Andrea Staňová

Early Zn2+-induced effects on membrane potential account for primary heavy metal susceptibility in tolerant and sensitive Arabidopsis species

BACKGROUND AND AIMS Uptake of heavy metals by plant root cells depends on electro-physiological parameters of the plasma membrane. In this study, responses of the plasma membrane in root cells were analysed where early reactions to the metal ion-induced stress are localized. Three different Arabidopsis species with diverse strategies of their adaptation to heavy metals were compared: sensitive Arabidopsis thaliana and tolerant A. halleri and A. arenosa. METHODS Plants of A. thaliana Col-0 ecotype and plants of A. arenosa and A. halleri originating from natural metallicolous populations were exposed to high concentrations of Zn(2+). Plants were tested for root growth rate, cellular tolerance, plant morphology and cell death in the root apex. In addition, the membrane potential (E(M)) of mature cortical root cells and changes in the pH of the liquid culture media were measured. KEY RESULTS Primary roots of A. halleri and A. arenosa plants grew significantly better at increased Zn(2+) concentrations than A. thaliana plants. Elevated Zn(2+) concentrations in the culture medium induced rapid changes in E(M). The reaction was species-specific and concentration-dependent. Arabidopsis halleri revealed the highest insensitivity of the plasma membrane and the highest survival rate under prolonged treatment with extra-high concentrations. Plants were able to effectively adjust the pH in the control, but much less at Zn(2+)-induced lower pH. CONCLUSIONS The results indicate a similar mode of early reaction to Zn(2+), but with different extent in tolerant and sensitive species of Arabidopsis. The sensitivity of A. thaliana and a high tolerance of A. halleri and A. arenosa were demonstrated. Plasma membrane depolarization was lowest in the hyperaccumulator A. halleri and highest in A. thaliana. This indicates that rapid membrane voltage changes are an excellent tool to monitor the effects of heavy metals.

Determination of selected biogenic amines in red wines by automated on-line combination of capillary isotachophoresis–capillary zone electrophoresis

A screening analytical method based on an automated on-line combination of capillary isotachophoresis-capillary zone electrophoresis (cITP-CZE) in hydrodynamically closed separation system, equipped with photometric detection at 280 nm, was developed for a routine determination of the selected biogenic amines, namely histamine, 2-phenylethylamine and tyramine, in red wines. The evaluated limits of detection (LODs) were 0.35 mg L(-1) for histamine, 0.33 mg L(-1) for 2-phenylethylamine and 0.37 mg L(-1) for tyramine. The repeatability of the migration time and peak area for histamine were 1.1% and 2.6%, respectively, for 2-phenylethylamine 0.7% and 2.0%, respectively, and for tyramine 0.8% and 2.1%, respectively. The method recoveries were 92.1% for histamine, 96.4% for 2-phenylethylamine and 95.5% for tyramine. The developed automated cITP-CZE-UV method was applied for the determination of histamine, 2-phenylethylamine and tyramine in seven red wine samples originating from Czech Republic.

On-line capillary isotachophoresis-capillary zone electrophoresis analysis of bromate in drinking waters in an automated analyzer with coupled columns and photometric detection.

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 200 nm 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 0.6 μg/L was achieved. Good repeatabilities of migration time (RSD less than 0.3%) and peak area (RSD less than 4.0%) at concentration level 1 μg/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.

Analysis of therapeutic peptides in human urine by combination of capillary zone electrophoresis–electrospray mass spectrometry with preparative capillary isotachophoresis sample pretreatment

The presented study deals with the off-line coupling of preparative isotachophoresis (pITP) with on-line combination of capillary zone electrophoresis with electrospray mass spectrometric detection (CZE-ESI-MS) used for the analysis of therapeutic peptides (anserine, carnosine, and buserelin) in complex matrix (urine). Preparative capillary isotachophoresis, operating in a discontinuous fractionation mode in column-coupling configuration, served as a sample pretreatment technique to separation, and fractionation of mixture of therapeutic peptides present in urine at low concentration level. The fractions isolated by pITP procedure were subsequently analyzed by capillary zone electrophoresis with electrospray mass spectrometric detection. Acetic acid at 200 mmol L(-1) concentration served as background electrolyte in CZE stage and it is compatible with MS detection in positive ionization mode. In pITP fractionation procedure, sodium cation (10 mmol L(-1) concentration) as leading ion and beta-alanine as terminating ion (20 mmol L(-1) concentration) were used. While using CZE-ESI-MS, the limits of detection were 0.18 μg mL(-1) for carnosine, 0.17 μg mL(-1) for anserine and 0.64 μg mL(-1) for buserelin in water and 0.19 μg mL(-1) for carnosine, 0.50 μg mL(-1) for anserine and 0.74 μg mL(-1) for buserelin in 10 times diluted urine, respectively. The cleaning power of pITP sample pretreatment was proved as the peptides provided the higher MS signals at lower concentration levels resulting from the minimized matrix effects. The quality of obtained MS/MS spectra was very good so that they can provide information about the structure of analytes, and they were used for verification of the analytes identities. The pITP pretreatment improved the detection limits of the analyzed therapeutic peptides at least 25 times compared to the CZE-ESI-MS itself.

Determination of brominated phenols in water samples by on-line coupled isotachophoresis with capillary zone electrophoresis.

A method for determination of nine brominated phenols as environmental risk compounds was developed by on-line coupled capillary isotachophoresis and capillary zone electrophoresis (ITP-CZE). For ITP step, 1x10(-2) mol L(-1) hydrochloric acid with 3x10(-2) mol L(-1) ammediol pH 9.1 was used as the leading electrolyte, and 3x10(-2) mol L(-1) beta-alanine with 2x10(-2) mol L(-1) sodium hydroxide pH 10.05 was used as the terminating electrolyte. As the background electrolyte for CZE separation, 2.5x10(-2) mol L(-1) beta-alanine with 2.5x10(-2) mol L(-1) lysine pH 9.6 was used. All electrolytes contained 0.05% or 0.1% (m/v) hydroxyethylcellulose to suppress the electroosmotic flow. UV detection at wavelength 220 nm was used. Detection limits in order of tens of nmol L(-1) were achieved. Good repeatability of migration times (less than 0.33% RSD) and good repeatability of peak areas (less than 7.19% RSD) at concentration level 5x10(-8) mol L(-1) were observed. Developed ITP-CZE method was applied to determination of brominated phenols in spiked tap and river water samples.

Preparative isotachophoresis with surface enhanced Raman scattering as a promising tool for clinical samples analysis

A surface enhanced Raman scattering (SERS) spectrometry is an interesting alternative for a rapid molecular recognition of analytes at very low concentration levels. The hyphenation of this technique with advanced separation methods enhances its potential as a detection technique. Until now, it has been hyphenated mainly with common chromatographic and electrophoretic techniques. This work demonstrates for a first time a power of preparative isotachophoresis-surface enhanced Raman scattering spectrometry (pITP-SERS) combination on the analysis of model analyte (buserelin) in a complex biological sample (urine). An off-line identification of target analyte was performed using a comparison of Raman spectra of buserelin standard with spectra obtained by the analyses of the fractions from preparative isotachophoretic runs. SERS determination of buserelin was based on the method of standard addition to minimize the matrix effects. The linearity of developed method was obtained in the concentration range from 0.2 to 1.5 nmol L(-1) with coefficient of determination 0.991. The calculated limit of detection is in tens of pico mols per liter.

Some possibilities of an analysis of complex samples by a mass spectrometry with a sample pretreatment by an offline coupled preparative capillary isotachophoresis.

This work deals with an analysis of biologically important compounds in complex matrices using preparative isotachophoresis (pITP) in column coupling configuration as a sample pretreatment technique followed by a direct infusion mass spectrometry with nano‐electrospray ionization (DI‐nESI‐MS). Busereline was chosen as a model analyte, and urine was chosen as an example of complex matrix. In pITP experiments, sodium cation (10 mmol/L concentration) was used as a leading ion and β‐alanine as terminating ion (20 mmol/L concentration). The fractions, obtained by pITP pre‐separation with the assistance of the mixture of discrete spacers, were finally analyzed by DI‐nESI‐MS. It was shown that pITP performed before DI‐nESI‐MS analysis can significantly simplify complex matrix, and, due to its concentration power, pITP can consequently decrease the concentration limit of detection. The concentration of buserelin in the urine samples analyzed by pITP‐DI‐nESI‐MS was 10 μg/L (reflecting at a 8.10−9 mol/L concentration) in our work but from the ion intensities obtained in MS as well as MS/MS analyses, it is clear that this concentration level could be several orders of magnitude lower for reliable detection and identification of buserelin in urine analyzed using pITP with DI‐nESI‐MS detection.

Buffer salt effects in off‐line coupling of capillary electrophoresis and mass spectrometry

In this work, the impact of buffer salts/matrix effects on the signal in direct injection MS with an electrospray interface (DI‐ESI‐MS) following pITP fractionation of the sample was studied. A range of buffers frequently used in CE analyses (pH 3–10) was prepared containing 10, 50, and 90% v/v of ACN, respectively. The sets of calibration solutions of cetirizine (an antihistaminic drug with an amphiprotic character) within a 0.05–2.0 mg/L concentration range were prepared in different buffers. The greatest enhancements in the MS signal (in terms of change in the slope of the calibration line) were obtained for the beta‐alanine buffer (pH 3.5) in positive ionization and for the borate buffer (pH 9.2) in negative ionization, respectively. The procedure was successfully applied to the analysis of buserelin (a peptidic drug). The slope of the calibration line for solutions containing the beta‐alanine buffer with 50% of ACN was 4 times higher than for water or urine, respectively. This study clearly demonstrates that the buffer salt/matrix effects in an off‐line combination of pITP and DI‐ESI‐MS can also play a positive role, as they can enhance the signal in MS. A similar influence of the above effects can also be presumed in the CE techniques combined on‐line with ESI‐MS.

Methodological aspects of an off-line combination of preparative isotachophoresis and high-performance liquid chromatography with mass spectrometry in the analysis of biological matrices

This work reports on some methodological aspects of an off‐line combination of preparative ITP and HPLC with mass spectrometric detection (pITP‐HPLC‐MS) and its potential applications to the analysis of high molecular mass compounds present in complex biological matrices from the analytical chemistry perspective. Lysozyme served as the model analyte and human saliva as the complex biological matrix in this study. A mixture of five low‐molecular mass compounds was found and successfully used in the pITP experiments as discrete spacers to isolate the analyte from the interferents present in the complex biological matrix and to minimize their disturbance effect on the final MS analysis. The experiments at the pITP stage were performed in the cationic mode. On‐column conductivity detectors were used for the detection of ITP zones. Lysozyme was found in the human saliva samples using just deconvolution of the MS data after background correction. The MS data obtained from HPLC‐MS analysis of pITP fractions exhibited the great analytical potential of the combination of pITP‐HPLC‐MS resulting from the ITP clean‐up effect as well as the ITP preconcentration of the analyte present at low concentration levels in complex biological matrices.