Emil Havránek

Ecotoxicological effects and uptake of metals (Cu+, Cu2+, Mn2+, Mo6+, Ni2+, V5+) in freshwater alga Scenedesmus quadricauda

Abstract The ecotoxicological effects of six metals (Cu+, Cu2+, Mn2+, Mo6+, Ni2+, V5+) expressed as growth inhibition, respiratory rate, and total chlorophyll, chlorophyll a and b contents by alga Scenedesmus quadricauda were determined. Besides these parameters, the uptake of tested metals by alga was determined by using RXFA. On the base of the calculated EC50 values, the following rank orders of inhibition were arranged: for algal growth: Cu2+>Cu+>Ni2+>V5+-Mo6+>Mn2+; for total chlorophyll content: Cu2+>Ni2+>Cu+>Mn2+>V5+>-Mo6+; for chlorophyll a content: Cu2+>Ni2+>Cu+>V5+>Mn2+>Mo6+; for chlorophyll b content: Cu 2+=Cu+>Ni2+>Mn2+>Mo6+>-V5+. Respiratory rate was measured for metal concentrations corresponding with EC50 values for growth inhibition by oxygen electrode. The most intensive respiration was observed for V5+ and less for Mo6+. When the uptake of metals from the medium was determined, in the highest percentage were uptaken Ni2+ (20%), Cu2+ (17,6%) and Mn2+ (15,19%). Other metals were only uptaken in the amounts lower than 5.5 % from the metal concentration added into the cultivation medium. For uptake determination as well as for respiratory rate measurements, concentrations corresponding with calculated EC50 values for growth inhibition were used.

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.

Analysis of enantiomers in biological matrices by charged cyclodextrin- mediated capillary zone electrophoresis in column- coupling arrangement with capillary isotachophoresis

The possibility to apply charged chiral selector as buffer additive in capillary zone electrophoresis (CZE) on-line coupled with capillary isotachophoresis (CITP) was studied. Enantioseparations and determinations of trace (ng/ml) antihistaminic drugs [pheniramine (PHM), dimethindene (DIM), dioxopromethazine (DIO)] present in samples of complex ionic matrices (urine) served as model examples. A negatively charged carboxyethyl-beta-cyclodextrin (CE-beta-CD) was used as a chiral selector in analytical CZE stage following upon a sample pretreatment by CITP (preconcentration of the analytes from 5 to 20-times diluted urine samples, partial sample clean up removing macroconstituents from the sample matrices). A high recognition capability of the oppositely charged CE-beta-CD was demonstrated by enantioselective retardation of the drugs in presence of micro-and semi-macroconstituents migrating in CZE stage and detectable by UV detector. In this way, enantiomers of the drugs could be easily separated and determined. Due to lack of interferences between the drugs and sample-matrix constituents in presence of charged CE-beta-CD, demands on both spacers in CITP step and multiple column-switching were minimized. CITP-CZE method with charged selector appeared to be a useful analytical approach for the trace enantiomers in complex ionic matrices as it combined enhanced separation selectivity and sample loadabitlity with high separation efficiency and provided favorable performance parameters including sensitivity, linearity, precision, accuracy/recovery and robustness with minimal demands on sample preparation. Analysis of urine sample taken from a patient treated by PHM, showing concentration profile of PHM enantiomers and their metabolites, illustrated potentialities of the method in clinical research.

Copper compounds in nuclear medicine and oncology

There have been some notable developments in several areas of inorganic pharmaceuticals that have potentially far-reaching importance for future medical applications and research. One significant development in the field of oncology and hematology is the application of copper complexes. Copper ions play an important role in biological systems, and without their catalytic presence in trace or ultra trace amounts many essential co-factors for biochemical reactions would not take place. Copper is an essential component of several endogenous antioxidant enzymes. Copper affects the transcription of multiple defense and repair genes to protect against metal-induced pathologies. Copper creates stable complexes with a wide variety of organic molecules that can provide required biological affinity and therapeutical activity suitable for targeting specific locations in the body (when using radioactive copper isotopes, 60Cu, 61Cu, 62Cu, 64Cu, 67Cu), as well as non-radioactive copper. The aim of this review is a critical evaluation of copper complexes used for therapy or diagnosis of various diseases in oncology from 2000 to the present. Graphical Abstract

Determination of fexofenadine in tablets by capillary electrophoresis in free solution and in solution with cyclodextrins as analyte carriers.

ABSTRACT Capillary electrophoresis (CE) methods for the determination of fexofenadine (FEX) in commercial pharmaceuticals were developed. It was demonstrated that FEX could be effectively analyzed in free solution cationic CE at low pH. Another analytical approach studied was based on cyclodextrin (CD) modified CE where highly charged CD derivatives served as analyte carriers. In this way, the separation range was spread to physiological pH region and a CE analysis of FEX, present actually in its zwitterionic form, could be accomplished. Several parameters affecting the separations were studied, including the type and concentration of carrier ion, counterion, analyte carrier, and pH of the buffer. The methods based on the free solution CE and CD-modified CE were compared each other, validated, and applied for the determination of FEX in tablets.

Composition of the Oil from the Flowerheads of Anthemis tinctoria L. Cultivated in Slovak Republic

Abstract The chemical composition of the essential oil obtained from the flowerheads of Anthemis tinctoria L(Asteraceae) was analyzed by capillary GC/MS. 86 components of the oil were separated, 48 of which were identified. The major constituents were 1,8-cineole (7.9%), β-pinene (7,3%), decanoic acid (5.4%) and α-pinene (4.4%).

Determination of terbinafine in pharmaceuticals and dialyzates by capillary electrophoresis

A capillary electrophoresis method has been developed for the separation and determination of terbinafine (TER) in various pharmaceutically relevant matrices. Capillary zone electrophoresis (CZE) separation and UV absorbance photometric detection were carried out in a 160mm capillary tube with a 300mum i.d., hydrodynamically (membrane) closed. The influences of pH, carrier cation and counterion on migration parameters of TER were studied and the following conditions were selected: a 20mmoll(-1) glycine running buffer adjusted to pH 2.7 with acetic acid, 0.2% (w/v) methylhydroxyethylcellulose (m-HEC) as an electro-osmotic flow (EOF) suppressor, a 250muA driving current, and 20 degrees C. The optimized separation conditions were convenient for the determination of TER in commercial tablets and spray and in dialyzates. Here, the dialysis was used to investigate in vitro permeation of TER through the skin from the gel. The samples of dialyzates were examined with and without simple extraction procedure and the results were compared. A permeation profile of the drug present in the gel of given composition was obtained analyzing pretreated samples. The proposed electrophoretic method was successfully validated. It was suitable for the simple, sensitive, rapid and highly reproducible assay of TER. CZE analysis was completed within 5.5min. The detection limit of TER was 1.73mumoll(-1) at a 224nm detection wavelength. The intra- and inter-laboratory precisions over the concentration range 6.0-60.0mumoll(-1) were between 0.32-0.69% and 1.04-1.44% including R.S.D. of migration times and peak areas, respectively. The mean absolute recoveries of drugs from samples were found to be 98.34 (tablets) and 99.47% (spray). It is suggested that there are potentialities to determine TER present in unpretreated complex samples, as CZE in a hydrodynamically closed separation system may be easily on-line combinable with purification and preconcentration CE modes (e.g., isotachophoresis, ITP).

Composition of the essential oil from Melissa officinalis L. cultivated in Slovak Republic

Abstract The leaf oil obtained by hydrodistillation from Melissa officinalis L. c. v. citra cultivated at Nitra locality (Slovak Republic) was analyzed by GC and GC/MS. M. officinalis oil was found to contain geranial (33.60%), neral (22.18%), citronellal (11.30%), caryophyllene oxide (8.35%), geranyl acetate (5.89%), and β-caryophyllene (4.20%) as the major components. More than 50 components were identified in the oil.

Chiral separation of dioxopromethazine in eye drops by CZE with charged cyclodextrin.

Capillary zone electrophoresis (CZE) with carboxyethyl-beta-cyclodextrin (CE-beta-CD) dissolved in the operating buffer was used for the separation and determination of enantiomers of phenothiazine antihistaminic, dioxopromethazine, in commercial pharmaceutical preparation, eye drops. This chiral selector, negatively charged under given separating conditions (20 mmol/l epsilon -aminocaproic acid, acetic acid, pH 4.5), was effective in enantioresolution of the antihistamine even at its low concentrations (3-6 mg/ml) in the buffer solution. CZE identification and quantitation of the relevant constituents present in the preparation (dioxopromethazine enantiomers, phenylephrine) were based on the response of photometric absorbency detector, operating at a 275 nm detection wavelength. Changes in pH, type and concentration of chiral selector were studied in relation to chiral resolution. Acceptable validation criteria for sensitivity, precision, linearity and repeatability are included.

Multidrug analysis of pharmaceutical and urine matrices by on-line coupled capillary electrophoresis and triple quadrupole mass spectrometry

The present work illustrates potentialities of CE hyphenated with MS/MS for the simultaneous determination and identification of a mixture of simultaneously acting drugs in pharmaceutical and biological matrices. Here, the hyphenation was provided by ESI interface, while the MS/MS technique was based on the triple quadrupole configuration. Three drugs, namely pheniramine, phenylephrine, and paracetamol were determined and identified with high reliability due to their characterization in three different dimensions, i.e. electrophoresis and MS/MS, that prevented practically any interference. Appropriately selected transitions of the analytes (parent ion-quantifier product ion-qualifier product ion) provided their selective determination at maximum S/N. The proposed CE-MS/MS method was validated (LOD/LOQ, linearity, precision, recovery, accuracy) and applied for (i) the multidrug composition pharmaceuticals, namely Theraflu®, and (ii) human urine taken after per-oral administration of the same pharmaceutical preparation. The method was applied also for the investigation of potential weak associates of the drugs and monitoring of predicted (bio)degradation products of the drugs. Successful validation and application of the proposed method suggest its routine use in highly effective and reliable advanced drug control and biomedical research.