A generic approach based on long-lifetime fluorophores for the assessment of protein binding to polymer nanoparticles by fluorescence anisotropy.

Quantitation of protein-nanoparticle interactions is essential for the investigation of the protein corona around NPs in vivo and when using synthetic polymer nanoparticles as affinity reagents for selective protein recognition in vitro. Here, a method based on steady-state fluorescence anisotropy measurement is presented as a novel, separation-free tool for the assessment of protein-nanoparticle interactions. For this purpose, a long-lifetime luminescent Ru-complex is used for protein labelling, which exhibits low anisotropy when conjugated to the protein but displays high anisotropy when the proteins are bound to the much larger polymer nanoparticles. As a proof of concept, the interaction of lysozyme with poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid) nanoparticles is studied, and fluorescence anisotropy measurements are used to establish the binding kinetics, binding isotherm and a competitive binding assay.

Simultaneous determination of low molecular weight nitrosamines in pharmaceutical products by fast gas chromatography mass spectrometry.

Control of N-nitrosamines has been in the focus of health authorities in recent years because many of these compounds are probable human carcinogens. In July 2018 the U.S. Food and Drug Administration (FDA) announced a recall for valsartan-containing medicines due to contamination with the carcinogenic low molecular weight nitrosamine, N-nitrosodimethylamine (NDMA). It has become clear that the problem can not only exist in the case of sartans, but in any active pharmaceutical ingredient (API)/drug product in which secondary or tertiary amines are present (as API or as impurities) and a nitrosating agent is available. The decision was made by regulators, according to which manufacturers of pharmaceutical products are obliged to perform a risk assessment for the potential presence of nitrosamines in active pharmaceutical ingredients and drug products. This resulted in a high demand for validated analytical methods that are able to quantify N-nitrosamines at low ppb levels in pharmaceutical products. In this work we have developed and validated a generic fast GC-MS method suitable for the quantitative determination of a wide range of low molecular weight nitrosamines, which include N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitroso-diphenylamine (NDPh), N-nitrosodipropylamine (NDPA), N-nitrosomethylethylamine (NMEA), N-nitrosomorpholine (NMOR), N-nitrosopiperidine (NPIP), N-nitroso-ethylisopropylamine (EIPNA), N-nitroso-diisopropylamine (DIPNA), N-nitroso-N-methylaniline (NMPA), 1-Methyl-4-nitrosopiperazine (MeNP) and N-nitroso-pyrrolidine (NPYR). The advantage of the method is that it is possible to screen low molecular weight nitrosamines in low concentrations with a short analysis time in a wide range of APIs and drug products.

Comparative Analysis of pH and Target-Induced Conformational Changes of an Oxytetracycline Aptamer in Solution Phase and Surface-Immobilized Form.

To date, numerous aptamer-based biosensing platforms have been developed for sensitive and selective monitoring of target analytes, relying on analyte-induced conformational changes in the aptamer for the quantification of the analyte and the conversion of the binding event into a measurable signal. Despite the impact of these conformational rearrangements on sensor performance, the influence of the environment on the structural conformations of aptamers has rarely been investigated, so the link between parameters directly influencing aptamer folding and the ability of the aptamer to bind to the target analyte remains elusive. Herein, the effect a number of variables have on an aptamer's 3D structure was examined, including the pH of the buffering medium, as well as the anchoring of the aptamer on a solid support, with the use of two label-free techniques. Circular dichroism spectroscopy was utilized to study the conformation of an aptamer in solution along with any changes induced to it by the environment (analyte binding, pH, composition and ionic strength of the buffer solution), while quartz crystal microbalance with dissipation monitoring was employed to investigate the surface-bound aptamer's behavior and performance. Analysis was performed on an aptamer against oxytetracycline, serving as a model system, representative of aptamers selected against small molecule analytes. The obtained results highlight the influence of the environment on the folding and thus analyte-binding capacity of an aptamer and emphasize the need to deploy appropriate surface functionalization protocols in sensor development as a means to minimize the steric obstructions and undesirable interactions of an aptamer with a surface onto which it is tethered.

Stabilization of PE with Pomegranate Extract: Contradictions and Possible Mechanisms.

Dry pomegranate peel was extracted with acetone and the extract was added to a Phillips type polyethylene. The concentration of the extract was changed from 0 to 1000 ppm in six steps and stabilization efficiency was checked by the multiple extrusion of the polymer followed by the characterization of chemical structure, processing, and residual stability. The results confirmed the excellent processing stabilization efficiency of the extract, but also the poor long-term stability of PE containing it in accordance with previously published results. The extract is amorphous and its solubility is relatively large in the polymer; thus, these factors cannot be the reason for the poor stabilization efficiency in an oxygen-rich environment. Chemical factors like the self-interaction of the polyphenol molecules, the stability of the radicals forming after hydrogen abstraction, and the lack of hydrogens with the necessary reactivity must be considered during the evaluation of the efficiency of the extract. These factors as well as the insufficient number of active hydrogens hinder the reaction of the additive molecules with oxygen-centered radicals, thus leading to inferior long-term stability. The extract can be used for the processing stabilization of polymers, but for applications requiring long-term stability, it must be combined with other natural antioxidants like flavonoids or Vitamin E.

Temperature-Responsive Magnetic Nanoparticles for Bioanalysis of Lysozyme in Urine Samples.

Highly selective multifunctional magnetic nanoparticles containing a thermoresponsive polymer shell were developed and used in the sample pretreatment of urine for the assessment of lysozymuria in leukemia patients. Crosslinked poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) was grown onto silica-coated magnetic nanoparticles by reversible addition fragmentation chain transfer (RAFT) polymerization. The lysozyme binding property of the nanoparticles was investigated as a function of time, protein concentration, pH, ionic strength and temperature and their selectivity was assessed against other proteins. High-abundant proteins, like human serum albumin and γ-globulins did not interfere with the binding of lysozyme even at elevated concentrations characteristic of proteinuria. A sample cleanup procedure for urine samples has been developed utilizing the thermocontrollable protein binding ability of the nanoparticles. Method validation was carried out according to current bioanalytical method validation guidelines. The method was highly selective, and the calibration was linear in the 25 to 1000 µg/mL concentration range, relevant in the diagnosis of monocytic and myelomonocytic leukemia. Intra- and inter-day precision values ranged from 2.24 to 8.20% and 1.08 to 5.04%, respectively. Intra-day accuracies were between 89.9 and 117.6%, while inter-day accuracies were in the 88.8 to 111.0% range. The average recovery was 94.1 ± 8.1%. Analysis of unknown urine samples in comparison with a well-established reference method revealed very good correlation between the results, indicating that the new nanoparticle-based method has high potential in the diagnosis of lysozymuria.

The influence of antibiotics on transitory resistome during gut colonization with CTX-M-15 and OXA-162 producing Klebsiella pneumoniae ST15.

Great efforts have been made to limit the transmission of carbapenemase-producing Enterobacteriaceae (CPE), however, the intestinal reservoir of these strains and its modulation by various antibiotics remain largely unexplored. Our aim was to assess the effects of antibiotic administration (ampicillin, ceftazidime, ciprofloxacin) on the establishment and elimination of intestinal colonization with a CTX-M-15 ESBL and OXA-162 carbapenemase producing Klebsiella pneumoniae ST15 (KP5825) in a murine (C57BL/6 male mice) model. Whole genome sequencing of KP5825 strain was performed on an Illumina MiSeq platform. Conjugation assays were carried out by broth mating method. In colonization experiments, 5 × 106 CFU of KP5825 was administered to the animals by orogastric gavage, and antibiotics were administered in their drinking water for two weeks and were changed every day. The gut colonization rates with KP5825 were assessed by cultivation and qPCR. In each of the stool samples, the gene copy number of blaOXA-162 and blaCTX-M-15 were determined by qPCR. Antibiotic concentrations in the stool were determined by high pressure liquid chromatography and a bioanalytical method. The KP5825 contained four different plasmid replicon types, namely IncFII(K), IncL, IncFIB and ColpVC. IncL (containing the blaOXA-162 resistance gene within a Tn1991.2 genetic element) and IncFII(K) (containing the blaCTX-M-15 resistance gene) plasmids were successfully conjugated. During ampicillin and ceftazidime treatments, colonization rate of KP5825 increased, while, ciprofloxacin treatments in both concentrations (0.1 g/L and 0.5 g/L) led to significantly decreased colonization rates. The gene copy number blaOXA-162 correlated with K. pneumoniae in vivo, while a major elevation was observed in the copy number of blaCTX-M-15 from the first day to the fifteenth day in the 0.5 g/L dose ceftazidime treatment group. Our results demonstrate that commonly used antibiotics may have diverse impacts on the colonization rates of intestinally-carried CPE, in addition to affecting the gene copy number of their resistance genes, thus facilitating their stable persistance and dissemination.

Synthesis, Molecular Recognition Study and Liquid Membrane-Based Applications of Highly Lipophilic Enantiopure Acridino-Crown Ethers.

New highly lipophilic enantiopure crown ethers containing a heterocyclic unit have been synthesized. Phase transport, UV-Vis- and fluorescence spectrophotometric investigations as well as electrochemical studies on the complexation of the new macrocycles with several amine and amino acid derivatives were also carried out. Achiral amines were used for studying the structural preference of the new macrocycles. Among the studied structural features of the guest molecules, the intermolecular π-π interaction showed the most significant effect on complexation, which made the aralkylamine-type compounds the most preferable guest molecules. The studied liquid membrane-based applications and photophysical investigations showed appreciable enantiomeric recognition toward some aralkylamine model compounds with homochiral preferences. New crown ether derivatives (R,R)-2 and (S,S)-2 were successfully applied as enantioselective carrier and sensor molecules.

Salt and solvent effects in the microscale chromatographic separation of heparan sulfate disaccharides.

The analysis of heparan sulfate disaccharides poses a real challenge both from chromatographic and mass spectrometric point of view. This necessitates the constant improvement of their analytical methodology. In the present study, the chromatographic effects of solvent composition, salt concentration, and salt type were systematically investigated in isocratic HILIC-WAX separations of heparan sulfate disaccharides. The combined use of 75% acetonitrile with ammonium formate had overall benefits regarding intensity, detection limits, and peak shape for all salt concentrations investigated. Results obtained with the isocratic measurements suggested the potential use of a salt gradient method in order to maximize separation efficiency. A 3-step gradient from 14 mM to 65 mM ammonium formate concentration proved to be ideal for separation and quantitation. The LOD of the resulting method was 0.8-1.5 fmol for the individual disaccharides and the LOQ was between 2.5-5 fmol. Outstanding linearity could be observed up to 2 pmol. This novel combination provided sufficient sensitivity for disaccharide analysis, which was demonstrated by the analysis of heparan sulfate samples from porcine and bovine origin.

Fast Potentiometric Analysis of Lead in Aqueous Medium under Competitive Conditions Using an Acridono-Crown Ether Neutral Ionophore.

Lead is a particularly toxic heavy metal that is present above acceptable levels in the water of many countries. This article describes a quick detection method of lead(II) ions using a polyvinyl chloride (PVC)-based ion-selective membrane electrode containing an acridono-crown ether ionophore by potentiometry. The electrochemical cell exhibits a Nernstian response for lead(II) ions between the concentration range of 10−4 to 10−2 M, and can be used in the pH range of 4⁻7. The applicability of this sensor was verified by measuring a multicomponent aqueous sample. Under the given conditions, this electrode is suitable for the selective quantitative analysis of lead(II) ions in the presence of many additional metal ions.

Preparation of Molecularly Imprinted Microspheres by Precipitation Polymerization.

Molecularly imprinted polymers (MIPs) gained an expansively growing interest in the past few decades. After an initial, explorative period of preparing MIPs exclusively with bulk polymerization, new polymer synthesis routes have been adapted to overcome the drawbacks of the traditional method. Among these the most appealing is precipitation polymerization that results in nano- and microspheres with narrow size distribution and makes the production of MIPs more straightforward. Here, we describe a precipitation polymerization protocol for a common small molecule template, propranolol that is carried out in the conventional way, in dilute monomer solution. Moreover, a modified precipitation polymerization protocol from concentrated monomer solution is presented for a diclofenac imprinted polymer which makes the synthesis even more versatile and circumvents the disadvantages of the dilute solution conditions.

Molecularly imprinted polymer microspheres containing photoswitchable spiropyran-based binding sites.

A versatile approach for the preparation of photoswitchable molecularly imprinted polymers (MIPs) is proposed where the selective recognition and the photoresponsive function are assumed by two different monomers. As a proof of concept, MIP microspheres were synthesized by precipitation polymerization for recognizing terbutylazine, a triazine-type herbicide. Formation of the selective binding sites was based upon H-bonding interactions between the template and the functional monomer methacrylic acid, whereas a polymerizable spiropyran unit was incorporated into the polymer matrix to provide light-controllable characteristics. A trifunctional monomer, trimethylolpropane trimethacrylate, was used as a cross-linker. The imprinted particles exhibited considerable morphological differences compared to their nonimprinted counterparts as observed by scanning electron microscopy. The imprinting effect was confirmed by equilibrium rebinding studies. The photoresponsiveness of the polymer particles was visualized by fluorescence microscopy and further characterized by spectroscopy. The template binding behavior could be regulated by alternating UV and visible light illumination when analyte release and uptake was observed, respectively. Binding isotherms fitted by the Freundlich model revealed the photomodulation of the number of binding sites and their average affinity. This facile synthetic approach may give an attractive starting point to endow currently existing highly selective MIPs with photoswitchable properties, thereby extending the scope of spiropyran-based photoresponsive smart materials.

In situ synthesis of molecularly imprinted nanoparticles in porous support membranes using high-viscosity polymerization solvents.

There is a growing need in membrane separations for novel membrane materials providing selective retention. Molecularly imprinted polymers (MIPs) are promising candidates for membrane functionalization. In this work, a novel approach is described to prepare composite membrane adsorbers incorporating molecularly imprinted microparticles or nanoparticles into commercially available macroporous filtration membranes. The polymerization is carried out in highly viscous polymerization solvents, and the particles are formed in situ in the pores of the support membrane. MIP particle composite membranes selective for terbutylazine were prepared and characterized by scanning electron microscopy and N2 porosimetry. By varying the polymerization solvent microparticles or nanoparticles with diameters ranging from several hundred nanometers to 1 µm could be embedded into the support. The permeability of the membranes was in the range of 1000 to 20,000 Lm⁻² hr⁻¹ bar⁻¹. The imprinted composite membranes showed high MIP/NIP (nonimprinted polymer) selectivity for the template in organic media both in equilibrium-rebinding measurements and in filtration experiments. The solid phase extraction of a mixture of the template, its analogs, and a nonrelated compound demonstrated MIP/NIP selectivity and substance selectivity of the new molecularly imprinted membrane. The synthesis technique offers a potential for the cost-effective production of selective membrane adsorbers with high capacity and high throughput.

Selective solid phase extraction of propranolol on multiwell membrane filter plates modified with molecularly imprinted polymer.

Molecularly imprinted polymers (MIPs) were synthesized in 24-well glass fiber membrane filter plates to obtain a novel type of solid phase extraction device for the cleanup of propranolol. Sample processing parameters like residence time during sample loading, sample volume, pH, sample solvent, type and amount of washing and elution solvents were investigated and optimized. Important differences from the traditional molecularly imprinted solid phase extraction (MISPE) cartridges have been identified. The MIP modified composite membrane suits well for the sample preparation of low volume biological samples. A protocol has been elaborated for the quantitation of propranolol from urine and plasma samples in the clinically relevant concentration ranges. Preliminary validation results indicate that the composite MIP membrane filter plates offer a viable alternative to existing MISPE cartridges and at the same time have advantages like much easier and faster synthesis method and high-throughput analysis.

Preparation of terbutylazine imprinted polymer microspheres using viscous polymerization solvents.

Spherical micron sized terbutylazine imprinted polymers have been obtained by copolymerization of acrylates in solvent mixtures containing a highly viscous ('oil') component. The new method requires much less organic solvent than precipitation polymerization to produce spherical MIP particles. Different proportions of oil have been used to clarify its role on the morphology. The particles obtained with the optimal composition could be easily packed into short HPLC columns. Chromatographic retention of the template and other compounds has been determined on the novel ('OMIP') columns and was compared to MIP prepared by bulk polymerization. In 70% aqueous acetonitrile eluent the OMIP shows a higher imprinting factor (i. e. less non-selective binding) and lower retention than the bulk MIP. The chromatographic selectivity against template analogs is similar on OMIP and bulk MIP, while the selectivity against non-related substances is better on the OMIP. The effect of oil on the polymer structure appears to be due to its high viscosity.

Accelerated development procedure for molecularly imprinted polymers using membrane filterplates.

A novel technique for the synthesis and testing of large numbers of molecularly imprinted polymers is described requiring much less time than the commonly used miniMIP approach. Instead of vials, the polymers are synthesized on the surface of microfiltration membranes in multiwell filterplates. The thin polymeric films enable accelerated template removal. The MIP development procedure is thereby shortened to two days. Performance of the system was demonstrated by creating a combinatorial library of MIPs selective for cimetidine, an antiulcer drug. The polymer composition has been optimized. An experimental design combined with a multivariate analysis (i.e., response surface modeling) was used to minimize the number of experiments in the optimization process. The highest imprinting factor was obtained using a MAA/EDMA/template molar ratio of 3.5:19.5:1.

Analytical followup of the gamma initiated synthesis of a molecularly imprinted polymer.

A molecularly imprinted polymer (MIP) for the template phenytoin has been prepared by gamma initiated copolymerization of methacrylamide and ethylene glycol dimethacrylate. The progress of polymerization was studied by measuring the monomer conversions and the template binding properties of the resulting polymers, respectively. The consumption rate of the two monomers showed different course. There was no difference observed in the polymerization rates of the MIP and the control polymer (NIP). The template binding properties of the MIP and the NIP changed considerably with the progress of the polymerization process and became similar to those of the thermally initiated polymers after full conversion.

Measurement of sodium ion concentration in undiluted urine with cation-selective polymeric membrane electrodes after the removal of interfering compounds.

The measurement of sodium ion concentration in urine can provide diagnostic information and guide therapy. Unfortunately, neutral-carrier-based ion-selective electrodes show a large positive drift and loss in selectivity in undiluted urine. The extraction of electrically neutral lipids from the urine into the sensing membrane was suggested as the main source of the drift, loss of selectivity and the consequent incorrect concentration readings. In this work, (i) solvent-solvent extraction, (ii) membrane-immobilized solvent extraction and (iii) solid phase extraction were used to remove interfering compounds from urine samples. The "cleaned" urine samples were subsequently analyzed using a calixarene (sodium ionophore X)-based, solid-contact, sodium-selective electrode in a flow-through manifold. The solid-contact sodium sensors had excellent stability in cleaned urine and an acceptable bias compared to commercial clinical analyzers.

High-performance liquid chromatographic-tandem mass spectrometric method for the determination of clemastine in human plasma.

A highly sensitive high-performance liquid chromatographic-tandem mass spectrometric method (HPLC-MS-MS) has been developed to quantitate clemastine in human plasma for the purpose of pharmacokinetic studies. Sample preparation was carried out by liquid-liquid extraction using deuterated clemastine as an internal standard. Chromatographic separation used a C18 reversed phase polymer column giving an extremely fast total run time of 2 min. The method was validated and used for the bioequivalence study of clemastine tablets in healthy male volunteers (n=28). The lower limit of detection proved to be 0.01 ng/ml for clemastine.

Influence of food on the oral bioavailability of deramciclane from film-coated tablet in healthy male volunteers.

The effect of a high-fat meal on the oral bioavailability of deramciclane 30 mg tablet was evaluated in 18 healthy male volunteers in a randomised, single dose, two-way crossover study. The drug was administered following an overnight fast or a standardised high-fat breakfast. The plasma concentrations of deramciclane and N-desmethylderamciclane were determined by using a validated HPLC-MS -MS/MS method. An effect of food on the bioavailability was indicated if the 90% confidence interval (CI) for the ratio of geometric means of fed and fasted treatments was not contained in the equivalence limit of 0.8-1.25 for AUC and C(max). The ratios of the mean C(max) and AUC(0-infinity) values of deramciclane were 1.24 (90% CI 1.12-1.38) and 1.31 (90% CI 1.21-1.41) in fed versus fasted subjects, which overlapped but exceeded the equivalence limit. In contrast to the parent compound, the 90% CI of the mean ratios for AUC(0-infinity) and C(max) of N-desmethylderamciclane were within the predefined range. The 24 and 31% increase in C(max) and AUC(0-infinity) of deramciclane, respectively, under fed condition is modest and probably has no clinical significance since it is relatively small compared to the inter-individual variability of these parameters.

Effect of solvents on the selectivity of terbutylazine imprinted polymer sorbents used in solid-phase extraction.

A solid-phase extraction sample preparation method using a molecularly imprinted polymer (MIP) selective for the triazine type pesticide terbutylazine has been developed. The method involves preconcentration from large volumes of water samples on a C18 disk coupled to selective clean-up on the MIP. The method has been optimised by studying the recovery and retention of terbutylazine and some other structurally related triazine derivates as a function of the selective washing solvent used. The effect of the water content of the selective washing solvent was also investigated on the recovery of the MIP. River water samples were analysed with the coupled technique, and efficient clean-up of the samples was observed.