Lars I. Andersson

Molecular imprinting : developments and applications in the analytical chemistry field

In analytical separation science, molecularly imprinted polymers have been applied in several analytical techniques, such as liquid chromatography, capillary electrochromatography and capillary electrophoresis, solid phase extraction, immunoassay, and as a selective sorbent in chemical sensors. A benefit of imprinted polymers is the possibility to prepare sorbents with selectivity pre-determined for a particular substance, or group of structural analogues. The application most close to a wider acceptance is probably that of solid phase extraction for clean-up of environmental and biological samples. The improved selectivity of imprinted polymers compared with conventional sorbents may lead to cleaner chromatographic traces in the subsequent analytical separation. Furthermore, the solid phase extraction application does not suffer from drawbacks generally associated with imprinted polymers in chromatography, such as peak broadening and tailing. Most liquid chromatographic studies have focused on using imprinted polymers as chiral stationary phases for enantiomer separations. Also, the use of imprinted polymers as selective sorbents in capillary electrochromatography has been presented. For this purpose, a protocol to prepare superporous, monolithic imprinted polymer-based capillary columns has been developed. Due to the high affinities and selectivities often achievable, imprinted polymers have been considered as alternative binding entities in biosensors and in immunoassay type protocols. Here, high stability, easy preparation and ability to be used for assay of both aqueous and organic solvent based samples are advantages of the polymers.

Enantiomeric resolution on molecularly imprinted polymers prepared with only non-covalent and non-ionic interactions.

Molecular imprints were prepared utilizing only weak bonds between the print molecule and functional monomers; the bonding forces used in the imprinting process were only those weaker than covalent and ionic bonds. Methacrylate-based molecular imprints were prepared using a number of chiral compounds, including N-protected amino acid derivatives, as print molecules. Methacrylic acid was used as the functional monomer because the acid function of the monomer forms hydrogen bonds with a variety of polar functionalities, such as carboxylic acids, carbamates, heteroatoms and carboxylic esters, of the print molecule. Bulk polymers were prepared, ground and sieved to particles of size less than 25 microns, packed into high-performance liquid chromatographic (HPLC) columns and used for enantiomeric separations in the HPLC mode. The polymers were shown to effect efficient enantiomeric resolution of a racemate of the print molecule in addition to substrate selectivity for the print molecule in a mixture of substrates with very similar structures. For example, the enantiomers of Cbz-aspartic acid and Cbz-glutamic acid (Cbz = carbobenzoxy) were resolved with separation factors of 1.9 and 2.5, respectively, on polymers with molecular imprints of the L-form of the respective compounds. In addition, these polymers, prepared against Cbz-L-aspartic acid and Cbz-L-glutamic acid, respectively, had the ability to bind selectively the print molecule from a mixture of both racemates, although the two compounds differ only by one methylene group. The results presented represent a substantial widening of the scope of molecular imprinting in that it may now be possible to prepare molecular imprints against a very large number of compounds.

Some studies of molecularly-imprinted polymer membranes in combination with field-effect devices

Investigations of the usefulness of molecularly-imprinted polymers in sensor applications were undertaken. Thin polymer membranes containing molecular imprints against l-phenylalanine anilide were prepared and applied as sensing layer in field-effect capacitors. In this report some experimental results obtained for C-V measurements when the polymer membranes were exposed to l-phenylalanine anilide (FAA), tyrosinanilide (TA), and phenylalaninol (FA) in ethanol are presented. Despite some difficulties, it was shown that in ethanol solution the compounds FAA and TA can be measured and distinguished from FA using a imprinted polymer membrane.

Multiple sclerosis: Identification and clinical evaluation of novel CSF biomarkers.

Multiple sclerosis (MS) is a neuro-inflammatory and neurodegenerative disease that results in damage to myelin sheaths and axons in the central nervous system and which preferentially affects young adults. We performed a proteomics-based biomarker discovery study in which cerebrospinal fluid (CSF) from MS and control individuals was analyzed (n=112). Ten candidate biomarkers were selected for evaluation by quantitative immunoassay using an independent cohort of MS and control subjects (n=209). In relapsing-remitting MS (RRMS) patients there were significant increases in the CSF levels of alpha-1 antichymotrypsin (A1AC), alpha-1 macroglobulin (A2MG) and fibulin 1 as compared to control subjects. In secondary progressive MS (SPMS) four additional proteins (contactin 1, fetuin A, vitamin D binding protein and angiotensinogen (ANGT)) were increased as compared to control subjects. In particular, ANGT was increased 3-fold in SPMS, indicating a potential as biomarker of disease progression in MS. In PPMS, A1AC and A2MG exhibit significantly higher CSF levels than controls, with a trend of increase for ANGT. Classification models based on the biomarker panel could identify 70% of the RRMS and 80% of the SPMS patients correctly. Further evaluation was conducted in a pilot study of CSF from RRMS patients (n=36), before and after treatment with natalizumab.

Probing the molecular basis for ligand-selective recognition in molecularly imprinted polymers selective for the local anaesthetic bupivacaine

Probing the molecular basis for ligand-selective recognition in molecularly imprinted polymers selective for the local anaesthetic bupivacaine

Recognition and enantioselection of drugs and biochemicals using molecularly imprinted polymer technology

Recognition and enantioselection of drugs and biochemicals using molecularly imprinted polymer technology

Efficient sample pre-concentration of bupivacaine from human plasma by solid-phase extraction on molecularly imprinted polymers

The ability to use imprinted polymers for solid-phase extraction is demonstrated in a model pre-concentration of bupivacaine from human plasma samples prior to gas chromatography. Imprinting of the structural analogue pentycaine yielded a sorbent which efficiently extracted analyte and internal standard, while possible interference on analyte quantification from leakage of remaining template molecules was eliminated. Human plasma samples were diluted with citrate buffer pH 5, and applied onto solid phase extraction columns containing 15 mg of imprinted sorbent. Wash steps with 20% methanol in water followed by acetonitrile preceded elution with 2% triethylamine in acetonitrile. A direct comparison with conventional sample pre-treatment methods showed the high selectivity of the imprinted sorbent resulted in distinctly cleaner chromatographic traces than were obtained both after liquid-liquid extraction and C18-based solid-phase extraction.

Recent advances in the preparation and use of molecularly imprinted polymers for enantiomeric resolution of amino acid derivatives

Abstract Acrylate-based molecular imprints were prepared, using l -phenylalanine anilide as the print molecule and methacrylic acid as the functional monomer, which is believed to interact both ionically and through hydrogen bonding with the print molecule. Several aspects of the polymer preparation were investigated, including the solvent of polymerization, the initiation system and the effect of the molar ratio of functional monomer to print molecule on the ability of the polymers to separate the enantiomers of the print molecule. Polymers were analysed by high-performance liquid chromatography where the effect of particle size and eluent composition were investigated for enantiomeric resolution of the print molecule. The ability of the polymers to separate racemic mixtures of amide derivatives of amino acids other than phenylalanine anilide (print molecule) was also investigated. It was thus shown that efficient enantiomeric resolution of amide derivatives of amino acids including the anilides, p-nitroanilides, β-naphthylamides and amides of amino acids, ranging from alanine to trytophan, was possible on a single polymer imprinted with l -phenylalanine anilide. Such separations were highly specific and dependent on the presence of both the print molecule and functional monomer in the polymerization mixture. The mechanism of recognition was shown to involve ionic bonding to the primary amine and hydrogen bonding to the amide function of the substate by acid groups of the polymer, “immobilized” in a stereospecific manner. Both interactions were necessary for efficient enantiometric resolution. Molecular imprints, prepared from l -phenylalanine anilide, were also shown to give efficient enantiomeric resolution of phenylalanine-containing dipeptides and may be useful for preparative-scale separations.

Enantiomeric resolution of amino acid derivatives on molecularly imprinted polymers as monitored by potentiometric measurements

Potentiometric measurements have been applied to the detection of enantiomeric separations on molecularly imprinted polymers. A flow-through column electrode, based on the use of polymers imprinted against L-phenylalanine anilide, is described. The electrode consisted of a glass column in which the polymer was packed and where the end frits constituted the electrodes. The flow stream potential across the column can be continuously recorded as solvent is pumped through the system. The column resolved the enantiomers of phenylalanine anilide as detected by both UV absorption and potentiometric measurements and the recorded signals could be correlated with the concentration of phenylalanine anilide. The calibration graphs obtained for the UV absorption of phenylalanine anilide were linear over the concentration range investigated, whereas the potentiometric signal was shown to be exponentially linear with concentration. The application of molecular imprints to the preparation of supports suitable for chromatographic separations of enantiomers and for the preparation of specific electrodes is discussed.

Microextraction in Packed Syringe Online with Liquid Chromatography‐Tandem Mass Spectrometry: Molecularly Imprinted Polymer as Packing Material for MEPS in Selective Extraction of Ropivacaine from Plasma

Abstract The excellent performance of a new sample preparation method, microextraction in packed syringe (MEPS), was recently illustrated by online LC‐MS and GS‐MS assays of local anaesthetics in plasma samples. In the method, approximately 1 mg of solid packing material was inserted into a syringe (100–250 µL) as a plug. Sample preparation took place on the packed bed. The new method was easy to use, fully automated, of low cost, and rapid in comparison with previously used methods. This paper presents the use of molecularly imprinted polymers (MIPs) as packing material for higher extraction selectivity. Development and validation of a method for MIP‐MEPS online with LC‐MS‐MS using ropivacaine in plasma as model compound were investigated. A bupivacaine imprinted polymer was used. The method was validated and the standard curves were evaluated by means of quadratic regression and weighted by inverse of the concentration: 1/x for the calibration range 2–2000 nM. The applied polymer could be used more than 100 times before the syringe was discarded. The extraction recovery was 60%. The results showed high correlation coefficients (R2>0.999) for all runs. The accuracy, given as a percentage deviation from the nominal concentration values, ranged from −6% to 3%. The precision, given as the relative standard deviation, at three different concentrations (QC samples) was consistently about 3% to 10%. The limit of quantification was 2 nM.