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Edible insects are widely consumed in Africa, Asia, Oceania and Latin America, but less commonly so in Western countries. Since the turn of the millennium, however, entomophagy has aroused growing interest worldwide in response to the increasing scarcity of food resources. In fact, edible insects can be a source of high-quality protein, and also of fat, energy, minerals and vitamins. However, the lack of regulatory guidelines for microbiologically or chemically hazardous agents potentially present in these new foods (e.g., mycotoxins) may make their consumption unsafe. In this work, we developed an environmentally friendly analytical method using natural deep eutectic solvents (NADES or natural DES) in combination with ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of six mycotoxins of great concern owing to their toxic effects on humans and animals (namely, fumonisin B1, fumonisin B2, T-2 toxin, HT-2 toxin, ochratoxin A and mycophenolic acid) in insect-based food products. The target mycotoxins were co-extracted from cricket flour by using the optimum DES composition (namely, a mixture of choline chloride and urea, in a 1:2 mole ratio, containing 15% water which resulted in the highest extraction recoveries for all toxins). An experimental design method (Fractional Factorial Design (FFD) was used to examine the influence of the operational variables DES volume and water content, amount of sample, extraction time and extraction temperature on the extraction efficiency for each mycotoxin. Under optimum conditions, extraction recoveries were close to 100% except for fumonisin B2 (70%) and T-2 toxin (50%), with relative standard deviations (RSDs) below 13% in all cases. The proposed NADES-UHPLC–MS/MS method was validated in accordance with the European Commission 2002/657/EC and 2006/401/EC decisions, and used to determine the target compounds in cricket flour, silkworm pupae powder and black cricket powder., This work was funded by the Spanish Ministry of Economy and Competitiveness (MINECO, Projects RTI2018-096410-B-C21 and AGL2017-89417-R). F.P. acknowledges award of a pre-doctoral FPU grant by the Ministry of Education and Vocational Training, and G.A.-R. a “Juan de la Cierva” postdoctoral grant by MINECO.

Due to increasing food safety standards, the analysis of mycotoxins has become essential in the food industry. In this work, we have developed a competitive upconversion-linked immunosorbent assay (ULISA) for the analysis of zearalenone (ZEA), one of the most frequently encountered mycotoxins in food worldwide. Instead of a toxin-conjugate conventionally used in competitive immunoassays, we designed a ZEA mimicking peptide extended by a biotin-linker and confirmed its excellent suitability to mimic ZEA by nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analysis. Upconversion nanoparticles (UCNP, type NaYF4:Yb,Tm) served as background-free optical label for the detection of the peptide mimetic in the competitive ULISA. Streptavidin-conjugated UCNPs were prepared by click reaction using an alkyne-PEG-neridronate linker. The UCNP conjugate clearly outperformed conventional labels such as enzymes or fluorescent dyes. With a limit of detection of 20 pg mL−1 (63 pM), the competitive ULISA is well applicable to the detection of ZEA at the levels set by the European legislation. Moreover, the ULISA is specific for ZEA and its metabolites (α- and β-zearalenol) without significant cross-reactivity with other related mycotoxins. We detected ZEA in spiked and naturally contaminated maize samples using liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) as a reference method to demonstrate food analysis in real samples.

The development of a bioluminescent immunosensor is reported for the determination of zearalenone (ZEA) based on a peptide mimetic identified by phage display. The peptide mimetic GW, with a peptide sequence GWWGPYGEIELL, was used to create recombinant fusion proteins with the bioluminescent Gaussia luciferase (GLuc) that were directly used as tracers for toxin detection in a competitive immunoassay without the need for secondary antibodies or further labeling. The bioluminescent sensor, based on protein G–coupled magnetic beads for antibody immobilization, enabled determination of ZEA with a detection limit of 4.2 ng mL−1 (corresponding to 420 μg kg−1 in food samples) and an IC50 value of 11.0 ng mL−1. The sensor performance was evaluated in spiked maize and wheat samples, with recoveries ranging from 87 to 106% (RSD

Homogeneous immunoassays represent an attractive alternative to traditional heterogeneous assays due to their simplicity, sensitivity, and speed. On the basis of a previously identified epitope-mimicking peptide, or mimotope, we developed a homogeneous fluorescence quenching immunoassay based on gold nanoparticles (AuNPs) and a recombinant epitope-mimicking fusion protein for the detection of mycotoxin fumonisin B1 (FB1). The fumonisin mimotope was cloned as a fusion protein with a yellow fluorescent protein that could be used directly as the tracer for FB1 detection without the need of labeling or a secondary antibody. Furthermore, owing to the fluorescence quenching ability of AuNPs, a homogeneous immunoassay could be performed in a single step without washing steps to separate the unbound tracer. The homogeneous quenching assay showed negligible matrix effects in 5% wheat extract and high sensitivity for FB1 detection, with a dynamic range from 7.3 to 22.6 ng mL-1, a detection limit of 1.1 ng mL-1, and IC50 value of 12.9 ng mL-1, which was significantly lower than the IC50 value of the previously reported assay using the synthetic counterpart of the same mimotope in a microarray format. The homogeneous assay was demonstrated to be specific for fumonisins B1 and B2, as no significant cross-reactivity with other mycotoxins was observed, and acceptable recoveries (86% for FB1 2000 μg kg-1 and 103% for FB1 4000 μg kg-1), with relative standard deviation less than 6.5%, were reported from spiked wheat samples, proving that the method could provide a valuable tool for simple analysis of mycotoxin-contaminated food samples.

This paper reports the development of a novel strategy for the easy immobilization of low molecular weight haptens to microarray platforms by coating with modified lipase molecules. The chimers consist of a dextran network covalently coupled to bacterial thermoalkalophilic lipase (BTL2). The relative high surface hydrophobicity of lipase allows easy immobilization of the conjugates onto glass planar waveguides previously modified with 1,1,1,3,3,3-hexamethyldisilazane, via nonspecific hydrophobic interactions, while the dextran layer provides a three-dimensional network that can be easily modified with different functional groups for further hapten immobilization on the glass substrate. The conjugates have been applied to the development of microarray biosensors for the simultaneous detection of three water pollutants namely, atrazine (ATR), a triazine pesticide, enrofloxacin (ENRO), a fluoroquinolone antimicrobial, and the hepatotoxin microcystin LR (MCLR). In an alternative approach, the chimeric hapten-dextran-BTL2 constructs were synthesized, purified and further immobilized on the hydrophobic transducer surface through their hydrophobic faces (Janus character). Detection of the three model targets was based in a competitive immunoassay format. A combination of detection and tracer antibodies was optimized to avoid significant cross-reactivity. Evanescent wave excitation was used to excite the fluorescent immunocomplexes formed on the planar waveguide and the identity of the target analyte was encoded by its location in the detection platform. The developed microarray allows the simultaneous detection of the target pollutants with IC 50 values, of 4.4 ± 0.7, 75 ± 9, 18 ± 4 ng L −1 for ATR, ENRO and MCLR, respectively and relative standard deviations (RSDs) lower than 15%. Waveguide functionalization using the dextran-BTL2 chimers is rapid, simple and allows the development of highly sensitive microarrays that can be easily applied to the multiplexed detection of pollutants in environmental samples.

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-6 H -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 = 258 nm; λ em = 440 nm) 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 1.1–2.8 µg kg −1 range in both samples.

Mycophenolic acid (MPA) is an immunosuppressant drug commonly used to prevent organ rejection in transplanted patients. MPA monitoring is of great interest due to its small therapeutic window. In this work, a phage-displayed peptide library was used to select cyclic peptides that bind to the MPA-specific recombinant antibody fragment (Fab) and mimic the behavior of MPA. After biopanning, several phage-displayed peptides were isolated and tested to confirm their epitope-mimicking nature in phage-based competitive immunoassays. After identifying the best MPA mimetic (ACEGLYAHWC with a disulfide constrained loop), several immunoassay approaches were tested, and a recombinant fusion protein containing the peptide sequence with a bioluminescent enzyme, NanoLuc, was developed. The recombinant fusion enabled its direct use as the tracer in competitive immunoassays without the need for secondary antibodies or further labeling. A bioluminescent sensor, using streptavidin-coupled magnetic beads for the immobilization of the biotinylated Fab antibody, enabled the detection of MPA with a detection limit of 0.26 ng mL-1 and an IC50 of 2.9 ± 0.5 ng mL-1. The biosensor showed good selectivity toward MPA and was applied to the analysis of the immunosuppressive drug in clinical samples, of both healthy and MPA-treated patients, followed by validation by liquid chromatography coupled to diode array detection.

In this work, we report the synthesis of novel magnetic molecularly imprinted polymers (m-MIPs) and their application to the selective extraction of the mycotoxin citrinin (CIT) from food samples. The polymers were prepared by surface imprinting of Fe3O4 nanoparticles, using 2-naphtholic acid (2-NA) as template molecule, N-3,5-bis(trifluoromethyl)phenyl-N'-4-vinylphenyl urea and methacrylamide as functional monomers and ethyleneglycol dimethacrylate as cross-linker. The resulting material was characterized by transmission electron microscopy (TEM), and X-ray diffraction (XRD) and Fourier transform infrared spectroscopies (FT-IR). The polymers were used to develop a solid-phase extraction method (m-MISPE) for the selective recovery of CIT from rice extracts prior to its determination by HPLC with UV diode array detection. The method involves ultrasound-assisted extraction of the mycotoxin from rice samples with (7:3, v/v) methanol/water, followed by sample cleanup and preconcentration with m-MIP. The extraction (washing and elution) conditions were optimized and their optimal values found to provide CIT recoveries of 94-98 % with relative standard deviations (RSD) less than 3.4 % (n = 3) for preconcentrated sample extracts (5 mL) fortified with the analyte at concentrations over the range 25-100 μg kg(-1). Based on the results, the application of the m-MIPs facilitates the accurate and efficient determination of CIT in rice extracts.

In this Article, we describe a fluorescence polarization immunoassay (FPIA) using a new label-near-infrared fluorescent dye. The developed FPIA method was optimized for the rapid analysis of free mycophenolic acid (MPA) in plasma of transplanted patients. The approach is based on the fluorescence competitive assay between the target immunosuppressant and a novel emissive near-infrared fluorescent dye-tagged MPA and MPA-AO for the binding sites of the anti-MPA antibody. The fluorescent analogue of MPA exhibits emission at 654 nm upon excitation at 629 nm (λ

This work describes the development and application of class-selective molecularly imprinted polymers (MIPs) for the analysis of beta-lactamase-resistant penicillins, namely cloxacillin (CLOXA), oxacillin (OXA), and dicloxacillin (DICLOXA), in milk samples. Our method is based on molecularly imprinted solid-phase extraction (MISPE) coupled to high-performance liquid chromatography (HPLC) with diode-array detection (DAD). 2-Biphenylylpenicillin (2BPEN), a surrogate with a close resemblance to beta-lactamase-resistant penicillins in terms of size, shape, hydrophobicity, and functionality, was synthesized and used as the template for the polymer synthesis. A MIP library was prepared and screened to select the optimum functional monomer, N-(2-aminoethyl)methacrylamide, and cross-linker, trimethylolpropane trimethacrylate, that provided the best recognition for the target antibiotics. For the MISPE application, the MIPs were prepared in the form of microspheres, using porous silica beads (40-75 μm) as sacrificial scaffolds. The developed MISPE method enables efficient extraction from aqueous samples and analysis of the antimicrobials, when followed by a selective washing with 2 mL acetonitrile-water (20:80 v/v) and elution with 1 mL 0.05 mol L(-1) tetrabutylammonium in methanol. The analytical method was validated according to EU guideline 2002/657/EC. The limits of quantification (S/N = 10) were in the 5.3-6.3 μg kg(-1) range, well below the maximum residue limits (MRLs) currently established. Inter-day mean recoveries were in the range 99-102 % with RSDs below 9 %, improving on the performance of previously reported MISPE methods for the analysis of CLOXA, OXA, or DICLOXA in milk samples.

A sensitive and reliable method using pressurised liquid extraction (PLE) followed by molecularly imprinted solid phase extraction (MISPE) and high performance liquid chromatography with fluorescence detection (HPLC–FLD) has been developed for the analysis of alternariol (AOH) and alternariol monomethyl ether (AME) in tomato samples. Influence of several extraction parameters that affect PLE efficiency were evaluated for the simultaneous extraction of both mycotoxins in the selected samples. AOH and AME were optimally extracted using MeOH/water (25:75, v/v) at 70°C as solvent, a pressure of 1000 psi and a single extraction cycle. The resulting PLE extracts were pre-concentrated by molecularly imprinted solid phase extraction (MISPE) cartridges followed of analysis by HPLC with fluorescence detection (λexc = 258, λem = 440 nm). The proposed method was applied to the analysis of AOH and AME in fortified tomato samples (20–72 µg· kg–1) with recoveries of 84–97% (RSD n = 6) for AOH and 67–91% (RSD n = 6) for AME. The detection limit for AOH and AME were 7 and 15 µg· kg–1, respectively. The ensuing PLE–MISPE–HPLC–FLD method was validated for the analysis of both mycotoxins in tomato samples in accordance with European Commission Decision 2002/657/EC.

In this article we describe a new class of high-density optical microarrays based on molecularly imprinted microsphere sensors that directly incorporate specific recognition capabilities to detect enrofloxacin (ENRO), an antibiotic widely used for both human and veterinary applications. This approach involves the preparation of highly cross-linked polymer microspheres by thermal precipitation–polymerization in the presence and absence of the target analyte ENRO to generate either molecularly imprinted (MIP) or non-imprinted polymer (NIP) microspheres, respectively. Each polymer type of tailor-made microsphere is fluorescently encoded with either coumarin-30 or tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride [Ru(dip)3]Cl2 to enable the microspheres to be distinguished. The new MIP-based sensing platform utilizes an optical fiber bundle containing approximately 50000 individual 3.1 μm diameter fibers that are chemically etched to create microwells in which MIP and NIP microspheres can be deposited and imaged using an epi-fluorescence microscope. The method enables multiplexed detection by independently addressing both types of beads through their separate light channels. The unique response to the presence of ENRO is manifested on the basis of a competitive immunoassay. A red-fluorescent dye-tagged ENRO, labeled with BODIPY® TR Cadaverine, competes with ENRO for specific binding sites. The developed immuno-like assay displayed a limit of detection (LOD) of 0.04 μM (10% binding inhibition) and a dynamic range of 0.29–21.54 μM (20–80% binding inhibition). The selectivity of the assay was evaluated by measuring the cross-reactivity of other fluoroquinolones (ciprofloxacin, norfloxacin, danofloxacin, and flumequine) and non-related antibiotics (penicillin G and doxycycline). This work demonstrates, for the first time, the applicability of MIPs, as an alternative to biomolecule receptors, for the development of multiplexed detection fiber-optic microarrays paving the way for a new generation of biomimetic sensors.

This paper describes the synthesis of novel molecularly imprinted polymers (MIPs), prepared by a noncovalent imprinting approach, for cleanup and preconcentration of curcumin (CUR) and bisdemethoxycurcumin (BDMC) from medicinal herbal extracts and further analysis by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Two molecular mimics, a mixture of reduced BDMCs and 4-(4-hydroxyphenyl)-2-butanone (HPB), have been synthesized and applied as templates for MIP synthesis. The polymers were prepared using N-(2-aminoethyl) methacrylamide (EAMA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as the cross-linker (in a 1:5 molar ratio), and a mixture of acetonitrile/dimethylsulfoxide (90 %, v/v) as porogen. MIPs prepared using a mixture of reduced BDMCs as template showed higher selectivity for CUR and BDMC than those obtained with HPB, with imprinting factors of 3.5 and 2.7 for CUR and BDMC, respectively, using H2O/acetonitrile (65:35, v/v) as mobile phase. The adsorption isotherms for CUR in the MIP and the nonimprinted polymer (NIP) were fitted to the Freundlich isotherm model, and the calculated average binding affinities for CUR were (17 ± 2) and (8 ± 1) mM−1 for the MIP and the NIP, respectively. The polymers were packed into solid-phase extraction (SPE) cartridges, and the optimized molecularly imprinted solid-phase extraction (MISPE-HPLC) with fluorescence detection (FLD) method allowed the extraction of both curcuminoids from aqueous samples (50 mM NH4Ac, pH 8.8) followed by a selective washing with acetonitrile/NH4Ac, 50 mM at pH 8.8 (30:70 %, v/v), and elution with 3 × 1 mL of MeOH. Good recoveries and precision ranging between 87 and 92 %, with relative standard deviation (RSD) of

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.

This paper reports a sensitive, highly selective flow injection (FI) method for the chemiluminescence-based determination of enrofloxacin (ENRO), a fluoroquinolone (FQ) antibiotic, in natural water samples. The detection principle is the on-line monitored photoreaction of tris(1,10-phenantroline)ruthenium(II) [Ru(phen)32 +] with peroxydisulphate ion to give Ru(phen)33 +, which is then reduced by the antimicrobial producing the chemiluminescence (CL) signal. The low selectivity of the CL reaction required using solid-phase extraction with molecularly imprinted polymers (MISPE) in combination with the FI-CL system. Fluoroquinolone-selective MIP beads were obtained by acid etching of silica/MIP composites and packed into SPE cartridges to clean-up and preconcentrate samples prior injection into the FI-CL system. Various parameters influencing the CL signal intensity including pH, flow rate, and Ru(phen)32 + and peroxydisulphate concentrations, were optimized. The variables affecting the extraction efficiency of the polymers were also optimized in order to minimize non-specific interactions and enable the selective uptake of the antibiotics from real samples. CL measurements made over the pH range 3.8–4.2 allowed the selective determination of enrofloxacin, and measurements at pH 4.8–5.8 the quantitation of total FQs (enrofloxacin, ciprofloxacin, norfloxacin, sarafloxacin, lomefloxacin and danofloxacin). The limit of detection for enrofloxacin was 0.27 μg L− 1 when 10 mL water samples were preconcentrates. The proposed method was used to determine trace amounts of FQs in mineral and tap water, and also in water samples from the river Quibu (Cuba), all with good recoveries: 84–119% (RSD 2.1–9.3%, n = 3) for samples fortified with enrofloxacin at concentrations from 4 to 40 μg L− 1. The method was validated by HPLC with fluorescence detection.

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.

We have developed disposable color-changing polymeric films for quantification of furfural-a freshness indicator-in beer using a smartphone-based reader. The films are prepared by radical polymerization of 4-vinylaniline, as a furfural-sensitive indicator monomer, 2-hydroxymethyl methacrylate as a comonomer, and ethylene dimethyl methacrylate (EDMA) as a cross-linker. The sensing mechanism is based on the Stenhouse reaction in which aniline and furfural react in acidic media with the generation of a deep red cyanine derivative, absorbing at 537 nm, which is visible to the naked eye. The colorimetric response has been monitored using either a portable fiber-optic spectrophotometer or the built-in camera of a smartphone. Under the optimized conditions, a linear response to furfural in beer was obtained in the 39 to 500 μg L(-1) range, with a detection limit of 12 μg L(-1), thus improving the performance of other well-established colorimetric or chromatographic methods. The novel films are highly selective to furfural, and no cross-reactivity has been observed from other volatile compounds generated during beer aging. A smartphone application (app), developed for Android platforms, measures the RGB color coordinates of the sensing membranes after exposure to the analyte. Following data processing, the signals are converted into concentration values by preloaded calibration curves. The method has been applied to determination of furfural in pale lager beers with different storage times at room temperature. A linear correlation (r0.995) between the storage time and the furfural concentration in the samples has been confirmed; our results have been validated by HPLC with diode-array detection.

A surface plasmon resonance (SPR) biosensor for the detection of microcystins (MCs) in drinking water has been developed. Several assay formats have been evaluated. The selected format is based on a competitive inhibition assay, in which microcystin-LR (MCLR) has been covalently immobilized onto the surface of an SPR chip functionalized with a self-assembled monolayer. The influence of several factors affecting sensor performance, such as the nature and concentration of the antibody, the composition of the carrier buffer, and the blocking and regeneration solutions, has been evaluated. The optimized SPR biosensor provides an IC(50) 0.67 ± 0.09 µg L(-1), a detection limit of 73 ± 8 ng L(-1), and a dynamic range from 0.2 to 2.0 µg L(-1) for MCLR. Cross-reactivity to other related MCs, such as microcystin-RR (88%) and microcystin-YR (94%), has also been measured. The SPR biosensor can perform four simultaneous determinations in 60 min, and each SPR chip can be reused for at least 40 assay-regeneration cycles without significant binding capacity loss. The biosensor has been successfully applied to the direct analysis of MCLR in drinking water samples, below the provisional guideline value of 1 µg L(-1) established by the World Health Organization for drinking water.

Antibiotics are a class of pharmaceuticals that are of great interest due to the large volumes of these substances that are consumed in both human and veterinary medicine, and due to their status as the agents responsible for bacterial resistance. They can be present in foodstuffs and in environmental samples as multicomponent chemical mixtures that exhibit a wide range of mechanisms of action. Moreover, they can be transformed into different metabolites by the action of microorganisms, as well as by other physical or chemical means, resulting in mixtures with higher ecotoxicities and risks to human health than those of the individual compounds. Therefore, there is growing interest in the availability of multiclass methods for the analysis of antimicrobial mixtures in environmental and food samples at very low concentrations. Liquid chromatography (LC) has become the technique of choice for multiclass analysis, especially when coupled to mass spectrometry (LC-MS) and tandem MS (LC-MS(2)). However, due to the complexity of the matrix, in most cases an extraction step for sample clean-up and preconcentration is required before analysis in order to achieve the required sensitivities. This paper reviews the most recent developments and applications of multiclass antimicrobial determination in environmental and food matrices, emphasizing the practical aspects of sample preparation for the simultaneous extraction of antimicrobials from the selected samples. Future trends in the application of LC-MS-based techniques to multiclass antibiotic analysis are also presented.

A novel and simple method for the selective cleanup and preconcentration of fluoroquinolone antibiotics in environmental water samples has been developed using molecularly imprinted polymer solid-phase extraction (MISPE). The molecularly imprinted polymer (MIP) has been prepared using enrofloxacin (ENR) as the template and a stoichiometric quantity of urea-based functional monomer to target the single oxyanionic moieties in the template molecule. The selectivity of the material for enrofloxacin, and structurally related and non-related compounds, has been evaluated using it as stationary phase in liquid chromatography. The novel polymer and the corresponding non-imprinted material (NIP) have been characterised using nitrogen adsorption-desorption isotherms and scanning electron microscopy. Various parameters affecting the extraction efficiency of the materials in the MISPE procedure were evaluated in order to achieve optimal preconcentration and to reduce non-specific interactions. The optimized MISPE/HPLC with fluorescence detection (FLD) method allows direct extraction of the antibiotics from the aqueous samples followed by a selective washing with acetonitrile/water (0.1M 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer, pH 7.5) (10/90, v/v) and elution with 2% trifluoracetic acid in methanol. Good recoveries and precision, ranging between 66 and 100% (RSD: 2-12%, n=3) for danofloxacin, enrofloxacin, oxolinic acid and flumequine, and moderate recoveries (15-40%, RSD 4-9%, n=3) for norfloxacin, ciprofloxacin, lomefloxacin and sarafloxacin, have been obtained for river water samples fortified with 0.50, 0.75 and 1.0microgL(-1) of all the antibiotics. The method detection limits ranged between 0.01 and 0.30microgL(-1) for all the antibiotics tested, when 100mL water samples were processed. The results demonstrate the applicability of the optimized method for the selective extraction of fluoroquinolones in environmental water samples at the ngL(-1) level.

A molecularly imprinted polymer (MIP)-based optode has been developed for zearalenone (ZON) mycotoxin analysis. The automated flow-through assay is based on the displacement of tailor-made highly fluorescent tracers by the analyte from a MIP prepared by UV irradiation of a mixture of cyclododecyl 2,4-dihydroxybenzoate (template, ZON mimic), 1-allyl piperazine (functional monomer), trimethylolpropane trimethacrylate (crosslinker) and 2,2′-azobisisobutyronitrile in acetonitrile (porogen). Three fluorescent analogues of ZON, namely 2,4-dihydroxybenzoic acid 2-[methyl(7-nitro-benzo[1,2,5]oxadiazol-4-yl)amino]ethyl ester (NBDRA), 2,4-dihydroxy- N -pyren-1-ylmethylbenzamide (PMRA) and of 2,4-dihydroxybenzoic acid 2-[(pyrene-l-carbonyl)amino] ethyl ester (PARA), have been molecularly engineered for the assay development. The pyrene-containing tracers also inform on the characteristics of the microenvironment of the MIP binding sites. PARA has been finally selected to optimize the ZON displacement fluorosensor, that shows a limit of detection of 2.5 × 10 −5 M in acetonitrile. A positive cross-reactivity has been found for β-zearalenol, but not for resorcinol, resorcylic acid, 17β-estradiol, estrone or bisphenol-A.

Molecularly imprinted porous polymer microspheres have been prepared for selective binding of alternariol (AOH), a phenolic mycotoxin produced by Alternaria fungi. In order to lead the synthesis of recognition materials, four original AOH surrogates have been designed, prepared and characterized. They bear different number of phenol groups in various positions and different degree of O-methylation on the dibenzo[b,d]pyran-6-one skeleton. A comprehensive library of mixtures of basic, acidic or neutral monomers, with divinylbenzene or ethyleneglycol dimethacrylate as cross-linkers, were polymerized at a small scale in the presence of the four molecular mimics of the toxin molecule. This polymer screening has allowed selection of the optimal composition of the microbeads (N-(2-aminoethyl)methacrylamide, EAMA, and ethylene glycol dimethacrylate). The latter are able to bind AOH in water-acetonitrile (80:20, v/v) with an affinity constant of 109±10mM(-1) and a total number of binding sites of 35±2μmolg(-1), being alternariol monomethylether the only competitor species. Moreover, (1)H NMR titrations have unveiled a 1:2 surrogate-to-EAMA stoichiometry, the exact interaction sites and a binding constant of 1.5×10(4)M(-2). A molecularly imprinted solid phase extraction (MISPE) method has been optimized for selective isolation of the mycotoxin from aqueous samples upon a discriminating wash with 3mL of acetonitrile/water (20:80, v/v) followed by determination by HPLC with fluorescence detection. The method has been applied, in combination to ultrasound-assisted extraction, to the analysis of AOH in tomato samples fortified with the mycotoxin at five concentration levels (33-110μgkg(-1)), with recoveries in the range of 81-103% (RSD n=6). To the best of our knowledge, this is the first imprinted material capable of molecularly recognizing this widespread food contaminant.

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.

A novel and simple method has been developed for the simultaneous determination of beta-lactam antibiotics (BLAs) (penicillin G, amoxicillin, ampicillin, penicillin V, oxacillin, cloxacillin, dicloxacillin and nafcillin) in wastewater. The method is based on solid-phase extraction (SPE) and high performance liquid chromatography with UV-diode array detection (UV-DAD). Two SPE cartridges have been compared for sample clean up and preconcentration: a reversed-phase silica-based cartridge (Bond Elut C18, Varian Inc.) and a strong polymeric mixed mode anion exchanger (Oasis MAX, Waters). The penicillins have been separated using a LUNA™ C18 (2) (150 mm × 4.6 mm, 5 μm) HPLC column and gradient elution with mobile phases consisting of aqueous trifluoroacetic acid and acetonitrile. The analytical wavelength was set at 220 nm. Under optimised conditions it was possible to preconcentrate up to 1000 mL of Milli-Q water in the Oasis MAX cartridges with recoveries in the range 82–97% (R.S.D. 2–9%) for all the antibiotic tested, except amoxicillin (52%, R.S.D. 8%), and limits of detection in the range of 8–24 ng L−1. The matrix components in industrial and urban wastewater samples reduce the preconcentration efficiency in both sorbents, especially for the Bond Elut C18. The use of the Oasis MAX allowed detection limits between 2.9–25.6, 2.5–12.4 and 2.2–12.7 μg L−1, when processing 250 mL of industrial, influent and effluent sewage treatment plant (STP) samples. Recoveries ranged between 46–91, 28–91 and 39–114% (industrial, influent and effluent STP, respectively) for samples spiked with all the antibiotics at 25 and 75 μg L−1 (n = 3 for each level).

A fast and accurate method for the determination of zearalenone (ZON) and α-zearalenol (α-ZOL) in ground wheat samples has been optimised using accelerated solvent extraction (ASE) and liquid chromatography (LC) with fluorescence detection. The effects of the experimentally controllable ASE parameters (solvent composition, temperature, pressure, number of extraction cycles and sample cell size) on the extraction efficiencies of the target analytes have been investigated using wheat samples fortified at the ng g−1 level. The optimum extraction conditions were obtained using a 11 mL ASE cell, methanol/acetonitrile (50:50 v/v) as the extraction solvent, 1500 psi, 50 °C, a 5 min static time and a 60% flush volume. The extracted samples were analysed using LC with fluorescence detection (λexc=271 and λem=452 nm). The stationary phase was a polar endcapped C18 column and acetonitrile/methanol/water 10/55/35 (v/v/v, 15 mM ammonium acetate) at a flow rate of 1.0 mL min−1 was used as the mobile phase. The injection volume was set at 8 μL. Mean recovery rates of 98.4% for α-ZOL (RSD

A bisphenol A (BPA) molecularly imprinted polymer, the composition of which was optimised using a chemometric approach, has been applied to the selective preconcentration of the template from aqueous samples. The selectivity of the polymer toward BPA and related compounds was evaluated chromatographically. The BPA-imprinted polymer was packed in a column and used for continuous on-column solid-phase extraction (MISPE) of aqueous samples followed by subsequent analysis by HPLC with fluorescence detection of the eluted fractions. The composition of the washing solvent applied in the MISPE procedure was optimised to favour the specific interactions of the MIP with BPA and to remove the non-selectively bound matrix components. The MISPE method has proven to be effective for selective preconcentration of BPA in aqueous samples (recoveries84% obtained in the eluate for 10-100 mL sample volumes) enabling detection and quantification limits of 1.0 and 3.3 ng mL(-1), respectively (based on 25 mL sample size). Analytical recoveries were between 92 and 101% for river water samples spiked with known amounts of BPA (30, 60, and 80 ng mL(-1)); relative standard deviations (RSD) were lower than 5.0%.

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/C 8 reversed-phase sorbent. The fluoroquinolones (FQs) were separated on a polar endcapped column (AQUA™ C 18 ). 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 10 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.

A key issue in the synthesis of molecularly imprinted polymers (MIPs) is the identification and optimisation of the main factors that affect the material structure and its molecular recognition properties. This paper describes the application of an experimental design and multivariate analysis method for the synthesis of bisphenol A (BPA)-selective MIPs. Six factors with a large impact on the MIP synthesis and its analytical performance have been optimised: the amount of template, the type and the percentage of functional and cross-linking monomers, the polymerisation method (i.e. thermal or UV initiation) and the porogenic solvent. The polymers have been prepared in small-scale (mini-MIPs) and, after careful removal of the template, their BPA rebinding capacity has been evaluated and related to the MIP composition. Among the two functional monomers tested, namely 4-vinylpyridine (4-vpy) and methacrylic acid (MAA), the former rendered the best selectivity for BPA analysis. The partial least squares (PLS) models revealed that the photoinitiated polymers with a 1:1 ratio of 4-vinylpyridine to cross-linker (EDMA or TRIM) yield the highest specific binding. Such procedure is time and cost effective and can be used as a general tool in the preparation of MIPs for different analytes.

An optical fiber biosensor for choline-containing phospholipids monitoring in serum samples is described. Phospholipids are hydrolyzed by the enzyme phospholipase- D to choline which is analyzed with a fiber-optic biosensor based on choline oxidase and oxygen transduction. Several supports have been evaluated for enzyme immobilization. The optimum biosensing layer consists of the enzyme immobilized on a nylon membrane and placed onto an oxygen sensitive layer formed by a luminescent Ru(II) complex, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II), dispersed in a silicone membrane. The performance of the biosensor has been optimized using a flow injection system. The linear dynamic range of the biosensing membranes is found to be 0.08–3.00 mg ml−1 phosphatidylcholine. Studies of the reproducibility, stability and interferences of the device, as well as the application of the sensor to measurements in serum samples, are reported.

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 50 mM 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.

This paper describes the development of a novel on-line biosensor based on a fluorescently labeled human calmodulin (CaM), hCaM M124C-mBBr, immobilized on controlled-pore glass (CPG), for the analysis of trifluoroperazine (TFP); a phenothiazine drug in human urine samples. The device was automated by packing hCaM M124C-mBBr-CPG in a continuous-flow microcell connected to a monitoring system, composed of a bifurcated optical fiber coupled to a spectrofluorometer. Operating parameters of the on-line biosensor (flow rate, sample injection volume, and carrier solution and buffer pH) were studied and optimized. Under the optimal conditions, the biosensor provides a detection and a quantification limit of 0.24 and 0.52 μg mL−1, respectively, and a dynamic range from 0.52 to 61.05 μg mL−1 TFP (n = 5, correlation coefficient 0.998). The response time (t 100) was shorter than 42 s (recovery time

The present work describes the development of a sensitive and highly selective innovative method for the simultaneous detection of six fluoroquinolone (FQ) antimicrobials (enrofloxacin, ciprofloxacin, norfloxacin, levofloxacin, danofloxacin, and sarafloxacin) in water samples. This detection is based on online solid phase extraction, coupled to liquid chromatography (LC), using for the first time tailor-made molecularly imprinted microspherical polymer particles prepared via precipitation polymerization. Various parameters affecting the extraction efficiency of the polymer have been optimized to reduce nonspecific interactions and to achieve selective uptake of the antibiotics from real samples. The method shows good recoveries ranging between 62% and 102% (V = 25 mL) for the different FQs tested and excellent interday and intraday precision with relative standard deviation (RSD) values between 2-5% and 2-6%, respectively. The detection limits were between 1-11 ng L(-1) (drinking water) and 1-12 ng L(-1) (fish farm water) when 25 mL samples were processed. The polymer showed selectivity for FQs containing a piperazine moiety whereas no retention was found for other antibiotics or nonrelated compounds. The method has been applied to the analysis of trace amounts of the FQs tested in drinking and fish farm water samples with excellent recoveries (91%) and good precision (RSDs5%).

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 (50 mM, 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

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 50 mM pH 3.0 (80:20, v/v), 80 °C, 2000 psi and three extraction cycles of 5 min. 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.

A novel technique for the synthesis and testing of large numbers of molecularly imprinted polymers is described requiring much less time than the commonly used miniMIP approach. Instead of vials, the polymers are synthesized on the surface of microfiltration membranes in multiwell filterplates. The thin polymeric films enable accelerated template removal. The MIP development procedure is thereby shortened to two days. Performance of the system was demonstrated by creating a combinatorial library of MIPs selective for cimetidine, an antiulcer drug. The polymer composition has been optimized. An experimental design combined with a multivariate analysis (i.e., response surface modeling) was used to minimize the number of experiments in the optimization process. The highest imprinting factor was obtained using a MAA/EDMA/template molar ratio of 3.5:19.5:1.

Three microalgal species (Dictyosphaerium chlorelloides (D.c.), Scenedesmus intermedius (S.i.) and Scenedesmus sp. (S.s.)) were encapsulated in silicate sol-gel matrices and the increase in the amount of chlorophyll fluorescence signal was used to quantify simazine. Influence of several parameters on the preparation of the sensing layers has been evaluated: effect of pH on sol-gel gelation time; effect of algae density on sensor response; influence of glycerol (%) on the membrane stability. Long term stability was also tested and the fluorescence signal from biosensors remained stable for at least 3 weeks. D.c. biosensor presented the lowest detection limits for simazine (3.6 microg L(-1)) and the broadest dynamic calibration range (19-860 microg L(-1)) with IC(50) 125+/-14 microg L(-1). Biosensor was validated by HPLC with UV/DAD detection. The biosensor showed response to those herbicides that inhibit the photosynthesis at photosystem II (triazines: simazine, atrazine, propazine, terbuthylazine; urea based herbicides: linuron). However, no significant increases of fluorescence response was obtained for similar concentrations of 2,4-D (hormonal herbicide) or Cu(II). The combined use of two biosensors that use two different genotypes, sensitive and resistant to simazine, jointly allowed improving microalgae biosensor specificity.

A novel water-compatible molecularly imprinted polymer (MIP), prepared with enrofloxacin (ENR) as the template, has been optimised for the selective extraction of fluoroquinolone antibiotics in aqueous media. The results of a morphological characterisation and selectivity tests of the polymer material for ENR and related derivatives are reported. High affinity for the piperazine-based fluoroquinolones marbofloxacin, ciprofloxacin, norfloxacin and ofloxacin was observed, whereas no retention was found for nonrelated antibiotics. Various parameters affecting the extraction efficiency of the polymer have been optimised to achieve selective extraction of the antibiotics from real samples and to reduce nonspecific interactions. These findings resulted in a MISPE/HPLC-FLD method allowing direct extraction of the analytes from aqueous samples with a selective wash using just 50% (v/v) organic solvent. The method showed excellent recoveries and precision when buffered urine samples fortified at five concentration levels (25-250 ng mL(-1) each) of marbofloxacin, ciprofloxacin, norfloxacin, enrofloxacin and sarafloxacin were tested (53-88%, RSD 1-10%, n = 3). Moreover, the biological matrix of the aqueous samples did not influence the preconcentration efficiency of the fluoroquinolones on the MIP cartridges; no significant differences were observed between the recovery rates of the antibiotics in buffer and urine samples. The detection limits of the whole process range between 1.9 and 34 ng mL(-1) when 5-mL urine samples are processed. The developed method has been successfully applied to preconcentration of norfloxacin in urine samples of a medicated patient, demonstrating the ability of the novel MIP for selective extraction of fluoroquinolones in urine samples.

A rapid and simple method is proposed for the routine determination of amoxicillin (AMOX) and penicillin V (PENV) in swine feedingstuffs. The method is based on pressurised liquid extraction (PLE) followed by high performance liquid chromatography with ultraviolet detection (PLE-HPLC-UV) for antibiotic analysis. Parameters affecting PLE procedure, such as temperature, solvent composition, number of extraction cycles and sample cell size, were evaluated in order to achieve the highest extraction efficiency. The optimised method employed 11mL extraction cells, acetonitrile-water mixtures (25:75, v/v) for AMOX and (50:50, v/v) for PENV, as extraction solvent, 102.07atm of extraction pressure, 50 degrees C of extraction temperature, 5min of static time and 60% flush volume of the cell size. Extracts were filtered and directly analysed by HPLC-DAD/UV without further clean-up. Mean recovery rates for feed samples fortified with 200-500mgkg(-1) of both antibiotics were 86% for AMOX (RSD< or =6%) and 95% for PENV (RSD< or =3%). The method was successfully applied to the analysis of a commercial medicated swine feedingstuff, and the results were in good agreement with those obtained using mechanical shaking or ultrasonic extraction combined with solid phase extraction (UE-SPE), previously applied in the literature for feed analysis. The extraction efficiencies were evaluated by statistical comparison (analysis of variance, ANOVA-single factor) of the results obtained using the different extraction methods. Compared to the alternative techniques, PLE offers several practical advantages: easy to perform, fast, savings in solvent volume and in time, all steps are fully automated and further clean-up is not necessary for penicillin analysis.

Specific polyclonal antibodies against s-triazine herbicides were obtained by preparing immunogens coupling home-synthesized haptens derivatives of simazine (6-chloro-N-ethyl-N'-ethyl-1,3,5-triazine-2,4-diamine) to lysine groups of hemocyanin from keyhole limpets and bovine serum albumin carrier proteins. Three highly sensitive rabbit antisera were obtained and evaluated with a battery of six enzyme tracers derived from triazine structures in an optimized ELISA format. The antiserum As8 and the HRP-2f tracer, which yield the best assay sensitivity for simazine (detection limit 0.11 +/- 0.02 microg L(-1), IC(50) 0.88 +/- 0.04 microg L(-1)), were applied to the development of a sensitive flow-through immunoassay for the analysis of this herbicide. The automated assay was based on a direct competitive immunosorbent assay and fluorescence detection. The optimized method presents an IC(50) value of 0.35 +/- 0.04 microg L(-1) with a detection limit of 1.3 +/- 0.9 ng L(-1) and a dynamic range from 0.010 to 7.5 microg L(-1) simazine. The generic nature of the antiserum was shown by good relative cross-reactivities with other triazines such as atrazine (420%) or propazine (130%) and a lower response to terbutylazine (6.4%) and desethyl-atrazine (2.2%). No cross-reactivity was obtained for nonrelated pesticides such as 2,4-dichlorophenoxyacetic acid or linuron and the assay could be applied as a screening method for triazine herbicides. The total analysis time was 30 min per determination and the immunosensor could be reused for more than 150 cycles without significant loss of activity. The immunosensor has been successfully applied to the direct analysis of simazine in surface water samples at the nanogram per liter level. The results obtained by comparative analysis of the immunosensor with a chromatographic procedure for triazines showed a close correspondence.

An automated molecularly imprinted sorbent based assay (MIA) for the rapid and sensitive analysis of penicillin-type beta-lactam antibiotics (BLAs) has been developed and optimized. The polymers were prepared using penicillin G procaine salt as template (PENGp) and a stoichiometric quantity of a urea-based functional monomer to target the single oxyanionic species in the template molecule. Highly fluorescent competitors (emission quantum yields of 0.4-0.95), molecularly engineered to contain pyrene labels while keeping intact the 6-aminopenicillanic acid moiety for efficient recognition by the cross-linked polymers, have been tested as analyte analogues in the competitive assay. Pyrenemethylacetamido penicillanic acid (PAAP) was the tagged antibiotic providing for the highest selectivity when competing with PenG for the specific binding sites in the molecularly imprinted polymer (MIP). Upon desorption from the MIP, the emission signal generated by the PAAP was related to the antibiotic concentration in the sample. The 50% binding inhibition concentration of penicillin G standard curves was at 1.81 x 10(-6) M PENG, and the detection limit was 1.97 x 10(-7) M. The sensor showed a dynamic range (normalized signal in the 20 to 80% range) from 6.80 x 10(-7) to 7.21 x 10(-6) M (20-80% binding inhibition) PENG in acetonitrile:HEPES buffer 0.1 M at pH 7.5 (40:60, v/v) solutions. Competitive binding studies demonstrated various degrees of cross-reactivity with penicillin-type beta-lactam antibiotics such as ampicillin (71%), oxacillin (66%), penicillin V (56%), amoxicillin (13%), and nafcillin (46%) and a lower response to other isoxazolyl penicillins such as cloxacillin (27%) and dicloxacillin (16%). The total analysis time was 14 min per determination, and the MIP reactor could be reused for more than 150 cycles without significant loss of recognition. The automatic MIA has been successfully applied to the direct analysis of penicillin G in spiked urine samples with excellent recoveries (mean value 92%). Results displayed by comparative analysis of the optimized MIA with a chromatographic procedure for penicillin G showed excellent agreement between both methods.

A molecularly imprinted polymer (MIP) prepared using penicillin G procaine salt as the template (PENGp) and a stoichiometric quantity of urea-based functional monomer to target the single oxyanionic species in the template molecule has been applied to the development of a molecularly imprinted solid-phase extraction (MISPE) procedure for the selective preconcentration of beta-lactam antibiotics (BLAs) from environmental water samples. Various parameters affecting the extraction efficiency of the polymer have been evaluated to achieve the selective preconcentration of the antibiotics from aqueous samples and to reduce nonspecific interactions. This resulted in an MISPE-HPLC method allowing the direct extraction of the analytes from the sample matrix with a selective wash using just 10% (v/v) organic solvent. On the basis of UV detection only, the method showed good recoveries and precision, ranging between 93% and 100% (RSD 3.8-8.9%, n = 3) for tap water and between 90% and 100% (RSD 4.2-9.1%, n = 3) for river water fortified with 30 or 60 microg L-1 (50 mL samples) penicillin G, penicillin V, nafcillin, oxacillin, cloxacillin, and dicloxacillin, suggesting that this MIP can be successfully applied to the direct preconcentration of BLAs in environmental water samples.

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 50 mM, pH 3.0/acetonitrile (50:50, v/v). The optimized procedure employed 50% flush volume, 5 min of static time and three extraction cycles at 70 °C and 1500 psi. 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 50–1000 ng g −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 (CC α ) and detection capability (CC β ) of the analytical method were found to be within the range 17–24 ng g −1 and 30–41 ng g −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.

A molecularly imprinted polymer prepared using 1-allylpiperazine (1-ALPP) as the functional monomer, trimethyltrimethacrylate (TRIM) as the crosslinker and the zearalenone (ZON)-mimicking template cyclododecanyl-2,4-dihydroxybenzoate (CDHB) has been applied to the clean-up and preconcentration of this mycotoxin (zearalenone) and a related metabolite, α-zearalenol (α-ZOL), from cereal and swine feed sample extracts. The extraction of ZON and α-ZOL from the food samples was accomplished using pressurized liquid extraction (PLE) with MeOH/ACN (50:50, v/v) as the extraction solvent, at 50 °C and 1500 psi. The extracted samples were cleaned up and preconcentrated through the MIP cartridge and analyzed using HPLC with fluorescence detection (λ exc=271/ λ em=452 nm). The stationary phase was a polar endcapped C18 column, and ACN/MeOH/water 10/55/35 (v/v/v, 15 mM ammonium acetate) at a flow rate of 1.0 mL min−1 was used as the mobile phase. The method was applied to the analysis of ZON and α-ZOL in wheat, corn, barley, rye, rice and swine feed samples fortified with 50, 100 and 400 ng g−1 of both mycotoxins, and it gave recoveries of between 85 and 97% (RSD 2.1–6.7%, n=3) and 87–97% (RSD 2.3–5.6%, n=3) for α-ZOL and ZON, respectively. The method was validated using a corn reference material for ZON.

The development of a sensitive flow-though immunosensor for the analysis of the mycotoxin zearalenone in cereal samples is described. The sensor was completely automated and was based on a direct competitive immunosorbent assay and fluorescence detection. The mycotoxin competes with a horseradish-peroxidase-labeled derivative for the binding sites of a rabbit polyclonal antibody. Control pore glass covalently bound to Prot A was used for the oriented immobilization of the antibody-antigen immunocomplexes. The immunosensor shows an IC 50 value of 0.087 ng mL -1 (RSD = 2.8%, n = 6) and a dynamic range from 0.019 to 0.422 ng mL -1 . The limit of detection (90% of blank signal) of 0.007 ng mL -1 (RSD = 3.9%, n = 3) is lower than previously published methods. Corn, wheat, and swine feed samples have been analyzed with the device after extraction of the analyte using accelerated solvent extraction (ASE). The immunosensor has been validated using a corn certificate reference material and HPLC with fluorescence detection.

An optical fiber biosensor for the determination of the pesticides propoxur (Baygon®) and carbaryl, two of the most commonly used carbamate insecticides in vegetable crops, is described. A pH indicator, chlorophenol red, is used as optical transducer of the inhibition of the enzyme acetylcholinesterase by the analytes. The biorecognition element is covalently immobilized onto controlled pore glass beads (CPG) and packed in a thermostatized bioreactor connected to a flow-through cell that contains CPG- immobilized chlorophenol red placed at the common end of a bifurcated fiber optic bundle. In the presence of a constant acetylcholine concentration, the colour of the pH sensitive layer changes and the measured reflectance signal can be related to the carbamate concentration in the sample solution. The performance of the biosensor has been optimized using a flow injection system. The linear dynamic range for the determination of carbaryl and propoxur spans from 0.8 to 3.0 mg l-1 and from 0.03 to 0.50 mg l-1, respectively. The detection limit (3 s) of the biosensor for propoxur (0.4 ng) is lower than that measured for carbaryl (25 ng). Reproducibility, stability and interference studies of the optical device are reported. The biosensor has been applied to the determination of propoxur in spiked vegetables (onion and lettuce) using ultrasound extraction, achieving recovery values between 93 and 95% for onion samples at the different concentration levels assayed. (C) 2000 Elsevier Science S.A. An optical fiber biosensor for the determination of the pesticides propoxur (Baygon) and carbaryl, two of the most commonly used carbamate insecticides in vegetable crops, is described. A pH indicator, chlorophenol red, is used as optical transducer of the inhibition of the enzyme acetylcholinesterase by the analytes. The biorecognition element is covalently immobilized onto controlled pore glass beads (CPG) and packed in a thermostatized bioreactor connected to a flow-through cell that contains CPG-immobilized chlorophenol red placed at the common end of a bifurcated fiber optic bundle. In the presence of a constant acetylcholine concentration, the colour of the pH sensitive layer changes and the measured reflectance signal can be related to the carbamate concentration in the sample solution. The performance of the biosensor has been optimized using a flow injection system. The linear dynamic range for the determination of carbaryl and propoxur spans from 0.8 to 3.0 mg l-1 and from 0.03 to 0.50 mg l-1, respectively. The detection limit (3 s) of the biosensor for propoxur (0.4 ng) is lower than that measured for carbaryl (25 ng). Reproducibility, stability and interference studies of the optical device are reported. The biosensor has been applied to the determination of propoxur in spiked vegetables (onion and lettuce) using ultrasound extraction, achieving recovery values between 93 and 95% for onion samples at the different concentration levels assayed.

An optical fiber biosensor for free cholesterol monitoring in serum samples is described. Silicone-entrapped tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) complex, the luminescence of which is sensitive to oxygen changes, is used as an optical transducer of the oxidation of cholesterol by cholesterol oxidase. The biocatalyst is entrapped in a graphite powder layer deposited onto the dyed silicone film. Optimization of some interdependent chemical variables which affect the performance of the biosensor has been achieved by application of a super-modified simplex method. The dynamic range of the biosensing membranes is found to be 0.15-3.0 mM of free cholesterol. Studies of the reproducibility, stability and interferences of the device, as well as the application of the sensor to measurements in serum samples, are reported. Simplex optimization has proven to be a very useful tool in the search for the optimal conditions for performing analyses with the optical fiber biosensor.