Complex formation and enantioselectivity studies of triazole fungicide and organophosphorus pesticide enantiomers using capillary electrophoresis

Several cyclodextrin modified-micellar electrokinetic chromatography (CDMEKC) methods were developed for the successful triazole fungicides separation. In the first part, an efficient method was developed for the simultaneous enantioseparation of cyproconazole (4 stereoisomer), bromuconazole (4 stereoisomer) and diniconazole (2 stereisomer) enantiomers using CD-MEKC with a dual mixture of neutral cyclodextrins as chiral selector. The best simultaneous separation of cyproconazole, bromuconazole, and diniconazole enantiomers was achieved with a mixture of 27 mM HP-β-CD and 3 mM HP-γ-CD in 25 mM phosphate buffer (pH 3.0) containing 40 mM sodium dodecyl sulfate (SDS) and 15% iso-propanol as organic modifier. Complete separation of 10 stereoisomer of triazole fungicides were obtained in a single run with good resolution (Rs 1.74“26.31) and high peak efficiency (N > 400 000). In the second part of the study, enantioseparation of hexaconazole, penconazole, myclobutanil, and triadimefon was investigated. Simultaneous enantioseparation of penconazole, myclobutanil, and triadimefon was achieved under acidic condition (pH 3.0) using 25 mM phosphate buffer, 50 mM SDS, and 30 mM HP-γ-CD, with Rs greater than 0.9 whereas, simultaneous enantioseparation of hexaconazole, penconazole, and myclobutanil was successfully achieved under neutral condition (pH 7.0) using 25 mM phosphate buffer, 40 mM SDS, and 40 mM HP-γ-CD, with Rs greater than1.6. In order to improve detection sensitivity, on-line preconcentration technique was investigated. It was found that sweeping technique as an on-line preconcentration technique improved the detection sensitivity of the enantioseparation of cyproconazole, bromuconazole, and diniconazole by 30 to 60-fold, with good repeatabilities in the migration time, peak area and peak height were obtained with RSDs in the range of 0.08“0.32%, 0.03“ 2.44%, and 2.13“8.44% respectively. Furthermore, sweeping technique improved the detection sensitivity of the enantioseparation of hexaconazole, penconazole and myclobutanil by 62- to 67-fold. Good repeatabilities in the migration time, peak area and peak height were obtained with RSDs in the range of 2.39“3.90%, 1.96€“6.15%, and 2.80“6.64% respectively. Finally, the formation constant of diniconazole enantiomers with HP-γ-CD under neutral and acidic condition was investigated using CD-MEKC

Chiral micellar electrokinetic chromatography

The potential of Micellar Electrokinetic Chromatography to achieve enantiomeric separations is reviewed in this article. The separation principles and the most frequently employed separation strategies to achieve chiral separations by Micellar Electrokinetic Chromatography are described. The use of chiral micellar systems alone or combined with other micellar systems or chiral selectors, as well as of mixtures of achiral micellar systems with chiral selectors is discussed together with the effect of different additives present in the separation medium. Indirect methods based on the derivatization of analytes with chiral derivatizing reagents and the use of achiral micelles are also considered. Preconcentration techniques employed to improve sensitivity and the main approaches developed to facilitate the coupling with Mass Spectrometry are included. The most recent and relevant methodologies developed by chiral Micellar Electrokinetic Chromatography and their applications in different fields are presented

Mono-\u3cem\u3eN\u3c/em\u3e-acyl-2,6-diaminopimelic Acid Derivatives: Analysis by Electromigration and Spectroscopic Methods and Examination of Enzyme Inhibitory Activity

Thirteen mono-N-acyl derivatives of 2,6-diaminopimelic acid (DAP)—new potential inhibitors of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18)—were analyzed and characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and two capillary electromigration methods: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Structural features of DAP derivatives were characterized by IR and NMR spectroscopies, whereas CZE and MEKC were applied to evaluate their purity and to investigate their electromigration properties. Effective electrophoretic mobilities of these compounds were determined by CZE in acidic and alkaline background electrolytes (BGEs) and by MEKC in acidic and alkaline BGEs containing a pseudostationary phase of anionic detergent sodium dodecyl sulfate (SDS) or cationic detergent cetyltrimethylammonium bromide (CTAB). The best separation of DAP derivatives, including diastereomers of some of them, was achieved by MEKC in an acidic BGE (500 mM acetic acid [pH 2.54] and 60 mM SDS). All DAP derivatives were examined for their ability to inhibit catalytic activity of DapE from Haemophilus influenzae (HiDapE) and ArgE from Escherichia coli (EcArgE). None of these DAP derivatives worked as an effective inhibitor of HiDapE, but one derivative—N-fumaryl, Me-ester-DAP—was found to be a moderate inhibitor of EcArgE, thereby providing a promising lead structure for further studies on ArgE inhibitors

Quantification of antithrombin isoform proportions in plasma samples of healthy subjects, sepsis patients, and in antithrombin concentrates

Antithrombin (AT) circulates in plasma in two isoforms, AT-alpha (90-95%) and AT-beta (5-10%). AT isoform proportions were measured in plasma samples of 17 healthy subjects and 26 posttraumatic or postoperative septic patients, as well as in 4 commercially available AT concentrates. Total AT was immune-purified from plasma and concentrates. Micellar electrokinetic chromatography was used to analytically separate and quantify the isoforms. Compared with plasma samples of healthy donors, septic plasmas revealed significantly reduced AT activity (p < 0.001) and beta-isoform content (p < 0.05). AT-beta correlated inversely with urea and creatinine serum concentrations (p < 0.01), indicating a relationship between better renal function and higher beta-isoform content. beta-Isoform neither correlated with age, gender, and 28-day mortality, nor with plasma concentrations of various inflammatory and organ function parameters. The commercial AT concentrate, which is equivalent to the current WHO standard, had an AT-beta content close to that found in plasma of healthy subjects. The availability of this novel quantitative AT isoform assay allows, for the first time, a closer look at the role of AT isoforms in hemostasis and sepsis pathophysiology. Copyright (C) 2002 S. Karger AG, Basel

Separation of polycyclic aromatic hydrocarbons by using micellar electrokinetic chromatography

In this work, the use of micellar electrokinetic chromatography (MEKC) for the separation of five polycyclic aromatic hydrocarbon (PAH) compounds is investigated. All of the compounds studied are listed by the United States Environmental Protection Agency as priority pollutants. In micellar electrokinetic chromatography, solutes partition between the aqueous phase and the micellar phase. This partition is based on the hydrophobicity of each compound. In some cases, organic modifiers are added to the electrophoretic solution to enhance the separation efficiency. The organic modifier used in this study is y-cyclodextrin (y-CD), and sodium dodecyl sulfate (SDS) is used as the micellar phase. When both y-CD and SDS are in the electrophoretic solution, a water insoluble, hydrophobic solute is partitioned between the micelles and the y-CD cavity. When the solute is included in the y-CD cavity, which is neutral, it migrates toward the cathode with the electroosmotic velocity. When a solute is incorporated into the SDS micelle, it migrates with the micellar velocity. This differential partition of the solute between the y-CD cavity and the SDS micelles enables the separation to be achieved. The Hewlett Packard HP3DCE system was used to perform the separation. Satisfactory separation was achieved by using an electrophoretic solution comprised of phosphate-borate buffer of pH 7.0 with 10 millimoles of SDS and 2 millimoles of y-CD, in a 50 gm x 56 cm capillary at 15 kilovolts

Applications of partial filling micellar electrokinetic chromatography: from electrospray mass spectrometry to fluorescence polarization immunodetection

Mechanistic studies of partial filling micellar electrokinetic chromatography PF-MEKC) and a theoretical model for the prediction of separation behavior in PF-MEKC was developed in this work. Along with the theoretical development of PF-MEKC, two different detection modes were applied to PF-MEKC, in contrast to the traditional UV detection used in micellar electrokinetic chromatography (MEKC). The first detection mode coupled PF-MEKC with eletrospray ionization mass spectrometry. The second detection mode developed in this work is PF-MEKC coupled with fluorescence polarization immunodetection;First, in the mechanistic studies of PF-MEKC a theoretical model was proposed for predicting the separation behavior of triazine herbicides. Comparisons between conventional and partial filling MEKC in terms of separation efficiency and resolution of the triazines herbicides are discussed;Subsequently, an on-line combination of PF-MEKC and electrospray ionization mass spectrometry (ESI-MS) is demonstrated for the analysis of triazine herbicides. This work offers a mechanism for the separation and mass detection of neutral molecules without the interference of surfactant in the mass spectrometer;Also presented is on-line fluorescent polarization immunodetection of triazine herbicides as a alternative to the traditional immunoassay. This detection schemes demonstrates that the cross-reactivity of an antibody can be utilized to actualize multiresidue detection of cross-reacting analytes

Field-Amplified Sample Injection-Micellar Electrokinetic Chromatography for the Determination of Benzophenones in Food Simulants

A field-amplified sample injection-micellar electrokinetic chromatography (FASI-MEKC) method for the determination of 14 benzophenones (BPs) in a food simulant used in migration studies of food packaging materials was developed, allowing almost baseline separation in less than 21 min. The use of a 10 mM sodium dodecyl sulfate (SDS) solution as sample matrix was mandatory to achieve FASI enhancement of the analyzed BPs. A 21- to 784-fold sensitivity enhancement was achieved with FASI-MEKC, obtaining limits of detection down to 5.168.4 µg/L, with acceptable run-to-run precisions (RSD values lower than 22.3%) and accuracy (relative errors lower than 21.0%). Method performance was evaluated by quantifying BPs in the food simulant spiked at 500 µg/L (bellow the established specific migration limit for BP (600 µg/L) by EU legislation). For a 95% confidence level, no statistical differences were observed between found and spiked concentrations (probability at the confidence level, p value, of 0.55), showing that the proposed FASI-MEKC method is suitable for the analysis of BPs in food packaging migration studies at the levels established by EU legislation

Principles of Micellar Electrokinetic Capillary Chromatography Applied in Pharmaceutical Analysis

Since its introduction capillary electrophoresis has shown great potential in areas where electrophoretic techniques have rarely been used before, including here the analysis of pharmaceutical substances. The large majority of pharmaceutical substances are neutral from electrophoretic point of view, consequently separations by the classic capillary zone electrophoresis; where separation is based on the differences between the own electrophoretic mobilities of the analytes; are hard to achieve. Micellar electrokinetic capillary chromatography, a hybrid method that combines chromatographic and electrophoretic separation principles, extends the applicability of capillary electrophoretic methods to neutral analytes. In micellar electrokinetic capillary chromatography, surfactants are added to the buffer solution in concentration above their critical micellar concentrations, consequently micelles are formed; micelles that undergo electrophoretic migration like any other charged particle. The separation is based on the differential partitioning of an analyte between the two-phase system: the mobile aqueous phase and micellar pseudostationary phase. The present paper aims to summarize the basic aspects regarding separation principles and practical applications of micellar electrokinetic capillary chromatography, with particular attention to those relevant in pharmaceutical analysis

Assay of empagliflozin tablets by a stability-indicating micellar electrokinetic chromatography method and cytotoxicity study of degraded samples

The first method by micellar electrokinetic chromatography (MEKC) for the determination of empagliflozin in tablets was developed and validated following the ICH guidelines. The separation was achieved in a fused silica capillary with 50 µm x 40 cm (inner diameter x effective length) at 28 ºC, +28 kV voltage, hydrodynamic injection 4s (50 mBar), detection at 225 nm and paracetamol was the internal standard. The running electrolyte was a mixture of 20 mM tris(hydroxymethyl) aminomethane (pH 10) and 100 mM sodium dodecyl sulphate (1:1). Specificity was evaluated by the stress testing and the method was specific, with no interference of the degradation product. Linearity was observed in the range of 50 to 150 μg/mL (r=0.9999). The method showed adequate accuracy (recovery value=100.60±0.60%), precision (RSD values &lt;2%) and robustness, which was evaluated by a full factorial design 23. Drug degradation kinetics was evaluated in alkaline andacidic conditions and first-order kinetic was observed in both conditions. The cytotoxicity of sample solutions degraded by UVA and UVC radiation, alkaline and acid media were studied as well. A similar cellular viability profile was observed with a slight decrease only in samples degraded by UVC radiation and basic medium

Fundamentals of micellar electrokinetic chromatography (MEKC)

Micellar electrokinetic chromatography (MEKC) is a useful branch of capillary electrophoresis (CE) that utilizes surfactant above critical micelle concentration (CMC) as pseudo-stationary phase. MEKC can be employed to separate both charged and neutral molecules, individually or simultaneously, including chiral compounds. MECK benefits from high peak efficiency due to electroosmotic flow (EOF) in the separation capillary, compounded with large variety of synthetic surfactants, organic modifiers, temperature and variable separation voltage has made MECK the method of choice for separation scientists. In this review, we present the introduction of CE, fundamentals of surfactant chemistry as it relates to MEKC, separation principles in MECK including equations involved in calculating separation parameters (capacity factor, resolution etc.)