In this work, we describe the introduction of a post-column solid-state reactor in the HPLC system used for the analyses of amino acids. The reactor used was filled with copper(II) oxide. Passage of the analytes through the reactor leads to the formation of Cu(II) complexes. Unlike free amino acids, the Cu-complexes show significant absorbance in the UV region and accordingly sensitivity of UV-VIS detection is increased by two to three orders of magnitude. As a result of this improvement in sensitivity, we have obtained LOD values in micromolar range and good linearity over the studied concentration range (5.0×10(-5) to 2.0×10(-3) mol/L). The method exhibits advantages typical of solid-state reactors, such as negligible loss of efficiency due to the derivatization, simplicity of realization and a long-term durability. The presented system brings an easy and versatile solution for UV-VIS detection of coordinating compounds, which do not normally absorb well in the UV-VIS region.
A new method for the electrophoretic separation of nine phenolic acids (derivatives of benzoic and cinnamic acids) with contactless conductometric detection is presented. Based on theoretical calculations, in which the mobility of the electrolyte co- and counterions and mobility of analytes are taken into consideration, the electrolyte composition and detection mode was selected. This approach was found to be especially valuable for optimization of the electrolyte composition for the separation of analytes having medium mobility. Indirect conductometric detection mode was superior to the direct mode as predicted theoretically. The best performance was achieved with 150 mM 2-amino-2-methylpropanol electrolyte at pH 11.6. The separation was carried out in a counter-electroosmotic mode and completed in less than 6 min. The LODs achieved were about 2.3-3.3 microM and could be further improved to 0.12-0.17 microM by using a sample stacking procedure. The method compares well to the UV-Vis detection.
Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was applied to separation and sensitive determination of red food colorants. Diode pumped frequency-doubled Nd:YAG laser (532 nm) was used as an excitation source in a laboratory-built CE-LIF system. For highly fluorescent erythrosine B (E127), an extrapolated limit of detection (LOD) of 0.4 ng mL(-1) (S/N=3) was achieved. Extrapolated LODs of other tested red additives, such as carmoisine, E122 (0.5 microg mL(-1)); amaranth, E123 (0.2 microg mL(-1)); ponceau 4R, E124 (0.3 microg mL(-1)) and red 2G, E128 (0.3 microg mL(-1)) were about one-order lower compared to results obtained with CE with absorbance detection in UV/vis (CE-UV/vis). The main advantages of using CE-LIF for analysis of food samples are high selectivity and minimization of matrix effect. To our knowledge, this is the first use of CE-LIF for determination of red food colorants.
CE/frontal analysis (CE/FA) is probably one of the most frequently used modes of CE for studying affinity interactions. It is typically performed with classic UV-Vis detection that suffers from low concentration sensitivity. To overcome this limitation, the applicability of CE/FA in combination with ESI-MS detection for the investigation of drug-HSA interactions was demonstrated. The developed new method combines the advantages of CE/FA, such as low sample consumption and no labeling or immobilization of interacting partners, with the benefits of MS detection, such as higher selectivity and sensitivity; moreover, it can be used for molecules lacking a fluorophore or chromophore. The binding parameters of tolbutamide (TL) and glimepiride (GLP), first- and second-generation antidiabetics that differ strongly in their solubility in aqueous solutions, were investigated by this CE/FA-MS method. This method, in contrast to the CE/FA method with the most commonly used UV-Vis detection, is more sensitive; an almost three times lower LOD was reached. The binding parameters of TL and GLP were investigated by this CE/FA-MS method and compared with the literature data. The binding constant value of TL obtained by UV-Vis detection was lower than the value obtained by the method hyphenated with MS detection, which is probably given by the influence of the ESI parameters on the stability of drug-HSA complex. In addition, the ratio of TL and HSA concentrations was divergent in both of the experimental approaches. Finally, it can be concluded that both detection methods have their strengths and weaknesses.
Flow injection analysis (FIA) is an analytical method where the reaction mixture is injected into flow of liquid. The reaction product is monitored by a suitable detector such as ultraviolet/visible (UV/VIS) spectrophotometric or electrochemical detector. Mass spectrometric detectors (MS) are coming to be a standard equipment of analytical laboratories in the present time. This work is focused on application of FIA-MS instrumentation for monitoring of Ellman's reaction where both reactants (acetylthiocholine and 5,5’-dithiobis-2-nitrobenzoic acid, DTNB) and the reaction product (5-mercapto-2-nitrobenzoic acid) are monitored. This reaction is usually used for monitoring of acetylcholinesterase and butyrylcholinesterase. Due to its simplicity, the developed method is generally applicable for monitoring of enzymatic reactions of cholinesterases. The main advantage of this method is high selectivity and reduction of influence of compounds, which are reacting with DTNB, resulting in a color product of Ellman's reaction.
Catalytic properties and high adsorption affinity of nucleic acids and proteins to silver amalgam electrode surface make this kind of electrified interface perspective for bioanalytical and biomedical applications. For the first time, a basal-plane pyrolytic graphite electrode (bPGE) has been used as a substrate for electrodeposition of silver amalgam particles (AgAPs). Optimization of the resulting composition, surface morphology and electrochemical properties of the AgAPs was done by scanning electron microscopy with energy disperse X-ray spectroscopy, image processing software and voltammetric detection of electrochemically reducible model organic nitro compound, 4-nitrophenol. Spectro-electrochemical applicability of bPGE-AgAP has been demonstrated by electrolysis of 4-nitrophenol. Simultaneous UV-Vis-chronoamperometry provided information on the number of exchange electrons and the reduction rate constants. Preferential adsorption of the fluorescently labelled calf thymus DNA and the green fluorescent protein (GFP) on the surface of AgAPs was observed by fluorescence microscopy. In contrast to previously studied indium-tin oxide and vapour-deposited gold decorated by AgAPs, herein the presented bPGE-AgAP has provided sufficiently wide negative potential window allowing direct electroanalysis of non-labelled DNA and GFP using intrinsic electrochemical signals independently of the fluorescent labelling. The bPGE-AgAP can thus be expected to find application opportunities in protein electrochemistry, (bio)sensor development or in-situ spectro-electrochemical studies.
Edukační publikace se zabývá v první části využitím čipů v imunochemii pro mikroanalýzu. Je uveden přehled podkladových materiálů a značek, jakož i senzitivita jednotlivých technologií. Vysvětluje se možnost imobilizace a detekce proteinů pomocí planárního vlnovodu. Ukazuje se používání čipů při paralelní kompetitivní imunoanalýze, sendvičové imunoanalýze a fluorescenční imunoanalýze na mikrokuličkách. Druhá část je věnována kapilární elektroforéze a jejímu využití k separaci po kompetitivní a nekompetitivní homogenní imunoanalýze, a heterogenní imunoanalýze. Diskutují se možnosti detekce analytu jako laserem indukovaná fluorescence, enzymové značky, chemiluminiscence, ampérometrie, UV/VIS absorbance, hmotnostní spektrometrie a povrchová plazmonová rezonance.
The first part of the educational article covers chip application for microarray in immunochemistry. An outline of surface chemistries and labelings is presented, as well as the sensitivity of various technologies. Protein immobilization and detection using planar waveguide technology is clarified. Chip applications are shown in parallel competitive immunoassay, sandwich immunoassay and fluorescent microsphere immunoassay. The second part covers capillary electrophoresis and its use for separation after competitive and non-competitive homogeneous immunoassay or heterogeneous immunoassay. The following possibilities of analyte detection are discussed: laser-induced fluorescence, enzyme labels, chemiluminiscence, amperometry, UV/VIS absorbance, mass spectrometry and surface plasmon resonance.
Twenty three strains of Penicillium expansum, as a predominant species, were isolated from 23 (92%) out of 25 grape samples of 17 different grape varieties. The results of the identification of P. expansum strains were confirmed by a PCR method. Most of the isolates of P. expansum (21/23, 91%), when tested for toxigenicity, were bi-toxigenic: they produced citrinin (CIT) and particularly high amounts of patulin (PAT). A validated UPLC-MS/MS method for the determination of PAT and CIT was applied. The limits of quantification (LOQ) for PAT and CIT in grape must and toxigenicity testing samples were 100 and 2 ng/g, respectively. The results of PAT and CIT quantification in 23 grape must samples demonstrated the occurrence of PAT in 10 (43%) grape must samples (mean: 171 ng/g; median: 50 ng/g; and range: 143-644 ng/g) and the occurrence of CIT in two (9%) grape must samples (mean: 1 ng/g; median: 1 ng/g; and range: 2.5-3.5 ng/g). This is the first report on the natural occurrence of CIT in grape must. A validated HPLC-UV-VIS method for the determination of PAT in wine samples was applied, and concentrations in all 23 wine samples were below the LOQ (<10 ng/g).
- MeSH
- Citrinin analysis MeSH
- Dietary Exposure MeSH
- Species Specificity MeSH
- Limit of Detection MeSH
- Patulin analysis MeSH
- Penicillium classification isolation & purification MeSH
- Polymerase Chain Reaction MeSH
- Reproducibility of Results MeSH
- Spectrophotometry, Ultraviolet MeSH
- Tandem Mass Spectrometry MeSH
- Wine analysis MeSH
- Vitis chemistry MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
Pevná bismutová kapková elektroda (SBiDE – solid bismuth drop electrode) je nová pracovní elektroda, kterou od roku 2020 komerčně nabízí na českém trhu společnost Metrohm. Cílem této práce bylo ověřit použitelnost SBiDE při voltametrickém stanovení modelové organické látky reprezentující elektrochemicky redukovatelné biologicky aktivní sloučeniny – léčiva metronidazolu (antibiotika používaného k léčbě onemocnění způsobených gram-pozitivními i gram-negativními anaerobními bakteriemi). Podle dostupných informací se jedná o vůbec první publikovanou výzkumnou práci využívající SBiDE. Za optimálních podmínek (Brittonův-Robinsonův pufr o pH 12,0 byl použitý jako základní elektrolyt a povrch pracovní elektrody nebyl elektrochemicky regenerován) byla získána pomocí diferenční pulzní voltametrie (DPV) lineární kalibrační závislost metronidazolu v koncentračním rozmezí od 1 do 600 μmol l−1, přičemž dosažená mez detekce (LOD) byla 0,41 μmol l−1 a mez stanovitelnosti (LOQ) byla 1,4 μmol l−1. Cyklická voltametrie (CV) na SBiDE byla použita pro charakterizaci elektrodového děje při ireverzibilní katodické redukci metronidazolu. Nově vyvinutá DPV metoda byla rovněž úspěšně použita při stanovení metronidazolu v autentických vzorcích pitné vody (LOD = 1,8 μmol l−1 a LOQ = 5,8 μmol l−1) a v různých lékových formách (jako srovnávací analytická metoda byla použita UV-Vis spektrofotometrie).
Solid bismuth drop electrode (SBiDE) is a new working electrode commercially available on the Czech market since 2020 by the company Metrohm. The aim of this work was to verify the applicability of SBiDE for the voltammetric determination of a model organic substance representing electrochemically reducible biologically active compounds, namely, the drug metronidazole (an antibiotic used to treat diseases caused by both Gram-positive and Gram-negative anaerobic bacteria). To the best of our knowledge, this is the very first published research work using SBiDE. Under optimum conditions (Britton-Robinson buffer of pH 12.0 was used as the supporting electrolyte and the working electrode surface was not electrochemically regenerated), a linear calibration dependence of metronidazole was obtained using differential pulse voltammetry (DPV) in the concentration range from 1 to 600 μmol L–1, with the limits of detection (LOD) and quantification (LOQ) of 0.41 μmol L–1 and 1.4 μmol L–1, respectively. Cyclic voltammetry (CV) on SBiDE was used to characterize the electrode process of the irreversible reduction of metronidazole. The newly developed DPV method was also successfully applied for the determination of metronidazole in authentic drinking water samples (LOD = 1.8 μmol L−1 and LOQ = 5.8 μmol L−1) and in various dosage forms (UV-Vis spectrophotometry was used as a comparative analytical method).
The oxidation mechanism of hematoxylin was studied in phosphate buffers and 0.1 M KCl by cyclic voltammetry and UV-Vis spectroscopy under deaerated conditions. The redox potential of hematoxylin in buffered solution strongly depends on pH. A two electron oxidation is preceded by deprotonation. The homogeneous rate of deprotonation process of hematoxylin in 0.1 M phosphate buffer is kd = (2.5 ± 0.1) × 104 s–1. The cyclic voltammetry under unbuffered conditions shows the distribution of various dissociation forms of hematoxylin. The dissociation constants pK1 = 4.7 ± 0.2 and pK2 = 9.6 ± 0.1 were determined using UV-Vis spectroscopy. The final oxidation product was identified by gas chromatography with mass spectrometry detection as hemathein. The distribution of oxidation products differs under buffered and unbuffered conditions. The dye degradation in natural unbuffered environment yields hemathein and hydroxyhematoxylin, which is absent in buffered solution.
