Jozef Marák

Capillary electrophoresis of inorganic anions

This review deals with the separation mechanisms applied to the separation of inorganic anions by capillary electrophoresis (CE) techniques. It covers various CE techniques that are suitable for the separation and/or determination of inorganic anions in various matrices, including capillary zone electrophoresis, micellar electrokinetic chromatography, electrochromatography and capillary isotachophoresis. Detection and sample preparation techniques used in CE separations are also reviewed. An extensive part of this review deals with applications of CE techniques in various fields (environmental, food and plant materials, biological and biomedical, technical materials and industrial processes). Attention is paid to speciations of anions of arsenic, selenium, chromium, phosphorus, sulfur and halogen elements by CE.

Capillary zone electrophoresis of complex ionic mixtures with on-line isotachophoretic sample pretreatment

Separation modes provided by the column-coupling configuration of the separation unit in an on-line combination of capillary isotachophoresis (ITP) with capillary zone electrophoresis (CZE) were studied from the point of view of their potential in the (trace) determination of ions present in complex ionic matrices. Urine was arbitrarily chosen as such a matrix while sulphanilate and 3,5-dinitrosalicylate (currently not present in urine) served as model analytes. In one of these modes, ITP was employed to remove only the most abundant sample constituent (chloride) and concentrate the rest of those migrating between the leading and terminating zones for injection into the ZE stage. In the other mode, ITP was employed for maximum sample clean-up. Here, only the analyte(s) with a minimum of the matrix constituents was transferred for a final separation in the ZE stage. The fraction to be transferred was defined via a pair of discrete spacers added to the sample. Although a highly efficient sample clean-up was typical in this instance, the use of identical migration regimes in both stages (the separations according to ionic mobilities) did not prove the resolution of one of the analytes (sulphanilate) from the matrix constituent(s) in the ZE stage. A considerable improvement in this respect was achieved easily when the ITP clean-up was based on the separation according to pK values while the constituents present in the transferred fraction were finally separated via differences in their ionic mobilities. This two-dimensional approach provided a way to achieve a 150 ppb (10−6 mol 1−1) concentration detection limit for sulphanilate in a 1-μl volume of urine taken for the electrophoretic run.

Biological and Structural Characterization of the Mycobacterium Smegmatis Nitroreductase Nfnb, and its Role in Benzothiazinone Resistance

Tuberculosis is still a leading cause of death in developing countries, for which there is an urgent need for new pharmacological agents. The synthesis of the novel antimycobacterial drug class of benzothiazinones (BTZs) and the identification of their cellular target as DprE1 (Rv3790), a component of the decaprenylphosphoryl‐β‐d‐ribose 2′‐epimerase complex, have been reported recently. Here, we describe the identification and characterization of a novel resistance mechanism to BTZ in Mycobacterium smegmatis. The overexpression of the nitroreductase NfnB leads to the inactivation of the drug by reduction of a critical nitro‐group to an amino‐group. The direct involvement of NfnB in the inactivation of the lead compound BTZ043 was demonstrated by enzymology, microbiological assays and gene knockout experiments. We also report the crystal structure of NfnB in complex with the essential cofactor flavin mononucleotide, and show that a common amino acid stretch between NfnB and DprE1 is likely to be essential for the interaction with BTZ. We performed docking analysis of NfnB‐BTZ in order to understand their interaction and the mechanism of nitroreduction. Although Mycobacterium tuberculosis seems to lack nitroreductases able to inactivate these drugs, our findings are valuable for the design of new BTZ molecules, which may be more effective in vivo.

Determination of sorbic acid in food products by capillary zone electrophoresis in a hydrodynamically closed separation compartment

Abstract A simple, selective and rapid capillary zone electrophoresis method for the determination of sorbic acid in food products is described. The determination was carried out in a hydrodynamically closed separation compartment at pH 5.2 and for the samples taken into this study (soft drinks, wine, fruit and juice concentrates, margarine and marmalade) only simple sample preparation procedures were needed. A very good reproducibility in the migration time of the analyte (R.S.D. = 0.61%) was achieved and as could be expected no negative effect of matrix constituents on this performance parameter was detected. The recoveries of sorbic acid in the spiked samples ranged from 98-102% and 2-3% R.S.D.s in parallel determinations of the acid were typical.

Direct quantitative determination of amlodipine enantiomers in urine samples for pharmacokinetic study using on-line coupled isotachophoresis-capillary zone electrophoresis separation method with diode array detection.

The present work illustrates possibilities of column-coupling capillary electrophoresis (CE-CE) combined with chiral selector (2-hydroxypropyl-beta-cyclodextrin, HP-beta-CD) and fiber-based diode array detection (DAD) for the direct quantitative enantioselective determination of trace drug (amlodipine, AML) in biological multicomponent ionic matrices (human urine). Capillary isotachophoresis (ITP) served as an ideal injection technique in CE-CE. Moreover, the ITP provided an effective on-line sample pretreatment prior to the capillary zone electrophoresis (CZE) separation. Enhanced separation selectivity due to the combination of different separation mechanisms (ITP vs. CZE-HP-beta-CD) enabled to obtain pure zones of the analytes, suitable for their detection and quantitation. The DAD, unlike single wavelength UV detection, enabled to characterize the purity (i.e. spectral homogeneity) of the analytes zones. A processing of the raw DAD spectra (the background correction and smoothing procedure) was essential when a trace analyte signal was evaluated. Obtained results indicated pure (i.e. spectrally homogeneous) zones of interest confirming effective ITP-CZE separation process. The proposed ITP-CZE-DAD method was characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, selectivity) and successfully applied to an enantioselective pharmacokinetic study of AML.

Capillary zone electrophoresis in a hydrodynamically closed separation system with enhanced sample loadability

Some phenomena linked with capillary zone electrophoresis (CZE) performed in a hydrodynamically closed separation system with enhanced sample load capacity via the use of capillary tubes of larger I.D.s were studied. Calculations of the plate heights for varying amounts of the analytes loaded onto 50 and 300 μm I.D. columns under identical CZE separating conditions revealed that the analytical advantages of using columns of larger I.D. include significantly reduced contributions of electromigration dispersion. At higher concentrations of the carrier electrolytes this gain, however, can be partially lost due to increased thermal dispersive effects. Calculated resolutions for a varying ratio of a pair of the analytes loaded onto 50 and 300 μm I.D. columns favoured the use of the latter I.D. in situations when the ratio of the analytes was higher than ca. 102:1. CZE experiments were carried out in a 300 μm I.D. capillary tube made of fluorinated ethylene–propylene copolymer (FEP) with a porous cellophane membrane serving as a hydrodynamic barrier to prevent a flow of the solution in the separation compartment due to a pressure difference between the electrode vessels. Movement of the membrane was found to be a source of undesired flows in the separation compartment, which adversely affected both the reproducibilities of migration times of the analytes and their separation efficiencies. Mechanical support eliminated these problems. Dispersive phenomena associated with electroosmosis in the closed separation compartment were effectively suppressed by using high molecular weight derivatives of water soluble polymers (methylhydroxyethylcellulose and polyethyleneglycol) in the carrier electrolyte solutions. Examples from the separations of various groups of analytes (synthetic food colourants, some inorganic anions, alkali and alkaline earth metal cations and glycoforms of erythropoietin) are given to illustrate a practical utility of the studied CZE approach.

Preparative capillary isotachophoresis as a sample pretreatment technique for complex ionic matrices in high-performance liquid chromatography

Abstract Preparative capillary isotachophoresis (ITP) was studied for sample pretreatment in the high-performance liquid chromatography (HPLC) of ionogenic analytes present in complex ionic matrices (urine and humic substances). Sulphanilate, methyl (4-aminobenzenesulphonyl)carbamate (asulam), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) served as model analytes. A high sample load of the ITP pretreatment was achieved by performing the preparative separations in tubes of 2.0 and 1.0 mm I.D. in the column-coupling configuration of the separation unit. The ITP separation according to ionic mobilities was combined with gradient elution HPLC in the ion-suppression mode to achieve highly dissimilar (orthogonal) separation systems in both techniques. The pretreatment provided the sample fraction for the HPLC analysis containing in addition to the analyte (sulphanilate) only ca. 2–3% of the urine matrix (spread along the complete elution profile) when a pair of discrete spacers defined the trapped constituents. Under these conditions the limit of detection for sulphanilate in urine could be reduced by more than two orders of magnitude. A high recovery of the pretreatment procedure [99 ± 1.5% for a 1.7 ppm (w/w) concentration of sulphanilate] was typical. For asulam, 2,4-D and MCPA present in a humic matrix it was shown that the ITP pretreatment may also be effective for multi-residue procedures while favourable analytical characteristics of the pretreatment such as recovery and efficient sample clean-up are maintained.

Capillary zone electrophoresis of orotic acid in urine with on-line isotachophoresis sample pretreatment and diode array detection

Potentialities of capillary zone electrophoresis with on-line isotachophoresis sample pretreatment and diode array detection (ITP-CZE-DAD) to the separation, detection and identification of trace analytes present in biological matrices were investigated. Urine represented a multicomponent, variable and high ionic strength matrix while orotic acid was chosen as a model analyte of a practical clinical relevance in this investigation. Using the ITP-CZE combination in the column-coupling configuration of the separation system ITP provided an enhanced sample load capacity to the separation system (a 30 microl sample injection volume), concentrated the analyte and served as an on-line sample clean up technique. On the other hand, CZE performed a final separation of the analyte from matrix constituents present in the ITP pretreated sample and provided favorable conditions for its detection and identification by DAD. Using current correction and smoothing procedures analytically relevant DAD spectra of orotic acid could be obtained also in instances when this was injected in a model sample at a 2 x 10(-7) mol/l concentration (an estimated limit of determination of orotic acid at a 218 nm detection wavelength). ITP-CZE separations of urine samples (based on differences in acid-base properties and host-guest complexations of the analyte and matrix anionic constituents) led to significant sample clean ups. Consequently, DAD spectra of orotic acid matching its reference spectrum, could be acquired also in instances when the acid was present in urine matrices (loaded in 30 microl injection volumes of 20-fold diluted urine samples) at 4-6 x 10(-7) mol/l concentrations. Here, residual trace matrix interferents prevented a closer approach to the above value attainable for model samples. Although this work was focused only on one analyte and urine matrix it implies very promising potentialities of the ITP-CZE-DAD combination in the identification and quantitation of trace analytes present in biological matrices, in general.

Isotachophoretic separation of alkali and alkaline earth metal cations in water—polyethylene glycol mixtures

Abstract The influence of polyethylene glycol (PEG) on the effective mobilities of alkali and alkaline earth metal cations was investigated. It is shown that appropriate proportions of water and PEG in the leading electrolyte enable a complete separation of this group of cations to be achieved by capillary isotachophoresis (ITP). The high dynamic concentration ranges of the ITP determinations achieved in the operational systems based on water—PEG mixtures are illustrated by practical examples (water samples, urine, aqueous extract of apple flesh).

Photometric detection at 405 nm in trace analysis by capillary isotachophoresis

The possibilities of photometric detection at 405 nm were investigated for trace isotachophoretic analysis of various ionic constituents. Increased selectivity of the detection enabled, for example, the detection and determination of nitrophenols at 10(-8) M in a 25-microliter sample. 5-Nitrofurylacrylic acid, a wine stabilizer, could be detected with confidence at ca. 0.1 ppm in wine without any sample pretreatment. Approximately 250-fmol amounts of amino acids, labelled with 2,4-dinitrophenyl, were detectable with the technique employed. This is almost three decades lower in comparison to what is achievable with an high resolution universal detector. The adsorption of the analytes in the sample handling devices as well as in the separation compartment were problems in the analysis at this concentration level. The detection limit given above was achieved when these phenomena were suppressed by the addition of naphthalene-1,3,6-trisulphonate or pyrophosphate to the sample solution.