Zbigniew Fijałek

Determination of selected trace elements in herbs and their infusions.

The following macro- and microelements were determined in the leaf of peppermint (Mentha piperitae folium) and nettle (Urticae folium) (as tea bags) and in their infusions: As, Ba, Ca, Cd, Co, Cr, Cu, Fe, I, Li, Mg, Mn, Ni, Pb, Se, Sn, Sr, Ti, V and Zn. The determinations were performed using inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS). From all the determined microelements the highest content found was that of iron, 244 and 107 mg/kg in the leaf of peppermint and nettle, respectively. However, the lowest content found was that of cobalt, 0.10 and 0.08 mg/kg for the leaf of mint and nettle, respectively. The most readily water eluting elements were strontium, selenium and iodine, the most difficult ones - barium and iron.

Determination of chromium and selected elements in multimineral and multivitamin preparations and in pharmaceutical raw material.

The content of elements in pharmaceutical preparations is one of the indispensable factors of the evaluation of their quality. In the present work, the following macro- and microelements Ca, Cr, Cu, Fe, Mg, Mn, Mo, P, Se and Zn were determined in multimineral and multivitamin preparations and in pharmaceutical raw material. Inductively coupled plasma mass spectrometry (ICP-MS) and electrothermal atomic absorption spectrometry (ET AAS) were used throughout the study. The examined samples were dissolved in a high-pressure microwave system using concentrated nitric acid. The effect of the carbon residue in the digest solution on the determination result was eliminated by introducing an equation correcting the ArC+ interference with 52Cr.

Determination of neomycin and related substances in pharmaceutical preparations by reversed-phase high performance liquid chromatography with mass spectrometry and charged aerosol detection

A new, simple and repeatable liquid chromatographic method with charged aerosol detection (LC-CAD) for determination of neomycin and related substances has been developed. Analysis of neomycin or other aminoglycosides is problematic due to a lack of chromophores. Universal response of CAD enables direct quantification of neomycin and related substances, for which no reference standard are available. Separation was performed on C18 Hypersil(®) Gold aQ column using water, methanol and heptaflurobutyric acid as mobile phase. Under developed chromatographic conditions all impurities were well separated from neomycin B. Peaks identification was evaluated by electrospray ionization mass spectrometry. The proposed method was validated according to ICH guidelines and applied to the content determination of neomycin and related substances in pharmaceutical preparations.

Comparative cytotoxicity studies of carbon-encapsulated iron nanoparticles in murine glioma cells.

Carbon-encapsulated iron nanoparticles (CEINs) have recently emerged as a new class of magnetic nanomaterials with a great potential for an increasing number of biomedical applications. To address the current deficient knowledge of cellular responses due to CEIN exposures, we focused on the investigation of internalization profile and resulting cytotoxic effects of CEINs (0.0001-100 μg/ml) in murine glioma cells (GL261) in vitro. The studied CEIN samples were characterized (TEM, FT-IR, Zeta potential, Boehm titration) and examined as raw and purified nanomaterials with various surface chemistry composition. Of the four type CEINs (the mean diameter 47-56 nm) studied here, the as-synthesized raw nanoparticles (Fe@C/Fe) exhibited high cytotoxic effects on the plasma cell membrane (LDH, Calcein AM/PI) and mitochondria (MTT, JC-1) causing some pro-apoptotic evens (Annexin V/PI) in glioma cells. The effects of the purified (Fe@C) and surface-modified (Fe@C-COOH and Fe@C-(CH2)2COOH) CEINs were found in quite similar patterns; however, most of these cytotoxic events were slightly diminished compared to those induced by Fe@C/Fe. The study showed that the surface-functionalized CEINs affected the cell cycle progression in both S and G2/M phases to a greater extent compared to that of the rest of nanoparticles studied to data. Taken all together, the present results highlight the importance of the rational design of CEINs as their physicochemical features such as morphology, hydrodynamic size, impurity profiles, and especially surface characteristics are critical determinants of different cytotoxic responses.

Assessing carbon-encapsulated iron nanoparticles cytotoxicity in Lewis lung carcinoma cells

Carbon‐encapsulated iron nanoparticles (CEINs) have been considered as attractive candidates for several biomedical applications. In the present study, we synthesized CEINs (the mean diameter 40–80 nm) using a carbon arc route, and the as‐synthesized CEINs were characterized (scanning and transmission electron microscopy, dynamic light scattering, turbidimetry, Zeta potential) and further tested as raw and purified nanomaterials containing the carbon surface modified with acidic groups. For cytotoxicity evaluation, we applied a battery of different methods (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide, lactate dehydrogenase, calcein AM/propidium iodide, annexin V/propidium iodide, JC‐1, cell cycle assay, Zeta potential, TEM and inductively coupled plasma mass spectrometry) to address the strategic cytotoxic endpoints of Lewis lung carcinoma cells due to CEIN (0.0001–100 µg ml–1) exposures in vitro. Our studies evidence that incubation of Lewis lung carcinoma cells with CEINs is accompanied in substantial changes of zeta potential in cells and these effects may result in different internalization profiles. The results show that CEINs increased the mitochondrial and cell membrane cytotoxicity; however, the raw CEIN material (Fe@C/Fe) produced higher toxicities than the rest of the CEINs studied to data. The study showed that non‐modified CEINs (Fe@C/Fe and Fe@C) elevated some pro‐apoptotic events to a greater extent compared to that of the surface‐modified CEINs (Fe@C‐COOH and Fe@C‐(CH2)2COOH). They also diminished the mitochondrial membrane potentials. In contrast to non‐modified CEINs, the surface‐functionalized nanoparticles caused the concentration‐ and time‐dependent arrest of the S phase in cells. Taken all together, our results shed new light on the rational design of CEINs, as their geometry, hydrodynamic and, in particular, surface characteristics are important features in selecting CEINs as future nanomaterials for nanomedicine applications. Copyright

Development of high performance liquid chromatography methods with charged aerosol detection for the determination of lincomycin, spectinomycin and its impurities in pharmaceutical products

Novel and simple liquid chromatography methods with charged aerosol detection (LC-CAD) for simultaneous quantitation of lincomycin and spectinomycin and its related substances have been developed and tested. This type of analysis is complicated due to the different chromatographic behavior of these two agents and the lack of chromophores in spectinomycin complex. CAD seems to be a promising alternative to overcome these difficulties. It shows a consistent inter-analyte response, independent of chemical structure of an analyte. It also enables the direct quantification of related substances for which no reference standards were available, with good accuracy and precision. Chromatographic separations were achieved using a C18 Hypersil(®) Gold column, with mobile phases consisting of water, acetonitrile and trifluoroacetic acid. All impurities were identified using time-of-flight mass spectrometry with electrospray ionization. The developed methods have been successfully used in the routine quality control analysis of pharmaceutical preparations.

Determination of flibanserin and tadalafil in supplements for women sexual desire enhancement using high-performance liquid chromatography with tandem mass spectrometer, diode array detector and charged aerosol detector

The new compound - flibanserin - begun to appear as a synthetic adulterant in counterfeit herbal supplements used to stimulate women sexual drive. It was detected in two samples submitted to the Polish National Medicines Institute for analysis. The second sample contained also tadalafil. This study presents the LC method development which enables the determination of flibanserin and tadalafil. It employs three different detectors charged aerosol detector (CAD), diode array detector (DAD) and mass spectrometer (MS). The conditions of the elaborated method were optimized to obtain the highest sensitivity and the best resolution, especially the separation of icariin - the natural compound observed often in supplements for sexual disorders. The validation of the method proved good linearity, good accuracy and precision of the measurements recorded by all three detectors. Additionally, for CAD data, an alternative calculation method using a unified calibration function was presented and evaluated. It seems that this is the way to overcome the problem of non-availability of the reference standard of a target compound. Flibanserin content was quantified using the data of other reference standard (tadalafil). The inaccuracy of proposed indirect determination was found to be ±3%. A statistical evaluation proved that the results obtained with all detection modes and the results calculated using a unified calibration were not significantly different (p>0.05).

Determination of Selected Trace Elements in Dietary Supplements Containing Plant Materials

Determination of Selected Trace Elements in Dietary Supplements Containing Plant Materials The need for analytical methods enabling determining trace metals concentration in herbal medicinal products and dietary supplements has increased in recent years due to the globalisation of international market and also due to the risk of using herbs contaminated by heavy metals. To this end, an analytical method was developed and validated for the accurate determination of trace elements (As, Cd, Co, Cr, Cu, Mn, Pb, Sr, Zn) in products in the form of tablets or capsules, herbal teas and their aqueous infusions. The validation of described procedure of digestion samples and determination metals by ICP-MS method, shows its usability in determination of trace elements in herbal products. Recovery percentage based on CRMs ranged from 90.1% to 110.7%, and the precision of the method was below 3%. Out of all microelements determined, the highest concentration was found for Mn in herbal teas. The daily intake of Mn with infusions accounted for 53.3% and 126.4%, which corresponded to 41.7% and 98.9% of the AI values for adult women and men, respectively. The method was also used for quality control of the labelled level of chromium and zinc. In all analysed samples, the concentrations of cadmium and lead were below the EC maximum levels stipulated for Cd and Pb in dietary supplements.

Internalization and cytotoxicity effects of carbon-encapsulated iron nanoparticles in murine endothelial cells: Studies on internal dosages due to loaded mass agglomerates

Carbon-encapsulated iron nanoparticles (CEINs) qualified as metal-inorganic hybrid nanomaterials offer a potential scope for an increasing number of biomedical applications. In this study, we have focused on the investigation of cellular fate and resulting cytotoxic effects of CEINs synthesized using a carbon arc route and studied in murine endothelial (HECa-10) cells. The CEIN samples were characterized as pristine (the mean diameter between 47 and 56nm) and hydrodynamic (the mean diameter between 270 and 460nm) forms and tested using a battery of methods to determine the cell internalization extent and cytotoxicity effects upon to the exposures (0.0001-100μg/ml) in HECa-10 cells. Our studies evidenced that the incubation with CEINs for 24h is accompanied with substantial changes of Zeta potential in cells which can be considered as a key factor for affecting the membrane transport, cellular distribution and cytotoxicity of these nanoparticles. The results demonstrate that CEINs have entered the endothelial cell through the endocytic pathway rather than by passive diffusion and they were mainly loaded as agglomerates on the cell membrane and throughout the cytoplasm, mitochondria and nucleus. The studies show that CEINs induce the mitochondrial and cell membrane cytotoxicities in a dose-dependent manner resulting from the internal dosages due to CEIN agglomerates. Our results highlight the importance of the physicochemical characterization of CEINs in studying the magnetic nanoparticle-endothelial cell interactions because the CEIN mass agglomerates can sediment more or less rapidly in culture models.

Application of high-performance liquid chromatography with charged aerosol detection (LC–CAD) for unified quantification of synthetic cannabinoids in herbal blends and comparison with quantitative NMR results

PurposeThe analysis of products which contain synthetic cannabinoids (SCs) is very challenging due to their diversity and rapidly changing SC structures, variable herbal matrices and, above all, inaccessibility of reference standards. Therefore, the aim of this study was to develop a method which allows quantification of SCs’ contents in herbal blends without their reference standards.MethodsIdentification of SCs was performed using liquid chromatography–high-resolution tandem mass spectrometry with a quadrupole time-of-flight analyser (LC–QTOF-MS/MS). A liquid chromatography–charged aerosol detection (LC–CAD) method with unified calibration for the quantification of SCs was developed and validated. Two available reference standards were used as universal standards. Quantitative analysis using a nuclear magnetic resonance spectroscopy method was also performed to externally validate the developed LC–CAD method.ResultsAll peaks of SCs observed in LC–CAD chromatograms were identified by LC–QTOF-MS/MS. Validation data and results from a CAD response evaluation indicated that the elaborated quantitative method was sufficiently accurate for the determination of SCs belonging to various chemical families. The LC–CAD method turned out to be very flexible, because it was successfully applied for the analysis of 19 herbal products.ConclusionsIn this study, methods which enable identification and quantification of currently known SCs as well as novel unknown derivatives without their reference standards were developed. These methods can be applied to the control of suspect SC products and may support the risk assessment of SC presence on the market. To our knowledge, this is the first trial to use LC–CAD for unified quantification of SCs without their reference standards.