Michał Woźniakiewicz

Microwave-assisted extraction of tricyclic antidepressants from human serum followed by high performance liquid chromatography determination.

A microwave-assisted extraction (MAE) method has been developed and optimized for the extraction of six tricyclic antidepressants (TCAD; nordoxepin, nortriptyline, imipramine, amitriptyline, doxepin, dezypramine) from human serum. Optimal parameters of MAE (solvent and extraction temperature) for water solution of these drugs were defined. The microwave-assisted procedure developed was validated by extraction of serum samples at two concentration levels and then successfully applied to the analysis of reference material. Limit of quantification, precision and recovery were found for the studied compounds in the ranges 0.04-0.15 microg/mL, 1.57-4.34% (RSD) and 94-105%, respectively.

Application of capillary electrophoresis in determination of acid dissociation constant values.

The chemical groups undergoing protonation or deprotonation in solution are described by the acid dissociation constant value, the key parameter for physicochemical characterization of biologically- and pharmacologically-important compounds. Capillary electrophoresis (CE) proved to be suitable technique for its determination: it enables automated and accurate measurements even for minute amount of sample, does not require the information about concentration, and handle both the impure and complex samples. In this review, a number of contributions reporting on the application of CE in pKa prediction has been summarized and critically discussed. The reader will find herein the brief introduction of theory, summary of all works published in the last decade, considerations on the most important innovations and achievements, and the discussion of pKa-related issues as e.g. the role of pKa-shifts in the chiral separation mechanism or the elucidation of migration order reversals observed during CE-mediated separations.

Application of capillary electrophoresis to examination of color inkjet printing inks for forensic purposes

In the process of questioned document examination, the discrimination of inkjet printing inks is becoming more necessary due to increasingly frequent counterfeiting of documents printed by inkjet printers. Therefore, a method based on micellar electrophoretic capillary chromatography (MECC) has been developed and applied to analysis of such inks extracted from paper. With the use of an optimized and validated analytical procedure, multielectropherograms of inks taken from various models of printers made by various producers (Hewlett-Packard, Epson, Brother, Lexmark and Canon) were created. It was shown that effective differentiation of individual inks was possible in terms of migration time, order and specific shapes of characteristic peaks. By comparison of recorded UV-Vis spectra, the identification of main dyes was also achievable. The usefulness of the method was confirmed by an intralaboratory test of utility, in which several forged printouts were successfully examined. The obtained results proved that the proposed procedure is a useful tool that could be applied to ink discrimination and group identification of dyes originating from inkjet printing inks. Consequently, the developed method can be applied in the forensic field, including investigation of the authenticity of documents.

An overview of on-line systems using drug metabolizing enzymes integrated into capillary electrophoresis

Enzymatic assays using CE are now among the most noteworthy applications of this analytical technique in pharmacology‐related investigations. Studies of metabolic pathways of new chemical entities mediated by drug metabolizing enzymes are attracting particular attention. Conventional CE‐based enzymatic in vitro assays are generally restricted to the separation of reagents after incubation performed off‐line. EMMA represents an alternative and fully prospective approach, allowing injection, reaction, separation, and detection to be conducted in a single capillary. Such an on‐line system—in contrast to the standard approach—enables automation, miniaturization, and a significant reduction in reagent volumes, resulting in a very robust and cost‐efficient method. Hence, EMMA could be a method of choice for the screening of new drugs, enzymatic inhibitors, and putative drug–drug interactions. This review provides a summary of reports covering the area of EMMA‐based and related methods implemented into in vitro studies of drug metabolizing enzymes. A general description of the EMMA framework, enzyme families, and a concise discussion of the prognosis for the development of this methodology are given as well.

Tyrosinase-based biosensor for determination of bisphenol A in a flow-batch system

A tyrosinase-based amperometric biosensor is proposed for determination of bisphenol A (BPA) in a flow-batch monosegmented sequential injection system. The enzyme was entrapped in a sol-gel TiO2 matrix modified with multi-walled carbon nanotubes (MWCNTs), polycationic polymer poly(diallyldimethylammonium chloride), (PDDA) and Nafion. Morphology of TYR/TiO2/MWCNTs/PDDA/Nafion matrix composite was studied via scanning electron microscopy (SEM). Electrochemical behavior of the developed biosensor towards bisphenol A was examined and analytical characteristics were assessed with respect to linear range, biosensor sensitivity, limit of detection, long term stability, repeatability and reproducibility. Linear range of biosensor response was found between 0.28 and 45.05 µM with high sensitivity of 3263 µA mM(-1) cm(-2) and detection limit 0.066 µM. The approach was successfully employed for determination of BPA in natural samples.

Examination of colour inkjet printing inks by capillary electrophoresis

The possibility of comparing inkjet printing inks by micellar electrokinetic capillary electrophoresis (MECC) with diode array detection was studied. An analytical procedure was designed and successfully applied to discriminate between the electrophoretic profiles of inks (extracted from paper) produced by five well-known manufacturers. The separation process was conducted in a polyimide-coated fused silica capillary (ID 50 μm, 60 cm total/50 cm effective length) with +30 kV high voltage applied. Background electrolyte was used of the following optimum composition: 40 mM sodium borate buffer, 20mM sodium dodecyl sulphate(IV) (SDS) and 10% (v/v) acetonitrile (pH 9.56). The experimental conditions were adjusted in terms of resolution and analysis time. The best results were obtained at 10 and 25°C storage and capillary temperature, respectively, using 25 dots (ø 0.8mm) cut from printouts as the sample and BGE diluted with water (1:99, v/v) as the injecting solution. The MECC separation of main printing ink components by the proposed method showed excellent precision - the RSD value of the migration time calculated for each of the investigated peaks did not exceed 3.3%. The optimized method was applied to group identification and differentiation of: (a) three colours of printing inks, (b) inks from different manufacturers (Hewlett-Packard, Epson, Brother, Lexmark and Canon) and (c) inks from different printer models. In all these cases, inks were successfully differentiated on the basis of position (migration time) and shape of their characteristic peaks.

n-3 Fatty acids as resolvents of inflammation in the A549 cells

BACKGROUND Fatty acids and their derivatives are one of the most crucial inflammation mediators. The aim of our study was to evaluate the impact of polyunsaturated fatty acids as eicosanoids precursors on the A549 cell line. METHODS Cells were incubated with 40 μM of arachidonic, eicosapentaenoic or docosahexaenoic acid for 24h, then activated with LPS. Fatty acids content in the cell membranes were determined using gas chromatography. COX-2, cPGES and FP-receptor quantities were determined by Western blot. 8-Isoprostane F2α concentrations were determined by EIA. Maresin and protectin D1 contents were analyzed by UHPLC/MS-TOF method. RESULTS Significant differences in membrane fatty acids and levels of 8-isoPGF2α in the activated cells were detected. Elevated expression of COX-2 and FP-receptor was observed in cells treated with AA and activated with LPS. Moreover, compared to AA and AA+LPS groups, cells incubated with EPA, DHA, EPA+LPS and DHA+LPS showed decreased expression of COX-2, cPGES and FP-receptor. In cells incubated with EPA or DHA and activated with LPS maresin and protectin D1 were detected. CONCLUSIONS The results of the study have revealed the pro-inflammatory properties of AA, while the EPA and DHA had the opposite, resolving effect. Interestingly, FP-receptor inhibition by EPA and DHA demonstrated the unique role of the FP-receptor as a potential target for antagonists, in the diseases of inflammatory character. This study provides new information about n-3 fatty acids and their pro-resolving mediators, which can be used in the process of developing new anti-inflammatory drugs.

Analytical aspects of achiral and cyclodextrin-mediated capillary electrophoresis of warfarin and its two main derivatives assisted by theoretical modeling

Several distinct analytical issues have been addressed by performing capillary electrophoresis-based separations of the warfarin, 7-hydroxywarfarin and 10-hydroxywarfarin in an achiral and cyclodextrin-containing media. The measurements were conducted across a range of pH in order to find optimum conditions for achiral and chiral separations. The values of acid dissociation constant (pKa) have been determined and compared. Subsequently, after performing a series of mobility shift assays at different pH and cyclodextrin concentration, the pKa values ascribed to diastereomeric complexes with methyl-β-cyclodextrin have been estimated. The significant pKa shifts upon complexation have been noticed for warfarin - up to 1.5 pH units, and only subtle for 10-hydroxywarfarin. A new approach that allows the estimation of association percentage based on the electrophoretic mobility curves has been also demonstrated. The complex mechanism of chiral separation has been found to be responsible for the observed migration profile, relying on a combined equilibrium between complexation/partition and protonation/deprotonation phenomena. The occurrence of the pKa-related migration order reversal has been demonstrated in achiral medium between warfarin and 7-hydroxywarfarin, and in chiral medium between enantiomers, causing a drop in enantioselectivity at specific pH. In parallel, the density functional theory-based calculations have been performed in order to obtain the structures of warfarin and its derivatives as well as to rationalize the shifts in pKa values.

Application of microwave irradiation to fast and efficient isolation of benzodiazepines from human hair

The aim of this research was to develop, optimize and validate a modern and rapid method of preparation of human hair samples using microwave irradiation for the purpose of determination of six benzodiazepines: alprazolam, estazolam, lorazepam, clonazepam, diazepam and tetrazepam, with lormetazepam as an internal standard (IS). Extracts were analyzed ultrahigh performance liquid chromatography with mass spectrometry time of flight detection (UHPLC-MS-TOF). Optimal parameters of microwave-assisted extraction (type of extraction solvent, extraction temperature, extraction time and pH of borate buffer) for these drugs in spiked hair samples were defined. The limit of detection ((0.003-0.025 ng/mg), intra- (1.5-4.3%) and interday (2.3-8.3%) precision (CV) and accuracy of the assay (89.8-105.7%) at three concentration levels: 0.22, 2.22 and 5.56 ng/mg were calculated. The optimized and validated MAE/UHPLC-MS-TOF method was then applied for analysis of clinical hair sample.

Determination of acid dissociation constant of 20 coumarin derivatives by capillary electrophoresis using the amine capillary and two different methodologies.

In this work capillary electrophoresis has been used to determine acid dissociation constant of 20 structurally diverse coumarin derivatives. For a majority of compounds pKa value has been determined for the first time. The obtained values vary between 4.16-9.10pH unit, pointing to the interesting structure-acidity relationships. The amine permanently coated capillary has been applied for that purpose, because it has turned out to be more effective in pKa determination than the bare silica and other coated capillaries, ensuring good precision and shorter migration times. A traditional methodology relying on measurements in a broad pH range and fitting of a sigmoidal function has been compared to an alternative simplified approach, reported for the first time, where only two electrophoretic mobility values suffice for pKa estimation. The first value corresponds to the partially ionized form and it is measured experimentally, while the second one to the totally ionized form - it is measured experimentally (two-values method) or estimated directly from molecular mass (one-value method). We show that despite a limited measurements number, the alternative approach may be consistent with the traditional methodology, yielding the relatively low pKa deviation. Its reliability has also been confirmed by the analytical predictions, comprising resolution, migration order, migration times and peaks overlapping. Therefore, combination of the amine capillary with the simplified calculation method is an attractive tool for fast and reliable pKa estimation.