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Concept: Micellar electrokinetic chromatography

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The physical/chemical stability and potential interactions after diluting two immunoglobulin G1 monoclonal antibodies (mAb), pertuzumab (Perjeta®) and trastuzumab (Herceptin®), in a single intravenous (i.v.) infusion bag containing 0.9% saline (NaCl) solution was evaluated. As commercial products, pertuzumab and trastuzumab are administered through i.v. infusion to patients sequentially, that is, one drug after the other. To increase convenience and minimize the in-clinic time for patients, the compatibility of coadministering pertuzumab (420 and 840 mg) mixed with either 420 or 720 mg trastuzumab, respectively, in a single 250 mL polyolefin or polyvinyl chloride i.v. bag stored for up to 24 h at 5°C or 30°C was determined. The controls (i.e., pertuzumab alone in an i.v. bag, trastuzumab alone in an i.v. bag) and the mAb mixture were assessed using color, appearance, and clarity, concentration, and turbidity by ultraviolet spectroscopy, particulate analysis by light obscuration, size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate, analytical ultracentrifugation, and ion-exchange chromatography. Additionally, capillary zone electrophoresis, imaged capillary isoelectric focusing, and potency were utilized to measure the stability of the admixtures containing 1:1 mixtures of pertuzumab/trastuzumab and their respective controls (420 mg pertuzumab alone and 420 mg trastuzumab alone). No observable differences were detected by the above methods in the pertuzumab/trastuzumab mixtures stored up to 24 h at either 5°C or 30°C. The physicochemical methods as listed above were able to detect both molecules as well as the minor variants in the drug mixture, even though some overlap of mAb species were seen in the chromatograms and electropherograms. Furthermore, biophysical analysis also did not show any interactions between the two mAbs or any physical instability under these conditions. Additionally, the drug mixture tested by the pertuzumab-specific inhibition of cell proliferation bioassay showed comparable potency before and after storage. On the basis of these results, pertuzumab and trastuzumab admixture in a single i.v. bag is physically and chemically stable for up to 24 h at 5°C or 30°C and can be used for clinical administration. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.

Concepts: Monoclonal antibodies, Gel electrophoresis, Mixture, Chromatography, Capillary electrophoresis, Electrophoresis, Micellar electrokinetic chromatography, Affinity electrophoresis

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A new, sensitive, and robust analytical method based on capillary zone electrophoresis with on-line capillary isotachophoresis sample pretreatment (ITP-CZE) using a column-coupling (CC) arrangement of automated capillary electrophoretic analyzer was developed for determination of bromate in different type of drinking water samples. Both columns were provided with contact-less conductivity detectors and in CZE step UV detection at 200nm wavelength was used. Electroosmotic flow of the buffer solutions was suppressed with the addition of 0.1% or 0.05% (m/v) methylhydroxyethylcellulose into the leading and terminating electrolyte, respectively. Hydrodynamic and electroosmotic flows of the buffer solutions were successfully suppressed and therefore, only the electrophoretic transport of ions was significant. Limit of detection for bromate approaching 0.6μg/L was achieved. Good repeatabilities of migration time (RSD less than 0.3%) and peak area (RSD less than 4.0%) at concentration level 1μg/L were obtained. Robustness of proposed ITP-CZE method and validation parameters were evaluated. Developed automated ITP-CZE method was applied to the determination of bromate in drinking water samples with different content of inorganic macroconstituents without the need of further sample preparation.

Concepts: Water, Gel electrophoresis, Capillary electrophoresis, Electrophoresis, Micellar electrokinetic chromatography, Affinity electrophoresis, Capillary fringe, Isotachophoresis

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INTRODUCTION: Seeds of Aesculus hippocastanum L. are used in European phytotherapy to treat inflammatory and vascular problems, and also to help in the regulation of the microcirculation. Thus, the quality control of herbal medicines using this species is important. OBJECTIVE: To develop and to optimise a capillary zone electrophoresis method to determine total β-escin in different extracts of A. hippocastanum L. METHODS: The optimal condition found through chemometric approach was: 25 mmol/L of bicarbonate-carbonate buffer, pH 10.3; +20 kV of voltage; 20°C of cartridge temperature; direct ultraviolet detection at 226 nm; 13 mbar injection for 5 s and analysis time within 6 min. RESULTS: Repeatability, coefficient of variation (CV; %) = 3.19, 3.07 and 1.89 (n = 12), and intermediate precision, CV (%) = 3.05, 3.53 and 2.99 (n = 24) for dry, hydroalcoholic and hydroglycolic extracts, respectively were achieved. The accuracy was evaluated through recovery tests in concentration levels of 100, 150 and 200 g/L, ranging from 98.17 to 104.68%. The proposed method exhibited linearity (r = 0.9983) in the concentration range from 101.4 to 907.2 g/L and limits of detection and quantification equal to 11.63 and 38.76 g/L respectively. CONCLUSION: A fast and reliable methodology for determination of total β-escin was successfully validated and applied on extracts of A. hippocastanum L. demonstrating its usefulness to quality control of medicines containing this plant species. Copyright © 2013 John Wiley & Sons, Ltd.

Concepts: Gel electrophoresis, Capillary electrophoresis, Electrophoresis, Micellar electrokinetic chromatography, Affinity electrophoresis, Capillary fringe, Aesculus hippocastanum, Aesculus

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Micellar electrokinetic capillary chromatography with electrochemical detection has been used to quantify biogenic amines in freeze-dried Drosophila melanogaster brains. Freeze drying samples offers a way to preserve the biological sample while making dissection of these tiny samples easier and faster. Fly samples were extracted in cold acetone and dried in a rotary evaporator. Extraction and drying times were optimized in order to avoid contamination by red-pigment from the fly eyes and still have intact brain structures. Single freeze-dried fly-brain samples were found to produce representative electropherograms as a single hand-dissected brain sample. Utilizing the faster dissection time that freeze drying affords, the number of brains in a fixed homogenate volume can be increased to concentrate the sample. Thus, concentrated brain samples containing five or fifteen preserved brains were analyzed for their neurotransmitter content, and five analytes; dopamine N-acetyloctopamine, N-acetylserotonin, N-acetyltyramine, N-acetyldopamine were found to correspond well with previously reported values.

Concepts: Micellar electrokinetic chromatography, Food preservation, Freeze drying, Vacuum, Sublimation

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An ionic liquid (IL)-modified micellar electrokinetic chromatography (MEKC) method was proposed for the separation and determination of eight phenolic acids. In order to increase separation efficiency and selectivity, the micelle system consisting of aqueous mixtures of ILs, Tween 20 and borate was optimized using a D-optimal design. A 16-run experimental plan was carried out. The results indicated that the addition of ILs in background electrolyte could significantly alter the electrophoretic behavior and improve the resolution of target analytes. By evaluating the electropherograms obtained, a satisfactory separation condition for all analytes was achieved in 10min with optimized buffer composed of 0.70% (w/w) 1-butyl-3-methylimidazolium tetrafluoroborate, 8.1% (w/w) polyoxyethylene sorbitan monolaurate (Tween 20) and 10mM sodium borate at pH 9.2. Under these conditions, all calibration curves showed good linearity (r(2)>0.9969), and accuracy (recoveries ranging from 94.71 to 106.85%). Finally, the proposed method was successfully applied to determine the phenolic acids in a Chinese medicine compound, compound danshen dripping pills.

Concepts: Chromatography, Analytical chemistry, PH, Capillary electrophoresis, Micellar electrokinetic chromatography, Borax, Buffer solution, Polysorbate

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Nanoparticle characterization is gaining importance in food technology, biotechnology, medicine, and pharmaceutical industry. An instrument to determine particle electrophoretic mobility (EM) diameters in the single-digit to double-digit nanometer range receiving increased attention is the gas-phase electrophoretic mobility molecular analyzer (GEMMA) separating electrophoretically single charged analytes in the gas-phase at ambient pressure. A fused-silica capillary is used for analyte transfer to the gas-phase by means of a nano electrospray (ES) unit. The potential of this capillary to separate analytes electrophoretically in the liquid phase due to different mobilities is, at measurement conditions recommended by the manufacturer, eliminated due to elevated pressure applied for sample introduction. Measurements are carried out upon constant feeding of analytes to the system. Under these conditions, aggregate formation is observed for samples including high amounts of non-volatile components or complex samples. This makes the EM determination of individual species sometimes difficult, if not impossible. With the current study we demonstrate that liquid phase electrophoretic separation of proteins (as exemplary analytes) occurs in the capillary (capillary zone electrophoresis, CE) of the nano ES unit of the GEMMA. This finding was consecutively applied for on-line desalting allowing EM diameter determination of analytes despite a high salt concentration within samples. The present study is to our knowledge the first report on the use of the GEMMA to determine EM diameters of analytes solubilized in the ES incompatible electrolyte solutions by the intended use of electrophoresis (in the liquid phase) during sample delivery. Results demonstrate the proof of concept of such an approach and additionally illustrate the high potential of a future on-line coupling of a capillary electrophoresis to a GEMMA instrument.

Concepts: Gel electrophoresis, Capillary electrophoresis, Electrophoresis, Liquid, Micellar electrokinetic chromatography, Affinity electrophoresis

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High-resolution capillary zone electrophoresis is used to assess the transferrin profile in serum of patients with eight different congenital disorders of glycosylation that represent type I, type II and mixed type I/II disorders. Capillary zone electrophoresis data are compared to patterns obtained by gel isoelectric focusing. The high-resolution capillary zone electrophoresis method is shown to represent an effective tool to assess the diversity of transferrin patterns. Hypoglycosylated disialo-, monosialo- and asialo-transferrin in type I cases can be distinguished from the corresponding underdesialylated transferrin glycoforms present in type II disorders. The latter can be separated from and are detected ahead of their corresponding hypoglycosylated forms of type I patients. Both types of glycoforms are detected in sera of mixed type I/II patients. The assay has the potential to be used as screening method for congenital disorders of glycosylation. It can be run with a few μL of serum when microvials are used. This article is protected by copyright. All rights reserved.

Concepts: Type I and type II errors, Gel electrophoresis, Capillary electrophoresis, Electrophoresis, Micellar electrokinetic chromatography, Affinity electrophoresis, All rights reserved, Copyright

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Transferrin is the major plasma transport protein for iron. We aimed to investigate the characteristics of transferrin variant by carbohydrate-deficient transferrin (CDT) test using capillary zone electrophoresis.

Concepts: Gel electrophoresis, Capillary electrophoresis, Electrophoresis, Micellar electrokinetic chromatography, Affinity electrophoresis, Capillary fringe

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A simple cyclodextrin-mediated capillary zone electrophoresis method equipped with a laser-induced fluorescence detector was developed for chiral analysis of the excitatory amino acids aspartate (Asp) and glutamate (Glu). Plasma and cerebrospinal fluid (CSF) samples were pretreated with centrifugal filter devices before analysis to remove high-molecular-weight proteins (molecular weight cut-off: 3000) and then derivatized using 10 mM 6-carboxyfluorescein N-hydroxysuccinimide ester in DMSO under sonication at 25 °C for 2 h. After the derivatization reaction, reacted samples were diluted 100-fold with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride (DTAF) solution and then hydrodynamically subjected to capillary electrophoresis (0.5 psi for 5 s, injection volume 8.27 nL). The separation buffer consisted of 50 mM borate buffer (pH 9.0) with 6 mM γ-CD and 0.1% polyvinylpyrrolidone, and the separation voltage was set at 20 kV. In the linearity calculations for the determination of d/l-Asp and d/l-Glu in plasma and CSF, a standard addition method was utilized to spike solutions with 0-20.0 μg mL-1l-Glu and 0-2.0 μg mL-1d-Glu and d/l-Asp to construct calibration curves. Correlation coefficients were above 0.998 for every analyte. The limits of detection (S/N = 3) for d/l-Asp and d/l-Glu standard solutions were 0.85-0.96 μg mL-1. The proposed method was applied successfully to determine d/l-Asp and d/l-Glu concentrations in the plasma and CSF samples of 26 patients with Alzheimer’s disease (AD), and the association between these concentrations and disease severity was investigated. Statistical analysis showed a moderately negative correlation (r = -0.158) between plasma l-Asp concentration and AD severity.

Concepts: Amino acid, Gel electrophoresis, Analytical chemistry, Capillary electrophoresis, Electrophoresis, Micellar electrokinetic chromatography, Affinity electrophoresis

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Rising interest in ellagic acid (EA) present in functional foods is supported by its antimutagenic, anticarcinogenic, antiviral, antibacterial and antioxidative effects. The present approach presents for the first time the determination of ellagic acid and other phenolics in wines by miniaturized solid phase extraction prior to capillary zone electrophoresis with UV. The extraction was performed using a home-made miniaturized pipette-tip column. The procedure allowed a significant reduction in conditioning/sample/washing/elution volumes. The effects of important factors affecting the extraction efficiency as well as electrophoretic performance were investigated to acquire optimum conditions. The analytes were separated within 10 min with a BGE containing 30  mmol L-1 sodium tetraborate 10 % v/v MeOH pH 9.10. The optimized method was applied to the determination of ellagic acid in commercial and pilot-scale wines. Indeed, the content of EA was correlated with viticultural parameters such as grape varietal, production area, and aging conditions (oak wood guard and glass bottle ward). In order to validate the results, a comparison between the CZE and HPLC data was made. This article is protected by copyright. All rights reserved.

Concepts: Gel electrophoresis, Capillary electrophoresis, Electrophoresis, Micellar electrokinetic chromatography, Wine, Affinity electrophoresis