A specificity-enhanced time-resolved fluoroimmunoassay for zeranol employing the dry reagent all-in-one-well principle

A simple dry chemistry time-resolved fluorescence immunoassay (TR-FIA) method was developed for the measurement of zeranol in bovine urine samples. The samples were purified by immunoaffinity chromatography and a specificity-enhanced zeranol antibody was employed in the immunoassay. This resulted in a highly selective method, which had only negligible reactivity with Fusarium spp, toxins. The all-in-one-well dry chemistry concept made the assay very simple to use because all the assay-specific reagents were already present in the reaction wells in dry form. Only the addition of diluted sample extract was required to perform the competitive one-step TR-FIA and the results were available in less than 1 h. The analytical limit of detection (mean + 3s) for the immunoassay was 0.16 ng ml(-1) (n=12) and the functional limit of detection for the whole method, estimated by the analysis of zeranol-free samples, was 1.3 ng ml(-1) (n=20). The recovery of zeranol at the level of 2 ng ml(-1) was 99% (n=18) and the within-assay variation ranged between 4.5 and 9.0%.

Interlaboratory ring test of time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol and comparison with zeranol test kits

Many zeranol immunoassay test kits cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the evaluation and application of recently published, dry reagent time-resolved fluoroimmunoassays (TR-FIA) for zeranol and the toxin alpha-zearalenol. A ring test of bovine urine fortified with zeranol and/or alpha-zearalenol in four European Union National Reference Laboratories demonstrated that the TR-FIA tests were accurate and robust. The alpha-zearalenol TR-FIA satisfactorily quantified alpha-zearalenol in urine fortified at 10-30 ng ml(-1) . The specificity-enhanced zeranol TR-FIA accurately quantified zeranol in the range 2-5 ng ml(-1) and gave no false-positive results in blank urine, even in the presence of 30 ng ml(-1) alpha-zearalenol. Zeranol TR-FIA specificity was demonstrated further by analysing incurred zeranol-free urine samples containing natural Fusarium spp. toxins. The TR-FIA yielded no false-positive results in the presence of up to 22 ng ml(-1) toxins. The performance of four commercially available zeranol immunoassay test kits was more variable. Three kits produced many false-positive results. One kit produced only one potential false-positive using a protocol that was longer than that of the TR-FIA. These TR-FIAs will be valuable tools to develop inspection criteria to distinguish illegal zeranol abuse from contamination arising from in vivo metabolism of Fusarium spp. toxins.

Profiling the ionome of rice and its use in discriminating geographical origins at the regional scale, China

Element profile was investigated for their use to trace the geographical origin of rice (Oryza sativa L.) samples. The concentrations of 13 elements (calcium (Ca), potassium (K), magnesium (Mg), phosphorus (P), boron (B), manganese (Mn), iron (Fe), nickel (Ni), copper (Cu), arsenic (As), selenium (Se), molybdenum (Mo), and cadmium (Cd)) were determined in the rice samples by inductively coupled plasma optical emission and mass spectrometry. Most of the essential elements for human health in rice were within normal ranges except for Mo and Se. Mo concentrations were twice as high as those in rice from Vietnam and Spain. Meanwhile, Se concentrations were three times lower in the whole province compared to the Chinese average level of 0.088 mg/kg. About 12% of the rice samples failed the Chinese national food safety standard of 0.2 mg/kg for Cd. Combined with the multi-elemental profile in rice, the principal component analysis (PCA), discriminant function analysis (DFA) and Fibonacci index analysis (FIA) were applied to discriminate geographical origins of the samples. Results indicated that the FIA method could achieve a more effective geographical origin classification compared with PCA and DFA, due to its efficiency in making the grouping even when the elemental variability was so high that PCA and DFA showed little discriminatory power. Furthermore, some elements were identified as the most powerful indicators of geographical origin: Ca, Ni, Fe and Cd. This suggests that the newly established methodology of FIA based on the ionome profile can be applied to determine the geographical origin of rice.

Quid petis? um estudo sobre o processo de doutoramento

"Sem resumo feito pelo autor"; - O grau de doutor é, na maioria dos países, o mais elevado grau concedido pelas universidades. Sendo considerado como um grau de "excelência", pauta-se, geralmente, por elevados padrões de exigência, certificando que o seu detentor, não apenas tem capacidade para realizar investigações de forma autónoma, como pode, dessa forma, contribuir de forma original e inovadora para o progresso do conhecimento. Enquanto grau de "excelência" tem servido como critério de certificação de professores universitários, sendo também um referente de qualidade dos cursos ministrados nas universidades e indicativo do desenvolvimento técnico e científico das diferentes sociedades. Considerada como potenciadora do desenvolvimento científico, cultural e tecnológico das sociedades, a formação de doutores envolve avultados recursos financeiros e humanos, num investimento nem sempre "rentável" a curto prazo. Ao mesmo tempo, numa sociedade globalizada e caracterizada pela ideia de "aprendizagem ao longo da vida", o número de pessoas que frequentam cursos de pós-graduação tem vindo a crescer de forma sistemática, colocando novos desafios e exigências às instituições de formação. Estas novas realidades têm obrigado a uma reflexão sobre a natureza e finalidades do doutoramento, numa tentativa de responder às necessidades sociais e económicas e aos interesses das pessoas em formação, mantendo embora o carácter de excelência do grau e os requisitos necessários à sua obtenção. No entanto, e talvez devido ao seu carácter ainda exclusivista e minoritário, a educação pós-graduada tem sido relativamente pouco estudada. Ao contrário do que acontece relativamente a outros niveis de ensino (mesmo superior), e apesar de gozar de um certo prestígio, a educação pós-graduada tem sido votada a um certo "esquecimento", o que levou mesmo um professor emérito da Universidade de Harvard a considerá-la como o "soft under-belly of the research university" (Bowen & Rudenstine,1992, p. xv). Mas a educação pós-graduada pode ser considerada como um fértil campo de investigação (Kogan, 1993), quer no domínio da Psicologia, quer no dominio de outras áreas científicas, susceptivel de um vasto campo de pesquisas, desde definição de padrões de qualidade à análise de processos desenvolvimentais em jogo ao longo do processo de doutoramento. 0 processo de doutoramento configura-se como um processo moroso e exigente, obrigando a um grande investimento intelectual e pessoal, ao longo de vários anos e, muitas vezes, em detrimento da vida familiar e profissional e de outros interesses sociais e culturais. 0 intenso envolvimento da pessoa nos trabalhos, assim como a sua longa duração podem fazer com que o processo de doutoramento seja vivenciado de forma problemática, originando sentimentos de desânimo e dúvidas relativamente às capacidades para obter o grau. Mas, por outro lado, os trabalhos de doutoramento, proporcionando conhecimentos especializados numa dada área do saber e dotando a pessoa de maiores competências científicas e intelectuais, conduzem a uma gradual e crescente autonomia, transformando o estudante de doutoramento num académico, inserido numa comunidade cientifica e de investigação. Assim, mais do que um conjunto de trabalhos e actividades que, ao fim de um tempo mais ou menos longo, conduzem à redacção de uma tese e à obtenção de um grau académico, o doutoramento pode ser encarado um processo de desenvolvimento, que, implicando a totalidade da pessoa e da sua vida, conduz a mudanças nas formas como concebe a realidade e se relaciona com ela. Envolve uma opção de vida — a vida académica, devotada ao estudo e à investigação — e, como tal, obriga a abdicar de outras opções, num processo que pode ser doloroso. Implica um estudo aprofundado de um dado tema, possibilitando que a pessoa se vá tornando especialista, alargando os seus conhecimentos e competências, num processo de aprendizagem que implica a (re)construção de significados, de um modo pessoal e idiossincrático (Entwistle, 1986; Salmon,1992); é, assim, um processo de construção de conhecimento, que pode proporcionar à pessoa concepções mais relativizadas do mundo teórico e conceptual que a rodeia. Relaciona-se com o desenvolvimento profissional e, como tal, tem repercussões na identidade da pessoa. Ao longo deste processo, os problemas sentidos (derivados essencialmente do intenso envolvimento nos trabalhos, que decorrem geralmente num grande isolamento físico e intelectual) podem contribuir para vivências negativas e pouco satisfatórias da experiência do doutoramento. É neste quadro que o papel do orientador pode ser decisivo, não apenas no apoio científico que pode prestar, mas também, e essencialmente, no suporte emocional, ajudando a transformar as crises em factores de desenvolvimento. Tendo em conta estes diferentes aspectos, procurou-se com este trabalho contribuir para uma melhor compreensão do processo de doutoramento, tal como ele é vivenciado pelos próprios doutorandos. Centrando-se nos estudantes de doutoramento, privilegia, naturalmente, a sua percepção do processo, em detrimento de outras pessoas envolvidas (orientadores, fia, ... ); não se esquece, contudo, a sua importância na forma como o processo é vivenciado. 0 primeiro capítulo - 0 Grau de Doutor está dividido em quatro grandes partes. Na primeira procura-se fazer um breve historial da evolução desde grau académico, desde o seu aparecimento nas universidades europeias até à reforma da Universidade de Berlim, implementada por Wilhelm von Humboldt, que lança as bases para o estabelecimento da "universidade de investigação". A segunda parte centra-se na universidade portuguesa, desde a sua fundação à actualidade, numa breve análise da sua evolução e com particular ênfase no processo de atribuição do grau de doutor e dos cerimoniais associados. Na terceira parte procura-se fazer uma análise comparativa do grau de doutor em diferentes países, dando relevo aos requisitos necessários à sua atribuição, à organização dos estudos doutorais, à ligação entre os sistemas universitário e de investigação e, ainda, a diversas problemáticas relacionadas com o processo de doutoramento nesses países. Finalmente, a quarta parte debruça-se sobre alguns problemas e perspectivas do grau de doutor na actualidade, com particular ênfase na discussão da sua natureza e objectivos e na análise dos requisitos exigidos e do tempo necessário à sua obtenção. 0 segundo capítulo - 0 Processo de Doutoramento - centra-se fundamentalmente em questões relacionadas com a forma como estudantes de doutoramento experienciam os trabalhos conducentes ao grau de doutor e divide-se em duas grandes partes. A primeira tem a ver directamente com as vivências e perspectivas dos estudantes ao longo do processo, em torno de duas grandes vertentes: problemas experienciados e a relação de orientação. Com base na revisão bibliográfica efectuada e tendo em conta os resultados de pesquisas realizadas em alguns países com estudantes de doutoramento, procurou-se sistematizar, o mais exaustivamente possível, os principais aspectos relacionados com estas temáticas, nomeadamente os que se prendem com vivências sentidas como problemáticas ao longo do processo, quer as de índole pessoal, quer as relacionadas com os trabalhos, e a relação de orientação.

Electroanalysis of urinary L-dopa using tyrosinase immobilized on gold nanoelectrode ensembles

The performance of an amperometric biosensor constructed by associating tyrosinase (Tyr) enzyme with the advantages of a 3D gold nanoelectrode ensemble (GNEE) is evaluated in a flow-injection analysis (FIA) system for the analysis of l-dopa. GNEEs were fabricated by electroless deposition of the metal within the pores of polycarbonate track-etched membranes. A simple solvent etching procedure based on the solubility of polycarbonate membranes is adopted for the fabrication of the 3D GNEE. Afterward, enzyme was immobilized onto preformed self-assembled monolayers of cysteamine on the 3D GNEEs (GNEE-Tyr) via cross-linking with glutaraldehyde. The experimental conditions of the FIA system, such as the detection potential (−0.200 V vs. Ag/AgCl) and flow rates (1.0 mL min−1) were optimized. Analytical responses for l-dopa were obtained in a wide concentration range between 1 × 10−8 mol L−1 and 1 × 10−2 mol L−1. The limit of quantification was found to be 1 × 10−8 mol L−1 with a resultant % RSD of 7.23% (n = 5). The limit of detection was found to be 1 × 10−9 mol L−1 (S/N = 3). The common interfering compounds, namely glucose (10 mmol L−1), ascorbic acid (10 mmol L−1), and urea (10 mmol L−1), were studied. The recovery of l-dopa (1 × 10−7 mol L−1) from spiked urine samples was found to be 96%. Therefore, the developed method is adequate to be applied in the clinical analysis.

Biomimetic norfloxacin sensors made of molecularly-imprinted materials for potentiometric transduction

A biomimetic sensor for norfloxacin is presented that is based on host-guest interactions and potentiometric transduction. The artificial host was imprinted into polymers made from methacrylic acid and/or 2-vinyl pyridine. The resulting particles were entrapped in a plasticized poly(vinyl chloride) (PVC) matrix. The sensors exhibit near-Nernstian response in steady state evaluations, and detection limits range from 0.40 to 1.0 μgmL−1, respectively, and are independent of pH values at between 2 and 6, and 8 and 11, respectively. Good selectivity was observed over several potential interferents. In flowing media, the sensors exhibit fast response, a sensitivity of 68.2 mV per decade, a linear range from 79 μM to 2.5 mM, a detection limit of 20 μgmL−1, and a stable baseline. The sensors were successfully applied to field monitoring of norfloxacin in fish samples, biological samples, and pharmaceutical products

Development of electrochemical methods for determination of tramadol - analytical application to pharmaceutical dosage forms

A square-wave voltammetric (SWV) method and a flow injection analysis system with amperometric detection were developed for the determination of tramadol hydrochloride. The SWV method enables the determination of tramadol over the concentration range of 15-75 µM with a detection limit of 2.2 µM. Tramadol could be determined in concentrations between 9 and 50 µM at a sampling rate of 90 h-1, with a detection limit of 1.7 µM using the flow injection system. The electrochemical methods developed were successfully applied to the determination of tramadol in pharmaceutical dosage forms, without any pre-treatment of the samples. Recovery trials were performed to assess the accuracy of the results; the values were between 97 and 102% for both methods.

Electroanalytical determination of codeine in pharmaceutical preparations

A square wave voltammetric (SWV) method and a flow injection analysis systemwi th electrochemical detection (FIA-EC) using a glassy carbon electrode were evaluated for the determination of codeine in pharmaceutical preparations. The interference of several compounds, such as acetaminophen,guaiacol, parabens, ephedrine, acetylsalicylic acid and caffeine, that usually appear associated with codeine pharmaceutical preparations was studied. It was verified that these electroanalytical methods could not be used with acetaminophen present in the formulations and that with guaiacol, parabens or ephedrine present the use of the FIA-EC system was impracticable. A detection limit of 5 µmol L- 1 and a linear calibration range from 40 to 140 µmol L- 1 was obtained with the SWV method. For the flow injection analysis procedure a linear calibration range was obtained from 7 to 50 µmol L- 1 with a detection limit of 3 µmol L- 1 and the FIA-EC systemallowed a sampling rate of 115 samples per hour. The results obtained by the two methods, SWV and FIA-EC, were compared with those obtained using reference methods and demonstrated good agreement, with relative deviations lower than 4%.

Electroanalytical study of fluvoxamine

Fluvoxamine (FVX) can be reduced at a mercury- drop electrode, with a maximum peak current intensity being obtained at a potential of -0.7 V vs. Ag/ AgCl, in an aqueous electrolyte solution of pH 2. The compound was determined in a pharmaceutical product and in spiked human serum by square-wave adsorptivestripping voltammetry (SWAdSV) after accumulation at the electrode surface, under batch conditions. Because the presence of dissolved oxygen did not interfere significantly with the analysis, it was also possible to determine FVX in the pharmaceutical product by use of a flow-injection analysis (FIA) system with SWAdSV detection. The methods developed were validated and successfully applied to the quantification of FVX in a pharmaceutical product. Recoveries between 76 and 89% were obtained in serum analysis. The FIA– SWAdSV method enabled analysis of up to 120 samples per hour at reduced cost, implying the possibility of competing with the chromatographic methods usually used for this analysis.

Electroanalytical determination of paroxetine in pharmaceuticals

Electroanalytical methods based on square-wave adsorptive-stripping voltammetry (SWAdSV) and flow-injection analysis with SWAdSV detection (FIA-SWAdSV) were developed for the determination of paroxetine (PRX). The methods were based on the reduction of PRX at a mercury drop electrode at −1.55V versus Ag/AgCl, in a borate buffer of pH 8.8, and the possibility of accumulating the compound at the electrode surface. Because the presence of dissolved oxygen did not interfere significantly with the analysis, it was also possible to determine PRX using FIASWAdSV. This method enables analysis of up to 120 samples per hour at reduced costs. Both methods developed were validated and successfully applied to the quantification of PRX in pharmaceutical products.

Citrate selective electrodes for the flow injection analysis of soft drinks, beers and pharmaceutical products

Aiming the establishment of simple and accurate readings of citric acid (CA) in complex samples, citrate (CIT) selective electrodes with tubular configuration and polymeric membranes plus a quaternary ammonium ion exchanger were constructed. Several selective membranes were prepared for this purpose, having distinct mediator solvents (with quite different polarities) and, in some cases, p-tert-octylphenol (TOP) as additive. The latter was used regarding a possible increase in selectivity. The general working characteristics of all prepared electrodes were evaluated in a low dispersion flow injection analysis (FIA) manifold by injecting 500µl of citrate standard solutions into an ionic strength (IS) adjuster carrier (10−2 mol l−1) flowing at 3ml min−1. Good potentiometric response, with an average slope and a repeatability of 61.9mV per decade and ±0.8%, respectively, resulted from selective membranes comprising additive and bis(2-ethylhexyl)sebacate (bEHS) as mediator solvent. The same membranes conducted as well to the best selectivity characteristics, assessed by the separated solutions method and for several chemical species, such as chloride, nitrate, ascorbate, glucose, fructose and sucrose. Pharmaceutical preparations, soft drinks and beers were analyzed under conditions that enabled simultaneous pH and ionic strength adjustment (pH = 3.2; ionic strength = 10−2 mol l−1), and the attained results agreed well with the used reference method (relative error < 4%). The above experimental conditions promoted a significant increase in sensitivity of the potentiometric response, with a supra-Nernstian slope of 80.2mV per decade, and allowed the analysis of about 90 samples per hour, with a relative standard deviation <1.0%.

Electrochemical determination of dihydrocodeine in pharmaceuticals

Two analytical methods for the quality control of dihydrocodeine in commercial pharmaceutical formulations have been developed and compared with reference methods: a square wave voltammetric (SWV) method and a flow injection analysis system with electrochemical detection (FIA-EC). The electrochemical methods proposed were successfully applied to the determination of dihydrocodeine in pharmaceutical tablets and in oral solutions. These methods do not require any pretreatment of the samples, the formulation only being dissolved in a suitable electrolyte. Validation of the methods showed it to be precise, accurate and linear over the concentration range of analysis. The automatic procedure based on a flow injection analysis manifold allows a sampling rate of 115 determinations per hour.

Amperometric and spectrophotometric determination of carbaryl in natural waters and commercial formulations

The work presented describes the development and evaluation of two flow-injection analysis (FIA) systems for the automated determination of carbaryl in spiked natural waters and commercial formulations. Samples are injected directly into the system where they are subjected to alkaline hydrolysis thus forming 1-naphthol. This product is readily oxidised at a glassy carbon electrode. The electrochemical behaviour of 1-naphthol allows the development of an FIA system with an amperometric detector in which 1-naphthol determination, and thus measurement of carbaryl concentration, can be performed. Linear response over the range 1.0×10–7 to 1.0×10–5 mol L–1, with a sampling rate of 80 samples h–1, was recorded. The detection limit was 1.0×10–8 mol L–1. Another FIA manifold was constructed but this used a colorimetric detector. The methodology was based on the coupling of 1-naphthol with phenylhydrazine hydrochloride to produce a red complex which has maximum absorbance at 495 nm. The response was linear from 1.0×10–5 to 1.5×10–3 mol L–1 with a detection limit of 1.0×10–6 mol L–1. Sample-throughput was about 60 samples h–1. Validation of the results provided by the two FIA methodologies was performed by comparing them with results from a standard HPLC–UV technique. The relative deviation was <5%. Recovery trials were also carried out and the values obtained ranged from 97.0 to 102.0% for both methods. The repeatability (RSD, %) of 12 consecutive injections of one sample was 0.8% and 1.6% for the amperometric and colorimetric systems, respectively.

Electroanalytical study of the pesticide asulam

The electrochemical behaviour of the herbicide Asulam was studied by cyclic and square wave voltammetry. Asulam may be irreversibly oxidised at a glassy carbon electrode. Maximum currents were obtained at pH=1.9 in aqueous electrolyte solution. Based on the electrochemical behaviour of Asulam, two analytical methodologies were developed for its determination in water samples, using square wave voltammetry (SWV) and flow injection analysis (FIA) coupled with an amperometric detector. Limits of detection of 7.1x10-6 mol L-1 and 1.2x10-8 mol L-1 for SWV and FIA respectively, were achieved. Repeatability was calculated by assessing the relative standard deviation (%) for 10 consecutive determinations of one sample. The found values were 2.1% for SWV and 5.0% for FIA. Validation of the results provided by SWV and FIA methodologies was performed by comparison with results from an HPLC-DAD technique. Good relative deviations were found (<5%). Recovery trials were performed to assess the accuracy of the results and the obtained values were between 84% and 107% for both methods.