Application of molecularly imprinted polymers to solid-phase extraction of analytes from real samples

A review is presented of recent developments in the use of molecularly imprinted polymers (MIPs) as selective materials for solid-phase extraction. Compared with traditional sorbents, MIPs can not only concentrate but also selectively separate the target analytes from real samples, which is crucial for the quantitatively determination of analytes in complex samples. Consequently, as one of the most effective sorbents, MIPs have been successfully applied to the pretreatment of analytes in foods, drugs, and biological and environmental samples in the past five years.     

Application of molecularly imprinted polymers to solid-phase extraction of analytes from real samples

A review is presented of recent developments in the use of molecularly imprinted polymers (MIPs) as selective materials for solid-phase extraction. Compared with traditional sorbents, MIPs can not only concentrate but also selectively separate the target analytes from real samples, which is crucial for the quantitatively determination of analytes in complex samples. Consequently, as one of the most effective sorbents, MIPs have been successfully applied to the pretreatment of analytes in foods, drugs, and biological and environmental samples in the past five years.     

Solid-phase extraction of tramadol from plasma and urine samples using a novel water-compatible molecularly imprinted polymer

In this study, a novel method is described for the determination of tramadol in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as the sample clean-up technique combined with high-performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and tramadol as template molecule. The novel imprinted polymer was used as a solid-phase extraction (SPE) sorbent for the extraction of tramadol from human plasma and urine. Various parameters affecting the extraction efficiency of the polymer have been evaluated. The optimal conditions for the MIP cartridges were studied. The MIP selectivity was evaluated by checking several substances with similar molecular structures to that of tramadol. The limit of detection (LOD) and limit of quantification (LOQ) for tramadol in urine samples were 1.2 and 3.5 μg L⁻¹, respectively. These limits for tramadol in plasma samples were 3.0 and 8.5 μg L⁻¹, respectively. The recoveries for plasma and urine samples were higher than 91%.     

Selective solid-phase extraction of bio- and environmental samples using molecularly imprinted polymers

Of the many applications of molecular imprinting in analytical separation science, the one with highest potential of soon being used in routine analysis is that of solid-phase extraction. Already several examples of selective pre-concentration of biological and environmental samples have been reported. The interest in imprinted extraction sorbents originates from the high selectivities and affinities obtainable, properties which can be qualitatively and quantitatively pre-determined for a particular analyte and separation by the imprinting process. This review summarises work published on molecular imprinted solid-phase extraction and discusses some imprinted-sorbent specific method development issues.     

A new molecularly imprinted polymer for the selective extraction of naproxen from urine samples by solid-phase extraction

A non-covalent molecularly imprinted polymer (MIP) was synthesised using naproxen (a non-steroidal, anti-inflammatory drug (NSAID)) as a template molecule. The MIP was chromatographically evaluated to confirm the imprinting effect, and was then applied as a selective sorbent in solid-phase extraction (SPE) to selectively extract naproxen. After this study, the MIP was used to extract naproxen from urine samples; it was demonstrated that by applying a selective washing step with acetonitrile (ACN) the compounds in the sample that were structurally related to naproxen could be eliminated.     

Novel biphasic separations utilising highly selective molecularly imprinted polymers as biorecognition solvent extraction agents

Molecularly imprinted polymers (MIPs) represent a class of artificial receptors that promise an environmentally robust alternative to naturally occurring biorecognition elements of biosensing devices and systems. However, in general, the performance of conventional MIPs in aqueous environments is poor. In the study reported here, this limitation has been addressed by the novel application of MIPs as a solvent extraction solid phase in a biphasic solvent system. This paper describes a previously unreported use of MIPs as solvent extraction reagents, their successful application to aqueous sample media and the opportunities for utilisation of this unique system in novel biosensing and separation procedures. This study demonstrates the development of a novel biphasic solvent system utilising MIP in the extracting phase to enhance both efficiency and selectivity of a simple two phase liquid extraction. Monodisperse propranolol imprinted polymer microspheres [p(divinylbenzene-co-methacrylic acid)] were prepared by precipitation polymerisation. Initially, the affinity of the polymers for (R,S)-propranolol was assessed by established techniques whereby the MIP demonstrated greater affinity for the template than did the non-imprinted control polymer (NIP). Importantly, MIP performance was also assessed using the novel dual solvent system. The depletion of (R,S)-propranolol from the aqueous phase into the polymer containing organic phase was determined. When compared to control extractions containing no polymer the presence of MIP in the extracting solvent phase resulted in an increased extraction of (R,S)-propranolol from the aqueous phase. Importantly, this extraction was significantly greater in the presence of MIP when compared to NIP. This unique principle generates opportunities for MIP based extractions and chemical enrichments in industrial applications, offering commercial, ecological and practical advantages to traditional solvent extraction techniques. The technique is readily transferable to analytical microsystems utilising MIP recognition elements generating promising opportunities for MIP based sensing of aqueous sample media.     

Use of molecularly imprinted polymers in the solid-phase extraction of clenbuterol from animal feeds and biological matrices

Clenbuterol molecularly imprinted polymers (MIPs) as chromatographic stationary phase for the solid-phase extraction (SPE) of the drug from biological samples have been prepared. Propylene columns filled with 500 mg of clenbuterol MIPs have been tested with respect to their loading capacity, memory effects, selectivity toward related drugs (mabuterol, clenproperol, clenisopenterol, ritodrine) and specificity toward interferences arising from heterogeneous matrices such as animal feeds, bovine urine and liver. Analytes were concentrated on Extrelut 20 columns and the residues resuspended in 70% acetonitrile. Application, washing and elution fractions were collected and analyzed by HPLC–diode array detection. Results indicate this MIP approach in SPE is extremely selective for clenbuterol, mabuterol, clenproperol and clenisopenterol (>95% found in the eluate), with a loading capacity of about 20 μg/100 mg of stationary phase. Ritodrine showed a recovery rate of 51%. The molecular recognition mechanism is so specific to allow clenbuterol detection and identification by conventional detectors at level of interest (ppb) also from complex matrices such as feeds, urine and liver.     

Design of a new cartridge for selective solid phase extraction using molecularly imprinted polymers: Selective extraction of theophylline from human serum samples

This paper describes design of a new cartridge for selective solid phase extraction (SPE) using molecularly imprinted polymers (MIPs). The apparatus which is termed solvent extraction-MISPE (SE-MISPE) cartridge, consisted of a modified conventional micro test tube and has been developed to perform simultaneous forward-extraction of analyte from aqueous sample solution to an organic phase and back-extraction to MIP solid phase. In order to evaluate the performance of the proposed method, extraction of theophylline (THP) from human serum sample was investigated. An appropriate amount of THP-imprinted polymer was placed in the bottom of the micro tube and an organic solvent pipetted onto it and left to swell the polymer completely. A polyethylene frit to secure MIP particles was positioned by two Teflon rings such that it was fixed below the level of the organic layer. Then, aqueous sample solution containing THP was layered over the organic phase and the lid was closed. After completion of extraction, the organic and aqueous phases were removed and the adsorbed analyte was desorbed using a polar organic solvent. In order to reach the highest recovery, the experimental parameters such as the type of organic solvent, pH and ionic strength of aqueous phase, organic to aqueous volume ratio, time of extraction, type and amount of desorbent solvent were optimized. Under the experimental conditions, a plot of HPLC peak areas vs. initial concentrations of THP in the concentration interval of 0.5–30 μg ml⁻¹ showed a good linearity (r = 0.9974). The limit of detection (LOD) and limit of quantification (LOQ) based on three and ten times of the noise of HPLC profile were 0.09 and 0.3 μg ml⁻¹, respectively. The relative standard deviation (RSD) of the proposed method for the extraction and determination of 5 μg THP from 200 μl standard sample solution for 3 replicate measurements was 3.5%. The results showed that by means of the proposed cartridge, THP could significantly separate from the other structurally related compounds such as theobromine (THB) and caffeine (CAF). The added THP could be quantitatively recovered (79–83%) from the serum samples by the proposed procedure, being thus a guarantee of the accuracy of the SE-MISPE procedure. In addition, the loss of capability of the SE-MISPE cartridge was not considerably observed after 10 times loading and elution cycles.     

Selective solid-phase extraction using molecularly imprinted polymer for analysis of methamidophos in water and soil samples

An analytical methodology for the analysis of methamidophos in water and soil samples incorporating a molecularly imprinted solid-phase extraction process using methamidophos-imprinted polymer was developed. Binding study demonstrated that the polymer exhibited excellent affinity and high selectivity to the methamidophos. Evidence was also found by FT-IR analysis that hydrogen bonding between the CO(2)H in the polymer cavities and the NH(2) and P=O of the template was the origin of methamidophos recognition. The use of molecularly imprinted solid-phase extraction improved the accuracy and precision of the GC method and lowered the limit of detection. The recovery of methamidophos extracted from a 10.0 g soil sample at the 100 ng/g spike level was 95.4%. The limit of detection was 3.8 ng/g. The recovery of methamidophos extracted from 100 mL tap and river water at 1 ng/mL spike level was 96.1% and 95.8%, and the limits of detection were 10 and 13 ng/L respectively. These molecularly imprinted solid-phase extraction procedures enabled selective extraction of polar methamidophos successfully from water and soil samples, demonstrating the potential of molecularly imprinted solid-phase extraction for rapid, selective, and cost-effective sample pretreatment.     

Evaluation of molecularly imprinted solid-phase extraction for a 1,2:3,4-diepoxybutane adduct to valine

A molecularly imprinted polymer, MIP, was prepared and evaluated as SPE sorbent for a cyclicized adduct formed to N-terminal valine (Pyr-Val) in hemoglobin from 1,2:3,4-diepoxybutane (DEB). This metabolite plays an important role in the carcinogenesis of 1,3-butadiene. The hydrazide of Pyr-Val, formed after hydrazinolysis of hemoglobin, as well as necessary standards was synthesized. The MIP was prepared from methacrylic acid with a structure analogue to the investigated adduct as template and the method was developed for aqueous conditions. Selective desorption was achieved when the sample was washed with water after loading in 10% acetonitrile. The primary interaction with the binding sites in the imprints was most likely of ionic character. Quantification of the Pyr-Val adduct was performed with LC/ESI-MS/MS, yielding an instrumental LOD of 150 pg injected amount.     

Retention mechanism of analytes in the solid-phase extraction process using molecularly imprinted polymers. Application to the extraction of triazines from complex matrices

The influence of sampling variables on the concentration of the dopamine metabolites 3-methoxytyramine (3MT), dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) was examined in equine urine. A logarithmic transformation of the data for all horses gave distribution which approximated the normal distributions for each metabolite. The mean urinary concentration of 3 MT in horses was 214 ng/mL and the application of a threshold with a probability of 1 in 10,000 gave an actionable level of 4 microg/mL. Environmental variables were not forensically significant in determining the population distribution. HVA was not found to be a reliable indicator of dopamine or levodopa administration.     

Class-specific molecularly imprinted polymers for the selective extraction and determination of sulfonylurea herbicides in maize samples by high-performance liquid chromatography–tandem mass spectrometry

A novel method based on the molecularly imprinted solid-phase extraction (MISPE) procedure has been developed for the simultaneous determination of concentrations of sulfonylurea herbicides such as chlorsulfuron (CS), monosulfuron (MNS), and thifensulfuron methyl (TFM) in maize samples by liquid chromatography–tandem quadrupole mass spectrometry (LC–MS/MS). The molecularly imprinted polymer (MIP) for sulfonylurea herbicides was synthesized by precipitation polymerization using chlorsulfuron as the template molecule, 2-(diethylamino)ethyl methacrylate (DEAMA) as the functional monomer, and trimethylolpropane trimethacrylate (TRIM) as the cross-linker. The selectivities of the chlorsulfuron template and its analogs on the molecularly imprinted polymer were evaluated by high-performance liquid chromatography (HPLC). The extraction and purification procedures for the solid-phase extraction (SPE) cartridge with a molecularly imprinted polymer as the adsorbent for the selected sulfonylurea herbicides were then established. A molecularly imprinted solid-phase extraction method followed by high-performance liquid chromatography–tandem mass spectrometry for the determination of chlorsulfuron, monosulfuron, and thifensulfuron methyl was also established. The mean recoveries of these compounds in maize were in the range 75–110% and the limits of detection (LOD) of chlorsulfuron, monosulfuron, and thifensulfuron methyl were 0.02, 0.75, and 1.45 μg kg⁻¹, respectively. It was demonstrated that the MISPE–HPLC–MS/MS method could be applied to the determination of chlorsulfuron, monosulfuron, and thifensulfuron methyl in maize samples.     

Novel molecularly imprinted polymers based on multi-walled carbon nanotubes with binary functional monomer for the solid-phase extraction of erythromycin from chicken muscle

A new surface imprinting technique was reported to synthesize multi-walled carbon nanotubes-molecularly imprinted polymers (MWNTs-MIPs) using erythromycin as the template, acryloyl-β-cyclodextrin (acryloyl-β-CD) and methacrylic acid (MAA) as the binary functional monomers. The MWNTs-MIPs were characterized by transmission electron microscopy (TEM), scanning electron micrograph (SEM) and Fourier transform-infrared spectroscopy (FT-IR). Adsorption experiments indicated the MWNTs-MIPs prepared with acryloyl-β-CD and MAA have high selective for erythromycin. The feasibility of the MWNTs-MIPs as solid-phase extraction (SPE) sorbent was evaluated, and the results showed that it can selectively extract erythromycin from chicken muscle samples with the recoveries ranging from 85.3% to 95.8%. The molecularly imprinted solid-phase extraction (MISPE) method could be applied for preconcentration and purification of erythromycin from chicken muscle samples.     

Magnetic molecularly imprinted polymers for improved extraction of tanshinones from herbs via integrated extraction and cleanup system

A novel separation technology at room temperature for traditional Chinese medicines was proposed in this work by adding magnetic molecularly imprinted polymers (M-MIPs) into extraction solution and sample matrix. The M-MIPs show a more adsorption capacities and higher selectivity for the template than magnetic non-molecularly imprinted polymers (M-NMIPs) without the specific binding sites. Addition of the M-MIPs to the extraction solution provides one-step extraction and cleanup, the improvement of extraction rate and extraction yields of three tanshinones (from 0.40, 0.23 and 0.12 mg g⁻¹ in 240 min by solvent extraction to 0.52, 0.27 and 0.19 mg g⁻¹ in 5 min by 200 mg sorbent), and reusability of extraction solvent. The extraction yields of three tanshinones by this technology at room temperature in 5 min were higher than those by ultrasonic extraction in 30 min, by heat reflux extraction in 45 min and by solvent extraction at room temperature in 4 h. The integrated technology has the advantages of one-step extraction and cleanup, high extraction efficiency, low solvent consumption and room temperature.     

Isolation and detection of steroids from human urine by molecularly imprinted solid-phase extraction and liquid chromatography

Naturally occurring steroids such as progesterone, testosterone and 17β-estradiol were analyzed in this study. These bio-identical molecules paradoxically can be either beneficial or harmful. Unfortunately as growth promoters can be toxic and cancerogenic at elevated levels. Due to difficulty in monitoring at trace quantities of these hormones in biological matrices specific adsorption materials molecularly imprinted polymers (MIPs) were used for preconcentration and clean up in sample preparation step. A non-covalent imprinting approach was used for bulk polymerization of progesterone, testosterone and 17β-estradiol imprinted polymers. Synthesis of MIPs was achieved by thermal, UV and γ irradiation initiated polymerization whereby were used methacrylic acid (MAA), 4-vinylpyridine (4-VP) as functional monomers, ethylene glycol dimethacrylate (EDMA), trimethylolpropane trimethacrylate (TRIM) as cross-linking agents and acetonitrile, isooctane–toluene (1:99, v/v) and chloroform as porogen solvents. It was also used as initiator 2,2′-azobis(2-methylpropionitrile) (AIBN) or benzyl methyl ether (BME). The MIPs were applied as selective sorbents in solid-phase extraction (SPE). Molecularly imprinted solid-phase extraction (MISPE) considered as hyphenated technique were applied in extraction step before HPLC-DAD analysis of steroids from human urine.     

Crown ethers as a tool for the preparation of molecularly imprinted polymers

Molecularly imprinted polymers have been prepared against aniline and a bis-aniline compound, making use of a crown ether (18-crown-6) to solubilize the monomer-template complexes. Subsequent chromatographic rebinding studies in the absence of crown ether revealed regioselectivity for the templates in the respective polymers. This study indicates that crown ethers can be potentially useful in conjunction with molecular imprinting to solubilize and imprint organic solvent-insoluble compounds.     

Group-selective molecularly imprinted polymer solid-phase extraction for the simultaneous determination of six sulfonamides in aquaculture products

Group-selective molecularly imprinted polymers (MIPs) made from sulfonamides (SAs) using functional monomer methacrylic acid (MAA) were synthesized. The derived molecularly imprinted solid-phase extraction (MISPE) cartridges were developed for the purification and enrichment of aquatic products. The optimum template molecule and the ratio of the functional monomer to the template for obtaining group selectivity to SAs were sulfadimethoxine (SDM) and 4:1, respectively. The MIPs were characterized by Brunauer-Emmett-Teller (BET), scatchard plot, and chromatography analysis, all of which demonstrate better chromatographic behavior and group-selectivity of MIPs for SAs compared with those of corresponding NIPs. The extraction conditions of MISPE for six SAs were optimized; the method precision and accuracy were satisfactory for the fish and shrimp samples at 0.05, 0.1, and 0.2 mg kg(-1) spiked levels. Recoveries ranging from 85.5% to 106.1% (RSD, 1.2-7.0%, n=3) were achieved. The limits of detection (S/N=3) and quantitation (S/N=10) in the shrimp and fish samples were achieved from 8.4 to 10.9 μg kg(-1) and from 22.4 to 27.7 μg kg(-1), respectively. Therefore, the obtained MIPs and MISPE can be employed for the enrichment and clean-up of SAs. This paper presents a new analytical method which enables the simultaneous determination and quantification of SAs in aquaculture products.     

Development of molecularly imprinted polymers for the binding of nitrofurantoin

Novel molecularly imprinted polymers (MIPs) for the recognition of nitrofurantoin (NFT) were prepared by photoinitiated polymerisation in polar solvent using 2,6-bis(methacrylamido) pyridine (BMP) as the functional monomer and carboxyphenyl aminohydantoin (CPAH) as the analogue of the template. The binding constants of the complex between BMP and nitrofurantoin or CPAH in DMSO were determined with 1H NMR titration to be 630 ± 104 and 830 ± 146 M⁻¹, respectively. To study the influence of the functional monomer, two polymer compositions were prepared containing the template, the functional monomer and the crosslinker in the molar ratio 1:1:12 for MIP1 and 1:4:20 for MIP2, respectively. The imprinting factor at saturation concentration of nitrofurantoin, which is the ratio of the amount bound to the MIP and the non-imprinted control polymer (NIP), was determined to be 2.47 for MIP1 and 2.49 for MIP2. The cross reactivity of the imprinted polymers seems to be determined by the ability to form hydrogen bonds to the functional monomer while the shape of the molecule has no real influence.     

Simultaneous determination of clomipramine and its desmethyl and hydroxy metabolites in plasma of patients by high-performance liquid chromatography after solid-phase extraction

Clomipramine (CMI) is a typical tricyclic antidepressant with a wide clinical spectrum, being used in major depressive, panic and obsessive-compulsive disorders. The relationship between clinical response and plasma levels of clomipramine and its N-desmethylated (N-desmethylclomipramine, DMCMI) and hydroxy-metabolites remains unclear. In particular, limited information is available on the correlation with clinical response in patients with obsessive-compulsive disorder (OCD). This study describes a new sensitive method to simultaneously determine CMI and its major N-desmethylated and hydroxy-metabolites present in human plasma by HPLC with a UV detector. After a solid-phase extraction from plasma (Isolute C2 columns) the separation of the compounds was performed on a Lichrospher CN column (250 x 4 mm, 5 microm with a 2-cm pre-column) by an eluent consisting of 10 mM K(2)HPO(4)-acetonitrile-methanol (35:25:40 v/v/v) at a flow of 1.5 ml/min. UV detector was set at 214 nm. The lower limit of quantification for all the analytes was at least 5 ng/ml. The coefficients of variation ranged between 2.0 and 4.9% with recovery rates between 97.0 and 100.3%. Linear regression analyses showed correlation coefficients between 0.98 and 0.99. This method is simple, fast and reliable with good specificity and sensitivity. Solid phase extraction is efficient and rapid, allowing the extraction of several plasma samples on the same day and may therefore be usefully and realistically applied in the clinical context. We thus investigated the relevance of plasma levels of CMI and its metabolites as a predictor of clinical outcome in a group of 15 patients with OCD.     

Preparation and evaluation of a molecularly imprinted polymer for the selective recognition of testosterone--application to molecularly imprinted sorbent assays

Biomimetic testosterone receptors were synthesized via molecular imprinting for use as antibody mimics in immunoassays. As evaluated by radioligand binding assays, imprinted polymers prepared in acetonitrile were very specific for testosterone because the nonimprinted control polymers bound virtually no radiolabeled testosterone. The polymers present an appreciable affinity, with association constants of K(a) = 3.3 x 10(7) M(- 1) (high-affinity binding sites). The binding characteristics of the polymers were also evaluated in aqueous environment to study their viabilities as alternatives to antibodies in molecularly imprinted sorbent assays. Compared with the testosterone-specific antibodies present in commercial kits, our molecularly imprinted polymers are somewhat less sensitive but show a high selectivity.