Molecularly imprinted polymers synthesized via template immobilization on fumed silica nanoparticles for the enrichment of phosphopeptides.

Phosphorylation is a protein post-translational modification (PTM) that plays an important role in cell signaling, cell differentiation, and metabolism. The hyper phosphorylated forms of certain proteins have been appointed as biomarkers for neurodegenerative diseases, and phosphorylation-related mutations are important for detecting cancer pathways. Due to the low abundance of phosphorylated proteins in biological fluids, sample enrichment is beneficial prior to detection. Thus, a need to find new strategies for enriching phosphopeptides has emerged. Molecularly imprinted polymers (MIPs) are synthetic polymeric materials manufactured to exhibit affinity for a target molecule. In this study, MIPs have been synthesized using a new approach based on the use of fumed silica as sacrificial support acting as solid porogen with the template (phosphotyrosine) immobilized on its surface. Phosphotyrosine MIPs were tested against a mixture of peptides and phosphopeptides by performing micro-solid phase extraction using MIPs (µMISPE) packed in a pipette tip. First, the capability of the materials to preferentially enrich phosphopeptides was evaluated. In a next step, the enrichment of phosphopeptides from a whole-cell lysate of human embryonic kidney (HEK) 293T cells was performed. The eluates were analyzed using MALDI-MS in the first case and with nano-HPLC-ESI-MS/MS in the second case. The results showed that the MIPs provided affinity for phosphopeptides, binding preferentially to multi-site phosphorylated peptides. The MIPs could enrich phosphopeptides in over 10-fold compared with the number of phosphopeptides found in a cell lysate without enrichment.

Studies towards enantioselective surface imprinted polymers.

Molecularly imprinted polymers (MIPs) are tailor-made materials that can specifically rebind desired target molecules. Usually, the template is in solution with monomers that form a specific polymeric cavity around the template. Here, a model system with an amino acid (Fmoc-L-Histidine) is presented. This new approach entails the imprinting of templates that are bound to support materials. It is shown that the new approach leads to materials that exhibit symmetric peak shapes for both enantiomers under isocratic conditions.

Use of molecularly imprinted solid-phase extraction sorbent for the determination of four 5-nitroimidazoles and three of their metabolites from egg-based samples before tandem LC-ESIMS/MS analysis.

A nitroimidazole, molecularly imprinted polymer (MIP) was tested to extract four 5-nitroimidazoles (i.e., dimetridazole (DMZ), ipronidazole (IPZ), metronidazole (MNZ), and ronidazole (RNZ)) and three of their metabolites (i.e., DMZOH, IPZOH, and MNZOH) from egg powder samples. Various MIP templates were produced, and their selectivity was assessed on nitroimidazole standard solutions using liquid chromatography coupled with ultraviolet detection. The optimal cleanup was then used for the extraction of nitroimidazole in egg powder samples, and their quantification was achieved by isotope dilution LC-ESIMS/MS. The sample preparation entails a solubilization of the samples with water and acetonitrile followed by a MISPE cleanup step before LC-ESIMS/MS analysis. Data acquisition was achieved using selected reaction monitoring, and quantification was done with five deuterated analogues (i.e., DMZ- d(3), RNZ- d(3), IPZ- d(3), DMZOH- d(3), and IPZOH- d(3)). DMZOH- d(3) was used to quantify MNZ and MNZOH since they do not have their corresponding internal standards. The method was validated according to the European Union criteria by spiking experiments at concentration levels of 1, 2, and 3 microg/kg. At these three levels and for compounds having their own internal standards, acceptable performance data were obtained, with internal standard corrected recoveries ranging from 91 to 111%, and decision limits (CCalpha) and detection capabilities (CCbeta) were below 0.34 and 0.39 microg/kg, respectively.

Development of an improved method for trace analysis of chloramphenicol using molecularly imprinted polymers.

A confirmatory method is described for the determination of the illegal antibiotic chloramphenicol using a specifically developed molecularly imprinted polymer (MIP) as the sample clean-up technique. The newly developed MIP was produced using an analogue to chloramphenicol as the template molecule. Using an analogue of the analyte as the template avoids a major traditional drawback associated with MIPs of residual template leeching or bleeding. The MIP described was used as a solid-phase extraction phase for the extraction of chloramphenicol from various sample matrices including honey, urine, milk and plasma. A full analytical method with quantification by LC-MS/MS is described. The method was fully validated according to the European Union (EU) criteria for the analysis of veterinary drug residues.

Solid-phase extraction of the alcohol abuse biomarker phosphatidylethanol using newly synthesized polymeric sorbent materials containing quaternary heterocyclic groups.

Phosphatidylethanol (PEth) is an interesting biomarker finding increased use for detecting long term alcohol abuse with high specificity and sensitivity. Prior to detection, sample preparation is an unavoidable step in the work-flow of PEth analysis and new protocols may facilitate it. Solid-phase extraction (SPE) is a versatile sample preparation method widely spread in biomedical laboratories due to its simplicity of use and the possibility of automation. In this work, SPE was used for the first time to directly extract PEth from spiked human plasma and spiked human blood. A library of polymeric SPE materials with different surface functionalities was screened for PEth extraction in order to identify the surface characteristics that control PEth retention and recovery. The plasma samples were diluted 1:10 (v/v) in water and spiked at different concentrations ranging from 0.3 to 5µM. The library of SPE materials was then evaluated using the proposed SPE method and detection was done by LC-MS/MS. One SPE material efficiently retained and recovered PEth from spiked human plasma. With this insight, four new SPE materials were formulated and synthesized based on the surface characteristics of the best SPE material found in the first screening. These new materials were tested with spiked human blood, to better mimic a real clinical sample. All the newly synthetized materials outperformed the pre-existing commercially available materials. Recovery values for the new SPE materials were found between 29.5% and 48.6% for the extraction of PEth in spiked blood. A material based on quaternized 1-vinylimidazole with a poly(trimethylolpropane trimethacrylate) backbone was found suitable for PEth extraction in spiked blood showing the highest analyte recovery in this experiment, 48.6%±6.4%.

Utilizing the Cross-Reactivity of MIPs.

The crossreactivity of molecularly imprinted polymers (MIPs) and its practical implications are discussed. Screening of MIP libraries is presented as a fasttrack route to discovery of resins selective towards new targets, exploiting the fact that MIPs imprinted with one type of template molecule also show recognition to related and sometimes also to apparently unrelated molecules. Several examples from our own and others' studies are presented that illustrate this crossreactivity and the pattern of recognition is discussed for selected examples.

Fast identification of selective resins for removal of genotoxic aminopyridine impurities via screening of molecularly imprinted polymer libraries.

This study describes the identification and evaluation of molecularly imprinted polymers (MIPs) for the selective removal of potentially genotoxic aminopyridine impurities from pharmaceuticals. Screening experiments were performed using existing MIP resin libraries to identify resins selective towards those impurities in the presence of model pharmaceutical compounds. A hit resin with a considerable imprinting effect was found in the screening and upon further investigation, the resin was found to show a broad selectivity towards five different aminopyridines in the presence of the two model active pharmaceutical ingredients (APIs) piroxicam and tenoxicam.

Selective extraction of triazine herbicides based on a combination of membrane assisted solvent extraction and molecularly imprinted solid phase extraction.

A selective extraction technique based on the combination of membrane assisted solvent extraction and molecularly imprinted solid phase extraction for triazine herbicides in food samples was developed. Simazine, atrazine, prometon, terbumeton, terbuthylazine and prometryn were extracted from aqueous food samples into a hydrophobic polypropylene membrane bag containing 1000µL of toluene as the acceptor phase along with 100mg of MIP particles. In the acceptor phase, the compounds were re-extracted onto MIP particles. The extraction technique was optimised for the type of organic acceptor solvent, amount of molecularly imprinted polymers particles in the organic acceptor phase, extraction time and addition of salt. Toluene as the acceptor phase was found to give higher triazine binding onto MIP particles compared to hexane and cyclohexane. Extraction time of 120min and 100mg of MIP were found to be optimum parameters. Addition of salt increased the extraction efficiency for more polar triazines. The selectivity of the technique was demonstrated by extracting spiked cow pea and corn extracts where clean chromatograms were obtained compared to only membrane assisted solvent extraction or only molecularly imprinted solid phase extraction. The study revealed that this combination may be a simple way of selectively extracting compounds in complex samples.

Analysis of permethrin isomers in composite diet samples by molecularly imprinted solid-phase extraction and isotope dilution gas chromatography-ion trap mass spectrometry.

Determination of an individual's aggregate dietary ingestion of pesticides entails analysis of a difficult sample matrix. Permethrin-specific molecularly imprinted polymer (MIP) solid-phase extraction cartridges were developed for use as a sample preparation technique for a composite food matrix. Vortexing with acetonitrile and centrifugation were found to provide optimal extraction of the permethrin isomers from the composite foods. The acetonitrile (with 1% acetic acid) was mostly evaporated and the analytes reconstituted in 90:10 water/acetonitrile in preparation for molecularly imprinted solid-phase extraction. Permethrin elution was accomplished with acetonitrile and sample extracts were analyzed by isotope dilution gas chromatography-ion trap mass spectrometry. Quantitation of product ions provided definitive identification of the pesticide isomers. The final method parameters were tested with fortified composite food samples of varying fat content (1%, 5%, and 10%) and recoveries ranged from 99.3% to 126%. Vegetable samples with incurred pesticide levels were also analyzed with the given method and recoveries were acceptable (81.0-95.7%). Method detection limits were demonstrated in the low ppb range. Finally, the applicability of the MIP stationary phase to extract other pyrethroids, specifically cyfluthrin and cypermethrin, was also investigated.

Selective determination of acidic pharmaceuticals in wastewater using molecularly imprinted solid-phase extraction.

The determination of acidic pharmaceuticals, such as non-steroidal anti-inflammatory drugs NSAIDs and clofibric acid (metabolite of clofibrate), at low ngL(-1) levels in wastewater requires highly selective and sensitive analytical procedures. The removal of matrix components during sample preparation results in significant benefits towards reducing the matrix effects during LC-MS analysis. Therefore this work describes a simple method to enrich and clean up NSAIDs and clofibric acid from sewage water using molecularly imprinted solid-phase extraction (MISPE). Final analysis was performed by liquid chromatography-tandem mass spectrometry. The performance of this method has been evaluated in fortified tap and sewage water in terms of recovery, precision, linearity, and method quantification limit. Recovery for all compounds ranged in all matrices between 84 and 116% with intra-day R.S.D. values below 11.5%. Matrix effect evaluation demonstrated that even complex sample matrixes, such as pond or sewage water did not showed significant ion suppression/enhancement compared to tap water. The performance of the method was further emphasized by the study of pond water, which receives treated water from a sewage treatment plant in south Sweden. Raw sewage and treated water were also tested. In those samples, all acidic pharmaceuticals were detected in concentration above method quantification limits ranging from 5.1 to 5153.0ngL(-1).

Advantages of molecularly imprinted polymers LC-ESI-MS/MS for the selective extraction and quantification of chloramphenicol in milk-based matrixes. comparison with a classical sample preparation.

A simple and fast selective extraction of the antibiotic chloramphenicol (CAP) from milk (raw milk, skimmed milk, and milk powder) using a molecularly imprinted polymer (MIP) sorbent is described. The method entails a single centrifugation step prior to loading the supernatant onto the MIP cartridge and subsequent elution with a mixture of solvents. CAP was further analyzed by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) operating in negative ionization acquisition mode. The advantages of the MIP approach were assessed by comparing the data generated from a classical solid-phase and liquid-liquid extractions procedure, previously developed in our laboratory. A better recovery of CAP due to an enhanced selectivity and a faster turnaround time (18 samples processed within 3 h compared to 8 h with the classical approach) were evidenced when using the MIP cleanup. The analysis of CAP in raw milk was further validated according to the 2002/657/EC European Union criteria for the analysis of veterinary drug residues at the minimum required performance limit (MRPL) of 0.3 microg/kg, using CAP-d(5) as internal standard. Non-internal-standard corrected recovery values ranged between 50% and 87% over the range of concentrations considered. The decision limit (CCalpha) and detection capability (CCbeta) were calculated to be 0.06 and 0.10 microg/kg, respectively.

Analysis of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in urine by extraction on a molecularly imprinted polymer column and liquid chromatography/atmospheric pressure ionization tandem mass spectrometry.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is present in the urine of tobacco users and, at lower concentrations, in the urine of nonsmokers exposed to secondhand smoke. NNAL is a valuable biomarker of human exposure to the carcinogenic nitrosamines in tobacco and tobacco smoke, but its presence at low concentrations in urine requires sensitive and often complex analytic procedures. In this report, we describe the development of an efficient method for the analysis of NNAL in human urine using liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/MS/MS) combined with a novel sample cleanup based on a molecularly imprinted polymer (MIP) column developed specifically for this assay. Our results suggest that this combination of MIP column extraction and LC/MS/MS can provide a sensitive and relatively simple analytical method suitable for application to epidemiologic investigations of health risks associated with the exposure to tobacco smoke or SHS in both smokers and nonsmokers.

Chiral recognition and separation of beta2-amino acids using non-covalently molecularly imprinted polymers.

Non-covalently molecularly imprinted polymers (MIPs) for beta2-amino acids were prepared for the first time. N-(2-chlorobenzyloxycarbonyl)-(R)-beta2-homophenylalanine (N-2-ClZ-(R)-beta2-HPhe) was imprinted with methacrylic acid (MAA) and/or 4-vinylpyridine (4-VPy) as the functional monomers, with ethylene glycol dimethacrylate (EDMA) as the cross-linker. The MIPs made with different ratios of MAA:4-VPy were studied in HPLC mode. The results show that MIPs made with 4-VPy yielded the best chiral separation factor (alpha= 1.86) for the template molecule. The importance for an efficient separation of pi-stacking interactions between the MIPs and the template molecule is demonstrated. Racemates of Z-alpha-amino acids and beta-amino acid analogues of the template were either not or poorly resolved by the MIPs, thus demonstrating the close three-dimensional complementarity of the MIPs' recognition sites with the template.