Viola Horváth

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.

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.

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.

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.

Cyclic voltammetric experiments with plasticized PVC membranes

Abstract Cyclic voltammetry, which is widely used in studying liquid/ liquid interfaces, was applied to plasticized PVC-based membranes in order to understand the nature of ion transfer processes at the interfaces of the membrane. Dummy membranes containing a lipophilic salt additive (tetrahexylammonium tetraphenylborate) were examined with this method. By varying the tetrahexylammonium concentration of the bathing solution from 0 to 10 -3 M, different behaviours of the interfaces could be observed with symmetric and asymmetric bathing.

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.

Sensitive high-performance liquid chromatographic determination of nifedipine in dog plasma using an automated sample preparation system with laboratory robot

Nifedipine, a calcium-channel blocking drug was analysed in dog plasma after oral dosing with two different formulations. Sample preparation was automated with a laboratory robot. Quantitative determination of the drug was performed on a reversed-phase HPLC system with electrochemical detection (ED) using an internal standard. Validation of the analytical method showed that the system is well suited for pharmacokinetic studies on dogs. The assay was linear in the range 1-50 ng/ml. Inter-day and intra-day variability were between 6.43-18.15% C.V. and 1.57-5.53% C.V., respectively.

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.

Electrochemical template synthesis of protein-imprinted magnetic polymer microrods

A novel method for the electrochemical template synthesis of surface-imprinted magnetic polymer microrods for protein recognition is proposed. The polymer was electrodeposited into sacrificial cylindrical microreactors, the internal walls of which were previously modified with a target model protein, avidin, by simple physisorption. The electropolymerization was performed from a mixture of 3,4-ethylenedioxythiophene, poly(styrenesulfonate) (PSS), and PSS-coated superparamagnetic nanoparticles resulting in the formation of inherently electroconductive polymers confined to the volume of the microreactor. Here we show that: (i) the template synthesis within cylindrical microreactors results in polymer rods with dimensions matching that of the reactor, (ii) the incorporation of superparamagnetic particles induces magnetic properties that allow for efficient collection and manipulation of the microrods released from the microreactors in magnetic field even from dilute solution, and (iii) the protein coating on the internal walls of the microreactors is shown to generate molecular imprints on the surface of the polymeric rods. This latter property was demonstrated by comparative binding experiments of a fluorescent avidin derivative to the surface-imprinted and non-imprinted magnetic polymer microrods.

Novel type of flow injection immunoassay for the determination of phenytoin in serum

Abstract A novel flow injection immunoassay using fluorimetric detection successfully measured an antiepileptic drug, phenytoin, from a serum background. The assay was based on the competitive binding of fluorescein labelled and unlabelled phenytoin followed by the separation of bound and unbound antigens with gel filtration chromatography. The immunoreaction and subsequent detection of products was performed in a newly designed stopped flow injection system employing a simple two injector system for reagent injection, mixing and rigorous timing of the incubation. Two experimental set-ups were tested. The optimal parameters and configurations of the systems were established and the results were verified against an independent method. The calibration curves were linear showing good sensitivity and reproducibility in the concentration range required for clinical analyses suggesting that this technique could be a viable alternative for the measurement of the phenytoin in serum samples.