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Molecularly imprinted porous polymer microspheres selective to Alternaria mycotoxins, alternariol (AOH) and alternariol monomethyl ether (AME), were synthesized and applied to the extraction of both mycotoxins in food samples. The polymer was prepared using 4-vinylpiridine (VIPY) and methacrylamide (MAM) as functional monomers, ethylene glycol dimethacrylate (EDMA) as cross-linker and 3,8,9-trihydroxy-6H-dibenzo[b,d]pyran-6-one (S2) as AOH surrogate template. A molecularly imprinted solid phase extraction (MISPE) method has been optimized for the selective isolation of the mycotoxins from aqueous samples coupled to HPLC with fluorescence (λ ex =258nm; λ em =440nm) or MS/MS analysis. The MISPE method was validated by UPLC-MS/MS for the determination of AOH and AME in tomato juice and sesame oil based on the European Commission Decision 2002/657/EC. Method performance was satisfactory with recoveries from 92.5% to 106.2% and limits of quantification within the 1.1-2.8µgkg -1 range in both samples., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This work reports the preparation of molecularly imprinted polymers (MIPs) selective to cephalosporin (CF) antibiotics, and their application as molecularly imprinted solid-phase extraction (MISPE) sorbents for the determination of these antimicrobials in milk samples. Several functional monomers and cross-linkers have been screened to select the best combination that provides high selectivity for the simultaneous multiresidue extraction of cefthiofur (THIO), cefazolin (AZO), cefquinome (QUI), cephapirin (API), cephalexin (ALE) and cephalonium (ALO) from the samples. The novel MIPs were prepared by a non-covalent imprinting approach in the form of spherical microparticles using the synthetic surrogate molecule sodium 7-(2-biphenylylcarboxamido)-3-methyl-3-cepheme-4-carboxylate, N-3,5-bis(trifluoromethyl)phenyl-N'-4-vinylphenyl urea (VPU) as functional monomer, and divinylbenzene (DVB) as crosslinking agent in a 1:2:20 molar ratio. The optimized MISPE method allowed the extraction of the target antimicrobials from raw cow milk samples using a selective washing with 5mL methanol/2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (0.1M, pH 7.5) (2:98, v/v) to remove the non-specifically retained compounds, followed by elution with 1mL of trifluoroacetic acid (TFA) in methanol (0.1:99.9, v/v). The extracts have been analysed by UHPLC-MS/MS and the analytical method has been validated according to EU guideline 2002/657/EC. The limits of quantification (S/N=10) were in the 1.7-12.5μgkg -1 range, well below the maximum residue limits (MRLs) currently established for the quantified cephalosporins in milk samples. The developed MIP allows mutiresidual determination of the six cephalosporin antibiotics mentioned above, significantly broadening the application to food analysis of MISPE methods., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This paper describes the synthesis of novel molecularly imprinted polymer (MIP) micro-beads for the selective extraction (MISPE) of six fluoroquinolone (FQ) antibiotics (enrofloxacin, ciprofloxacin, lomefloxacin, danofloxacin, sarafloxacin and norfloxacin) from chicken muscle samples and further analysis by high-performance liquid chromatography (HPLC) with fluorescence (FLD) or mass spectrometry (MS) detection. A combinatorial screening approach has been applied to select the optimal functional monomer and cross-linker formulation for polymer synthesis. The MIP prepared using enoxacin (ENOX) as the template - a mixture of methacrylic acid (MAA) and trifluoromethacrylic acid (TFMAA) as functional monomers and ethylene glycol dimethacrylate (EDMA) as the cross-linker - showed superior FQ recognition properties than the rest of the materials generated. MIP spherical particles were prepared using silica beads as sacrificial scaffolds. The polymers were packed in solid phase extraction (SPE) cartridges. The optimized MISPE-HPLC method allows the extraction of the antimicrobials from aqueous samples followed by a selective washing with acetonitrile/water (0.005% TFA, pH=3.0), 20:80 (v/v) and elution with 5% trifluoroacetic acid in methanol. Optimum MISPE conditions led to recoveries of the target FQs in chicken muscle samples ranging between 68 and 102% and precisions in the 3-4% range (RSD, n=18). The method has been validated according to European Union Decision 2002/657/EC, in terms of linearity, accuracy, precision, selectivity, decision limit (CCα) and detection capability (CCβ) by HPLC-FLD and HPLC-MS/MS. The limits of detection were improved using HPLC-MS/MS analysis and ranged between 0.2 and 2.7μgkg(-1) (S/N=3) for all the FQs tested., (Copyright © 2014 Elsevier B.V. All rights reserved.)

An automated array biosensor based on evanescent-wave excitation has been developed for the detection of microcystins (MCs) in freshwater samples. The sensing surface consisted of microcystin-leucine-arginine (MCLR) covalently immobilized onto a planar waveguide (microscope slide). The binding of anti-MCLR monoclonal antibodies, spiked in the sample, to the immobilized MCLR was competitively inhibited by MCLR in solution and the amount of antibody bound to the patterned antigens was revealed using Cy5-labeled rabbit anti-mouse IgG. Surface chemistry has been optimized to improve biosensor performance in terms of sensitivity, regeneration ability and to avoid non specific binding for further application to environmental monitoring. The optimized biosensor assay presents an IC(50) value of 0.34 ± 0.01 μg/L, a detection limit of 16 ± 3 ng/L and a dynamic range from 0.06 to 1.5 μg/L MCLR, improving the performance of previously reported devices. Cross-reactivity to other related MCs, such as microcystin-RR (MCRR, 90%), microcystin-RR desmethylated (dm-MCRR, 95%) and microcystin-YR (MCYR, 91%), was also evaluated. The automated microarray can assay up to six different samples in parallel, with a total analysis time of about 60 min. The sensing surface was regenerated with 50mM NaOH and each chip was reused for, at least, 15 assay-regeneration cycles without significant binding capacity loss. The immunosensor has been successfully applied to the direct analysis of MCs in surface water samples and the results were in close agreement with those provided by LC-MS/MS., (Copyright © 2011 Elsevier B.V. All rights reserved.)

According to the current EU legislation, the presence of antimicrobial residues in baby foods is forbidden. Nevertheless, there is a lack of analytical methods to determine veterinary antimicrobials in baby foods and support the zero tolerance policy for this type of foods. This paper describes a simple method based on molecularly imprinted solid phase extraction (MISPE) and liquid chromatography with fluorescence detection (LC-FLD) for the determination of residues of fluoroquinolones (FQs) in baby foods. The method involves sample extraction with a solution of o-phosphoric acid (50mM, pH 3.0)/ACN (20:80, v/v) and further clean-up by loading the extracts onto MIP cartridges. Optimum MISPE conditions led to recoveries of the target FQs in the range of 92-106%, with RSDs <8%. Method validation has been performed according to Commission Decision 2002/657/EC, in terms of linearity, accuracy, precision, selectivity, decision limit (CCα) and detection capability (CCβ). The proposed method has been successfully applied to the analysis of baby foods of different compositions bought in local supermarkets and pharmacies. The results did not show the presence of residual amounts of FQs in the analysed samples above the method's decision limits (CCα between 5 and 151μgkg(-1))., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Micropatterned molecularly imprinted polymer (MIP) transmissive 2D diffraction gratings (DGs) are fabricated and evaluated as label-free antibiotic bio(mimetic)sensors. Polymeric gratings are prepared by using microtransfer molding based on SiO(2)/Si molds. The morphology of the MIP gratings is studied by optical and atomic force microscopes. MIP 2D-DGs exhibit 2D optical diffraction patterns, and measurement of changes in diffraction efficiency is used as sensor response. The refractive index of the micropatterned MIP material was estimated, via solvent index matching experiments, to be 1.486. Immersion of a MIP 2D-DG in different solutions of target-antibiotic enrofloxacin leads to significant variations in diffraction efficiency, demonstrating target-molecule detection. On the other hand, no significant response is observed for both control experiments: MIP grating exposed to a non-retained analyte and an equivalent non-imprinted polymer grating exposed to the target analyte, showing highly specific antibiotic label-free optical recognition., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In the present study, we have developed a method based on pressurized liquid extraction (PLE) and liquid chromatography with fluorescence detection (LC-FLD) for the determination of residues of fluoroquinolones (FQs) in infant food products. PLE extraction has been optimized by the application of experimental design methodologies. Initially, a fractional factorial design (FFD) was used to screen the significance of four extraction parameters: solvent composition, temperature, pressure and number of cycles. The most significant factors, identified by ANOVA analysis, were the solvent composition, temperature and pressure, which were further optimized with the aid of a face centred design (FCD) and the desirability function. The optimized operating PLE conditions were as follows: ACN/o-phosphoric acid 50mM pH 3.0 (80:20, v/v), 80 degrees C, 2000psi and three extraction cycles of 5min. Under these conditions, recoveries of the target FQs varied between 69% and 107% with RSDs below 9%. The whole method was validated according to the Commission Decision 2002/657/EC guidelines. The proposed method has been successfully applied to the analysis of different infant food products bought in local supermarkets and pharmacies. The results showed the presence of residues of enrofloxacin in a non-compliant baby food sample corresponding to a chicken-based formulation, which were also confirmed and quantified by LC-MS/MS analysis., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

This paper describes a new method for the effective extraction, clean-up and chromatographic analysis of residues of four fluoroquinolones (ciprofloxacin, enrofloxacin, danofloxacin and sarafloxacin) in powdered infant formulae and follow-on preparations. Samples were reconstituted following the manufacturer's recommendations and treated with trichloroacetic acid in methanol 10% (w/v) for deproteinization. Two solid-phase extraction cartridges have been evaluated for sample clean-up and preconcentration, Strata Screen A and Strata X and the later provided the best recoveries for all the analytes tested. Chromatographic analysis has been carried out using a polar endcapped column (AQUA C(18)) and fluorescence detection, with lomefloxacin (LOME) as internal standard. Method validation has been performed according to European Commission Decision 2002/657/EC criteria, in terms of linearity, recovery, precision, specificity, decision limit (CC(alpha)) and detection capability (CC(beta)). Typical recoveries ranged between 70 and 110% at levels below and above the maximum residue limits of the target analytes in bovine milk, with an excellent intralab reproducibility (RSDs<7%). Matrix effects did not significantly affect method accuracy, as evidenced by analyzing different brands of milk. The method has been successfully applied to the analysis of 100 samples of infant and follow-on formulae of the Spanish and Latin American market, using LC-MS/MS as confirmatory technique.

A new method for the simultaneous determination of three fluoroquinolones (FQs) enrofloxacin (ENRO) ciprofloxacin (CIPRO) and sarafloxacin (SARA) in table eggs has been developed, applying pressurized liquid extraction (PLE) and liquid chromatography (LC) with fluorescence detection (LC-FLD). The influence of several extraction parameters (e.g. solvent mixture, temperature and extraction time) on FQs extraction efficiency and coextracted matrix interferents was evaluated using fortified control eggs and matrix matched standard curves. The results showed that FQs extraction efficiency depends mainly on solvent composition and the optimum extraction mixture was found to be phosphate 50mM, pH 3.0/acetonitrile (50:50, v/v). The optimized procedure employed 50% flush volume, 5min of static time and three extraction cycles at 70 degrees C and 1500psi. Method validation was performed according to the guidelines of the Directive 96/23/EC, using control egg samples, fortified with the target FQs in the range 50-1000ngg(-1) and applying the optimized extraction conditions on three different days, providing recoveries between 67-90% with RSDs lower than 11% in all cases. The decision limit (CCalpha) and detection capability (CCbeta) of the analytical method were found to be within the range 17-24ngg(-1) and 30-41ngg(-1), respectively. The method was successfully applied to the determination of ENRO and its metabolite CIPRO in incurred egg samples from ENRO-treated hens and LC-MS has been used and for confirmatory purposes.

Molecularly imprinted polymers (MIPs) with selective recognition properties for zearalenone (ZON), an estrogenic mycotoxin, and structurally related compounds have been prepared using the non-covalent imprinting approach. A rationally designed ZON analogue, cyclododecyl 2,4-dihydroxybenzoate (CDHB), that exhibits resemblance to ZON in terms of size, shape and functionality has been synthesized and used as template for MIP preparation instead of the natural toxin. Several functional monomers have been evaluated to maximize the interactions with the template molecule during the polymerization process. The polymer material prepared with 1-allylpiperazine (1-ALPP) as functional monomer, trimethyl trimethacrylate (TRIM) as cross-linker and acetonitrile as porogen (in a 1:4:20 molar ratio) displayed superior binding capacities than any other of the MIPs tested. Selectivity of this material for ZON and structurally related and non-related compounds has been evaluated using it as stationary phase in liquid chromatography. Our results demonstrate that the imprinted polymer shows significant affinity in the porogenic solvent for the template mimic (CDHB) as well as for the ZON and other related target metabolites in food samples, dramatically improving the performance of previously reported MIPs for ZON recognition. Therefore, MIPs can be an excellent alternative for clean-up and preconcentration of the mycotoxin in contaminated food samples.

Column liquid chromatography with fluorescence (FLD) and UV-diode array detection (UV-DAD) was used for the simultaneous determination of ciprofloxacin (CIPRO), enrofloxacin (ENRO), marbofloxacin (MARBO), danofloxacin (DANO) and sarafloxacin (SARA) residues in milk, using norfloxacin (NOR) as internal standard. Two solid-phase extraction (SPE) cartridges, were evaluated for sample clean-up and preconcentration, Strata X, based on a modified styrene-divinylbenzene polymer, and Strata Screen A, a mixed anion exchanger/C8 reversed-phase sorbent. The fluoroquinolones (FQs) were separated on a polar endcapped column (AQUA C18). The recoveries for raw milk spiked with the antibiotics at three concentrations close to the maximum residue limit (MRL), were 80-103% for ENRO, CIPRO and DANO, with relative standard deviations (R.S.D.) lower than 6.6%. SARA recoveries were 70% (R.S.D. = 7%) and values in the order of 95% (R.S.D. = 1.5%) were obtained for MARBO at the MRL level. The quantification limits ranged from 2.4 to l0 ng ml(-1) and are below the MRL established for these drugs by the European Union. The method was successfully applied to the analysis of ENRO and its metabolite CIPRO in an incurred milk sample.

We describe a novel method and instrumental setup for regenerating antibodies immobilized on a fiberoptic probe of an immunosensor using ultrasonic irradiation with broadband imaging transducers. The instrumental setup and irradiation conditions for antibody regeneration using ultrasound are described. The results of the measurements with antibody against breast cancer antigen illustrate the effectiveness and potential of the regenerable immunosensor. A 65% removal of the antigens bound to the Mab immobilized on the fiber surface is attained after ultrasound regeneration.

An optical fiber oxygen sensor, based on the dynamic quenching of the luminescence of tris(1,10-phenanthroline)-ruthenium(II) cation by molecular oxygen, is presented. The complex is adsorbed onto silica gel, incorporated in a silicone matrix possessing a high oxygen permeability, and placed at the tip of the optical fiber. Oxygen has been monitored continuously in the 0-750 Torr range, with the detection limit being as low as 0.7 Torr. The device has been applied to the development of a fast responding and highly sensitive fiber-optic glucose biosensor based on this highly sensitive oxygen transducer. The sensor relates oxygen consumption (as a result of enzymatic oxidation) to glucose concentration. The enzyme is immobilized on the surface of the oxygen optrode; carbon black is used as an optical isolation in order to prevent ambient light and sample fluorescence to interfere. Measurements have been performed in a flow-through cell in air-equilibrated glucose standard solutions of pH 7.0. The effects of enzyme immobilization procedures (including enzyme immobilization on carbon black) as to response times (around 6 min), analytical ranges (0.06-1 mM glucose), reproducibility in sensor construction, and long-term stability have been studied as well.