George Horvai

Determination of phenytoin in plasma by molecularly imprinted solid-phase extraction

A molecularly imprinted polymer (MIP) using phenytoin as template and methacrylamide as the functional monomer was prepared. The selectivity was measured by comparing capacity factors of phenytoin and other structurally related compounds. The polymer was evaluated as a selective sorbent in molecularly imprinted solid-phase extraction (MISPE). Several washing solvents were tested to study their ability to disrupt the non-specific interactions occurring between the sample and the polymer matrix and the role of water in the recognition process was also investigated. It was shown that the key step of successful sample extraction is the right choice of the washing solvent. Plasma samples spiked with phenytoin were analyzed by the MISPE methodology developed in this work. Method validation (intra- and inter-day precision, recovery, specificity) was carried out. The calibration curve showed good linearity in the 2.5-40 microg/ml range corresponding to therapeutically relevant plasma levels. The intra- and inter-day precision values were below the 15% limit established for bioanalytical methods. The results showed that the method could be successfully applied for the determination of phenytoin in plasma samples.

A new method for determining the interfacial molecules and characterizing the surface roughness in computer simulations. Application to the liquid–vapor interface of water

A new method is presented to identify the truly interfacial molecules at fluid/fluid interfaces seen at molecular resolution, a situation that regularly occurs in computer simulations. In the new method, the surface is scanned by moving a probe sphere of a given radius along a large set of test lines that are perpendicular to the plane of the interface. The molecules that are hit by the probe spheres are regarded as interfacial ones, and the position of the test spheres when they are in contact with the interfacial molecules give an estimate of the surface. The dependence of the method on various parameters, in particular, on the size of the probe sphere is discussed in detail. Based on the list of molecules identified as truly interfacial ones, two measures of the molecular scale roughness of the surface are proposed. The bivariate distribution of the lateral and normal distances of two points of the interface provides a full description of the molecular scale morphology of the surface in a statistical sense. For practical purposes two parameters related to the dependence of the average normal distance of two surface points on their lateral distance can be used. These two parameters correspond to the frequency and amplitude of the surface roughness, respectively. The new method is applied for the analysis of the molecular level structure of the liquid–vapor interface of water. As an immediate result of the application of the new method it is shown that the orientational preferences of the interfacial water molecules depend only on the local curvature of the interface, and hence the molecules located at wells of concave curvature of the rippled surface prefer the same orientations as waters located at the surface of small apolar solutes. The vast majority of the truly interfacial molecules are found to form a strongly percolating two‐dimensional hydrogen bonded network at the surface, whereas no percolation is observed within the second molecular layer beyond the surface.

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.

New insight into the orientational order of water molecules at the water/1,2-dichloroethane interface: A Monte Carlo simulation study

The preferential orientation of the water molecules near the water/1,2-dichloroethane interface is analyzed in detail at different distances from the interface on the basis of a grand canonical ensemble Monte Carlo simulation. The orientation of the individual water molecules is described by the angular polar coordinates of the interface normal vector in a local coordinate frame fixed to the particular water molecule, and the bivariate joint distribution of the two polar angles is calculated. It is found that water molecules have two distinct orientational preferences, and these two preferences exist simultaneously among the water molecules penetrating farthest into the organic phase. In the first preferred orientation the plane of the molecule is parallel to the interface, whereas in the second the molecular plane is aligned perpendicularly to the interface and the molecular dipole vector declines from the plane parallel to the interface by about 30° pointing toward the organic phase. The first of the two preferred orientations is found to be present in the entire interfacial region and also, to a smaller extent, in the subsurface water layer adjacent to the interface. The second orientational preference is only present among the water molecules penetrating farthest into the organic phase. The two orientations correspond to the alignment of a hydrogen bonded pair of water molecules, in which the molecule located toward the aqueous phase has the first, whereas the one on the organic side the second of the two preferred orientations. The obtained picture is in a clear contrast with the findings of previous studies, in which the orientation of the water molecules was described by monovariate distributions of the alignment of one or more selected molecule-fixed vectors. In order to understand the origin of the difference between the present results and earlier findings we also calculate the monovariate distributions of the direction of three of such molecular vectors, i.e., the dipole vector of the water molecule, the vector joining the two H atoms, and the vector perpendicular to the molecular plane. The comparison of the obtained monovariate distributions with the bivariate joint distribution of the two polar angles reveals that the averaging of the bivariate distribution over any of its two angles completely obscures the dual orientational preference. The present study clearly points out the importance of choosing appropriate statistical distributions in the analysis of simulation results and demonstrates the pitfalls of averaging over too many variables.

Donnan exclusion failure in low anion site density membranes containing valinomycin

Abstract The theory of Donnan Exclusion Failure of carrier-containing, low-fixed site density membranes has been derived, for high carrier loadings. The precarious character of commercial PVC samples for membrane applications, because of the low site densities is illustrated by the ease of Donnan Failure induced by bromide, nitrate, iodide, and thiocyanate. The theory is checked by a correlation between potentiometric response maxima (the onset of Failure), and independently determined salt extraction coefficients. Massive uptake of salt, under Donnan Failure conditions, was illustrated by comparing impedance spectra of KSCN with KCl as bathing electrolytes.

Automated determination of levodopa and carbidopa in plasma by high-performance liquid chromatography-electrochemical detection using an on-line flow injection analysis sample pretreatment unit

An automated analytical procedure is described for the parallel determination of L-3,4-dihydroxyphenylalanine (levodopa, L-dopa, LD) and the analogous hydrazine compound carbidopa (CD) in dog plasma by ion-pair high-performance liquid chromatography with electrochemical detection (HPLC-ED). After deproteinization of the plasma samples with perchloric acid the catecholamines were extracted from the supernatant by adsorption on a small column filled with alumina. The extraction and redissolution were automatically performed in a flow injection analysis unit (FIA) coupled to the HPLC system. The performance of the whole system was tested on dog plasma samples including specimens taken after oral administration of the anti-Parkinsonism drug Duellin, which is a combination tablet of levodopa and carbidopa.

Multiple Forms of Endocannabinoid and Endovanilloid Signaling Regulate the Tonic Control of GABA Release

Persistent CB1 cannabinoid receptor activity limits neurotransmitter release at various synapses throughout the brain. However, it is not fully understood how constitutively active CB1 receptors, tonic endocannabinoid signaling, and its regulation by multiple serine hydrolases contribute to the synapse-specific calibration of neurotransmitter release probability. To address this question at perisomatic and dendritic GABAergic synapses in the mouse hippocampus, we used a combination of paired whole-cell patch-clamp recording, liquid chromatography/tandem mass spectrometry, stochastic optical reconstruction microscopy super-resolution imaging, and immunogold electron microscopy. Unexpectedly, application of the CB1 antagonist and inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], but not the neutral antagonist NESS0327 [8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-5,6-dihydro-4H-benzo[2,3]cyclohepta[2,4-b]pyrazole-3-carboxamine], significantly increased synaptic transmission between CB1-positive perisomatic interneurons and CA1 pyramidal neurons. JZL184 (4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate), a selective inhibitor of monoacylglycerol lipase (MGL), the presynaptic degrading enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), elicited a robust increase in 2-AG levels and concomitantly decreased GABAergic transmission. In contrast, inhibition of fatty acid amide hydrolase (FAAH) by PF3845 (N-pyridin-3-yl-4-[[3-[5-(trifluoromethyl)pyridin-2-yl]oxyphenyl]methyl]piperidine-1-carboxamide) elevated endocannabinoid/endovanilloid anandamide levels but did not change GABAergic synaptic activity. However, FAAH inhibitors attenuated tonic 2-AG increase and also decreased its synaptic effects. This antagonistic interaction required the activation of the transient receptor potential vanilloid receptor TRPV1, which was concentrated on postsynaptic intracellular membrane cisternae at perisomatic GABAergic symmetrical synapses. Interestingly, neither AM251, JZL184, nor PF3845 affected CB1-positive dendritic interneuron synapses. Together, these findings are consistent with the possibility that constitutively active CB1 receptors substantially influence perisomatic GABA release probability and indicate that the synaptic effects of tonic 2-AG release are tightly controlled by presynaptic MGL activity and also by postsynaptic endovanilloid signaling and FAAH activity. SIGNIFICANCE STATEMENT Tonic cannabinoid signaling plays a critical role in the regulation of synaptic transmission. However, the mechanistic details of how persistent CB1 cannabinoid receptor activity inhibits neurotransmitter release have remained elusive. Therefore, electrophysiological recordings, lipid measurements, and super-resolution imaging were combined to elucidate those signaling molecules and mechanisms that underlie tonic cannabinoid signaling. The findings indicate that constitutive CB1 activity has pivotal function in the tonic control of hippocampal GABA release. Moreover, the endocannabinoid 2-arachidonoylglycerol (2-AG) is continuously generated postsynaptically, but its synaptic effect is regulated strictly by presynaptic monoacylglycerol lipase activity. Finally, anandamide signaling antagonizes tonic 2-AG signaling via activation of postsynaptic transient receptor potential vanilloid TRPV1 receptors. This unexpected mechanistic diversity may be necessary to fine-tune GABA release probability under various physiological and pathophysiological conditions.

Enantiomer-selectivity of ion-selective electrodes based on a chiral crown-ether ionophore

Abstract A newly synthesized crown ether ionophore can differentiate between R-(+) and S-(-)-1-phenylethylammonium ions when incorporated into a plasticized PVC based electrode membrane, thus providing enantiomer-selectivity. Different membrane compositions were studied. The selectivity coefficient found was 0.71, independent of the membrane composition. This proves the robustness of the ISE method for testing the enantiomeric selectivity of newly synthesized ionophores. Selectivities of the new ionophore towards different hydrophilic cations were also determined.

Properties of free surface of water-methanol mixtures. Analysis of the truly interfacial molecular layer in computer simulation.

Molecular dynamics simulations of the vapor-liquid interface of water-methanol mixtures of five different compositions were performed on the canonical (N,V,T) ensemble at 298 K. In addition, the vapor-liquid interface of the two neat systems was simulated, as well. The obtained configurations were analyzed by means of the novel identification of truly interfacial molecules method, which provides a full list of the molecules that are right at the surface (i.e., at the boundary of the two phases). The molecular level roughness of the surface, the adsorption of the methanol molecules at the surface layer, the orientation of the surface molecules, the residence time of the molecules at the surface layer, as well as the surface aggregation of the molecules were analyzed in detail. Both the frequency and the amplitude of the surface roughness were found to become larger with an increasing methanol content. This effect was found to be stronger for the amplitude, which falls in the range of 2-4 A, depending on the composition of the system. Methanol was found to be adsorbed at the surface layer, being preferentially at the humps of the molecularly rough surface. Surface methanol prefers to orient in such a way that the O-CH(3) bond remains perpendicular to the macroscopic plane of the surface, pointing the methyl group to the vapor phase. The main orientational preference of the water molecules is to lie parallel to the surface. Methanol was found to remain considerably longer at the surface layer of the mixed systems than water. Thus, contrary to the fact that the residence times of the two molecules were found to be rather similar to each other at the surface of their neat liquids, the residence time of the methanol molecules was an order of magnitude larger than that of water molecules at the surface of their mixtures. A strong lateral microscopic segregation of the molecules was observed at the surface layer; the minor component of the system (irrespective of whether it was water or methanol) was found to form two-dimensional aggregates, leaving the rest of the surface empty for the major component. The effect of the vicinity of the vapor phase on the properties of the molecules was found to vanish very quickly: the composition of the second layer as well as the properties of the molecules of this layer (e.g., dynamics and orientation) did not differ considerably from those in the bulk liquid phase.

Molecular level properties of the free water surface and different organic liquid/water interfaces, as seen from ITIM analysis of computer simulation results

Molecular dynamics simulations of the interface of water with four different apolar phases, namely water vapour, liquid carbon tetrachloride, liquid dichloromethane (DCM) and liquid dichloroethane (DCE) are performed on the canonical ensemble at 298 K. The resulting configurations are analysed using the novel method of identification of the truly interfacial molecules (ITIM). Properties of the first three molecular layers of the liquid phases (e.g. width, spacing, roughness, extent of the in-layer hydrogen bonding network) as well as of the molecules constituting these layers (e.g., dynamics, orientation) are investigated in detail. In the analyses, particular attention is paid to the effect of the polarity of the non-aqueous phase and to the length scale of the effect of the vicinity of the interface on the various properties of the molecules. The obtained results show that increasing polarity of the non-aqueous phase leads to the narrowing of the interface, in spite of the fact that, at the same time, the truly interfacial layer of water gets somewhat broader. The influence of the nearby interface is found to extend only to the first molecular layer in many respects. This result is attributed to the larger space available for the truly interfacial than for the non-interfacial molecules (as the shapes of the two liquid surfaces are largely independent of each other, resulting in the presence of voids between the two phases), and to the fact that the hydrogen bonding interaction of the truly interfacial water molecules with other waters is hindered in the direction of the interface.