Analysis of alternariol and alternariol monomethyl ether in foodstuffs by molecularly imprinted solid-phase extraction and ultra-high-performance liquid chromatography tandem mass spectrometry.

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 (λex=258nm; λem=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.

Multiresidue analysis of cephalosporin antibiotics in bovine milk based on molecularly imprinted polymer extraction followed by liquid chromatography-tandem mass spectrometry.

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.

Prospecção fitoquímica e avaliação da citotoxicidade e genotoxicidade de Helenium cf. amarum (Raf.) H. Rock / Phytochemical screening and evaluation of the cytotoxicity and genotoxicity of Helenium cf. amarum (Raf.) H. Rock / Tamizaje fitoquímico y evaluación de la citotoxicidad y genotoxicidad del Helenium cf. amarum (Raf.) H. Rock

Introduction: Helenium cf. amarum (Raf.) H. Rock is a plant of the family Asteraceae. Its common name is yellow camomile. It is used as tranquilizer, stimulant and digestive, and for the treatment of nausea, fever and skin disorders. Objectives: carry out a phytochemical screening and evaluate the cytotoxic and genotoxic effect of a crude hydroalcoholic extract of Helenium amarum using the Allium cepa test. Methods: seeds of Allium cepa were subjected to germination at four concentrations (0.6, 1.0, 2.0 and 3.0 mg/ml) of H. amarum crude leaf extract. After being dried in an oven for 5 days, they were pulverized and macerated in 70 % ethanol at room temperature for 72 hours. The extract was then filtered and the liquid phase subjected to a rotary evaporator. Two sorts of treatment were applied: 1) continuous treatment: the seeds were germinated directly in the extract at different concentrations. 2) intermittent treatment: the seeds were first germinated in Milli-Q water until they grew 2 cm long rootlets, and were then exposed to different extract concentrations. Results: the germination rate was affected by extract concentration, and was lower than that of the negative control in all treatments. The mitotic index for all concentrations was lower than that of controls for both treatments. In batch processing, the aneugenic effects index at the assayed concentrations was lower than that of controls, whereas the clastogenic index was 1 % for the control and treatments 1 and 3 mg/ml, lower than 1 % for treatments 0.6 and 2 mg/ml, and 20 % for the positive control. Phytochemical screening showed positive results for tannins and steroids. Conclusions: Helenium amarum has toxic and cytotoxic effects and allelopathic action, but not genotoxic effects at the assayed concentrations.

Molecular recognition with nanostructures fabricated by photopolymerization within metallic subwavelength apertures.

The first demonstration of fabrication of submicron lateral resolution molecularly imprinted polymer (MIP) patterns by photoinduced local polymerization within metal subwavelength apertures is reported. The size of the photopolymerized MIP features is finely tuned by the dose of 532 nm radiation. Rhodamine 123 (R123) has been selected as a fluorescent model template to prove the recognition capability of the MIP nanostructures, which has been evaluated by fluorescence lifetime imaging microscopy (FLIM) with single photon timing measurements. The binding selectivity provided by the imprinting effect has been confirmed in the presence of compounds structurally related to R123. These results pave the way to the development of nanomaterial architectures with biomimetic artificial recognition properties for environmental, clinical and food testing.

Multiresidue analysis of fluoroquinolone antimicrobials in chicken meat by molecularly imprinted solid-phase extraction and high performance liquid chromatography.

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 (CCß) 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.

Immuno-like assays and biomimetic microchips.

Biomimetic assays with molecularly imprinted polymers (MIPs) are bound to be an alternative to the traditional immuno-analytical methods based on antibodies. This is due to the unique combination of advantages displayed by the artificial materials including the absence of animal inoculation and sacrifice, unnecessary hapten conjugation to a carrier protein for stimulated production, the possibility of manufacturing MIPs against toxic substances, excellent physicochemical stability, reusability, ease of storage, and recognition in organic media. If the selectivity and affinity of MIPs are increased, many more immuno-like assays will be developed using radioactive, enzymatic, colorimetric, fluorescent, chemiluminescent, or electrochemical interrogation methods. This chapter provides a comprehensive comparison between the bio- and biomimetic entities and their usage.

Quantitative determination of penicillin V and amoxicillin in feed samples by pressurised liquid extraction and liquid chromatography with ultraviolet detection.

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.

Molecularly imprinted polymers with a streamlined mimic for zearalenone analysis.

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.

Allergy to dermatophagoides in a group of Spanish gypsies: genetic restrictions.

BACKGROUND: Spanish gypsies have traditionally lived as nomads, a reason why few epidemiological studies were done in this ethnic group. However, the high prevalence of asthmatic diseases demonstrated in a population residing in the North of Spain induces us to analyse whether it was due to the influence of genetic loci previously implicated in other population studies as causing the disorders. METHODS: DRB1* and DQB1* HLA class II, TCR-Valpha8.1, FcepsilonRI-beta Rsa I exon 7 and intron 2, TNF-beta (LTalpha-Nco I) and CD14, were tested for association with asthma and atopy by multiple regression analysis, in 5 families comprising 87 individuals. RESULTS: Significant associations were found with DQB1*02 (p = 0.02) and DQB1*0301 (p = 0.008) and elevated levels of total serum IgE. A negative association (p = 0.02) was found between total serum IgE and DRB1*14. FcepsilonRI-beta Rsa I-In2 allele 1 was associated with high levels of total serum IgE (p = 0.04). Levels of Der p 1 IgE antibodies were negatively associated with DRB1*11-DQB1*0301 (p = 0.007), and positively with TCR Valpha-8 allele 1 (p = 0.04) and with FcepsilonRI-beta Rsa I-In2 allele 1 (p = 0.009). CONCLUSIONS: Our results do not show any association between asthma and the genetic loci studied although they do suggest the existence of multiple genetic influences on the allergic response in these families.

[An analysis of the level of accuracy of the official diagnosis of temporary work incapacity]. / Análisis del nivel de exactitud del diagnóstico oficial de la incapacidad laboral transitoria.

OBJECTIVE: To assess the precision of the Sickness Certificate (SC) issued for Temporary Unfitness for Work (TUW). To measure the percentage of TUW where the diagnoses in the medical records and in the TUW Sickness Certificate do not coincide. To seek objective criteria to determine TUW. DESIGN: A descriptive, prospective and observational study. SETTING: Health Centre in an urban area in Vitoria. PATIENTS AND OTHERS PARTICIPANTS: All the sickness occasioning time off work between May and July, 1991, recorded at the above Health Centre: a sample of 224 TUW. MEASUREMENTS AND MAIN RESULTS: In 11.6% of the TUW the diagnoses did not coincide. The main reasons for the lack of correspondence were: initial ignorance of the diagnosis (34.6%); confidentiality before a third party (25.9%); pretense (14.8%). There was a notable difference between the psychological diagnoses (12) and those found in the official certificate 3 for TUW (p = 0.032). The most common diagnostic group was the locomotive one (29%). CONCLUSIONS: There is considerable inexactness in the diagnoses on the official TUW certificate. A lot of time off for psychological reasons is covered up by organic complaints. The locomotive group of complaints is the most commonly found one. We propose that the CIAP classification should be adopted to standardise diagnoses in this area.