Capillary electrophoretic methods
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This article gives an overview of the applications of capillary electrophoretic methods to investigate the non-covalent interactions of peptides (peptide complexes) with variable middle- and high-molecular-mass receptors (ligands) as well as with small ions and molecules in the period 2007-2014. Different modes of capillary electrophoretic methods, such as mobility shift (vacancy) affinity capillary electrophoresis, multiple injection affinity capillary electrophoresis, partial filling affinity capillary electrophoresis, Hummel-Dryer method, vacancy peak method and (continuous) frontal analysis capillary electrophoresis, are briefly described and their applicability to determination of binding constants of peptide complexes is discussed. In addition, the detailed experimental conditions of individual applications and the values of binding constants of the particular peptide complexes are presented.
- MeSH
- elektroforéza kapilární metody MeSH
- peptidy analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Capillary electrophoresis represents a promising technique in the field of pharmaceutical analysis. The presented review provides a summary of capillary electrophoretic methods suitable for routine quality control analyses of small molecule drugs published since 2015. In total, more than 80 discussed methods are sorted into three main sections according to the applied electroseparation modes (capillary zone electrophoresis, electrokinetic chromatography, and micellar, microemulsion, and liposome-electrokinetic chromatography) and further subsections according to the applied detection techniques (UV, capacitively coupled contactless conductivity detection, and mass spectrometry). Key parameters of the procedures are summarized in four concise tables. The presented applications cover analyses of active pharmaceutical ingredients and their related substances such as degradation products or enantiomeric impurities. The contribution of reported results to the current knowledge of separation science and general aspects of the practical applications of capillary electrophoretic methods are also discussed.
Intoxikace metanolem může způsobit diagnostické potíže, pokud neexistuje údaj o požití závadné látky. V úvodu epidemie metanolových otrav v ČR v roce 2012 jsme se s negativní anamnézou konzumace alkoholu potýkali hned několikrát. Hladiny metanolu byly u těchto několika pacientů stopové, někdy dokonce nulové. Pacienti trpěli četnými přidruženými nemocemi, někteří z nich museli být během přijetí resuscitováni. Jednoduchá metoda stanovení hladiny kyseliny mravenčí nebyla v prvních dnech vlny otrav běžně dostupná. Intoxikace metanolem byla definitivně potvrzena v těchto případech až dodatečným vyšetřením kyseliny mravenčí v séru ve specializované laboratoři s odstupem několika dalších dnů. Tyto skutečnosti nás vedly k ověření možnosti detekce nadlimitní koncentrace formiátu v séru rychlým screeningovým vyšetřením. Analýza vzorků krevního séra, odebraných během léčby intoxikace metanolem, kapilární elektroforézou ukázala, že lze dobře monitorovat hladinu formiátu v séru jak před zahájením terapie, tak během ní. Klíčová slova: metanol – kyselina mravenčí – acidóza – kapilární elektroforéza – hemodialýza
Methanol intoxication can be difficult to diagnose, especially if there is no supporting evidence of ingestion of the toxic compound. At the begining of the methanol intoxication epidemic in the Czech Republic (2012) we faced a negative anamnesis of alcohol consumption several times. The measured levels of methanol in these patients were very low, sometimes methanol was even not detected. The patients were suffering from concomitant illnesses and some of them had to be resuscitated during admission. Simple screening methods able to detect formic acid were not available at the beginning of the epidemic. Eventually, methanol intoxication was confirmed a few days later in a specialized toxicological laboratory. Therefore, we tested a novel capillary electrophoretic method for the rapid screening of concentration of formic acid in the serum above its normal physiological range. Analysis of the serum of the patients intoxicated with methanol proved that monitoring of formate concentration in the serum with the newly developed method before and during the medical management is possible. Keywords: methanol – formic acid – acidosis – capillary electrophoresis – haemodialysis
- Klíčová slova
- kyselina mravenčí,
- MeSH
- acidóza etiologie patofyziologie terapie MeSH
- antidota terapeutické užití MeSH
- časná diagnóza MeSH
- časové faktory MeSH
- dialýza ledvin MeSH
- elektroforéza kapilární * metody statistika a číselné údaje využití MeSH
- ethanol aplikace a dávkování terapeutické užití MeSH
- fomepizol MeSH
- formiáty * krev metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- methanol * krev metabolismus otrava MeSH
- otrava diagnóza patofyziologie terapie MeSH
- péče o pacienty v kritickém stavu MeSH
- pyrazoly terapeutické užití MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
- práce podpořená grantem MeSH
Kapilární elektroforéza (CZE) je alternativní metodou separace a semikvantitativního hodnocení sérových proteinů. Krátké sdělení se zabývá rutinním použitím multikapilárních systémů pro elektroforézu sérových bílkovin a dopadem výměny přístroje Paragon 2000 (Beckman-Coulter) za Capillarys 2 (Sebia) provázené změnou referenčních mezí. Dlouhodobé sledování cyklů externího hodnocení kvality dokládá plynulý přechod a srovnatelnost od elektroforézy na agarózovém nosiči k multikapilárním systémům. Výsledky jsou mezilaboratorně dobře srovnatelné, lze rovněž vyvodit závěr, že změny analytického systému pro rutinní elektroforézu sérových bílkovin v časovém intervalu dvou let nepřinesly komplikace ani v oblasti kvantitativního hodnocení gamapatií.
Capillary zone electrophoresis (CZE) is an alternative method for the separation and quantitative evaluation of serum proteins. CZE is very effective, quick and namely a precise method. Two specific automated multichannel (multicapillary) instruments are available, the Paragon 2000 (Beckman-Coulter) and the Capillarys 2 (Sebia), characterized by different number of assessed protein fractions are mentioned and newly tested reference intervals. Long-term observation of the cycles of external quality assurance (EQA) programs exemplifies smooth transition from agarose electrophoresis to multichannel capillary systems and comparability of analysis. The interlaboratory comparisons indicated good comparability of the results and conclusion may therefore be drawn that the changes in analytical systems for routine electrophoresis of serum proteins within the time interval of two years have not complicated the situation in the area of quantitative evaluation of gammapathies as well.
Purine and pyrimidine nucleotides influence many metabolic pathways and their analogs have been widely used in medicine. A capillary electrophoretic method was developed for measuring intracellular nucleotides. The final BGE consisted of 40 mM citric acid with addition of 0.8 mM CTAB titrated by gamma-aminobutyric acid to pH 4.4. The electrophoretic separations were carried out in an uncoated silica capillary (id/od - 75/375 microm; effective/total length - 90/97 cm). The method allows a complete separation of 21 nucleotides and deoxynucleotides within 15 min with separation efficiencies up to 400,000 theoretical plates per meter. Due to the use of an acidic separation medium, the method offers a high selectivity toward the studied analytes versus possible interferences from matrices. Sample preparation was optimized in order to shorten work-time and prevent analyte degradation. The method was applied for analyzing nucleotides in human erythrocytes and Chinese hamster ovary cells. Diagnostic potential for inherited metabolic disorders of nucleotide metabolism is presented.
- MeSH
- cetrimoniové sloučeniny chemie MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- elektroforéza kapilární metody MeSH
- erytrocyty chemie MeSH
- financování organizované MeSH
- GABA chemie MeSH
- koncentrace vodíkových iontů MeSH
- křečci praví MeSH
- kyselina citronová chemie MeSH
- lidé MeSH
- purinové nukleotidy analýza MeSH
- pyrimidinové nukleotidy analýza MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- zvířata MeSH
Plant vascular tissue is essential for the exchange of water, nutrients, metabolic products, and signals among distant organs in cormophytes. The compositions of phloem and xylem saps are highly dependent on many internal and external factors, and thus their analysis provides a valuable insight into plant physiology, growth, and development as well as nutrition status or presence of biotic or abiotic stresses. Capillary electrophoresis characterized by highly efficient separations and minuscule sample requirements represents a suitable analytical technique for this purpose because the sap constitutes a complex mixture with generally minimal availability. This review aims at providing a comprehensive overview of published capillary electrophoretic methods for the analysis of primary components present in the phloem and xylem saps of higher plants.
Alzheimer's disease is the most common cause of dementia, afflicting over 34 million patients worldwide. Since β-secretase is a rate-limiting enzyme of the production of neurotoxic β-amyloid peptide oligomers abnormally accumulated in the affected brain tissue, its specific inhibition appears to be a promising approach to slowing down or even stopping the progression of the disease. Hence two on-line capillary electrophoretic methods for studies of β-secretase activity based on the principles of transverse diffusion of laminar flow profiles and electrophoretically mediated microanalysis were developed, both using a simple unlabeled peptide substrate and UV detection. The optimized procedures were thoroughly validated and applied for determining the enzyme's kinetic parameters and the inhibition characteristics of two potent probe inhibitors. The resulting values were found to be comparable to literature data obtained with other analytical techniques. The suitability of the employed methodologies for different experimental designs is discussed on the basis of a statistical evaluation of the experimental data. The presented methods constitute a miniaturized and fully automated tool, which should be suitable for kinetic and inhibition studies of β-secretase as a target for Alzheimer's disease drug discovery in the early stages of the development of a new drug.
- MeSH
- aktivace enzymů účinky léků MeSH
- Alzheimerova nemoc enzymologie MeSH
- elektroforéza kapilární normy MeSH
- enzymatické testy přístrojové vybavení metody MeSH
- inhibitory enzymů farmakologie MeSH
- kinetika MeSH
- lidé MeSH
- objevování léků * MeSH
- sekretasy antagonisté a inhibitory MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Despite being commensal bacterium involved in the maintenance of healthy skin, Cutibacterium acnes is also associated with inflammatory diseases. Since inflammatory and immunogenic properties vary between C. acnes phylotypes, reliable classification of clinical C. acnes isolates is important for determining their pathogenicity. Combination of optimized separation methods, polymer-enhanced transient isotachophoresis and sweeping of the charged bacterial cells in micellar electrokinetic chromatography in the roughened fused silica capillary, was used for the separation of twenty clinical C. acnes isolates. Their correct classification into the individual phylotypes was achieved in 20 min at laboratory temperature. In addition, decrease in the separation temperature to 15 °C led to the separation of the individual isolates of some phylotypes. Relative standard deviations of migration times of both intra- and inter-day analyses did not exceed 1.7%. Linearity of the proposed method in the concentration range from 5 × 105 to 1 × 107 cells mL-1 was characterized by the coefficient of determination R2 = 0.9985. Limit of detection of 5 × 105 cells mL-1 (50 cells in 100 nL of the injected sample) was determined for all the examined bacteria.
- MeSH
- acne vulgaris * MeSH
- chromatografie micelární elektrokinetická kapilární * MeSH
- kůže MeSH
- lidé MeSH
- micely MeSH
- oxid křemičitý chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Capillary electrophoresis coupled to mass spectrometry (CE/MS) is gaining its space among the most powerful tools in modern (bio)analytical laboratory. The most challenging instrumental aspect in CE/MS is striking the balance between the stability and reproducibility of the signal and sensitivity of the analysis. Several interface designs have been published in the past decade addressing the variety of instrumental aspects and ease of operation. Most of the interfaces can be categorized either into the sheath flow arrangement (considered to be a de facto standard), or sheathless interface, often expected to provide the ultimate sensitivity. In this work we have explored an "interface-free" approach, where the CE/MS analysis was performed in narrow bore (<20 μm ID) electrospray capillary. The separation capillary and electrospray tip formed one entity and the high voltage, applied at the injection end of the capillary served for both the separation and electrospray ionization. Thus the separation voltage was defined as the product of the electrospray current and resistivity of the separation electrolyte. Optimum conditions for the separation and electrospray ionization were achieved with voltage programming. The performance of this simplest possible CE/MS system was tested on peptide separations from the cytochrome c tryptic digest. The subnanoliter sample consumption and sensitivity in the attomole range predetermines such a system for analysis of limited samples.
This chapter deals with the application of affinity capillary electrophoresis (ACE) to investigation of noncovalent interactions (complexes) of valinomycin, a macrocyclic dodecadepsipeptide antibiotic ionophore, with ammonium and alkali metal ions (lithium, sodium, potassium, rubidium, and cesium). The strength of these interactions was characterized by the apparent binding (stability, association) constants (K b) of the above valinomycin complexes using the mobility shift assay mode of ACE. The study involved measurements of effective electrophoretic mobility of valinomycin at variable concentrations of ammonium or alkali metal ions in the background electrolyte (BGE). The effective electrophoretic mobilities of valinomycin measured at ambient temperature and variable ionic strength were first corrected to the reference temperature 25 °C and constant ionic strength (10 or 25 mM). Then, from the dependence of the corrected valinomycin effective mobility on the ammonium or alkali metal ion concentration in the BGE, the apparent binding constants of the valinomycin-ammonium or valinomycin-alkali metal ion complexes were determined using a nonlinear regression analysis. Logarithmic form of the binding constants (log K b) were found to be in the range of 1.50-4.63, decreasing in the order Rb(+) > K(+) > Cs(+) > > Na(+) > NH4 (+) ~ Li(+).
