Peter Mikuš

Advanced CE for chiral analysis of drugs, metabolites, and biomarkers in biological samples.

An analysis of recent trends indicates that CE can show real advantages over chromatographic methods in ultratrace enantioselective determination of biologically active compounds in complex biological matrices. It is due to high separation efficiency and many applicable in‐capillary electromigration effects in CE (countercurrent migration, stacking effects) enhancing significantly (enantio)separability and enabling effective sample preparation (preconcentration, purification, analyte derivatization). Other possible on‐line combinations of CE, such as column coupled CE–CE techniques and implementation of nonelectrophoretic techniques (extraction, membrane filtration, flow injection) into CE, offer additional approaches for highly effective sample preparation and separation. CE matured to a highly flexible and compatible technique enabling its hyphenation with powerful detection systems allowing extremely sensitive detection (e.g. LIF) and/or structural characterization of analytes (e.g. MS). Within the last decade, more as well as less conventional analytical on‐line approaches have been effectively utilized in this field and their practical potentialities are demonstrated on many new application examples in this article. Here, three basic areas of (enantioselective) drug bioanalysis are highlighted and supported by a brief theoretical description of each individual approach in a compact review structure (to create integrated view on the topic), including (i) progressive enantioseparation approaches and new enantioselective agents, (ii) in‐capillary sample preparation (preconcentration, purification, derivatization), and (iii) detection possibilities related to enhanced sensitivity and structural characterization.

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.

Separation of multicomponent mixtures of 2,4-dinitrophenyl labelled amino acids and their enantiomers by capillary zone electrophoresis

The use of capillary zone electrophoresis (CZE) for the separation of a group of 33 2,4‐dinitrophenyl labeled amino acids (DNP‐AA), including DNP‐AA racemates, DNP‐L‐AA enantiomers and achiral DNP‐AAs, was investigated. α‐, β‐ and γ‐cyclodextrins (CDs) and their derivatives (hydroxypropyl derivatives of α‐, β‐ and γ‐CDs, polymeric β‐CD and 6A‐methylamino‐β‐cyclodextrin (MA‐β‐CD)) served as complexing agents and chiral selectors in this investigation. Although native α‐ and γ‐CDs and their derivatives influenced the effective mobilities of the studied DNP‐AAs in different ways, they generally failed to resolve enantiomers of the individual DNP‐AAs. On the other hand, β‐CD and all of its derivatives were found to be effective in this respect. Of these, the best results were achieved with a positively charged MA‐β‐CD and this chiral selector resolved enantiomers of ten DNP‐AA racemates available for this study. However, a complete resolution of these enantiomers in one CZE run required that the effect of the chiral selector be complemented by complexing effects of polyvinyl pyrrolidone (PVP) or γ‐CD. Complexing and chiral recognition capabilities of MA‐β‐CD combined with complexing effects of γ‐CD and PVP provided separating conditions suitable for the CZE separations of multicomponent mixtures of DNP‐AAs with preserved resolutions of the enantiomers. For example, a mixture consisting of 43 DNP‐AA constituents was resolved using an MA‐β‐CD/γ‐CD combination with three peak overlaps.

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).

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.

Comparison of hydrodynamically closed isotachophoresis-capillary zone electrophoresis with hydrodynamically open capillary zone electrophoresis hyphenated with tandem mass spectrometry in drug analysis: pheniramine, its metabolite and phenylephrine in human urine.

The advanced two dimensional isotachophoresis (ITP)-capillary zone electrophoresis (CZE) hyphenated with tandem mass spectrometry (MS/MS, here triple quadrupole, QqQ) was developed in this work to demonstrate analytical potentialities of this approach in the analysis of drugs in multicomponent ionic matrices. Pheniramine (PHM), phenylephrine (PHE), paracetamol (PCM) and their potential metabolic products were taken for the analysis by the ITP-CZE-ESI-QqQ technique working in hydrodynamically closed CE separation system and then a comparison with the conventional (hydrodynamically open) CZE-ESI-QqQ technique was made. The ITP-CZE-ESI-QqQ method was favorable in terms of obtainable selectivity (due to highly effective heart-cut analysis), concentration limits of detection (LOD at pgmL(-1) levels due to enhanced sample load capacity and ITP preconcentration), sample handling (on-line sample pretreatment, i.e. clean-up, preconcentration, preseparation), and, by that, possibilities for future automation and miniaturization. On the other hand, this experimental arrangement, in contrast to the CZE-ESI-QqQ arrangement supported by an electroosmotic flow, is principally limited to the analysis of uniformly (i.e. positively or negatively) charged analytes in one run without any possibilities to analyze neutral compounds (here, PCM and neutral or acidic metabolites of the drugs had to be excluded from the analysis). Hence, these general characteristics should be considered when choosing a proper analytical CE-MS approach for a given biomedical application. Here, the analytical potential of the ITP-CZE-ESI-QqQ method was demonstrated showing the real time profiles of excreted targeted drugs and metabolite (PHM, PHE, M-PHM) in human urine after the administration of one dose of Theraflu(®) to the volunteers.