Achiral and chiral high-performance liquid chromatographic determination of flubendazole and its metabolites in biomatrices using UV photodiode-array and mass spectrometric detection

Flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, belongs to the group of benzimidazole anthelmintics, which are widely used in veterinary and human medicine. The phase I flubendazole biotransformation includes a hydrolysis of the carbamoyl methyl moiety accompanied by a decarboxylation (hydrolysed flubendazole) and a carbonyl reduction of flubendazole (reduced flubendazole). Flubendazole is a prochiral drug, hence a racemic mixture is formed during non-stereoselective reductions at the carbonyl group. Two bioanalytical HPLC methods were developed and validated for the determination of flubendazole and its metabolites in pig and pheasant hepatic microsomal and cytosolic fractions. Analytes were extracted from biomatrices into tert-butylmethyl ether. The first, achiral method employed a 250 mm x 4 mm column with octylsilyl silica gel (5 microm) and an isocratic mobile phase acetonitrile-0.025 M KH(2)PO(4) buffer pH 3 (28:72, v/v). Albendazole was used as an internal standard. The whole analysis lasted 27 min at a flow rate of 1 ml/min. The second, chiral HPLC method, was performed on a Chiralcel OD-R 250 mm x 4.6 mm column with a mobile phase acetonitrile-1 M NaClO(4) (4:6, v/v). This method enabled the separation of both reduced flubendazole enantiomers. The enantiomer excess was evaluated. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at lambda=246 nm). Each of the analytes under study had characteristic UV spectrum, in addition, their chemical structures were confirmed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) experiments. Stereospecificity in the enzymatic carbonyl reduction of flubendazole was observed. While synthetic racemic mixture of reduced flubendazole was separated to equimolar amounts of both enantiomers, practically only one enantiomer was detected in the extracts from all incubates.     

C-2/c-3 annulation and C-2 alkylation of indoles with 2-alkoxycyclopropanoate esters

The annulation reaction between various indoles and 2-alkoxycyclopropanoate esters is reported. Both high efficiency and complete stereochemical control were observed in some cases with this annulation process. A single stereocenter on the cyclopropane controls the diastereoselective formation of up to four new stereocenters. A different reaction course was observed with 3-substituted indole substrates, and an intervening C-3 to C-2-migration process arose that gives synthetically useful C-2 alkylation indole products.     

The Use of Carrier Ampholyte-Free Isoelectric Focusing for Proteomic Analysis

Carrier ampholyte-free isoelectric focusing was applied for pre-concentration, purification and micropreparation of phycobiliproteins (C-phycocyanin, allophycocyanin, B-phycoerythrin) extracted from cyanobacteria Anabeana doliolum and from red microalga Porphyridium cruentum. The extraction of phycobiliproteins was carried out in deionized water. The sonication in the ultrasonic bath and liquid nitrogen freeze grind was used for extraction of proteins of interest. Pre-concentrated and pre-separated proteins were collected and analyzed via MALDI-TOF-TOF mass spectrometer after their proteolytic digestion via trypsin. Based on tandem mass spectrometric analysis, the C-phycocyanin, allophycocyanin and B-phycoerythrin were identified unambiguously.     

Resistance of polyvinyl alcohol blends stabilized by sodium and ammonium salts of lignite humic acids against γ-irradiation

The dried blends containing sodium and ammonium salts of lignite humic acids (humates, 0.5–10% w/w) in polyvinyl alcohol (PVA) were exposed to high dosage of γ-irradiation in the range of doses 127–806 kGy. Resulted products were then tested for their stability using thermogravimetrical analysis. As a reference the non-treated blends were used since the pure PVA exposed to γ-irradiation very quickly lost its stability and resulting consistence did not allow the stability tests. Stabilities showed a strong concentration and counterion dependency. While sodium counterion caused mostly destabilization with increasing dose, the ammonium counterion acted in an opposite way. The tests carried out in a moisturizing container revealed the changes in water absorbing capacity of irradiated samples and allowed partial explanation of humate stabilizing effect. Generally, at lower concentration of a humate the increase was observed with an increase in the γ-irradiation dose and vice versa. The results confirmed the antioxidant and stabilizing effect of humic acids added to some synthetic polymers and their applicability in materials exposed to γ-irradiation.     

Interaction of cells with polymers

This paper summarizes the results of our experiments studying the effect of functional chemical groups of synthetic hydrophilic polymers on the recognition of these materials by macrophages in in vivo experiments. The interaction of keratinocytes with synthetic hydrophilic polymers in vitro is also demonstrated. The implants containing anionic groups (carboxylates) were shown to be better tolerated by nonspecific immunity. The keratinocytes are able to grow on hydrophilic polymer supports and migrate to surfaces with improved adhesivity (in vitro). The clinical applications of these experiments are demonstrated.     

Ligand-driven light-induced spin transition in spin crossover compounds

Spin crossover (SCO) coordination compounds that show bistability between low spin and high spin states are promising light-controllable molecular switches. Selective wavelength irradiation of the coordination centre at low temperatures is known as a light-induced excited spin state trapping (LIESST effect) and it leads to the modulation of physical properties of SCO materials on the macroscopic as well as on the molecular level. Another way to trigger the spin state conversion by light is based on the isomerization of photoactive ligand moieties. The ligand field strength is changed due to light-induced photoisomerization and, therefore, corresponding cis–trans or ring-closing/ring-opening isomeric couples might exhibit different spin states at isothermal conditions. Such an approach is called as ligand driven light-induced spin change (LD LISC effect). From the application point of view, it presents a promising alternative to the LIESST effect because it can operate at room temperature. This article is focused on the most interesting iron and cobalt SCO compounds with photoisomerizable ligands and provides the overview of achieving results based on the LD LISC effect.     

Dye decolorisation by laccase immobilised in lens-shaped poly(vinyl alcohol) hydrogel capsules

In this study, laccase (from Trametes versicolor, 8.3 U mg _enz ^?1 ) was used for the decolorisation of Saturn Blue L4G (10 mg L^?1). The efficiency of the decolorisation (ratio between the amount of decolorised dye and initial amount of dye) by a free enzyme was 48 % and the decolorisation rate was determined at 2.11 × 10^?3 mg_dye mg _enz ^?1 min^?1. After immobilisation in lens-shaped poly(vinyl alcohol) hydrogel capsules LentiKats® Biocatalyst (LB) (concentration of immobilised enzyme: 4 mg per g of particles; volume-loading rate of LB: 10 g per 100 mL of medium), the enzyme retained 16.1 % of its original activity (1.34 U mg _enz ^?1 ). Immobilised laccase was used for the dye decolorisation in 130 repeated batch tests with 71 % efficiency (LB activity: 7 × 10^?3 mgdye min^?1 g _LB ^?1 ). In continuous mode (after 716.5 h), the efficiency of the dye decolorisation was 48 % (LB activity: 3.3 × 10^?4 mg_dye min^/?1 g _LB ^?1 ).     

Fast and Sensitive Screening of Oxandrolone and Its Major Metabolite 17-Epi-Oxandrolone in Human Urine by UHPLC—MS/MS with On-Line SPE Sample Pretreatment

Oxandrolone, a synthetic testosterone analog, is used for the treatment of several diseases associated with weight loss. Unfortunately, oxandrolone is abused by many athletes and bodybuilders due to its strong anabolic effect. We have developed and validated a highly sensitive and rapid on-line SPE-UHPLC-MS/MS method for the determination of oxandrolone and simultaneous identification of its major metabolite 17-epi-oxandrolone in urine matrices. Enrichment of the analytes via an integrated solid-phase extraction was achieved using an Acquity UPLC BEH C18 Column. Subsequently, the chromatographic separation of the on-line preconcentrated sample fraction was achieved using an Acquity HSS T3 C18 Column. For the structural identification of these analytes, a high-resolution mass spectrometer Synapt-G2Si coupled to the Acquity M-class nano-LC system with ionKey source was used. A highly sensitive determination of oxandrolone was achieved using a tandem quadrupole mass spectrometer XEVO TQD. The method was successfully validated in the linear range of oxandrolone from 81.63 pg·mL⁻¹ (limit of quantification, LOQ) to 5000 pg·mL⁻¹ in the human urine matrix. It was applied to the analysis of real urine samples obtained from a healthy volunteer after the oral administration of one dose (10 mg) of oxandrolone. Concentration vs. time dependence was tested in the time interval of 4 h–12 days (after oral administration) to demonstrate the ability of the method to detect the renal elimination of oxandrolone from the human body. Favorable performance parameters along with successful application indicate the usefulness of the proposed method for its routine use in antidoping control labs.     

Protein-resistant surfaces through mild dopamine surface functionalization

The synthesis and evaluation of new dopamine-based catechol anchors coupled to poly(ethylene glycol) (PEG) for surface modification of TiO(2) are reported. Dopamine is modified by dimethylamine-methylene (7) or trimethylammonium-methylene (8) groups, and the preparation of mPEG-Glu didopamine polymer 11 is presented. All these PEG polymers allow stable adlayers on TiO(2) to be generated through mild dip-and-rinse procedures, as evaluated both by variable angle spectroscopic ellipsometry and X-ray photoelectron spectroscopy. The resulting surfaces substantially reduced protein adsorption upon exposure to full human serum.