals in terms of resolution and economy, taking into account that the limits of detection are not a handicap in pharmaceutical samples.

Analysis of amphetamine, methamphetamine and methylenedioxy‐methamphetamine by micellar capillary electrophoresis using cation‐selective exhaustive injection

Cation-selective exhaustive injection (CSEI) is used as an on-line concentration method for the high-sensitivity analysis of illicit amphetamines using CE. Optimum conditions for the determination of amphetamine, methamphetamine and methylenedioxy-methamphetamine were investigated. Sodium dodecyl sulfate (25 mM) in 100 mM phosphate buffer (pH 2.9) with 20% methanol as organic additive was used as the background electrolyte for CE separation. The LOD, based on an S/N of 3:1, was about 0.01 microg/mL using normal capillary micellar electrokinetic chromatography, while by using CSEI in combination with micellar sweeping the sensitivity increased up to 1000-fold with the LOD lower than 50 pg/mL. The reproducibility of CSEI combined with micellar sweeping for analyzing amphetamines was satisfactory (relative standard deviation around 10% by using area ratios against an internal standard). This method is highly sensitive and can be used to analyze trace amount amphetamines in human hair.

Development of a smokeless powder reference material for propellant and explosives analysis

The accurate analysis of the additives in smokeless powder is often an integral part of investigations of improvised explosives devices (IEDs) and in the evaluation of organic gunshot residues (OGSR). To help assure the quality of smokeless powder measurements, the US National Institute of Standards and Technology (NIST) has developed a Reference Material, RM 8107 Additives in Smokeless Powder. The RM, a smokeless rifle powder, was chosen after evaluation of three candidate powders. The additives were determined using solvent extraction with ultrasonic agitation followed by micellar capillary electrophoresis (CE) and liquid chromatography (LC). RM 8107 provides reference values obtained by LC analysis for the key additives nitroglycerin, diphenylamine (and its nitration product, N-nitrosodiphenylamine), and ethylcentralite.

Enantiomeric separation of furan derivatives and fused polycycles by cyclodextrin-modified micellar capillary electrophoresis

The enantiomeric separations of highly hydrophobic furan derivatives and polycycles were performed and optimized using CD-modified micellar CE. The most effective chiral selector for the enantiomeric separation of these analytes was hydroxypropyl-gamma-CD. The effects of CD and SDS concentration and organic modifier were examined in order to optimize the separation conditions. The ratio of CD to surfactant concentration affected the enantiomeric separation significantly, with increases in the derivatized CD concentration generally enhancing resolution. Addition of an organic solvent modifier to the run buffer served to increase the analytes' solubility and enhance the separation efficiency. A highly acidic pH was necessary to effectively suppress the EOF when operating in the reverse polarity mode.

In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography.

A method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin-permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed-polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast-twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest.

Effects of partial/asymmetrical filling of micelles and chiral selectors on capillary electrophoresis enantiomeric separation: generation of a gradient.

The effect of several experimental parameters on enantiomeric separations in micellar capillary electrophoresis (MCE) was studied. A model separation system was tested. It was composed of an acidic phosphate buffer with sodium dodecyl sulfate (SDS) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as the chiral selector. A substituted angelicin was used as a chiral analyte. Changes in the concentration of SDS micelles/SDS monomers in the presence of HP-beta-CD and their impact on the enantioselective separation were investigated. Variation of the composition of electrolytes in the individual compartments of the separation system (inlet vial, capillary, and outlet vial) affected both the migration times and the resolution of the enantiomers. Current vs. time dependencies also were monitored during the separations. A mathematical model of electromigration in micellar systems with chiral selector present was proposed and a computer simulation was used to explain the observed phenomena and to confirm the generation of a CD/SDS-micelle concentration gradient under certain experimental conditions. This is the reported first attempt of a computer simulation of the complex, dynamic chiral environment of the CD-SDS-MCE system.

Enantiomeric separation of neutral hydrophobic dihydrofuroflavones by cyclodextrin-modified micellar capillary electrophoresis.

The enantiomeric separations of several very hydrophobic dihydrofuroflavones were performed and optimized using cyclodextrin-modified micellar capillary electrophoresis. Overall, the greatest enantiomeric peak-to-peak separations for the greatest number of flavones were obtained with hydroxypropyl-gamma-cyclodextrin. The effects of cyclodextrin and sodium dodecyl sulfate concentration and pH were examined in order to optimize the separation conditions. The ratio of surfactant-to-cyclodextrin concentration affected the chiral discrimination of the system significantly, with increases in the derivatized cyclodextrin concentration generally enhancing resolution. Higher efficiencies were obtained with lower concentrations of surfactant and cyclodextrin, although enantioseparation optimization often required higher concentrations to be used. A highly acidic pH was necessary to effectively suppress the electroosmotic flow when operating in the reversed polarity mode. Experiments utilizing the normal polarity mode and higher pH produced no separations.

Simultaneous quantitation of zidovudine and zidovudine monophosphate from plasma, amniotic fluid and tissues by micellar capillary electrophoresis.

Zidovudine (AZT) therapy given during pregnancy has been shown to reduce the vertical transmission of the human immunodeficiency virus (HIV) from mother to fetus. In order to investigate the efficacy of AZT, it is important to know the concentration of its active phosphorylated metabolites. We have developed the first CE method for the simultaneous quantitation of AZT and zidovudine monophosphate (AZT-MP) from rat plasma, amniotic fluid and fetal tissues. Sample extractions were performed by protein precipitation using acetonitrile for the plasma and amniotic fluids, while in fetal tissues solid phase extraction using Waters Oasis HLB extraction cartridges was used. Recoveries ranged from 78 to 92% for AZT, AZT-MP and 3'-azidouridine (internal standard, AZDU), in the three matrices. The optimum separation conditions were achieved using a 40 mm sodium dodecylsulfate (SDS) in 50 mm phosphate buffer (pH 7) with a run voltage of 15 kV. The CE system consists of a 75 microm i.d., 50 cm effective length uncoated fused silica capillary. The method was validated over the range 0.5-100 microg/ml ( micro g/g for tissues). Intra-day precision (RSD) and accuracy (%error) for AZT ranged from 0.13 to 11 and 0.68 to 11.1%, respectively, while for AZT-MP it ranged from 2.05 to 11.1 and 4.22 to 11.7%. Inter-day precision and accuracy for AZT ranged from 3.82 to 11.2 and 3.14 to 9.01%, while for AZT-MP it ranged from 3.9 to 9.32 and 3.44 to 9.37%, respectively. We also report the enzymatic dephosphorylation of AZT-MP in the placental tissue of rats. This new enzymatic pathway provides increased understanding of the mechanism of anti-viral transport in the rat during pregnancy.

Optimization of the separation of a group of triazine herbicides by micellar capillary electrophoresis using experimental design and artificial neural networks.

The micellar electrokinetic chromatography separation of a group of triazine compounds was optimized using a combination of experimental design (ED) and artificial neural network (ANN). Different variables affecting separation were selected and used as input in the ANN. A chromatographic exponential function (CEF) combining resolution and separation time was used as output to obtain optimal separation conditions. An optimized buffer (19.3 mM sodium borate, 15.4 mM disodium hydrogen phosphate, 28.4 mM SDS, pH 9.45, and 7.5% 1-propanol) provides the best separation with regard to resolution and separation time. Besides, an analysis of variance (ANOVA) approach of the MEKC separation, using the same variables, was developed, and the best capability of the combination of ED-ANN for the optimization of the analytical methodology was demonstrated by comparing the results obtained from both approaches. In order to validate the proposed method, the different analytical parameters as repeatability and day-to-day precision were calculated. Finally, the optimized method was applied to the determination of these compounds in spiked and nonspiked ground water samples.