Spectrofluorimetric determination of atenolol from human urine using high‐affinity molecularly imprinted solid‐phase extraction sorbent

This study presents a novel, sensitive and selective molecularly imprinted solid‐phase extraction (MISPE)–spectrofluorimetric method for the removal and determination of atenolol from human urine. Molecularly imprinted and non‐imprinted polymers were synthesized thermally using a radical chain polymerization technique and used as solid‐phase extraction sorbents. Acrylic acid ethylene glycol dimethacrylate, dibenzoyl peroxide and dichloroethane were used as a functional monomer, cross‐linker, initiator and porogen, respectively. The calibration curve was in the range of 0.10–2.0 μg/ml for the developed method. Limit of detection and limit of quantification values were 0.032 and 0.099 μg/ml, respectively. Owing to the selectivity of the MISPE technique and the sensitivity of spectrofluorimetry, trace levels of atenolol have been successfully determined from both organic and aqueous media. Relatively high imprinting factor (4.18) and recovery results (74.5–75.3%) were obtained. In addition, intra‐ and interday precision values were 0.38–1.03% and 0.47–2.05%, respectively, proving the precision of the proposed method. Thus, a selective, sensitive and simple MISPE–spectrofluorimetric method has been developed and applied to the direct determination of atenolol from human urine.     

Molecularly imprinted polymer of 5-methyluridine for solid-phase extraction of pyrimidine nucleoside cancer markers in urine

Normal and modified urinary nucleosides represent potential biomarkers for cancer diagnosis. To selectively extract modified nucleosides, we developed a molecularly imprinted polymer (MIP) of 5-methyluridine as selective material for molecularly imprinted solid-phase extraction (MISPE). The MIPs were obtained from vinyl-phenylboronate ester derivative of the template, acrylamide and pentaerythritol triacrylate co-polymer, and were tested in batch and cartridge experiments with aqueous samples. Our results indicated that the imprinted polymer was selective for pyrimidine nucleosides with a K(d) and a B(max) of 46 microM and 18 micromol/g, respectively. Finally, a MISPE of the most common pyrimidine nucleoside cancer markers in urine sample was realized.     

Evaluation of water-compatible molecularly imprinted polymers as solid-phase extraction sorbents for the selective extraction of sildenafil and its desmethyl metabolite from plasma samples

The evaluation of molecularly imprinted polymers (MIPs) as selective sorbents for the solid-phase extraction of sildenafil and its principal metabolite, desmethylsildenafil, was investigated. Two MIPs were synthesised using structural analogues of sildenafil as templates, and a comparison of the performance of the two MIP sorbents in organic and aqueous media was performed. Additionally, the feasibility of applying molecularly imprinted solid-phase extraction (MISPE) to the clean-up of plasma samples containing sildenafil and desmethylsildenafil was investigated. A preliminary, quantitative MISPE for the determination of both compounds in plasma was also performed. The results showed that the MIPs used for the selective extraction of sildenafil gave better compound recovery when aqueous samples were used in comparison to organic-based samples. A preliminary, quantitative MISPE of sildenafil and desmethylsildenafil indicated that the imprinted materials could be used successfully as SPE sorbents for sample pre-treatment for the determination of sildenafil, and related compounds, in plasma.     

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%.     

Molecularly imprinted polymer cartridges coupled on-line with high performance liquid chromatography for simple and rapid analysis of dextromethorphan in human plasma samples

In this paper, a novel method is described for automated determination of dextromethorphan in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as a 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 dextromethorphan as template molecule. These imprinted polymers were used as solid-phase extraction sorbent for the extraction of dextromethorphan from human plasma samples. Various parameters affecting the extraction efficiency of the MIP cartridges were evaluated. The high selectivity of the sorbent coupled to the high performance liquid chromatographic system permitted a simple and rapid analysis of this drug in plasma samples with limits of detection (LOD) and quantification (LOQ) of 0.12 ng/mL and 0.35 ng/mL, respectively. The MIP selectivity was evaluated by analyzing of the dextromethorphan in presence of several substances with similar molecular structures and properties. Results from the HPLC analyses showed that the recoveries of dextromethorphan using MIP cartridges from human plasma samples in the range of 1-50 ng/mL were higher than 87%.     

Rational design and synthesis of molecularly imprinted polymers (MIP) for purifying tylosin by seeded precipitation polymerization

A useful approach was proposed to easily synthesize molecularly imprinted adsorbent for the purification of tylosin from broth. Firstly, by molecular simulation based on density functional theory, methacrylic acid was chosen as a functional monomer by comparing the binding energy. Second, a novel method of polymerization based on precipitation polymerization with added seeds was used in water-mixed solvent for the preparation of water-compatible micron-sized MIP. Its static adsorption capacity for tylosin in aqueous solution was estimated to be 106.5 mg/g with the highest imprinting factor (IF) of 3.6. The selectivity coefficient (α) of tylosin to desmycosin was 3.3. The antibiotic in fermentation broth could be purified by means of molecularly imprinted solid phase extraction (MISPE), which allows MIP to be used for the purification of tylosin from a complex sample.     

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.     

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.     

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.     

Characterization of molecularly imprinted polymers using a new polar solvent titration method

A new method of characterizing molecularly imprinted polymers (MIPs) was developed and tested, which provides a more accurate means of identifying and measuring the molecular imprinting effect. In the new polar solvent titration method, a series of imprinted and non‐imprinted polymers were prepared in solutions containing increasing concentrations of a polar solvent. The polar solvent additives systematically disrupted the templation and monomer aggregation processes in the prepolymerization solutions, and the extent of disruption was captured by the polymerization process. The changes in binding capacity within each series of polymers were measured, providing a quantitative assessment of the templation and monomer aggregation processes in the imprinted and non‐imprinted polymers. The new method was tested using three different diphenyl phosphate imprinted polymers made using three different urea functional monomers. Each monomer had varying efficiencies of templation and monomer aggregation. The new MIP characterization method was found to have several advantages. To independently verify the new characterization method, the MIPs were also characterized using traditional binding isotherm analyses. The two methods appeared to give consistent conclusions. First, the polar solvent titration method is less susceptible to false positives in identifying the imprinting effect. Second, the method is able to differentiate and quantify changes in binding capacity, as measured at a fixed guest and polymer concentration, arising from templation or monomer aggregation processes in the prepolymerization solution. Third, the method was also easy to carry out, taking advantage of the ease of preparing MIPs. Copyright © 2014 John Wiley & Sons, Ltd.     

Molecularly imprinted polymer microspheres for solid-phase extraction of chloramphenicol residues in foods

Preparation of molecularly imprinted polymer microspheres (MIPMs) for chloramphenicol (CAP) by aqueous suspension polymerization is reported for the first time in this study. The resulting MIPMs had the ability to specifically adsorb CAP, and the molecularly imprinted solid phase extraction (MISPE) based on the MIPMs was shown to be applicable for clean-up and preconcentration of trace CAP in milk and shrimp samples with high recoveries of 92.7% and 84.9%, respectively. Combined with MISPE, the conventional HPLC-UV analysis sensitivity for CAP in foods could be significantly increased.     

Multidimensional on-line sample preparation of verapamil and its metabolites by a molecularly imprinted polymer coupled to liquid chromatography-mass spectrometry

A new molecularly imprinted polymer (MIP) material was synthesized selective for verapamil and utilized for on-line metabolic screening of this common calcium antagonist in biological samples. Since some metabolites of verapamil have also shown pharmacological properties, a selective and sensitive sample preparation approach that provides a metabolic profile in biologically relevant samples is important. The MIP material was coupled on-line to a restricted access material (RAM) precolumn. The multidimensional nature of this set-up removed large matrix interferents such as proteins from the sample, while the selectivity of the MIP enabled further cleanup of the smaller analytes. The selectivity and extraction efficiency of the MIP for verapamil and its metabolites was evaluated in various biological matrices, such as cell cultures and urine. The experimental set-up with the developed method enabled the direct injection of biological samples for the selective isolation, preconcentration, identification and analysis of verapamil and its phase I metabolites by LC-MS(n). This multidimensional approach provided much qualitative information about the metabolic profile of verapamil in various biological matrices. An analytical method was developed for the quantification of verapamil and gallopamil in urine, plasma and cell culture. Acceptable linearity (R(2)=0.9996, 0.9982 and 0.9762) with an average injection repeatability (n=3) of 10, 25 and 15% R.S.D. was determined for urine, plasma and cell culture, respectively. This is the first application of the procedure for the selective metabolic screening of verapamil in biological samples.     

PREPARATION OF GLUTATHIONE IMPRINTED POLYMER

Glutathione imprinted polymer was prepared using 1-vinyl imidazole and ethylene glycol dimethacrylate as the functional monomer and crosslinker, respectively, in dimethyl sulfoxide. The adsorption selectivity of glutathione-imprinted polymer was tested by reduced glutathione, oxidized glutathione, and L-Gly-Leu-Tyr in 30% phosphate buffer (0.01 M, pH 5.0)–70% acetonitrile and binding affinity values were compared. Reusability of molecularly imprinted polymer particles was also investigated. Molecularly imprinted polymer particles were found to be stable and to maintain glutathione adsorption capacity at 95% when washed with methanol–acetic acid (10%) after seven usages. Functional monomer 1-vinyl imidazole and cross linker ethylene glycol dimethacrylate-based glutathione imprinted polymer could be used as solid phase extraction material for recognition of glutathione in biological samples.     

High-performance liquid chromatographic determination of hippuric acid in human urine

A method is described for the determination of urinary hippuric acid by high-performance liquid chromatography. The method used ethyl acetate extraction for partial clean-up of the urine. The separation was carried out on a reversed-phase column using 20% methanol in 0.01 M aqueous potassium phosphate containing 0.5% acetic acid as a mobile phase. The column effluent was monitored with a UV detector at 254 nm. Hippuric acid was separated from other normal urine constituents in less than 10 min. Metabolites of xylene and styrene did not interfere with the assay. Analytical recoveries from urine were excellent and peak height and concentration were linearly related.     

Determination of urinary hippuric acid by micellar electrokinetic capillary chromatography

We propose a method for the simultaneous determination of hippuric acid (HA) and creatinine based on capillary micellar electrokinetic chromatography. Experimental conditions were 20 mM sodium phosphate, pH 7.20, 25 mM sodium dodecyl sulfate, 5% (v/v) acetonitrile. Electropherograms evidenced HA and creatinine peaks in less than 12 min. The method showed good linearity for both analytes and satisfactory within-day precision. The present method, which is accurate, sensitive, rapid and simple, may be applied to single-spot urine samples.     

Liquid chromatography for iothalamate in biological samples

We have previously reported an iothalamate assay for the assessment of the glomerular filtration rate (GFR) that required a long column equilibration time and 22 min run time per sample. We now report a simpler assay that requires a run time of only 5.5 min and is more precise and accurate than the earlier technique. The mobile phase consisted of methanol-acetonitrile-50 mM sodium monobasic phosphate (10:5:85, v/v) at pH 4.4, pumped at a rate of 1.5 ml/min on a C(18) reversed-phase column. Samples of plasma and urine were deproteinized with 1 volume of 4% perchloric acid or 9 volumes of 2% perchloric acid, respectively. No internal standard was used. The diode array detection system collected absorbance at 240 nm and the peak height areas of iothalamate were determined. The iothalamate peak appeared at 3.5 min. Detector response was linear over the range tested (10-2000 microg/ml). Within-run precision was <3% for both plasma and urine and accuracy was 96-102%. Between-day precision for plasma and urine analyses were <7%. The recovery of iothalamate in urine and plasma were 102% and 91%, respectively. There was excellent thermal and pH stability of iothalamate. No interference was found with para-amino hippuric acid (PAH) or N-acetyl PAH, which can be simultaneously assayed, if desired.     

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.     

Investigation of the nature of MIP recognition: the development and characterisation of a MIP for Ibuprofen

This paper describes the rational design, generation and testing of a molecularly imprinted polymer specific for Ibuprofen. Ibuprofen is a member of the class of drugs termed non-steroidal anti-inflammatory drugs (NSAIDS). In the present study, Ibuprofen was used as a template molecule for the preparation of molecularly imprinted polymers. A MIP has been produced which is capable of recognising Ibuprofen in aqueous media. Furthermore, Ibuprofen can be selectively extracted from aqueous conditions by molecularly imprinted solid phase extraction (MISPE). Recoveries were typically high (>80%) and good selectivity for Ibuprofen over structurally related analogues was seen. Moreover, the nature of the recognition between MIP and template has been investigated by NMR and molecular modelling to analyse whether or not it is possible to predict how well a given MIP will perform under set conditions. In addition, the physical characteristics of the MIP have been investigated including the particle size distribution on exposure of the MIP to different solvents. This has been related to the ability of the MIP to rebind Ibuprofen under the same conditions. The data from the characterisation of the MIP has been used to further enhance the understanding of the nature of MIP recognition.     

‘Bite-and-Switch’ approach using computationally designed molecularly imprinted polymers for sensing of creatinine

A method for the selective detection of creatinine is reported, which is based on the reaction between polymerised hemithioacetal, formed by allyl mercaptan, o-phthalic aldehyde, and primary amine leading to the formation of fluorescent isoindole complex. This method has been demonstrated previously for the detection of creatine using creatine-imprinted molecularly imprinted polymers (MIPs) Since MIPs created using traditional methods were unable to differentiate between creatine and creatinine, a new approach to the rational design of a molecularly imprinted polymer (MIP) selective for creatinine was developed using computer simulation. A virtual library of functional monomers was assigned and screened against the target molecule, creatinine, using molecular modelling software. The monomers giving the highest binding score were further tested using simulated annealing in order to mimic the complexation of the functional monomers with template in the monomer mixture. The result of this simulation gave an optimised MIP composition. The computationally designed polymer demonstrated superior selectivity in comparison to the polymer prepared using traditional approach, a detection limit of 25 μM and good stability. The ‘Bite-and-Switch’ approach combined with molecular imprinting can be used for the design of assays and sensors, selective for amino containing substances.     

Hydrophilic interaction liquid chromatography and accurate mass measurement for quantification and confirmation of morphine, codeine and their glucuronide conjugates in human urine

A hydrophilic interaction liquid chromatography-time-of-flight mass spectrometry (HILIC-TOFMS) method for the quantification and confirmation of morphine (M), codeine (C), morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and codeine-6-glucuronide (C6G) is presented. The method was validated in terms of specificity, selectivity, extraction recovery, accuracy, repeatability, linearity and matrix effect. After a straightforward sample preparation by solid phase extraction (SPE) the compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. The HILIC technique provided good chromatographic separation which was critical for isomers M3G and M6G. The analytes were detected after electrospray ionization (ESI) in positive mode with mass accuracies below 2 mDa using a 5-mDa window. A measurement range of 50-5000 ng/ml was applied for calibration using deuterated analogs as internal standards. The precision of the method was 5.7% and 10.2% (RSD) within and between days, respectively. The applicability of the method was demonstrated with authentic urine samples known to contain codeine and/or morphine and their intact glucuronide conjugates. Identification of the analytes was based on in-source collision induced dissociation (ISCID), applying three diagnostic ions with accurate mass.